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28158437 | PMC5604590 | pmc | 5,678 | {
"abstract": "Corallimorpharians (coral-like anemones) have a close phylogenetic relationship with scleractinians (hard corals) and can potentially provide novel perspectives on the evolution of biomineralization within the anthozoan subclass Hexacorallia. A survey of the transcriptomes of three representative corallimorpharians led to the identification of homologs of some skeletal organic matrix proteins (SOMPs) previously considered to be restricted to corals. Carbonic anhydrases (CAs), which are ubiquitous proteins involved in CO 2 trafficking, are involved in both coral calcification and photosynthesis by endosymbiotic Symbiodinium (zooxanthellae). These multiple roles are assumed to place increased demands on the CA repertoire and have presumably driven the elaboration of the complex CA repertoires typical of corals (note that “corals” are defined here as reef-building Scleractinia). Comparison of the CA inventories of corallimorpharians with those of corals reveals that corals have specifically expanded the secreted and membrane-associated type CAs, whereas similar complexity is observed in the two groups with respect to other CA types. Comparison of the CA complement of the nonsymbiotic corallimorph Corynactis australis with that of Ricordea yuma , a corallimorph which normally hosts Symbiodinium , reveals similar numbers and distribution of CA types and suggests that an expansion of the CA repertoire has been necessary to enable calcification but may not be a requirement to enable symbiosis. Consistent with this idea, preliminary analysis suggests that the CA complexity of zooxanthellate and nonzooxanthellate sea anemones is similar. The comparisons above suggest that although there are relatively few new genes in the skeletal organic matrix of corals (which controls the skeleton deposition process), the evolution of calcification required an expanded repertoire of secreted and membrane-associated CAs.",
"introduction": "Introduction Corallimorpharia is a small and enigmatic anthozoan order closely related to the hard corals (order Scleractinia) but differing from them in that its representatives lack a skeleton. The relationship between corals and corallimorpharians has been equivocal, one factor in this being that—skeletons aside—they are essentially indistinguishable on morphological grounds ( den Hartog 1980 ; Medina et al. 2006 ; Daly et al. 2007 ; Kitahara et al. 2014 ; Lin et al. 2014 ). Although it has been argued that the corallimorpharian ancestor was a coral that underwent skeleton loss ( Medina et al. 2006 ), this idea has not been generally accepted (see, e.g., Budd et al. 2010 ; Barbeitos et al. 2010 ). Whole transcriptome scale phylogenomics implies that the Scleractinia and Corallimorpharia are distinct monophyletic groups ( Lin et al. 2016 ), thus the ability to deposit a massive aragonite skeleton evolved after the two orders diverged. However, the close relationship between these orders implies that corallimorpharians could be uniquely informative with respect to the evolution of the biomineralization process within the Hexacorallia. One approach to understanding the evolution of taxon-specific traits is provided by comparative genomics, and this has been employed to investigate some aspects of coral biology. For example, comparisons between the coral Acropora and the sea anemone Nematostella imply that a more complex immune repertoire is mandatory for the establishment and maintenance of symbionts by the former ( Shinzato et al. 2011 ; Hamada et al. 2013 ). Similar approaches indicate that the (noncalcifying) sea anemone has homologs of a number of the genes involved in skeleton deposition in corals ( Ramos-Silva et al. 2013 ), suggesting that relatively few new genes may have been required to enable calcification on scales characteristic of reef-building Scleractinia. Although the sea anemone genome has provided some important insights into coral biology, the depth of the coral/sea anemone divergence (around 500 Myr; Shinzato et al. 2011 ) limits the usefulness of such comparisons. The closer relationship between corals and corallimorpharians suggests that the latter may be more informative comparators, but until recently corallimorphs have been poorly represented in terms of available molecular data, whereas whole genome sequences ( Shinzato et al. 2011 ) and large transcriptome data sets (e.g. Moya et al. 2012 ) have been available for some time for corals, with at least twenty of varying quality and completeness now available ( Bhattacharya et al. 2016 ). Calcification has arisen independently many times during animal evolution. Within the Cnidaria, many octocorals deposit spicules composed of calcium carbonate in the form of calcite, whereas the skeletons of Scleractinia are composed exclusively of aragonite. Because calcification has arisen independently on multiple occasions, some of the components involved are unique to each lineage, but the chemistry of the process dictates that there is also a conserved component ( Moya et al. 2012 ). The latter category of genes includes those involved in ion transport and in controlling carbonate chemistry, for example, bicarbonate transporters ( Zoccola et al. 2015 ) and carbonic anhydrases (CAs) ( Jackson et al. 2007 ; Grasso et al. 2008 ; Bertucci et al. 2013 ). The nonconserved category of the calcification repertoire typically includes many of the genes whose products control the deposition of calcium carbonate to form the skeleton—for example, the heterogeneous skeletal organic matrix proteins (SOMPs) involved in mollusk calcification lack orthologs in other phyla. Considerable progress has recently been made in characterizing the calcification repertoire of corals. The first SOM protein to be identified in a coral is known as galaxin, and was originally identified by proteomic analyses of the skeleton of Galaxea fascicularis ( Fukuda et al. 2003 ). To date, four distinct galaxin-related sequences have been identified in Acropora ; two “adult-type” galaxins ( Reyes-Bermudez et al. 2009 ; Ramos-Silva et al. 2013 ) and two divergent but related “galaxin-like” sequences ( Reyes-Bermudez et al. 2009 ), which are not in the skeleton ( Ramos-Silva et al. 2013 ) but which, from their spatial expression, may be involved in laying it down. Galaxins are cysteine-rich repetitive proteins, each repeat containing one or more di-cysteine motifs. Each of the Acropora galaxins possesses an N-terminal signal peptide. After signal peptide cleavage, however, the adult-type galaxin proteins consist entirely of di-cysteine-rich repeat units, whereas acidic domains precede the repetitive regions in the mature forms of both of the Acropora millepora “galaxin-like” proteins ( Ramos-Silva et al. 2013 ). Galaxin-related sequences have been reported from a range of other animals, but these typically have low sequence similarity (e.g., Esgal1 from the squid Euprymna scolopes is involved in the establishment and maintenance of its bacterial symbiont Vibrio fischeri ; Heath-Heckman et al. 2014 ) and resemble each other only in containing di-cysteine repeat motifs. So it has been suggested that galaxins sensu stricto may be restricted to corals ( Reyes-Bermudez et al. 2009 ; Ramos-Silva et al. 2013 ). By applying proteomic approaches, Ramos-Silva et al. (2013 ) identified 36 SOMPs in the coral A. millepora . A similar study on Stylophora pistillata , another coral ( Drake et al. 2013 ), implicated some of the same components, but also regarded some nonskeletal proteins as SOMPs (Ramos-Silva, Marin, et al. 2013b). Two galaxins were amongst the SOMPs identified by Ramos-Silva et al. (2013 ), but the most surprising aspect of this analysis was that most (28) of the 36 SOMPs identified in Acropora have homologs that are either widespread or are present in Nematostella vectensis or Hydra spp, noncalcifying cnidarians for which whole genome data are available. Thus only 8 (SAP1, SAP2, SOMP1, SOMP2, SOMP3, SOMP4, SOMP6, and cephalotoxin-like) of the 36 SOMPs identified in Acropora were coral-specific (not found in anemones or other organisms), although note that the last of these had a surprising level of similarity to a mollusk protein ( Ramos-Silva et al. 2013 ). Based on immunolocalization to the calicoblastic ectoderm of S. pistillata , it has recently been suggested that a specific solute carrier—the bicarbonate active transporter (BAT) SLC4γ—plays a key role in the deposition of the coral skeleton by facilitating movement of inorganic carbonate to the site of calcification ( Zoccola et al. 2015 ). The presence of SLC4γ orthologs in a range of corals, but not in sea anemones, was taken as evidence that this gene played a key role in the evolution of biomineralization in the Scleractinia ( Zoccola et al. 2015 ). For this reason, the presence or absence of SLC4γ orthologs in corallimorpharians is important in terms of understanding the origins of calcification in corals. CAs are ubiquitous enzymes that catalyze the interconversion of HCO 3 − and CO 2 and are involved in a wide range of functions that includes pH buffering. In calcifying organisms, CAs have important additional roles in transporting carbonate to the site of calcification, hence these enzymes are a conserved component of the calcification repertoire ( Weis and Reynolds, 1999 ; Jackson et al. 2007 ; Moya et al. 2012 ). In symbiotic animals such as corals, CAs also function in ensuring the supply of CO 2 to the photosynthetic symbionts; note that a large proportion of CO 2 fixed by Symbiodinium in corals is derived from (coral) respiration ( Furla et al. 2000 ), and a large part of the fixed carbon may be exported back to the host (reviewed in Davy et al. 2012 ). These various demands have presumably driven the elaboration of complex CA repertoires that are typical of corals (see for review Bertucci et al. 2013 ). We recently reported ( Lin et al. 2016 ) the assembly of large transcriptome data sets for three corallimorpharians; Rhodactis indosinensis , Ricordea yuma , and Corynactis australis . To better understand the origins of the coral calcification repertoire, the transcriptomes of the corallimorpharians and those of representatives of other cnidarian groups were surveyed, focusing specifically on known components of the skeletal organic matrix, proteins associated with supplying carbonate to the site of calcification, or implicated in calcification on the basis of expression patterns in coral development. The results are consistent with the evolution of calcification requiring relatively few genomic changes in corals.",
"discussion": "Discussion To better understand how the ability to secrete an aragonite skeleton arose within the Scleractinia, we searched the transcriptomes of three representative corallimorpharians for homologs of genes implicated in coral calcification. Several caveats apply in interpretation of the comparative data. First, although considerable bodies of data from three corallimorpharian species are presented and the assembly statistics are good ( Lin et al. 2016 ; see also below), these data sets are incomplete. Thus, presences are more significant than absences. Second, comprehensive genome and transcriptome data are as yet available only for a very small number of coral species and it is unclear how well these reflect corals in general. One relatively robust conclusion from the comparative analyses is that corallimorpharian genomes encode clear homologs of some genes previously considered to be coral-specific. The identification of homologs of SOMP1 and B036-D5 in corallimorpharians means that surprisingly few of the genes known or suspected to be involved in the deposition of the skeleton are actually unique to corals. Many of those genes that are unique to corals are cysteine-rich (SOMP2, galaxins sensu stricto , SCRiPs) and are likely to have been recruited from structural ECM proteins ( Reyes-Bermudez et al. 2009 ). Subject to the caveats above, the apparent differences between corals and corallimorpharians in terms of the machinery involved in transport of inorganic carbon across membranes have important evolutionary implications. Although corallimorpharians lack skeletons, most of the tropical shallow-water species (28 of the ∼34 valid species) host the same photosynthetic symbionts as corals ( Symbiodinium spp.), as do 2 of the 3 species studied here ( Rhodactis and Ricordea ), hence their CA repertoires are of particular interest. The transcriptome surveys clearly imply that the evolution of biomineralization in the Scleractinia required expansion of the carbonic anhydrase repertoire, particularly of the secreted and membrane-associated type. Whereas a maximum of two sequences of this type was detected in the corallimorpharians surveyed ( fig. 2 ), ≥ 9 were detected in A. millepora and Porites lutea (see supplementary material S2, Supplementary Material online). Although only a smaller number (four) could be identified in the Pocillopora damicornis transcriptome (see supplementary fig. S3 , Supplementary Material online; Traylor-Knowles et al. 2011 ), this almost certainly reflects the incomplete nature of the assembly. Searching the P. damicornis transcriptome assembly revealed that 390 of the 753 genes comprising the BUSCO core metazoan set (i.e. 46%) were missing, whereas far fewer were missing from the corallimorpharian data sets (see supplementary table S1 , Supplementary Material online). Within the large clade of secreted and membrane-associated sequences, the branching pattern of coral sequences—distinct clades for A. millepora, P. lutea (and, in supplementary fig. S3 , Supplementary Material online, also for P. damicornis )—contradicted expectations. The most likely explanation for this branching pattern is that the sequences have undergone concerted evolution in each species, but alternative interpretations, including independent expansion of CA repertoires, cannot be rejected. As in the case of the Scleractinia, the repertoires of secreted and membrane-associated CAs have likewise been independently expanded in other calcifying invertebrates; this phenomenon has been documented in the case of calcisponges ( Voigt et al. 2014 ). The analyses presented as supplementary figure S3 , Supplementary Material online, imply that a similar expansion has occurred in the mollusk, Lottia gigantea , but the incomplete nature of many of the sequences means that it is unclear whether signal peptides and transmembrane domains are present in the sequences that group with the secreted and membrane-associated CAs from Cnidaria (both features are predicted in the case of one member of this clade, Lottia XP_009053021). Similar numbers of CAs, and a similar distribution across the various types, between Corynactis , a nonsymbiotic corallimorpharian, and Ricordea , which normally hosts Symbiodinium , suggests that, whereas an expansion of the CA repertoire was necessary to enable calcification, it may not be a requirement to enable symbiosis. Consistent with this idea, preliminary analysis suggests that the CA complexity of symbiotic and nonsymbiotic sea anemones is similar. Conversely, on the basis of coral-sea anemone comparisons, it has previously been suggested that the recognition and maintenance of appropriate symbionts may require a more sophisticated innate immune repertoire ( Shinzato et al. 2011 ). With the availability of data for symbiotic and nonsymbiotic corallimorpharians (this paper) and the symbiotic sea anemone Exaiptasia ( Baumgarten et al. 2015 ) this idea can now be more thoroughly investigated. Are corallimorpharians simply corals that have lost their skeletons, as has been suggested ( Medina et al. 2006 ), or did calcification evolve after the Scleractinia diverged from Corallimorpharia? Data presented here and elsewhere imply that the evolution of calcification required at least one novel bicarbonate transport protein (SLC4γ; Zoccola et al. 2015 ) and an expansion of the carbonic anhydrase repertoire, particularly the secreted and membrane-associated types, as well as the recruitment of some ECM-derived genes to control the deposition process. If the corallimorpharian ancestor lost the ability to calcify, those genes—including a large number of carbonic anhydrase isoforms—have been lost, which is a less parsimonious explanation than if calcification post-dates the coral-corallimorpharian divergence. However, fewer loss events may be required if coral CAs are encoded by linked loci, and linkage seems likely given their apparent concerted evolution."
} | 4,169 |
35275725 | PMC8916725 | pmc | 5,680 | {
"abstract": "Reef-building corals maintain an intracellular photosymbiotic association with dinoflagellate algae. As the algae are hosted inside the symbiosome, all metabolic exchanges must take place across the symbiosome membrane. Using functional studies in Xenopus oocytes, immunolocalization, and confocal Airyscan microscopy, we established that Acropora yongei Rh (ayRhp1) facilitates transmembrane NH 3 and CO 2 diffusion and that it is present in the symbiosome membrane. Furthermore, ayRhp1 abundance in the symbiosome membrane was highest around midday and lowest around midnight. We conclude that ayRhp1 mediates a symbiosomal NH 4 + -trapping mechanism that promotes nitrogen delivery to algae during the day—necessary to sustain photosynthesis—and restricts nitrogen delivery at night—to keep algae under nitrogen limitation. The role of ayRhp1-facilitated CO 2 diffusion is less clear, but it may have implications for metabolic dysregulation between symbiotic partners and bleaching. This previously unknown mechanism expands our understanding of symbioses at the immediate animal-microbe interface, the symbiosome.",
"introduction": "INTRODUCTION Photosymbiotic associations between invertebrates and microalgae are widespread in aquatic environments. Perhaps the most well known of these partnerships is that of reef-building corals (phylum: Cnidaria) and dinoflagellate symbiotic algae (family: Symbiodiniaceae), which is key to the evolutionary success of coral reef ecosystems ( 1 ). In an otherwise oligotrophic environment, the cnidarian host satisfies the majority of its energetic needs using photosynthates derived from its symbiotic algae ( 2 ). The host cells are believed to exercise considerable control over the metabolism of their symbionts, which favors both the production and release of algal photosynthates. This control is possible because of an architectural arrangement whereby coral gastrodermal cells host the algal symbionts intracellularly within an arrested phagosome known as the symbiosome [reviewed in ( 3 )]. Because the symbiosome isolates the alga from the cytosol of the host cell, the symbiosome membrane necessarily mediates all metabolic exchanges between the symbiotic partners. In addition, the symbiosome membrane may serve as an interface for the coral to manipulate the alga’s microenvironment. For example, the coral symbiosome is markedly acidic (pH ~4) because of active H + pumping by V-type H + -ATPases (VHAs) located in the symbiosome membrane ( 4 ). The acidic nature of the symbiosome drives CO 2 accumulation as part of a carbon concentrating mechanism (CCM) that helps overcome the low affinity of algal Rubisco for CO 2 , thereby promoting algal photosynthesis ( 4 ). This H + gradient has been proposed to additionally energize the movement of other essential nutrients and metabolites into or out of the symbiosome including nitrogen, phosphorus, and sugars ( 3 , 4 ). However, no additional molecular players or regulatory mechanisms have been definitely identified to date. The vast majority of the symbiotic algae’s nitrogen demand is supplied by protein catabolism by their animal host, which produces waste as ammonia gas (NH 3 ) and ammonium ion (NH 4 + ), which exist in a pH-dependent equilibrium [collectively referred to as “total ammonia” (Tamm)] ( 2 , 5 ). Rather than excreting its nitrogenous waste into the environment like most other aquatic animals ( 6 ), the coral symbiosis recycles a substantial portion of Tamm via the glutamine synthase/glutamate dehydrogenase/glutamine oxoglutarate aminotransferase pathways (GS/GDH/GOGAT) ( 5 , 7 , 8 ). In addition, corals are able to take up NH 4 + from seawater and transport it to their algal symbionts ( 9 ), and isolated algal symbionts take up and use NH 4 + ( 10 ). Moreover, coral host cells are known to regulate Tamm delivery to their symbionts, and as a result, the algae accumulate significantly more nitrogen in the light than in the dark ( 9 , 11 ). The diel regulation of Tamm delivery by corals allows for host control over the carbon and nitrogen metabolisms of symbionts ( 12 ) and, by extension, the growth rate and biomass of the symbiont population to prevent symbiont overgrowth that would disrupt the symbiosis ( 13 ). Transcriptomic analyses on whole coral colonies have identified candidate transporters proposed to mediate Tamm delivery to symbionts ( 14 ), but a lack of localization studies precludes a definite assessment of their involvement in symbiosis. Overall, the mechanisms that mediate and regulate nitrogen transport to symbionts across the symbiosome membrane remain unknown. NH 3 and NH 4 + exist in pH-dependent equilibrium with pKa ~9.25, and thus >96% of Tamm is found as NH 4 + both in seawater (pH ~8) and in coral host cells (pH ~7.4) ( 15 ). However, the much lower pH in the symbiosome space has three critical and interlinked implications: first, a virtually nil NH 3 partial pressure ( p NH 3 ) in the symbiosome space that should drive NH 3 gas diffusion from the host cytoplasm; second, the immediate “trapping” of NH 3 as NH 4 + in the symbiosome space, which can be taken up by the alga, thus maintaining the inwardly directed NH 3 diffusion gradient; and lastly, an unfavorable electrochemical gradient for NH 4 + transport into the symbiosome. However, despite being a gas, NH 3 has limited permeability through lipidic membranes because of its strong dipole moment that makes it a polar molecule [reviewed in ( 16 )]. In some plant-bacteria symbioses, NH 3 transport across the symbiosome membrane is facilitated by nodulin-intrinsic proteins ( 17 , 18 ); however, this protein family is exclusive to plants. In addition, NH 3 diffusion across biological membranes can be significantly enhanced by Rhesus (Rh) channels, a family of evolutionary conserved proteins present in eubacterial, invertebrate, and vertebrate lineages ( 19 – 21 ). On the basis of the observed up-regulation of an Rh-like mRNA transcript upon establishment of symbiosis in anemones ( 22 – 24 ), Rh channels have been suggested to play important roles in cnidarian-algae symbioses. However, the Rh-like mRNA was expressed in many soft coral cell subtypes ( 25 ), and therefore, the coded protein probably plays multiple physiological roles. In addition, Rh channels are typically present in the cell outer plasma membrane [reviewed in ( 16 , 26 )], and few studies have localized Rh-like proteins to intracellular compartments or organelles ( 27 , 28 ). Last, the various Rh protein isoforms have different substrate specificity: some may transport both NH 3 and NH 4 + ( 16 ), some act as dual NH 3 and CO 2 gas channels ( 19 , 29 , 30 ), and others do not facilitate Tamm/CO 2 transport across membranes at all and have structural functions instead ( 31 , 32 ). However, detailed functional studies about transport properties by “primitive” Rh proteins from invertebrate animals (termed “Rhp”) are very scarce. As a result, assessing the physiological role of the coral Rh-like coded protein and its potential involvement in delivering Tamm to algal endosymbionts requires elucidating its actual function as well as its cellular and subcellular localizations. Furthermore, if coral Rh facilitated CO 2 diffusion and was present on the symbiosome membrane, it would provide a pathway for CO 2 backflow from the symbiosome into the coral gastrodermal cells and affect interactions between nitrogen transport and the CCM. Given that NH 3 diffusion through biological membranes is limited, we hypothesized that corals use Rh-like proteins to deliver NH 3 to their algae across the symbiosome membrane, which would subsequently get trapped as NH 4 + in the acidic symbiosome. To investigate this possibility, we cloned an Rh-like gene from the coral Acropora yongei ( ayRhp1 ) and determined its phylogenetic relationship to other Tamm-transporting proteins. Then, we heterologously expressed ayRhp1 protein in Xenopus oocytes and measured Tamm transport under a range of pHs to determine whether it transports NH 3 , NH 4 + , or both. In additional oocyte experiments, we determined whether ayRhp1 facilitates CO 2 diffusion. Using custom-made antibodies and immunocytochemistry, we established the localization of ayRhp1 protein in the various cell subtypes throughout the coral colony, and, using confocal Airyscan microscopy, we investigated whether ayRhp1 was specifically located in the symbiosome membrane. Last, we quantified the subcellular localization of ayRhp1 within algae-containing coral gastrodermal cells throughout a diel cycle to explore a potential mechanism whereby coral host cells could regulate Tamm delivery to their algal symbionts.",
"discussion": "RESULTS AND DISCUSSION Rhp1 genes are widespread in corals The cloned ayRhp1 cDNA open reading frame contains 1440 base pairs encoding a protein with a predicted molecular weight of 51.8 kDa. BLAST searches in genomic and transcriptomic databases revealed predicted ayRhp1 homologs in multiple coral species from both the robust and complex clades, which diverged from each other 300 million to 400 million years ago ( 33 ). These coral Rh proteins clustered together with Rhp1 genes from invertebrate animals (fig. S1). The protein features of ayRhp1 are similar to those of well-studied Rh channels from mammals (fig. S2). It has 12 transmembrane helices and an N-linked glycosylation site (N61), which differentiate all animal Rh50 channels capable of Tamm transport (Rhag-cg, Rhp1-2) ( 16 ) from the Rh30 proteins involved in structural functions ( 27 ). Crystallography and simulation studies have identified several key amino acid residues that are required for NH 3 transport across mammalian RhCG ( 20 , 21 ): a phenylalanine gate (F130 and F235) and a cytosolic shunt (L193, T325, L328, I334, N341, and N342), which recruit NH 4 + at the external and internal vestibules, respectively, twin histidines that deprotonate NH 4 + to NH 3 (H185 and H344), two highly conserved aspartic acid residues that help shuttle the H + back to the original compartment (D177 and D336), and a hydrophobic transmembrane channel that selectively conducts NH 3 but not NH 4 + ( 32 , 34 ). An alignment of ayRhp1 with RhCG reveals that the phenylalanine gate (F147 and F251), the twin histidines (H202 and H364), and analogous aspartate residues (D195 and D356) are all conserved in ayRhp1, while the cytosolic shunt and hydrophobic channel-lining residues are highly conserved (~83 and ~70%, respectively). In addition, ayRhp1 contains the nine residues that form the putative cytoplasmic CO 2 binding pocket of mammalian RhCG [which facilitates CO 2 transport ( 19 )]: L91, F94, D234, A237, M238, M299, V300, Q303, and N304. Six of these residues are also conserved in the Rh protein from the bacterium Nitrosomonas europaea , where the CO 2 binding pocket was definitely identified using x-ray crystallography ( 35 ). In summary, the overall high conservation of these key structures suggests that ayRhp1 can facilitate both NH 3 and CO 2 transport; this was experimentally tested through functional studies. ayRhp1 facilitates NH 3 and CO 2 diffusion ayRhp1 was functionally characterized by measuring Tamm uptake rates in Xenopus oocytes injected with ayRhp1 cRNA. To avoid potential artifacts resulting from using radiolabeled [ 14 C]-methylammonium as a Tamm analog ( 16 ), we used a hypochlorite-salicylate-nitroprusside–based colorimetric assay to directly measure Tamm accumulation in oocytes and estimate Tamm uptake rate. The bath solutions contained 1 mM Tamm at pH 6.5, 7.5, or 8.5, resulting in 10-fold pKa-dependent [NH 3 ] increases for every pH unit (1.8, 17.4, and 150.5 μM, respectively). ayRhp1-expressing oocytes had consistently higher Tamm uptake rates than those of controls in all conditions tested ( P < 0.001; fig. S3). In addition, Tamm uptake rate in ayRhp1 oocytes significantly increased from 10.2 ± 1.4 pmol Tamm min −1 at pH 6.5, to 36.9 ± 2.8 pmol Tamm min −1 at pH 7.5, and to 49.6 pmol Tamm min −1 at pH 8.5 ( Fig. 1A ), an apparent J max and K m of 51.93 ± 1.45 pmol Tamm min −1 and 7.14 ± 0.91 μmol Tamm liter −1 , respectively ( Fig. 1B ). These results indicate that ayRhp1 transports NH 3 following the partial pressure difference. In addition, Tamm uptake rate of oocytes incubated in a solution with 10 mM Tamm at pH 7.5 was 50.3 ± 10.0 pmol Tamm min −1 (i.e., indistinguishable from the rate in the 1 mM Tamm pH 8.5 solution) ( Fig. 1B ). These two solutions have similar [NH 3 ] (174.0 versus 150.5 μM), but the former has >10-fold greater [NH 4 + ] than the latter (9826.0 μM versus 849.5 μM). Together, these results established that ayRhp1 can facilitate NH 3 diffusion following pH-dependent partial pressure gradients and that Tamm transport is not directly dependent on the [NH 4 + ] difference. Fig. 1. Functional characterization of total ammonia (Tamm) and CO 2 transport by Acropora yongei Rhesus protein (ayRhp1). ( A ) Effect of [NH 3 ] on Tamm uptake rate in Xenopus oocytes expressing ayRhp1. Control Tamm uptake rates have been subtracted. Data show means ± SEM of six to eight oocytes; the letters denote significant differences [one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test; pH 6.5 versus pH 7.5, P < 0.0001; pH 7.5 versus pH 8.5, P = 0.0222; pH 6.5 versus pH 8.5, P < 0.0001]. ( B ) Michaelis-Menten Tamm uptake kinetics calculated from the data shown in (A) (black dots to the left of the dotted line). Apparent J max = 51.93 ± 1.45 pmol Tamm min −1 and K m = 7.14 ± 0.91 μmol Tamm liter −1 . The red triangle indicates Tamm uptake rate obtained in a solution with 10 mM Tamm at pH 7.5 (175 μM NH 3 and 9.825 mM NH 4 + ) (i.e., similar [NH 3 ] to the previous data point, but ~10-fold higher [NH 4 + ]). ( C ) Functional characterization of CO 2 transport by ayRhp1. Xenopus oocytes expressing ayRhp1 (ayRhp1) display a higher rate of CO 2 release than control oocytes after equal CO 2 preloading. Data show means ± SEM of n = 8, 25 oocytes per n ; ** denotes significant differences (Welch’s t test; P = 0.0019). Next, we preloaded control and ayRhp1 cRNA-injected oocytes with 5% CO 2 and measured CO 2 release into normocapnic media using a custom-built CO 2 analyzer ( 36 ). These experiments revealed that ayRhp1-expressing oocytes released CO 2 at a rate ~50% faster than control oocytes ( P = 0.0019; Fig. 1C ). As wild-type Xenopus oocytes lack notable HCO 3 − efflux ( 37 ), this demonstrates that ayRhp1 can facilitate the diffusion of CO 2 in addition to NH 3 . ayRhp1 protein is present in multiple coral cell types Immunofluorescence microscopy using custom-made–specific antibodies revealed high ayRhp1 protein expression throughout A. yongei coral tissue sections ( Fig. 2A ). In the epidermis, ayRhp1 was present in the apical membrane of columnar cells along the seawater-coral interface ( Figs. 2B 1 and 3A ). Although corals recycle most of their nitrogen waste through their algal symbionts, they also excrete some Tamm to the environment ( 2 , 7 ). Thus, we hypothesize that ayRhp1 in epidermal cells aids in nitrogenous waste excretion as previously described in gills and skin from fish and aquatic invertebrates ( 38 , 39 ). Moreover, Tamm excretion may be facilitated by stirring of the boundary layer by ciliary beating, akin to mussels and polychaetes ( 38 , 40 ). Fig. 2. Immunolocalization of Acropora yongei Rhesus protein (ayRhp1). ( A ) Overview of A. yongei tissues; the boxes indicate regions of interest shown at higher magnification below, and the white arrowhead indicates ayRhp1-labeled calcifying cells. ( B 1 ) Apical membrane of columnar cells in the oral epidermis. ( C 1 ) Desmocyte with intense signal in its apical region. ( D 1 ) Alga-containing gastrodermal cells. ( B 2 , C 2 , and D 2 ) Corresponding bright-field differential interference contrast images; the white arrowheads mark corresponding locations in (B), (C), and (D). Coral and algal nuclei are shown in blue, and ayRhp1 immunofluorescence is shown in green. Several algal nuclei are marked with asterisks in (B 1 ) and (D 1 ) for clarity. This coral was sampled at midday. sw, seawater; co, coelenteron; sk, skeleton. Scale bars, 20 μm. Fig. 3. Confocal Airyscan immunolocalization of Acropora yongei Rhesus protein (ayRhp1) in the oral epidermis, desmocytes, and calcifying cells. ( A 1–3 ) ayRhp1 on the apical membrane of columnar cells in the oral epidermis. ( B 1–3 ) Desmocyte with intense ayRhp1 signal in its apical region. ( C 1–3 ) Calcifying cells displaying ayRhp1 signal on membranes and in the cytosol. Corresponding areas between panels are marked with arrowheads. [(A 1 ) to (C 1 )], [(A 2 ) to (C 2 )], and [(A 3 ) to (C 3 )] show ayRhp1, Na + /K + -ATPase (NKA), and 4′,6-diamidino-2-phenylindole (DAPI) signals, ayRhp1 signal alone, or NKA signal alone, respectively. Nuclei (DAPI) are shown in blue, ayRhp1 in green, and the NKA in purple. Scale bars, 5 μm. In the calicodermis, ayRhp1 was expressed in both calcifying cells that deliver dissolved inorganic carbon (DIC), Ca 2+ , and matrix proteins for skeletal formation and in desmocytes that anchor living coral tissue to the skeleton. The ayRhp1 signal in desmocytes was very intense, especially at the apical membrane adjacent to the skeleton ( Figs. 2C 1 and 3B and fig. S4A 1 ). Previous studies have provided morphological descriptions of coral desmocytes ( 41 , 42 ); however, to our knowledge, this is the first description of any protein specifically expressed in this cell type. As an NH 3 channel, ayRhp1 may contribute to coral calcification by enhancing NH 3 diffusion to buffer the pH of the extracellular calcifying medium (ECM) and maintain conditions favorable for calcification. Metabolic NH 3 has been proposed to promote biological calcification of avian egg shells ( 43 ), land snail shells ( 44 ), and coral skeletons ( 45 ) by buffering H + produced during CaCO 3 precipitation as NH 4 + . As a CO 2 channel, ayRhp1 could help deliver DIC to the ECM following the outwardly directed p CO 2 gradient favoring CO 2 diffusion from calicodermis cells into the ECM, which is an important source of DIC for calcification in multiple coral species ( 46 , 47 ). In the gastroderm, ayRhp1 was highly expressed in alga-hosting coral cells surrounding the symbiotic algae ( Fig. 2D 1 ), in a pattern that resembled that of VHA in the symbiosome membrane ( 4 ). This was explored in further detail. ayRhp1 is present in the symbiosome membrane Confocal Airyscan microscopy and coimmunostaining of ayRhp1 and Na + /K + -ATPase (NKA) allowed us to definitively establish ayRhp1’s subcellular localization within the tightly packaged host cells in coral tissues. Consistent with its universal presence in the plasma membrane ( 48 ), NKA outlined the perimeter of all alga-containing host cells ( Fig. 4, A and B ). The ayRhp1 signal was also present around the algae; but in most cells, it was internal to that of NKA and also present in the thin region between the host cell nucleus and the alga ( Fig. 4 A 1 ). Since these cells are tightly packed, this region is occupied by the symbiosome membrane ( 4 , 49 ). This was most readily evident in the region adjacent to the coral nucleus where a region of cytosol separates the external NKA and internal ayRhp1 signals, further indicating their respective presence in the plasma and symbiosome membranes ( Figs. 4 A 2 and 3). The symbiosomal localization of ayRhp1 was further confirmed in host cells containing two algae (fig. S5C), which are rather scarce but contain a larger cytoplasmic region that allows for better visualization of subcellular compartments. In addition, a minority of cells lacked ayRhp1 in the region between the host nucleus and the algae ( Fig. 4B ), which instead colocalized with or appeared slightly internal to NKA around the nuclear periphery, indicating ayRhp1’s presence in the host plasma membrane, cytosolic vesicles, or both. We attempted to quantify the two ayRhp1 subcellular localization patterns using tissue sections; however, the high density of tightly packed gastrodermal cells coupled with their intense NKA signal confounded imaging and prevented an unbiased approach. To circumvent these limitations, we immunostained isolated coral cells, an approach we previously used to confirm the presence of VHA in the symbiosome membrane ( 4 ). Similar to tissue sections, a majority of isolated cells displayed ayRhp1 signal in the thin region between the host cell nucleus and the alga, indicative of ayRhp1 symbiosomal localization (fig. S5, B, C, and E to I). We also observed a minority of cells with ayRhp1 signal around the host cell nucleus (fig. S5, D and J to N), indicative of ayRhp1’s presence in the cytoplasm or plasma membrane of the host cell. Free algal cells released during the isolation procedure, identified by the lack of an adjacent host nucleus, did not have ayRhp1 signal (fig. S5A). Fig. 4. Confocal Airyscan immunolocalization of Acropora yongei Rhesus protein (ayRhp1) in alga-containing coral cells. ( A 1 ) Cells displaying ayRhp1 in the symbiosome membrane of tissue sections. ( B 1 ) Cells displaying nonsymbiosomal ayRhp1 of tissue sections. ( A 2 and B 2 ) Higher magnification of the region denoted by the white boxes in (A 1 ) and (B 1 ). ( A 3 and B 3 ) Three-dimensional renderings of (A 2 ) and (B 2 ). Corresponding areas between (A 2 ) and (B 2 ) and (A 3 ) and (B 3 ) are marked. Nuclei are shown in blue, ayRhp1 in green, and the NKA in purple. Notice the separation (A 1–3 ) or colocalization (B 1–3 ) of ayRhp1 and NKA corresponding to symbiosomal or nonsymbiosomal ayRhp1 localizations, respectively. Host nuclei are denoted with an asterisk, and algal nuclei with arrowheads. Scale bars, 5 μm (A 1 and B 1 ), 0.5 μm (A 2 and B 2 ), and 0.5 μm (A 3 and B 3 ). ( C ) Percentage of total alga-containing A. yongei host cells with symbiosomal ayRhp1 over a diel cycle. Data show means ± SEM. n = 3 per time point, 50 cells per n , 900 cells total. The asterisks indicate significant differences with the 1300-hour time point (two-way repeated-measures ANOVA followed by Dunnett’s posttest; ** P < 0.01; *** P < 0.0001). Diel trafficking of ayRhp1 On the basis of established patterns of nitrogen delivery to coral algal symbionts ( 9 , 11 ), we hypothesized that the ayRhp1 subcellular localization would change in a diel fashion. We therefore quantified ayRhp1 subcellular localization patterns using epifluorescence microscopy on isolated alga-containing coral cells over a diel cycle. This allowed us to achieve sufficient replication (50 cells from 18 coral branches, 3 coral branches at each of six time points, for a total of 900 cells observed in blind fashion). Once an algal-containing coral host cell was identified, the observer rapidly and continuously shifted the focal plane and alternated between fluorescence and bright-field DIC while looking through the microscope eyepiece. This technique allowed the observer to determine whether the ayRhp1 signal was present in between the host cell nucleus and the alga (classified as “symbiosomal localization”; Fig. 4A and fig. S5, B, C, and E to I), or around the periphery of the host cell’s nucleus (“nonsymbiosomal localization”; Fig. 4B and fig. S5, D and J to N). The percentage of cells displaying ayRhp1 symbiosomal localization was significantly higher during the day, with a maximum of 61.3 ± 4.4% cells displaying this pattern at 1300 hours in contrast to only 26.0 ± 2.0% of cells at 2300 hours ( P < 0.001) ( Fig. 4C ). These results indicate that ayRhp1 is preferentially present in the symbiosome membrane during the day. To our knowledge, this is the first report of diel changes in proteomic makeup of the cnidarian symbiosome membrane and furthers the notion that this interface that separates symbiotic partners can be dynamically modified by the host cell to control the physiology of the alga. A putative host-controlled nitrogen concentrating mechanism The pKa for Tamm combined with the pH difference between the host cell’s cytosol and the symbiosome dictates >2000 higher p NH 3 in the former. Although this establishes a steep partial pressure gradient favoring NH 3 diffusion into the symbiosome, diffusion across lipidic membranes is generally limited [reviewed in ( 16 )]. The presence of ayRhp1 in the symbiosome membrane is poised to overcome this limitation, thus enhancing NH 3 delivery to the algal symbionts in an analogous manner to nodulin-intrinsic proteins in plant- Rhizobium symbioses ( 17 , 18 ). Once inside the highly acidic symbiosome, NH 3 will be immediately converted into NH 4 + , which cannot move across the plasma membrane, or through ayRhp1. This mechanism is known as “NH 4 + acid trapping” and is well documented in diverse excretory epithelia from humans ( 20 ), teleost fishes ( 39 ), cephalopod and bivalve mollusks ( 38 , 50 ), and crustaceans [reviewed in ( 26 ); ( 20 , 38 , 39 , 50 )]. Moreover, the uptake of NH 4 + by the alga will ensure the continuous conversion of NH 3 into NH 4 + in the symbiosome, which in turn will maintain NH 3 diffusion from the host cytosol. By analogy to the CCM that facilitates symbiont photosynthesis ( 4 ), NH 3 transport by ayRhp1 coupled to acid trapping of NH 4 + in the symbiosome can be considered a host-controlled nitrogen concentrating mechanism (NCM). The high degree of conservation among cnidarian Rh channels (fig. S1) and the presence of an acidic symbiosome in anemones and corals from both the complex and robust clades ( 4 ) suggest that Rh-mediated NCMs are widespread in cnidarians. However, species- and environment-specific differences in the NCM contribution to Tamm transport may exist and must be explored. In addition, CO 2 -facilitated diffusion by ayRhp1 has implications for the host-controlled symbiosomal CCM ( 4 ). In this model, H + transport by VHA generates an acidic symbiosome that drives the DIC equilibrium toward CO 2 accumulation in the symbiosome space, which then diffuses into the alga where it is fixed by algal Rubisco. The presence of ayRhp1 in the symbiosome membrane may initially seem counterproductive for the CCM, as it provides a pathway for CO 2 to leak back into the host cell cytoplasm. Continued carbon fixation by the algae, however, ensures a more favorable gradient for CO 2 diffusion into the algae compared with the host cell. The CO 2 that backflows into the coral host cell would be hydrated by cytosolic carbonic anhydrases (CA) into H + and HCO 3 − ( 51 ), which can then be used as substrates for VHA and HCO 3 − transporters in the symbiosome membrane. Furthermore, the fluid in the coelenteron and mitochondria in the gastrodermal cells ( 4 ) are additional sources of CO 2 that can fuel symbiosome acidification ( Fig. 5 ). This mechanism is akin to the human kidney collecting duct, where VHA, CAs, HCO 3 − transporters, and Rh channels interact with each other for the purposes of HCO 3 − reabsorption and Tamm excretion ( 52 ). Fig. 5. Model of the coral nitrogen concentrating mechanism in alga-containing coral host cells. ( 1 ) Coral mitochondria produce NH 3 and CO 2 . ( 2 ) Cytosolic CA catalyzes CO 2 hydration into H + and HCO 3 − . ( 3 ) H + and ( 4 ) HCO 3 − are moved into the symbiosome space by VHA and an unidentified HCO 3 − transporter, respectively. Inside the symbiosome, H + and HCO 3 − dehydrate into CO 2 , which diffuses into and is photosynthetically fixed by the alga. ( 5 ) NH 3 diffuses via the A. yongei Rhesus protein (ayRhp1) into the symbiosome space, where it is immediately protonated and trapped as NH 4 + . An unidentified algal transporter imports NH 4 + into the alga, where it is assimilated. Some CO 2 also diffuses via ayRhp1 back into the host cell’s cytosol, where it is rehydrated and transported back to the symbiosome space. ATP, adenosine 5′-triphosphate. The increased proportion of cells displaying symbiosomal ayRhp1 localization during the day matches established patterns of increased Tamm delivery from host to symbiont ( 53 ) and symbiont nitrogen assimilation ( 9 ) during light conditions. The diurnal nitrogen supply is primarily used not to advance growth but to sustain a high turnover of photosystem proteins and pigments damaged by ultraviolet radiation and electron transfer, which is essential for continued and efficient photosynthesis ( 9 , 54 , 55 ). This situation highlights the need for unique regulatory mechanisms in photosymbiotic associations compared with symbioses with nonphotosymbiotic microbes, such as those in plant roots. Conversely, the removal of ayRhp1 from the symbiosome membrane at night would serve to restrict nitrogen supply to symbiotic algae, thus limiting the synthesis of nonphotosynthetic proteins that would be essential to sustain their growth and reproduction ( 55 ). This mechanism gains additional significance when we consider that the coral symbiosome is highly acidic in both light and dark conditions ( 4 ), and this implies a continued steep inwardly directed p NH 3 gradient. Moreover, alga-containing gastrodermal cells are in contact with the gastrovascular cavity or coelenteron. This compartment contains Tamm at concentrations that can be several hundred-fold higher compared with seawater ( 56 ) and experiences steep diel pH fluctuations that can reach pH values as high as 9 during the day and as low as 6.75 at night ( 56 , 57 ). The presence of ayRhp1 channels in the host plasma membrane at night would facilitate the removal of NH 3 from the host cell into the coelenteron, where it would be trapped as NH 4 , further restricting nitrogen supply to the algae at night. The regulation of nitrogen delivery via changes in ayRhp1 subcellular localization is not mutually exclusive with regulation via the GS/GDH/GOGAT pathway ( 11 , 58 , 59 ), and they complement each other. The involvement of this pathway is largely based on changes in gene expression or enzyme activity upon transitioning from symbiotic to aposymbiotic stages ( 22 ) or during long-term environmental disturbances ( 60 , 61 ). But despite diel mRNA expression patterns ( 62 ), the abundance of most metabolic enzymes in coral cells, including that of GS, does not seem to change on a diel basis ( 63 ). However, this does not preclude diel regulation of enzyme activity by posttranslational modifications or substrate availability that could act synergistically with ayRhp1 to control nitrogen to symbiotic algae. Moreover, ayRhp1 subcellular localization was not identical in all cells at any time period, indicating finer regulation based on position on the coral colony, symbiotic stage, or some other unidentified factors. Perspectives and limitations The ayRhp1- and VHA-dependent NCM identified here together with diel changes in ayRhp1 subcellular distribution provide a potential mechanism whereby coral host cells can supply nitrogen to their algal symbionts while still maintaining them in a nitrogen-limited state to control their growth under oligotrophic conditions. Alterations in nitrogen delivery to coral symbiotic algae have been linked to eutrophication and other environmental stressors that result in disruption of the symbiosis at the colony level, commonly known as coral bleaching ( 64 – 69 ). For example, heat stress may promote coral amino acid catabolism as a means to meet increased metabolic demand in a warmed environment, which has been suggested to trigger a feedback loop that releases symbionts from nitrogen limitation, uncouples the symbiotic relationship, and leads to bleaching ( 70 ). In addition, future studies must take into account that ayRhp1 is present in multiple cell types throughout coral tissues, which cannot be discerned using transcriptomics, proteomics, or metabolomics assays on bulk coral colony samples. Specifically, changes in ayRhp1 abundance could be driven by ayRhp1 in cells in the epidermis, gastrodermis, calicodermis, or combinations, and each of these conditions would reflect a unique coral response, in some cases with opposite implications for coral health. With this in mind, techniques that allow the investigation of coral biology at the cellular and molecular levels such as nanoscale secondary ion mass spectrometry (“nano-SIMS”) ( 11 , 70 ) and confocal microscopy ( 71 – 73 ) are essential complements to “-omics” techniques. In particular, confocal Airyscan microscopy will allow studying physiological processes in calcifying cells and at the symbiosome membrane in unprecedent detail. Further work is required to determine whether the observed ayRhp1 symbiosomal localization is widespread among scleractinian corals. In addition, future studies should examine wild corals to ascertain the importance of this putative Rh channel–dependent NCM in the field. Future studies could also explore the role of other environmental nitrogen sources (i.e., urea and NO 3 − ) on host-symbiont metabolism. These are time- and materials-intensive tasks but are necessary to fully contextualize the coral NCM at the ecophysiological level."
} | 8,336 |
30774712 | PMC6367845 | pmc | 5,684 | {
"abstract": "Background The production of biohydrogen (H 2 ) as a promising future fuel in anaerobic hyperthermophiles has attracted great attention because H 2 formation is more thermodynamically feasible at elevated temperatures and fewer undesired side products are produced. However, these microbes require anoxic culture conditions for growth and H 2 production, thereby necessitating costly and time-consuming physical or chemical methods to remove molecular oxygen (O 2 ). Therefore, the development of an O 2 -tolerant strain would be useful for industrial applications. Results In this study, we found that the overexpression of frhAGB -encoding hydrogenase genes in Thermococcus onnurineus NA1, an obligate anaerobic archaeon and robust H 2 producer, enhanced O 2 tolerance. When the recombinant FO strain was exposed to levels of O 2 up to 20% in the headspace of a sealed bottle, it showed significant growth. Whole transcriptome analysis of the FO strain revealed that several genes involved in the stress response such as chaperonin β subunit, universal stress protein, peroxiredoxin, and alkyl hydroperoxide reductase subunit C, were significantly up-regulated. The O 2 tolerance of the FO strain enabled it to grow on formate and produce H 2 under oxic conditions, where prior O 2 -removing steps were omitted, such as the addition of reducing agent Na 2 S, autoclaving, and inert gas purging. Conclusions Via the overexpression of frhAGB genes, the obligate anaerobic archaeon T. onnurineus NA1 gained the ability to overcome the inhibitory effect of O 2 . This O 2 -tolerant property of the strain may provide another advantage to this hyperthermophilic archaeon as a platform for biofuel H 2 production. Electronic supplementary material The online version of this article (10.1186/s13068-019-1365-3) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions In this study, we demonstrated that the overexpression of frhAGB -encoding hydrogenase genes significantly enhanced O 2 tolerance of obligate anaerobe T. onnurineus NA1. This engineered strain overcame the inhibitory effects of O 2 and showed growth and H 2 production under oxic condition. This study gives an insight into the development of biotechnologically useful anaerobic H 2 producer and identification of an unknown function of the hydrogenase in T. onnurineus NA1.",
"discussion": "Discussion When the frhAGB gene cluster was overexpressed in T. onnurineus NA1, a recombinant strain (FO) showed better growth than the wild-type strain. The molecular mechanism underlying the enhanced cell growth is not yet understood. Instead, we attempted to exploit the potential of the FO strain using information from whole transcriptomic analysis. The expression of antioxidant-related genes encoding peroxiredoxin and alkyl hydroperoxide reductase subunit c was 2.5- and 4.1-fold up-regulated under the anaerobic conditions, respectively. The transcriptional up-regulation of antioxidant-related genes is usually associated with cellular responses to oxidative stress. However, the sources for oxidative stress are unidentified. Extracellular O 2 is usually removed by the addition of a reducing agent, autoclaving the medium, and inert gas purging. The absence of O 2 in our experiment was evidenced by minimal or no change in the transcriptional expression of other antioxidant-related genes, such as thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), and thioredoxin reductase (TON_1603), which were identified to be strongly up-regulated in the presence of O 2 (manuscript forthcoming). We speculated that the FO strain, with two antioxidant-related genes up-regulated, might exhibit changes in O 2 tolerance. To verify this hypothesis, the O 2 sensitivities of the FO strain, wild-type strain, and ΔfrhA mutant were compared. The expression level of the frhAGB gene cluster had a strong influence on oxidative stress defense. In the ΔfrhA mutant, however, the transcript levels of genes encoding peroxiredoxin and alkyl hydroperoxide reductase subunit c were similar to those in the wild-type strain [ 28 ]. This result implies that there are more genes that contribute to oxidative stress defense in the FO strain. O 2 tolerance in obligate anaerobes by means of hydrogenases has been reported previously. In obligate anaerobic bacteria Desulfovibrio vulgaris strains, H 2 -consuming periplasmic soluble hydrogenase and c -type cytochrome couples were identified to play a role in O 2 reduction, thereby protecting against oxidative stress [ 29 , 30 ]. It has been reported that the hydrogenase was up-regulated when D. vulgaris Hildenborough was exposed to O 2 [ 30 ]. In another obligate anaerobic bacteria Geobacter sulfurreducens , a periplasmic hydrogenase Hya was identified to be necessary for growth after exposure to oxidative stress. While the Hya-deficient strain was more sensitive to the presence of superoxide or hydrogen peroxide, overexpression of Hya enabled the strain to endure oxidative stress better than the wild-type strain. However, the mechanism by which this hydrogenase contributed to the defense against oxidative stress relative to the promotion of antioxidant enzyme activity was not elucidated [ 31 ]. In this study, the FO strain was distinct in that it was able to achieve similar growth yields under oxic conditions, where most anaerobes and the wild-type T. onnurinues NA1 strain are incapable of growth due to the high O 2 level, compared with those of anoxic conditions. In addition, the maximum H 2 production rates and maximum specific H 2 production rates of the FO strain cultured under both oxic and anoxic conditions were quite similar to the high values for the WTF-156T strain, which had been engineered by adaptive evolution on formate-supplemented medium [ 23 ]. This feature can ameliorate the need to tightly maintain the anaerobic environment prior to cultivation in a batchwise or continuous system, where a certain level of O 2 contamination is inevitable, by operating with the addition of carbon sources or other nutrients, or by maintaining a constant pH using strong acid or bases. Therefore, the ability of the FO strain to tolerate O 2 would make it even more suitable for industrial applications. Our study implicates that other anaerobic H 2 producers might be relieved from the strict control of O 2 during growth through augmentation of the defense against oxidative stress. It would be helpful if we could get high amounts of whole cell biocatalysts and even purified enzymes from them easily by growing those strains under oxic conditions while the activity of enzymes, such as hydrogenases, FHLs, and HDCRs, essential for H 2 production [ 12 , 14 , 16 , 17 ], is untouched. This study helps to understand the physiological role of the frhAGB -encoding hydrogenase, which is distinct from Frh hydrogenases of methanogens and is thus far poorly understood. In our previous report on the frhA -deletion mutant, it was shown that the hydrogenase might be associated with the regulation of gene expression in a non-methanogen [ 28 ]. For example, frhA gene deletion caused up-regulation of the codh – mch – mnh3 gene cluster essential for CO-dependent H 2 production even in the absence of external CO and led to significant increases in cell growth (2.8-fold) and H 2 production (3.4-fold) [ 18 ]. In this study, overexpression of frhAGB genes also up-regulated the expression of antioxidant-related genes without exposing them to an oxidizing agent such as O 2 . Furthermore, the FO strain was superior to the wild-type strain with respect to growth under oxic conditions. Regulatory hydrogenases HupUV and HoxBC have been reported to participate in the transcriptional regulation of gene expression [ 25 ]. These hydrogenases function as H 2 sensors in two-component regulatory systems, which consist of a protein histidine kinase and a response regulator [ 32 ]. Two-component regulatory systems are distributed widely in bacteria, but only a few are found in archaea [ 33 ]. Therefore, if the soluble hydrogenase is playing a regulatory role, its mechanism in T. onnurineus NA1 awaits further studies. It is noteworthy that two genes encoding transcriptional regulatory proteins (TON_0836 and TON_1663) were down-regulated by overexpression of frhAGB genes. Further studies will be required to address whether these regulators are involved in the regulation of gene expression by the frhAGB -encoding hydrogenase. Contributions of other up- or down-regulated genes by overexpression of frhAGB genes cannot be ruled out; therefore, this issue requires further investigation."
} | 2,197 |
32046260 | PMC7077370 | pmc | 5,685 | {
"abstract": "In this paper, the anticipated challenges and future applications of self-healing composite materials are outlined. The progress made, from the classical literature to the most recent approaches, is summarized as follows: general history of current self-healing engineering materials, self-healing of structural composite materials, and self-healing under extreme conditions. Finally, the next stage of research on self-healing composites is discussed.",
"conclusion": "4. Concluding Remarks In addition to the cracks on aircraft fuselage and nuclear power plant walls mentioned above, numerous structures require protection as human activities expand to such areas as submarine tunnels and spacecraft. As the space age accelerates, it is expected that individuals will actually travel through space and explore unknown worlds. For example, as events such as a micro-meteor impacting personal spacesuits at 50 km/s [ 33 ] or piles of stones hitting undersea tunnels for years no longer exist only in the imagination, the performance and circumstances of self-healing in the future should be considered under more severe conditions. Very fortunately, for the past 30 years, materials and successful approaches have been found that enlarge self-healing applications. Hopefully, the core principles will be understood soon, and the fundamental solutions will be revealed.",
"introduction": "1. Introduction In nature, self-healing is an autonomous and a fascinating phenomenon that can be observed in most living organisms. Wounds such as scratched skin or fractured bones are easily healed through the activity of the vascular system [ 1 ]. The survival of animals and plants depends on their expansion owing to their restorative capacity. Such bio-inspired recovery of engineering materials, namely, \"self-healing\" following external damage, has been studied for the past 30 years [ 2 , 3 ]. When blood is released from scratched skin, healing substances are released, solidify, and aggregate along the ruptured area. Nature-inspired self-healing features have been explored in biomimetic designs and healing strategies. Structural damage is repaired through the systematic transportation of the healed material and the polymerization-based curing process of the damaged area. The first generation of self-healing studies were performed using microcapsules [ 4 ]. The capsule was certainly viable and did not require external energy to begin the healing process. However, the layer with the capsule was thick owing to the bulky microcapsules. Moreover, in the view of repeatable healing, these capsules were used only once. Therefore, new approaches for small healing substances and multiple healing abilities were required. As observed in mammals or plants, the vascular network enables rapid and continuous transportation of healing substances to the damaged area. This effective microvascular system is composed of a network structure and perfectly covers the entire body/surface. For the Gen. II self-healing research, the main mechanism of such self-healing, the vascular capillary network, which carries the healing substance, was studied [ 5 , 6 , 7 ]. Although numerous self-healing materials have been developed, it remains unclear whether the methods proposed for fabricating these materials are economically feasible and scalable to the industrial level. For example, capsule-based self-healing methods exhibit several disadvantages, such as low uniformity of the dispersed capsules and complicated fabrication processes [ 8 ]. To overcome these drawbacks, several fiber-based self-healing approaches have been introduced in recent years, one of which, solution blowing, has already been scaled up. Nevertheless, the range of materials that can be used as a shell for encasing the core materials in these core-shell nanofibers is severely limited. Capsule-based methods allow for the use of a wider range of materials within their limitations. Moreover, hybrid methods, including both capsule-based and fiber-based approaches, require further development to maximize their advantages. For example, self-healing composites consisting of fast-healing capsules, and small-sized self-healing core-shell nanofibers can be used to repair damage in a wide range of cracks, such as those that are several nanometers in size (owing to the nanofibers). Moreover, thanks to capsules that are hundreds of nanometers wide, such a hybrid approach will not exhibit the limitations associated with slow healing in terms of low uniformity owing to the capsule use and the presence of nanofibers. Finally, the addition of corrosion inhibitors or the use of pH and redox polymers can further improve the self-healing performance [ 9 ]. Applications including the interfacial strengthening of composite laminates for the aviation and automotive industries remain significant as a means of protection against impact damage and fatigue cracking. Therefore, a nano-textured self-healing interleaved structure aimed at interfacial enhancement is required. Furthermore, the development of innovative technologies, such as the proliferation of soft robots and actuators, as well as products based thereon, requires innovative flexible self-healing composites that can withstand multiple operation cycles without fatigue crack growth."
} | 1,319 |
28841373 | null | s2 | 5,686 | {
"abstract": "DNA nanostructures assembled on living cell membranes have become powerful research tools. Synthetic lipid membranes have been used as a membrane model to study the dynamic behavior of DNA nanostructures on fluid soft lipid bilayers, but without the inherent complexity of natural membranes. Herein, we report the assembly and disassembly of DNA nanoprisms on cell-mimicking micrometer-scale giant membrane vesicles derived from living mammalian cells. Three-dimensional DNA nanoprisms with a DNA arm and a cholesterol anchor were efficiently localized on the membrane surface. The assembly and disassembly of DNA nanoprisms were dynamically manipulated by DNA strand hybridization and toehold-mediated strand displacement. Furthermore, the heterogeneity of reversible assembly/disassembly of DNA nanoprisms was monitored by Förster resonance energy transfer. This study suggests the feasibility of DNA-mediated functional biomolecular assembly on cell membranes for biomimetics studies and delivery systems."
} | 252 |
38384856 | PMC10879693 | pmc | 5,687 | {
"abstract": "Summary Coral conservation requires a mechanistic understanding of how environmental stresses disrupt biomineralization, but progress has been slow, primarily because corals are not easily amenable to laboratory research. Here, we highlight how the starlet sea anemone, Nematostella vectensis , can serve as a model to interrogate the cellular mechanisms of coral biomineralization. We have developed transgenic constructs using biomineralizing genes that can be injected into Nematostella zygotes and designed such that translated proteins may be purified for physicochemical characterization. Using fluorescent tags, we confirm the ectopic expression of the coral biomineralizing protein, SpCARP1, in Nematostella . We demonstrate via calcein staining that SpCARP1 concentrates calcium ions in Nematostella , likely initiating the formation of mineral precursors, consistent with its suspected role in corals. These results lay a fundamental groundwork for establishing Nematostella as an in vivo system to explore the evolutionary and cellular mechanisms of coral biomineralization, improve coral conservation efforts, and even develop novel biomaterials.",
"introduction": "Introduction Coral reefs represent some of the most biodiverse ecosystems on Earth 1 , 2 , 3 and are necessary for maintaining healthy coastlines. 4 , 5 The backbone of these marine ecosystems are stony corals that, due to increased environmental stresses, are rapidly in decline. 6 Conservation efforts have been hampered, at least in part, by our limited understanding of the basic biology of corals and their ability to biomineralize and generate a diverse array of calcium carbonate skeletons that are susceptible to demineralization from changing ocean temperatures and acidification. Any meaningful effort to reverse the decline of corals requires a mechanistic understanding of 1) the molecular and biochemical processes of coral biomineralization and 2) how biomineralization is disrupted by environmental stresses. Unfortunately, efforts to probe the molecular basis of biomineralization in corals have proven difficult because of a general lack of genetic tools and difficulties culturing corals in laboratory settings. Biomineralization is the production of inorganic minerals through biological mechanisms. This ability has evolved independently many times, resulting in unique structures such as bivalve shells, 7 , 8 , 9 , 10 sea urchin spicules, 10 , 11 , 12 , 13 , 14 and coral skeletons. 15 , 16 , 17 In marine organisms, the most studied mineralization pathways involve the absorption of Ca 2+ 18 , 19 into cells expressing membrane-associated alpha carbonic anhydrases that convert CO 2 to bicarbonate. 20 , 21 , 22 , 23 This results in the production of amorphous calcium carbonate (ACC) precursors that are stabilized to form crystal structures secreted into the extracellular microenvironment. 24 , 25 , 26 , 27 ACC precursors attach to the growing surface of the coral skeleton and crystallize into aragonite under the control of highly acidic biomineralization proteins. 27 , 28 The acid-rich regions localize calcium ions, increasing the ionic strength within the calcification microenvironment. 29 These proteins, together with other molecules, behave as organic substrate that appears to serve as a nucleation site. 17 Energetically favorable conditions for biomineralization can arise spontaneously and rapidly, suggestive of a mechanism by which ACC biomineralization could have evolved independently through the use of non-homologous proteins with similar physicochemical characteristics that result in similar mineralized materials. 24 , 27 , 30 Secreted proteins within mineralizing cells have been shown to catalyze nucleation 29 , 31 and/or interact with ACC precursors to provide stability as mineralizing tissue becomes more structured and complex. 13 , 27 , 32 , 33 , 34 These proteins are considered to be “intrinsically disordered” (IDP) because they have no set tertiary structures. 35 , 36 , 37 , 38 Although biomineralizing species may not share homologous IDPs, many of these proteins contain similar properties such as highly acidic residues 8 , 39 , 40 , 41 , 42 and post-translational modifications that modify their folding and biomineralizing activity. 32 , 34 , 42 , 43 , 44 , 45 Existing biomineralization models have distinct advantages and disadvantages. 32 , 44 , 45 Bacterial expression systems help clarify the role of carbonic anhydrases in biomineralization, 44 , 45 , 46 yet they are unable to modify proteins endogenously after translation and therefore cannot be used for elucidating the role of post-translational modifications in biomineralization. Eukaryotic cell cultures may be useful for testing the functional role of post-translational modifications of marine proteins, with the most success coming from mollusk nacre proteins expressed in insect lines. 47 , 48 However, to our knowledge, few groups have been able to establish stable cell cultures derived from marine invertebrates. 49 , 50 Existing cell lines rely on media that are not compatible with marine ecosystems, meaning any inferred insight into the evolutionary and biological mechanisms of biomineralization would need to be validated in vivo within a marine system. Marine invertebrates offer several advantages to in vivo assays of biomineralization. Sea urchins are useful as developmental models to understand the dynamics of spicule growth during embryonic skeleton formation 34 , 51 and syncytial mineralization. 52 Mollusks can be used to test the effects of novel, taxon-specific proteins on shell formation. 37 , 40 Corals are useful for characterizing how matrix proteins stabilize biominerals in extracellular microenvironments. 15 , 27 In each of these in vivo systems, mechanistic studies of the dynamic processes of biomineralization can be difficult to interpret due to the complexity of the interacting biomineralizing processes. 53 Here, we present the starlet sea anemone ( Nematostella vectensis ) as a model for studying the dynamic processes of biomineralization. Despite being in the same class (Anthozoa) as scleractinian corals, N. vectensis does not naturally mineralize, eliminating potential confounding factors of interacting mineralization reactions. 53 Comparative genomics reveals that N. vectensis retains much of the molecular machinery believed to be necessary for biomineralization, including carbonic anhydrases as well as SpCARP1-related proteins such as Calumenin. 29 , 54 \n N. vectensis is a powerful developmental model that can easily produce thousands of embryos on demand with simple light and temperature cues. Many techniques for manipulating gene expression are already well established in N. vectensis, including CRISPR/Cas9 genome editing, 55 , 56 , 57 , 58 stable and transient cell-type-specific transgenesis, 59 , 60 , 61 , 62 and various forms of gene knockdown techniques (e.g., antisense morpholinos, RNAi, shRNA, dominant-negative approaches). 60 , 63 , 64 Together, these attributes make N. vectensis well-suited for investigating gene function during biomineralization. In this article, we show that N. vectensis can express transgenic proteins involved in biomineralization in other taxa and present a novel in vivo system to evaluate the ability of IDPs to self-assemble into hierarchical structures to interact with calcium ions to further understand biomineralization.",
"discussion": "Discussion We have demonstrated how N. vectensis , a soft-bodied anthozoan, may be utilized to study biomineralization in vivo . The putative promoters presented here were selected for optimizing the quantity and secretion of target biomineralization IDPs. Ubiquitin, as a regulatory protein that is highly conserved across eukaryotes, should be found in virtually every animal cell. Indeed, the cis- regulatory sequence we identified as a ubiquitin promoter appears to drive the broad expression of mCherry in a variety of cell types by 24 hpf ( Figures 2 A and 2B). Such selective expression could be due to an incomplete regulatory sequence or selective protein degradation. Our data shows the putative mucin promoter drives the expression of SpCARP1::mCherry within 48 hpf in secretory cells of the aboral ectoderm, with strong mosaic expression throughout the body column and into the tentacles of unfed polyps ( Figures 2 C and 2D), consistent with the expression of mucin. 66 Mucin-secreting cells are extremely abundant in the aboral ectoderm, and because these animals are excellent regenerators a stable transgenic line with the mucin promoter driving the expression of SpCARP1::mCherry should provide abundant material for future analyses of the interactions between SpCARP1 and putative IDPs. Corals have specialized cells that control the chemistry of seawater in a confined space where skeleton deposition occurs, otherwise defined as the “calcifying space.” Corals concentrate calcium and carbonate ions in this calcifying space, and IDPs such as SpCARP proteins control the nucleation of aragonite. 29 \n N. vectensis , as a non-calcifying organism, does not possess such specialized calcifying cells. By simulating the biomineralization-favorable conditions of high calcium and high carbonate concentrations, we were able to assess the responsiveness of SpCARP1 and detect regions within N. vectensis polyps where biomineralization may be most likely to occur. Analysis of the full length NvCaluF shows that it contains the characteristic acidic domain extension seen in SpCARP1 ( Figure S3 ; Table S1 ). NvCaluF mRNA has been shown to be exclusively expressed in stinging cells in late planulae and early primary polyps via in situ hybridization. 58 This expression pattern is consistent with reports of SpCARP1 being located in the oral epidermis and in association with stinging cells in S. pistillata tentacles. 67 By supplementing our 1/3X FSW with calcium- and/or carbonate-rich solutions and evaluating calcium sequestration with calcein staining ( Figures 3 , 4 , and 5 ; see also Figure S4 ), we show that the calcium-binding activity of SpCARP1 is primarily concentrated in the tentacles of N. vectensis polyps and seems to be enhanced with increased concentrations of carbonate ions in seawater ( Figure 4 ), a critical requirement for biomineral nucleation. Our results are consistent with the initial stages of the formation of amorphous calcium carbonate and suggestive of a gradual self-assembly mechanism that concentrates calcium as a function of exogenous expression of SpCARP1 in N. vectensis . Future studies can further assess the presence of mineral structures in N. vectensis tentacles using scanning electron microscopy or polarized light optical microscopy. We demonstrate that our experimental system is versatile and may be adapted to other forms of biomineralization. We show that N. vectensis can express IDPs involved in the CaCO 3 biomineralization of sea urchin spicules and CaPO 4 precipitation in vertebrate tooth enamel ( Figure S5 ). The persistence of fluorescent signal in dissociated cells ( Figure S3 ) suggests it should be possible to investigate the matrix-mediated polarization of IDPs and to test the role of intercellular interactions by taking advantage of 3D sculpting of dissociated transgenic N. vectensis cells. Given the amount of embryonic material provided in a single spawning cycle and the ease of injections, N. vectensis may be used as an expression system to generate large amounts of cells expressing biomineralizing IDPs that can be isolated, purified, and assayed in controlled in vitro crystallization environments. In all, our results hint at the possibility to expand the use of the N. vectensis system to other forms of biomineralization and perhaps even develop novel biomineralized materials for biomedical research. The primary focus of this study was to show how N. vectensis may be utilized to understand the molecular mechanisms that drive coral biomineralization to assist future conservation efforts. This study is the first to attempt to induce biological mineralization in a novel in vivo system. We chose the coral acidic protein SpCARP1 because it has been shown to induce rapid mineralization in vitro, 29 and to concentrate calcium ions leading to the formation of aragonite crystals in coral proto-polyps derived from cell cultures. 68 However, the calcium ion-concentration activity of such a protein has never been reported in live adult organisms, like we show here in Nematostella small polyps. We demonstrate that N. vectensis can both tolerate the transgenic expression of intrinsically disordered proteins involved in biomineralization from a range of taxa that can sequester and concentrate calcium ions in a carbonate-enriched seawater solution, providing compelling evidence for the initiation of the biomineralizing process in a non-mineralizing organism. These results highlight the potential of N. vectensis in examining the capacity of various cell types to secrete biominerals, opening up opportunities to understand the capacity of cells to acquire novel functions. Our model system may be used as a proxy to coral systems in the lab to test the molecular components of biomineralization that may improve stress tolerance and resilience to native coral populations, thereby filling a much-needed gap in coral research and aiding restoration efforts. Limitations of the study A single transgenic IDP is likely insufficient to lead to the formation of a mature skeleton. Nevertheless, this study lays the groundwork to establish N. vectensis as a tool to interrogate other coral IDPs, transporters, ion pumps, and so forth that are implicated in coral biomineralization and that can be co-expressed in the same or adjacent cell types in vivo . For example, another coral acid-rich protein, SpCARP4, is of particular interest because it is one of the most abundant proteins in the coral skeleton and has been suggested to guide the formation of calcium carbonate crystals to specific orientations. 67 SpCARP4 is the most abundant protein in the coral skeletal organic matrix 54 that localizes with mineral nanoparticles in early mineralization zones, desmocytes (modified cells that anchor tissue to the skeleton), and oral epidermis. 67 The expression of SpCARP4 has also been detected in calicoblasts, the cells involved in the production of calcium carbonate. 69 We predict that N. vectensis will be able to tolerate SpCARP4 transgenesis and, if expressed together with SpCARP1, reveal new insights into the interaction between different IDPs and their respective functions in biomineralization. Future studies should evaluate whether N. vectensis produces the post-translational modifications on SpCARP1 believed to promote biomineralizing activity. Such experiments may be cross-checked with site-directed mutagenesis and biochemical analyses of modified proteins to clarify position-specific roles of post-translational modifications on biomineralizing activities. These studies may help delineate the mechanisms that led calcifying cells to evolve independently in many organisms from a patchwork of nonhomologous proteins and cellular pathways. Such mechanistic studies are necessary to understand how biomineralizing organisms have responded to environmental changes in the past and how they may respond in the future, thereby elucidating how CaCO 3 biomineralization shapes Earth’s surface environment. 53 , 70 , 71 , 72 , 73"
} | 3,892 |
36061740 | PMC9434775 | pmc | 5,688 | {
"abstract": "The anode is considered to be a key factor to improve\nthe single-chamber\nbioelectrochemical system’s efficiency to degrade oily sludge\nin sediment while generating electricity. There are few studies on\nthe effect of the anode structure on the performance of oily sludge\nMFCs systematically. In this paper, an oily sludge bioelectrical system\nwas constructed using carbon felt and carbon plate as anode materials,\nadjusting the anode material arrangement as transverse and longitudinal,\nand using different anode materials from single to sextuple anodes.\nThe results of this study showed that the rate of degradation of oily\nsludge was greater with carbon felt (17.04%) than with the carbon\nplate (13.11%), with transverse (23.61%) than with the longitudinal\n(19.82%) arrangement of anodes, and with sextuple anodes (33.72%)\nthan with a single anode (25.26%) in the sediment microbial fuel cells\n(SMFCs). A similar trend was observed when the voltage, power density,\nand electromotive force (EMF) of SMFCs were estimated between the\ncarbon felt and carbon plate, transverse and longitudinal arrangements,\nsingle and sextuple anodes. It is concluded that the proper adjustment\nof anode arrangements, using carbon felt as an anode material, and\nincreasing the number of anodes to six may accelerate the rate of\ndegradation of oily sludge in oily sludge sediment microbial fuel\ncells (SMFCs). Furthermore, the electricity generation performance\nwas also improved.",
"conclusion": "4 Conclusions Oily sludge SMFCs can effectively\ndegrade crude oil and convert\nit into electricity. The anode structure will affect the oil removal\nand electricity generation performances of oily sludge SMFCs. The\nresearch results are as follows: (a) The crude oil removal rate (17.04%)\nof the oily sludge SMFCs using carbon felt as the electrode material\nwas better than that of the carbon plate and so were production voltage\n(215.33 mV), power density (81.49 mW/m 3 ), and electromotive\nforce (288.94 mV). (b) The crude oil removal rate (23.61%)\nof the SMFCs with horizontally arranged anodes was better than that\nof the SMFCs with vertically arranged anodes and so were production\nvoltage (290.40 mV), power density (104.49 mW/m 3 ), electromotive\nforce (429.29 mV). (c) When using multiple anodes as electrodes,\nwith the increase of the number of anodes, the voltage, power density,\nelectromotive force, and crude oil removal of SMFCs first increase\nand then tend to be stable. The maximum voltage, power density, electromotive\nforce, and crude oil removal rate were 408.82 mV, 145.32 mW/m 3 , 516.39 mV, and 33.72%, respectively. It can be seen that the proper adjustment of the anode\nstructure\ncan well improve the oil removal and power generation performances\nof oily sludge deposition SMFCs, which will become an effective strategy\nto improve the performance of oily sludge SMFCs.",
"introduction": "1 Introduction Oily sludge is a mixture\nof crude oil and water emulsion, which\nis produced during the process of crude oil exploration. Naturally,\noily sludge is a hazardous environmental pollutant and may cause severe\nhealth risks in human beings and animals once exposed to such contamination. 1 The treatment methods of oily sludge include\nincineration, solvent extraction, biological treatment technology,\netc. 2 For energy-positive waste treatment,\nnovel microbial electrochemical systems are developed such as microbial\nfuel cells (MFCs), microbial electrolysis cells (MEC), microbial desalination\ncells (MDCs), microbial reverse electrodialysis cells (MRCs), etc. 3 − 7 MFCs are new bioelectrochemical systems in which a microorganism\ncatalyzes and oxidizes the organic compounds and converts the chemical\nenergy stored by the organic matter into electrical energy. 8 − 11 Sediment microbial fuel cells (SMFCs) are special battery systems\nevolved from the traditional MFC structure. The anode is buried in\nthe anaerobic bottom sludge, and the microorganisms in the bottom\nsludge are used as catalysts to degrade organic matter into CO 2 and H 2 O and generate electrons. The cathode is\nsuspended on the surface of the catholyte, and electrons flow to the\ncathode through an external circuit. O 2 is used as the\nelectron acceptor to generate clean electricity. 12 − 14 The main advantage\nof the SMFC is that it cannot be used only for waste or wastewater\ntreatment, but its structure makes it particularly suitable for river,\nlake, or marine sediment restoration. In the early 1990s, MFCs have\nbeen used in wastewater treatment. In recent years, the use of SMFCs\nto process oily sludge and simultaneous production capacity has become\na hot topic for MFC research. 15 Guo et\nal. showed that the oily sludge is suitable for SMFC operation, with\na maximum output voltage of 299.13 mV. 15 , 16 However, a\nkey issue that currently limits the practical application of SMFCs\nis the low power generation efficiency. There are many limiting factors\ninfluencing internal resistance. 17 − 20 The cathode, anode, pH, appropriate\ncatalysts (activated carbon, cobalt-nitrogen framework), and external\nresistance are important limiting factors for the performance of the\nMFC. 21 − 23 Among the many factors that affect its electricity\nproduction, the anode is considered to be a key factor because it\naffects the microorganism growth and metabolism of full stop. Anode\nperformance can be enhanced using a better catalyst, anode structure,\nand anode configuration. 24 − 29 It is well known that electrode materials have a great influence\non the power generation and pollution control of MFCs. Research has\nshown that properly changing the number of anodes can improve the\npower generation capacity. 30 − 32 Different arrangements (voltage\nseries and parallel and biomass series and parallel) can increase\nthe voltage of microbial fuel cells to different degrees. 33 However, the effects of the anode structure\n(electrode material, anode number, and arrangement) on the performance\nof oily sludge have not been systematically studied. Therefore,\nto explore the influence of the anode structure on the\nperformance of oily sludge SMFCs, in this paper, a series of SMFCs\nwere constructed using oily sludge as the anode substrate, with different\nelectrode materials (carbon felt and carbon plate), different anode\narrangements (transverse and longitudinal arrangement), and different\nanode multiples (single anode, double anode, quadruple anode, quintuple\nanode, and sextuple anode), and their effects on SMFC power generation\nperformance and oil sludge degradation were systematically studied,\nproviding basic research for oily sludge performance improvement strategies.",
"discussion": "3 Results and Discussion 3.1 Influence of Electrode Materials on the Performance\nof Oily Sludge SMFCs 3.1.1 Influence of Electrode Materials on the\nDegradation Performance of SMFCs The crude oil removal rate\nof oily sludge SMFCs with carbon felt (17.04%) is higher than that\nwith a carbon plate electrode (13.11%) as shown in Figure 2 . Using carbon felt as an electrode\nmaterial has a higher removal rate of crude oil, maybe because the\ncarbon felt electrode has a larger specific surface area than the\ncarbon plate electrode under the same projection area. 40 Carbon felt is conducive to the adhesion of\nelectricity-producing bacteria, which improves the oily sludge degradation\nperformance of SMFCs. 41 Figure 2 Different electrode materials’\nSMFC crude oil removal effect. 3.1.2 Influence of Electrode Materials on the\nElectricity Production Performance of SMFCs As shown in Figure 3 , the voltage of\nthe SMFC using a carbon felt electrode is higher than that with a\ncarbon plate in the early stage of electricity generation. This is\nbecause although the carbon plate is resistant to corrosion, acid,\nand alkali, its purity and density are high and not conducive to the\nadhesion of electricity-generating bacteria. 42 In contrast, carbon felt is disorderly arranged and has a loose\ntexture and large surface pores, which are more conducive to the attachment\nof electricity-generating bacteria. 43 During\nthe stable and declining periods of electricity production, the output\nvoltage of the carbon felt electrode SMFC, up to 215.33 mV, is significantly\nhigher than that of the carbon plate. It shows that compared to carbon\nplate electrodes, carbon felt electrodes are also more conducive to\nincreasing the output voltage of oily sludge SMFCs. 43 Carbon felt is conducive to the adhesion of not only electricity-producing\nbacteria but also more petroleum-degrading bacteria, which improve\nthe oily sludge degradation performance of SMFCs. 41 Figure 3 SMFC voltage–time curves of different anode materials. It can be seen from Figure 4 that the maximum power density of the oily\nsludge SMFC using\nthe carbon felt electrode is much higher than that with the carbon\nplate electrode, up to 81.49 mW/m 3 . Compared with the carbon\nplate electrode, the carbon felt electrode has better electricity\ngeneration performance. It can be seen from the SMFC polarization\ncurves ( Figure 4 B)\nthat the EMF of the carbon felt electrode SMFC (288.94 mV) is significantly\nhigher than that of the carbon plate electrode SMFC (213.40 mV), and\nthe apparent internal resistance (222.61 Ω) is much lower than\nthat of the carbon plate electrode SMFC (310.54 Ω). This means\nthat carbon felt has the lower mass transfer resistance, which can\nimprove the electron transfer efficiency. It is because the fiber\nsurface of carbon felt is relatively smooth, which is conducive to\nthe diffusion of microbial metabolites. 44 Figure 4 SMFC\npower density and polarization curves of different electrode\nmaterials. 3.2 Influence of the Anode Arrangement on the\nPerformance of Oily Sludge SMFCs 3.2.1 Influence of the Anode Arrangement on the\nDegradation Performance of Oily Sludge SMFCs The crude oil\nremoval rate of the SMFC with horizontally arranged anodes (23.61%)\nis obviously higher than that of the SMFC with longitudinally arranged\nones (19.82%), as shown in Figure 5 . It may be because the horizontal arrangement of anodes\nis more conducive to the electrochemical activity of microorganisms, 45 and the better the electrochemical activity\nof the microorganisms, the more thoroughly the crude oil in the oily\nsludge is degraded. Thereby, the horizontal arrangement improves the\ncrude oil removal rate of SMFCs. Figure 5 SMFC removal effect of crude oil with\ndifferent anode arrangements. 3.2.2 Influence of the Anode Arrangement on the\nElectricity Generation Performance of SMFCs In the SMFC voltage–time\ncurve of different anode arrangements ( Table 1 ), the output voltage of the SMFC with the\ntransverse anode arrangement is higher than that with the longitudinal\nanode arrangement. It indicates that the power generation performance\nof the SMFC with the transverse anode arrangement is better than that\nof the SMFC with the longitudinal anode arrangement. Table 1 Voltage of SMFCs with Transverse and\nLongitudinal Anode Arrangements in Different Times arrangement voltage/mV time/day transverse longitudinal 1 188.6 ± 0.2 181.1 ± 0.5 2 203.7 ± 0.4 171.1 ± 0.2 3 206.1 ± 0.6 179.8 ± 0.7 4 226 ± 0.3 171.6 ± 1.2 5 216.3 ± 1.0 173.2 ± 0.8 6 216.5 ± 0.9 186.8 ± 1.1 7 213.5 ± 0.8 177.8 ± 0.3 8 226.3 ± 0.4 191.9 ± 0.5 9 217.7 ± 0.2 180.1 ± 0.2 10 234.6 ± 0.3 208.2 ± 0.3 11 265.2 ± 1.2 197.8 ± 1.0 12 282.9 ± 0.2 191.4 ± 0.4 13 286 ± 0.5 194.3 ± 0.6 14 285.1 ± 0.5 198.6 ± 0.7 15 295.4 ± 0.7 217.3 ± 0.5 16 290.9 ± 1.5 231.5 ± 1.4 17 282.1 ± 0.6 265.8 ± 0.6 18 261.6 ± 0.8 261.4 ± 0.7 19 263.2 ± 1.0 249.7 ± 0.8 20 249.5 ± 1.7 250 ± 1.5 21 291.9 ± 1.8 269.2 ± 1.4 The power density curves of the SMFCs with different\nanode arrangements\nare shown in Figure 6 A. The maximum power densities of the SMFCs with horizontally and\nlongitudinally arranged anodes are 104.49 and 33.00 mW/m 3 , respectively. The EMF values of the SMFCs with horizontally and\nlongitudinally arranged anodes are 429.29 mV and 324.46 mV, and the\napparent internal resistance is 467.75 and 727.97 Ω, respectively.\nThe maximum power density and EMF of the SMFC with horizontally arranged\nanodes are higher than those with longitudinally arranged anodes,\nand the apparent internal resistance is lower than that with longitudinally\narranged anodes. It further shows that the power generation performance\nof the SMFC with horizontally arranged anodes is better than that\nwith the vertically arranged anodes. Figure 6 SMFC power density and polarization curves\nwith different anode\narrangements. 3.3 Influence of Multiple Anodes on the Performance\nof Oily Sludge SMFCs 3.3.1 Influence of Multiple Anodes on the Degradation\nPerformance of Oily Sludge SMFCs After 30 days of oily sludge\nSMFC treatment, the effect of multiple anodes on the crude oil removal\nrate in the oily sludge SMFCs is shown in Figure 7 . With the increase of the number of anodes,\nthe crude oil removal rate of the anode substrate increases. With\nsextuple anodes, the oily sludge SMFC anode substrate has the highest\ncrude oil removal rate (33.72%). Therefore, the degradation performance\nof the SMFC is best with sextuple anodes. Figure 7 SMFC crude oil removal\neffect of multiple anodes. 3.3.2 Influence of Multiple Anodes on the Power\nGeneration Performance of Oily Sludge SMFCs As shown in Table 2 , with the increase\nof the number anodes, the output voltage increases, and the output\nvoltage of the sextuple anode is the highest, up to 408.8 mV. It is\nbecause the anode of the SMFC is the hub for the accumulation of electricity-generating\nmicroorganisms and electron transfer, which plays a vital role in\nimproving the electricity-generating performance of the SMFC. Increasing\nthe anode number will make more oily sludge participate in the battery\nreaction. In this process, more microorganisms are concentrated on\nthe anode surface, producing more electrons and increasing the output\nvoltage of the SMFC. 46 Increasing the anode\nnumber can increase the output voltage of the SMFC, but it does not\nmean that the output voltage will always increase with the anode number\nincrease. When the anode number increases excessively, the adhesion\nof electricity-generating microorganisms on the anode is insufficient,\nand the increase in the amount of adhesion is not large or small,\nresulting in an insignificant or low output voltage. It can be seen\nfrom the figure that when the anode is increased from quintuple and\nsextuple, the output voltage increase is not obvious, and the sextuple\nanode is the most suitable value for this experiment. 47 Table 2 Voltage of Multianode SMFCs in Different\nTimes multianode voltage/mV time/day sextuple quintuple quadruple double single 1 227.4 ± 0.4 221.9 ± 0.2 188.6 ± 0.2 177.4 ± 0.4 129.3 ± 0.2 2 235.5 ± 0.1 266.7 ± 0.1 203.7 ± 0.4 166.8 ± 0.5 140.9 ± 0.6 3 254.5 ± 0.5 252.2 ± 0.4 206.1 ± 0.4 189 ± 0.4 141.1 ± 0.4 4 303.5 ± 0.3 291.1 ± 0.4 226 ± 0.2 195.3 ± 0.6 155.2 ± 0.2 5 302.7 ± 0.2 308.3 ± 0.3 216.3 ± 0.4 173.7 ± 0.7 142.1 ± 0.5 6 318.7 ± 1.1 331.2 ± 1.2 216.5 ± 0.2 198.5 ± 0.5 172.1 ± 0.6 7 322.5 ± 1.2 309.1 ± 1.0 213.5 ± 0.4 194.9 ± 0.4 190.4 ± 0.4 8 318.5 ± 0.1 305.1 ± 0.2 226.3 ± 0.4 203.5 ± 0.4 171.7 ± 0.4 9 333.2 ± 0.2 302 ± 0.5 217.7 ± 0.3 208.3 ± 1.2 195.5 ± 1.0 10 308.4 ± 0.3 320.6 ± 0.4 234.6 ± 0.4 202.3 ± 0.2 179.2 ± 0.5 11 346.6 ± 0.4 349.6 ± 0.7 265.2 ± 0.8 202.2 ± 0.4 193.3 ± 0.9 12 373.1 ± 1.2 362.7 ± 0.5 282.9 ± 0.6 214.6 ± 0.1 197.2 ± 0.8 13 378.6 ± 0.4 367.5 ± 0.2 286 ± 0.3 222.2 ± 0.4 180 ± 0.7 14 386.1 ± 0.6 375.8 ± 0.4 285.1 ± 0.1 218 ± 0.3 179.8 ± 0.4 15 386.5 ± 0.2 376.4 ± 0.3 315.4 ± 1.1 223.6 ± 0.2 190.9 ± 0.5 16 408.8 ± 0.7 382.9 ± 0.4 320.9 ± 0.2 231 ± 0.3 198.1 ± 0.4 17 403.4 ± 0.9 400.9 ± 0.5 282.1 ± 0.8 225.3 ± 0.8 213.7 ± 0.3 18 394 ± 1.2 384 ± 1.1 261.6 ± 0.6 239.2 ± 0.9 225.2 ± 0.2 19 379.9 ± 1.3 381 ± 0.3 263.2 ± 0.5 233.9 ± 0.1 239.8 ± 0.5 20 395.3 ± 1.5 370.2 ± 0.2 249.5 ± 0.4 256.3 ± 0.2 230.9 ± 0.4 21 387.1 ± 0.4 385.8 ± 0.4 321.9 ± 0.2 267.8 ± 0.1 250.4 ± 0.3 From the power density ( Figure 8 A) and polarization ( Figure 8 B) curves of the SMFC with different anode\nmultiples, it can be seen that as the anode multiple increases, the\npower density and EMF of the SMFC gradually increase, and the apparent\ninternal resistance gradually decreases. When the number of anodes\nis six, the power density and electromotive force are the largest,\nup to 145.32 mW/m 3 and 516.39 mV, respectively, and the\napparent internal resistance is the smallest, 417.08 Ω. The\ninternal resistance is similar to the external resistance (500 Ω),\nindicating that the external resistance is reasonable. 33 Therefore, the power generation performance\nof the SMFC with sextuple anodes is the best. It shows that with appropriately\nincreasing the number of anodes, the contact range of the anode oily\nsludge and the anode in the SMFCs increases. It helps more electricity-producing\nmicroorganisms adhere to the surface of the anode and reduce the mass\ntransfer resistance between oily sludge and the anode. 48 Figure 8 Power density and polarization curve of multianode SMFCs. It shows that when the crude oil removal rate of\nSMFCs is small,\nthe output voltage and power density are also small. After adjusting\nthe anode structure of oily sludge SMFCs, the oil removal rate increases,\nand the power production also increases gradually. The most proper\nadjustment of anode arrangements is using carbon felt as an anode\nmaterial and increasing the number of anodes to six, and the highest\ncrude oil removal rate reaches 33.72%; meanwhile, the output voltage\nof the oily sludge SMFCs is the highest, up to 408.82 mV, and the\npower density is also the highest (145.32 mW/m 3 ). The output voltage and the power density of oily sludge SMFCs increase\nwith the crude oil removal rate, indicating that the electricity generation\nof SMFCs and the crude oil removal rate are in the same direction,\nthat is, the degradation of crude oil by SMFCs can be efficiently\nconverted into electricity. It is because the arrangement of the anodes\nis more conducive to the electrochemical activity of microorganisms;\nthe better the electrochemical activity of the microorganisms, the\nmore completely the crude oil in the oily sludge can be degraded,\nthereby increasing the crude oil removal rate of the SMFCs, and the\nbetter the power generation performance of the system. 49 − 51"
} | 4,580 |
30678297 | PMC6384945 | pmc | 5,689 | {
"abstract": "Environmental pressures caused by population growth and consumerism require the development of resource recovery from waste, hence a circular economy approach. The production of chemicals and fuels from organic waste using mixed microbial cultures (MMC) has become promising. MMC use the synergy of bio-catalytic activities from different microorganisms to transform complex organic feedstock, such as by-products from food production and food waste. In the absence of oxygen, the feedstock can be converted into biogas through the established anaerobic digestion (AD) approach. The potential of MMC has shifted to production of intermediate AD compounds as precursors for renewable chemicals. A particular set of anaerobic pathways in MMC fermentation, known as chain elongation, can occur under specific conditions producing medium chain carboxylic acids (MCCAs) with higher value than biogas and broader applicability. This review introduces the chain elongation pathway and other bio-reactions occurring during MMC fermentation. We present an overview of the complex feedstocks used, and pinpoint the main operational parameters for MCCAs production such as temperature, pH, loading rates, inoculum, head space composition, and reactor design. The review evaluates the key findings of MCCA production using MMC, and concludes by identifying critical research targets to drive forward this promising technology as a valorisation method for complex organic waste.",
"conclusion": "12. Conclusions MCCAs, such as caproic and caprylic acid, are compounds of interest due to their broad range of potential applications. In contrast to chemical or single-culture biotechnological processes, using the consorted action of MMC allows to produce MCCA from complex organic feedstocks, such as food waste, in open, non-sterile systems via the natural process of chain elongation. However, the yields, concentrations and selectivity of this process must be improved in order to increase its viability. Therefore, we have summarised the current knowledge on the underlying mechanism of chain elongation by MMC, discussed the current state of the art on the use of complex organic feedstock and reviewed key operational parameters, and their interactions. Some of the key findings lie with the fact that with complex substrates and microbial cultures, there must be a greater emphasis on managing competing reactions and positively selecting for chain elongation microbiomes. Since the microbial diversity of MMC ecosystems has been shown to be distinct from pure cultures and clean substrates, existing thermodynamic and kinetic models should be expanded to include complex feedstock and mixed cultures. Advances in microbial culture analysis, such as improved implementation of various “-omics” methods on complex samples, will boost current understanding of MMC fermentation. Most common complex feedstocks trialled so far include residues from the bio-ethanol and dairy industries, different types of cellulosic wastes, syngas fermentation effluent and different types of organic food waste. These type of feedstocks have resulted in maximum production rates up to 8.02 g COD L −1 d −1 . Supplementation of complex waste-derived feedstock with chain elongation substrates such as ethanol increased production rates, with maxima reported up to 62.8 g COD L −1 d −1 . However, the negative environmental effects from chemical addition have also been reported. The use of synthetic substrates allowed production rates up to 115.2 g COD L −1 d −1 . Through an extensive review of the literature, including studies targeting MCCAs or reporting MCCAs as by-products, various key operational parameters were identified and discussed to highlight the research gaps. Mesophilic temperatures are so far a preferred choice for chain elongation, yet there is little justification for this. The preferred operational pH seems to lie in a slight acidic range from pH 5 to 7, in order to limit the activity of methanogens. The relationship between organic loading rate (OLR) and MCCA production rates showed a positive correlation to some extent, however this is complicated by the degree of biodegradability of the feedstock. Linked to the organic load is the substrate-inoculum ratio (F/M) at the start-up of the process which favours the accumulation of intermediate compounds instead of methane production when F/M > 5. In addition, whilst increased OLR tends to improve chain elongation, this must be coupled with sufficiently long residence times and biomass retention. OLR and retention times will have to be optimized depending on whether the reactor design has included mechanisms for biomass retention, and the biodegradability of the feedstock. The literature study revealed very little information is available on some specific operational parameters that have been studied for other MMC applications. For example, in similar MMC fermentation processes a minimal alkalinity was beneficial to stabilise the process and reduce the need for pH controlling agents. However, the buffer capacity required to stimulate chain elongation has not been thoroughly investigated. The partial pressures of CO 2 and H 2 in the reactor headspace have been identified to influence chain elongation, however production of these gases during fermentation, and their accumulation in reactor headspace is rarely considered. In order to circumvent the antimicrobial limitations imposed by MCCAs on the microbiome, in situ extraction is often proposed, but the alternative strategies which promote the development of biofilm or granule formation, and MMC adaptation, are worthy of further research. Finally, the development of down-stream processing methods, and integration within a bio-refinery context, are crucial issues to transform MCCA production from organic waste streams into a competitive waste valorisation technology that will contribute to the development of a circular economy.",
"introduction": "1. Introduction In 2016 nearly 58% of the organic fraction of municipal solid waste (OFMSW) in the EU was sent directly to landfill or incineration (estimated using the Eurostat database accessed 28/11/2018: recycling of bio-waste (cei_wm030), generation of waste by waste category (ten00108) and population on 1 January (tps00001)), resulting in undesirable environmental effects, little to no value recovery, and hence a loss of resources. However, recycling in the EU is now increasing [ 1 ], and hence separately collected organic waste is becoming more available for resource recovery or waste valorisation, i.e., the process of converting waste into energy, chemicals or materials. Technologies for bio-waste valorisation can be categorised as thermal or thermochemical such as hydrothermal liquefaction, pyrolysis and gasification, physicochemical like extraction and transesterification or biological conversion processes [ 2 , 3 ]. Biomass gasification has been proposed to homogenise various substrates to syngas and further process this for chemical production [ 4 ]. Reviews are available regarding technologies for waste to energy [ 5 , 6 ], or waste to chemicals and materials [ 7 , 8 , 9 , 10 ]. The choice of treatment method will depend on several factors such as type and availability of organic waste streams, e.g., the waste’s organic strength measured by chemical oxygen demand (COD) [ 11 ], relative content of biopolymers (i.e., cellulose, hemicellulose or lignin) [ 12 ], or biomass type (woody biomass, types of agricultural residues, household organic waste and sewage sludge) [ 13 , 14 ]. Development of a circular economy where waste is used as resource for renewable energy and chemicals will require the integration of different types of conversion processes to deal with the complexity of bio-waste and maximize resource recovery [ 15 ]. Established bio-waste valorisation technologies are composting and anaerobic digestion (AD), which each produce fertilizer and methane-rich biogas as end-products. However, the final products have relatively low economic value. For instance only € 2 worth of compost is obtained per tonne food waste [ 16 ]. AD generates a slightly more valuable product: assuming the OFMSW typically contains 306.4 g COD kg −1 of anaerobic biodegradable content [ 17 ] and that biogas conversion yields € 0.25 worth of biogas per kg of COD [ 18 ], then a tonne of food waste will produce about € 76 worth of biogas. However, the intermediate fermentation compounds produced during AD have a higher market value. Fermentation to accumulate the intermediate carboxylates is known as the carboxylate platform. Producing carboxylates through fermentation forms a sustainable alternative to their current production from fossil fuels or extraction in small amounts from natural oils [ 19 ]. Compared to AD, the carboxylate platform shows lower conversion yields, yet the higher product value and broader applications can result in a higher economic value [ 20 ]. In the last decade, particular interest has grown in medium chain carboxylic acids (MCCAs). They are defined as carboxylic acids with an aliphatic straight carbon chain of 6 to 12 carbon atoms, e.g., n -caproic acid has a straight chain of 6 carbon atoms (C6). MCCAs are more hydrophobic compared to shorter chain carboxylates, which makes them a more interesting fermentation product as it facilitates recovery from the fermentation broth [ 21 ]. In terms of potential value, C6 has a market size of 25,000 tonne per year, with an unrefined value of $1000, and refined value of $2000 to $3000 per tonne [ 22 , 23 ]. Overall, MCCAs have a wide range of applications: they can be applied as growth-promoting antibiotic replacements in animal feed [ 24 , 25 ], or be converted via various bio-, thermo-, or electro-chemical processes into bulk fuels or solvents [ 14 , 26 , 27 , 28 ]. The production of MCCAs as higher value products from organic waste can incentivise for improved recycling while simultaneously replacing current unsustainable production processes. MCCAs are produced by certain bacteria in a strongly reduced anaerobic environment, via a metabolic pathway that has been recently reviewed by Spirito et al. [ 29 ]. The bacteria gain energy by combining the oxidation of an electron donor, i.e., lactic acid or ethanol, to acetyl-CoA with the reductive elongation of acetyl-CoA with acetic acid (C2), propionic acid (C3), butyric acid (C4), pentanoic acid (C5), or caproic acid (C6) generating a carboxylic acid with 2 additional carbons at each step ( Figure 1 ). The reduction step is required to provide sufficient Gibbs free energy (ΔG) to generate ATP in the initial oxidation step, restore the NAD + /NADH balance in the cell, and contribute to further energy generation via electron-transport phosphorylation. Ethanol and lactic acid have similar thermodynamic capacity to act as electron donors [ 30 ]. This chain elongation pathway is called the reverse β-oxidation pathway, since it is seen as the reversed biochemical degradation or β-oxidation of fatty acids. Instead of using pure or engineered cultures, a consortium of microorganisms has more potential to deal with complex and variable feedstock such as organic waste. Mixed microbial cultures (MMC), also referred to as microbiomes, are communities of microorganisms within a well-defined environment of specific physicochemical properties [ 31 ]. Microbiomes are employed in biotechnology, for example in anaerobic digestion (AD), and in bioremediation by cultivating communities within contaminated soils [ 32 , 33 ]. The term “microbiome” is used to describe the mixed microbial communities related to the human and animal gut, mouth or skin, or plant rhizospheres. The first report of MMC that produced MCCAs dates back to the mid-19th Century, where Béchamp attributed the production of approx. 6 g COD L −1 C6 from ethanol, meat extract and chalk in a fermentation reactor to microbial activity [ 34 ]. A few decades later in the early 20th Century, an oily, immiscible layer comprising 5.3 g COD L −1 C4 and 6.4 g COD L −1 C6 was produced in a 30-day fermentation with impure cultures from a nutrient medium containing 24 g COD L −1 ethanol [ 35 ]. Further microscopic study of the fermentation sludge revealed a consortia of microorganisms comprising methanogenic archaea and spore-forming bacteria [ 35 ]. By contrast with pure cultures, MMC do not require sterilisation, can degrade a complex feedstock, show a resilience to operational upsets [ 36 ], and allow continuous, long-term operation [ 37 ]. These advantages provide a strong argument for utilising microbiomes. In MMC, conversion of organic substrates occurs following a cascade of steps catalysed by different microorganisms that form synergistic and competitive interactions, resulting in a complex microbial ecosystem with a versatile metabolic capacity [ 38 ]. The different microbial groups can convert organic molecules into substrates available for chain elongating bacteria. In general, biodegradable organics are hydrolysed and fermented to intermediate compounds that acidify the medium, i.e., acidogenesis, including hydrogen gas (H 2 ), lactic acid, ethanol, formic acid (C1) and volatile fatty acids (VFAs), i.e., straight short chain carboxylic acids with 2 to 4 carbon atoms. The accumulated intermediates can undergo several secondary bioconversion steps, including chain elongation to produce MCCAs ( Figure 2 ) [ 26 ]. For instance, co-culture of the chain elongating bacteria Clostridium kluyveri with specific cellulolytic species or a rumen microbiome showed chain elongation potential from a cellulose substrate and ethanol [ 39 , 40 ]. The supporting community can even be designed or selected to allow chain elongation from a specific compound, such as glycerol or syngas (CO) [ 41 , 42 , 43 ], or allow the use of alternative electron donors such as, for instance, the cathode in a bio-electrochemical system [ 44 , 45 ]. While it is generally believed that specific operational conditions allow development of a MMC for a functional and stable process [ 46 ], the broad metabolic capacity also gives rise to a set of various competitive reactions and by-products, especially when utilising a complex feedstock. Manipulating the environmental conditions, by regulating operation, allows some control to be exerted on the product spectrum, as it affects the thermodynamics of conversion processes, and therefore the microbiome composition that catalyses these conversions. However, current knowledge of control over the product outcome to improve MCCA yields in MMC fermentation is limited since experiments that use complex feedstock for MCCAs production have only emerged in the past few years. While the operational conditions that select for other MMC fermentation products such as volatile fatty acids (VFAs) [ 47 ] and hydrogen (H 2 ) [ 48 ] have been reviewed, the operational conditions or process set-up that allow MCC to be steered towards MCCA formation have to be further evaluated. A recent review is available regarding the use of bio-electrochemical systems for MCCA production as a complementary technology to AD [ 49 ]. Certain other reviews include a section on MCCAs as potential MMC fermentation products, either in the context of operational control applied in AD [ 50 ], or the contexts of a biorefinery [ 51 ], wastewater treatment [ 11 ] or food waste treatment [ 21 , 52 , 53 , 54 ]. However, a focussed analysis of the literature to identify and connect key operational parameters to target MCCA production from MMC fermentation of complex feedstocks is lacking. Therefore, this work aims to analyse the current literature, and hence complement existing reviews. For this, studies were included that specifically target chain elongation, but the scope was extended to include other MMC-based studies that have noted MCCA as by-products from, for instance, VFA or H 2 production. Concentrations and production rates are converted to a COD-basis to allow comparison between studies using different reporting concentrations ( Appendix A ). The review evaluates the key operational parameters for MCCA production from complex substrates using MMC, with the objective of stimulating and accelerating research to produce sustainable, bio-based fuels and chemicals from organic waste. In addition, a database was generated from the experimental data available in the literature regarding MCCA production using MMC fermentation [ 55 ]."
} | 4,125 |
33187833 | null | s2 | 5,690 | {
"abstract": "Intricate gene regulatory networks control the transition between the planktonic and biofilm lifestyles in bacteria. New evidence from Mhatre et al. uncovers how various adaptive mutations that arose in a key gene at the nexus of signaling networks in Burkholderia cenocepacia led to the emergence of lineages with different ecological roles, enabling stable coexistence of multiple genotypes and increasing productivity of the community."
} | 109 |
39496599 | PMC11535438 | pmc | 5,691 | {
"abstract": "Organometal halide perovskite (OHP) composites are flexible and easy to synthesize, making them ideal for ambient mechanical energy harvesting. Yet, the output current density from the piezoelectric nanogenerators (PENGs) remains orders of magnitude lower than their ceramic counterparts. In prior composites, high permittivity nanoparticles enhance the dielectric constant (ϵ r ) but reduce the dielectric strength (E b ). This guides our design: increase the dielectric constant by the high ϵ r nanoparticle while enhancing the E b by optimizing the perovskite structure. Therefore, we chemically functionalize the nanoparticles to suppress their electrically triggered ion migration for an improved piezoelectric response. The polystyrene functionalizes with FAPbBr 2 I enlarges the grains, homogenizes the halide ions, and maintains their structural integrity inside a polymer. Consequently, the PENG produces a current density of 2.6 µAcm −2 N −1 . The intercalated electrodes boost the current density to 25 µAcm −2 N −1 , an order of magnitude enhancement for OHP composites, and higher than ceramic composites.",
"introduction": "Introduction Piezoelectric nanogenerators use the piezoelectric effect for harnessing ambient vibrations to trickle charging modern electronic devices and sensing networks. Due to their unique merits in compact size, weight, and stability in harsh environments, PENGs are considered one of the most suitable energy harvesting technologies in recent years to be used with wearable, flexible, and implantable sensing platforms. Piezoceramics such as Pb(ZrTi)O 3 (PZT) 1 , BaTiO 3 (BTO) 2 , 3 , Pb (Zn 1/3 Nb 2/3 )O 3 -PbTiO 3 (PZN-PT) 4 , Sm-Pb(Mg 1/3 Nb 2/3 )O 3 -PbTiO 3 (Sm-PMN-PT) 5 are the dominant materials of choice for PENG applications due to their large piezoelectric charge constants. Yet, their brittle nature and cost pose challenges in various applications. Consequently, flexible polymers like poly (vinylidene fluoride) (PVDF) 6 , 7 , poly(vinylidene fluoride-co-trifluoro ethylene) (PVDF-TrFE) 8 , polydimethylsiloxane (PDMS) 9 , etc. are used as host matrices in conjunction with piezoceramic fillers. The successful utilization of PENG as an energy source hinges on attaining both sufficiently high voltage and current outputs. While the output voltage from PENG exceeds 3 V (sufficient for most charging applications), a significant challenge in addressing the charging needs of most electronic devices persists in their low output current densities. Diverse strategies have been implemented to boost current densities, including the exploration of innovative device structures and the selection of materials with high piezoelectric charge constant (d 33 ). For instance, by stacking PZT NW arrays Gu et al. fabricated PENG with a current density of 23 μAcm −2 in 2013 10 , in 2014, Park et al. developed PZT thin film-based PENG with interdigitated electrodes and reported a current density of 150 µAcm −2 \n 11 . Materials with high piezoelectric charge constants yield high output current density in a PENG while reducing the piezoelectric voltage coefficient (g 33 ) and the output voltage according to the relation of (g 33 = d 33 /ε r ; ε r is the relative permittivity). However, if the high d 33 ceramic material is dispersed in a high g 33 polymer matrix such as PVDF, PVDF-TrFE, it can yield a high output current density while retaining a standard output voltage level above 3 V. For instance, Sm-PMN-PT@PVDF piezoceramic composite has a current density of 15 µAcm −2 (1.24 µAcm −2 N −1 ), and a voltage of 7 V. In 2020, Gu et al. marked another record of output current density to 290 µAcm −2 (24 µAcm −2 N −1 ) by stacking 72-layers of Sm-PMN-PT@PVDF films, which is still the best-reported piezoceramic composite 7 . The critical metric for assessing the PENG performance lies in the normalized current density, denoted in µAcm −2 N −1 . This parameter considers the applied force necessary for the generation of the reported current, offering a comprehensive gauge of the device’s operational efficiency. Organometal halide perovskites (OHPs) are currently a focal point of extensive research for their application in PENG devices 12 – 16 . This interest is driven by multiple advantages, including their solution processability, flexibility, and low-temperature synthesis when compared to traditional piezoceramics. These features enable the fabrication of OHP-based PENG devices on large-area flexible substrates, making them suitable for applications in flexible, wearable, and implantable electronics. Recent research has demonstrated that the piezoelectric charge constants (d 33 ) of OHPs are now comparable to those of piezoceramics. For example, TMCM-MnCl 3 (TMCM, tri-methylchloromethyl ammonium) (d 33 ~185 pCN −1 ), TMCM-CdCl 3 (d 33 ~220 pCN −1 ) 17 , (TMFM) x (TMCM) 1-x -CdCl 3 (TMFM, tri-methylfluoromethyl ammonium; 0 ≤ x ≤ 1) (d 33 ~1540 pCN −1 ) 18 , etc. Despite the use of high piezoelectric charge constant OHPs, the challenge of achieving high current density persists. For instance, the maximum current density for the TMCM-CdCl 3 @PDMS composite was reported as 3.45 µAcm −2 (equivalent to 0.69 µAcm −2 N −1 ) 19 . Like piezoceramics, a prevalent strategy to improve the performance of OHP-based PENGs (and others) involves utilizing a matrix of piezoelectric polymers such as PVDF in combination with OHP nanomaterial. Poling in such composites induces the formation of the electroactive β-phase through electrostatic interaction with the nanomaterial 20 . Despite achieving hundreds of volts in output voltage 21 , the normalized current density of such PENGs remains notably low, with the highest reported value at 0.72 µAcm −2 N −1 \n 13 . This figure is roughly 30 times lower than the best-performing PENGs utilizing piezoceramics. A challenge in such composites arises from the trade-off between achieving a high dielectric constant and their low dielectric strength. While a high dielectric constant enhances polarization (resulting in a higher piezoelectric charge constant), the lower dielectric strength limits the poling of these composites at sufficiently high electric fields for extended durations 22 . Consequently, the unidirectional alignment of piezoelectric domains cannot be achieved, compromising optimal performance. In the case of OHPs, the breakdown is induced by ion migration effects under the applied poling electric field, leading to perovskite structure degradation. Grain boundaries play a critical role as preferred pathways for ion migration. Due to the higher electrical conductivity of OHPs, they act as current pathways, and alterations to their structure, such as the formation of PbI 2 or local p-n junctions, reduce conductivity and, consequently, current density in the PENGs. In this study, we present a FAPbBr 2 I-PVDF-based PENG where polystyrene (PS) is employed to control the structure and compositional variation of FAPbBr 2 I. This ternary piezocomposite in a PENG generates a high output current density of 11 µAcm −2 (2.6 µAcm −2 N −1 ). The piezocomposites were further used as a building block to vertically assemble them. The multiple layers of piezocomposite films separated through intercalated copper electrodes enhance the output current density. The cascade-type piezoelectric nanogenerator (CPENG) architecture (14 layers) exhibits an impressive output current density of approximately 105 µAcm −2 (peak to peak) at 30 Hz and 4.2 N, with a corresponding normalized current density of 25 µAcm −2 N −1 . This performance surpasses OHP-based PENGs by an order of magnitude and outperforms the best ceramic-based composites, as reported with a normalized current density of 24.17 µAcm −2 N −1 in a 72-layer PENG. The basis of this improvement is due to (1) The use of PS leads to improved grain size which reduces the density of grain boundaries, as a result, ion migration is reduced by an order of magnitude compared to plain FAPbBr 2 I-PVDF. (2) With the use of PS, a uniform distribution of halide ions (Br − and I − ) is achieved in the composite, this leads to a homogenous band structure and prevents the formation of local energy barriers (due to variation in band structure from changes in halide composition) which could impede current collection. (3) The dielectric constant of the PS-FAPbBr 2 I-PVDF composite is improved by over five times compared to plain FAPbBr 2 I-PVDF, which will increase the piezoelectric charge constant. (4) X-ray diffraction results reveal increased lattice spacing with the use of PS, indicating strain relaxation in the OHP structure. This further reduces ion migration, improves carrier mobility, and lowers defect concentration, contributing to the stabilization of the PENG devices and enhancing charge collection 23 . These effects collectively contribute to a higher breakdown strength in PS-FAPbBr 2 I-PVDF-based PENGs, sustaining up to 30 min of poling at a field strength of ~50 Vµm −1 compared to plain FAPbBr 2 I-PVDF devices that typically breakdown in less than 1 min under similar conditions. Thus, our work demonstrates the successful mitigation of challenges associated with OHP-based piezoelectric nanogenerators through a synergistic approach involving optimized material design and effective device engineering by cascading multiple layers. This integrated strategy achieves a record-normalized current density.",
"discussion": "Results and discussion Design and characterization of polymer functionalized perovskite composites Polystyrene was used to functionalize the OHP (FAPbBr 2 I) because of its specific interactions with, the A site cation (FA + ) and the lead halide species. Additionally, its high dielectric strength, along with low dielectric losses 24 , makes it particularly advantageous for PENG applications. Previous studies have demonstrated that integrating PS into the perovskite matrix results in the modulation of both nucleation and the growth rate of perovskite crystal grains, accompanied by a reduction in defect density 25 . The functionalization is grounded in the typical cation-π interaction between the FA + cation of the FAPbBr 2 I and π -electrons of aromatic styrene in PS, as illustrated in Fig. 1a . The specific molecular-level interaction between perovskite precursor and PS was characterized by Raman spectroscopy (Fig. S1 ). The pristine FAPbBr 2 I-PVDF and PS-FAPbBr 2 I-PVDF films exhibit sharp characteristic peaks of Pb–X (X = I, Br) lattice mode centered at 80 cm −1 and a broad peak (160–250 cm −1 ) corresponding to the organic moiety 26 . As observed in Fig. 1b , adding 1 wt/vol% PS in the FAPbBr 2 I, the peak corresponding to the Pb–X (X = I, Br) lattice mode shifts to a lower wavenumber. Such variation in the Raman active modes of the perovskite signifies an enhanced molecular-level interaction between PS and perovskite precursors. A similar interaction between PS and the organometal halide perovskite precursors has been previously confirmed by gel permeation chromatography and the growth of single crystal perovskite 27 . The X-ray diffraction pattern in Fig. 1c shows the presence of the dominant peaks corresponding to (0 0 1) (0 0 2) and (2 1 0) lattice planes and highlights the formation of the mixed halide perovskite phase in both pristine FAPbBr 2 I-PVDF and PS-FAPbBr 2 I-PVDF films 28 , 29 . A stronger anisotropic orientation along the (0 0 1) plane is evident in pristine FAPbBr 2 I compared to PS-FAPbBr 2 I. From the view of thermodynamics, orientation diversity offers increased entropy and a more stable perovskite phase 30 . The narrowing of the peak (reduction in the full width and half maximum value) and increase in the peak intensity in the PS-FAPbBr 2 I-PVDF XRD pattern indicates improved crystallinity and an increase in the crystallite size (Tables S1, S2 ) 31 . An additional shoulder peak of residual PbX 2 (X = I, Br) is observed at 2θ of 12.4 ° exclusively in the pristine FAPbBr 2 I. The absence of the PbX 2 (X = I, Br) peak in the PS-perovskite composite film can be ascribed to the ability of PS chains to interact with PbI 2 and PbBr 2 , which are weak Lewis acids and formed intermittently during solvation of perovskite precursors. This leads to enhanced perovskite phase conversion from the combined interaction between PS, FA, and PbX 2 . Additionally, there is a consistent shift in the XRD peaks toward lower 2θ values in PS-FAPbBr 2 I-PVDF films compared to FAPbBr 2 I-PVDF films. This shift indicates a lattice expansion in the PS-FAPbBr 2 I-PVDF films, signifying relaxation in the perovskite lattice 32 . The lattice constants calculated from the peak positions in the XRD pattern further illustrate this expansion (Tables S3 , S4 and Note S 1 ). Fig. 1 Surface functionalization of organometal halide perovskite. a Cation−π interaction mechanism between FAPbBr 2 I and the PS. b Raman spectroscopy and c XRD patterns of the pristine FAPbBr 2 I-PVDF and 1% PS-FAPbBr 2 I-PVDF films. d ToF-SIMS depth profiling of pristine and e 1% PS-FAPbBr 2 I-PVDF films. f FE-SEM images of the grains of plain FAPbBr 2 I and g 1% PS-functionalized FAPbBr 2 I thin film (insets show the grain size distribution curves). h AFM surface topography image of the 1% PS-FAPbBr 2 I film. Source data are provided as a Source Data file. Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) depth profiling (Fig. 1d, e ) was conducted to see the variation in the distribution of the halide ions between PS-FAPbBr 2 I-PVDF and FAPbBr 2 I -PVDF films. We observe that a uniform concentration of I − and Br − is present across the PS-FAPbBr 2 I-PVDF composite film. In comparison, for the FAPbBr 2 I-PVDF film, the I − distribution is lower and non-uniform (especially during the initial 0–200 s of sputtering) relative to the Br − profile, hence showing phase segregation in the top layer. Phase segregation of the halide ions can lead to detrimental effects on the device’s performance due to structural changes 33 . In the perovskite solution system, the atoms, ions, and solvent molecules can coordinate with each other, forming intermediate adducts or complexes. Since the trend of Lewis acidity in lead (II) halide follows the order of PbI 2 > PbBr 2 , the stronger interaction of PbI 2 with PS allows more iodide-rich perovskite phase formation, which leads to its uniform distribution across the film 34 . This observation, combined with the shift of the peak position in the X-ray diffraction patterns for PS-FAPbBr 2 I-PVDF, confirms that the perovskite lattice is larger due to the uniform inclusion of the higher ionic radii of the 6-coordinated I − ( r I − = 2.06 Å) along with Br − ( r Br − = 1.82 Å). The larger lattice will also have a smaller strain which has been shown to improve the stability of the perovskite phase, along with decreased defect density and increased carrier mobility 35 , 36 . The depth profiling data also reveals that the signal intensity of the I − ion is maintained at the same level as that of the F − ion (representative of PVDF) across the entire depth of the sputtering time (Fig. 1d ) in the PS-FAPbBr 2 I-PVDF film. This shows that the composite is homogenous, and the interaction between PVDF and the perovskite phase will be uniform across the film. The direct interaction of PS chains with the perovskite precursors leads to relatively larger grain size in the PS-FAPbBr 2 I films in comparison to that of pristine FAPbBr 2 I as evident in the lateral field emission scanning electron microscopy images (Fig. 1f, g ). Due to the decreased nucleation rate, average grain sizes increase from 400 to 600 nm (in FAPbBr 2 I) to 600–900 nm in the PS-FAPbBr 2 I film. The distribution curve in Fig. 1f, g represents the probability density function at each of the values in x using the normal distribution with mean μ and standard deviation σ . The corresponding equation is given by: \\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$f(x\\,|\\mu,\\sigma )=\\frac{1}{\\sigma \\sqrt{2\\pi }}{{{\\rm{e}}}}^{\\frac{{-(x-{{\\mathrm{\\mu }}})}^{2}}{{2\\sigma }^{2}}}$$\\end{document} f ( x ∣ μ , σ ) = 1 σ 2 π e − ( x − μ ) 2 2 σ 2 The AFM surface topology (Fig. 1h ) also confirms the large grain formation on the PS-functionalized film. Therefore, based on these combined effects we expect greater stability of the perovskite phase in the PS-FAPbBr 2 I-PVDF films, along with lower ion migration effects. Dielectric and piezoelectric properties of the piezocomposite An electric field was applied across the composite film to align the dipoles unidirectionally to generate a macroscopic dipole moment. The electric field applied for a sufficiently longer time causes 60/180° rotation of the molecular chains and a conversion of the α to the β- phase even without the mechanical stretching of the chains 37 . The most widely used PVDF polymer requires a high electric field of at least 50 Vµm −1 to completely harness its piezoelectric capabilities 38 . Such a requirement imposes serious restrictions in manipulating OHP composites, as a high electric field proliferates dynamic point defects and causes faster breakdown due to ion migration, as illustrated in Fig. 2a . In an OHP perovskite structure, FA + , Br/I − , and Pb 2+ are all considered mobile ions, and this results in high ionic conductivity, and a large leakage current in the perovskite-based devices 31 , 39 , 40 . We find that, with an external bias in the dark, an electronic current is instantly observed owing to the fast movement of the electronic charge carriers. At the same time, the mobile ions with low activation energy also slowly drift toward metal electrodes. As the oppositely charged ions begin accumulating at the metal electrodes, the ion-induced electric field partially cancels the external bias and reduces the overall current. This transient decay in current is observed until the ion accumulation reaches an equilibrium condition (Fig. 2b ). The pristine FAPbBr 2 I film owing to the higher density of mobile ionic defects and halide ion segregation exhibits a large decay in the current 41 . As seen in Fig. 2b , the dark current in the pristine FAPbBr 2 I film decays rapidly from its initial value of 3.10 × 10 −6 and reaches 1.86 × 10 −6 mA/cm 2 (~40% decay) within 35 s. In contrast, the PS-FAPbBr 2 I films exhibit a much lower dark current (~5.0 × 10 −7 mA/cm 2 ) which decays by a smaller magnitude to ~4.20 × 10 −7 mA/cm 2 . The smaller magnitude of decay in the dark current implies that the PS reduces the ion migration effect by more than an order of magnitude, based on the structural results discussed above. Furthermore, the electric field strength-dependent leakage current density 42 of pure FAPbBr 2 I-PVDF-based devices is observed to be 5.44 × 10 −4 mA/cm 2 at 0.1 kV/cm and 1.49 × 10 −2 mA/cm 2 at 3 kV/cm (Fig. S2 ). In contrast, the leakage currents in the 1 wt% PS-FAPbBr 2 I-PVDF composite films are reduced to 1.67 × 10 −5 and 1.35 × 10 −3 mA/cm 2 , respectively, at the same electric fields. The multiple-fold reduction in the leakage current in the presence of PS further corroborates its beneficial role in providing insulation against the leakage paths in the composite film while simultaneously allowing improved grain growth and homogenized distribution of FAPbBr 2 I nanoparticles across the PVDF matrix. Fig. 2 Electric-field induced Ion-migration in an OHP composite film. a Schematic representation of ion-migration mechanism and role of the PS in a FAPbBr 2 I-PVDF composite film. The arrows indicate the direction of ion migration, while the red cross symbols represent the PS chains blocking the ion migration paths. b The dark-current density of pristine and 1% PS-functionalized composites. Source data are provided as a Source Data file. The structural integrity of the OHP perovskite in the composite is crucial to preventing undesirable breakdown during poling. We compared the average dielectric strength of 12 different locations on each of the composite devices (Experimental data in Table S5 ). The 1% PS devices had an average breakdown strength of 191 Vµm −1 , 130% times that of the films without PS (Fig. S3 ). Based on the average poling electric field of composites being between 40 and 120 Vµm −1 , we applied a dc bias of 3 kV to the pristine (without any PS) and 1% PS-containing composites (thickness of ~ 65 and ~58 µm, respectively in Fig. S4 ) and noted the breakdown time (Fig. 3a ) 38 . With the electric field of ~46 Vµm −1 , all the pristine films reached their breakdown point in less than 5 min, while 50% of them could withstand only 1 min of poling. In contrast, the PS-containing composites could survive up to 30 min of poling with an electric field of ~51 Vµm −1 , where the lowest-performing devices have a breakdown point higher than all the pristine composite samples. Fig. 3 Piezoelectric and dielectric response of the OHP composite film. a Variation of electrical breakdown time of the pristine and 1% PS included composites of FAPbBr 2 I-PVDF with an applied electric bias of 3 kV. The red-shaded region represents the composite films without PS, while the green-shaded region corresponds to the composite films containing 1% PS. b Measured permittivity (ϵ r ) of the composites. c Output current density of the PENGs fabricated with varying PS concentration. d – f 3D representation (NanoScope Analysis 1.8) of the piezoelectric response in a 1% PS-FAPbBr 2 I-PVDF film with different tip biases (area of 2 × 2 µm 2 ). Source data are provided as a Source Data file. A striking difference was observed in the relative dielectric permittivity (ϵ r ) of the plain, and PS-functionalized composite films. In contrast to the ϵ r of 7.5 in pristine FAPbBr 2 I, the 1% PS-functionalized films exhibit a ϵ r value of 38 (Fig. 3b ), representing an increase of more than five times. The more homogeneous distribution of perovskite species and improved crystallinity with larger grain size are anticipated to increase the dielectric constant of 1% PS composites (Note S 2 ) 43 . Since grain boundaries typically have smaller dielectric permittivity than the grains in perovskite materials 44 , 45 . The observed peak shift to smaller angles in the XRD data suggests a relaxation in the perovskite lattice. This relaxation is also expected to enhance dipole alignment and polarizability 46 . We varied the PS concentration and observed its influence on the output current generation in the PENG devices. The output current density from the PENG devices improved, especially for the 1% PS-containing FAPbBr 2 I film, which has the highest current density of 11 µA/cm 2 . This is 2.4 times higher than the pristine perovskite PENGs, and 7.3 times higher than pristine PVDF-based devices (Fig. 3c ). This is attributed to the structural effects which include larger grain size, defect passivation of perovskites, and enhanced dielectric constant with a more homogenous composition of the composite 47 , 48 . The interaction between PS-FAPbBr 2 I and PVDF was studied from the absorbance spectra of pure PS, pure PVDF, PVDF-PS, and PVDF-PS-FAPbBr 2 I films (Fig. S5 ). In the FTIR spectra (Fig. S5a ), the characteristic peaks of PS appear at 696 cm −1 (aromatic ring bending), 1492 cm −1 (aromatic ring mode), 2850 and 2923 cm −1 (CH 2 symmetric and asymmetric stretching), and 3025, 3059, and 3081 cm −1 (aromatic C-H stretching). Similarly, distinctive peaks of PVDF centered at 487 cm −1 (CF 2 bending and wagging), 613 cm −1 (CF 2 bending and CCC skeletal vibration), 763 cm −1 (CH 2 and CF 2 in-plane rocking or bending), 875 cm −1 (CC symmetric stretching and skeletal bending) and 1184 cm −1 (CF 2 stretching) are observed 49 , 50 . In the PVDF-PS film, the characteristics peaks for PVDF show no change which indicates little interaction between PS and PVDF. The intensity of PS-related peaks is reduced due to its minimal concentration (1 wt%). In the PS-FAPbBr 2 I-PVDF film, while the peaks observed in the PVDF-PS film are retained, a new peak corresponding to the CN stretching in the formamidinium group of perovskite appears at 1715 cm −1 . Furthermore, the IR peak corresponding to CC stretching in PVDF shifts from 875 cm −1 to a higher wavenumber of 880 cm −1 indicating interaction with the perovskite (Fig. S5b ). The critical feature is that with PS-OHP in the PVDF matrix, the β-phase content in PVDF increases from 36 to 71.5%. This is based on the diminishing of the α-phase PVDF peaks at 614, 763, and 974 cm −1 in the PS-PVDF-Perovskite film. An enhancement in the relative intensity of the IR peaks at 840 and 1276 cm −1 is observed which correspond exclusively to the β-phase of PVDF 51 . Nevertheless, at higher PS concentrations, effective perovskite content reduces in the film, and this decreases the overall film quality and the effective beta-phase amount in the PVDF. So, a decreasing trend in output current density with larger PS loading is observed (Fig. S6 ). The output voltage from the 0% PS film is slightly lower than the 1% PS film, while PS loading beyond 1% further decreases the output voltage (Fig. S7 ). To assess its flexibility 52 , Young’s modulus (YM) of the composites with different PS additions was also measured from the tensile stress-strain curves (Fig. S8 ). The Young’s modulus was calculated for the 0% PS film as 1.35 GPa, while for the 1 and 10% PS-added films it was reduced to 1.3 and 1.16 GPa. Thus, obtaining a delicate balance with the amount of PS in the composite is critical. We measured the microscopic piezoelectric response with the applied electric bias (+5 V, +10 V) in a piezoelectric force microscopy (PFM) (Fig. 3 d– f ). The 1% PS composite film poled with +5 V dc bias for ~10 min exhibits an amplitude response of ~1.5 V, which is ~10 times more than the non-poled film (0 V). As we increased the dc bias to +10 V for 10 min, amplitude response further increased to ~1.8 V. It is evident that with the rise in electrical poling voltage and poling time, more and more dipoles are unidirectionally aligned and resulting in a higher piezoelectric response in the composite film. This experiment further depicts the importance of electrical poling of the composite film for obtaining an enhanced piezoelectric response. Design of the CPENG The enhanced current density and stability observed in the 1% PS composites served as motivation to adopt it as a foundational element for the assembly of the multilayer cascade-type piezoelectric nanogenerator (CPENG). In a PENG, the piezoelectric polarization charges are utilized to produce output current to the external circuit. When subjected to mechanical stress, opposite polarization charges emerge at the interfaces of the top and bottom electrodes. The prospect of creating numerous interfaces through two-dimensional electrodes within a piezoelectric film holds the potential for an enhancement in output current density 53 . To achieve this cascade device, we employed intercalated copper electrodes positioned between two oppositely poled composite films (Fig. 4a ). The electrodes were interconnected in a parallel electrical configuration, where positive polarization surfaces were linked to create a common positive (+ve) terminal, and the negative surfaces of the films formed a shared negative (−ve) terminal. When subjected to force, the current generated in each unit accumulated through the interfacial electrodes, resulting in a larger combined current output. A solvent-free urethane-based prepolymer was employed as an adhesive owing to its outstanding initial adhesion, workability, and bonding capacity 54 . Briefly, the adhesive was blend-coated on a thin copper electrode and tightly pressed with the composite film for 48–72 h in ambient conditions. Subsequently, another composite with opposite polarity was coated with adhesive and securely bonded to the copper electrode, forming a single-electrode intercalated two-layer PENG device (further details can be found in the Experimental section). This process was repeated to fabricate CPENGs with 1, 4, 8, 14, and 21 layers. Figure 4b presents a photograph of a CPENG with 21 layers, accompanied by cross-sectional scanning electron microscopy images and cross-section images obtained through energy-dispersive X-ray spectroscopy (EDS) analysis. The elemental mapping images corresponding to copper (Fig. 4b-ii ) and fluorine (Fig. 4b-iii ) illustrate the copper electrodes and PVDF composite films, respectively. Other representative elements of the perovskite (Pb, Br, I, and N) from the EDS analysis are also shown in Fig. S9 . This represents the homogeneous distribution of perovskite nanoparticles within the piezocomposite and well-intercalated copper electrodes between the composite films. Fig. 4 Design of the cascade-type piezoelectric nanogenerator (CPENG). a The basic structure of the CPENG. The blue spheres represent the repeating composite films, while the polyhedral shapes depict the perovskite structure embedded in the PVDF. Each of the composites is connected in parallel electrical connection. Two consecutive films have opposite polarization directions and are intercalated with a copper electrode. Intercalated electrodes adjacent to the same polarization direction are connected. b Photograph of a 21-layer CPENG (inset shows a bent device by a metallic tweezer) (i) cross-sectional FE-SEM (ii-iii) EDS elemental mapping clearly showing the intercalated copper electrodes (red) and composite films (green). We conducted a mechanistic investigation using finite element analysis to understand how the assembly of multiple layers could optimize the output performance (Fig. S10 ). We simulated the piezo potential of various models with 1, 4, 8, 14, and 21 layers of films, maintaining opposite polarization directions for two consecutive films. With an increase in the number of layers, the piezo potential decreases from 30 V (single layer) to 3 V for a 21-layer PENG. When a force is applied to the PENG, the dipole moments become smaller along the thickness direction, leading to the cancellation of the piezo potential due to the opposite polarization direction of two consecutive films 55 . However, the change in polarization across each layer translates to output current, which is then multiplied through intercalated electrodes. Output performance and application of the CPENG To assess the practical mechanical energy harvesting capabilities of the devices, we applied a force of 4.2 N (0.42 kg) using a steel block and systematically examined their respective performance. The single-layer PENG produced a maximum output voltage of approximately 29 V. This voltage gradually decreased to 17, 7, 3.5, and 2.6 V for the 4, 8, 14, and 21-layer devices, respectively (Fig. 5a ). This behavior can be elucidated through the simple capacitor model, wherein the parallel connection of capacitors leads to an increase in overall capacitance, subsequently causing a decrease in voltage (V = Q/C; V voltage, Q charge, and C capacitance) 56 . Fig. 5 Output performance of the CPENGs. a Measured output voltage and b current density of CPENGs made with 1, 4, 8, 14, and 21 layers. Comparison of experimental ( c ) and simulated ( d ) variation of output voltage and current density with total number of layers. Source data are provided as a Source Data file. The output current density of the devices was measured under short-circuit conditions, as illustrated in Fig. 5b . As the number of layers increased to 1, 4, 8, 14, and 21, the output current density rose from 11 to 29 µA/cm 2 , 64, 105, and then decreased to 55 µA/cm 2 , respectively. Figure 5c demonstrates that while the output voltage decreases nonlinearly, the output current density increases almost linearly with the number of layers. Moreover, the finite element simulation model reveals that the calculated total charge density in short-circuit conditions (which corresponds to current density) follows a similar increasing trend with the number of layers in a PENG, as depicted in Fig. 5d . The simulation results in Fig. 5d align with our experimental findings, except for the current density for the 21-layer CPENG. This is due to the strong stress-buffering effect as the number of layers increases 7 . The use of a higher number of copper/adhesive electrodes will reduce the transmitted stress to each composite, which was not included in the simulation model 57 , 58 . The CPENG demonstrated resilience during testing for ~1000 mechanical cycles without deterioration in its performance, as shown in Fig. S11 . To validate its practical energy harvesting capabilities, we connected the CPENG through a full bridge rectifier to different capacitors (Fig. 6a ). The CPENG was used to charge different capacitors of 4.7, 10, 22, and 47 µF (Fig. 6b ). At 30 Hz and 4.2, N it can charge a 4.7 µF capacitor to ~1 V in 16 s, 10 µF to 1 V in 38 s, 22 µF to 0.95 V in 50 s, and 47 µF to 0.6 V in 1 min. Fig. 6 Application of the CPENG. a Schematic of an electrical circuit for energy storing to a capacitor. b Mechanical energy is converted to electrical energy by the CPENG to charge different capacitors. c The CPENG is charging an LTC 3588-2 system on a chip (SoC). d Comparison of normalized current density with CPENG and other representative PENGs. Source data are provided as a Source Data file. Gently triggering it with a finger generated and stored electricity, with 24 touches charging a 1 µF capacitor to 0.8 V, showcasing a high charging rate for a PENG at low frequency (Fig. S12 ). Additionally, the CPENG (four layers) powered a System on Chip (SoC) at 30 Hz (Fig. 6c ), enabling it to activate a radio frequency (RF) transmitter module embedded in the SoC every 2 min. It’s worth emphasizing that the defect-passivated composite films developed in this work can be scaled up for more practical applications. We normalized the output current density of the 14-layer CPENG with the applied force of 4.2 N and compared it with state-of-the-art PENGs (Fig. 6d ). The output current density per unit force in the CPENG is notably higher, surpassing the reported OHP-based PENGs by an order of magnitude. Among ceramic-based composites, the 72-layer PENG holds the highest reported normalized current density of 24.17 µAcm −2 N −1 . In contrast, the 14-layer CPENG in this study, with PS-FAPbBr 2 I composites, generates 25 µAcm −2 N −1 . This finding underscores that the normalized current density of the PS-functionalized perovskite composite system in this work is the best-reported among PENG devices (Table S6 ). We anticipate that this discovery further underscores the economic and technological advantages of using organometal halide perovskites over ceramic piezoelectrics in PENG applications and will pave the way for new developments to enhance the performance of PENG-based devices. In summary, the innovative approach of functionalizing organometal halide perovskite with polystyrene has been successfully employed to enhance the output current density of the piezoelectric nanogenerator. The optimization of polystyrene concentration in FAPbBr 2 I precursors has been instrumental in reducing defects, increasing grain size, and achieving a more homogeneous distribution of halide ions, resulting in a smaller lattice strain. Consequently, the PS-functionalized organometal halide perovskite (PS-OHP) exhibits greater structural integrity, reducing ion migration under an electric field and overcoming the “dielectric constant vs. dielectric strength” limit. The optimized concentration of 1% PS significantly suppresses leakage current by one order of magnitude, demonstrating effective control over ion migration. Additionally, the controlled nucleation of perovskites through PS incorporation leads to a twofold increase in grain sizes compared to pristine perovskites. This innovative ternary composite design and cascading them help to elevate the output current density of the extensively studied perovskite PENG by one order of magnitude thus setting a record. The high-performance PENG with ultrahigh current density achieved in this study represents a stride towards establishing a sustainable power source for portable and flexible electronics."
} | 9,150 |
25048697 | PMC4105467 | pmc | 5,692 | {
"abstract": "Horizontal gene transfer often leads to phenotypic changes within recipient organisms independent of any immediate evolutionary benefits. While secondary phenotypic effects of horizontal transfer (i.e., changes in growth rates) have been demonstrated and studied across a variety of systems using relatively small plasmids and phage, little is known about the magnitude or number of such costs after the transfer of larger regions. Here we describe numerous phenotypic changes that occur after a large-scale horizontal transfer event (∼1 Mb megaplasmid) within Pseudomonas stutzeri including sensitization to various stresses as well as changes in bacterial behavior. These results highlight the power of horizontal transfer to shift pleiotropic relationships and cellular networks within bacterial genomes. They also provide an important context for how secondary effects of transfer can bias evolutionary trajectories and interactions between species. Lastly, these results and system provide a foundation to investigate evolutionary consequences in real time as newly acquired regions are ameliorated and integrated into new genomic contexts.",
"introduction": "Introduction \n H orizontal G ene T ransfer (HGT), the movement of genetic material between individuals without reproduction, is a major evolutionary force within microbial communities and impacts genome dynamics across all life [1] , [2] . Although HGT events often provide direct fitness benefits to recipient cells, such as antibiotic resistance, integration of foreign DNA is an inefficient process [3] – [5] . As a result, newly acquired regions often interfere with physiological, genetic, and regulatory pathways to cause changes independent of phenotypes under immediate or direct selection pressures [6] . Numerous studies have demonstrated the existence of such costs by documenting changes to fitness, growth rate, or other phenotypes after the transfer of relatively small genomic regions. However, few studies have examined costs associated with megaplasmid transfer. A variety of non-mutually exclusive mechanisms potentially contribute to costs of HGT. For instance, recently acquired genes are typically expressed at inefficient levels leading to limitations in resources such as ribonucleotides, amino acids, or ATP [7] , [8] . Additional genes can occupy molecular machines required for basic cellular functions, such as polymerases and ribosomes, and sequester these limiting enzymatic resources from more critical activities [9] , [10] . Foreign proteins may not fold correctly in their new cellular contexts, which could lead to disruption or triggering of stress responses [4] , [11] . Recently acquired regions may disrupt flux through cellular systems, leading to the buildup of toxic intermediates [12] , [13] . While such costs have been directly observed in laboratory experiments, retrospective studies across genomes add an additional layer of complexity as there exists an inverse correlation between gene retention after HGT and number of protein-protein interactions affected [14] . In most cases the precise molecular mechanisms underlying observed costs of HGT have not been identified, however, both the magnitude and molecular basis for costs could be greatly affected by both the size and gene content of the acquired region. Costs of HGT have typically been studied by focusing on phenotypic changes after HGT of relatively small plasmids and lysogenic phage, even though large-scale transfers (>60,000 bp) occur at appreciable rates throughout bacteria [6] , [15] – [17] . We have developed an experimental system to investigate the costs of large-scale HGT by using a ∼1Mb megaplasmid which is self-transmissible throughout Pseudomonas species [18] . Transfer of this megaplasmid occurs in both liquid and solid media and requires a type IV secretion system. This HGT event introduces approximately 700 ORFs into recipient cells, including many “housekeeping” genes as well as almost a full complement of tRNA loci as is characteristic of chromids [16] , [19] . Importantly, it does not appear as though full pathways are present for megaplasmid encoded housekeeping gene pathways, so function very likely requires direct interaction with chromosomal networks. In a parallel manuscript [18] , we demonstrate that acquisition of this megaplasmid lowers fitness of Pseudomonas stutzeri by ∼20% and here we report on multiple additional phenotypes affected by large-scale HGT. Specifically, we find that megaplasmid acquisition leads to sensitivity to quinolone antibiotics, DNA intercalating agents, temperature, and killing by other bacterial species. We further find that HGT changes bacterial behavior in that biofilm formation is decreased and motility is increased. This widespread pleiotropy could signal that multiple phenotypic costs occur throughout transfer events [6] . Moreover, that such pleiotropic relationships between phenotypes are mediated at a systems level by single HGT events creates a unique situation where phenotypic evolution occurs as a by-product of evolutionary amelioration after transfer rather than direct selection on phenotypes themselves. In sum, we document the significant potential for secondary effects of HGT to alter phenotypic evolution and adaptive trajectories across microbial populations. This system further underscores the indirect power of costs of HGT to rapidly generate phenotypic diversity across closely related bacteria.",
"discussion": "Discussion For horizontally transferred regions to be maintained within a population, they must either provide a large enough benefit or be transmitted at high enough rates across individuals to avoid loss due to selection or genetic drift [26] , [27] . That such benefits may be the primary target of strong selective pressures within a given environment, as with antibiotic resistance, doesn't preclude the existence of neutral secondary phenotypic changes or HGT-associated costs which are deleterious in other environments [6] . Although such costs of HGT appear to be widespread, there have been few efforts to investigate phenotypic effects of megaplasmid transfer. Furthermore, even when costs are observed, measurements are often limited to single phenotypes even though multiple cellular systems could be affected [5] , [6] , [13] , [28] – [30] . Here we explore a system where HGT increases bacterial genome size by ∼20%, using a megaplasmid which is self-transmissible throughout pseudomonads [18] , [19] . We report that megaplasmid acquisition alters numerous phenotypes within P. stutzeri in unprecedented ways, which highlights the potential for large-scale transfers to shape evolutionary dynamics within natural populations. This system provides a unique foundation to explore how evolution affects pleiotropic interactions between phenotypes altered as a result of large-scale HGT events, but also a powerful model to dissect individual interactions underlying these costs at a molecular level. In a separate manuscript [18] , we have demonstrated that megaplasmid acquisition by P. stutzeri impacts competitive fitness and bacterial growth under standard laboratory conditions. Here we show that megaplasmid acquisition is also accompanied by secondary changes to a variety of phenotypes affecting cellular physiology, environmental survival, and interactions with other species. For instance, megaplasmid acquisition increases sensitivity to quinolone antibiotics as well as stresses such as heat. To the best of our knowledge this is the first report of horizontal gene transfer directly lowering antibiotic resistance, and is especially interesting given that environmental stress can increase quinolone resistance [31] . That these responses are specifically affected by the test environments, as opposed to correlated effects on slower growth across a range of conditions, is highlighted by lack of sensitivity to a variety of other tested conditions ( File S1 ). We also demonstrate that megaplasmid acquisition increases sensitivity to a substance present within the supernatant of P. aeruginosa cultures. As with quinolone sensitivity, to the best of our knowledge this is the first report of a plasmid mediating sensitivity to supernatant from bacterial cultures. Although we currently do not know the molecule(s) responsible for this effect, multiple bacteriocins and other antimicrobial targets known to be found within the supernatant will be the target of future studies [24] , [25] . We have further shown that acquisition of the megaplasmid increases bacterial motility (or lowers the chemotaxis threshold) within soft agar and decreases biofilm formation in liquid culture. It is important to note that all of these phenotypic changes are effectively neutral under the defined laboratory conditions, which we use to select for successful conjugation, because this megaplasmid is engineered to provide tetracycline resistance. Therefore, under selective conditions in the lab, cells that can't acquire the megaplasmid will die due to antibiotic selection. While selective pressures in nature are likely more complex, these secondary changes could have dramatic effects on ecological strategies or niches between closely related bacterial populations. This megaplasmid is representative of other large-scale gene transfers in terms of coding capacity, genetic content, and divergence from the recipient genome [15] , [16] , [19] . In spite of data demonstrating a negative bias for retention of highly connected genes after HGT events, megaplasmids often contain and can transfer numerous housekeeping genes [16] . The megaplasmid within our system itself contains 38 tRNA loci, polymerase subunits, DNA recombination and repair systems, a putative ribosomal protein, as well as other proteins that could be involved in housekeeping functions [19] . At the moment we don't know whether HGT associated costs are dependent upon any of these genes interfering with chromosomal pathways. However, results obtained herein could represent general outcomes after HGT events or may only be emergent properties of HGT by specific types of larger vectors like chromids. One major question arising from these results concerns the independence of phenotypic shifts after HGT. Do all observed changes result from a single protein-protein interaction, numerous individual detrimental interactions, or the disruption of complex and interwoven regulatory networks? Furthermore, is the breadth of altered phenotypes a general property of HGT events as a whole or does the number of changes increase with size or gene content of transferred region? One candidate pathway does stand out as a potential mediator of these phenotypes a priori . Acquisition of the megaplasmid brings with it hundreds of new genes, the protein products of many of which are membrane localized [19] . Since cell size is limited, the incorporation of additional membrane bound proteins likely disrupts molecular signatures and dynamics of the membrane and could easily trigger or disrupt the envelope stress response [32] . The envelope stress response is conserved throughout bacteria, and responds to a variety of membrane stresses through the action of proteases, anti-sigma factors, as well as a host of other regulators. Since membrane integrity is critical for bacterial survival, the envelope stress response often sits at the top of regulatory cascades that control numerous phenotypes [33] . That previous results suggest quinolone sensitivity and sensitivity to P. aeruginosa supernatant are correlated with cell membrane integrity provides support for this model [34] – [36] . Furthermore, chaperone function links both heat and envelope stress responses, since one of the main determinants for both pathways is improperly folded proteins [32] , [37] , [38] . Lastly, motility and biofilm formation are directly regulated by the envelope stress response in Pseudomonas , as both the flagellum and pili are critical membrane bound structures [39] , [40] . The addition of so extra DNA through HGT could also affect gene regulation at a global level through modification of chromosomal conformation. Proteins like HN-S play important roles in limiting detrimental effects of HGT by silencing transferred regions [41] , [42] , but are also critical for packaging the chromosome [43] . HN-S like proteins encoded by IncHI plasmids potentially mediate multiple phenotypic effects in after HGT in Salmonella including: increasing competitive fitness at low temperatures, increasing survival at high temperatures, and a reduction of motility [44] . MvaT is a Pseudomonas analog of HN-S, and an MvaT homolog (Pmr) found within the Pseudomonas pCAR1 plasmid mediates multiple phenotypic changes including increased resistance to chloramphenicol [13] . Plasmids often encode nucleiod associated proteins (NAPs) such as HN-S, so that regulatory alterations of chromosomally encoded pathways may be a fairly general feature of plasmid driven HGT [45] . It is striking that the phenotypes associated with acquisition of pMPPla107 by P. stutzeri are nearly the exact opposite of changes reported after HGT within for other systems. The pMPPla107 megaplasmid does contain at least three loci that resemble nucleoid associated proteins (including two divergent copies of MvaT/Pmr and a locus similar to IHF), but it is currently unknown whether interactions between these loci and chromosomal counterparts are responsible for observed costs. It is also possible that similar interactions between chromosome and plasmid occur across IncHI, pCAR1, and pMPPla107, but that P. stutzeri DBL332 regulates downstream pathways opposite of Salmonella and other pseudomonads. Along these lines, quinolone antibiotics are known to disrupt gyrase function and alter the level of DNA supercoiling within the cell [36] . Sensitivity to quinolones could therefore arise after HGT because chromosomal maintenance requires a threshold amount of functional gyrase. Presence of the megaplasmid could increase this threshold or lower the amount of available gyrase, either of which would lower the concentration of quinolone required for antibiotic effects. Changes in DNA supercoiling are known to affect cellular responses to various stresses including heat shock [46] . However, one should note that a complex feedback loop exists between DNA supercoiling and the envelope stress response [47] , and both may be important contributors to costs of HGT demonstrated within this manuscript. Alternatively, a variety of other independent physiological and regulatory pathways could be responsible for these changes including nutrient limitation triggering the stringent response or the disruption of multiple quorum sensing pathways altering regulation across the genome [48] . Outside of any direct fitness benefits from HGT, that multiple phenotypic changes are linked through single HGT events could skew evolutionary dynamics in unpredictable ways. For example, it is well known that adaptive trajectories and “evolvability” can be influenced by the order that beneficial or compensatory mutations fix [49] , [50] . Secondary effects of HGT could bias future evolutionary paths within populations by altering magnitude or direction of epistatic interactions between adaptive mutations [51] . Furthermore, since recently acquired regions often function sub-optimally, the total number of potential beneficial mutations within an environment could be increased due to compensatory changes for the HGT event [52] . This influx of beneficial mutations correlated to HGT could impact both the order and magnitude of fixed adaptive mutations through clonal interference [53] . Along these lines, significant differences in evolutionary potential could arise based on, for instance, whether antibiotic resistance is introduced through HGT or de novo mutation. Such a balance could be further impacted in positive or negative ways by environment specific feedbacks on costs of HGT [54] . We also note that pMPPla107 conjugates across strains at high rates within the laboratory environment, on both solid media and in shaking liquid cultures, with transfer dependent on a type IV secretion system [18] . Therefore, the costs observed after HGT of this megaplasmid may not need to be compensated in order for pMPPla107 to be maintained within a population. These observations suggest an interesting line of research exploring interactions between costs of HGT, transmission rates, and megaplasmid persistence within populations through time. Looking forward, amelioration of costs of HGT could lead to dramatic phenotypic differences between strains descended from a recent common ancestor due strictly to differences in paths of evolution and pleiotropic relationships from HGT [55] . These pleiotropic relationships could be reinforced or disrupted depending on which suites of mutations fix over time. For instance, amelioration of megaplasmid associated costs could produce one cluster of strains that is phenotypically indistinguishable from the non-megaplasmid ancestor while another cluster compensates only for costs at 27 o c and is unable to grow at 37°C. Furthermore, as a result of costs of the megaplasmid to P. stutzeri , increased resistance to quinolone antibiotics such as nalidixic acid or ciprofloxacin could evolve solely due to compensation for the megaplasmid in the absence of direct selection by quinolones. Since HGT introduces foreign genes and pathways into novel genomic contexts, each transfer event brings with it great potential to disrupt existing genetic and physiological networks within the recipient cell. Although numerous results have analyzed and dissected costs after the transfer of relatively small plasmids and phage, we have developed a model system with which to explore phenotypic costs associated with large-scale HGT (∼20%). The phenotypic shifts we see are dramatic and include changes of bacterial tolerance to numerous stresses as well as alterations of behavior. Individually, a subset of these changes have been observed in other systems as a result of HGT or as a pleiotropic result of de novo adaptive mutations. However, taken as a whole, these results highlight the power of epistasis between the recipient genome and recently acquired regions to completely shift genetic and phenotypic expectations between closely related organisms. Furthermore, our results demonstrate the amazing breadth of phenotypes potentially affected by one HGT event and emphasize how singular evolutionary events can re-wire, reshape, and influence even the most well-studied genetic pathways."
} | 4,685 |
39741005 | PMC11741001 | pmc | 5,693 | {
"abstract": "Wetland methane emissions are the primary natural contributor\nto\nthe global methane budget, accounting for approximately one-third\nof total emissions from natural and anthropogenic sources. Anaerobic\noxidation of methane (AOM) serves as the major sink of methane in\nanoxic wetland sediments, where electron acceptors are present, thereby\neffectively mitigating its emissions. Nevertheless, environmental\ncontrols on electron acceptors, in particular, the ubiquitous iron\noxides, involved in AOM are poorly understood. Here, we explored methane\nsinks within a hypersaline pool situated in a coastal wetland. The\ngeochemical profiles reveal a tiering, where microbial sulfate reduction\ndominates in the organic-rich surface sediment, yielding to iron reduction\nin the deeper organic-poor yet sulfate-rich subsurface sediment. This\nshift is attributed to the drilling-induced depression and subsequent\ndiagenetic transformation of the surface sediment. Radiotracer incubations\ndemonstrate a strong association of AOM with sulfate in surface sediment\nand with iron oxides in subsurface sediment. Despite high concentrations\nof sulfate in coastal wetlands, Fe-dependent AOM may play a significant,\nyet often under-considered, role as a sink for methane emissions.",
"introduction": "Introduction Methane is a potent greenhouse gas and\nis the second-largest contributor\nto global warming after carbon dioxide. 1 − 3 It arises from both natural\nsources and human activities. Approximately 60% of current methane\nemissions stem from human activities, with the remaining portion originating\nfrom natural processes. 4 Notably, wetlands\nstand out as the predominant natural contributor to atmospheric methane\nglobally, making them a significant focal point in addressing climate\nchange concerns. 5 Methane emissions from\nwetlands are dictated by its microbial production and oxidation. Methanogenic\narchaea use a range of substrates such as H 2 /CO 2 , acetate, and methylated compounds to produce methane under anoxic\nconditions, 6 while a group of aerobic bacteria\ncan also generate methane in oxic water bodies. 7 Once formed in anoxic sediments, methane can be oxidized\nby anaerobic oxidation of methane (AOM), depending on the availability\nof electron acceptors. 8 , 9 Methane entering the water column\nor surface soil may undergo aerobic oxidation before eventually being\nreleased into the atmosphere. 10 , 11 Common electron\nacceptors for AOM include sulfate, nitrate, nitrite,\nhumic substances, and metal oxides. 8 , 12 − 15 Sulfate-driven AOM is ubiquitous in coastal and marine environments\nand is typically performed by consortia of anaerobic methanotrophic\n(ANME) archaea and sulfate-reducing bacteria. 16 − 19 Nitrate- and nitrite-dependent\nAOM has been observed in freshwater environments. 20 Nitrate-dependent AOM is mediated by specific members of\nthe ANME clade (ANME-2d, “ Ca. Methanoperedens ”), operating in a syntrophic relationship with nitrite consumers\nor performing denitrification independently. 15 , 21 Nitrite-dependent AOM, on the other hand, is performed by oxygenic\nbacteria known as “ Methylomirabilis oxyfera ” from the NC10 group, which reduce nitrite and concurrently\nproduce oxygen as an intermediate, facilitating the oxidation of methane. 14 Humic substances, such as anthraquinone 2,6-disulfonate\n(AQDS), have been employed as electron sinks for AOM in short-term\nexperiments, whereas their environment significance is yet to be thoroughly\ninvestigated. 13 , 22 , 23 AOM can also be coupled to the reduction of various heavy\nmetals,\nsuch as Fe(III), Mn(IV), Cr(VI), As(V), and Se(VI). 12 , 24 , 25 Notably, Fe stands out as the most prevalent\nheavy metal with its oxides widely distributed in natural environments,\nparticularly in wetlands. Fe-mediated AOM can be performed by methanotrophs,\nsuch as ANME-2d archaea (“ Ca. Methanoperedens ”), as well as by methanogens, which oxidize methane nonsyntropically,\nexploiting soluble, nanophase, or solid-phase ferric Fe as electron\nacceptors. 13 , 26 − 30 In particular, incubation experiments demonstrated\nthat Fe-AOM has the potential to reduce a range of solid-phase ferric\nFe, from highly reactive Fe oxides (FeOx) such as ferrihydrite to\npoorly reactive Fe minerals such as hematite and magnetite. 12 , 31 By employing tracer incubation of sediment slurries, modeling of\nporewater profiles, and the identification of authigenic minerals,\nthe presence of Fe-AOM has been documented in diverse aquatic settings,\nencompassing both marine and freshwater environments. 12 , 32 − 39 For example, in marine sediments, Fe-AOM has been identified beneath\nthe sulfate–methane transition zone (SMTZ) where sulfate-driven\nAOM effectively oxidizes the majority of the upward methane flux.\nThe high dissolved Fe 2+ concentration and vivianite authigenesis\nbelow the SMTZ have been attributed to the occurrence of Fe-AOM. 33 , 34 , 38 , 40 , 41 In freshwater lakes, the role of Fe-AOM\ncan be more predominant due to low sulfate concentrations. 32 , 35 , 42 , 43 Nonetheless, the role of Fe-AOM as a methane sink in wetlands,\na significant contributor to methane emissions, has been poorly studied. 9 , 44 Given the importance of AOM in mitigating methane emissions, knowledge\nabout Fe-AOM in wetland sediments is essential. Enhancing our comprehension\nof methane oxidation pathways will enable more accurate predictions\nof methane emissions from wetlands, especially in the context of eutrophication\nand climate change. In this study, we investigate the interplay of\ncarbon–sulfur–iron biogeochemical cycles, placing particular\nemphasis on the dynamic interaction between methane and Fe(III) minerals\nwithin the Carpinteria Salt Marsh Reserve. Through radiotracer and\nbatch sediment slurry incubations, Krause and Treude 45 demonstrated that methylated substrates drive active methanogenesis\nwithin a hypersaline pool in the saltmarsh. They also inferred the\npotential occurrence of Fe-AOM at the study site based on ex situ\nAOM rates. 45 Combining porewater and solid-phase\nanalyses with the determination of microbial turnover rates, we present\ncompelling biogeochemical evidence confirming the occurrence of Fe-AOM\nin the subsurface sediment from the hypersaline pool. In light of\nthe concurrent methane production and oxidation observed at the study\nsite, 45 a comprehensive understanding of\nAOM pathways offers novel perspectives on the cryptic methane cycling\nand its pivotal role in regulating methane emissions from wetlands.",
"discussion": "Results and Discussion Geochemical Profiles The hypersaline pool sediment\nwas characterized by high sulfate (>86 mM) and low methane (<33\nμM) concentrations ( Figure 1 B). The decrease in the sulfate concentration downcore\nwas likely caused by a combination of microbial sulfate reduction\nand the diffusion of additional sulfate from the supernatant water\nduring the dry season, when the water becomes hypersaline and enriched\nin sulfate. Methane concentrations throughout the sampled sediment\nremained constant at 21 ± 5 μM (one standard deviation,\n1σ). The TOC content was highest (9.4 wt %) at the sediment\nsurface, then decreased to an average of 0.9 ± 0.2 wt % below\nthe upper 6 cm, reflecting strong organic matter degradation in the\nupper 6 cm ( Figure 1 C). The δ 13 C TOC was invariant below 2\ncm (−24.3 ± 0.5‰), whereas its values peaked up\nto −3.9‰ at the sediment surface where microbial mats\nwere present ( Figure 1 C). This extremely high δ 13 C TOC is consistent\nwith a minimal isotope fractionation seen in many hypersaline microbial\nmats due to diffusion-limited DIC uptake and active bicarbonate transport. 63 The organic matter degradation resulted in the\nrelease of phosphate into the porewater, 64 leading to a progressive increase of its concentration in the upper\n6 cm ( Figure 1 G). Porewater\nnitrate and nitrite were detectable only in the upper 2 cm, totaling\n6.5 and 5.9 μM at 0–1 and 1–2 cm, respectively\n( Figure 1 G). Vertical\ngeochemical profiles further revealed two distinct mineralogical zones:\nan FeS-rich layer between 0 and 6 cm overlying an FeOx-rich layer\nbetween 6 and 15 cm ( Figure 1 H–J). Sulfate reduction rates, which were determined\nin the hypersaline pool in 2018, 45 peaked\nat the sediment surface and decreased sharply with depth in the upper\n6 cm ( Figure 1 F). Active\nsulfate reduction fueled aqueous sulfide accumulation in the FeS-rich\nlayer ( Figure 1 E),\nleading to the formation of Fe sulfide minerals up to 40 μmol\ng –1 and the depletion of poorly crystalline Fe(III)\nminerals down to 0.5 μmol g –1 ( Figure 1 H–J). In the FeOx-rich\nlayer, the dissolved Fe 2+ concentration formed a broad\npeak at 6–13 cm, reaching its maximum up to 278 μM at\n10 cm ( Figure 1 E).\nA similar pattern was observed in the abundance of poorly crystalline\nFe(III) minerals, with peak values up to 68 μmol g –1 , while the content of Fe sulfides was consistently low (3.6 ±\n1.8 μmol g –1 ) below 6 cm ( Figure 1 H–J). In this layer,\nsulfate reduction rates were below the detection limit, suggesting\nthat the dissolved Fe 2+ was not sourced from abiotic sulfide-mediated\nreductive dissolution of Fe oxides. Accordingly, the cooccurrence\nof abundant solid-phase Fe(III) phases and dissolved Fe 2+ implies microbial iron reduction. 65 The\nreduction of Fe oxides prompted the liberation of phosphate previously\nadsorbed onto these oxides into the porewater. 64 Nevertheless, a decline in the phosphate concentration\nwas observed at 10 cm where dissolved Fe 2+ reached its\npeak, indicating the potential precipitation of Fe(II)-phosphate minerals,\nsuch as vivianite. 38 , 41 Conversely, in the upper 6 cm,\nFe was predominantly sequestered as Fe sulfide, leading to a buildup\nof phosphate in the porewater ( Figure 1 G). Notably, the highest AOM rates 45 peaked\nand aligned with the broad dissolved Fe 2+ peak between\n6 and 12 cm ( Figure 1 E,F), pointing strongly to the occurrence of Fe-dependent AOM. Aside\nfrom Fe(III), other electron acceptors coupled to AOM include sulfate,\nnitrate/nitrite, humic substances, and other oxidized metal species\n(e.g., Mn(IV)). 8 , 12 , 13 , 15 Given that sulfate reduction rates and nitrate/nitrite\nconcentrations were below detection limits in this depth section,\nAOM is likely not coupled to sulfate and nitrate/nitrite reduction.\nThe extremely low TOC content at 6–12 cm can further eliminate\nhumic substances, which are a major part of the TOC pool in sediments, 66 , 67 as electron acceptors. Mn(IV)-dependent AOM is a possible process,\nalthough it remains a minor contributor to metal-catalyzed AOM in\nnatural settings. This limited impact is attributed to the relatively\nlow abundance of manganese in sediment, with mass ratios to aluminum\naveraging approximately 0.005 (for Mn/Al) compared to approximately\n0.6 for iron (Fe/Al) in the studied region. 68 Accordingly, Mn(IV) and its coupling to AOM were not determined\nin this study. It should be noted that salinity and sulfate\nconcentrations in\nthe core from the Krause and Treude study 45 were different from this study. The porewater concentrations of\nmethane, sulfate, and salinity in the sediment core during the 2018\nsampling were 26 ± 7 μM, 68 ± 3 mM, and 119 ±\n7 PSU, respectively ( Figure S2 ). Methane\nconcentrations were similar to those of the 2022 core (21 ± 5\nμM), whereas sulfate concentrations and salinity were substantially\nhigher in the 2022 core (114 ± 28 mM and 264 ± 47 PSU, respectively),\npointing to an advanced evaporation phase in the present study. However,\nwe are confident that the vertical distribution of AOM and sulfate\nreduction remained very similar between the sampling events as the\ncoloring of the sediment layers and their vertical extension did not\nchange between sampling ( Figure S2 ). In\nthe following sections, we will focus on the environmental conditions\nunder which Fe-dependent AOM occurs as well as the relationship between\nAOM and the biogeochemical cycling of carbon, sulfur, and iron in\nthe hypersaline pool. Evolution of the Sediment Property and Microbial Activity in\nthe Hypersaline Pool The hypersaline pool was created by\nhuman activity. In 1945, an oil company conducted an exploratory drilling\noperation in the CSMR. 46 Although the well\nnever entered sustained production and the derrick was dismantled,\nthe drilling process inadvertently created a depression that served\nas a reservoir for water accumulation, comprising both freshwater\nfrom precipitation and seawater from storm events. The original sediment\nin the pool was composed of organic-poor but Fe-oxide-rich deposits,\nas ubiquitously observed in the saltmarsh. Some of the Fe oxides are\nsourced from riverine input from neighboring mountains, while others\nmay be due to oxidative precipitation of groundwater-derived ferrous\niron. 69 Over time, the pool started to\naccumulate labile organic matter such as plant debris from the surroundings\nand dead algae that grew in the pool, in addition to the microbial\nmat at the sediment surface, which has fueled enhanced organic matter\ndegradation in surface sediment. Among all of the degradation\npathways, microbial iron reduction and sulfate reduction are particularly\nimportant for the changes in the mineral composition of the sediment.\nSulfate reduction competes with microbial iron reduction for the same\nelectron donors, which are typically fermentation products. 70 Although iron reducers may be favored by the\nhigher energy yield of their metabolism, they are limited by the availability\nof solid-phase Fe(III) minerals, while sulfate reducers are not electron-acceptor-limited\nin shallow marine sediment settings. 56 , 65 The aqueous\nsulfide produced through microbial sulfate reduction is highly reactive\nand reduces Fe oxides abiotically and thereby competes with microbial\niron reduction for thermodynamically favorable, poorly crystalline\nFe oxides. 55 , 71 The reductive dissolution of\nFe oxides subsequently leads to the formation of Fe monosulfide (FeS)\nand ultimately pyrite (FeS 2 ), 72 as shown by the high AVS and CRS contents in the uppermost sediment\n( Figure 1 J). This diagenetic\ntransformation results in the exhaustion of microbially reducible\nFe(III), 73 , 74 eliminating the occurrence of microbial\niron reduction in the FeS-rich layer where sulfate reduction dominates\norganic matter mineralization ( Figure 1 F,H). The highest sulfate reduction rates detected\nat the sediment surface are driven by the recent deposition of labile\norganic matter, as indicated by the extremely high TOC content ( Figure 1 C,F). Figure 1 Core image and geochemical depth profiles in sediment from the\nhypersaline pool in the Carpinteria Salt Marsh Reserve. (A) Image\nof the solid-phase sediment core collected in November 2021. (B, E,\nand G) Data from the porewater core collected in September 2022: (B)\nporewater sulfate and methane concentrations, along with the sulfate-to-chloride\nratio; (E) porewater sulfide and ferrous iron concentrations; and\n(G) porewater phosphate and combined nitrate and nitrite concentrations.\n(F) Rates of sulfate reduction (SR) and anaerobic oxidation of methane\n(AOM) analyzed by Krause and Treude 45 in\nJune 2018. Note that rates were calculated based on sulfate and methane\nconcentrations collected during the same sampling campaign (see Figure S2 ). (C, D, and H–J) Data from\nthe solid-phase sediment core collected in November 2021: (C) total\norganic carbon (TOC) content and carbon isotopic composition of TOC;\n(D) total nitrogen (TN) content and C/N ratio; (H) contents of poorly\ncrystalline Fe(III) and Fe(II) minerals; (I) contents of highly crystalline\nFe oxide minerals; and (J) contents of Fe sulfide minerals. We synchronized\nthe depth axis of the porewater core with the solid-phase and ex situ\nrate cores, using the identified lithological transition as our reference\npoint. Details regarding the sampling of sediment cores are provided\nin the Materials and Methods section. Sulfate reduction is below the detection limit\nin the subsurface\nFeOx-rich sediment, whereas the peak in the dissolved Fe 2+ concentration indicates the occurrence of iron reduction ( Figure 1 E,F). This upside-down\nredox cascade is likely due to the relatively low organic carbon content\nbelow 6 cm (0.9 ± 0.2 wt %). Here, the recalcitrant nature of\nthe organic matter hinders its degradation rate, and the high availability\nof microbially reducible Fe(III) allows microbial iron reduction to\ncompete with, and therefore suppress, sulfate reduction. 70 , 73 , 74 The upward Fe 2+ flux\nfurther fuels the formation of Fe sulfides at the transition between\nthe two layers, commonly referred to as the sulfidization front in\nthe Baltic and Black Seas. 73 − 75 We observed a small amount of\npyrite in the FeOx-rich sediment, despite sulfate reduction being\nbelow the detection limit for the core collected in June 2018 ( Figure 1 F,J). It is possible\nthat sulfate reduction occurs at extremely low rates, with seasonal\nvariability in response to changes in sulfate concentration and salinity.\nThis nonsteady-state diagenesis could result in the extremely slow\npyritization of subsurface organic-poor sediment. Alternatively, considering\nthe proximity (∼3 km) of the saltmarsh to the neighboring mountains,\nthe pyrite may have been introduced from an external source, such\nas flooding events, during deposition. 76 Taken together, the hypersaline pool presents an example of\nhow\nhuman activity significantly changed the biogeochemical cycles of\ncarbon, sulfur, and iron in a coastal wetland and how the timing of\ndiagenetic transformations of sulfur and iron leads to very distinct\nmicrobial activity in the sediment where the FeS- and FeOx-rich layers\nare dominated by microbial sulfate and iron reduction, respectively. Electron Acceptors for Anaerobic Oxidation of Methane Based on the geochemical profiles, we suggest that AOM is primarily\ncoupled to sulfate reduction and iron reduction in the FeS-rich and\nFeOx-rich layers, respectively. Accordingly, we designed and conducted\nsediment slurry incubation experiments to elucidate the electron acceptors\ninvolved in AOM in each layer. In previous studies, 13 C-labeled\nmethane was more commonly used as a tracer to track AOM activity in\nlong-term incubations with a series of substrates and inhibitors. 12 , 31 , 32 , 34 , 42 , 43 The use of 13 C-labeled methane has the advantage of enriching the microbial\nbiomass and allowing the track of microbial activity over an extended\nperiod of time, while the rates may not remain stable over the same\nperiod. On the other hand, 14 C- and 35 S-radiotracer\ntechniques are highly sensitive and effective in quantifying microbial\nturnover rates in short-term incubations (e.g., 6 h to 2 days) and\nthe rate is expected to be more stable. 9 , 37 , 77 One should note that environmental conditions in\nsediment slurries are strongly altered compared to the intact sediment,\nand hence, the balance and rates of processes may be affected. By\nfurther disrupting the balance of microbial processes using substrates\nand inhibitors, however, we can gain insights into the reactions linked\nto AOM. In this study, we employed 14 C-methane and 35 S-sulfate radiotracers to track the activity of AOM and sulfate\nreduction. We set up a series of incubations, using amorphous Fe(III)\noxyhydroxide and ferrihydrite to provide microbially reducible Fe(III),\nadding molybdate to inhibit sulfate reduction, and adding aqueous\nsulfide to remove microbially reducible Fe oxides and thereby inhibit\niron reduction. High sulfate reduction rates, up to 149 nmol SO 4 2– per cm 3 of diluted sediment\nslurry per day, were detected in the FeS-rich layer in incubations\nwithout molybdate, while sulfate reduction was nearly completely inhibited\n(down to 0.1% of the noninhibited rate) in the molybdate incubation\n( Figure 2 A). In the\nFeOx-rich layer, extremely low sulfate reduction activity was detected\n(10–100 pmol SO 4 2– cm –3 d –1 ) in incubations without molybdate, and activity\nwas undetectable in incubations with molybdate. These results are\nconsistent with the ex situ rates determined by the whole-round core\ntechnique ( Figure 1 F), where sulfate reduction was detected only in the FeS-rich layer.\nIn addition to inhibitors, the introduction of electron acceptors\ncan also lead to a reduction in the sulfate reduction rate. For example,\nintroducing Fe oxides, in particular ferrihydrite, to the incubations\ntends to lower sulfate reduction rates ( Figure 2 A) because the microbial reduction of newly\nadded Fe oxides yields more free energy than sulfate reduction, and\nthereby partially suppress sulfate reduction. 56 , 57 Figure 2 Rates\nof sulfate reduction (SR; panel A) and anaerobic oxidation\nof methane (AOM; panel B) from slurry incubation experiments. The\nincubations lasted for 2 days. Control refers to the unamended sediment\nslurry, i.e., without the addition of inhibitors or substrates. Am-Fe(III),\nAq-sulfide, and BDL represent amorphous Fe(III) oxyhydroxide, aqueous\nsulfide, and below detection limits, respectively. Amorphous Fe(III)\noxyhydroxide and ferrihydrite are termed Fe oxides in the main text.\nThe y -axes are displayed using a logarithmic scale.\nError bars are 1σ error. Similar to sulfate reduction, AOM rates in the\nFeS-rich layer were\nthe highest in the incubation without molybdate (∼848 pmol\nCH 4 cm –3 d –1 ), whereas\nadding molybdate reduced the AOM rate by 80%, indicating that AOM\nis mostly coupled to sulfate reduction ( Figure 2 B). Notably, however, AOM was still active\non the order of 100 pmol CH 4 cm –3 d –1 in incubations with molybdate, implying the use of\nother electron acceptors in parallel to sulfate. Two potential electron\nacceptor candidates are humic substances and nitrate/nitrite. Indeed,\nthe ex situ AOM rates from 2018 45 showed\none peak at the sediment surface ( Figure 1 F), where the highest TOC and nitrate/nitrite\nconcentrations were found ( Figure 1 C,G). Introducing Fe oxides to the FeS-rich layer did\nnot stimulate AOM activity ( Figure 2 B), suggesting that the microbial communities here\nwere not able to switch to Fe-driven AOM in the course of the incubation,\npossibly because of the sluggish growth rate of minority microbial\npopulations performing Fe-AOM and/or the involvement of special structures\nsuch as cytochrome c proteins and pili for iron reduction. 17 , 18 As discussed, the addition of Fe oxides decreased the sulfate reduction\nrates in the FeS-rich layer ( Figure 2 A). Since AOM was largely coupled to sulfate reduction,\nintroducing Fe oxides thereby reduced AOM rates in incubations without\nmolybdate. Another unexpected observation was that adding aqueous\nsulfide (5 mM) did not change the rate of sulfate reduction but significantly\ndecreased AOM activity ( Figure 2 ), implying a differential toxicity of aqueous sulfide to\nthe two groups of microorganisms. In the FeOx-rich layer, the\nAOM rates were nearly constant (on\nan average of 159 pmol cm –3 d –1 ) across the four sets of incubations without molybdate ( Figure 2 B). The addition\nof Fe oxides did not stimulate AOM activity, possibly because in situ\nFe oxides were already available in nonlimiting concentrations. AOM\nactivity was not reduced in incubations with molybdate, suggesting\nthat the process was not coupled to sulfate reduction but likely to\niron reduction, as implied by geochemical profiles ( Figure 1 ). The addition of aqueous\nsulfide showed different results with and without molybdate. When\nsediment slurries were treated with both aqueous sulfide and molybdate,\nAOM was below the detection limit, suggesting that both iron and sulfate\nreduction coupled to AOM were inhibited. On the other hand, when aqueous\nsulfide was added without molybdate, only iron reduction was inhibited,\nallowing sulfate reduction coupled to AOM to gain more advantage and\nthereby maintaining AOM activity. Overall, the incubation data from\nthe FeOx-rich layer demonstrates that AOM is indeed coupled to iron\nreduction but can be switched to sulfate reduction when Fe oxides\nare not available. Further, comparing the AOM rates in both layers,\nwe infer that the transitioning from Fe-driven AOM to sulfate-driven\nAOM is more feasible than the reverse scenario. A recent study\nusing 16S rRNA gene sequencing reported the relative\nabundances of methanogens and ANME archaea across multiple sites within\nthe CSMR. 78 The study identified ANME-2a,\n2b, and 2c exclusively in the upper 2.5 cm of the FeS-rich layer,\ncoinciding with sulfate-driven AOM activity ( Figure 1 F). 78 In contrast,\nonly methanogens—predominantly halophilic Methanonatronarchaeaceae ( 79 ) and, to a lesser extent, Methanosarcinaceae —were detected in the subsurface FeOx-rich layer, with no\nANME archaea present. 78 Recent studies\nhave demonstrated that canonical methanogens, including members within Methanosarcinaceae , are capable of growth as methanotrophs,\nwith their growth dependent on ferrihydrite reduction, highlighting\nthe potential role of methanogens in Fe-AOM. 28 , 29 Collectively, we propose that Fe-AOM in the FeOx-rich layer may\nbe mediated by methanogens. Environmental Implications Coastal wetlands have been\nregarded as a minor source of methane emissions compared to freshwater\nwetlands, primarily because high sulfate concentrations facilitate\nsulfate-driven AOM. 80 − 82 Here, we show that despite the high concentrations\nof sulfate, AOM is coupled with the reduction of an unconventional\nelectron acceptor—iron oxides—in subsurface sediment\nof a coastal wetland. Fe-AOM in sulfate-free sediments has been extensively\nstudied. 31 , 33 , 34 , 38 Extending these observations into sulfate-rich sediments\nsignificantly advances the earlier observations and hypotheses, while\nsuggesting that Fe-AOM is an under-considered sink for methane in\nwetlands. To evaluate the importance of each AOM pathway at\nthe study site, we convert the measured ex situ volumetric rates 45 to depth-integrated areal rates, albeit with\ncaveats due to potential seasonal variations in AOM rates. The depth-integrated\nsulfate- and Fe-driven AOM rates were calculated to be 0.22 and 0.78\nmmol m –2 d –1 , respectively, under\nthe assumption that sulfate and Fe oxides serve as the sole electron\nacceptors within each layer. This comparison indicates that Fe-AOM\nis the major anaerobic pathway of methane oxidation in the hypersaline\npool. This finding is in contrast to offshore marine sediments where\nmost of the methane is oxidized by sulfate-driven AOM in the SMTZ. 8 , 19 In the deep subsurface of marine sediments, Fe-AOM is commonly found\nbelow the SMTZ, but it only accounts for a very minor fraction of\ntotal methane removal in typical marine sediments (e.g., ∼3%\nin the Baltic Sea). 34 , 83 This can be attributed to the\nlow reactivity of Fe oxides in the deep subsurface. As Fe(III)-bearing\nminerals are buried down through the surface sediments, the most reactive\nfraction is readily consumed by organoclastic iron reduction and abiotic\nreductive dissolution by sulfide, leaving the Fe(III)-minerals below\nthe SMTZ to be less reactive and bioavailable compared to the ones\nat the sediment surface. 33 , 84 In coastal wetlands,\nhowever, fresh reactive Fe oxides are pervasive from terrestrial inputs,\nproviding ideal conditions for the occurrence of Fe-AOM. In addition\nto sulfate-driven AOM, Fe-AOM may act as a major sink for methane\nin wetland sediments, significantly mitigating methane emissions from\nwetlands. Given the 8:1 Fe–CH 4 stoichiometry,\nthe strong\nFe 2+ production from Fe-AOM could enhance the sequestration\nof phosphorus through the formation of Fe(II)-phosphate minerals (e.g.,\nvivianite; Figures 1 J), potentially limiting the degree of coastal eutrophication. 34 , 38 We stress that the importance of Fe-AOM likely varies between diverse\ntypes of wetlands due to different environmental conditions, such\nas the availability of organic matter, sulfate, and reactive Fe oxides.\nAlthough the biogeochemical zonation observed in this study is specific\nto organic-rich sediment overlying organic-poor sediment (e.g., the\nBaltic and Black Seas), the interaction between methane and electron\nacceptors is broadly relevant to wetlands and other natural environments.\nGiven that iron oxides are pervasive in wetlands, 85 we conclude that Fe-AOM in FeOx-rich wetlands has the potential\nto significantly impact the biogeochemical cycles of carbon, sulfur,\niron, and phosphorus, with intrinsic connections to climate change\nand eutrophication."
} | 7,142 |
39670638 | PMC11639387 | pmc | 5,694 | {
"abstract": "Abstract Serpentine soils are characterized as a unique environment with low nutrient availability and high heavy metal concentrations, often hostile to many plant species. Even though these unfavorable conditions hinder the growth of various plants, particular vegetation with different adaptive mechanisms thrives undisturbed. One of the main contributors to serpentine adaptation represents serpentine bacteria with plant growth-promoting properties that assemble delicate interactions with serpentine plants. Robinia pseudoacacia L. is an invasive but adaptive species with phytoremediation potential and demonstrates extraordinary success in this environment. To explore more in-depth the role of plant growth-promoting serpentine bacteria, we isolated them and tested their various plant growth-promoting traits both from the rhizosphere and roots of R. pseudoacacia . Based on the demonstrated plant growth-promoting traits such as siderophore production, phosphate solubilization, nitrogen fixation, indole-3-acetic acid production, and ACC deaminase production, we sequenced overall 25 isolates, 14 from the rhizosphere and 11 from the roots. Although more efficient in exhibiting plant growthpromoting traits, rhizospheric bacteria showed a low rate of diversity in comparison to endophytic bacteria. The majority of the isolates from the rhizosphere belong to Pseudomonas , while isolates from the roots exhibited higher diversity with genera Pseudomonas, Bacillus, Staphylococcus, Lysinibacillus and Brevibacterium/Peribacillus/Bacillus . The capacity of the described bacteria to produce siderophores, solubilize phosphate, and fix nitrogen highlights their central role in enhancing nutrient availability and facilitating R. pseudoacacia adaptation to serpentine soils. The findings highlight the potential significance of serpentine bacteria, particularly Pseudomonas , in contributing to the resilience and growth of R. pseudoacacia in serpentine environments.",
"introduction": "Introduction As firmly rooted life forms, plants concurrently cope with the adverse array of biotic and abiotic stress factors. They developed an intricate set of adaptive mechanisms to survive and thrive, altering their biochemical and genetic activity and adjusting physical barriers ( Hu et al. 2018 ; Iqbal et al. 2021 ). In addition, many plant species improve their defense mechanisms by forming discrete interactions with beneficial microorganisms from soil ( Trivedi et al. 2020 ). A particularly significant group of microorganisms for favorable interactions are the plant growth-promoting bacteria (PGPB), which mainly colonize roots, their surface, and the rhizosphere ( Pii et al. 2015 ). PGPB can enhance overall plant health and resistance to different biotic and abiotic stressors through various mechanisms such as phosphate solubilization ( Alori et al. 2017 ), production of siderophores ( Saha et al. 2016 ), indole acetic acid (IAA) ( Duca et al. 2014 ), deaminase 1-aminocyclopropane-1-carboxylic acid (ACC) ( Glick 2014 ) and nitrogen fixation ( Pii et al. 2015 ; Goswami and Deka 2020 ). In heavy metal-burdened soils with low nutrient availability, such as serpentine, these plant-microbe interactions are even more evident in a plant’s stress response. Serpentine outcrops represent distinctive types of ecosystems with specific chemical characteristics. Serpentine soils generally have a low concentration of macronutrients, Ca:Mg ratio, and high concentrations of heavy metals, especially Ni ( Brooks 1987 ). An unfavorable environment conditioned the growth of particular vegetation resistant to the abovementioned drawbacks. Due to the extreme mineral composition, several authors underline the importance of serpentine bacteria. In various studies, PGPB from serpentine outcrops showed resistance to high concentrations of heavy metals and/or enhanced plant resilience ( Rajkumar et al. 2009 ; Ma et al. 2009 ; Fan et al. 2018 ). Robinia pseudoacacia L. is an originally North American woody plant species from the Fabaceae family, which was introduced in Europe a few centuries ago ( Kolbek et al. 2004 ; Vítková et al. 2017 ). As a pioneer species, it inhabits many habitats, such as sandy and rocky outcrops, dry forests, alluvial habitats, and farm fields ( Chytrý et al. 2008 ). Although it is considered an invasive species, the negative influence on the native plant communities is unclear. Many findings indicate clear homogenization and reduced herbaceous diversity, especially in the understory area ( Peloquin and Heibert 1999 ; Benesperi et al. 2012 ). However, the results of a few other studies did not reveal any significant invasive properties of R. pseudoacacia ( Von Holle et al. 2006 ; Sitzia et al. 2012 ). Despite its invasiveness, R. pseudoacacia is still used for the afforestation of anthropogenically-degraded habitats ( Yüksek and Yüksek 2011 ; Hu et al. 2021 ). It is a promising alternative as a phytoremediation technique due to its large biomass and the potential to thrive in heavy metal-contaminated areas ( Vlachodimos et al. 2013 ). Other than the physical advantages (ways of dispersal, root system, and fast growth) the overall mechanisms of R. pseudoacacia resilience are not fully covered. In this study, we shed light on serpentine bacterial isolates with plant growth-promoting characteristics both from the rhizosphere and roots as an essential puzzle in an R. pseudoacacia success. To achieve this, we isolated bacterial strains from both the rhizosphere and roots of R. pseudoacacia and evaluated their plant growth-promoting (PGP) traits. These properties included siderophore production, phosphate solubilization, nitrogen fixation, indole-3-acetic acid (IAA) production, and ACC deaminase activity-essential functions that are known to enhance plant growth and stress tolerance in nutrient-poor and heavy-metal environments. We sequenced 25 isolates, comprising 14 from the rhizosphere and 11 from the roots, to further characterize their diversity. This study provides insight into the functional diversity of serpentine-associated PGPB and their potential engagement in R. pseudoacacia resilience in harsh habitats.",
"discussion": "Discussion With the extreme edaphic conditions and plant communities that differently influence soil properties, serpentines represent a unique complex for microbial studies. Even though soil chemistry is a crucial factor in plant survival, the effects of plant-microbe interactions contribute significantly to overall plant health ( Kumar and Verma 2018 ). Statistical analysis of HMs in soil showed high concentrations of Ni, Fe, Co, and Cr that exceeded TV. These results were expected since serpentine soils are abundant with heavy metals such as Ni, Fe Co, and Cr ( Kierczak et al. 2021 ). Serpentine soils are especially rich in Ni ( Brooks 1987 ) and extreme soil contamination revealed by the Igeo index was anticipated. In contrast, Cd and Pb are usually found in scarcity, which is also confirmed in our results. As serpentine soils are derived from ultramafic rocks, which contain ferromagnesian minerals susceptible to weathering, extremely high Mg and Fe concentrations were also expected. One of the essential hallmarks of serpentine soils is also the low concentration of Ca, K, and P. As the analyzed Ca concentration is well below the average concentration in soil, Ca/Mg ratio showed to be very low (0.0063). Loew and May (1901) were the first ones to conclude that the low ratio of these two elements is the main limiting factor for the plant growth on the serpentine type of substrate. This was confirmed by later studies where the addition of Ca to the soil improved plant growth ( Kruckeberg 1954 ; Vlamis 1949 ; Walker et al. 1955 ). Considering the above-mentioned, Donja Paklenica represents a real serpentine habitat, although more studies related to plant and microbial communities are necessary for a comprehensive perspective. Bacterial isolates were collected solely from the R. pseudoacacia rhizosphere and roots. This plant species was selected based on its success in expansion and resilience to thrive in intolerable environmental conditions ( Vlachodimos et al. 2013 ). Heavy metal analysis generally showed Rb isolates tolerate Ni, Cu and Co. Serpentine environment likely exerts selective pressure on microbial communities, favoring those that can withstand metal toxicity ( Sazykin et al. 2023 ). On the other hand, only a few endophytic isolates were resistant to the tested concentrations, which suggests endophytes may not be as well adapted to heavy metal stress as rhizobacteria. This could imply that while endophytes can benefit the host plant, they may be more sensitive to metal toxicity than rhizobacteria, which can thrive in metal-contaminated environments. Siderophore production as a PGP trait in PGPB is well characterized in numerous studies ( Rajkumar et al. 2010 ; Lee et al. 2012 ; Zhang et al. 2023 ). In our study, most high producers were from the Pseudomonas genus. Pseudomonas species as a plant growthpromoting bacteria can produce pyoverdine and induce plant growth ( Gamalero and Glick 2011 ). In addition, they produce pyochelin, which can chelate various HMs such as Al, Cd, Co, Cr, Hg, Mn, Pb, Zn, and Ni ( Braud et al. 2009 ). Although more research is required regarding physiological response, this may be one of the crucial components of R. pseudoacacia resilience in the serpentine environment. In some cases, the comparison between qualitative and quantitative analysis showed differences in siderophore production of the same isolate. This could be attributed to an inhibitory effect of HDTMA in CAS agar plates. Gram-positive bacteria are usually susceptible to HDTMA toxicity and can inhibit growth ( Pérez-Miranda et al. 2007 ). Nye et al. (1994) also reported the toxicity of HDTMA to gram-negative soil bacteria in high concentrations. A Fe-deficient environment like an iron-free succinate medium employed in the quantitative method may stimulate siderophore production in some isolates ( Kumar et al. 2017 ). Despite the occasional differences in results, Spearman’s Rank Correlation Coefficient (R s ) revealed a positive correlation between the results of qualitative and quantitative analysis (R s = 0.64; p < 0.05). As one of the least available macronutrients in soil, phosphorus is a crucial element in plant metabolism. Considering the rapid transformation of P into insoluble forms unavailable to plants and the generally low concentration of P in serpentine soils, phosphate solubilizing bacteria (PSB) play a substantial role in phosphorus uptake by plants. Although no significant difference was observed between Rb and Eb, only four endophytic bacteria showed phosphate solubilizing properties. This case could be explained through the difference in contact availability of phosphorus in the rhizosphere and the inner tissue of the plant. In the screening of 82 isolates, Fan et al. (2018) discovered only three endophytic isolates with phosphate solubilization activity. To recruit PSB, roots exude diverse compounds necessary for the development of a productive microbial community in the rhizosphere ( Berendsen et al. 2012 ; Wang et al. 2020 ). Previous studies showed less PSB presence than other PGPB isolates. Research conducted by Antoun et al. (1998) showed that out of 266 isolates, 54% had phosphate solubilization ability. At the same time, 83% of isolates showed siderophore production ability. Given that R. pseudoacacia is a plant species from Fabaceae , it was obvious that many isolates exhibit nitrogen fixation ( Masson-Boivin et al. 2009 ; Alemneh et al. 2020 ). Even though only the qualitative method was employed, we could monitor the difference between Rb and Eb. Firstly, the rapid change in color and formation of the pellicle was observed in Rb and then in Eb afterward. Eventually, almost all isolates from Rb and Eb expressed nitrogen fixation. It seems that Rb localization, composed mainly of Pseudomonas spp., and the production of nitrogenase simulates the intense response to nitrogen fixation through more accessible contact with N-forms (Haahtela et al. 1987; Wang et al. 2020 ). All isolates produced IAA to a specific concentration, thus underlying the importance of this compound in R. pseudoacacia resilience. In our study, Pseudomonas and Staphylococcus isolates exhibit the highest concentration of IAA. Similarly, in the research of Acacia farnesiana , Herrera-Quiterio et al. (2020) found that these two genera, among 12%, identified with IAA production ability. In contrast, Yahaghi et al. (2018) investigated Brassica juncea and found that identified Staphylococcus isolates were not among IAA producers. Serpentine soil in our study was more similar to Herrera-Quiterio et al. (2020) while differed from Yahaghi et al (2018) . It is also evident that the rate of IAA synthesis depends on locality and plant species ( Barriuso et al. 2005 ). The connection between IAA and ACC primarily involves their ability to regulate ethylene. The connection between IAA and ACC primarily involves their ability to regulate ethylene. As an ethylene precursor, ACC is involved in many processes, including abscission, fruit ripening, and response to various environmental stressors. The balance between IAA and ACC is crucial for plant overall health, and ACC deaminase plays a significant role in maintaining that balance in ethylene production. Almost all Rb isolates expressed ACC deaminase activity, while only five Eb showed this ability. Gamalero et al. (2023) highlight the common capacity of rhizospheric bacteria to produce ACC deaminase. Concurrently, in our study, many isolates produced both IAA and ACC deaminase. Fan et al. (2018) discovered the same trend, with 43 isolates from R. pseudoacacia roots exhibiting ACC deaminase activity and 50 producing IAA. Similarly to our study where few Pseudomonas isolates revealed this trait, Shaharoona et al. (2006) also discovered this ability in Pseudomonas , where the improved growth of maize was observed following by Pseudomonas inoculation. This pattern, however, is not limited to Pseudomonas strains. In Eb isolates, this trait was detected in Bacillus and Staphylococcus sp. and other studies related to PGP properties ( Araya et al. 2020 ; Misra and Chauhan 2020 ). In the context of genus diversity, the difference was evident among rhizobacteria and endophytic bacteria. Endophytic bacteria represent a remarkable diversity reservoir and their significance in plant stress response has been mentioned in several studies ( Brígido et al. 2019 ; Xu et al. 2019 ; Alibrandi et al. 2020 ). Rhizobacteria, on the other hand, demonstrated low diversity in our study, which may be explained by plant-soil feedback, which affects the microbial community in the soil. In addition, due to several challenging environmental factors, serpentine habitats are difficult for many plant species to thrive. Similarly, as a selective pressure, heavy metals and soil nutrients act as a driving force in the composition of rhizobacteria. Long-term exposure of soil bacteria to high concentrations of heavy metals leads to the emergence of heavy metal tolerance in the bacterial community. Soil bacteria are sensitive to heavy metal pollution; however, due to their great potential for adaptation, heavy metal-resistant strains manage to survive in these adverse conditions. As the abundance and the diversity of sensitive bacteria decrease while only resistant bacterial strains survive, heavy metals negatively affect the taxonomic structure and the diversity of the soil bacterial community ( Sazykin et al. 2023 ). Sun et al. (2022) found relatively lower rhizosphere microbial diversity in severely contaminated soil, affecting microbial groups from Proteobacteria, Basidiomycota, Ascomycota, and Chloroflexi. In addition to high heavy metal concentrations, other properties of serpentine soils (texture, pH, low nutrient concentration, moisture, vegetation cover, etc.) may also negatively affect soil diversity. Root exudates strongly impact the soil microbiota, acting as an attractant or repellent to particular bacterial and fungal strains ( Hu et al. 2018 ). Plant-specific root and rhizosphere microbial communities may thus be defined by root exudates ( Bulgarelli et al. 2013 ). The serpentine bacteria exhibit remarkable plant growth-promoting (PGP) properties, such as nitrogen fixation, phosphate solubilization, and the production of growth-enhancing hormones like IAA, which may significantly contribute to R. pseudoacacia resilience in serpentine soils. These mechanisms improve nutrient uptake and promote plant vigor, particularly in the nutrient-poor and metal-rich conditions characteristic of serpentine environments. Although the effects of individual microbes on plant growth have been thoroughly studied, the impact of the serpentine microbiome has yet to be uncovered. In future studies, metagenomics could reveal specific microbial interactions and metabolic pathways that directly enhance R. pseudoacacia ability to cope with heavy metal stress and other environmental challenges. R. pseudoacacia is an alien plant species with established invasiveness success. In addition to PGP serpentine bacteria, other factors could also support its spread. To have a broader perspective regarding its resilience, the following studies should encompass multiple serpentine localities and the physiological response of R. pseudoacacia to heavy metal stress in investigating potential phytoremediation applications."
} | 4,423 |
39557655 | PMC11636273 | pmc | 5,697 | {
"abstract": "Abstract Microbial soil habitats are characterized by rapid shifts in substrate and nutrient availabilities, as well as chemical and physical parameters. One such parameter that can vary in soil is oxygen; thus, microbial survival is dependent on adaptation to this substrate. To better understand the metabolic abilities and adaptive strategies to oxygen-deprived environments, we combined genomics with transcriptomics of a model organism, Acidobacterium capsulatum , to explore the effect of decreasing, environmentally relevant oxygen concentrations. The decrease from 10 to 0.1 µM oxygen (3.6 to 0.036 pO 2 % present atmospheric level, respectively) caused the upregulation of the transcription of genes involved in signal transduction mechanisms, energy production and conversion and secondary metabolites biosynthesis, transport, and catabolism based on clusters of orthologous group categories. Contrary to established observations for aerobic metabolism, key genes in oxidative stress response were significantly upregulated at lower oxygen concentrations, presumably due to an NADH/NAD + redox ratio imbalance as the cells transitioned into nanoxia. Furthermore, A. capsulatum adapted to nanoxia by inducing a respiro-fermentative metabolism and rerouting fluxes of its central carbon and energy pathways to adapt to high NADH/NAD + redox ratios. Our results reveal physiological features and metabolic capabilities that allowed A. capsulatum to adapt to oxygen-limited conditions, which could expand into other environmentally relevant soil strains.",
"conclusion": "Conclusions In this study, we examined the transcriptional response of A. capsulatum 161 to diminishing O 2 oncentrations in the low nanomolar range. Overall, O 2 -limiting conditions invoked a significant stress response in A. capsulatum 161. Our data indicate that A. capsulatum 161 has the genomic potential for multiple routes for the early steps of glucose catabolism. Under O 2 -limited but glucose-unlimited conditions, A. capsulatum 161 reroutes fluxes through its central metabolism from glycolysis to fermentative end products to counteract NADH/NAD + imbalances building up due to loss of respiratory capacities under electron acceptor-limiting conditions. Understanding these capacities advances the knowledge on the metabolic responses A. capsulatum 161 is capable of in order to successfully thrive and persist under fluctuating substrate availabilities in terrestrial environments. The investigated oxygen range (10 to 0.1 µmol O 2 l −1 or 3.6–0.036 pO 2 % present atmospheric level) is environmentally relevant (Sexstone et al. 1985 ), presumably seen in various soil niches. Coping with dynamic O 2 tensions is therefore vital for aerobic bacteria dwelling in (temporarily) O 2 -deprived habitats. During “spring snowmelt” or in the rhizosphere, catabolism and ATP yields can be uncoupled due to O 2 limitation and carbon availability. To survive reductive stress during O 2 deprivation, soil bacteria depend on metabolic strategies to maintain a proton motive force and redox balance. But modifications in metabolic routes at trace O 2 levels extend beyond soils; these findings have implications in other environments, such as oxygen minimum zones (OMZs) in the Earth’s oceans. OMZs are large water masses with low oxygen concentrations, thus favoring anaerobic metabolism (Kalvelage et al. 2015 ). Interestingly, aerobic metabolism was previously detected in regions of apparent anoxic conditions (“anoxic” OMZs) (Garcia-Robledo et al. 2017 ), along with the presence of terminal oxidases (Kalvelage et al. 2015 , Tsementzi et al. 2016 ) and production of O 2 at trace levels (Canfield and Kraft 2022 ). These regions could provide a niche where bacteria transition from a respiratory to a respiro-fermentative metabolism to maximize energy yield, prior to using less favorable electron acceptors, such as nitrate. Taken together, the transition from aerobic respiration to a respiro-fermentative metabolism could provide bacteria the flexibility to generate energy during periods of limiting O 2 in fluctuating environments for maintenance and survival of their populations.",
"introduction": "Introduction Microorganisms face a multitude of fluctuating and often limiting conditions across various environments, such as soils, human gut, and aquatic environments. Carbon, electron acceptors (such as oxygen (O 2 )), and/or nutrients can vary over space and time. As such, microorganisms need to compensate and employ strategies to survive during these potentially growth-restricting conditions. One such strategy is respiratory flexibility. The utilization of both high- and low-affinity terminal oxidases enables exploitation of the full range of O 2 concentrations for oxidative phosphorylation and energy conservation, providing a great benefit in the ever-changing O 2 concentrations across environments. This can be attained by inducing branched respiratory chains that terminate in multiple oxidases with different affinities for O 2 (Bueno et al. 2012 ) recently shown in members of ubiquitous soil bacteria, the Acidobacteriota (Eichorst et al. 2018 , Trojan et al. 2021 ). Other strategies by which cells respond to limitations are, e.g. modifying enzyme synthesis to take up growth-limiting nutrients or by modulating uptake rates for nutrients available in excess (Roszak and Colwell 1987 ). Alternatively, they can reroute metabolic fluxes, which enables them to shift to alternative sources of energy and building blocks while avoiding possible blockages due to specific nutrient limitations (Roszak and Colwell 1987 , Bergkessel et al. 2016 ). Catabolism and ATP production are often incongruent during these periods of limitation (Stouthamer 1979 ). As a result of this incongruency, a trade-off can occur between catabolic rate and ATP yield, whereby bacteria utilize pathways for the most efficient production of molar ATP yield (Y ATP : mole of ATP/mole of oxidized substrate). For example, when catabolic rates are high but O 2 limiting, fermentative pathways (when available) are employed, together with respiratory pathways, commonly referred to as respiro-fermentation physiology (Pfeiffer et al. 2001 , Vemuri et al. 2007 ) allowing bacteria to maximize ATP production during electron acceptor limitation. This respiro-fermentative physiology has been observed in Escherichia coli, Bacillus subtilis , and Saccharomyces cerevisiae , yet the evolution and regulation of this metabolism is still under debate (Molenaar et al. 2009 ). Presumably, bacteria have evolved to harbor greater metabolic flexibility for ATP production, rather than pathways yielding optimal growth yield (Stouthamer 1979 ). Members of the phylum Acidobacteriota are ubiquitous across numerous soils (Fierer 2017 , Delgado-Baquerizo et al. 2018 ) with a central role in carbon mineralization and plant decomposition (Fierer 2017 , Crowther et al. 2019 ). Still very little is known about factors controlling their abundance in the environment or their effects on biogeochemical cycles under changing environmental conditions. In this study, we investigated the adaptive capability to O 2 -limited conditions with a model member of the phylum Acidobacteriota, Acidobacterium capsulatum 161. It is a member of the family Acidobacteriaceae that is commonly found across many environments, such as soils. Acidobacterium capsulatum 161 has originally been documented to be capable of microaerophilic growth and only later of weak fermentative growth as well (Pankratov et al. 2012 , Myers and King 2016 ). Recently, its capacity for respiratory flexibility due to the presence and functionality of high- and low-affinity terminal oxidases was demonstrated (Trojan et al. 2021 ). Here, we expanded our investigation of this strain to ascertain if it has additional abilities to alter its metabolism, such as the rerouting of metabolic fluxes, by profiling the whole transcriptomic response of A. capsulatum 161 to decreasing O 2 concentrations in the micro- and nanomolar range, the latter referred to as nanoxic (<1 µmol O 2 l −1 ) (Berg et al. 2022 ). To date, no reports have closely documented the catabolic routes of carbon and energy metabolism of Acidobacteriota or have evaluated its global transcriptomic response to O 2 deprivations. By combining genomics and hypoxic culture incubations using highly sensitive optical O 2 sensors (Lehner et al. 2015 ), we were able to investigate transcription patterns at the oxic–anoxic interface and could observe a transition from respiratory to respiro-fermentative metabolism in A. capsulatum 161.",
"discussion": "Discussion Adaptations and fast responses to changes in environmental conditions often occur at the metabolic level and in this work, we gained new insights into the transcription response of A. capsulatum 161 to diminishing O 2 concentrations at low micro- and nanomolar levels. Our data indicate that diminishing O 2 played a pivotal role in regulating the expression of genes involved in central metabolism under C excess conditions (Figs 3 and 4 ). To counter the toxic accumulation of respiration byproducts building up from the lack of O 2 , it shifted its metabolism and rerouted fluxes from an energy favorable respiratory state (Fig. 4B ) to a respiro-fermentative condition, in which acetate together with ethanol seemed to be major end-products (Figs 3 and 4C ). Glucose transport, PP pathways, and pyruvate production were downregulated at low-nanomolar O 2 concentrations (Fig. 3 )—presumably to reduce the NADH/NAD + redox ratio, which is a critical regulator of cell metabolism ultimately controlling the onset of respiro-fermentative metabolism (Shen and Atkinson 1970 , Szenk et al. 2017 ). As A. capsulatum 161 transitioned from oxic to nanoxic conditions under C excess, the transcripts of many NADH-generating enzymes related to oxidative respiration were reduced (Fig. 4B ). Glucose import ( galP) exhibited reduced expression from 10 to 0.1 µmol O 2 l −1 , presumably as a means to limit the amount of available glucose. Yet, glucose 1-dehydrogenase (gdh ) was overexpressed, potentially modulated a great part of the carbon flow through the gluconate bypass, thus reducing glucose concentration in the cell (Fig. 3 ). However, it appears that the cell did not use gluconate, as gluconolaconase had very low transcript levels at low O 2 concentrations (<10 µmol O 2 l −1 ). This could suggest that the enzyme requires a certain oxygen concentration to function. Pyruvate oxidase (PoxB) was upregulated in A. capsulatum 161, which catalyzes the decarboxylation of pyruvate to acetate and CO 2 (Figs 3 and 4C ), suggesting that pyruvate catabolism is the major switch point between the respiratory and fermentative responses. The glycolytic flux was redirected toward the production of fermentation products, acetate (upregulation of pta and acyP ) and ethanol (upregulation of adhP ) (Figs 3 and 4C , Fig. S2 , Table S4 ), to prevent carbon intermediates to enter the TCA cycle (El-Mansi and Holms 1989 ). Cells can then convert acetyl-CoA through the Pta-AckA pathway, producing and excreting acetate while generating ATP (El-Mansi and Holms 1989 ). Since the flux from acetyl-CoA to acetate does not generate any NADH (while the flux from acetyl-CoA through the TCA cycle generates 8 NAD(P)H and 2 FADH 2 ), carbon flow diversion to acetate could be viewed as a means of A. capsulatum 161 to reduce or prevent further NADH accumulation (El-Mansi and Holms 1989 , Holms 2001 ). This is in congruence with previous work on Staphylococcus aureus , where acetate production was enhanced under low O 2 and glucose excess conditions (Ferreira et al. 2013 ). In addition, the conversion of acetaldehyde to ethanol via adhP was upregulated, consuming NADH and hence a way to counteract the NADH/NAD + imbalance (Figs 3 and 4C ). Taken together, we hypothesize that the concomitant rise in NADH levels from glucose excess and low O 2 conditions drove the onset of fermentative metabolism (acetate and ethanol production) to avoid toxic levels of NADH in the cell. Acetate and ethanol production stemming from pyruvate bypasses any energy-conserving steps associated with NADH, allowing a fast oxidation of pyruvate and efficient shuttling of protons/electrons to the ETC. Various studies have shown that in concentrated glucose environments, E. coli and other organisms switch to and obtain some of their energy anaerobically by acetate fermentation, even when O 2 is plentiful, if the rate of glucose consumption is greater than the capacity to reoxidize the reduced equivalents generated (Farmer and Jones 1976 , Hollywood and Doelle 1976 , Andersen and Meyenburg 1980 , Meyer et al. 1984 , Farmer and Liao 1997 , Kayser et al. 2005 , Vemuri et al. 2006 , Vazquez et al. 2008 , Molenaar et al. 2009 , Nahku et al. 2010 , Valgepea et al. 2011 , 2013 , Zhuang et al. 2011 , Basan et al. 2015 , Peebo et al. 2015 , Schütze et al. 2020 ). Although a major role of NADH is to supply electrons to the ETC thereby fueling the production of ATP, the strategy of A. capsulatum 161 was to reduce the NADH production stemming from respiratory pathways to avoid NADH imbalance while generating ATP (Szenk et al. 2017 ). The use of alternative pathways for NAD + regeneration concomitant was also reported in other facultative anaerobes such as E. coli (Vemuri et al. 2006 , Farhana et al. 2010 , Martínez-Gómez et al. 2012 , Szenk et al. 2017 ) and members of the genera Salmonella and Shigella (Gray et al. 1966 , Wolfe 2005 ). The metabolic flexibility would allow these bacteria to cope with varying concentrations of carbon and O 2 in such environments like soils. This respiro-fermentative strategy might extend into the Acidobacteriota , as many genomes harbor this potential as evidence by the presence of acetate kinase and alcohol dehydrogenase (Eichorst et al. 2018 ). Our experimental conditions also invoked a significant upregulation of the glycogen metabolism suggesting cells transform excess glucose to the storage compound glycogen (Fig. 3 , Fig. S2 , Table S4 ). The accumulation of glycogen provides a metabolic reserve for A. capsulatum 161 under potential carbon-limited conditions in the future, which could be an important strategy in environments such as soils allowing cells to cope with transient limiting conditions. Stress response to O 2 and reactive oxygen species (ROS) is crucial for the ability to exist in habitats that are characterized by fluctuating O 2 concentrations. Whether microbes can occupy such a habitat or a microniche within partly depends upon whether they are able to withstand local concentrations of high or low O 2 . Several universal stress proteins in A. capsulatum 161 were significantly upregulated by decreasing O 2 concentrations approaching anoxia; especially two of these usp genes were affected by the drop of O 2 to 0.1 µmol O 2 l −1 (Fig. 2A , Table S2 ). Furthermore, the drop of O 2 invoked a significant increase of transcription of a sensor histidine kinase (Fig. 2A , Table S2 ), which presumably allowed A. capsulatum 161 to sense environmental stimuli and manage various environmental changes by coupling environmental cues to gene expression (Stock et al. 2000 , Mascher et al. 2006 , Kaczmarczyk et al. 2014 ). Cellular stress can further lead to protein denaturation (Hightower 1991 ), and proteolytic removal of non‐functional proteins is crucial for optimal metabolic activities (Porankiewicz et al. 1999 ). We detected an upregulation of the ATP-dependent Clp proteases in the transcriptomic response of A. capsulatum 161 to diminishing O 2 concentrations (Fig. 2A , Table S2 ), suggesting that they are important in removing irreversibly damaged polypeptides that may interfere with metabolic pathways under O 2 -limited stress conditions. In A. capsulatum 161, a clear differential upregulation of genes involved in counteracting oxidative stress was observed upon the decrease of oxygenation from 10 to 0.1 µmol O 2 l −1 (Fig. 2A , Table S2 ), indicating that it is capable of adapting to different redox states. Oxidative stress defense genes such as manganese superoxide dismutase, thioredoxins, and glutaredoxins were highly expressed under stimulated at low O 2 (Fig. 2A , Table S2 ), as seen previously in Nitrosomonas europaea (Sedlacek et al. 2020 ). The increased demand for proteins involved in ROS defense could be caused by NADH/NAD + redox ratio imbalances, as NADH accumulates and becomes toxic. Under O 2 -limiting conditions, an increased level of NADH builds up, as it is less efficiently reoxidized to NAD + as a result of reduced aerobic respiration. The high-affinity bd -type oxidase ( cydAB) and NADH dehydrogenase ( ndh-II ) were upregulated upon the drop of oxygenation to 0.1 µmol O 2 l −1 (Fig. 2B , Table S3 ). We previously hypothesized that the upregulation of the bd -type oxidase could suggest a contribution to respiratory activity at trace O 2 conditions or favor the more faster electron flux than cbb 3 -type oxidases to permit more rapid reducing potential from carbon surplus (Trojan et al. 2021 ). The uncoupled NADH dehydrogenase NDH-II was highly upregulated at 0.1 µmol O 2 l −1 (Fig. 2B , Table S3 ), presumably to compensate for the slow regeneration of NAD + due to the low O 2 availability. NDH-II only catalyzes the oxidation of NADH and reduction of quinones without the ability to pump protons, which, based on our data, seemed to be beneficial under micro- and nanoxic conditions. Alternative electron‐transfer routes seem to allow A. capsulatum 161 adjusting its energy transduction efficiency to its needs and substrate availability. In A. capsulatum 161, electrons can flow from the NADH dehydrogenase and SDH (complex II of the ETC) to the quinone/quinol pool, from where the electrons may either bypass the cytochrome bc 1 complex (complex III) and directly flow to the bd -type quinol terminal oxidase or flow via a cytochrome c either to the low-affinity caa 3 ‐type or the high-affinity cbb 3 ‐type cytochrome c terminal oxidase. Under aerobic- or carbon-limiting conditions, the proton-coupled NDH-I might be the main driver of NADH oxidation and maintain the proton motive force required for ATP synthesis (Fig. 4A ). Under the reduced environment in our incubations, when the reduction state of the quinone/quinol pool increases, the pathway to the NDH-II and bd -type quinol oxidase seemed to increase, with NDH-II being then the dominant dehydrogenase to oxidize excess NADH and support redox balance (Figs 2B and 4C ). This strategy has been observed for E. coli , which may use NDH-II to counteract increasing NADH/NAD + triggered by faster metabolism due to increased glucose uptake in order to support fast growth (Vemuri et al. 2006 , Liu et al. 2019 )."
} | 4,762 |
39557655 | PMC11636273 | pmc | 5,697 | {
"abstract": "Abstract Microbial soil habitats are characterized by rapid shifts in substrate and nutrient availabilities, as well as chemical and physical parameters. One such parameter that can vary in soil is oxygen; thus, microbial survival is dependent on adaptation to this substrate. To better understand the metabolic abilities and adaptive strategies to oxygen-deprived environments, we combined genomics with transcriptomics of a model organism, Acidobacterium capsulatum , to explore the effect of decreasing, environmentally relevant oxygen concentrations. The decrease from 10 to 0.1 µM oxygen (3.6 to 0.036 pO 2 % present atmospheric level, respectively) caused the upregulation of the transcription of genes involved in signal transduction mechanisms, energy production and conversion and secondary metabolites biosynthesis, transport, and catabolism based on clusters of orthologous group categories. Contrary to established observations for aerobic metabolism, key genes in oxidative stress response were significantly upregulated at lower oxygen concentrations, presumably due to an NADH/NAD + redox ratio imbalance as the cells transitioned into nanoxia. Furthermore, A. capsulatum adapted to nanoxia by inducing a respiro-fermentative metabolism and rerouting fluxes of its central carbon and energy pathways to adapt to high NADH/NAD + redox ratios. Our results reveal physiological features and metabolic capabilities that allowed A. capsulatum to adapt to oxygen-limited conditions, which could expand into other environmentally relevant soil strains.",
"conclusion": "Conclusions In this study, we examined the transcriptional response of A. capsulatum 161 to diminishing O 2 oncentrations in the low nanomolar range. Overall, O 2 -limiting conditions invoked a significant stress response in A. capsulatum 161. Our data indicate that A. capsulatum 161 has the genomic potential for multiple routes for the early steps of glucose catabolism. Under O 2 -limited but glucose-unlimited conditions, A. capsulatum 161 reroutes fluxes through its central metabolism from glycolysis to fermentative end products to counteract NADH/NAD + imbalances building up due to loss of respiratory capacities under electron acceptor-limiting conditions. Understanding these capacities advances the knowledge on the metabolic responses A. capsulatum 161 is capable of in order to successfully thrive and persist under fluctuating substrate availabilities in terrestrial environments. The investigated oxygen range (10 to 0.1 µmol O 2 l −1 or 3.6–0.036 pO 2 % present atmospheric level) is environmentally relevant (Sexstone et al. 1985 ), presumably seen in various soil niches. Coping with dynamic O 2 tensions is therefore vital for aerobic bacteria dwelling in (temporarily) O 2 -deprived habitats. During “spring snowmelt” or in the rhizosphere, catabolism and ATP yields can be uncoupled due to O 2 limitation and carbon availability. To survive reductive stress during O 2 deprivation, soil bacteria depend on metabolic strategies to maintain a proton motive force and redox balance. But modifications in metabolic routes at trace O 2 levels extend beyond soils; these findings have implications in other environments, such as oxygen minimum zones (OMZs) in the Earth’s oceans. OMZs are large water masses with low oxygen concentrations, thus favoring anaerobic metabolism (Kalvelage et al. 2015 ). Interestingly, aerobic metabolism was previously detected in regions of apparent anoxic conditions (“anoxic” OMZs) (Garcia-Robledo et al. 2017 ), along with the presence of terminal oxidases (Kalvelage et al. 2015 , Tsementzi et al. 2016 ) and production of O 2 at trace levels (Canfield and Kraft 2022 ). These regions could provide a niche where bacteria transition from a respiratory to a respiro-fermentative metabolism to maximize energy yield, prior to using less favorable electron acceptors, such as nitrate. Taken together, the transition from aerobic respiration to a respiro-fermentative metabolism could provide bacteria the flexibility to generate energy during periods of limiting O 2 in fluctuating environments for maintenance and survival of their populations.",
"introduction": "Introduction Microorganisms face a multitude of fluctuating and often limiting conditions across various environments, such as soils, human gut, and aquatic environments. Carbon, electron acceptors (such as oxygen (O 2 )), and/or nutrients can vary over space and time. As such, microorganisms need to compensate and employ strategies to survive during these potentially growth-restricting conditions. One such strategy is respiratory flexibility. The utilization of both high- and low-affinity terminal oxidases enables exploitation of the full range of O 2 concentrations for oxidative phosphorylation and energy conservation, providing a great benefit in the ever-changing O 2 concentrations across environments. This can be attained by inducing branched respiratory chains that terminate in multiple oxidases with different affinities for O 2 (Bueno et al. 2012 ) recently shown in members of ubiquitous soil bacteria, the Acidobacteriota (Eichorst et al. 2018 , Trojan et al. 2021 ). Other strategies by which cells respond to limitations are, e.g. modifying enzyme synthesis to take up growth-limiting nutrients or by modulating uptake rates for nutrients available in excess (Roszak and Colwell 1987 ). Alternatively, they can reroute metabolic fluxes, which enables them to shift to alternative sources of energy and building blocks while avoiding possible blockages due to specific nutrient limitations (Roszak and Colwell 1987 , Bergkessel et al. 2016 ). Catabolism and ATP production are often incongruent during these periods of limitation (Stouthamer 1979 ). As a result of this incongruency, a trade-off can occur between catabolic rate and ATP yield, whereby bacteria utilize pathways for the most efficient production of molar ATP yield (Y ATP : mole of ATP/mole of oxidized substrate). For example, when catabolic rates are high but O 2 limiting, fermentative pathways (when available) are employed, together with respiratory pathways, commonly referred to as respiro-fermentation physiology (Pfeiffer et al. 2001 , Vemuri et al. 2007 ) allowing bacteria to maximize ATP production during electron acceptor limitation. This respiro-fermentative physiology has been observed in Escherichia coli, Bacillus subtilis , and Saccharomyces cerevisiae , yet the evolution and regulation of this metabolism is still under debate (Molenaar et al. 2009 ). Presumably, bacteria have evolved to harbor greater metabolic flexibility for ATP production, rather than pathways yielding optimal growth yield (Stouthamer 1979 ). Members of the phylum Acidobacteriota are ubiquitous across numerous soils (Fierer 2017 , Delgado-Baquerizo et al. 2018 ) with a central role in carbon mineralization and plant decomposition (Fierer 2017 , Crowther et al. 2019 ). Still very little is known about factors controlling their abundance in the environment or their effects on biogeochemical cycles under changing environmental conditions. In this study, we investigated the adaptive capability to O 2 -limited conditions with a model member of the phylum Acidobacteriota, Acidobacterium capsulatum 161. It is a member of the family Acidobacteriaceae that is commonly found across many environments, such as soils. Acidobacterium capsulatum 161 has originally been documented to be capable of microaerophilic growth and only later of weak fermentative growth as well (Pankratov et al. 2012 , Myers and King 2016 ). Recently, its capacity for respiratory flexibility due to the presence and functionality of high- and low-affinity terminal oxidases was demonstrated (Trojan et al. 2021 ). Here, we expanded our investigation of this strain to ascertain if it has additional abilities to alter its metabolism, such as the rerouting of metabolic fluxes, by profiling the whole transcriptomic response of A. capsulatum 161 to decreasing O 2 concentrations in the micro- and nanomolar range, the latter referred to as nanoxic (<1 µmol O 2 l −1 ) (Berg et al. 2022 ). To date, no reports have closely documented the catabolic routes of carbon and energy metabolism of Acidobacteriota or have evaluated its global transcriptomic response to O 2 deprivations. By combining genomics and hypoxic culture incubations using highly sensitive optical O 2 sensors (Lehner et al. 2015 ), we were able to investigate transcription patterns at the oxic–anoxic interface and could observe a transition from respiratory to respiro-fermentative metabolism in A. capsulatum 161.",
"discussion": "Discussion Adaptations and fast responses to changes in environmental conditions often occur at the metabolic level and in this work, we gained new insights into the transcription response of A. capsulatum 161 to diminishing O 2 concentrations at low micro- and nanomolar levels. Our data indicate that diminishing O 2 played a pivotal role in regulating the expression of genes involved in central metabolism under C excess conditions (Figs 3 and 4 ). To counter the toxic accumulation of respiration byproducts building up from the lack of O 2 , it shifted its metabolism and rerouted fluxes from an energy favorable respiratory state (Fig. 4B ) to a respiro-fermentative condition, in which acetate together with ethanol seemed to be major end-products (Figs 3 and 4C ). Glucose transport, PP pathways, and pyruvate production were downregulated at low-nanomolar O 2 concentrations (Fig. 3 )—presumably to reduce the NADH/NAD + redox ratio, which is a critical regulator of cell metabolism ultimately controlling the onset of respiro-fermentative metabolism (Shen and Atkinson 1970 , Szenk et al. 2017 ). As A. capsulatum 161 transitioned from oxic to nanoxic conditions under C excess, the transcripts of many NADH-generating enzymes related to oxidative respiration were reduced (Fig. 4B ). Glucose import ( galP) exhibited reduced expression from 10 to 0.1 µmol O 2 l −1 , presumably as a means to limit the amount of available glucose. Yet, glucose 1-dehydrogenase (gdh ) was overexpressed, potentially modulated a great part of the carbon flow through the gluconate bypass, thus reducing glucose concentration in the cell (Fig. 3 ). However, it appears that the cell did not use gluconate, as gluconolaconase had very low transcript levels at low O 2 concentrations (<10 µmol O 2 l −1 ). This could suggest that the enzyme requires a certain oxygen concentration to function. Pyruvate oxidase (PoxB) was upregulated in A. capsulatum 161, which catalyzes the decarboxylation of pyruvate to acetate and CO 2 (Figs 3 and 4C ), suggesting that pyruvate catabolism is the major switch point between the respiratory and fermentative responses. The glycolytic flux was redirected toward the production of fermentation products, acetate (upregulation of pta and acyP ) and ethanol (upregulation of adhP ) (Figs 3 and 4C , Fig. S2 , Table S4 ), to prevent carbon intermediates to enter the TCA cycle (El-Mansi and Holms 1989 ). Cells can then convert acetyl-CoA through the Pta-AckA pathway, producing and excreting acetate while generating ATP (El-Mansi and Holms 1989 ). Since the flux from acetyl-CoA to acetate does not generate any NADH (while the flux from acetyl-CoA through the TCA cycle generates 8 NAD(P)H and 2 FADH 2 ), carbon flow diversion to acetate could be viewed as a means of A. capsulatum 161 to reduce or prevent further NADH accumulation (El-Mansi and Holms 1989 , Holms 2001 ). This is in congruence with previous work on Staphylococcus aureus , where acetate production was enhanced under low O 2 and glucose excess conditions (Ferreira et al. 2013 ). In addition, the conversion of acetaldehyde to ethanol via adhP was upregulated, consuming NADH and hence a way to counteract the NADH/NAD + imbalance (Figs 3 and 4C ). Taken together, we hypothesize that the concomitant rise in NADH levels from glucose excess and low O 2 conditions drove the onset of fermentative metabolism (acetate and ethanol production) to avoid toxic levels of NADH in the cell. Acetate and ethanol production stemming from pyruvate bypasses any energy-conserving steps associated with NADH, allowing a fast oxidation of pyruvate and efficient shuttling of protons/electrons to the ETC. Various studies have shown that in concentrated glucose environments, E. coli and other organisms switch to and obtain some of their energy anaerobically by acetate fermentation, even when O 2 is plentiful, if the rate of glucose consumption is greater than the capacity to reoxidize the reduced equivalents generated (Farmer and Jones 1976 , Hollywood and Doelle 1976 , Andersen and Meyenburg 1980 , Meyer et al. 1984 , Farmer and Liao 1997 , Kayser et al. 2005 , Vemuri et al. 2006 , Vazquez et al. 2008 , Molenaar et al. 2009 , Nahku et al. 2010 , Valgepea et al. 2011 , 2013 , Zhuang et al. 2011 , Basan et al. 2015 , Peebo et al. 2015 , Schütze et al. 2020 ). Although a major role of NADH is to supply electrons to the ETC thereby fueling the production of ATP, the strategy of A. capsulatum 161 was to reduce the NADH production stemming from respiratory pathways to avoid NADH imbalance while generating ATP (Szenk et al. 2017 ). The use of alternative pathways for NAD + regeneration concomitant was also reported in other facultative anaerobes such as E. coli (Vemuri et al. 2006 , Farhana et al. 2010 , Martínez-Gómez et al. 2012 , Szenk et al. 2017 ) and members of the genera Salmonella and Shigella (Gray et al. 1966 , Wolfe 2005 ). The metabolic flexibility would allow these bacteria to cope with varying concentrations of carbon and O 2 in such environments like soils. This respiro-fermentative strategy might extend into the Acidobacteriota , as many genomes harbor this potential as evidence by the presence of acetate kinase and alcohol dehydrogenase (Eichorst et al. 2018 ). Our experimental conditions also invoked a significant upregulation of the glycogen metabolism suggesting cells transform excess glucose to the storage compound glycogen (Fig. 3 , Fig. S2 , Table S4 ). The accumulation of glycogen provides a metabolic reserve for A. capsulatum 161 under potential carbon-limited conditions in the future, which could be an important strategy in environments such as soils allowing cells to cope with transient limiting conditions. Stress response to O 2 and reactive oxygen species (ROS) is crucial for the ability to exist in habitats that are characterized by fluctuating O 2 concentrations. Whether microbes can occupy such a habitat or a microniche within partly depends upon whether they are able to withstand local concentrations of high or low O 2 . Several universal stress proteins in A. capsulatum 161 were significantly upregulated by decreasing O 2 concentrations approaching anoxia; especially two of these usp genes were affected by the drop of O 2 to 0.1 µmol O 2 l −1 (Fig. 2A , Table S2 ). Furthermore, the drop of O 2 invoked a significant increase of transcription of a sensor histidine kinase (Fig. 2A , Table S2 ), which presumably allowed A. capsulatum 161 to sense environmental stimuli and manage various environmental changes by coupling environmental cues to gene expression (Stock et al. 2000 , Mascher et al. 2006 , Kaczmarczyk et al. 2014 ). Cellular stress can further lead to protein denaturation (Hightower 1991 ), and proteolytic removal of non‐functional proteins is crucial for optimal metabolic activities (Porankiewicz et al. 1999 ). We detected an upregulation of the ATP-dependent Clp proteases in the transcriptomic response of A. capsulatum 161 to diminishing O 2 concentrations (Fig. 2A , Table S2 ), suggesting that they are important in removing irreversibly damaged polypeptides that may interfere with metabolic pathways under O 2 -limited stress conditions. In A. capsulatum 161, a clear differential upregulation of genes involved in counteracting oxidative stress was observed upon the decrease of oxygenation from 10 to 0.1 µmol O 2 l −1 (Fig. 2A , Table S2 ), indicating that it is capable of adapting to different redox states. Oxidative stress defense genes such as manganese superoxide dismutase, thioredoxins, and glutaredoxins were highly expressed under stimulated at low O 2 (Fig. 2A , Table S2 ), as seen previously in Nitrosomonas europaea (Sedlacek et al. 2020 ). The increased demand for proteins involved in ROS defense could be caused by NADH/NAD + redox ratio imbalances, as NADH accumulates and becomes toxic. Under O 2 -limiting conditions, an increased level of NADH builds up, as it is less efficiently reoxidized to NAD + as a result of reduced aerobic respiration. The high-affinity bd -type oxidase ( cydAB) and NADH dehydrogenase ( ndh-II ) were upregulated upon the drop of oxygenation to 0.1 µmol O 2 l −1 (Fig. 2B , Table S3 ). We previously hypothesized that the upregulation of the bd -type oxidase could suggest a contribution to respiratory activity at trace O 2 conditions or favor the more faster electron flux than cbb 3 -type oxidases to permit more rapid reducing potential from carbon surplus (Trojan et al. 2021 ). The uncoupled NADH dehydrogenase NDH-II was highly upregulated at 0.1 µmol O 2 l −1 (Fig. 2B , Table S3 ), presumably to compensate for the slow regeneration of NAD + due to the low O 2 availability. NDH-II only catalyzes the oxidation of NADH and reduction of quinones without the ability to pump protons, which, based on our data, seemed to be beneficial under micro- and nanoxic conditions. Alternative electron‐transfer routes seem to allow A. capsulatum 161 adjusting its energy transduction efficiency to its needs and substrate availability. In A. capsulatum 161, electrons can flow from the NADH dehydrogenase and SDH (complex II of the ETC) to the quinone/quinol pool, from where the electrons may either bypass the cytochrome bc 1 complex (complex III) and directly flow to the bd -type quinol terminal oxidase or flow via a cytochrome c either to the low-affinity caa 3 ‐type or the high-affinity cbb 3 ‐type cytochrome c terminal oxidase. Under aerobic- or carbon-limiting conditions, the proton-coupled NDH-I might be the main driver of NADH oxidation and maintain the proton motive force required for ATP synthesis (Fig. 4A ). Under the reduced environment in our incubations, when the reduction state of the quinone/quinol pool increases, the pathway to the NDH-II and bd -type quinol oxidase seemed to increase, with NDH-II being then the dominant dehydrogenase to oxidize excess NADH and support redox balance (Figs 2B and 4C ). This strategy has been observed for E. coli , which may use NDH-II to counteract increasing NADH/NAD + triggered by faster metabolism due to increased glucose uptake in order to support fast growth (Vemuri et al. 2006 , Liu et al. 2019 )."
} | 4,762 |
39557655 | PMC11636273 | pmc | 5,698 | {
"abstract": "Abstract Microbial soil habitats are characterized by rapid shifts in substrate and nutrient availabilities, as well as chemical and physical parameters. One such parameter that can vary in soil is oxygen; thus, microbial survival is dependent on adaptation to this substrate. To better understand the metabolic abilities and adaptive strategies to oxygen-deprived environments, we combined genomics with transcriptomics of a model organism, Acidobacterium capsulatum , to explore the effect of decreasing, environmentally relevant oxygen concentrations. The decrease from 10 to 0.1 µM oxygen (3.6 to 0.036 pO 2 % present atmospheric level, respectively) caused the upregulation of the transcription of genes involved in signal transduction mechanisms, energy production and conversion and secondary metabolites biosynthesis, transport, and catabolism based on clusters of orthologous group categories. Contrary to established observations for aerobic metabolism, key genes in oxidative stress response were significantly upregulated at lower oxygen concentrations, presumably due to an NADH/NAD + redox ratio imbalance as the cells transitioned into nanoxia. Furthermore, A. capsulatum adapted to nanoxia by inducing a respiro-fermentative metabolism and rerouting fluxes of its central carbon and energy pathways to adapt to high NADH/NAD + redox ratios. Our results reveal physiological features and metabolic capabilities that allowed A. capsulatum to adapt to oxygen-limited conditions, which could expand into other environmentally relevant soil strains.",
"conclusion": "Conclusions In this study, we examined the transcriptional response of A. capsulatum 161 to diminishing O 2 oncentrations in the low nanomolar range. Overall, O 2 -limiting conditions invoked a significant stress response in A. capsulatum 161. Our data indicate that A. capsulatum 161 has the genomic potential for multiple routes for the early steps of glucose catabolism. Under O 2 -limited but glucose-unlimited conditions, A. capsulatum 161 reroutes fluxes through its central metabolism from glycolysis to fermentative end products to counteract NADH/NAD + imbalances building up due to loss of respiratory capacities under electron acceptor-limiting conditions. Understanding these capacities advances the knowledge on the metabolic responses A. capsulatum 161 is capable of in order to successfully thrive and persist under fluctuating substrate availabilities in terrestrial environments. The investigated oxygen range (10 to 0.1 µmol O 2 l −1 or 3.6–0.036 pO 2 % present atmospheric level) is environmentally relevant (Sexstone et al. 1985 ), presumably seen in various soil niches. Coping with dynamic O 2 tensions is therefore vital for aerobic bacteria dwelling in (temporarily) O 2 -deprived habitats. During “spring snowmelt” or in the rhizosphere, catabolism and ATP yields can be uncoupled due to O 2 limitation and carbon availability. To survive reductive stress during O 2 deprivation, soil bacteria depend on metabolic strategies to maintain a proton motive force and redox balance. But modifications in metabolic routes at trace O 2 levels extend beyond soils; these findings have implications in other environments, such as oxygen minimum zones (OMZs) in the Earth’s oceans. OMZs are large water masses with low oxygen concentrations, thus favoring anaerobic metabolism (Kalvelage et al. 2015 ). Interestingly, aerobic metabolism was previously detected in regions of apparent anoxic conditions (“anoxic” OMZs) (Garcia-Robledo et al. 2017 ), along with the presence of terminal oxidases (Kalvelage et al. 2015 , Tsementzi et al. 2016 ) and production of O 2 at trace levels (Canfield and Kraft 2022 ). These regions could provide a niche where bacteria transition from a respiratory to a respiro-fermentative metabolism to maximize energy yield, prior to using less favorable electron acceptors, such as nitrate. Taken together, the transition from aerobic respiration to a respiro-fermentative metabolism could provide bacteria the flexibility to generate energy during periods of limiting O 2 in fluctuating environments for maintenance and survival of their populations.",
"introduction": "Introduction Microorganisms face a multitude of fluctuating and often limiting conditions across various environments, such as soils, human gut, and aquatic environments. Carbon, electron acceptors (such as oxygen (O 2 )), and/or nutrients can vary over space and time. As such, microorganisms need to compensate and employ strategies to survive during these potentially growth-restricting conditions. One such strategy is respiratory flexibility. The utilization of both high- and low-affinity terminal oxidases enables exploitation of the full range of O 2 concentrations for oxidative phosphorylation and energy conservation, providing a great benefit in the ever-changing O 2 concentrations across environments. This can be attained by inducing branched respiratory chains that terminate in multiple oxidases with different affinities for O 2 (Bueno et al. 2012 ) recently shown in members of ubiquitous soil bacteria, the Acidobacteriota (Eichorst et al. 2018 , Trojan et al. 2021 ). Other strategies by which cells respond to limitations are, e.g. modifying enzyme synthesis to take up growth-limiting nutrients or by modulating uptake rates for nutrients available in excess (Roszak and Colwell 1987 ). Alternatively, they can reroute metabolic fluxes, which enables them to shift to alternative sources of energy and building blocks while avoiding possible blockages due to specific nutrient limitations (Roszak and Colwell 1987 , Bergkessel et al. 2016 ). Catabolism and ATP production are often incongruent during these periods of limitation (Stouthamer 1979 ). As a result of this incongruency, a trade-off can occur between catabolic rate and ATP yield, whereby bacteria utilize pathways for the most efficient production of molar ATP yield (Y ATP : mole of ATP/mole of oxidized substrate). For example, when catabolic rates are high but O 2 limiting, fermentative pathways (when available) are employed, together with respiratory pathways, commonly referred to as respiro-fermentation physiology (Pfeiffer et al. 2001 , Vemuri et al. 2007 ) allowing bacteria to maximize ATP production during electron acceptor limitation. This respiro-fermentative physiology has been observed in Escherichia coli, Bacillus subtilis , and Saccharomyces cerevisiae , yet the evolution and regulation of this metabolism is still under debate (Molenaar et al. 2009 ). Presumably, bacteria have evolved to harbor greater metabolic flexibility for ATP production, rather than pathways yielding optimal growth yield (Stouthamer 1979 ). Members of the phylum Acidobacteriota are ubiquitous across numerous soils (Fierer 2017 , Delgado-Baquerizo et al. 2018 ) with a central role in carbon mineralization and plant decomposition (Fierer 2017 , Crowther et al. 2019 ). Still very little is known about factors controlling their abundance in the environment or their effects on biogeochemical cycles under changing environmental conditions. In this study, we investigated the adaptive capability to O 2 -limited conditions with a model member of the phylum Acidobacteriota, Acidobacterium capsulatum 161. It is a member of the family Acidobacteriaceae that is commonly found across many environments, such as soils. Acidobacterium capsulatum 161 has originally been documented to be capable of microaerophilic growth and only later of weak fermentative growth as well (Pankratov et al. 2012 , Myers and King 2016 ). Recently, its capacity for respiratory flexibility due to the presence and functionality of high- and low-affinity terminal oxidases was demonstrated (Trojan et al. 2021 ). Here, we expanded our investigation of this strain to ascertain if it has additional abilities to alter its metabolism, such as the rerouting of metabolic fluxes, by profiling the whole transcriptomic response of A. capsulatum 161 to decreasing O 2 concentrations in the micro- and nanomolar range, the latter referred to as nanoxic (<1 µmol O 2 l −1 ) (Berg et al. 2022 ). To date, no reports have closely documented the catabolic routes of carbon and energy metabolism of Acidobacteriota or have evaluated its global transcriptomic response to O 2 deprivations. By combining genomics and hypoxic culture incubations using highly sensitive optical O 2 sensors (Lehner et al. 2015 ), we were able to investigate transcription patterns at the oxic–anoxic interface and could observe a transition from respiratory to respiro-fermentative metabolism in A. capsulatum 161.",
"discussion": "Discussion Adaptations and fast responses to changes in environmental conditions often occur at the metabolic level and in this work, we gained new insights into the transcription response of A. capsulatum 161 to diminishing O 2 concentrations at low micro- and nanomolar levels. Our data indicate that diminishing O 2 played a pivotal role in regulating the expression of genes involved in central metabolism under C excess conditions (Figs 3 and 4 ). To counter the toxic accumulation of respiration byproducts building up from the lack of O 2 , it shifted its metabolism and rerouted fluxes from an energy favorable respiratory state (Fig. 4B ) to a respiro-fermentative condition, in which acetate together with ethanol seemed to be major end-products (Figs 3 and 4C ). Glucose transport, PP pathways, and pyruvate production were downregulated at low-nanomolar O 2 concentrations (Fig. 3 )—presumably to reduce the NADH/NAD + redox ratio, which is a critical regulator of cell metabolism ultimately controlling the onset of respiro-fermentative metabolism (Shen and Atkinson 1970 , Szenk et al. 2017 ). As A. capsulatum 161 transitioned from oxic to nanoxic conditions under C excess, the transcripts of many NADH-generating enzymes related to oxidative respiration were reduced (Fig. 4B ). Glucose import ( galP) exhibited reduced expression from 10 to 0.1 µmol O 2 l −1 , presumably as a means to limit the amount of available glucose. Yet, glucose 1-dehydrogenase (gdh ) was overexpressed, potentially modulated a great part of the carbon flow through the gluconate bypass, thus reducing glucose concentration in the cell (Fig. 3 ). However, it appears that the cell did not use gluconate, as gluconolaconase had very low transcript levels at low O 2 concentrations (<10 µmol O 2 l −1 ). This could suggest that the enzyme requires a certain oxygen concentration to function. Pyruvate oxidase (PoxB) was upregulated in A. capsulatum 161, which catalyzes the decarboxylation of pyruvate to acetate and CO 2 (Figs 3 and 4C ), suggesting that pyruvate catabolism is the major switch point between the respiratory and fermentative responses. The glycolytic flux was redirected toward the production of fermentation products, acetate (upregulation of pta and acyP ) and ethanol (upregulation of adhP ) (Figs 3 and 4C , Fig. S2 , Table S4 ), to prevent carbon intermediates to enter the TCA cycle (El-Mansi and Holms 1989 ). Cells can then convert acetyl-CoA through the Pta-AckA pathway, producing and excreting acetate while generating ATP (El-Mansi and Holms 1989 ). Since the flux from acetyl-CoA to acetate does not generate any NADH (while the flux from acetyl-CoA through the TCA cycle generates 8 NAD(P)H and 2 FADH 2 ), carbon flow diversion to acetate could be viewed as a means of A. capsulatum 161 to reduce or prevent further NADH accumulation (El-Mansi and Holms 1989 , Holms 2001 ). This is in congruence with previous work on Staphylococcus aureus , where acetate production was enhanced under low O 2 and glucose excess conditions (Ferreira et al. 2013 ). In addition, the conversion of acetaldehyde to ethanol via adhP was upregulated, consuming NADH and hence a way to counteract the NADH/NAD + imbalance (Figs 3 and 4C ). Taken together, we hypothesize that the concomitant rise in NADH levels from glucose excess and low O 2 conditions drove the onset of fermentative metabolism (acetate and ethanol production) to avoid toxic levels of NADH in the cell. Acetate and ethanol production stemming from pyruvate bypasses any energy-conserving steps associated with NADH, allowing a fast oxidation of pyruvate and efficient shuttling of protons/electrons to the ETC. Various studies have shown that in concentrated glucose environments, E. coli and other organisms switch to and obtain some of their energy anaerobically by acetate fermentation, even when O 2 is plentiful, if the rate of glucose consumption is greater than the capacity to reoxidize the reduced equivalents generated (Farmer and Jones 1976 , Hollywood and Doelle 1976 , Andersen and Meyenburg 1980 , Meyer et al. 1984 , Farmer and Liao 1997 , Kayser et al. 2005 , Vemuri et al. 2006 , Vazquez et al. 2008 , Molenaar et al. 2009 , Nahku et al. 2010 , Valgepea et al. 2011 , 2013 , Zhuang et al. 2011 , Basan et al. 2015 , Peebo et al. 2015 , Schütze et al. 2020 ). Although a major role of NADH is to supply electrons to the ETC thereby fueling the production of ATP, the strategy of A. capsulatum 161 was to reduce the NADH production stemming from respiratory pathways to avoid NADH imbalance while generating ATP (Szenk et al. 2017 ). The use of alternative pathways for NAD + regeneration concomitant was also reported in other facultative anaerobes such as E. coli (Vemuri et al. 2006 , Farhana et al. 2010 , Martínez-Gómez et al. 2012 , Szenk et al. 2017 ) and members of the genera Salmonella and Shigella (Gray et al. 1966 , Wolfe 2005 ). The metabolic flexibility would allow these bacteria to cope with varying concentrations of carbon and O 2 in such environments like soils. This respiro-fermentative strategy might extend into the Acidobacteriota , as many genomes harbor this potential as evidence by the presence of acetate kinase and alcohol dehydrogenase (Eichorst et al. 2018 ). Our experimental conditions also invoked a significant upregulation of the glycogen metabolism suggesting cells transform excess glucose to the storage compound glycogen (Fig. 3 , Fig. S2 , Table S4 ). The accumulation of glycogen provides a metabolic reserve for A. capsulatum 161 under potential carbon-limited conditions in the future, which could be an important strategy in environments such as soils allowing cells to cope with transient limiting conditions. Stress response to O 2 and reactive oxygen species (ROS) is crucial for the ability to exist in habitats that are characterized by fluctuating O 2 concentrations. Whether microbes can occupy such a habitat or a microniche within partly depends upon whether they are able to withstand local concentrations of high or low O 2 . Several universal stress proteins in A. capsulatum 161 were significantly upregulated by decreasing O 2 concentrations approaching anoxia; especially two of these usp genes were affected by the drop of O 2 to 0.1 µmol O 2 l −1 (Fig. 2A , Table S2 ). Furthermore, the drop of O 2 invoked a significant increase of transcription of a sensor histidine kinase (Fig. 2A , Table S2 ), which presumably allowed A. capsulatum 161 to sense environmental stimuli and manage various environmental changes by coupling environmental cues to gene expression (Stock et al. 2000 , Mascher et al. 2006 , Kaczmarczyk et al. 2014 ). Cellular stress can further lead to protein denaturation (Hightower 1991 ), and proteolytic removal of non‐functional proteins is crucial for optimal metabolic activities (Porankiewicz et al. 1999 ). We detected an upregulation of the ATP-dependent Clp proteases in the transcriptomic response of A. capsulatum 161 to diminishing O 2 concentrations (Fig. 2A , Table S2 ), suggesting that they are important in removing irreversibly damaged polypeptides that may interfere with metabolic pathways under O 2 -limited stress conditions. In A. capsulatum 161, a clear differential upregulation of genes involved in counteracting oxidative stress was observed upon the decrease of oxygenation from 10 to 0.1 µmol O 2 l −1 (Fig. 2A , Table S2 ), indicating that it is capable of adapting to different redox states. Oxidative stress defense genes such as manganese superoxide dismutase, thioredoxins, and glutaredoxins were highly expressed under stimulated at low O 2 (Fig. 2A , Table S2 ), as seen previously in Nitrosomonas europaea (Sedlacek et al. 2020 ). The increased demand for proteins involved in ROS defense could be caused by NADH/NAD + redox ratio imbalances, as NADH accumulates and becomes toxic. Under O 2 -limiting conditions, an increased level of NADH builds up, as it is less efficiently reoxidized to NAD + as a result of reduced aerobic respiration. The high-affinity bd -type oxidase ( cydAB) and NADH dehydrogenase ( ndh-II ) were upregulated upon the drop of oxygenation to 0.1 µmol O 2 l −1 (Fig. 2B , Table S3 ). We previously hypothesized that the upregulation of the bd -type oxidase could suggest a contribution to respiratory activity at trace O 2 conditions or favor the more faster electron flux than cbb 3 -type oxidases to permit more rapid reducing potential from carbon surplus (Trojan et al. 2021 ). The uncoupled NADH dehydrogenase NDH-II was highly upregulated at 0.1 µmol O 2 l −1 (Fig. 2B , Table S3 ), presumably to compensate for the slow regeneration of NAD + due to the low O 2 availability. NDH-II only catalyzes the oxidation of NADH and reduction of quinones without the ability to pump protons, which, based on our data, seemed to be beneficial under micro- and nanoxic conditions. Alternative electron‐transfer routes seem to allow A. capsulatum 161 adjusting its energy transduction efficiency to its needs and substrate availability. In A. capsulatum 161, electrons can flow from the NADH dehydrogenase and SDH (complex II of the ETC) to the quinone/quinol pool, from where the electrons may either bypass the cytochrome bc 1 complex (complex III) and directly flow to the bd -type quinol terminal oxidase or flow via a cytochrome c either to the low-affinity caa 3 ‐type or the high-affinity cbb 3 ‐type cytochrome c terminal oxidase. Under aerobic- or carbon-limiting conditions, the proton-coupled NDH-I might be the main driver of NADH oxidation and maintain the proton motive force required for ATP synthesis (Fig. 4A ). Under the reduced environment in our incubations, when the reduction state of the quinone/quinol pool increases, the pathway to the NDH-II and bd -type quinol oxidase seemed to increase, with NDH-II being then the dominant dehydrogenase to oxidize excess NADH and support redox balance (Figs 2B and 4C ). This strategy has been observed for E. coli , which may use NDH-II to counteract increasing NADH/NAD + triggered by faster metabolism due to increased glucose uptake in order to support fast growth (Vemuri et al. 2006 , Liu et al. 2019 )."
} | 4,762 |
39557655 | PMC11636273 | pmc | 5,698 | {
"abstract": "Abstract Microbial soil habitats are characterized by rapid shifts in substrate and nutrient availabilities, as well as chemical and physical parameters. One such parameter that can vary in soil is oxygen; thus, microbial survival is dependent on adaptation to this substrate. To better understand the metabolic abilities and adaptive strategies to oxygen-deprived environments, we combined genomics with transcriptomics of a model organism, Acidobacterium capsulatum , to explore the effect of decreasing, environmentally relevant oxygen concentrations. The decrease from 10 to 0.1 µM oxygen (3.6 to 0.036 pO 2 % present atmospheric level, respectively) caused the upregulation of the transcription of genes involved in signal transduction mechanisms, energy production and conversion and secondary metabolites biosynthesis, transport, and catabolism based on clusters of orthologous group categories. Contrary to established observations for aerobic metabolism, key genes in oxidative stress response were significantly upregulated at lower oxygen concentrations, presumably due to an NADH/NAD + redox ratio imbalance as the cells transitioned into nanoxia. Furthermore, A. capsulatum adapted to nanoxia by inducing a respiro-fermentative metabolism and rerouting fluxes of its central carbon and energy pathways to adapt to high NADH/NAD + redox ratios. Our results reveal physiological features and metabolic capabilities that allowed A. capsulatum to adapt to oxygen-limited conditions, which could expand into other environmentally relevant soil strains.",
"conclusion": "Conclusions In this study, we examined the transcriptional response of A. capsulatum 161 to diminishing O 2 oncentrations in the low nanomolar range. Overall, O 2 -limiting conditions invoked a significant stress response in A. capsulatum 161. Our data indicate that A. capsulatum 161 has the genomic potential for multiple routes for the early steps of glucose catabolism. Under O 2 -limited but glucose-unlimited conditions, A. capsulatum 161 reroutes fluxes through its central metabolism from glycolysis to fermentative end products to counteract NADH/NAD + imbalances building up due to loss of respiratory capacities under electron acceptor-limiting conditions. Understanding these capacities advances the knowledge on the metabolic responses A. capsulatum 161 is capable of in order to successfully thrive and persist under fluctuating substrate availabilities in terrestrial environments. The investigated oxygen range (10 to 0.1 µmol O 2 l −1 or 3.6–0.036 pO 2 % present atmospheric level) is environmentally relevant (Sexstone et al. 1985 ), presumably seen in various soil niches. Coping with dynamic O 2 tensions is therefore vital for aerobic bacteria dwelling in (temporarily) O 2 -deprived habitats. During “spring snowmelt” or in the rhizosphere, catabolism and ATP yields can be uncoupled due to O 2 limitation and carbon availability. To survive reductive stress during O 2 deprivation, soil bacteria depend on metabolic strategies to maintain a proton motive force and redox balance. But modifications in metabolic routes at trace O 2 levels extend beyond soils; these findings have implications in other environments, such as oxygen minimum zones (OMZs) in the Earth’s oceans. OMZs are large water masses with low oxygen concentrations, thus favoring anaerobic metabolism (Kalvelage et al. 2015 ). Interestingly, aerobic metabolism was previously detected in regions of apparent anoxic conditions (“anoxic” OMZs) (Garcia-Robledo et al. 2017 ), along with the presence of terminal oxidases (Kalvelage et al. 2015 , Tsementzi et al. 2016 ) and production of O 2 at trace levels (Canfield and Kraft 2022 ). These regions could provide a niche where bacteria transition from a respiratory to a respiro-fermentative metabolism to maximize energy yield, prior to using less favorable electron acceptors, such as nitrate. Taken together, the transition from aerobic respiration to a respiro-fermentative metabolism could provide bacteria the flexibility to generate energy during periods of limiting O 2 in fluctuating environments for maintenance and survival of their populations.",
"introduction": "Introduction Microorganisms face a multitude of fluctuating and often limiting conditions across various environments, such as soils, human gut, and aquatic environments. Carbon, electron acceptors (such as oxygen (O 2 )), and/or nutrients can vary over space and time. As such, microorganisms need to compensate and employ strategies to survive during these potentially growth-restricting conditions. One such strategy is respiratory flexibility. The utilization of both high- and low-affinity terminal oxidases enables exploitation of the full range of O 2 concentrations for oxidative phosphorylation and energy conservation, providing a great benefit in the ever-changing O 2 concentrations across environments. This can be attained by inducing branched respiratory chains that terminate in multiple oxidases with different affinities for O 2 (Bueno et al. 2012 ) recently shown in members of ubiquitous soil bacteria, the Acidobacteriota (Eichorst et al. 2018 , Trojan et al. 2021 ). Other strategies by which cells respond to limitations are, e.g. modifying enzyme synthesis to take up growth-limiting nutrients or by modulating uptake rates for nutrients available in excess (Roszak and Colwell 1987 ). Alternatively, they can reroute metabolic fluxes, which enables them to shift to alternative sources of energy and building blocks while avoiding possible blockages due to specific nutrient limitations (Roszak and Colwell 1987 , Bergkessel et al. 2016 ). Catabolism and ATP production are often incongruent during these periods of limitation (Stouthamer 1979 ). As a result of this incongruency, a trade-off can occur between catabolic rate and ATP yield, whereby bacteria utilize pathways for the most efficient production of molar ATP yield (Y ATP : mole of ATP/mole of oxidized substrate). For example, when catabolic rates are high but O 2 limiting, fermentative pathways (when available) are employed, together with respiratory pathways, commonly referred to as respiro-fermentation physiology (Pfeiffer et al. 2001 , Vemuri et al. 2007 ) allowing bacteria to maximize ATP production during electron acceptor limitation. This respiro-fermentative physiology has been observed in Escherichia coli, Bacillus subtilis , and Saccharomyces cerevisiae , yet the evolution and regulation of this metabolism is still under debate (Molenaar et al. 2009 ). Presumably, bacteria have evolved to harbor greater metabolic flexibility for ATP production, rather than pathways yielding optimal growth yield (Stouthamer 1979 ). Members of the phylum Acidobacteriota are ubiquitous across numerous soils (Fierer 2017 , Delgado-Baquerizo et al. 2018 ) with a central role in carbon mineralization and plant decomposition (Fierer 2017 , Crowther et al. 2019 ). Still very little is known about factors controlling their abundance in the environment or their effects on biogeochemical cycles under changing environmental conditions. In this study, we investigated the adaptive capability to O 2 -limited conditions with a model member of the phylum Acidobacteriota, Acidobacterium capsulatum 161. It is a member of the family Acidobacteriaceae that is commonly found across many environments, such as soils. Acidobacterium capsulatum 161 has originally been documented to be capable of microaerophilic growth and only later of weak fermentative growth as well (Pankratov et al. 2012 , Myers and King 2016 ). Recently, its capacity for respiratory flexibility due to the presence and functionality of high- and low-affinity terminal oxidases was demonstrated (Trojan et al. 2021 ). Here, we expanded our investigation of this strain to ascertain if it has additional abilities to alter its metabolism, such as the rerouting of metabolic fluxes, by profiling the whole transcriptomic response of A. capsulatum 161 to decreasing O 2 concentrations in the micro- and nanomolar range, the latter referred to as nanoxic (<1 µmol O 2 l −1 ) (Berg et al. 2022 ). To date, no reports have closely documented the catabolic routes of carbon and energy metabolism of Acidobacteriota or have evaluated its global transcriptomic response to O 2 deprivations. By combining genomics and hypoxic culture incubations using highly sensitive optical O 2 sensors (Lehner et al. 2015 ), we were able to investigate transcription patterns at the oxic–anoxic interface and could observe a transition from respiratory to respiro-fermentative metabolism in A. capsulatum 161.",
"discussion": "Discussion Adaptations and fast responses to changes in environmental conditions often occur at the metabolic level and in this work, we gained new insights into the transcription response of A. capsulatum 161 to diminishing O 2 concentrations at low micro- and nanomolar levels. Our data indicate that diminishing O 2 played a pivotal role in regulating the expression of genes involved in central metabolism under C excess conditions (Figs 3 and 4 ). To counter the toxic accumulation of respiration byproducts building up from the lack of O 2 , it shifted its metabolism and rerouted fluxes from an energy favorable respiratory state (Fig. 4B ) to a respiro-fermentative condition, in which acetate together with ethanol seemed to be major end-products (Figs 3 and 4C ). Glucose transport, PP pathways, and pyruvate production were downregulated at low-nanomolar O 2 concentrations (Fig. 3 )—presumably to reduce the NADH/NAD + redox ratio, which is a critical regulator of cell metabolism ultimately controlling the onset of respiro-fermentative metabolism (Shen and Atkinson 1970 , Szenk et al. 2017 ). As A. capsulatum 161 transitioned from oxic to nanoxic conditions under C excess, the transcripts of many NADH-generating enzymes related to oxidative respiration were reduced (Fig. 4B ). Glucose import ( galP) exhibited reduced expression from 10 to 0.1 µmol O 2 l −1 , presumably as a means to limit the amount of available glucose. Yet, glucose 1-dehydrogenase (gdh ) was overexpressed, potentially modulated a great part of the carbon flow through the gluconate bypass, thus reducing glucose concentration in the cell (Fig. 3 ). However, it appears that the cell did not use gluconate, as gluconolaconase had very low transcript levels at low O 2 concentrations (<10 µmol O 2 l −1 ). This could suggest that the enzyme requires a certain oxygen concentration to function. Pyruvate oxidase (PoxB) was upregulated in A. capsulatum 161, which catalyzes the decarboxylation of pyruvate to acetate and CO 2 (Figs 3 and 4C ), suggesting that pyruvate catabolism is the major switch point between the respiratory and fermentative responses. The glycolytic flux was redirected toward the production of fermentation products, acetate (upregulation of pta and acyP ) and ethanol (upregulation of adhP ) (Figs 3 and 4C , Fig. S2 , Table S4 ), to prevent carbon intermediates to enter the TCA cycle (El-Mansi and Holms 1989 ). Cells can then convert acetyl-CoA through the Pta-AckA pathway, producing and excreting acetate while generating ATP (El-Mansi and Holms 1989 ). Since the flux from acetyl-CoA to acetate does not generate any NADH (while the flux from acetyl-CoA through the TCA cycle generates 8 NAD(P)H and 2 FADH 2 ), carbon flow diversion to acetate could be viewed as a means of A. capsulatum 161 to reduce or prevent further NADH accumulation (El-Mansi and Holms 1989 , Holms 2001 ). This is in congruence with previous work on Staphylococcus aureus , where acetate production was enhanced under low O 2 and glucose excess conditions (Ferreira et al. 2013 ). In addition, the conversion of acetaldehyde to ethanol via adhP was upregulated, consuming NADH and hence a way to counteract the NADH/NAD + imbalance (Figs 3 and 4C ). Taken together, we hypothesize that the concomitant rise in NADH levels from glucose excess and low O 2 conditions drove the onset of fermentative metabolism (acetate and ethanol production) to avoid toxic levels of NADH in the cell. Acetate and ethanol production stemming from pyruvate bypasses any energy-conserving steps associated with NADH, allowing a fast oxidation of pyruvate and efficient shuttling of protons/electrons to the ETC. Various studies have shown that in concentrated glucose environments, E. coli and other organisms switch to and obtain some of their energy anaerobically by acetate fermentation, even when O 2 is plentiful, if the rate of glucose consumption is greater than the capacity to reoxidize the reduced equivalents generated (Farmer and Jones 1976 , Hollywood and Doelle 1976 , Andersen and Meyenburg 1980 , Meyer et al. 1984 , Farmer and Liao 1997 , Kayser et al. 2005 , Vemuri et al. 2006 , Vazquez et al. 2008 , Molenaar et al. 2009 , Nahku et al. 2010 , Valgepea et al. 2011 , 2013 , Zhuang et al. 2011 , Basan et al. 2015 , Peebo et al. 2015 , Schütze et al. 2020 ). Although a major role of NADH is to supply electrons to the ETC thereby fueling the production of ATP, the strategy of A. capsulatum 161 was to reduce the NADH production stemming from respiratory pathways to avoid NADH imbalance while generating ATP (Szenk et al. 2017 ). The use of alternative pathways for NAD + regeneration concomitant was also reported in other facultative anaerobes such as E. coli (Vemuri et al. 2006 , Farhana et al. 2010 , Martínez-Gómez et al. 2012 , Szenk et al. 2017 ) and members of the genera Salmonella and Shigella (Gray et al. 1966 , Wolfe 2005 ). The metabolic flexibility would allow these bacteria to cope with varying concentrations of carbon and O 2 in such environments like soils. This respiro-fermentative strategy might extend into the Acidobacteriota , as many genomes harbor this potential as evidence by the presence of acetate kinase and alcohol dehydrogenase (Eichorst et al. 2018 ). Our experimental conditions also invoked a significant upregulation of the glycogen metabolism suggesting cells transform excess glucose to the storage compound glycogen (Fig. 3 , Fig. S2 , Table S4 ). The accumulation of glycogen provides a metabolic reserve for A. capsulatum 161 under potential carbon-limited conditions in the future, which could be an important strategy in environments such as soils allowing cells to cope with transient limiting conditions. Stress response to O 2 and reactive oxygen species (ROS) is crucial for the ability to exist in habitats that are characterized by fluctuating O 2 concentrations. Whether microbes can occupy such a habitat or a microniche within partly depends upon whether they are able to withstand local concentrations of high or low O 2 . Several universal stress proteins in A. capsulatum 161 were significantly upregulated by decreasing O 2 concentrations approaching anoxia; especially two of these usp genes were affected by the drop of O 2 to 0.1 µmol O 2 l −1 (Fig. 2A , Table S2 ). Furthermore, the drop of O 2 invoked a significant increase of transcription of a sensor histidine kinase (Fig. 2A , Table S2 ), which presumably allowed A. capsulatum 161 to sense environmental stimuli and manage various environmental changes by coupling environmental cues to gene expression (Stock et al. 2000 , Mascher et al. 2006 , Kaczmarczyk et al. 2014 ). Cellular stress can further lead to protein denaturation (Hightower 1991 ), and proteolytic removal of non‐functional proteins is crucial for optimal metabolic activities (Porankiewicz et al. 1999 ). We detected an upregulation of the ATP-dependent Clp proteases in the transcriptomic response of A. capsulatum 161 to diminishing O 2 concentrations (Fig. 2A , Table S2 ), suggesting that they are important in removing irreversibly damaged polypeptides that may interfere with metabolic pathways under O 2 -limited stress conditions. In A. capsulatum 161, a clear differential upregulation of genes involved in counteracting oxidative stress was observed upon the decrease of oxygenation from 10 to 0.1 µmol O 2 l −1 (Fig. 2A , Table S2 ), indicating that it is capable of adapting to different redox states. Oxidative stress defense genes such as manganese superoxide dismutase, thioredoxins, and glutaredoxins were highly expressed under stimulated at low O 2 (Fig. 2A , Table S2 ), as seen previously in Nitrosomonas europaea (Sedlacek et al. 2020 ). The increased demand for proteins involved in ROS defense could be caused by NADH/NAD + redox ratio imbalances, as NADH accumulates and becomes toxic. Under O 2 -limiting conditions, an increased level of NADH builds up, as it is less efficiently reoxidized to NAD + as a result of reduced aerobic respiration. The high-affinity bd -type oxidase ( cydAB) and NADH dehydrogenase ( ndh-II ) were upregulated upon the drop of oxygenation to 0.1 µmol O 2 l −1 (Fig. 2B , Table S3 ). We previously hypothesized that the upregulation of the bd -type oxidase could suggest a contribution to respiratory activity at trace O 2 conditions or favor the more faster electron flux than cbb 3 -type oxidases to permit more rapid reducing potential from carbon surplus (Trojan et al. 2021 ). The uncoupled NADH dehydrogenase NDH-II was highly upregulated at 0.1 µmol O 2 l −1 (Fig. 2B , Table S3 ), presumably to compensate for the slow regeneration of NAD + due to the low O 2 availability. NDH-II only catalyzes the oxidation of NADH and reduction of quinones without the ability to pump protons, which, based on our data, seemed to be beneficial under micro- and nanoxic conditions. Alternative electron‐transfer routes seem to allow A. capsulatum 161 adjusting its energy transduction efficiency to its needs and substrate availability. In A. capsulatum 161, electrons can flow from the NADH dehydrogenase and SDH (complex II of the ETC) to the quinone/quinol pool, from where the electrons may either bypass the cytochrome bc 1 complex (complex III) and directly flow to the bd -type quinol terminal oxidase or flow via a cytochrome c either to the low-affinity caa 3 ‐type or the high-affinity cbb 3 ‐type cytochrome c terminal oxidase. Under aerobic- or carbon-limiting conditions, the proton-coupled NDH-I might be the main driver of NADH oxidation and maintain the proton motive force required for ATP synthesis (Fig. 4A ). Under the reduced environment in our incubations, when the reduction state of the quinone/quinol pool increases, the pathway to the NDH-II and bd -type quinol oxidase seemed to increase, with NDH-II being then the dominant dehydrogenase to oxidize excess NADH and support redox balance (Figs 2B and 4C ). This strategy has been observed for E. coli , which may use NDH-II to counteract increasing NADH/NAD + triggered by faster metabolism due to increased glucose uptake in order to support fast growth (Vemuri et al. 2006 , Liu et al. 2019 )."
} | 4,762 |
37503268 | PMC10370097 | pmc | 5,700 | {
"abstract": "Metabolomics is a powerful tool for uncovering biochemical diversity in a wide range of organisms, and metabolic network modeling is commonly used to frame results in the context of a broader homeostatic system. However, network modeling of poorly characterized, non-model organisms remains challenging due to gene homology mismatches. To address this challenge, we developed Metabolic Interactive Nodular Network for Omics (MINNO), a web-based mapping tool that takes in empirical metabolomics data to refine metabolic networks for both model and unusual organisms. MINNO allows users to create and modify interactive metabolic pathway visualizations for thousands of organisms, in both individual and multi-species contexts. Herein, we demonstrate an important application of MINNO in elucidating the metabolic networks of understudied species, such as those of the Borrelia genus, which cause Lyme disease and relapsing fever. Using a hybrid genomics-metabolomics modeling approach, we constructed species-specific metabolic networks for three Borrelia species. Using these empirically refined networks, we were able to metabolically differentiate these genetically similar species via their nucleotide and nicotinate metabolic pathways that cannot be predicted from genomic networks. These examples illustrate the use of metabolomics for the empirical refining of genetically constructed networks and show how MINNO can be used to study non-model organisms.",
"conclusion": "CONCLUSION Here, we introduce MINNO, a new software tool that allows researchers to integrate genomic and empirical metabolomics data into a single software environment in order to build and refine metabolic networks. We illustrate the utility of this tool for identifying missing reactions within multiple metabolic pathways for Borrelia species. Using MINNO, we identified 18 missing reactions from the KEGG database, of which nine were supported by the primary literature. The remaining reactions show good homology as in the NCBI-RefSeq database (see Table 1 ). MINNO provides a tool that can be applied to any organism to systematically refine or investigate metabolic pathways. MINNO was designed to be inherently flexible for these diverse applications and support a wide range of input formats. We anticipate that it will be a useful asset for analyzing genome-wide knockouts, studying novel organisms that are divergent from typical model organisms, metabolic flux analysis, and visualization of metabolic networks.",
"introduction": "INTRODUCTION Metabolomics has emerged as a mainstream approach for investigating a diverse range of biological phenomena and exploring the molecular underpinnings of disease ( 1 , 2 ). One of the core underlying tools used in metabolomics research is metabolic network modeling, which is used to place metabolic data in the context of an organism’s overall metabolic network ( 3 ). Although the core elements of metabolism are shared between most living organisms (e.g., central carbon metabolism), species-to-species diversity can contribute to significant differences in nutritional preferences ( 4 , 5 ). These differences are especially important in the context of evolution, where natural selection has driven organisms to streamline their metabolic networks according to the specific niches they inhabit ( 6 ). Currently, most metabolic networks are derived from a handful of model organisms such as Escherichia coli, Saccharomyces cerevisiae, or Mus musculus . Species-specific networks are then constructed from genomic homology searches using tools such as the Prokaryotic Genome Annotation Pipeline (PGAP) ( 7 , 8 ), which identify the most likely enzyme for each reaction in the network ( 9 ). Although this strategy works well in species that are closely related to these model organisms, it is less effective when applied to species that are highly divergent from the original model ( 10 ). Mismatches due to poor homology result in missing enzymes in the metabolic network, which, without further data refinement, can be misinterpreted as metabolic deficiencies ( 11 , 12 ). This is a critical problem for understanding the evolution of microbes and for making inferences about the metabolic architecture of non-model organisms. Another major challenge in investigating non-model organisms is that existing network visualization tools make it difficult to integrate multi-omics data to tune genomic networks. Although several visualization tools have been developed such as Escher ( 13 ), MetExploreViz ( 14 ), Omix ( 15 ), Cytoscape ( 16 ), CellDesigner ( 17 ), and PathVisio ( 18 ), they all suffer from a lack of scalability and reusability. None of these tools come with a generic base network architecture that can be used to build the network of any organism and they often require bioinformatics or coding skills to alter existing networks ( 15 , 17 ). When expanding metabolic networks by adding more pathways, users are typically required to manually add them one by one or reorganize the network if the initial layout is lacking ( 13 , 14 , 16 , 18 ). To address these challenges, we developed the JavaScript-based web application Metabolic Interactive Nodular Network for Omics (MINNO). MINNO promotes network reusability by offering base networks that serve as a foundation for overlaying organism-specific networks. Moreover, it enables the integration of diverse metabolic pathways in a modular fashion, eliminating the need for coding or extensive reorganization of the entire combined network. These capabilities enhance the scalability of network construction and facilitate the empirical data-driven refinement of metabolomics networks. As a proof-of-concept, we used MINNO to conduct an empirical refinement of three metabolic pathways for three species of Borrelia , spirochetes that cause Lyme disease and relapsing fever in humans and other vertebrates ( 19 , 20 ). Borrelia spirochetes follow a complex life cycle in which they are sequentially passed from ticks to a mammalian host ( 21 , 22 ). These species are obligate parasites, and selective pressure has streamlined their metabolic networks to dispense with the biosynthesis of many metabolites that can be obtained directly from their host ( 19 , 20 ). Using MINNO, we found evidence for metabolic streamlining and divergence among the Lyme disease and relapsing fever spirochetes resulting from these specific host/vector interactions. In summary, the construction of metabolic networks for non-model organisms using genomics analysis is hindered by homology mismatches, which present a critical challenge in understanding microbial evolution and inferring their metabolic architecture. Existing visualization tools lack the necessary scalability and reusability features to effectively integrate multi-omics data into the network, thereby impeding network refinement. To address these limitations, MINNO utilizes a hybrid genomics-metabolomics strategy that incorporates metabolic boundary flux analysis, genomic network projection, and empirical refinement based on metabolic data and will thus facilitate the study of a significantly broader range of non-model species.",
"discussion": "RESULTS and DISCUSSION Strategy: Network and data visualization using MINNO The MINNO visualization tool facilitates both investigation and understanding of the complex interplay between genotypic and phenotypic features in omics data. MINNO is a JavaScript-based web application compatible with Google Chrome and Mozilla Firefox browsers. It uses the D3.js JavaScript library to create dynamic interactive visualizations in web browsers ( 30 ). The tool can load files, such as network files and data files, in JSON, XML, and CSV file formats, while it exports data in JSON, XML, PNG, and SVG formats for multiple applications. It has numerous built-in features that facilitate the creation of detailed network visualizations without the need to switch to multiple editing software tools. More details about MINNO can be found in the user manual that includes a tutorial developed for users to familiarize themselves with many of the tool’s features. MINNO is available open-source (under the MIT open-access license) at www.lewisresearchgroup.org/software . MINNO comes with 66 base metabolic pathways from the KEGG database ( 31 ), covering all primary metabolic pathways that can be combined to build large-scale metabolic networks that include user-added reactions and features. Users can then superimpose an organism’s known metabolic pathway data from the KEGG database on these base metabolic pathways without the need to rebuild a network from scratch for each organism studied. The tool can also access metabolic network models from the Biochemical, Genetic and Genomic (BiGG) database ( 32 ). The tool accepts multi-omics data, such as metabolomics, proteomics, and fluxomics data, which can be integrated and visualized on the nodes and edges of the metabolic network. MINNO utilizes empirical data to facilitate the identification of missing reactions by providing users with the ability to investigate reactions pathway-by-pathway or by individual modules. The concept of modularity plays a crucial role in this process. Metabolic networks exhibit modularity as a network property, wherein a module or pathway consists of densely interconnected nodes compared to connections between different modules ( 33 , 34 ). This modular structure enables the detection of missing reactions within metabolic networks by ensuring that nodes within each module are interconnected either with each other or with the surrounding environment. The concept of modularity is a fundamental aspect of metabolic networks and can be applied to metabolic networks of any species. However, except for a handful of model organisms, there are thousands of understudied species that have poorly constructed metabolic networks due to homology mismatch issues. The KEGG database currently includes over 8,794 species along with their respective metabolic pathways ( 31 ). By providing access to this extensive information, MINNO allows users to refine metabolic networks and explore individual species or interactions among multiple species. Network refinement strategy: In this example, we used MINNO for our metabolic network refinement strategy to understand metabolic differences among related microbial pathogens ( Fig. 1 ). This strategy involves first culturing microbes in vitro and then sampling the cultures over specific time intervals so that metabolite intensities can be recorded as a function of time ( Fig. 1A ). Metabolites present in the cultures are then detected (in this example, by mass spectrometry) to generate temporal profiles of identified metabolites ( Fig. 1B ). The MINNO visualization tool takes metabolic base/ortholog network data (KGML files) from the KEGG database ( Fig. 1C ). Using the tool, users can superimpose an organism’s specific metabolic pathway onto base metabolic maps, which ultimately facilitates identification of potential missing reactions in the organism’s metabolic network as shown as grey edges. The user can then incorporate temporal metabolite intensity profiles and intra- or extracellular data onto the network to infer missing reactions by considering boundary fluxes and if available, the isotope labeling pattern, without resorting to complex mathematical modeling ( Fig. 1D ). In this figure, the dashed links represent the inferred missing reactions. Users can then search for genes and proteins corresponding to missing reactions using experimental or bioinformatics methods. The refined network with updated missing reactions can then be shared in KEGG format file ( Fig. 1E ). This approach allowed users to refine the networks of under-studied species such as Borrelia and find the necessary reactions to explain the metabolic profiles that were missing from their original annotated networks. MINNO can also be used for multi-omics data integration as it can incorporate gene, protein, metabolite, and flux data on the same metabolic network. MINNO User interface: Fig. 2 highlights some key features of the web-based application MINNO. The built-in menu is located on the left side of the screen, where users can select base metabolic pathways or specific species pathways from the KEGG database and BiGG Models database. Metabolic pathways from the BiGG Models database do not have positional data. However, MINNO possesses the ability to self-organize the network using node repulsion and edge attractions from the D3.js JavaScript library ( 30 ), which is commonly employed in various other network visualization tools ( 14 , 16 ). Later, users can customize the network by dragging and aligning nodes in the network. Additionally, the tool allows users to superimpose base and organism-specific KEGG metabolic networks, which show annotated reactions as dark nodes and edges, while unannotated/missing reactions are depicted by light grey nodes and edges. This enables determining the potential missing reactions after experimental data is uploaded onto the network. Refining nucleotide metabolic pathways using MINNO We used MINNO to perform a metabolic network refinement analysis of three Borrelia species known to cause Lyme and relapsing fever diseases. By leveraging the modularity concept of metabolic networks and employing boundary flux analysis, we were able to identify missing reactions from the KEGG database for these species. The purine metabolism of B. burgdorferi in the KEGG database is fragmented as depicted by solid links in Fig. 3A , as it lacks the classic purine salvage pathway ( 35 ). The consumption of both adenine and guanine by B. burgdorferi suggests the presence of purine transporters, which has recently been reported in the literature ( 36 , 37 , 38 ). By analyzing the boundary fluxes of cultured cells, we have identified missing reactions in purine metabolism from the KEGG database, indicated by dashed links in Fig. 3A . In contrast, the pyrimidine pathway for B. burgdorferi is relatively less fragmented in the KEGG database as shown by solid links in Fig. 3A . However, the boundary flux profile of this species suggests the presence of pyrimidine-nucleoside phosphorylase ( PnP ) based on the production of thymine from thymidine, as shown in Fig. 3A , which is missing in the KEGG database. Additionally, B. burgdorferi lacks ribonucleotide reductase, an enzyme responsible for converting ribonucleotides (for RNA synthesis) into deoxyribonucleotides (for DNA synthesis) ( 35 ). According to our data, PnP salvages deoxyribose sugars from thymidine for DNA synthesis. In summary, we used MINNO and empirical metabolomics data to identify eight reactions that are missing from the KEGG database. Subsequent publications have confirmed six of these missing purine reactions ( Table 1 ). Furthermore, MINNO predicted four missing pyrimidine metabolism reactions from the KEGG database, all of which are supported by primary literature ( Table 1 ). Metabolic distinction between Borrelia species causing Lyme disease and relapsing fever: To better understand metabolic differences between Borrelia species, we focused on refining the metabolic networks of Borrelia species associated with relapsing fever: B. parkeri and B. turicatae . These species share genetic similarities, and as expected, their boundary flux profiles exhibit similarities as well ( 24 ) ( Fig. 3 (B , C) ; Fig. 4 ). Similar to the purine metabolism of B. burgdorferi, purine metabolism of both B. parkeri and B. turicatae is fragmented as shown as solid links in Fig. 3 (B , C) . However, unlike B. burgdorferi , both possess the classic purine salvage pathway. They both consume adenosine and adenine, and any excess purine is excreted as hypoxanthine. Interestingly, neither of the isolates studied has an annotated ribonucleotide reductase in the KEGG database. However, based on their boundary flux profiles, we anticipate that both B. parkeri and B. turicatae harbor a ribonucleotide reductase ( rnr ) ( Fig. 3 B , C ). Metabolic similarities between Borrelia species causing Lyme and Relapsing fever diseases: It is worth noting that these three Borrelia species also shared metabolic similarities. One common feature observed in all three species studied is the absence of the thyX gene in the KEGG database, as shown in Fig. 3 . The thyX gene is essential in all three species for providing the necessary deoxyribonucleosides required for DNA synthesis. Another notable similarity is their deficiency in various biosynthetic pathways essential for the production of important vitamins and co-factors, such as nicotinate and nicotinamide. This deficiency highlights their reliance on salvaging precursors for NAD(P) synthesis from the host or surrounding environment. Our observations revealed that all three Borrelia species consume nicotinamide and NAD+ while excreting nicotinate out of the cells ( Fig. 5 ). Notably, the net excretion of nicotinate exceeds the levels of nicotinamide consumed for each isolate. This suggests the possible presence of nicotinamide-nucleotide amidase ( pncC ). This salvage process also leads to the generation of essential molecules like PRPP and ATP, as well as the accumulation of potentially toxic substances such as ammonia. In summary, we used MINNO to predict six reactions in purine metabolism for B. parkeri and B. turicatae that were missing from the KEGG database, with four of these predictions supported by primary literature ( Table 1 ). MINNO was also used to identify two missing reactions in the KEGG database from the pyrimidine metabolism, although none of them are currently supported by primary literature. However, these predictions are supported based on homology matches through the PGAP pipeline from NCBI-RefSeq. For nicotinate metabolism, we predicted one reaction shared by all three Borrelia species, which is missing from the KEGG database ( Table 1 ). Summary of functionality and applications Overall, MINNO enables users to refine metabolic networks and integrate multi-omics data to provide a system level view of metabolic homeostasis. MINNO’s modular approach, whereby discrete metabolic pathway modules can be easily merged together, facilitates the creation of metabolic networks in diverse non-model organisms. It also allows users to visualize data on these merged metabolic pathways quickly and easily, without any coding required, facilitating a deeper understanding of complex multi-omics data in the context of the broader metabolic system. MINNO can support a variety of applications, such as FBA visualization to model more sophisticated genome-scale behaviors ( 39 ), mapping metabolic architecture in complex microbiome communities ( 40 ), investigating interspecies “cross-talking” interactions ( 41 , 42 ), and determining the molecular mechanisms of novel antibiotics ( 43 )."
} | 4,742 |
36771814 | PMC9920240 | pmc | 5,701 | {
"abstract": "Kraft lignin, a side-stream from the pulp and paper industry, can be modified by laccases for the synthesis of high added-value products. This work aims to study different laccase sources, including a bacterial laccase from Streptomyces ipomoeae (SiLA) and a fungal laccase from Myceliophthora thermophila (MtL), for kraft lignin polymerization. To study the influence of some variables in these processes, a central composite design (CCD) with two continuous variables (enzyme concentration and reaction time) and three levels for each variable was used. The prediction of the behavior of the output variables (phenolic content and molecular weight of lignins) were modelled by means of response surface methodology (RSM). Moreover, characterization of lignins was performed by Fourier-transform infrared (FTIR) spectroscopy and different nuclear magnetic resonance (NMR) spectroscopy techniques. In addition, antioxidant activity was also analyzed. Results showed that lignin polymerization (referring to polymerization as lower phenolic content and higher molecular weight) occurred by the action of both laccases. The enzyme concentration was the most influential variable in the lignin polymerization reaction within the range studied for SiLA laccase, while the most influential variable for MtL laccase was the reaction time. FTIR and NMR characterization analysis corroborated lignin polymerization results obtained from the RSM.",
"conclusion": "4. Conclusions In order to produce the most appropriate lignin for each possible application, structural modification of this molecule is needed. A possible way to achieve this goal is by using laccase enzymes, which is considered a sustainable and environmentally friendly approach. Concerning this, this study confirmed the ability of a bacterial laccase from S. ipomoeae (SiLA) and a commercial fungal laccase from M. thermophila (MtL) to polymerize kraft lignin (referring to polymerization as lower phenolic content and higher molecular weight). Specifically, the enzyme dosage was the most influential variable in the kraft lignin polymerization reaction, within the range studied, when the bacterial SiLA laccase was used, while for MtL fungal laccase the most influential variable was the reaction time. FTIR and NMR characterization spectra verified lignin polymerization, observing new condensed structures such as α-5′, 5-5′, and 4-O-5′.",
"introduction": "1. Introduction After cellulose, lignin is the second most abundant biopolymer on the planet [ 1 ]. It is synthetized from p -coumaryl alcohol (H), coniferyl alcohol (G), and sinapyl alcohol (S) monomers by enzymatic polymerization, in which oxidoreductase enzymes such as laccases and peroxidases are involved [ 2 ]. As a result, a heterogeneous complex tridimensional macromolecule is formed, containing different types of both ether (e.g., β-O-4′, α-O-4′, 5-O-4′, etc.) and carbon–carbon bonds (e.g., β-β′, β-5′, β-1′, 5-5′, etc.), and a wide variety of reactive groups depending on the biomass source. Accordingly, dicots (e.g., eucalypt, birch, poplar) contain around 20–25% of lignin, which is mostly composed of G and S units and traces of H units. On the other hand, gymnosperms (e.g., pine, spruce) with approximately 20–35% of lignin, are mostly composed of G units and very low proportions of H units. Finally, monocot grasses (e.g., flax, hemp, sisal) have lower lignin content (9–20%) composed of G and S units, together with high levels of H units [ 3 ]. Moreover, along with their source, the lignin isolation technology used strongly affects their features and properties and, therefore, the valorization ways [ 4 ]. Actually, the main source of lignin is the pulp and paper industry. Approximately 100 million tons per year of lignin were produced in 2015 with a value of roughly USD 732.7 million. Moreover, it is expected to increase to USD 913.1 million in 2025 [ 5 ]. Among the different pulp and paper processes, the kraft process is the most extended pulping technology, with an average lignin production estimated at 55–90 million tons per year [ 6 ]. Most of this kraft lignin is normally combusted, due to their high calorific value, to produce energy that is partially used in the same pulp and paper mills. However, this process generates an excess of energy, making the valorization of this waste lignin more interesting as high-added-value chemicals and materials that guarantee the sustainability and competitiveness of these mills [ 7 ]. In addition, kraft lignin valorization is also expected to benefit the future circular bioeconomy, which aims to maximize the usage and value of all raw materials, products, and wastes. Although the inherent heterogeneity of kraft lignin (i.e., chemical composition, molecular structure, and molecular weight distribution) makes this waste material extraordinarily interesting, these features may be an obstacle for certain applications. To overcome this fact, the modification of the lignin structure is often a necessary step to produce the right lignin for each possible application [ 8 ]. The oxidative enzymes, such as laccases and peroxidases, involved in lignin biosynthesis in nature, can accomplish this modification [ 9 ]. Laccases (EC 1.10.3.2) are multicopper-containing oxidases with phenoloxidase activity, being widely expressed in nature, mainly in plants, insects, fungi, and bacteria [ 10 ]. The biological role of these enzymes is determined by their source and the phase of life of the organism producing them. For instance, fungal laccases participate in stress defense, morphogenesis, fungal plant–pathogen/host interactions, and lignin degradation, while bacterial laccases are involved in pigmentation, morphogenesis, toxin oxidation, and protection against oxidizing agents, and ultraviolet light [ 11 ]. These enzymes catalyze the oxidation of an extensive variety of phenolic and non-phenolic molecules, using oxygen as the final electron acceptor and releasing water as a by-product [ 11 ]. The catalytic site of laccases contains four copper ions. On the one hand, type-T1 copper, responsible for the characteristic blue color of the enzyme, is involved in the oxidation of the reducing substrate, acting as the primary electron acceptor. On the other hand, type-T2 copper together with two type-T3 coppers form a tri-nuclear copper cluster where oxygen is reduced to water [ 12 ]. The electrochemical potential of type-T1 copper is one of the most important properties of laccases, fluctuating between 0.4 and 0.8 V. Bacterial and plant laccases have low redox potential, whereas medium and high values are usually reported for fungal laccases [ 12 ]. The oxidative versatility, low catalytic requirements, and capacity of laccases to catalyze degradation or polymerization reactions make these enzymes suitable for a wide range of applications in different sectors, including lignocellulosic biorefinery, pulp and paper industry, food and textile sectors, bioremediation, and biosensor applications, among others [ 13 ]. More specifically, the enzymatic polymerization of lignin by laccases has been applied in the synthesis of new lignin-based polymeric materials [ 14 ] in, for example, the manufacture of green binders for fiberboard manufacturing [ 15 ], nanocomposite films formed by coating lignin nanoparticles along the microfibrilled cellulose fiber network [ 16 ], controlled-delivery fertilizer systems [ 17 ], and a pesticide release system [ 18 ]. In most of the studies on the enzymatic polymerization of lignin, fungal laccases are commonly used [ 15 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ]. Only in recent years, bacterial laccases have also gained attention for this purpose [ 16 , 24 , 25 ]. Hence, there is a necessity to explore the potential of novel laccases, including bacterial enzymes, for kraft lignin polymerization. This work aims to study the oxidative polymerization of Eucalyptus globulus kraft lignin by using different laccase sources, such as a bacterial laccase isolated from Streptomyces ipomoeae (SiLA) and a commercial fungal laccase from the ascomycete Myceliophthora thermophila (MtL). Laccase dosage and reaction time were the input variables evaluated at three levels to study laccase polymerization reactions, using a central composite design (CCD). The prediction of the behavior of the output variables (phenolic content and molecular weight of the resulting laccase-treated lignins) was modelled by means of response surface methodology (RSM). Moreover, structural characterization of the resulting lignins by Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, as well as their antioxidant activity, were also evaluated.",
"discussion": "3. Results and Discussion There is an increasing interest to evaluate the potential of novel laccases, including those from bacterial origin, for residual lignin polymerization. Presently, the fungal M. thermophila (MtL) laccase, with a high pH range and noticeable thermal stability, is widely employed for lignin polymerization [ 15 , 20 , 21 , 35 ]. S. ipomoeae (SiLA) laccase, with similar properties to MtL laccase, has been also assessed for this purpose [ 36 ], although to a lesser extent. 3.1. Effect of Laccase Dosage and Reaction Time on Total Phenolic Content As it can be observed in Figure 1 , both MtL and SiLA laccases showed the ability to reduce the phenolic content of the treated kraft lignins obtained from all experiments of the CCD. Nevertheless, the bacterial laccase achieved a higher reduction of total phenolic content compared to the fungal laccase, in spite of the similar low redox potential showed by both laccases (0.450 mV) [ 27 , 37 ]. The quadratic regression equation obtained from the phenolic content values ( Figure 1 a,b) when using MtL laccase (Equation (1)) and SiLA laccase (Equation (2)) over kraft lignin, and their regression coefficients are the following: mg GAE/g lignin = 434.8 − 0.306 · Time (min) − 0.786 · Dosage (IU/g) − 0.000339 · Time (min) · Time (min) + 0.00089 · Dosage (IU/g) · Dosage (IU/g) + 0.002381 · Time (min) · Dosage (IU/g)\n R-squared = 91.24% (1) mg GAE/g lignin = 222.5 + 0.1458 · Time (min) − 0.588 · Dosage (IU/g) − 0.000343 · Time (min) · Time (min) + 0.00157 · Dosage (UA/g) · Dosage (IU/g) − 0.000776 · Time (min) · Dosage (IU/g)\n R-squared = 92.93% (2) Figure 2 a,b show the response surface for lignin phenolic content as a function of enzyme concentration and reaction time using MtL or SiLA laccases, respectively. As it can be seen, the total phenolic content of lignin decreased with increasing reaction time in the studied region when MtL laccase was used. The maximum phenolic content reduction (53%) was obtained at 452 min using 100 IU/g of MtL laccase. In the case of SiLA laccase, the most influential variable on the total phenolic content reduction was the laccase dosage, observing a maximum decrease (77%) using 160 IU/g of SiLA laccase at 390 min. As it is widely known, laccases can oxidize free phenolic lignin units, yielding resonance-stabilized phenoxyl radicals via a single electron transfer process [ 38 ]. Thus, the establishment of new linkages between the formed phenoxyl radicals leads to a lower content of free phenols in lignin. Different studies have already described the ability of MtL laccase to reduce the phenolic content of both kraft lignin and lignosulfonates. Then, the phenolic content was decreased by this fungal laccase by around 66% in the case of eucalypt kraft lignin [ 15 ], whereas a reduction of 52% was described using lignosulfonates [ 35 ]. On the other hand, the phenolic content reduction in lignin by bacterial laccases has been also reported. In this regard, Mayr et al. [ 25 ] showed the ability of CotA laccase to decrease the phenolic content between 30% and 65% in different kraft lignins of softwood and hardwood origin, respectively. Similar results were described by Wang et al. [ 16 ] when a commercial bacterial laccase (Metzyme ® ) was used to polymerize alkali lignins from birch and spruce materials. Contrary to this study, the effects of laccase dosage and reaction time on the phenolic content of lignin are usually evaluated separately. Gillgren et al. [ 19 ] showed that longer reaction time resulted in higher reductions of phenolic content of both organosolv lignin and lignosulfonates when they were treated with a fungal laccase from Trametes (syn. Coriolus polyporus ). A similar trend was reported by Huber et al. [ 35 ], using a laccase from MtL to polymerize both eucalypt kraft lignin and lignosulfonates. Moreover, these authors also observed a higher decrease in the phenolic content using higher MtL laccase dosages, indicating that the amount of enzyme used, together with reaction time, are important factors for the lignin polymerization process. Finally, Mayr et al. [ 25 ] also reported the influence of reaction time on the phenolic content decrease when a bacterial CotA laccase was used to polymerize different kraft lignins, observing a decrease in phenolic content by extending the reaction time. 3.2. Effect of Laccase Dosage and Reaction Time on Molecular Weight The molecular weight distributions of laccase-treated lignins are displayed in Figure S1 . From them, weight-average (Mw) and number-average (Mn) molecular weights, as well as polydispersity (Mw/Mn) values were calculated ( Table S1 ). In general, both MtL and SiLA laccases produced an increment in the Mw values of the treated kraft lignins obtained from all experiments ( Figure 3 a,b). Polydispersity values also showed higher values compared to the untreated lignin ( Table S1 ). The quadratic regression equation obtained from the Mw values ( Figure 3 a,b) when using MtL laccase (Equation (3)) and SiLA laccase (Equation (4)) over kraft lignin, and their regression coefficients are the following: Mw (Da) = 3371 + 14.40 · Time (min) + 30.7· Dosage (IU/g) − 0.0050 · Time (min) · Time (min) − 0.0959 · Dosage (IU/g) · Dosage (UA/g) − 0.0223 · Time (min) · Dosage (IU/g)\n R-squared = 83.75% (3) Mw (Da) = 3936 + 7.42 · Time (min) + 25.46 · Dosage (IU/g) + 0.00040 · Time (min) · Time (min) + 0.1201· Dosage (IA/g) · Dosage (IU/g) − 0.0471 · Time (min) · Dosage (IU/g)\n R-squared = 98.38% (4) Figure 4 a,b show the response surface for lignin Mw as a function of enzyme concentration and reaction time of MtL and SiLA laccases, respectively. As can be observed, the molecular weight of the lignin increased by increasing the reaction time in the studied interval when MtL laccase was used, in agreement with the observed reduction in phenolic content ( Section 3.1 ). The maximum molecular weight increment (3.0-fold, which correspond to the value of Mw 10,865 Da) was obtained at 452 min using 100 IU/g of MtL laccase. In the case of SiLA laccase, the most influential variable on the molecular weight was the laccase dosage used, as also observed for the phenolic content of lignin ( Section 3.1 ). The maximum increment (3.5-fold, which corresponds to the value of Mw 12,545 Da) was obtained using 184.85 IU/g of SiLA laccase at 240 min. As previously commented, the stabilized phenoxyl radicals, generated from lignin by laccase oxidation, undergo radical–radical coupling through phenyl ether–carbon and carbon–carbon linkages, yielding the observed increase in Mw values of kraft lignin by both laccases. Moreover, the polydispersity increase is also expected due to the non-selective radical–radical coupling reactions, which link lignin end groups to each other spontaneously with low or no control and, consequently leading to higher polydispersity values [ 20 ]. MtL laccase has already shown its capability to increase the molecular weight of both kraft lignin and lignosulfonates. Thus, Gouveia et al. [ 15 ] reported a strong increase (17.0-fold) in the average molecular weight of laccase-treated eucalypt kraft lignin (80,000 Da) compared to the untreated lignin sample (4700 Da). Huber et al. [ 35 ] also described a 12.0-fold increase in Mw (22,400 Da) for enzymatic polymerization of lignosulfonates (1900 Da for untreated lignin), and only a 1.4-fold increase (2300 Da) when kraft lignin (1600 Da for untreated lignin) was used. On the other hand, the molecular weight increase by bacterial laccases has been also described. Thus, Wang et al. [ 16 ] reported a 2.9-fold increase (from 17,750 Da for untreated lignin to 52,000 Da for laccase treated lignin) when a Metzyme ® laccase was used to polymerize alkali spruce lignin. Mayr et al. [ 25 ] achieved 6.0-fold increases in molecular weight for softwood (from 21,600 Da for untreated lignin to 130,000 Da for laccase-treated lignin) and 19.2-fold for hardwood kraft lignins (from 3150 Da for untreated lignin to 60,000 Da for laccase-treated lignin) when they were treated with a CotA laccase. Similarly to phenolic content, the effects of laccase dosage and reaction time on the molecular weight of lignin are generally studied separately, reporting different results in function of both laccase and lignin sources. Gouveia et al. [ 21 ] observed that the major changes in molecular weight of kraft lignin treated with the fungal MtL laccase occurred during the first 2 h, although longer reaction time resulted in higher Mw values of the resulting treated lignins. These authors also showed a molecular weight increase as the enzyme dosage was augmented. In this regard, Areskogh et al. [ 39 ] determined that no significant increments in the molecular weight of lignosulfonates were observed at low MtL enzyme dosage, while the molecular weight increased by augmenting the enzyme concentration. Huber et al. [ 35 ] also demonstrated that the amount of biocatalyst used strongly influences the polymerization process. When 50 mg of MtL laccase was used, 4.0-fold and 1.7-fold molecular weight increments were determined for lignosulfonates and kraft lignin, respectively. However, when the MtL laccase was augmented to 100 mg, a 12.0-fold increase in the molecular weight was measured for enzymatic polymerization of lignosulfonates, and only a 1.4-fold increase was seen for kraft lignin. Finally, Mayr et al. [ 25 ] also achieved higher increases in the molecular weight of softwood and hardwood kraft lignins at longer reaction times using a bacterial CotA laccase. While the significant phenolic content of kraft lignin can translate into good reactivity for producing phenol-formaldehyde resins, epoxy resins, polyester systems, and polyurethanes, among others, the molecular weight increase of kraft lignin by laccase enzymes enables new applications as lignin-based dispersants providing better adsorption properties, stabilizer for emulsions, and in thermoplastic blends or copolymers enhancing thermal and mechanical performance [ 32 ]. 3.3. Antioxidant Activity The antioxidant ability of lignins (i.e., their capacity to act as radical scavengers) promotes their use as natural additives in food, cosmetics, pharmaceuticals, and polymeric formulations as an alternative to synthetic compounds such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), among others [ 40 ]. It is widely known that having a high phenolic content, low molecular weight, and narrow distribution seem to be favorable for the antioxidant capacity of lignin [ 40 ]. Nevertheless, in spite of the phenolic content decrease and molecular weight increase observed herein for the treated kraft lignins by both laccases, they still showed some antioxidant capacity, expressed as TEAC, (0.02–0.18) compared to the untreated sample (0.2) ( Figure 5 a,b for MtL and SilA laccases, respectively). 3.4. FTIR Characterization FTIR spectra of untreated and laccase-treated kraft lignins (MtL-KL and SiLA-KL resulting from experiments with maximum decrease in phenolic content and increase in molecular weight achieved) are displayed in Figure 6 . The observed bands were assigned in comparison with others previously reported in the literature [ 41 , 42 ] and are displayed in Table S2 . FTIR spectrum of kraft lignin showed the characteristic bands of lignin, which include those observed at 1610, 1515 and 1415 cm −1 associated with aromatic ring vibrations, and at 1455 cm −1 attributed to C−H asymmetric vibrations and deformations ( Figure 6 a). Bands attributed to syringyl (S) and guaiacyl (G) units were also identified, including those at 1315 cm −1 (S and G units), a shoulder at 1270 cm −1 (G units), 1220 cm −1 (G units), 1115 cm −1 (S units), 1025 cm −1 (G units) and 820 cm −1 (S units). The major change in the FTIR spectra of MtL-KL and SiLA-KL samples compared to untreated kraft lignin spectra was observed at the bands corresponding to the C=O stretching for conjugated (1650 cm −1 ) and unconjugated (1715 cm −1 ) linkages ( Figure 6 b,c), as a consequence of the lignin oxidation caused by both laccases, being more noticeable in the case of the bacterial laccase. This effect was supported by UV–Vis, observing a decrease in the two absorption maxima at λ 230–240 and 280 nm, attributed to non-conjugated phenolic groups, in both MtL-KL and SiLA-KL samples due to lignin oxidation ( Figure S2 ). Comparable results have been previously described by Gouveia et al. [ 15 , 21 ], when a laccase from M. thermophila was used for eucalypt kraft lignin polymerization, and Gillgren et al. [ 19 ], when a laccase from the white-fungus C. polyporus was employed to polymerize organosolv lignin and lignosulfonates. Moreover, both MtL-KL and SiLA-KL samples kept their characteristic triplet at 1610, 1515 and 1415 cm −1 , which is indicative of no modification of lignin aromatic backbone, as previously observed by Areskogh et al. [ 43 ] during polymerization of lignosulfonates by M. thermophila laccase. 3.5. NMR Characterization The HSQC spectra of untreated and laccase-treated kraft lignins (MtL-KL and SiLA-KL resulting from experiments with maximum decrease in phenolic content and increase in molecular weight achieved) are shown. They included the whole spectra (δ C /δ H 0.0–150.0/0.0–9.0) in Supplementary Figure S3 , and the spectra corresponding to the oxygenated aliphatic (δ C /δ H 45.0–95.0/2.5–6.0 ppm) and the aromatic (δ C /δ H 90.0–150.0/5.0–9.0 ppm) regions in Figure 7 and Figure 8 , respectively. The main 13 C– 1 H lignin correlation signals identified in HSQC spectra are displayed in Table S3 , endorsed according to those described by the literature [ 42 , 44 , 45 , 46 , 47 ]. The lignin substructures identified are depicted in Figures S4 and S5 . The oxygenated aliphatic region of the kraft lignin spectrum exhibited information about the different interunit linkages present ( Figure 7 a), including those from native and kraft-derived linkages. Despite the well-known lignin degradation under alkaline conditions during kraft pulping [ 48 ], several remaining signals from native β-O-4′ and β-β′ resinol substructures were observed, as well as correlation signals for spirodienones and cinnamyl alcohol end-groups. Signals from kraft-derived lignin linkages could also be recognized. Among them, signals from epiresinols and diaresinol, both diastereomers from the transformation of the native resinol substructure during kraft pulping [ 47 , 49 ]. An aryl-glycerol substructure could also be hesitantly identified, produced from the non-phenolic β-aryl ether linkage under alkaline conditions during kraft pulping [ 50 ]. Finally, a correlation signal of lignin terminal structures with a carboxyl group in C α (Ar–CHOH–COOH; F α ), overlapping with aryl-glycerol, could also be found. The aromatic region of kraft lignin spectra displayed the typical correlation signals of S, G, and H lignin units ( Figure 8 a), the usual pattern of hardwood lignins [ 51 ]. Moreover, a group of signals from lignin oxidation, such as oxidized S units, corresponding to syringaldehyde or acetosyringone, and oxidized G units, attributed to vanillin and acetovanillone, could also be detected. Signals from kraft-derived lignin linkages were also found in the aromatic region. Among them, correlation signals endorsed to β1 and β5 stilbene, derived from degradation of spirodienone and β-5′ phenylcoumaran during kraft pulping, respectively, were identified [ 46 , 47 ]. Finally, correlation signals from S 1-1′ (3,5-tetramethoxy-para-diphenol), G 1-1′ (3-dimethoxy-para-diphenol) and S 1 -G 1′ /G 5′ were tentatively identified as a result of C α -C 1 breakdown in a retro-aldol reaction, followed by a radical coupling reaction, during kraft pulping [ 32 , 45 , 46 ]. Significant changes in the oxygenated aliphatic region of MtL-KL and SiLA-KL spectra ( Figure 7 b,c for MtL-KL and SiLA-KL samples, respectively), compared to the kraft lignin spectrum ( Figure 7 a), were found. In general, a complete disappearance of signals assigned to native and kraft-derived linkages was observed for both MtL-KL and SiLA-KL lignin samples, probably due to the cleavage of interunit linkages by treatment with both laccases. Nevertheless, some signals from β-O-4′ and diaresinol were still found. In this sense, Prasetyo et al. [ 23 ] reported a decrease in the intensity signals of β-O-4′ linkages when lignosulfonates were treated with Trametes villosa and Trametes hirsuta laccases. Wang et al. [ 16 ] also described a cleaved of β-aryl ether and β-β′ resinol substructures during the treatment of alkali lignins with a commercial (MetZyme ® ) bacterial laccase. In addition to the cleavage of interunit linkages, a new signal could also be observed in the aliphatic oxygenated region of both MtL-KL and SiLA-KL spectra, which was tentatively attributed to α-5′ condensed structures. The appearance of this structure is probably due to lignin condensation/polymerization reactions by laccases action. In this sense, Wang et al. [ 16 ] already described the formation of this condensed structure during the treatment of alkali lignins with the MetZyme bacterial laccase. MtL-KL and SiLA-KL spectra also showed a near complete disappearance of the aromatic 13 C– 1 H correlation signals ( Figure 8 b,c) compared to the kraft lignin spectrum ( Figure 8 a). Nevertheless, some intensity of signals corresponding to G and H units as well as to lignin oxidation, such as oxidized S and G units, could still be found. This important loss of aromatic correlation signals observed in the HSQC spectra after the enzymatic treatment with both laccases could indicate a significant modification of the lignin aromatic backbone. However, when laccase-treated lignins were analyzed by 1D-NMR, it could be inferred that the loss of aromatic correlation signals observed by 2D-NMR was due to deprotonation of the lignin benzene rings, as revealed by the 1 H NMR MtL-KL and SiLA-KL spectra ( Figure S6 ). Meanwhile strong signals of aromatic carbons could be seen in the 13 C-NMR MtL-KL and SiLA-KL spectra ( Figure S7 ), proving that benzene rings were not degraded by laccase treatment, as previously seen by FTIR analysis ( Section 3.4 ). The decrease or complete disappearance of aromatic proton signals was not entirely unexpected, as it has been previously described in lignosulfonates by the action of T. villosa and T. hirsuta laccases [ 23 ], in eucalypt kraft lignin treated with M. thermophila laccase [ 15 ], in alkali lignins by the action of MetZyme bacterial laccase [ 16 ], and in organosolv lignin and lignosulfonates treated with C. polyporus laccase [ 19 ]. All these authors have related this effect with the formation of condensed structures such as 5-5′ or 4-O-5′. 13 C-NMR of laccase treated lignins ( Figure S7b,c ) also showed a remarkable increase in the signal at δ C 176 ppm (carbonyl groups), especially in the SiLA lignin sample as previously described by FTIR analysis ( Section 3.4 ), resulting from lignin oxidation caused by laccases action. A significant decrease in the signals at δc 147 ppm, corresponding to C 3 and C 5 of phenolic S units and C 3 and C 5 in phenolic G units, and at δc 134 ppm, endorsed to C 1 and C 4 in phenolic S units and C 1 in phenolic G units, was also observed, resulting from the phenolic lignin units’ oxidation by laccase treatment, which supports the phenolic content decrease observed in Section 3.1 . At a time, an increase in the shoulders at δc 152 ppm and at δc 130 ppm, from non-phenolic lignin units were also visible. Santos et al. [ 52 ] assigned the signal at δc 152 ppm to C 3 in new 5-5′ or C 3 (and C 4 /C 5 ) in new 4-O-5′ structures formed during laccase ( T. villosa ) treatment of lignosulfonates, whereas Magina et al. [ 53 ] endorsed the signal at δc 130 ppm to C 5 in 5-5′ structure during MtL laccase treatment of lignosulfonates. Then, these observations arising from NMR analysis suggest lignin condensation/polymerization reactions by laccases action, supporting the molecular weight increment observed by SEC ( Section 3.2 ). The phenoxy radicals formed by the action of the laccase enzymes on the phenolic units present in the initial kraft lignin together with those derived from the cleavage of interunit linkages underwent radical–radical coupling through phenyl ether–carbon and carbon–carbon links resulting in new condensed structures such as α-5′, 5-5′, and 4-O-5′ ( Figure 9 )."
} | 7,319 |
35994737 | PMC9453739 | pmc | 5,702 | {
"abstract": "The pairing of analytical chemistry with genomic techniques\nrepresents\na new wave in natural product chemistry. With an increase in the availability\nof sequencing and assembly of microbial genomes, interrogation into\nthe biosynthetic capability of producers with valuable secondary metabolites\nis possible. However, without the development of robust, accessible,\nand medium to high throughput tools, the bottleneck in pairing metabolic\npotential and compound isolation will continue. Several innovative\napproaches have proven useful in the nascent stages of microbial genome-informed\ndrug discovery. Here, we consider a number of these approaches which\nhave led to prioritization of strain targets and have mitigated rediscovery\nrates. Likewise, we discuss integration of principles of comparative\nevolutionary studies and retrobiosynthetic predictions to better understand\nbiosynthetic mechanistic details and link genome sequence to structure.\nLastly, we discuss advances in engineering, chemistry, and molecular\nnetworking and other computational approaches that are accelerating\nprogress in the field of omic-informed natural product drug discovery.\nTogether, these strategies enhance the synergy between cutting edge\nomics, chemical characterization, and computational technologies that\npitch the discovery of natural products with pharmaceutical and other\npotential applications to the crest of the wave where progress is\nripe for rapid advances."
} | 363 |
37938692 | PMC9723771 | pmc | 5,703 | {
"abstract": "Increased ocean temperature associated with climate change is especially intensified in coastal areas and its influence on microbial communities and biogeochemical cycling is poorly understood. In this study, we sampled a Baltic Sea bay that has undergone 50 years of warmer temperatures similar to RCP5-8.5 predictions due to cooling water release from a nuclear power plant. The system demonstrated reduced oxygen concentrations, decreased anaerobic electron acceptors, and higher rates of sulfate reduction. Chemical analyses, 16S rRNA gene amplicons, and RNA transcripts all supported sediment anaerobic reactions occurring closer to the sediment-water interface. This resulted in higher microbial diversities and raised sulfate reduction and methanogenesis transcripts, also supporting increased production of toxic sulfide and the greenhouse gas methane closer to the sediment surface, with possible release to oxygen deficient waters. RNA transcripts supported prolonged periods of cyanobacterial bloom that may result in increased climate change related coastal anoxia. Finally, while metatranscriptomics suggested increased energy production in the heated bay, a large number of stress transcripts indicated the communities had not adapted to the increased temperature and had weakened resilience. The results point to a potential feedback loop, whereby increased temperatures may amplify negative effects at the base of coastal biochemical cycling.",
"conclusion": "Conclusions A strength of this study was that it was carried out in the natural environment with seasonal fluctuations. Despite inherent complications such as potential differences in nutrient inputs into the two bays, this work showed that warmer temperatures in the heated bay over 50 years selected an altered sediment microbial community with decreased seasonal variation, resulting in more stable and diverse sediment communities at 0–1 cm below the seafloor. These included Cyanobacteria that result in a potential prolonged bloom period and increased coastal anoxia connected to climate change. Previous studies suggest warmer temperatures will result in increased energy production [ 25 ] and this was also supported in our study with more RNA transcripts associated with energy production and higher organic matter content in the heated bay. However, the stress and repair transcripts suggested the microbes’ temperature optima were below that of the water such that the increased productivity may be tempered and the community’s resilience may be weakened. This could result in a negative feedback loop where the increased warming related effects will lead to an increase in Cyanobacteria production, elevated organic matter degradation, lower oxygen concentrations, and shallowing of geochemical zones with potential methane release to the atmosphere in already oxygen deficient zones that exacerbates the ongoing climate change. However, it remains to be confirmed if such altered microbial processes would occur in coastal sediments worldwide and such studies could be carried out in additional thermally altered areas.",
"introduction": "Introduction Climate change related increases in average surface temperatures [ 1 ] and extreme weather events [ 2 ] has affected the global oceans [ 1 ], resulting in higher CO 2 concentrations and the concomitant acidification, increased salinity, stratification, de-oxygenation, and rising sea levels [ 3 , 4 ]. Ocean temperatures are predicted to further rise by up to 2.0 °C down to 100 m in depth [ 5 ] and European seas have increased by ~0.01 °C per year since 1860 [ 6 ]. Microbes are central to marine energy and nutrient cycles and changes in their community structure and ability to respond to warming will have profound effects on global biogeochemistry and ecosystems. How species diversity responds to these changes might depend on the ecosystem, scales, climate, and organisms considered [ 7 ]. In general, experimental warming predicts a reduction of local richness across terrestrial and aquatic ecosystems by 8.9% [ 7 ]. However, many global warming predictions use short time and fixed temperature laboratory studies that make it difficult to predict effects in long-term, naturally fluctuating systems [ 8 ]. The Baltic Sea is one of the largest brackish-water areas worldwide and is affected by anthropogenic nutrient loading that leads to accelerated eutrophication effects including higher biomass production and increased oxygen consumption that have resulted in a ten-fold expansion of hypoxic (<2 mg/L O 2 ) dead zones within the last century [ 9 – 11 ]. Increased temperature accelerates hypoxia by decreasing the oxygen solubility [ 4 ] that has accounted for 15% of the total oxygen decline [ 12 ] along with higher microbial metabolic rates further increasing oxygen loss [ 4 , 13 ]. Shallow coastal waters (0–200 m) are more sensitive to atmospheric CO 2 levels [ 14 ] and heat transfer to sediments will be more rapid, likely leading to longer periods of seasonal hypoxia [ 15 ]. At higher latitudes, seasonal changes play an important role on the ecosystem structure and nutrient cycling within coastal waters [ 16 ] including sediment organic matter (OM) concentrations [ 17 ]. Increased temperature and therefore perturbed seasonality could result in a decreased ability of the biological pump to remove carbon from the atmosphere [ 18 ]. Microbes in marine sediments play a key role through mineralizing organic matter that removes atmospheric carbon and by recycling nutrients for primary producers [ 19 ]. In addition, due to their short generation time coupled with changes in distribution and activity periods (phenological shifts) they are some of the first responders to climate change [ 20 ]. However, how these fluctuations will amplify effects through the food web of coastal sediment communities remains poorly understood [ 21 ]. Warm water has been discharged for the past 50 years from a nuclear power plant into a Baltic Sea bay, acting as a natural laboratory that can be compared to a non-impacted control bay. The study system serves as a unique opportunity to investigate potential future changes of global warming within a natural fluctuating system, while the control bay was unaffected and represents the contemporary conditions. These types of systems also provide the opportunity to observe coastal climate change effects. The intake for the cooling was from nearby open coastal water at 16–18 m below the sea surface that is heated up to ~10 °C above the ambient water temperature while it cools down the reactors, without being in direct contact with the reactors. This results in an average temperature increase in the heated bay within the predicted range for the RCP5-8.5 (Representative Concentration Pathway) scenario by the year 2100 of 3.3–5.7 °C [ 22 ]. However, this predicted temperature increase may also occur in less extreme RCP scenarios in northern hemisphere coastal waters. In this study, bottom sediment was collected on four occasions in 2018–2019 at three points within each bay generating geochemical parameters ( n = 9 per bay per sampling). In addition, 16S rRNA gene amplicon ( n = 9 per bay per sampling) and community RNA transcript ( n = 3 per bay per sampling; Table S1 ) data were generated for these samples. In this study, we investigated: (1) how does 50 years of warming affect microbial community diversity and structure; (2) how and to what degree does increased warming influence microbial energy and nutrient cycling; (3) if the warming resulted in phenological shifts; and 4) what consequences do these effects have in the face of future climate change.",
"discussion": "Results and discussion Geochemical parameters The heated bay was on average 5.1 °C (mean±s.d. control bay 10.9 ± 3.6 °C, heated bay 16.8 ± 4.3 °C) warmer than the control bay with greater temperature differences in October to February and little or no difference during June to August 2018 (Fig. 1 ). The surface and bottom water temperatures were not statistically different in the heated bay (General linear model (GLM), ANOVA, F 9,62 = 0.01, p = 0.929) that was likely due to mixing of the water column by the discharge water while a thermocline was observed with varying intensity in the control bay ( F 9,62 = 27.03, p < 0.0001). While these temperature increases were on the outer range predicted for 2100 (with 3.3–5.7 °C by year 2100 according to SSP5-8.5 [ 22 ]), they represent relevant temperature increases for coastal areas [ 23 , 24 ]. Fig. 1 Spatiotemporal variation in geochemical parameters in the heated and control bays. a Temperature measured within the heated (orange) and control (blue) bays using HOBO data loggers at three different sampling sites (1 m below the surface) between December 2017 and November 2019. Standard deviations ( n = 3) between the sampling sites in each bay are shown in light gray. Surface (gray) for heated bay (▲) and control bay (●) and bottom (black) water temperatures were also measured at each sampling time. b Mean sediment pore water (0–1 cm) and bottom water geochemical parameters from the heated (orange) and control (blue) bays are indicated with dots. Temperature, oxygen, and salinity were measured on bottom water with the other environmental variables measured on pore water from the collected sediments. Vertical standard deviations (s.d.) show spatial variation over sampling sites within each bay ( n = 3) while horizontal deviations (s.d.) represent the temporal variation over sampling occasions ( n = 4). Temperature of the sediment were taken June 2018 (heated bay 17.5–31 cm depth and control bay 23.5–33 cm depth). Red asterisks indicate significant changes between the bays (Supplementary Table 2). c Downward sulfate diffusive fluxes at 6 cm sediment depth for three sampling points across the heated (orange) and control (blue) bays at three different sampling points (June 2018, November 2018, and March 2019). Dashed lines show the average sulfate fluxes for the heated (dark orange; 1.74 × 10 –9 mmol/cm 2 /s) and control (dark blue; 0.83 × 10 –9 mmol/cm 2 /s) bays. Spatiotemporal differences in bio- and geochemical parameters at shallow depth below the seafloor (0–1 cm) were observed between the two bays (Fig. 1 and Table S2 ). Oxygen concentrations in water decreased with higher temperatures [ 13 ] while microbial metabolic rates increased [ 25 ], likely causing the observed lower oxygen concentration in the heated bay (mean ± s.d. = 9.76 ± 4.3) compared to the control bay (10.76 ± 3.6 mg/L; GLM, ANOVA, F 17,49 = 9.84, p = 0.0028). However, the bottom waters were not hypoxic as the sampling sites were likely too shallow (1.2–4.9 m; Supplementary Table 1) allowing permanent reoxygenation. The mean sediment organic matter content after loss on ignition analysis of 42.3 ± 8.9% in the heated bay was significantly higher than the control bay (37.2 ± 6.9%, ANOVA, F 17,49 = 11.39, p = 0.0144) potentially due to elevated growth rates and higher primary production as a result of e.g. increased temperatures plus necromass from algal blooms sinking to the sediment. The increased necromass led to higher use of electron donors that likely supported anaerobic nitrate reduction and was verified by an increased but highly variable mean sediment porewater nitrite concentration of 1.59 ± 1.5 µM in the heated bay compared to 0.98 ± 0.42 µM in the control bay with a concomitant significantly lower nitrate concentration in the heated versus control bay (42.85 ± 36.6 and 48.16 ± 24.1 µM; F 17,49 = 4.96, p = 0.03). In addition, the mean ferrous iron concentration, likely from ferric reduction, was higher but also varied largely over the year in the heated versus control bay (5.56 ± 3.2 and 5.12 ± 0.5 µM). Higher sulfate reduction rates likely resulted in significantly lower sulfate concentration (2.04 ± 1.2 mM) in the heated bay 0–1 cm sediment depth compared to the control bay (2.52 ± 1.3 mM; F 17,49 = 13.95, p = 0.005). The mean phosphate concentration was also lower in the heated bay (201.3 ± 109.4 µM heated bay and 223.1 ± 103.7 µM control bay) potentially due to increased microbial activity consuming phosphate for growth, precipitation of ferric phosphate [ 26 ], phosphate input in the control bay from e.g. Cyanobacteria (Fig. S2 ), and variations in anthropogenic phosphorus input into the two bays [ 27 ]. In summary, these data supported increased organic matter accumulation in the heated bay sediments linked to a decrease in electron acceptors associated with anaerobic energy conservation. Sulfate flux The geochemical data (Fig. 1 ) corroborated previous findings that sulfate concentration and flux in sediment pores are primarily controlled by the rate of organic matter mineralization via sulfate reduction [ 28 ], with higher temperatures leading to elevated reduction rates and a thinning/shallowing of the sulfate reduction zone towards the sediment-water interface. For instance, pore water samples at 6 cm below the seafloor in the heated bay showed lower average sulfate concentrations and a two-fold increase in the average sulfate flux in relation to the control bay (2.92 versus 4.01 mM and 1.74 versus 0.83 × 10 −9 mmol/cm 2 /s, respectively). A thinner/shallower sulfate reduction zone also implied that methanogenesis, which mostly occurs after sulfate in pores is consumed [ 29 ], will occur closer to the sediment-water interface and facilitate methane emissions from the seafloor. In summary, prolonged warming will likely lead to a compression of geochemical zones with sulfate reduction occurring closer to the sediment surface, which potentially facilitates sulfide release from the sediment. Therefore, this thinning/shallowing of anaerobic energy conservation may result in exacerbating the effects of climate change by initiating a negative feedback loop with increased dead zones. Microbial diversities Sediment microorganisms are commonly stratified according to their electron acceptor requirements for energy conservation from e.g. reduction of oxygen, nitrate, ferric iron, sulfate, and carbon dioxide [ 29 ] although this distribution is more complex in coastal areas where other environmental factors (including spatial heterogeneity plus episodic input of organic carbon or photosynthesis) influence the stratification [ 30 ]. The decreased oxygen concentration in the heated bay may be a driver of microbial community clusters [ 31 ] and seasonal temperature influences on microbial activities (Fig. 2 and Table S2 ). Within the dataset, doubletons made <4% of the overall relative abundance of the community and did not change the diversity differences of the bays (Fig. S3 and Table S2 ). The amplicon sequencing variant (ASV) Shannon´s H indices (6.22 ± 0.6 and 5.28 ± 0.9) and evenness (0.88 ± 0.04 and 0.77 ± 0.09) were significantly higher (ANOVA, Shannon´s H, F 17,49 = 59.78, p < 0.001 and evenness, F 17,49 = 234.93, p < 0.001) in the heated bay that had warmer and more stable (both in space and time) temperatures compared to the control bay (Fig. 3 and Table S2 and Fig. S3 ). The overall higher number of species found (Chao1, 1306.22 ± 566.01 heated bay and 1096.722 ± 496.11 control bay; Fig. S3 ) within the heated bay was contrary to the classical niche theory of spatial environmental heterogeneity harboring more species due to niche availability [ 32 ]. This was also counter to the expectation that the larger temperature fluctuations in the control bay and concomitant coexistence of multiple species with different optimum growth temperatures that become dominant at various times of the year (Figs. 3 , 4 and Fig. S4 ) would result in an overall higher microbial diversity [ 7 ]. The data supported that the thinner geochemical zones, increased respiration rates shown by augmented sulfate flux, and consumption of anaerobic electron acceptors selected for a greater microbial diversity in the 0–1 cm below seafloor heated bay sediment. Fig. 2 16S rRNA gene amplicon ASVs and RNA transcript microbial beta diversities. a Canonical correspondence analysis plot of microbial communities (based on relative abundance of ASVs) with explanatory environmental variables (vectors) for the four different sampling time points in the heated (orange) and control (blue) bays. Chemical parameters were measured in the pore water of the 0–1 cm sediment, organic matter (OM) was measured from the sediment, and temperature, salinity, and oxygen were measured in the bottom water. Red asterisks indicate parameters that significantly explain the microbial community variation (Supplementary Table 2). b Principal component analysis based on VST transformed RNA transcript counts filtered for at least five reads within at least three samples for the heated bay (orange; n = 12) and the control bay (blue; n = 12). Fig. 3 16S rRNA gene amplicon ASV diversity indices. Shannon´s H index and evenness diversity indices for the heated (orange) and control (blue) bays for each sampling month. Linear regression model for testing significant differences between bays ( n = 36) with pairwise comparison for testing differences between bays on each sampling month ( n = 9 per bay) was used (Supplementary Table 2). Asterisks indicate significant differences between diversity of microbial communities at each sampling month; *** p ≤ 0.001. Shown are the minimum relative abundance, first quartile (25%), third quartile (75%), and the maximum relative abundance. The upper and lower whisker extends from the hinge at most 1.5*IQR (interquartile range), data beyond are plotted individually and marked as outliers. Outliers are given as dots. Fig. 4 Abundances of ASVs and RNA transcripts in the heated and control bay for major taxa. a Balloon plot with significant (adjusted p -value < 0.05) differentially abundant ASVs with >0.5% abundance annotated on family level. The abundance was calculated on at least 0.5% relative abundance in a sample. The control bay with month May, June, and November 2018 plus March 2019 are shown on the left while the heated (temperature-affected) bay with sampling month are shown on the right. Shown are the sum of replicates ( n = 3) and sampling sites ( n = 3) per bay on the four different sampling occasions with circle sizes showing the abundance of families at each time point and location. b Balloon plot of top 100 TPM per sample of genes filtered for significant (adjusted p -values < 0.05) differential expression within the bacteria annotated on known family level (Cyanobacteria and unknown on family level filtered out) on the y -axis. The control bay with month May, June, and November 2018 plus March 2019 plotted on the left while the heated bay (temperature-affected) with sampling month are shown on the right. Shown are the TPM sum of sampling sites as replicates ( n = 3) per bay on the four differe n t sampling occasions; circle sizes show the TPM value of families at each time point and location. c Additional balloon plot of the data from b of the sum of TPM of the families Thiotrichaceae, Thiobacilliaceae, and Cryomorphaceae of sampling sites as replicates ( n = 3) per bay on the four different sampling occasions from the top 100 TPMs per sample. d Significant (adjusted p -values < 0.05) differential abundant ASVs related to the phylum Cyanobacteria. e Sum of top 100 TPM per sample of genes filtered for significant (adjusted p -values p < 0.05) differential expression related to Cyanobacteria. Microbial communities A comparison of the 16S rRNA gene amplicon and RNA transcript data suggested no noteworthy influence of the cooling water intake of the more open Baltic Sea into the heated bay. For example, the dominant species annotated from the RNA transcripts in the heated bay sediment could also be found within the control bay at lower numbers (e.g. Thiobacillus ). Additionally, comparing data from benthic and open water communities within the Baltic Sea showed evidence that the microbes from in-flowing cooling water containing open Baltic Sea water did not noticeably influence the overall sediment microbial community composition [ 11 , 33 , 34 ]. Canonical correspondence analysis (CCA) supported that the microbial community compositions were different between the two bays (PERMANOVA, F = 13.99, p = 0.001, Fig. 2 and Table S2 ). In addition to temperature, the main drivers separating the communities in the two bays were depth and the sampling location (Fig. 2 ). Further, discussion of the validity of the communities within the heated bay despite input of microbes from the cooling system water is provided in the method section of the Supplemental Information. The most abundant 16S rRNA gene ASVs from both bays aligned within the Proteobacteria (23.2% and 17.6%), Cyanobacteria (20.9% and 45.9%), and Bacteroidota (16.4% and 11.7%) that matched previous results from Baltic Sea sediments [ 11 ]. Dominant taxa in the control bay included chemolithotrophic or mixotrophic Hydrogenophilaceae [ 35 ], the oxygen positively correlated Flavobacteriaceae, and the facultative anaerobic Ignavibacteriaceae [ 36 ] compared to a more even distribution of a greater number of taxa including the sulfate reducing Desulfomicrobiaceae [ 37 ] and Desulfocapsaceae [ 38 ] plus the anoxic sediment family Woesiaceae [ 39 ] in the heated bay (Fig. 4 and Tables S3 – S4 ). The microbial communities were highly affected by the generally lower oxygen availability in the heated bay with the control bay following seasonal patterns with warmer summer temperatures favoring lower oxygen conditions in the bottom sediments. However, the heated bay also showed increased relative abundance over the year of some aerobic organic matter degrading bacteria including ASVs aligning with e.g. the Arenimonas genus (Fig. S4 ) that may have contributed to a higher and constant oxygen consumption [ 40 ]. This likely resulted in thinner geochemical zones, a switch to anaerobic respiration closer to the sediment-water interface, and a greater number of niches for anaerobic bacteria in the sampled top 1 cm below the seafloor. After oxygen was depleted, nitrate was potentially reduced by mainly Proteobacteria including Steroidobacteraceae [ 41 ] and Sulfurovaceae [ 42 ] leading to higher nitrite concentrations (Figs. 1 , 2 and 4 ). Iron reduction in the heated bay sediments may have been mediated by the significantly higher abundant Rhodobacteraceae [ 43 ] (differential abundance analysis, p < 0.05; Table S4 ) or by sulfate reducing bacteria also capable of ferric reduction [ 44 ] ( p < 0.05; Figs. 1 , 2 and 4 ). The lower sulfate concentration in the heated bay sediment over the whole year was likely associated with the concomitant higher abundance of sulfate reducing taxa [ 30 ] including Desulforhopalus , Desulfofustis , and Desulfomicrobium that only occurred in the heated bay (Fig. S4 ). Sulfate reduction results in reduced sulfur that was likely oxidized by the increased relative abundance over all sampling times in the heated bay of Chromatiaceae, Candidatus Thiodiazotropha, and the positively correlated with increasing temperature Sulfurovaceae family (all p < 0.05; Fig. 4 , Fig. S4 , and Tables S3 – S5 ). Cyanobacteria had a high relative abundance in both bays with a statistically higher relative abundance in the control bay ( p < 0.05; Table S4 ). While resolution at higher taxonomic levels was limited, Chroococcales was highly abundant at one sampling site in the heated bay compared to Oscillatoriales and Synecchocales in the control bay (Fig. S5 ). The high amounts of Cyanobacteria in the sediment of both bays increased organic carbon that consumes oxygen during mineralization and is suggested to lead to coastal hypoxia in the Baltic Sea as a whole [ 45 ]. In contrast, the shallow depth may have permitted photosynthesis by Cyanobacteria on the sediment surface that would produce oxygen. In summary, condensed geochemical layers in the heated bay led to selection of a high diversity of ASVs in the 0–1 cm sediment layer predominantly aligning with taxa characterized as anaerobic and cycling sulfur compounds. RNA transcript based activities The majority of the RNA transcripts originated from Bacteria (75.7%) followed by Eukaryotes (6.7%), Archaea (1.8%), and viruses (<0.001%; Fig. S6 ). The Bacteria transcripts generated 2268 unique genes from 404 families (Fig. S7 and Table S6 ). Principle component analysis (PCA) based upon RNA transcripts gave a very similar distribution between the bays as that observed for the 16S rRNA gene amplicon data (Fig. 2 ). Significantly different RNA transcripts with an annotated function (DESeq2 differential expression analysis; adjusted p < 0.05; Fig. S7 and Table S7 ) also showed fewer transcripts attributed to more diverse families within the heated bay compared to higher transcript numbers to fewer, more dominant families in the control bay (Fig. 4 ). This further supported the data as representative of the sediment microbial communities and activities. High numbers of RNA transcripts in both bays were annotated as taxa involved in sulfur cycling (Fig. 4 ). These included several sulfate reducing microbial families in the heated bay such as Desulfuromonadaceae, Desulfovibrionaceae, Desulfobulbaceae, and Desulfobacteraceae compared to fewer diverse microbial sulfate reducing families, but with a higher activity that included the Desulfobacteraceae in the control bay. The generated sulfide was likely oxidized by a broad range of taxa with different relative counts of RNA transcripts for e.g. Thiobacillaceae that can oxidize sulfur compounds in both oxic and anoxic conditions [ 46 ] predominantly identified in the heated bay and aerobic Thiotrichaeceae [ 47 ] largely in the control bay. In addition, the Chromatiaceae family was both present and active in both bays suggesting anoxic sulfide oxidation [ 48 ]. A further difference in RNA transcripts was observed for the phylum Cyanobacteria with high counts suggesting all year round activity in the heated bay compared to seasonal blooms of activity in the control bay (Fig. 4 ). While Cyanobacteria had a statistically higher 16S rRNA gene amplicon relative abundance in the control bay, the RNA transcripts showed temporal peaks in activity in the control bay, compared to all year-round activity in the heated bay. The data support that future prolonged warming related to climate change may result in a phenological shift with longer periods of Cyanobacteria activity leading to anoxic conditions in the sediment surface [ 49 ]. In addition, both bays exhibited evidence of cryptic sulfur cycling as has been observed in nearby Baltic Sea sediments [ 11 ]. Metabolic responses Comparison of highly statistically significant RNA transcripts (log fold change (LFC) ≥ 4 or ≥ −4 and p < 0.05; Fig. 5 and Table S7 ) identified 595 and 553 differentially abundant genes in the heated and control bays, respectively. Of these, differences in RNA transcripts between the two bays were annotated as related to nitrogen (5 and 8 genes), sulfur (13 and 10), and methane metabolism (21 and 23), photosynthesis (36 and 42), chaperones (27 and 26), and repair (20 and 18; Fig. 5 ). Transcripts with a high LFC annotated as involved in nitrogen metabolism lacked genes coding for dissimilatory nitrogen metabolism as both microbial communities were likely cycling nitrogen compounds. Transcripts coding for oxygen consuming methanotrophy ( pmo A1, mean LFC 3.18 heated bay vs. −3.47 control bay) and sulfur oxidation ( fcc B, 2.63 vs. −4.4) processes had higher LFC values supporting the overall higher oxygen concentration in the control bay [ 50 ]. In contrast, transcripts for dissimilatory sulfate reduction ( dsv B (4.26 vs. −2.75), apr AB (3.9 vs. −3.53, 3.8 vs. −3.09), and qrc BCD (5.01, 5.25, 5.35) were higher in the heated bay that supported the geochemical and 16S rRNA gene amplicon data of compressed geochemical zones. Further support for the compressed geochemical zones included transcripts annotated as involved in methanogenesis [ 51 ] ( fdh AB (5.1, 5.25), coo S (5.25 vs. −2.89), and acs AC (4.81 vs. −2.36, 4.47 vs. −3.57) mainly identified in the heated bay along with the key mcr BC genes (Table S6 ) attributed to Archaea that were mainly identified in the heated bay. Energy conservation processes such as the ATP-synthase with higher transcripts in the heated bay (particularly evident at one sampling site) supported that increased temperature due to climate change increases productivity [ 25 ]. In contrast, RNA transcripts related to photosynthesis genes (PSI and PSII) had generally higher numbers within the control bay, suggesting the dominance of cyanobacterial blooms during early summer and autumn [ 26 ] compared to the all year round activity of Cyanobacteria in the heated bay (Fig. 4 and Fig. S8 ). The increased RNA transcripts for photosynthesis genes suggested the Cyanobacteria were on the sediment surface at a depth where light penetrates. Further analysis identified a large number of chaperone transcripts with a LFC > 4 that were predominantly identified in the heated bay and included heat stress proteins such as hsp A (5.31 vs. −4.09), dna K (4.62 vs. −3.59), gro S (4.62 vs. −3.46), and ibp A (3.99 vs. −3.59) [ 52 ]. However, genes coding for stress proteins were also identified in the control bay, albeit with much fewer transcripts and partly during the summer when the temperature was similar to the heated bay (Fig. S8 ). In addition, a similar trend of RNA transcripts from the DNA repair and recombination proteins were identified in the heated bay, likely as a response to the described stress [ 53 ] (Fig. S8 ). Fig. 5 Metabolic responses of differential expressed genes. a Volcano plot of significantly differential expressed genes (adjusted p -value < 0.05) selected as related to energy metabolism, stress, and repair KEGG categories. LFC are shown on the x -axis while the –log10 adjusted p -values are shown on the y -axis for differential RNA transcripts in the control (●) and heated (▲) bays. b Selected RNA transcripts with an LFC > 4 for energy metabolism, stress, and repair. LFC is shown on the x -axis while genes are shown on the y -axis for the control ( n = 12; left side) and heated ( n = 12; right side) bays. The data represent mean LFCs of RNA transcripts of genes as separately calculated for each bay. The presence of over 500 statistically different RNA transcripts with a LFC ≥ 4 in each bay suggested that large changes had occurred between the respective microbial community activities and that future climate change would profoundly change Baltic Sea sediment communities. The results further supported condensed geochemical zones with a higher metabolic activity of sulfate reduction and methane production in the heated bay. Furthermore, more RNA transcripts associated with ATP production were identified in the heated bay suggesting higher metabolic rates and energy generation. While the heated bay organic matter content supported increased production, this was likely tempered by the additional energy demand to alleviate stress and repair cellular components such that it may not be completely translated into cellular reproduction [ 54 ]. The large number of heated bay RNA transcripts related to stress also suggested that while the microbial community had altered, the community members’ temperature optima were below that of the heated bay. Therefore, coastal sediment communities will likely take greater than 50 years to become adapted to the increased temperature such that the increased energy production is fully converted to higher replication rates."
} | 7,965 |
37242960 | PMC10221719 | pmc | 5,705 | {
"abstract": "Benzoxazine resins are new thermosetting resins with excellent thermal stability, mechanical properties, and a flexible molecular design, demonstrating promise for applications in marine antifouling coatings. However, designing a multifunctional green benzoxazine resin-derived antifouling coating that combines resistance to biological protein adhesion, a high antibacterial rate, and low algal adhesion is still challenging. In this study, a high-performance coating with a low environmental impact was synthesized using urushiol-based benzoxazine containing tertiary amines as the precursor, and a sulfobetaine moiety into the benzoxazine group was introduced. This sulfobetaine-functionalized urushiol-based polybenzoxazine coating (poly(U−ea/sb)) was capable of clearly killing marine biofouling bacteria adhered to the coating surface and significantly resisting protein attachment. poly(U−ea/sb) exhibited an antibacterial rate of 99.99% against common Gram negative bacteria (e.g., Escherichia coli and Vibrio alginolyticus ) and Gram positive bacteria (e.g., Staphylococcus aureus and Bacillus sp.), with >99% its algal inhibition activity, and it effectively prevented microbial adherence. Here, a dual-function crosslinkable zwitterionic polymer, which used an “offensive-defensive” tactic to improve the antifouling characteristics of the coating was presented. This simple, economic, and feasible strategy provides new ideas for the development of green marine antifouling coating materials with excellent performance.",
"conclusion": "4. Conclusions In summary, a benzoxazine precursor was synthesized by the Mannich condensation, which was reacted with 1,3−propanesultone to successfully prepare a poly(U−ea/sb) antifouling coating with dual “offensive and defensive” antifouling properties. Ex-periments revealed that the poly(U−ea/sb) coating not only efficiently killed the attached bacteria and algae but also exhibited a good inhibition effect for the adhesion of non−Specific proteins. By the attack of fouling organisms, the “active offensive” and “passive defensive” mechanisms were activated simultaneously, rendering an efficient and long-lasting antifouling performance to the coating ( Scheme 3 ). The use of the benzoxazine precursor rendered excellent heat resistance and long-term stability to the coating, while the poly(U−ea/sb) coating surface was smooth, dense, and non-porous as the poly(U−ea) coating surface. As a green, biodegradable coating, poly(U−ea/sb) was composed of the natural product urushiol, with non-polluting quaternary ammonium salts instead of traditional antifouling agents for antibacterial and algae inhibition, while simultaneously relying on a strong hydrophilic surface to form a hydrated layer that physically blocked the adhesion of non-specific proteins. As a marine antifouling coating, the poly(U−ea/sb) coating not only exhibited 99% antibacterial and antialgal rates, but also the water contact angle was reduced by 60.52% compared with that of the poly(U−ea) coating, and the hydrophilicity was considerably improved, which exhibited better resistance to the adhesion of foreign proteins and adhesion of bacteria. Therefore, considering its high efficiency and environmental friendliness, the poly(U−ea/sb) antifouling material demonstrates potential to solve the economic loss caused by marine biofouling and environmental pollution related to the abuse of antifouling agents.",
"introduction": "1. Introduction Marine biofouling refers to the adherence of marine organisms on the surfaces of marine equipment, leading to economic damage in terms of human marine activities [ 1 , 2 ]. Marine biofouling is a global issue with permanent detrimental consequences on machinery [ 3 , 4 ]. Currently, petroleum-based resin self-polishing antifouling coatings comprising cuprous oxide (Cu 2 O) and a powerful biocide (such as zinc thiopyridine, copper/zinc pyridine thione, etc.) are widely used at home and abroad [ 5 , 6 ]. Although these self-polishing antifouling coatings prevent marine organisms from fouling, the coatings exert serious toxic effects on non-target marine organisms and the environment [ 7 , 8 ]. Therefore, to solve the ecotoxicological problems caused by the use of toxic antifouling coatings, designing and building green, long-lasting, excellent performance, and environmentally friendly new antifouling coatings, such as hydrophilic antifouling coatings, is imperative [ 9 ]. Most of the hydrophilic coatings exhibit eco-friendly and marine eco-friendly properties, which are highly sought after by researchers [ 10 ]. Owing to their good biocompatibility, hydrophilicity, non-toxicity, and excellent resistance to protein adhesion, zwitterionic polymers were initially used in medical devices [ 11 ], cotton fabrics [ 12 ], and filter membranes [ 13 ], and later for marine antifouling applications [ 14 , 15 ]. Zwitterionic polymers comprise the same number of anions and cations, in which the quaternary ammonium salt (R 4 N + ) directly penetrates the cell membrane surface and binds to the negative charge of the phospholipid bilayer on the cell membrane via electrostatic interactions, resulting in cell membrane destruction and bacterial necrosis. On the other hand, zwitterionic polymers exhibit strong electrostatic-induced hydration; as a result, a tight water layer is easily formed on the material’s surface, which in turn prevents the adhesion of fouling organisms effectively [ 16 , 17 ]. Chiang et al., prepared an antiprotein adhesion filtration membrane with a water flux recovery of 95.5% by grafting sulfobetaine methacrylate (SBMA) onto a polyvinylidene fluoride (PVDF) membrane surface [ 18 ]. Hibbs et al., prepared a series of polysulfone and polyacrylate-based amphoteric coatings on an epoxy resin primer aluminum substrate with 70% antibacterial and algal inhibition rates [ 19 ]. Liu prepared an innovative polymer brush on a resin matrix that can effectively inhibit the adhesion of red sponge larvae by one-step surface activation ATRP (SSI-ATRP) [ 20 ]. Zwitterionic polymers play a key role in hydrophilic antifouling coatings because of their good antifouling and environmental protection properties [ 21 , 22 ]. At the same time, under the “double carbon,” background, green, natural, easily extractable, and renewable natural products are selected as raw materials to prepare hydrophilic bio-based antifouling coatings. In addition, hydrophilic bio-based antifouling coatings are promising polymers because of their good hydrophilicity, cost-effectiveness, and commercial feasibility. However, most of the methods to synthesize zwitterionic materials coating materials involve direct polymerization and grafting with zwitterionic ions on the coating surface, which may involve complex chemical reactions and necessitate specific requirements on the coating surface; hence, commercialization is difficult. Therefore, designing and building zwitterionic bio-based coatings with excellent antifouling performance and in agreement with the modern “green environmental protection” marine antifouling concept is still challenging. Meanwhile, raw lacquer is a type of lacquer juice harvested from lacquer trees, which can be used as a natural and durable coating. Urushiol (U) is the main film-forming substance in raw lacquer, with a content of 60–70% [ 23 ]. Urushiol-based polymers exhibit incomparable uniqueness of petroleum-based resin coatings in terms of their mechanical properties, substrate adhesion, antibacterial and aging resistance. By introducing other functional groups and compounding degradable components, the structure and properties of urushiol-based polymers can be regulated effectively, and a new type of a bio-based marine antifouling coating with excellent performance can be prepared [ 24 ]. Previously, our group has successfully synthesized a low-surface-energy marine antifouling coating, referred to as urushiol-based benzoxazine copper polymer (UBCP) with antifouling performance, using urushiol, octylamine, and a copper compound as the phenolic source, amine source, and catalyst, respectively [ 25 ]. The as-obtained UBCP was cured into films at room temperature, which exhibited strong substrate adhesion, a smooth and dense surface, and the controllable release of an effective copper ion at minimized concentrations; hence, excellent antifouling performance was imparted to UBCP. As a new type of a thermosetting resin, benzoxazine exhibits high temperature resistance, chemical stability, and high durability. In the cross-linking reaction, benzoxazine easily forms a chemical bond with the coating substrate, such as the ring-opening addition of the oxazine ring, in which active methylene reacts strongly with electrophilic groups; as most of the substrates contain electrophilic groups, the adhesion of benzoxazine to the substrate is good [ 26 , 27 , 28 ]. Based on the above premise, the introduction of sulfobetaine into benzoxazine monomers is assumed to not only improve the poor hydrophilicity of benzoxazine through anions and cations effectively, but also the amphoteric ionization of polymers is easily conducted in comparison to direct polymerization. Reactive polymers with an adjustable chemical structure can be prepared with a high zwitterionic function and good antifouling performance. In this study, urushiol-based benzoxazine (U−ea) is prepared by the Mannich condensation using urushiol, paraformaldehyde, and ethanolamine as the raw materials. Subsequently, sulfobetaine-functionalized benzoxazine (U−ea/sb) is prepared by the reaction of a tertiary amine on the oxazine ring with sulfonate. The synthesis is simple, with no by-product. The effects of zwitterionic groups on the surface wettability, thermal stability and physical and mechanical properties of the coating can be analyzed by testing the contact angle, droplet adhesion, thermogravimetric analysis and mechanical properties of the coating. In addition, the antifouling performance of poly(U−ea/sb) coatings is systematically investigated using typical Gram negative bacteria such as Escherichia coli ( E. coli ) and Vibrio alginolyticus ( V. alginolyticus ), Gram positive bacteria such as Staphylococcus aureus ( S. aureus ) and Bacillus. sp., and algal species such as Nitzschia closterium ( N. closterium ), Phaeodactylum tricornutum ( P. tricornutum ), and Dicrateria zhanjiangensis ( D. zhanjiangensis ). Bovine serum albumin ( BSA ) and γ-globulin are used to systematically evaluate the antifouling performance of the poly(U−ea/sb) coatings. In this paper, the development of antifouling coatings with significant hydrophilic and excellent antifouling properties is focused, and ideas are suggested for the preparation of new, efficient, eco-friendly, hydrophilic bio-based antifouling coatings with dual “offensive-defensive” antifouling mechanisms.\n\n2.4. Introduction of Sulfobetaine Groups into Polybenzoxazine Chains First, the U−ea monomer (0.01 mol, 4.01 g) was dissolved in 10 mL of tetrahydrofuran, and the mixture was added into a 100-mL three-neck round-bottom flask equipped with a thermometer, a reflux condenser, and a dropping funnel. After the addition of 1,3−propanesultone (0.01 mol, 1.22 g), the solution was reacted at 35 °C for 24 h. At the end of the reaction, the solvent was removed by vacuum distillation and dried under vacuum at room temperature for 24 h to obtain the sulfobetaine-functionalized benzoxazine monomer (U−ea/sb)."
} | 2,871 |
22752113 | PMC3477478 | pmc | 5,706 | {
"abstract": "Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene ( acs ) of Kuenenia stuttgartiensis , encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs , the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria. Electronic supplementary material The online version of this article (doi:10.1007/s00203-012-0829-7) contains supplementary material, which is available to authorized users.",
"introduction": "Introduction Bacteria capable of anaerobic ammonium oxidation (anammox) derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas, thereby constituting a significant sink for fixed nitrogen under anoxic conditions (Arrigo 2005 ; Lam and Kuypers 2011 ). Cellular carbon is hypothesized to be fixed via the acetyl-coenzyme A (CoA) pathway, suggesting a chemolithoautotrophic lifestyle (Schouten et al. 2004 ; Strous et al. 2006 ). Throughout this pathway, organic carbon is formed by reducing CO 2 to CO and subsequently to cellular components via acetyl-CoA. Interestingly, it was shown recently that anammox bacteria have a more versatile metabolism than previously assumed: several genera were able to oxidize organic compounds to CO 2 with nitrate and/or nitrite as electron acceptor, possibly refixing the CO 2 via the acetyl-CoA pathway and fueling the catabolic reaction with nitrite (Güven et al. 2005 ; Kartal et al. 2007b , 2008 ). Although the nitrate reduction pathway has been elucidated (Kartal et al. 2007a ), the underlying biochemical pathway for organic acid oxidation is still unknown. The abundance of genes potentially involved in organic acid conversion points to its importance in anammox metabolism. The metabolism of acetate is commonly initiated by its activation to acetyl-CoA that is an essential intermediate of various anabolic and catabolic pathways and has a central role in the carbon metabolism in all three domains of life (Wolfe 2005 ; Ingram-Smith et al. 2006a ). At least five different ways to synthesize acetyl-CoA are known at present (AMP-forming acetyl-CoA synthetase (ACS), ADP-forming acetyl-CoA synthetase (ACD), acetate kinase/phosphotransacetylase (ACKA and PTA), CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) and acetate-CoA transferase). One of these enzyme complexes, an AMP-forming acetyl-CoA synthetase, is essential for the synthesis and conversion of acetate to acetyl-CoA and was experimentally investigated in this study. ACS catalyzes the direct formation of acetyl-CoA from acetate, ATP and CoA and is present in nearly all organisms. In prokaryotes, it is known to operate often in an assimilatory route during growth on low acetate concentrations (≤10 mM) (Wolfe 2005 ). It is a member of a family of AMP-forming enzymes that catalyze two-step reactions in which an acyl-adenylate intermediate is formed and pyrophosphate is released (Starai and Escalante-Semerena 2004 ). The analysis of the genome sequence of the anammox bacterium Kuenenia stuttgartiensis revealed several open reading frames (ORFs) with similarity ≥30 % to known acetate-activating enzymes. Their presence gave a first indication about the route of acetate utilization in anammox bacteria, although the incorporation of acetate-derived carbon into cellular biomass could not be detected so far (Kartal et al. 2007b , 2008 ). The present study focused on the functional expression of a putative AMP-forming ACS, the most abundant acetate-activating enzyme in the proteome of K. stuttgartiensis encoded in ORF kustc1128 (Kartal et al. 2011 ). An ackA - pta - acs triple mutant of E. coli was complemented with the K. stuttgartiensis \n acs gene resulting in recovery of growth on acetate. To investigate the substrate specificity and kinetic properties of the putative ACS, the acs gene was overexpressed in the host E . coli Rosetta™ 2. The potential physiological role of acetate conversion in vivo was determined by colorimetric determination of acetyl-CoA formed from acetate by K. stuttgartiensis . This is the first time that an anammox enzyme could be functionally expressed in a heterologous host and that its properties could support an important role in the carbon assimilation metabolism of K. stuttgartiensis .",
"discussion": "Results and discussion Phylogeny and expression of genes encoding acetate-activating enzymes in K. stuttgartiensis A thorough analysis of the K. stuttgartiensis genome assembly revealed seven protein-coding ORFs (>30 % identity) that were possibly involved in acetate metabolism as well as key enzymes of the acetyl-CoA pathway (Supplementary Table S1). Such a redundant repertoire of acetate- and acetyl-CoA-converting enzymes has not been observed in chemolithotrophic bacteria before. The most highly expressed ORF associated with acetate conversion in K. stuttgartiensis was kustc1128 encoding a putative AMP-forming acetyl-CoA synthetase (589 aa, calculated molecular mass 67 kDa; Table 1 ). Using the K. stuttgartiensis kustc1128 sequence as a template, a similar gene could also be identified in several other available anammox metagenomes (Gori et al. 2011 ; Harhangi et al. 2012 ; van de Vossenberg et al. 2012 ) suggesting a central role in anammox bacteria. The identities among the anammox ACS were >60 %. Phylogenetically, kustc1128 and other anammox homologues clustered at maximum identities of 57 % with representatives of the Archaea and Firmicutes (Fig. 1 ). These and several proteo- and actinobacterial sequences were affiliated to a larger cluster of acetyl-CoA synthetases that share a distinct domain structure (cluster I). The amino acid sequence differs significantly (<40 % identity) from the commonly described ACS (cluster II). A homologue of this cluster II could not be identified in K. stuttgartiensis after extensive analysis that makes kustc1128 the only ACS-like encoding gene. Table 1 Relative gene expression and coverage in the proteome of potential acetate-activating enzymes in K. stuttgartiensis \n Locus Annotation Gene expression a \n Peptides b \n kusta0048 Acetate-CoA ligase (ADP-forming); β-domain ( acdB ) 0.70 0 kustb2015 Acetate-CoA synthetase/acetate-CoA ligase 0.63 0 kustc0502 Acetate-CoA ligase (ADP-forming); α-domain ( acdA ) 0.46 0 kustc1128 Acetyl-CoA synthetase ( acsA ) 1.40 6 (13 %) kuste3169 Acetyl-CoA synthetase (ADP-forming) 0.64 1 (2 %) kuste3170 Hypothetical phsophotransacetylase protein 0.50 0 kuste3344 Phenylacetate-CoA ligase ( paaK ) 0.31 0 \n a Relative expression: (# reads × read length/ORF length, relative to overall coverage) \n b Number of peptide hits (percentage coverage) \n Fig. 1 Neighbor-joining tree of phylogeny estimated by ClustalW included in the MEGA 5.05 software package, showing acetyl-CoA synthetase (AMP-forming) homologues with two different conserved domain architectures: cluster I and cluster II. Values at the internal nodes indicate bootstrap values based on 1,000 iterations \n Closest hits were obtained (>60 %) to the ACS of hydrogenotrophic methanogens of the rice cluster I (RC-1), which use the enzyme for acetate assimilation (Erkel et al. 2006 ). Like the RC-1 archaeon, K. stuttgartiensis also encodes a putative vacuolar-type H + -translocating inorganic pyrophosphatase (kustd1836) that might function as a transmembrane proton pump (Serrano et al. 2004 , 2007 ; Bielen et al. 2010 ). The pyrophosphate (PP i ) released as a by-product of acetate activation, could theoretically be used to establish a proton motive force and thereby recover a quantity of the energy previously invested (Jetten et al. 1992 ). Mutant complementation tests An E. coli strain (AJW807) deficient in all three pathways of acetate activation was used to determine the functionality of kustc1128 as an AMP-forming ACS. The pET30a-containing mutant expressing kustc1128 upon induction with IPTG was able to grow on acetate as carbon source. Protein was isolated from a LB-grown E. coli AJW807 control group and from three different complemented mutants, grown on M63 supplemented with acetate. In the complemented mutant clones, the ACS enzyme had a specific activity of 57 nmol min -1 mg protein −1 . This is in concert with earlier described ACS activity tests of the ack mutant of E. coli K12 (Brown et al. 1977 ). The mutant incapable of acetate activation showed significantly less activity (0.8 nmol min −1 mg protein −1 ). This remaining activity could be due to the background activity of phenylacetate-CoA ligase (Brunner et al. 1975 ) or long-chain acyl-CoA ligase (Kornberg and Pricer 1953 ). The expression of ORF kustc1128 restored the acetate-activating capacities in an E. coli \n ackA - pta - acs triple mutant, indicating its physiological role as an active AMP-forming ACS in K. stuttgartiensis . Substrate specificity and kinetic parameters The heterologous expression as a His-tagged protein allowed rapid purification of the K. stuttgartiensis ACS over a Ni–NTA column. The His-tagged ACS was loaded on 10 % SDS-PAGE (Supplementary Fig. S1). Only one prominent band was visible at the expected mass. MALDI-TOF MS analysis of the purified ACS after a trypsin digestion confirmed its identity as kustc1128 (Supplementary Fig. S1; sequence coverage 35.8 %). The purified enzyme was tested for substrate specificity and kinetic properties. Activity toward acetate was the highest among tested organic acids (130 ± 9 nmol min −1 mg protein −1 ; n = 6). The enzyme retained its activity over a wide pH range with an optimum around pH 7. Activity decreased with only 20 % between pH 6.5 and 8.5 (Supplementary Fig. S2). Relative to acetate, the propionate and formate conversion rates were reduced to 84 and 66 %, respectively, whereas butyrate (31 %) and isobutyrate (34 %) were converted at even lower rates (Table 2 ). The K \n m for acetate was estimated at 0.2 mM that is comparable with K \n m values of E. coli, Haloarcula marismortui and Azotobacter aceti ACS (O’Sullivan and Ettlinger 1976 ; Kumari et al. 1995 ; Bräsen and Schönheit 2005 a) and well within the range of other described ACS enzymes (0.003–1.2 mM, (Bräsen et al. 2005 b; Li et al. 2012 ) (Fig. 2 ). It has been reported previously that ACS could convert other organic substrates, in particular propionate, albeit with a significantly lower specific activity (Jetten et al. 1989 ; de Cima et al. 2007 ; Ingram-Smith and Smith 2007 ). The K. stuttgartiensis enzyme shows only a slightly reduced activity with propionate compared to acetate indicating a broad substrate range. Also, the conversion of longer substrates such as isobutyrate and butyrate is not a common characteristic and has been shown only for the archaeal ACS enzymes in Archaeoglobus fulgidus (ACS2) and Pyrobaculum aerophilum (Bräsen et al. 2005 ; Ingram-Smith and Smith 2007 ). It is hypothesized that the broad substrate specificity is established by a substitution in one of the four conserved residues in the acetate-binding pocket that determines the specificity of the acyl-substrate (Ingram-Smith et al. 2006b ). Based on sequence comparison, the Ile 312 in the K. stuttgartiensis ACS is replaced by Val, a trait conserved among the described organisms sharing similar catalytic properties. Table 2 Specific activity of the purified ACS-like enzyme with different organic acids Organic acid Rate (nmol min −1 mg protein −1 ) % of rate with acetate Acetate 130.3 100 Propionate 114.9 88 Formate 84.8 65 Butyrate 40.5 31 Iso-butyrate 39.1 30 \n Fig. 2 Rate dependence of the potential K. stuttgartiensis ACS activity at different acetate concentrations. The inset shows a plot of the reciprocal velocity against the reciprocal of the substrate concentration \n Acetate conversion in Kuenenia stuttgartiensis That anammox bacteria can use organic acids as electron acceptor has been shown previously (Kartal et al. 2007b , 2008 ). The fate of those organics is until now still speculative, but all known pathways of acetate or propionate conversion proceed via acetyl-CoA, which would also be the end product of carbon fixation in anammox bacteria. As incorporation of acetate-derived carbon has not be shown yet, whole cells of K. stuttgartiensis were incubated with acetate ATP, and HSCoA and the conversion into acetyl-CoA were determined by measuring the Fe 3+ -acetyl hydroxamate complex formation. The rate of acetyl-CoA formation was significantly higher than for the complemented E. coli mutant (7.7 μmol min −1 mg −1 ) (Fig. 3 ). The conversion of acetate to acetyl-CoA increased linearly with the amount of cells added, whereas boiled cells did show any activity. Considering this assay relies on total protein concentrations, this rate could very well fit with that of the heterologously expressed, His-tag purified enzyme. Fig. 3 Formation of acetyl-CoA from potassium acetate in response to the addition of different amounts of whole K. stuttgartiensis cells \n In the present study, we could show that acetate could be activated by kustc1128, an acs -like protein, as well as whole cells of K. stuttgartiensis suggesting that indeed the reductive acetyl-CoA pathway was used by anammox bacteria as previously suggested. Such acetate activation could also lead to the direct incorporation of acetate into cell biomass by anammox bacteria. Additionally, the PP i released upon the formation of acetyl-CoA could be used to translocate protons by an H + -translocating pyrophosphatases building up a proton motive force over the anammoxosomal membrane, which is central to the anammox catabolism (Kartal et al. 2011 ). Recently, it was shown that the ATP-consuming reaction of ACS could be coupled to ATP-producing processes, a possibility that gives interesting perspectives regarding further research on anammox carbon metabolism (Mayer et al. 2012 )."
} | 3,671 |
29642652 | PMC5951469 | pmc | 5,707 | {
"abstract": "Silica nanoparticles were dispersed in an aqueous emulsion of alkoxy silanes and organic fluoropolymer. The dispersion was sprayed onto white marble and sandstone. The deposited composite coatings exhibited (i) superhydrophobicity and superoleophobicity, as evidenced by the high (>150°) static contact angles of water and oil drops as well as (ii) water and oil repellency according to the low (<7°) corresponding tilt contact angles. Apart from marble and sandstone, the coatings with extreme wetting properties were deposited onto concrete, silk, and paper, thus demonstrating the versatility of the method. The siloxane/fluoropolymer product was characterized using Fourier Transform Infrared Spectroscopy (FT-IR), Raman spectroscopy and Scanning Electron Microscopy equipped with an Energy Dispersive X-ray Spectrometer (SEM-EDX). Moreover, SEM and FT-IR were used to reveal the surface structures of the composite coatings and their transition from superhydrophobicity to superhydrophilicity which occurred after severe thermal treatment. The composite coatings slightly reduced the breathability of marble and sandstone and had practically no optical effect on the colour of the two stones. Moreover, the coatings offered good protection against water penetration by capillarity.",
"conclusion": "4. Conclusions Superhydrophobic, water repellent, superoleophobic, and oil repellent properties were induced in marble and sandstone which were sprayed and coated with an aqueous dispersion that contained alkoxy silanes, organic fluoropolymer (revealed by FT-IR, Raman, and SEM-EDX studies) and silica nanoparticles. High static (>150°) and low tilt (<7°) contact angles of water and oil drops on coated stones were measured. Similar extreme wetting properties were achieved when the composite coatings were applied onto concrete, silk, and paper, demonstrating that the method can be effectively applied to treat various surfaces at ambient conditions. According to SEM images, the deposited coatings exhibited augmented roughnesses raised by surface structures at the micro/nano-meter scale. A severe transition of the coating wettability, from superhydrophobicity to superhydrophilicity, was observed upon extreme thermal treatment as hydrophobic (methyl) groups were replaced by hydrophilic (hydroxyl) groups according to a FT-IR study. The composite coatings slightly reduced the breathability of marble and sandstone by approximately 17 and 20% respectively, and had practically no optical effect on the colour of the two stones. Moreover, the coatings offered good protection against water penetration by capillarity. The distinctive role of the silica nanoparticles in the aforementioned properties was elucidated, as coatings without nanoparticles were deposited on marble and sandstone and studied on a comparative basis with the composite coatings. Overall, it was demonstrated that the nanoparticles improved the properties of the protective coatings.",
"introduction": "1. Introduction Atmospheric water and rain can penetrate the porosity channels of natural stones causing direct (e.g., through freezing–thawing cycles) or indirect (e.g., by the deposition of pollutants) degradation effects in cultural heritage monuments, buildings and objects. The application of hydrophobic materials as protective coatings has been suggested as a potential solution for the surface protection of natural stones, used in cultural heritage [ 1 , 2 , 3 , 4 , 5 ]. More recently, advanced hybrid and composite materials of special surface structures were produced for stone protection, offering enhanced hydrophobicity and in some cases superhydrophobicity [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. The static contact angle ( θ S ) of a water drop on a hydrophobic surface is 150° < θ S < 90°, whereas on a superhydrophobic surface, θ S becomes very large, typically > 150°. Consequently, superhydrophobic coatings can offer, in principle, better stone protection against the deteriorative activity of water, provided that superhydrophobicity is accompanied by water repellency. The hydrophobic/hydrophilic character of a surface is associated with the θ S but water repellency/adhesion is better assessed by the tilt contact angle ( θ t ), defined as the angle that a surface must be tilted to move a water drop. In a truly water repellent surface, θ t is very small, typically < 10°. Alternatively, instead of the θ t , the contact angle hysteresis, defined as the difference between the advancing and the receding contact angles, can be used to describe the dynamic wettability of a surface [ 7 ]. Large θ S (i.e., superhydrophobicity) is not necessarily accompanied by small θ t (i.e., water repellency). For example, a water drop on the surface of a rose petal corresponds to θ S > 150° [ 36 , 37 ]; yet, the drop cannot roll off even when the surface is turned upside down [ 36 ], implying that the drop is pinned [ 37 ] and adheres to the superhydrophobic surface of the rose petal. Consequently, both θ S and θ t are important to evaluate the protection efficacy which a coating offers to natural stone. Several methods to produce superhydrophobic and water repellent coatings can be found in the literature [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 ]. For example, the wetting properties of polymer surfaces can change dramatically from a usual inherent hydrophobicity (or slight hydrophilicity) to superhydrophobicity and water repellency by embedding nanoparticles into the polymer matrices [ 6 , 7 ]. The presence of nanoparticles results in the formation of rough surface structures which induce extreme wetting properties [ 6 , 7 ]. This method of controlled nanoparticle-embedding into macromolecular matrices has some important advantages for the effective and sustainable protection of the cultural heritage [ 32 ]. It is an easy, low-cost, and one-step method which can be applied to treat large surfaces in ambient conditions and it uses common silane/siloxane products (or organic polymers), nanoparticles, and solvents. Silane/siloxane products are extensively used in stone consolidation and conservation [ 38 ]. Based on the aforementioned advantages, polysiloxane + nanoparticles (or organic polymer + nanoparticles) coatings have recently received significant attention and have been suggested for the protection of natural stone [ 6 , 7 , 8 , 9 , 11 , 12 , 13 , 14 , 16 , 17 , 20 , 22 , 23 , 24 , 25 , 27 , 28 , 29 , 32 , 33 , 34 , 35 ]. We have recently shown that, with careful selection of the silane/siloxane product and the concentration of added nanoparticles, the resulting polysiloxane + nanoparticles coating can evince both superhydrophobicity and superoleophobicity as well as water and oil repellency [ 39 ]. This is a major improvement, as monuments and other cultural heritage objects are often exposed to urban air pollution. Consequently, designing coatings which can repel not only water but also other liquids of lower surface tension (e.g., oil) is of great importance. To the best of our knowledge, this was the first report describing the production and deposition of a superoleophobic and oil repellent coating onto calcareous stones such as marble and sandstone [ 39 ]. In this previously published study, attention was focused largely on the interaction of the composite coatings with drops of various liquids and the measurements of the θ S and θ t contact angles [ 39 ]. In the present study, the wetting properties of the composite coatings are further investigated. Other important evaluation tests in the field of stone conservation, such as capillary water absorption, vapour permeability, and colour change, are carefully carried out. Furthermore, the selected macromolecular product (Silres BS29A) is characterized using several spectroscopic techniques. Spectroscopy is also used to explain a severe transition from superhydrophobicity to superhydrophilicity which is induced by extreme thermal treatment. Finally, it is shown that the coatings can be easily deposited on several substrates, thus demonstrating the versatility of the method.",
"discussion": "3. Results and Discussion 3.1. Characterisation of Silres BS29A Figure 1 a shows the FT-IR spectrum of the Silres coating (Silres BS29A without nanoparticles) on marble. The spectrum of the uncoated marble is included in the figure for comparison. Observations including the coating spectrum of the hydroxyl group vibrations broad band at ~3350 cm −1 , the dominant bands appearing at around 2900 cm −1 (which were assigned to the C–H modes of the methylene groups), the intense carbonyl band at ~1710 cm –1 and various C–O, C–H, Si–O, Si–C modes appearing in the 1650–700 cm −1 region, indicate that the coating consisted primarily of an alkoxy silane material [ 40 , 41 ]. Moreover, the characteristic absorption bands at 1195 cm −1 and 955 cm −1 were probably due to the C–F 2 and C–F stretching modes, respectively [ 42 ], thus suggesting the presence of a fluoropolymer in the composition of the Silres coating. The FT-IR results ( Figure 1 a) are in agreement with the Raman spectrum which was obtained for the Silres coating, as shown in Figure 1 b. The dominant bands appearing at around 2900 cm −1 , which were assigned to the C–H modes of the methylene groups as well as the various C–O, C–H, Si–O, C–C, Si–C modes appearing in the low energy region (below 1500 cm −1 ), indicate that the Silres coating consists predominantly of an organo alkoxy silane [ 43 ]. Furthermore, the peak at 730 cm −1 may be attributed to symmetric stretching vibrations of C–F 2 scissoring and the band at ~1214 cm −1 is probably a result of asymmetric stretching vibrations of C–F 2 [ 44 ]. Consequently, the results of the FT-IR and Raman spectra in Figure 1 are in agreement with the product’s (Silres BS29A) description provided by the manufacturer (Wacker) and suggest that the Silres coating is a mixture of alkoxy silanes and fluoropolymer. The presence of silicon (Si) and fluorine F in the Silres composition was furthermore evidenced using SEM-EDX, as described in the Supplementary File . Fluoropolymer had a key role in the wetting properties of the coating as it resulted in a reduced surface energy and therefore enhanced the repulsive character of the coating against the deposition of any liquid. It was reported that the surface energy decreases in the order –CH 2 > –CH 3 > –CF 2 > –CF 2 H > –CF 3 [ 45 ]. Fluorinated and perfluorinated materials have low wettabilities and have therefore become the logical choice to produce superomniphobic materials, which are designed to repel any liquid [ 46 ]. However, fluorinated chemicals, including perfluoroalkylsilanes and fluoroacrylic polymers, have potentially effects on human health and on the environment [ 47 ]. Major attention has been focused on the hazardous properties of the short and long chain PFAAs (perfluoroalkyl acids) which have been recognized as contaminants of high concern owing to their high persistence, toxicity, bioaccumulation potential, and distribution in the environment [ 48 , 49 ]. 3.2. Superhydrophobicity, Superoleophobicity, Water and Oil Repellency Table 1 shows the θ S and θ t results of water and oil drops on Silres + nanoparticles coatings. For comparison, the corresponding results obtained on Silres coatings are included. Apart from marble and sandstone which were the target materials of the study, other materials including silk, corrugated paper, and concrete were coated. According to the results of Table 1 , superhydrophobicity and water repellency were induced in all materials coated by Silres + nanoparticles, as water drops on these composite coatings corresponded to θ S > 150° and θ t < 10°. Likewise, superoleophobicity and oil repellency were achieved on Silres + nanoparticles coatings as oil drops on these composite coatings corresponded to θ S > 150° and θ t < 10°, except for treated paper where θ S = 145°. The results of Table 1 suggest that the use of SiO 2 nanoparticles enhanced the hydrophobic and oleophobic character of the coatings and their repellency against both water and oil. Lower and higher θ S and θ t , respectively, of water and oil drops were measured on Silres than on Silres + nanoparticles coatings. According to the results of Table 1 , contact angles of water drops on Silres + nanoparticles coatings were nearly independent of the substrate material. Static contact angles ( θ S ) of water drops on the composite coatings were between a very small range from 158° to 165° and θ t values varied within 3°–5°. Larger variations of θ S and θ t are reported in Table 1 for water drops on Silres coatings. It was reported that the surface structure and the apparent wetting properties of a polysiloxane coating is affected by the roughness of the underlying substrate [ 7 ]. Accordingly, different θ S and θ t were measured on Silres coatings placed on different substrates ( Table 1 ). However, when nanoparticles were added, their role in the surface structure of the composite coating was dominant and therefore any effect from the underlying substrate became negligible [ 7 , 32 ]. This effect of the nanoparticles was previously shown [ 7 , 32 ] and it is herein revealed in the SEM images of Figure 2 . The surfaces of the Silres + nanoparticles coatings which were deposited on marble and sandstone are shown in Figure 2 a,b, respectively. It is observed that the surface structures of the two coatings are similar and therefore practically independent of the substrate morphology. Consequently, the different underlying substrates should have no effect on the interaction of the two coated surfaces of Figure 2 with the water drops, as supported by the results of Table 1 . According to the results of Table 1 , the θ S values of oil drops onto Silres + nanoparticles coatings on the different substrates were within 145°–160°. This is a much broader range compared to the 158°–165° range observed for the water drops. The surface tension of oil (=32 mN/m) is lower than that of water (=72 mN/m). Hence, oil drops are more sensitive in slight changes of the coating’s surface structure than water drops. This agrees with the results of a previously published paper [ 39 ]. It was reported that the effect of the nanoparticle concentration, which affected the resulting surface structure of the coating, was more dramatic on the θ S of oil drops compared to the θ S of water drops [ 39 ]. Consequently, small changes in the surface structure of the Silres + nanoparticles coatings induced by the underlying substrate had practically no impact on the shape of water drops but they have substantially affected the θ S of oil drops. Drops of water and oil on Silres + nanoparticles coatings which were deposited on silk, paper, and concrete are shown in Figure 3 . The superhydrophobic and superoleophobic properties of the composite coatings are demonstrated. The easy/self-cleaning ability of the composite coatings is shown in Figure 4 using a block of coated sandstone. Large drops of water contaminated with soil were placed on the treated sandstone specimen ( Figure 4 a,b). The large drops rolled off when the specimen was slightly tilted ( Figure 4 b). The surface could be easily cleaned with fresh water which removed the soil contaminants ( Figure 4 c,d) without leaving any visible stain. An interesting property of polysiloxane surfaces is their transition from hydrophobicity/superhydrophobicity to hydrophilicity/superhydrophilicity, which occurs after thermal treatment at high temperatures. This is a result of the degradation of the hydrophobic functional groups and their replacement by hydrophilic functional groups generated through oxidation [ 50 , 51 ]. For the Silres + nanoparticles coatings, this transition is reported in Figure 5 . In particular, Figure 5 a shows the FT-IR spectrum of the bare sandstone which was taken as a reference background. The hydrophilic nature of the stone is revealed in the corresponding photograph of Figure 5 a. The measurements in Figure 5 b,c were taken from a Silres + nanoparticles coating on sandstone prior and after treatment at a high temperature (750 °C), respectively. The methyl group bands at ~2900 cm −1 appeared to be strong in the fresh composite coating ( Figure 5 b). However, the intensities of these C−H peaks were substantially reduced after thermal treatment whereas an intensity increment of the hydroxyl group broad band was recorded ( Figure 5 c). This chemical change in the coating’s surface led to a severe transition from superhydrophobicity to superhydrophilicity, as evidenced by the photographs included in Figure 5 b,c. 3.3. Water Capillary Absorption The efficacy of the Silres + nanoparticles coatings to repel water absorbed by capillarity was evaluated for treated marble and sandstone specimens. For comparison, uncoated stone blocks and samples coated by Silres were included in the study. The amount of the absorbed water per unit area (Q i ) after leaving the specimen in contact with water for time t i was calculated as follows [ 52 , 53 ]: (1) Q i = w i − w o A \nwhere w i is the weight of the sample after being in contact with water for time t i , w o is the initial weight of the sample prior to the test and A is the sample’s area which had been in contact with water during the test. The calculated Q i values were plotted as a function of time t i for marble ( Figure 6 a) and sandstone ( Figure 6 b) specimens. Figure 6 a shows that the bare and coated marble specimens quickly became saturated in absorbed water, as evidenced by the recorded plateaus of the three Q i − t i curves. The amounts of water were absorbed according to the following order: uncoated sample -> sample coated by Silres -> sample coated by Silres + nanoparticles, with the latter being the sample that absorbed the least amount of water at each specific t i . Consequently, the use of nanoparticles in the coating had a positive effect in the protection of marble against water penetration by capillarity. However, according to the results of Figure 6 a, the difference in the Q i results between marble samples treated with hydrophobic Silres and superhydrophobic Silres + nanoparticles coatings, was within the experimental error bars. The results for the sandstone specimens in Figure 6 b qualitatively follow the same trend with the results of Figure 6 a (marble). In particular, both Silres and Silres + nanoparticles coatings offered protection to the sandstone, against water absorption through capillarity, as their use resulted in reduced Q i compared to the bare, uncoated sandstone sample ( Figure 6 b). Moreover, the superhydrophobic composite coating gave somewhat better results but its superiority over the hydrophobic Silres coating was within the experimental error ( Figure 6 b). A comparison of the two Figure 6 a,b, suggests that the saturation points were recorded in longer t i for sandstone specimens which absorbed larger amounts of water compared to the corresponding marble specimens. In particular, saturations of the absorbed amounts of water were recorded at ~160 ( Figure 6 b) and ~10 min ( Figure 6 a) for sandstone and marble, respectively. Notably, after 250 min of contact with water, the amounts of water absorbed by the sandstone specimens were as follows: 0.037 g/cm 2 for the uncoated sample, 0.021 g/cm 2 for the sample coated by Silres, and 0.019 g/cm 2 for the sample coated by Silres + nanoparticles ( Figure 6 b). The corresponding values for the marble specimens were lower at 0.022 g/cm 2 , 0.008 g/cm 2 , and 0.006 g/cm 2 , respectively ( Figure 6 a). Sandstone has a higher porosity and therefore a capacity to absorb larger amounts of water than marble. For this reason, the sandstone specimens needed a longer time to become saturated in absorbed water than the marble specimens, as revealed by the results of Figure 6 . For the maximum amounts of absorbed water corresponding to the plateaus of the curves in Figure 6 , the reduction percentage of water absorption by capillarity (RC%) was calculated using Equation (2) [ 2 , 5 ]: (2) R C % = ( m u w − m t w m u w ) × 100 \nwhere m uw and m tw are the masses of water absorbed by capillarity by the untreated/bare and treated/coated specimens, respectively. An ideal coating must eliminate the amount of water absorbed by capillarity (RC% = 100). The results are summarised in Table 2 . The application of the Silres coating resulted in reductions in the amounts of absorbed water by 66.5% and 44.7% for marble and sandstone samples, respectively. These values of RC% increased by roughly an additional 10%, when nanoparticles were added to the protective coatings. In particular, the RC became 75.1% and 53.8% for the marble and sandstone samples coated by Silers + nanoparticles. 3.4. Water Vapour Permeability A coating that is designed for the protection of stone should not affect the water vapour transport properties. The effects of the Silres and the Silres + nanoparticles coatings on water vapour permeability were evaluated for the treated marble and sandstone specimens. The reduction percentage of vapour permeability (RVP%) was calculated according to Equation (3) [ 5 ]: (3) R V P % = ( m u v − m t v m u v ) × 100 \nwhere m uv and m tv are the masses of water vapour penetrating the untreated/bare and treated/coated specimens, respectively. An ideal coating must have no effect on the water vapour permeability (RVP% = 0). The RVP% results are reported in Table 2 . The two coatings, Silres and Silres + nanoparticles, had roughly the same effect on the breathability of sandstone. A slight reduction of the RVP was noticed when nanoparticles were added to the protective coating, as the RVP% was reduced from 23.6 for sandstone coated by Silres to 20.0 for sandstone coated by Silres + nanoparticles. However, this was a very slight improvement on the transport of the water vapour. Sandstone has a high porosity which plays a dominant role in the vapour transport process. Therefore, the exact type of the surface protective coating had only a minor effect on the RVP% of the treated sandstone. This was demonstrated by the results reported for sandstone in Table 2 and is also supported by previously published reports. Rhodorsil 224 and Porosil VV Plus are two solvent-based siloxane products which were applied to sandstone and gave RVPs which were on the order of 20% [ 8 ]. Silica nanoparticles added to Rhodorsil and Porosil coatings did not have any major effect on the measured RVPs [ 8 ]. Consequently, three siloxane (Silres BS29A, Rhodorsil 224 and Porosil VV Plus) coatings on sandstone, with or without silica nanoparticles, gave roughly the same RVP, suggesting that the specific type of protective coating does not have any major effect on the breathability of sandstone. In contrast to the highly porous sandstone, the use of nanoparticles had a major effect on the breathability of marble, which is a stone of low porosity. A major improvement on vapour transport was noticed when nanoparticles were added to the protective coating; the RVP% was reduced from 42.8 for marble coated by Silres to 16.8 for marble coated by Silres + nanoparticles. The augmented RVP% (=42.8), which was recorded when Silres was applied, indicates that the marble’s small pores were blocked by the protective material. The positive role that the nanoparticles had on water vapour permeability can be attributed to two effects. First, nanoparticles enhanced the coating’s surface roughness. Therefore, nanoparticles increased the active coating’s surface which was exposed to air; this facilitated the transport of vapour. Second, it has been reported that the diffusion rate of water vapour through a porous network increases as the hydrophobic character of the pores is enhanced [ 54 ]. In a previously published report, higher diffusion rates of water vapour through hydrophobic pores were measured than through hydrophilic pores [ 55 ]. In the present study, the nanoparticles enhanced the hydrophobic character of the coating inducing superhydrophobicity and therefore they should have a positive effect on water vapour transport through marble. 3.5. Colour Change The optical effects of the Silres and Silres + nanoparticles coatings on marble and sandstone were evaluated through colourimetric measurements, as discussed below. The global colour differences (ΔΕ*) of marble and sandstone, induced upon coating application, was derived from Equation (4): (4) Δ E * = ( L t * − L u * ) 2 + ( a t * − a u * ) 2 + ( b t * − b u * ) 2 \nwhere L*, a* and b* are the brightness, the red–green component and the yellow–blue component of the CIE 1976 scale, respectively. The “u” and “t” subscript characters correspond to the untreated/bare and treated/coated specimens, respectively. The results are summarized in Table 3 , which shows that the application of the superhydrophobic and superoleophobic Silres + nanoparticles coatings did not have any considerable optical effect on the aesthetic appearances of both marble and sandstone. The applications of the composite coatings led to colour changes in marble (ΔΕ* = 0.48) and sandstone (ΔΕ* = 1.43) which are not perceived by the human eye (ΔΕ* < 1.5). According to the results of Table 3 , the highest ΔΕ* (=3.11) was measured for sandstone coated by Silres. This optical effect is detectable by the human eye and it was largely a result of the change of the L* component which was reduced from 58.57 (uncoated sandstone) to 56.15 (sandstone coated by Silres). Moreover, a major contribution to the recorded ΔΕ* came from the change of the b* component which was increased from 3.43 to 5.37 when Silres was applied onto sandstone. The effect of the change of the a* component on the ΔΕ* was negligible. When SiO 2 nanoparticles were embedded in the protective coating, their high L* (=88.00) and low b* (=−7.00) resulted in an increase of L* and decrease of b*, which became 58.00 and 4.74, respectively (sandstone coated by Silres + nanoparticles). Accordingly, this approached the corresponding values of the uncoated sandstone. Consequently, the use of the SiO 2 nanoparticles had a positive effect on the optical change of sandstone: a lower ΔΕ* was measured when sandstone was coated with the composite coating than with Silres. On the contrary, the use of the SiO 2 nanoparticles had a negative effect on the optical change of marble: a higher ΔΕ* was measured when marble was coated with the composite coating compared to the colour change recorded when Silres was deposited onto the marble. In particular, ΔΕ* increased from 0.10 (marble coated by Silres) to 0.48 (marble coated by Silres + nanoparticles). In relative terms, this is a major increase. However, both colour changes are negligible in terms of perception by the human eye."
} | 6,785 |
29411153 | PMC5801134 | pmc | 5,709 | {
"abstract": "Thiol groups grafted silicon surface was prepared as previously described. 1 H ,1 H ,2 H ,2 H -perfluorodecanethiol (PFDT) molecules were then immobilized on such a surface through disulfide bonds formation. To investigate the contribution of PFDT coating to antifouling, the adhesion behaviors of Botryococcus braunii ( B. braunii ) and Escherichia coli ( E. coli ) were studied through biofouling assays in the laboratory. The representative microscope images suggest reduced B. braunii and E. coli accumulation densities on PFDT integrated silicon substrate. However, the antifouling performance of PFDT integrated silicon substrate decreased over time. By incubating the aged substrate in 10 mM TCEP·HCl solution for 1 h, the fouled PFDT coating could be removed as the disulfide bonds were cleaved, resulting in reduced absorption of algal cells and exposure of non-fouled silicon substrate surface. Our results indicate that the thiol-terminated substrate can be potentially useful for restoring the fouled surface, as well as maximizing the effective usage of the substrate. Electronic supplementary material The online version of this article (10.1186/s13065-018-0385-6) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusion In summary, PFDT molecules were integrated onto thiol-terminated silicon substrate through the formation of disulfide bonds. The PFDT modified silicon substrate appeared to possess, to some extent, a micro-organism resistant property. However, as the time for the immersion test increased, the overall B. braunii cell density on the PFDT modified silicon substrate increased indicating its antifouling property cannot last forever. It was found that the adhered B. braunii on PFDT modified silicon substrate can be removed by applying TCEP·HCl solution. TCEP·HCl serves as a reducing reagent and can therefore break the disulfide bonds and detach the PFDT coating, along with the B. braunii cells adhered on it. This presented approach provides a rational design for removing antifouling coating that becomes aged, all without damaging the original substrate.",
"introduction": "Introduction Biofouling is a complex process that involves living organisms and cells probing and attaching to surfaces. Biofouling is a big challenge for the biomedical industry because biofilms form easily on surfaces such as door handles, surgical equipment, and many other medical devices and could increase the spread of disease in humans. Data have shown that an estimated 1.7 million infections are caused from healthcare-associated infections annually [ 1 ]. In addition, because the growth of marine organisms on ship hulls can cause a drag force, biofouling can also result in decreased fuel efficiency and increased fuel consumption [ 2 – 6 ]. One strategy to reduce biofouling adsorption is to passivate the substrate through the coupling of antifouling molecules such as poly(ethylene glycol) [ 7 ] or poly(ethylene glycol) dimethacrylate [ 8 ]. Various works in surface modification for antifouling purposes have been summarized and reported [ 9 , 10 ]. An important challenge in the field of antifouling is that an antifouling coating does not last forever; it becomes less effective as it ages. It also has been brought to our attention that once the deposition of foulants has taken place, the surface modification no longer effectively prevents fouling, which is understandable considering that the effect of solute/coating interaction is severely reduced once a layer of deposited foulants is formed [ 10 ]. Therefore, once the fouling layer is formed, the old antifouling coating needs to be removed, and a new antifouling coating needs to be applied. One way of removing antifouling coating is by scraping, a time-consuming process that might damage the surface. We must therefore explore a method by which to remove the fouled coating easily. Silicon materials are integral parts of our daily lives and have widespread applications in healthcare and manufacturing due to silicon’s unique material properties, including high flexibility, chemical and thermal stability, and ease of fabrication [ 9 ]. In addition, silicon materials are mechanically and chemically resilient-able to resist wear in aqueous and organic environments-and display good electrical properties. Therefore, in this study, silicon substrate was selected as a model. Previously, we had developed a technique that allowed us to coat thiol-terminated silicon substrate with PFDT molecules through disulfide bonds. Here, the antifouling property of the PFDT-coated silicon substrate was tested by aging the substrate in Escherichia coli ( E. coli ) and Botryococcus braunii ( B. braunii ) cultures respectively. A large amount of B. braunii colonies were found anchored on the substrate in a 30-day immersion test. However, by applying a reducing agent, the disulfide bonds could be cleaved and the fouled coating could be removed, therefore exposing a non-fouled silicon substrate.",
"discussion": "Results and discussion To evaluate the antifouling performance of PFDT modified silicon substrate, we immersed the PFDT-coated silicon wafer and Piranha solution (one part 98% H 2 SO 4 and two parts 30% hydrogen peroxide) cleaned silicon wafer in a B. braunii culture. As shown in Fig. 2 , after culturing for 1 week, there were large amount of algal cells adhered on the Piranha solution cleaned silicon wafer (Fig. 2 a), whereas fewer algal cells adhered on the PFDT modified silicon wafer (Fig. 2 b). B. braunii cells on Piranha solution cleaned silicon substrate grew in small groups, and some of them formed clusters (Fig. 2 a). Our observations are in agreement with the previous studies regarding the typical stages of bio-fouling development that bioorganisms can multiply locally and then assemble to form microcolonies [ 14 , 15 ]. The obvious reduction in the number of algal cells adhered to PFDT modified silicon substrate (Fig. 2 b) indicates such a surface has resistance to the adhesion of algal cells, and it is not “algal friendly”. This result is consistent with other published studies involving the fluorination of substrates being applied to minimize microbial adhesion [ 16 – 18 ]. Fig. 2 Representative microscope images of Piranha solution cleaned ( a ) and PFDT molecules modified silicon surface ( b ) after immersion test in B. braunii culture for 1 week. The population of B. braunii cells attached on the Piranha solution cleaned silicon surface is much higher than that on the PFDT molecules modified silicon surface. We can therefore conclude that PFDT coated silicon surface possesses fouling resistant properties regarding B. braunii \n Similar to the results obtained with B. braunii , E. coli cells readily adhere on Piranha solution cleaned silicon substrate as well (Fig. 3 a). After PFDT modification, a significant reduction in the number of adherent E. coli cells was observed (Fig. 3 b), confirming the antibacterial efficiency of the PFDT modified silicon substrate. Attached bacterial densities were calculated (Fig. 4 ). Five fields of view (0.25 mm 2 ) on five replicate substrates were analyzed for each surface condition. Fig. 3 Representative microscope images of Piranha solution cleaned ( a ) and PFDT molecules modified silicon surface ( b ) after immersion test in E. coli culture for 24 h. The population of E. coli cells attached on the Piranha solution cleaned silicon surface is much higher than that on the PFDT molecules modified silicon surface. We can therefore conclude that the PFDT coated silicon surface possesses fouling resistant properties regarding E. coli ( E. coli cells were stained by methylene blue) \n Fig. 4 Attachment of E. coli cells on Piranha solution cleaned and PFDT coated silicon surface \n Figure 5 a shows the micrograph of the PFDT modified silicon surface after 1 month of incubation in a B. braunii culture. As can be seen in the micrograph, the cell density, the percentage, and the average area of spread of B. braunii cells increased significantly throughout the test, indicating the reduced antifouling performance of PFDT modified silicon substrate. Our experimental results are in accordance with previous reports that pre-microbial attachment can provide specific binding sites for further attachment of microbes and growth. The B. braunii microcolonies formed on the PFDT modified silicon substrate during a 1-week immersion test in order to undergo further adaption and development into B. braunii macrocolonies. However, when the sample was incubated in Bristol medium containing 10 mM TCEP·HCl for 1 h at room temperature followed by rinsing with Bristol medium, the cell density decreased noticeably. It is because the reducing agent, TCEP·HCl, released the PFDT layer through breaking the disulfide bond, and therefore detached the algal cells from the surface. Although several B. braunii microcolonies remained on the surface after TCEP·HCl treatment, it might have been due to the uneven coverage of the PFDT coating. The oxidation reduced the amount of thiol groups on the surface that could graft PFDT molecules via disulfide bonds. The attachment of B. braunii cells to the parts where no PFDT molecules were grafted could not be interrupted by TCEP·HCl. Fig. 5 Representative microscope images of PFDT molecules modified silicon surface after immersion test in B. braunii culture for 1 month at room temperature ( a ) and such surface after immersing in Bristol medium containing 10 mM TCEP·HCl for 1 h at room temperature ( b ) \n As a control experiment, a PFDT modified silicon substrate without B. braunii immersion test was submerged in 10 mM TCEP·HCl solution for 1 h followed by rinsing with DI water, and then dried under a stream of N 2 (g) at room temperature. The lack of F peak in XPS survey spectra (Fig. 6 ) confirms that PFDT was detached from the surface after TCEP·HCl solution treatment. Peak-fitting of the S 2 s envelope was utilized to analyze a change in the chemical state of terminal sulfur on the silicon surface. The S 2 s peak instead of the S 2 p peak was used in this analysis because the S 2 p peak could avoid any possible overlap of the S 2 p (160–169 eV) region with the Si 2 s (155–165 eV) signal from the substrate [ 19 ]. It also because S 2 s appears as a simpler, single peak, and not a spin–orbit doublet as does S 2 p [ 20 ]. It was reported that the peak at nearly (227.6 ± 0.1) eV was assigned to the thiol group [ 20 ]. Based on the peak areas from the deconvolution exercise (Fig. 7 ), thiol groups decreased from 35.3% of the surface-bound sulfur (Fig. 7 a) to 1.6% (Fig. 7 b). This result indicates that thiol groups released from disulfide bonds (–S–S–) through TCEP·HCl treatment were oxidized to a significant extent. Fig. 6 XPS survey spectrum of PFDT molecules modified silicon surface after incubating in 10 mM TCEP·HCl solution for 1 h \n Fig. 7 High-resolution XPS spectra of S 2 s region of a freshly prepared thiol-terminated silicon surface ( a ) and a PFDT coated silicon surface after TCEP·HCl treatment ( b ). The black line is the raw data; colored lines denote individual fit components \n Besides XPS, the difference in the chemical compositions was also reflected in the surface wettability of a given sample. The representative water contact angle images of chemically-modified silicon substrates in different stages are shown in Fig. 8 . For the freshly prepared hydrogen-terminated silicon substrate (Fig. 8 a), the water contact angle was about 78.4 ± 1.1°. After thiolation, the water contact angle was about 23.6 ± 1.2° (Fig. 8 b). The surface wettability transformation from 78.4 ± 1.1° to 23.6 ± 1.2° can be attributed to the hydrogen bonds formed between thiol groups on thiolated silicon substrate and water molecules. After chemical modification with PFDT, the water contact angle was changed to about 70.0 ± 1.9°. This can be attributed to the introduction of the low surface energy of PFDT. After the TCEP·HCl treatment, the water contact angle was changed to about 22.1 ± 1.6° (Fig. 8 d), implying the release of the PFDT layer from the surface. Fig. 8 Water contact angle profiles captured on hydrogen-terminated ( a ), thiol-terminated ( b ), PFDT molecules modified ( c ), and PFDT molecules modified with TCEP·HCl treated ( d ) silicon substrates \n Then, PFDT modified Si substrate with TCEP·HCl treatment was incubated in 100 mmol L −1 PFDT anhydrous ethanol solution again for 2 h. After being thoroughly rinsed with anhydrous ethanol by sonicating, the XPS result (Fig. 9 ) presents a F1 s peak at 289 eV with weak intensity. This weak F1 s peak indicates that the surface was barely modified by PFDT molecules which correspond to the loss of surface-bound thiol groups due to oxidation. Fig. 9 XPS survey spectrum of silicon surface prepared by first being modified with PFDT molecules, then treated with TCEP·HCl solution, and finally immersed in PFDT anhydrous ethanol solution for 2 h \n The antifouling performance of the PFDT modified silicon substrate with TCEP·HCl treatment was investigated by following the procedures described previously. After 1 week of incubation in a B. braunii culture, the density of cells attached to the surface (PFDT modified silicon substrate with TCEP·HCl treatment) observed by microscope (Fig. 10 ) was greater than that on the PFDT modified silicon substrate without TCEP·HCl treatment. However, the cell density on the PFDT modified silicon substrate with TCEP·HCl treatment was lower than that on the Piranha solution cleaned silicon substrate. B. braunii cell clusters, which were formed on Piranha solution cleaned silicon substrate, were not observed on PFDT modified silicon substrate with TCEP·HCl treatment. Fig. 10 Representative microscope image of PFDT molecules modified silicon surface with TCEP·HCl solution treatment after immersion test in B. braunii culture for 1 week \n In order to evaluate the effect of TCEP·HCl on the attachment of B. braunii , a control experiment was conducted. A Piranha solution cleaned silicon substrate with B. braunii clusters (sample in Fig. 2 a) was incubated into Bristol medium containing 10 mM TCEP·HCl for 1 h. From the microscope image (Fig. 11 ), the attachment of B. braunii is similar to that of Fig. 2 a. This result indicates TCEP·HCl had no effect on the attachment of B. braunii . Fig. 11 Representative microscope image of Piranha solution cleaned silicon surface which was first incubated in B. braunii culture at room temperature for 1 week, and then incubated in Bristol medium containing 10 mM TCEP·HCl at room temperature for 1 h"
} | 3,680 |
33445528 | PMC7826872 | pmc | 5,710 | {
"abstract": "Soil fungi strongly influence ecosystem structure and functioning, playing a key role in many ecological services as decomposers, plant mutualists and pathogens. Arbuscular mycorrhizal fungi (AMF) establish mutualistic symbiotic associations with plant roots and act as biofertilizers by enhancing plant nutrients and water uptake. Information about the AMF association with Crocus sativus L. (saffron) and their impact on crop performances and spice quality has been increasing in recent years. Instead, there is still little data on the biodiversity of soil microbial communities associated with this crop in the Alpine environments. The aims of this study were to investigate the fungal communities of two Alpine experimental sites cultivated with saffron, and to rank the relative impact of two AMF inocula, applied to soil as single species (R = Rhizophagus intraradices , C. Walker & A. Schüßler) or a mixture of two species (M = R . intraradices and Funneliformis mosseae , C. Walker & A. Schüßler), on the resident fungal communities which might be influenced in their diversity and composition. We used Illumina MiSeq metabarcoding on nuclear ribosomal ITS2 region to characterize the fungal communities associated to Crocus sativus cultivation in two fields, located in the municipalities of Saint Christophe (SC) and Morgex (MG), (Aosta Valley, Italy), treated or not with AMF inocula and sampled for two consecutive years (Y1; Y2). Data analyses consistently indicated that Basidiomycota were particularly abundant in both sites and sampling years (Y1 and Y2). Significant differences in the distribution of fungal taxa assemblages at phylum and class levels between the two sites were also found. The main compositional differences consisted in significant abundance changes of OTUs belonging to Dothideomycetes and Leotiomycetes (Ascomycota), Agaricomycetes and Tremellomycetes (Basidiomycota), Mortierellomycetes and Mucoromycetes. Further differences concerned OTUs, of other classes, significantly represented only in the first or second year of sampling. Concerning Glomeromycota, the most represented genus was Claroideoglomus always detected in both sites and years. Other AMF genera such as Funneliformis, Septoglomus and Microdominikia, were retrieved only in MG site. Results highlighted that neither sites nor inoculation significantly impacted Alpine saffron-field fungal communities; instead, the year of sampling had the most appreciable influence on the resident communities.",
"conclusion": "5. Conclusions For the first time, we have characterized the fungal communities of an Italian alpine agroecosystem cultivated with saffron and treated with arbuscular mycorrhizal fungal inocula. Since saffron is the world’s highest priced spice, the increases in yield and quality obtained using AMF inoculum along with having no evident impact on resident microbial populations suggests that farms in marginal areas such as alpine sites can increase profitability by inoculating saffron bulbs with arbuscular mycorrhizal fungi. In addition to AMF, it would be advisable to investigate other endophytes to be co-inoculated with saffron bulbs, or directly recruited from soils, that could offer further advantages, for example, an increasing tolerance to biotic and/or abiotic stresses. This last aspect is of particular importance because, after a few years of continuous cultivation, saffron could exert allelopathic activity against many soil components [ 64 , 65 ], thus compromising both its replanting and the cultivation of alternative crops (i.e., lettuce) [ 66 ]. Lastly, the provided datasets may contribute to future searches on fungal bio-indicators to be applied as biodiversity markers of a specific site and/or agriculture cultivation.",
"introduction": "1. Introduction Soil has always been known to be a source of microorganisms and although studied for many years its microorganism diversity is far from being fully known. Microbial communities present in soil are responsible for carrying out key ecosystem services for life on our planet but unfortunately, many beneficial functions are threatened due to climate change, soil degradation and agricultural exploitation [ 1 , 2 ]. The availability of soil nutrients to plants is key to obtaining healthier and better fitted crops, as they are agricultural productivity dependent on a wide range of ecosystem services provided by the soil microbiota [ 3 ]. However, most crops of global and local interest are still heavily dependent on the use of fertilizers and other chemicals which are hazardous to human and animal health and to the soil itself [ 4 , 5 ]. As concerns have been raised about the impact on soil microbiome of herbicides, pesticides and inorganic fertilizers, and more generally on the soil nutrient availability and plant phytotoxicity, the interest in alternative strategies for ecosystem management has greatly increased [ 6 ]. Plant growth promoting microorganisms (PGPM), i.e., soil and rhizosphere-inhabiting microorganisms, in minute quantities, promote plant growth [ 7 ], and have become one of the most important components of bio-fertilizers for sustainable agriculture. They are applied due to their role in plant growth promotion by regulating the dynamics of various processes (e.g., decomposition of the organic matter), the accessibility of various nutrients to plants (iron, magnesium, nitrogen, potassium and phosphorus), as well as acting against pathogens [ 8 , 9 ]. Within the categories of PGPM are the plant growth-promoting rhizobacteria (PGPR) and among the beneficial fungi, the arbuscular mycorrhizal fungi (AMF) [ 10 , 11 ]. It is well known that fungi can interact with plant roots in different ways, from mutualistic mycorrhizal symbioses (i.e., when both organisms live in direct contact with each other and establish mutually beneficial relationships [ 12 ], to parasitism [ 13 ]). Among mycorrhizal symbioses, arbuscular mycorrhizal (AM) fungi represent a fungal mutualistic endophytic group which establishes symbioses with over 90% of all plant species since the origin of terrestrial plants [ 12 ]. There is an increasing interest for the use of AM fungi to promote sustainable agriculture, considering the widely accepted benefits of the association with plants to nutrition efficiency (for both macronutrients, especially phosphorus, and micronutrients), water balance, and biotic and abiotic stress protection [ 14 ]. Successful progresses in AMF inoculation have been achieved and reported worldwide, but studies regarding its application from laboratory and greenhouse to field trials are still encouraged. Agricultural practices strongly affect soil physical and chemical properties, and impact on the microbial communities affecting their abundance, diversity, and activity [ 15 ]. The effects of management practices could be positive or negative [ 16 , 17 ]. On the negative side, they may affect the interaction between different microbial communities, including bacteria and fungi [ 18 ], which are known to be key drivers for a more sustainable soil management [ 19 ]. High-throughput DNA sequencing techniques have greatly expanded our capability to characterize soil microbiome and identify the factors, including land management, that shape soil microbial communities across space and time [ 20 ]. In light of these studies, although most soil microorganisms still remain undescribed, some of them have recently been characterized based on their ecological strategies [ 21 ]. This aspect is of importance to identify and predict functional attributes of individual taxa that could be manipulated and managed to maintain or increase soil fertility and crop production under severe threats, including intensive exploitation [ 22 ]. While studying a soil sample it is important to consider that there is no “typical” soil microbiome, but the relative abundances of major prokaryotic and eukaryotic taxa found in the soil microbiome can vary considerably depending on the soil in question [ 23 ]. It has been widely reported that soil samples, collected from the same sampling sites just a few centimeters apart from each other, may retain very different microbiomes [ 20 , 24 ]. The microbiome variation can be attributed to spatial variability in the soil environment and to specific characteristics of the sampling site, sampling time and crop species and management [ 3 ]. For this reason, over the last years, some protocols have been endorsed by international projects, such as the Earth Microbiome Project ( http://earthmicrobiome.org/ ), to analyze and compare soil microbial diversity at a large scale [ 25 ]. Fungi are widely distributed among all terrestrial ecosystems with a huge biodiversity and ecological importance by their principal role in ecosystems processes such as carbon cycling, plant nutrition, and phytopathology [ 26 ]. However, the distribution of fungal species, phyla, and functional groups as well as the determinants of fungal diversity and biogeographic patterns are still poorly understood despite recent large-scale sampling campaigns [ 27 ]. So far, information on soil fungal biodiversity in ecosystems such as Italian Alpine cultivated areas is still scarce, with the exception of some studies on vineyards [ 28 , 29 ] and apple orchards [ 30 ], compared to those concerning European alpine meadows, pastures, woods or specific alpine endemic plants and environments [ 31 , 32 , 33 , 34 ]. In the Aosta Valley region (north west Italy), smallholder farming systems, such as saffron cultivation, lead to interesting and unique Alpine agricultural ecosystems. Indeed, they are characterized by a high level of agricultural diversity, being mainly focused on meeting farmers’ needs. In this regard, they could represent a valid means for increasing incomes of multifunctional farms, with a positive impact on the recovery and economy of these often remote areas [ 35 , 36 ]. In particular, saffron is gaining increasing attention as an alternative crop in sustainable agricultural systems due to its unique biological, physiological, and agronomic traits, such as the capability to exploit marginal land. The application of AMF or PGPR inocula has also found interest due to the possibility to increase the overall cultivation sustainability and quality [ 37 , 38 , 39 ] In this context, we have already gained insight into the impact of AMF inoculants on growth and secondary metabolites production in saffron plantations [ 40 ]. Specifically, we record an increasing interest in applying low-input cropping systems (e.g., saffron cultivation) in mountain regions. Nonetheless, this is accompanied by a scant data collection on soil microbial diversity in saffron productive areas along with limited information on the effect of AMF inoculation on field-grown Crocus sativus L. (saffron). To fill this knowledge gap, the aims of this study were to investigate the fungal communities of two Alpine experimental sites cultivated with saffron, and to rank the relative impact of two AMF inocula, applied to soil as single species (R = Rhizophagus intraradices , C. Walker & A. Schüßler) or a mixture of two species (M = R. intraradices and Funneliformis mosseae , C. Walker & A. Schüßler), on the resident fungal communities which might be influenced in their diversity and composition. We used Illumina MiSeq metabarcoding on nuclear ribosomal ITS2 region to characterize the fungal communities associated to Crocus sativus cultivation in two fields, located in the municipalities of Saint Christophe (SC) and Morgex (MG), (Aosta Valley, Italy), treated or not with AMF inocula and sampled for two consecutive years (Y1; Y2). In the frame of an increasing demand to reduce chemical inputs in agriculture, the results of this study could reveal useful information on the real impact of AMF inoculation on the resident fungal communities opening new perspectives on the possible roles of AMF and/or other most competitive beneficial microbes to be further exploited in a sustainable agriculture perspective.",
"discussion": "4. Discussion Despite growing interest, the variability of soil microbial fungal communities (fungi and bacteria) and the biotic and abiotic factors that drive their differentiation are still poorly understood in some remote, but still ecologically important, environments such as Alpine marginal cultivated fields. Soil microbiome could be of particular relevance in saffron ( Crocus sativus L.) cultivation, since soil has been shown to serve as a reservoir of microorganisms which, once colonizing roots, might contribute to saffron plant growth, nutrient availability and pathogen defense [ 38 , 39 , 52 ]. Besides bacteria, the major component of soil microbiota is represented by fungi which play crucial roles as saprotrophs, plant mutualists, symbionts and pathogens [ 53 ]. Moreover, fungi are key in controlling the soil structure and its water content and in regulating the aboveground biodiversity and productivity [ 54 ]. Due to their large number of species, specialization, and important ecological functions, fungi are also considered excellent bioindicators of soil quality [ 55 ]. This aspect is especially relevant in the case of marginal alpine areas where saffron yield and quality may vary greatly by site on the basis of several factors such as soil types, climatic conditions and cultivation techniques [ 35 , 36 ]. However, one of the most important factors, namely the soil microbiota associated with cultivation sites, has been poorly explored in C. sativus worldwide, in spite of its economic value both at national and international level. Here, for the first time, we reported results obtained by profiling the fungal components of soils cultivated with saffron through Illumina MiSeq metabarcoding on fungal ITS2 from the Valle d’Aosta agriculture sites of Saint Christophe (SC) and Morgex (MG), and covering two years of sampling (Y1, Y2). In general, the two sites were characterized by slightly different fungal phyla assemblages: in both sites and years (Y1 and Y2), Basidiomycota were particularly abundant in soil. This findings is not in line with previous reports identifying dominant fungal phylotypes as belonging to generalist Ascomycota that dominate soils globally [ 56 ]. Our results are also different from those obtained by Coller et al. [ 29 ] in Alpine vineyards soils showing Ascomycota (51.8%) and Zygomycota (20.1%) as dominant Phyla, while Basidiomycota representing only a small fraction (11.2%). The surprising percentage of Basidiomycota in saffron cultivated fields, where some of the OTUs were affiliated to ectomycorrhizal species (e.g., Inocybe vulpinella Bruylants; Suillus granulatus (L.) Roussel, S. viscidus (L.) Roussel), could be explained by the fact that most major genera of fungi, such as the ectomycorrhizal genera Russula , Boletus , Inocybe , Cortinarius and Amanita , seem to be present on all habitable continents [ 56 ]. It is worth noting that the site of SC is also characterized by: a shrubby fence, the presence of a tree inside the plot, several plants of birch just around the plot fence and very few grass ( Figure 1 ). We can speculate that our data on the belowground fungal community may provide useful elements on the aboveground features such as previous and actual vegetation coverage and/or agronomic procedures, allowing to assess the impact of anthropogenic land use to hidden diversity in soil [ 31 ]. Through the analysis of fungal ecological guilds, we indeed highlighted that fungal symbiotrophs were significant more in SC than in MG. On the other hand, the higher abundance of saprotroph, pathotroph and saprotroph/symbiotroph fungi in MG could be explained by some other site specific edaphic characteristics. Our results are in line with those obtained in the same experimental plots by Caser and colleagues [ 41 ]. Indeed, they clearly demonstrated that the largest difference in physiological and biochemicals flower-related traits and corm properties of saffron plants cultivated in the same sites (i.e., Morgex and Saint Christophe) were between the growing seasons (Y1 to Y2). In particular, they found that many more moldy corms (wilted) occurred in the first cultivation season (36.8%) than in the second (16.8%), and more in Morgex (52.4%) than Saint Christophe (39.1%). They argued that elevated percentage of wilted corms was probably due to the high relative humidity and precipitation rate (more than 550 mm/year), mainly occurring in MG than in SC and, to the absence of corm antifungal treatments. In accordance with those findings, our results showed that Morgex’s soil seems to thrive not only significantly higher saprotroph but more important, pathotroph and saprotroph/symbiotroph fungi. Even if a specific characterization of plant pathogens was not conducted, the high wilting rate found in MG could be related and favored by the presence, in this site, of several fungal species belonging to Ascomycetes such as Blumeria , Colletotrichum , Curvularia , Gibberella , Leptosphaeria , Plectosphaerella , Ramularia , Stigmina . Indeed, all these have been reported to be associated with saffron diseases [ 57 , 58 ]. We also detected some taxa, previously reported as endophytic fungal isolates, belonging to two Ascomycota lineages representing two orders (Helotiales and Pleosporales) and one order each in Basidiomycota and Mortierellomycota, namely Agaricales and Mortierellales, respectively. The presence in soil of microbial endophytes is very important. In fact, some endophytes (e.g., M. alpina ) showed positive effects on many growth parameters (i.e., total biomass, size of corms, number of apical sprouting buds, number of adventitious roots) and plant secondary metabolite production [ 59 ]. Furthermore, the endophyte can enhance biotic stress tolerance to corm rot fungus by releasing arachidonic acid [ 60 ]. Despite being also designed to rank the relative impact of two AMF inocula on the resident soil fungal communities, this study highlighted that the variable significantly affecting the fungal communities was instead only the year of sampling (Y1 and Y2). Neither the sites nor inoculum application significantly influenced soil fungal diversity and composition. In particular, our results also showed that apparently, the resident AM fungal communities found in the treated or control plots of Morgex and Saint Christophe were not significantly affected by the introduction of commercial AMF-based inoculum and that the AMF sequences retrieved from the soil metagenome very partially reflected the species inoculum composition. In addition, unlike what has been previously reported [ 61 ] the inoculation process seems to increase the dominance of a single species (i.e., Claroidoglomus ) and decrease diversity of the preexisting AMF communities. Unlike the results obtained in other Alpine environments of Northern Italy by Berruti et al. [ 28 ], on vineyards of Aosta Valley, and Turrini et al. [ 30 ], on apple orchards of South Tyrol, the most abundant genus retrieved in saffron alpine agriculture sites, was not Glomus but Claroideoglomus , representing up to 80% of the Glomeromycota sequences. Similar to previous reports, AMF taxa belonging to Septoglomus and Funneliformis corresponded to less than 4% of total sequences in these agriculture sites. However, we must point out that the differences found may be due to the different methods, which were used in the studies cited above, to investigate AM soil fungal communities and only partially overlapping with ours. Regarding the higher AMF abundance and biodiversity found in Morgex we could speculate that this site is characterized by some patchy areas dominated by grasses; an environment more favorable to AM Fungi such as surrounding herbaceous plants, from which AMF propagules and/or healthy AMF mycelial networks could gradually have colonized saffron cultivated fields. Another hypothesis could be that in MG the vegetation-mediated legacy effects on soil microbial communities is still maintained. Furthermore, no sequences of Rhizophagus/Rhizoglomus spp. that represented the main taxon of one of the applied inoculum (i.e., R) were found. Many factors can affect the success of inoculation and AMF persistence, including environmental and cultivation conditions, species compatibility, degree of spatial competition with other soil organisms, and the time of inoculation. Hence, it is important to assess the effects of AMF on crop traits both as early application and as residual persistence in the following crop cultivation seasons [ 61 ]. This aspect of particular importance is in accordance with previous results showing that AMF root colonization of C. sativus, treated with mixed or with single inoculum, during the two successive cultivation cycles (year Y1 and Y2), was very low in terms of both intensity of colonization (0.0–9.0%) and percentage of arbuscules (0.0–4.4%) [ 40 ]. These results are, however, in line with those reported by Ceballos et al. [ 62 ] and Berruti et al. [ 61 ] on cassava and maize plants inoculated by AMF, respectively. In fact, even if root mycorrhization was very low for both plants (which are usually very well mycorrhized in the field) they eventually produced higher yields anyway. Beside the results regarding the belowground aspect of our experiment, it is important instead to underline that in many experiments AM fungal applications exerted an important impact on the above-grown features [ 37 , 39 ]. In particular, the inoculation of saffron with single or multiple AM fungal isolates was demonstrated to increase either flower production and, saffron yield as well as spice antioxidant activity and the content of some important bioactive compounds (i.e., picrocrocin, crocin I, and quercitrin) [ 39 , 40 ]. We can assume that the AM fungal inoculum may have had a side stimulating effect on other resident soil microbial components (i.e., PGPR) that affect plant growth and physiology. Another possible explanation could be the presence of PGPR strictly associated with spores of some AM fungal species commonly used as inoculum [ 63 ]."
} | 5,554 |
20222824 | null | s2 | 5,713 | {
"abstract": "The combination of synthetic stable branched DNA and sticky-ended cohesion has led to the development of structural DNA nanotechnology over the past 30 years. The basis of this enterprise is that it is possible to construct novel DNA-based materials by combining these features in a self-assembly protocol. Thus, simple branched molecules lead directly to the construction of polyhedrons, whose edges consist of double helical DNA and whose vertices correspond to the branch points. Stiffer branched motifs can be used to produce self-assembled two-dimensional and three-dimensional periodic lattices of DNA (crystals). DNA has also been used to make a variety of nanomechanical devices, including molecules that change their shapes and molecules that can walk along a DNA sidewalk. Devices have been incorporated into two-dimensional DNA arrangements; sequence-dependent devices are driven by increases in nucleotide pairing at each step in their machine cycles."
} | 240 |
39690267 | PMC11652604 | pmc | 5,714 | {
"abstract": "Global climate change exacerbates abiotic stresses, as drought, heat, and salt stresses are anticipated to increase significantly in the coming years. Plants coexist with a diverse range of microorganisms. Multiple inter-organismic relationships are known to confer benefits to plants, including growth promotion and enhanced tolerance to abiotic stresses. In this study, we investigated the mutualistic interactions between three fungal endophytes originally isolated from distinct arid environments and an agronomically relevant crop, Solanum lycopersicum . We demonstrated a significant increase in shoot biomass under drought conditions in co-cultivation with Penicillium chrysogenum isolated from Antarctica, Penicillium minioluteum isolated from the Atacama Desert, Chile, and Serendipita indica isolated from the Thar Desert, India. To elucidate plant gene modules commonly induced by the different endophytes that could explain the observed drought tolerance effect in tomato, a comprehensive transcriptomics analysis was conducted. This analysis led to the identification of a shared gene module in the fungus-infected tomato plants. Within this module, gene network analysis enabled us to identify genes related to abscisic acid (ABA) signaling, ABA transport, auxin signaling, ion homeostasis, proline biosynthesis, and jasmonic acid signaling, providing insights into the molecular basis of drought tolerance commonly mediated by fungal endophytes. Our findings highlight a conserved response in the mutualistic interactions between endophytic fungi isolated from unrelated environments and tomato roots, resulting in improved shoot biomass production under drought stress. Supplementary Information The online version contains supplementary material available at 10.1007/s11103-024-01532-y.",
"introduction": "Introduction Plants are continuously subjected to changes in their environment, including biotic stresses, such as pathogen infections or herbivores, and abiotic stresses, such as drought, heat, cold, flooding, salinity, metal toxicity, or nutrient deficiency, which impact crop productivity. The severity of these abiotic stresses is exacerbated by the current climate change scenario and is projected to increase significantly in the coming years (Kumar 2016 ). Water deficit is a major abiotic stress that severely affects crop yields (Barnabás et al. 2008 ). The climate change report of the Intergovernmental Panel on Climate Change (IPCC) indicates that heatwaves and droughts are anticipated to increase in frequency in the coming years (IPCC 2023 ). Plants coexist with a diverse array of microorganisms, both detrimental and beneficial. Since the initial definition of symbiotic interactions (de Bary 1879 ) and the subsequent refinement of the interpretation (Hertig et al. 1937 ), plant–microbe interactions have been recognized to confer numerous benefits for plant performance in terms of growth promotion and abiotic stress tolerance (Rodriguez et al. 2004 ). Plant endophytes are conventionally defined as microorganisms residing within plant tissues, both aboveground and belowground, capable of existing for the majority or the entirety of their life cycle inside the plant without causing harm (Stone et al. 2000 ). However, the concept of plant endophyte has been redefined more recently by Hardoim et al. ( 2015 ) as microbes that colonize and inhabit plant tissues regardless of the outcome of the interaction. In this study, Penicillium chrysogenum , Penicillium minioluteum , and Serendipita indica , three root colonizing fungal endophytes isolated from extremely arid environments, have been investigated. Serendipita indica (formerly known as Piriformospora indica ) is an axenically root colonizing endophyte of the order Sebacinales, isolated from the Thar Desert in India, with a broad host range (Verma et al. 1998 ; Weiss et al. 2016 ; Mensah et al. 2020 ). The strain of P. chrysogenum utilized in this investigation was isolated in Antarctica from the roots of the vascular plant Colobanthus quitensis (Oses-Pedraza et al. 2020 ). This strain has been characterized as a root colonizing fungal endophyte capable of colonizing the roots of Lactuca sativa and S. lycopersicum (Molina-Montenegro et al. 2020 ). The P. minioluteum strain employed in this study originates from the Atacama Desert in Chile, where it was isolated from Chenopodium quinoa roots (González-Teuber et al. 2018 ). Numerous studies have previously investigated the effects of these fungal endophytes on plant performance. For instance, S. indica promotes plant performance and biomass production (Varma et al. 1999 ; Peškan-Berghöfer et al. 2004 ; Vadassery et al. 2009 ; De Rocchis et al. 2022 ; Pérez-Alonso et al. 2022 ), and enhances tolerance to biotic and abiotic stresses in its host plant (Jogawat et al. 2016 ; Sefloo et al. 2019 ; Tsai et al. 2020 ; Shukla et al. 2022 ). Penicillium chrysogenum has also been reported to promote plant growth and abiotic stress tolerance (Molina-Montenegro et al. 2020 ; Morsy et al. 2020 ; Morales-Quintana et al. 2022 ), while P. minioluteum enhances salt stress tolerance in soybean (Khan et al. 2011 ) and drought stress and salt stress tolerance in quinoa (González-Teuber et al. 2018 , 2022 ). The ability of endophytes to promote growth or enhance stress tolerance of their host plants is hypothesized to be a consequence of either direct or indirect mechanisms (Santoyo et al. 2016 ). They can facilitate nutrient acquisition, produce secondary metabolites, or modulate the levels or signaling pathways of phytohormones (Waqar et al. 2024 ). Phytohormones are key drivers in plant stress responses. They are well-established small signaling molecules that rapidly change in their abundance in response to environmental changes and act at sub-micromolar concentrations (Davies 2010 ). The classical five phytohormone classes include auxins, gibberellins, abscisic acid (ABA), ethylene, and cytokinins (Gaspar et al. 1996 ). However, jasmonic acid (JA), brassinosteroids, and salicylic acid (SA) have subsequently been added to this classification (Bari and Jones 2009 ). ABA, JA, and SA are recognized as the primary stress phytohormones in plants, due to their roles in plant stress responses. According to recent studies, S. indica enhances drought tolerance. It has been demonstrated that S. indica enhances water stress tolerance in rice by regulating stomatal behavior and reactive oxygen species (ROS) scavenging systems (Tsai et al. 2020 ). Stomatal aperture and associated physiological processes are largely controlled by ABA (Davies 2010 ). Furthermore, S. indica promotes proline accumulation in walnut roots in response to drought stress (Liu et al. 2021 ). It has also been observed that the P5CS gene related to proline biosynthesis is induced in tomato inoculated with S. indica under drought stress conditions (Azizi et al. 2021 ). In this study, we investigated the drought tolerance phenotype of tomato plants inoculated with three genetically unrelated fungal endophytes from diverse extreme environments to address the question of the existence of shared response modules in plants commonly activated by beneficial root-colonizing fungal symbionts. To this end, a comprehensive transcriptomics analysis of tomato roots under drought conditions, inoculated with either P. chrysogenum , P. minioluteum , or S. indica , followed by a weighted gene co-expression network analysis (WGCNA) was performed. The experiments enabled us to identify a core drought response module in tomato that is induced by at least two fungi isolated from the Antarctic region and the Thar Desert in India. The module contains genes related to ABA, auxin, and ion homeostasis that are differentially expressed in the roots upon fungal infection.",
"discussion": "Discussion Beneficial plant symbionts can play crucial roles in mitigating biotic and abiotic stresses in their hosts. In this investigation, we identified a core set of genes that potentially elucidate the observed increase in biomass in aboveground parts of tomato plants under drought conditions when inoculated with the fungal endophytes P. chrysogenum and S. indica . Numerous studies have previously corroborated the concept of improving drought tolerance in plants, including tomato, through inoculation with root-colonizing fungal endophytes, e.g., P. chrysoge num (Morsy et al. 2020 ; Morales-Quintana et al. 2022 ), P. minioluteum (González-Teuber et al. 2018 ), and S. indica (Sherameti et al. 2008 ; Tsai et al. 2020 ; Azizi et al. 2021 ; Liu et al. 2021 ; Boorboori and Zhang 2022 ). These fungi are reported to also increase the tolerance of their host plants to several other abiotic stresses, such as salinity. Penicillium chrysogenum in combination with P. brevicompactum increases salt stress resistance and plant growth in tomato, lettuce, and cayenne (Molina-Montenegro et al. 2020 ). Penicillium minioluteum enhances salinity resistance in soybeans (Khan et al. 2011 ), and S. indica increases plant resistance to salt stress (Lanza et al. 2019 ; Boorboori and Zhang 2022 ). Multiple studies have identified specific physiological, biological, and biochemical mechanisms that contribute to water deficit tolerance in plants induced by fungal endophytes (Dastogeer and Wylie 2017 ). This study describes the mutualistic interactions between three distinct fungal endophytes, isolated from unrelated arid locations, and the agriculturally significant crop plant, S. lycopersicum . All three fungal symbionts conferred increased drought tolerance to tomato plants, as evidenced by the significantly higher biomass production of the aerial plant parts of seedlings infected with the fungi compared to the non-infected control plants (Fig. 1 b). To elucidate whether the tested root colonizing endophyte fungi triggered the same molecular mechanism(s) to confer increased drought tolerance, a comprehensive RNA-Seq analysis was conducted on tomato roots inoculated and non-inoculated with the endophytes under two different water regime conditions. As illustrated in Fig. 2 a, the number of DEGs was greater in the comparison between non-inoculated plants (Ctrl 40% FC vs. Ctrl 100% FC) subjected to drought stress and fungus-inoculated plants (Fungi 40% FC vs. Fungi 100% FC) under similar conditions. This observation indicates a possibly attenuated stress response in tomato plants co-cultivated with the tested fungi. Furthermore, the analysis of the transcriptional effect of the fungal infections under control (Fungi 100% FC vs. Ctrl 100% FC) and drought conditions (Fungi 40% FC vs. Ctrl 40% FC) revealed only a minimal impact on gene expression levels. It is noteworthy that the number of DEGs for P. chrysogenum and S. indica under combined stress conditions (Pch 40% FC vs. Ctrl 100% FC, 5751 genes; Sind 40% FC vs. Ctrl 100% FC, 5017 genes) significantly exceeds the sum of the individual stress responses (Ctrl 40% FC vs. Ctrl 100% FC, 4259 genes; Fungi 100% FC vs. Ctrl 100% FC, 673 genes). This observation is likely attributable to an extensive reprogramming of the drought stress response when the plants were infected with the different symbionts. The response to P. minioluteum , however, was less pronounced, which separates this fungus from the two others. As demonstrated by the PCA (Fig. 2 b), the highest degree of explained variability in the RNA-Seq data is associated with drought conditions, as reflected in the first principal component, which substantially corroborates the significant impact of drought on the transcriptome. Our hypothesis that the fungi could induce general drought stress response modules in tomato was supported by the absence of clear differentiation between control samples and those of fungus-inoculated plants. To gain deeper insight into shared transcriptomic alteration between drought stress and combined drought stress and individual fungus infections, we conducted a Venn diagram analysis to elucidate intersections among the set of DEGs from control and inoculated roots under drought conditions compared to non-inoculated roots under well-watered conditions (Fig. 2 c). In this plot, the highest number of DEGs is shared between the inoculated and the non-inoculated conditions, indicating the existence of a large gene cluster related to drought stress response in tomato roots, regardless of the inoculation condition. However, this analysis did not adequately account for the possibility that the interaction with the symbionts merely potentiates the response of genes that are also induced under control conditions, albeit at a lower level. In this context, to further investigate the transcriptional analysis, a WGCN analysis was performed. The WGCNA examined the correlation patterns among DEGs under various inoculation and drought conditions (Fig. 3 ). In the absence of a module showing a significant correlation among all three inoculated conditions, we rejected the hypothesis of a shared common response to drought among P. chrysogenum , P. minioluteum , and S. indica -inoculated roots. However, we identified the turquoise module, which exhibited a significant correlation under conditions in which the plants were inoculated with either P. chrysogenum or S. indica . This observation highlights a common shared drought response between tomato plants inoculated with the two fungi. With regard to the RNA-Seq data obtained for P. minioluteum , it can be inferred that the fungus-induced drought tolerance enhancement of the tomato plants is likely achieved through an alternative mechanism, or by a more subtle modification of the transcriptome. The latter possibility is suggested by the positive, albeit statistically non-significant, correlation with the fungus. The correlation between this fungus and the two others was not significant, although the tomato plants inoculated with P. minioluteum demonstrated a similar increased biomass production under drought stress compared to those infected with the other two symbionts. It will be an intriguing future endeavor to elucidate the complex network of transcriptional alterations triggered by P. minioluteum in tomato plants under drought stress conditions and compare them with those responses observed for the two other fungi. However, given the substantial correlation between the responses triggered by P. chrysogenum and S. indica , we focused our investigation on the comparison of the interactions between those two fungi and tomato. With the objective of gaining a more comprehensive understanding of the shared processes elicited in drought-stressed tomato plants co-cultivated with P. chrysogenum and S. indica , we conducted further analysis of the associated genes found in the turquoise module (Fig. 4 a). To enhance the scope of the analysis and maximize the information extracted from the dataset, we utilized A. thaliana ortholog genes, leveraging Arabidopsis as the most well-developed model for translational research in plants (Yaschenko et al. 2024 ). The GO analysis (Fig. 4 b) revealed GO terms of particular interest, namely ‘response to hormone’ (GO:0009725), ‘response to water deprivation’ (GO:0009414), and ‘response to abiotic stimulus’ (GO:0009628), as they suggested a direct connection to genes associated with biological functions related to responses to drought stress and that were differentially expressed in the presence of fungi. The selected GO classifications encompassed 59 associated genes. To further investigate the differential regulation of these genes, we analyzed their expression in non-infected control plants (Ctrl) and in plants infected with either P. chrysogenum or S. indica under drought stress conditions (Fig. 5 a). Through the application of a hierarchical clustering approach, we identified three clusters based on the growth conditions and the expression levels of the genes. Based on the observed results, we conclude that the shared response to drought stress in plants infected with fungi is mediated by the same group of genes. However, it is noteworthy that the expression levels differ between P. chrysogenum and S. indica inoculated plants. These differences in expression levels may potentially account for the variations in the observed phenotypes. Plants inoculated with P. chrysogenum exhibited higher biomass compared to those inoculated with S. indica when grown under drought stress conditions (Fig. 1 b). The identified genes were subsequently utilized to construct a functional interaction network with the aim of identifying a core regulatory module within the 59 candidate genes (Fig. 5 b). The resulting network provided evidence for the existence of several gene clusters that share common biological functions, which appear to be regulated by a reduced subset of genes. Our findings are summarized in the model presented in Fig. 6 , which illustrates the proposed main drivers of the increased drought tolerance phenotype observed in the tomato plants co-cultivated with either P. chrysogenum or S. indica . Fig. 6 A model summarizing the core drought response modules identified in this work The largest group of nodes is associated with ABA-dependent processes. This observation aligns with several reports highlighting the pivotal role of ABA in enhancing resistance to drought stress in plants harboring S. indica in their roots (Peskan-Berghöfer et al. 2015 ; Xu et al. 2017 ). Furthermore, 4 out of the 5 nodes with the highest degree of connectivity in the network are ABA-related, underscoring its crucial role in conferring increased drought stress tolerance in plants interacting with P. chrysogenum or S. indica . Among the 13 ABA-related genes, we identified several TFs, including GBF3 , HB7 , and RD26 , as well as 5 PP2Cs, namely ABI1 , AFP1 , AFP2 , HAB2 , and HAI1 . An induction of GBF3 is reported to confer increased resistance to drought in A. thaliana (Ramegowda et al. 2017 ). The ectopic expression of HB7 has also been demonstrated to increase drought tolerance in tomato (Mishra et al. 2012 ), while the induced expression of RD26 is similarly known to enhance drought tolerance in plants (Nakashima et al. 2012 ; Duan et al. 2019 ). These three TFs appear to be transcriptionally activated in the roots of tomato plants that established a symbiosis with P. chrysogenum or S. indica under drought conditions compared to non-inoculated roots from control plants grown under drought conditions (Supplementary Table S3). Consequently, we conclude that these TFs are critical molecular components for the fungus-triggered drought tolerance mechanism in tomato plants. The promotion of proline biosynthesis is a well-established drought stress response. Proline serves critical functions as an osmoregulator, chemical chaperone, and ROS scavenger (Liang et al. 2013 ). Our network analysis revealed the induction of P5CS2 , a key gene in proline biosynthesis. ∆ 1 -pyrroline-5-carboxylate synthases (P5CSs) catalyze the conversion of glutamate to γ-glutamate-semialdehyde, and they are closely associated with osmotic and drought stress responses (Amini et al. 2015 ). The induction of P5CS1 and P5CS2 in Arabidopsis is mediated by ABA (Strizhov et al. 1997 ), suggesting a potential correlation between the ABA gene group and P5CS2 (Fig. 5 b). Furthermore, it has been demonstrated that S. indica infections stimulate the accumulation of several metabolites in tomato, including betaine, glycine, and proline (Ghorbani et al. 2018 ), and induce P5CS gene expression under drought conditions (Azizi et al. 2021 ). In tomato, SOS genes are involved in salt tolerance mechanisms (Huang et al. 2024 ). Specifically, in response to increases in cytoplasmic Ca 2+ concentration, the Ca 2+ sensor SOS3 is activated, which facilitates interaction with the kinase SOS2. Subsequently, the SOS3/SOS2 complex phosphorylates and thereby activates the plasma membrane Na + /H + antiporter SOS1, resulting in reduced Na + toxicity in plant cells (Martínez-Atienza et al. 2007 ). Recently, SOS3 has also been demonstrated to interact with the Na + transporter HKT1 (Gámez-Arjona et al. 2024 ). Our RNA-Seq data provided evidence for a more pronounced repression of SOS3 and HKT1 in P. chrysogenum and S. indica inoculated roots under drought conditions compared to the non-inoculated roots under drought conditions (Supplementary Table S3). This observation suggests that the inoculated plants may accumulate more Na + through the reduced abundance of SOS1 and HKT1. The regulation of Na + uptake and compartmentation has been reported to contribute to the preservation of cell turgor (Álvarez-Aragón and Rodríguez-Navarro 2017 ). As illustrated in Fig. 5 a, the gene encoding the potassium channel SKOR exhibits stronger induction in the P. chrysogenum and S. indica inoculated roots than in the non-inoculated roots. This potentially increases the delivery of K + from the stellar cells to the xylem (Liu et al. 2006 ), leading to higher K + accumulation in aboveground parts of the plant. This observation aligns with the previously described impact of S. indica on the distribution of K + in Arabidopsis (Pérez-Alonso et al. 2022 ). Consequently, this phenomenon may influence the regulation of stomatal aperture. In the plant hormone-related groups, TIR1 appears to be more strongly repressed in the inoculated roots than in the non-inoculated roots. TIR1 is part of the SCF TIR1/AFBs -Aux/IAA [SKP-Cullin-F box (SCF), TIR1/AFB (AUXIN SIGNALING F-BOX), AUXIN/INDOLE ACETIC ACID (Aux/IAA)] complex, thus forming part of the canonical auxin perception machinery (Salehin et al. 2015 ). The loss of TIR1 in Arabidopsis is reported to increase drought tolerance (Salehin et al. 2019 ). Along with the repression of TIR1 , we were also able to detect the stronger repression of the jasmonate (JA)-related gene COS1 . JA has a proven role in biotic and abiotic stress responses (Wang et al. 2021 ). COS1 acts as a CORONATINE INSENSITIVE 1 ( COI1 ) suppressor that is essential for JA perception and the regulation of JA-mediated plant defense and senescence (Xiao et al. 2004 ). It will be an important future task to investigate these findings through reverse genetics experiments. In conclusion, we propose a molecular mechanism, shared by P. chrysogenum and S. indica , that involves the transcriptional regulation of a central core module of genes, responsible for the increased drought tolerance phenotype of tomato plants inoculated with the fungal endophytes. In this context, it is noteworthy that the two endophytes were isolated from two geographically distinct desert environments but appear to trigger the same conserved gene cluster in a non-specific host plant that is not endemic to the habitats where the fungi were isolated. This observation strongly supports the hypothesis that the tested root-colonizing beneficial fungi acquired the required properties to increase drought tolerance in plants by addressing highly conserved mechanisms in plants independently from each other over the course of coevolution with their host plants in similar extreme environments."
} | 5,842 |
31555065 | null | s2 | 5,715 | {
"abstract": "No abstract available"
} | 5 |
38319112 | PMC10936437 | pmc | 5,716 | {
"abstract": "ABSTRACT The soil-root interface harbors complex fungal communities that play vital roles in the fitness of host plants. However, little is known about the assembly rules and potential functions of rhizospheric and endospheric mycobiota. A greenhouse experiment was conducted to explore the fungal communities inhabiting the rhizosphere and roots of 87 rice cultivars at the tillering stage via amplicon sequencing of the fungal internal transcribed spacer 1 region. The potential relationships between these communities and host plant functional traits were also investigated using Procrustes analysis, generalized additive model fitting, and correlation analysis. The fungal microbiota exhibited greater richness, higher diversity, and lower structural variability in the rhizosphere than in the root endosphere. Compared with the root endosphere, the rhizosphere supported a larger coabundance network, with greater connectivity and stronger cohesion. Null model-based analyses revealed that dispersal limitation was primarily responsible for rhizosphere fungal community assembly, while ecological drift was the dominant process in the root endosphere. The community composition of fungi in the rhizosphere was shown to be more related to plant functional traits, such as the root/whole plant biomass, root:shoot biomass ratio, root/shoot nitrogen (N) content, and root/shoot/whole plant N accumulation, than to that in the root endosphere. Overall, at the early stage of rice growth, diverse and complex rhizospheric fungal communities are shaped by stochastic-based processes and exhibit stronger associations with plant functional traits. IMPORTANCE The assembly processes and functions of root-associated mycobiota are among the most fascinating yet elusive topics in microbial ecology. Our results revealed that stochastic forces (dispersal limitation or ecological drift) act on fungal community assembly in both the rice rhizosphere and root endosphere at the early stage of plant growth. In addition, high covariations between the rhizosphere fungal community compositions and plant functional trait profiles were clearly demonstrated in the present study. This work provides empirical evidence of the root-associated fungal assembly principles and ecological relationships of plant functional traits with rhizospheric and root endospheric mycobiota, thereby potentially providing novel perspectives for enhancing plant performance.",
"conclusion": "Conclusions Stochastic processes dominate the assembly of both rhizosphere (dispersal limitation) and root endosphere (ecological drift) fungal communities at the soil-root interface during the early stage of plant growth. The degree of association between complex and diverse rhizosphere fungal communities and plant functional traits was greater than that between the simplified root endosphere fungal community and plant functional traits. Our work goes beyond simply describing the diversity patterns of rhizospheric and root endospheric fungal communities to decipher the assembly mechanisms structuring rhizospheric and root endospheric mycobiota and to highlight the stronger links between rhizosphere fungal microbiota and plant functional traits. These results could inform future efforts to engineer beneficial root-associated microbiomes for improving plant performance.",
"introduction": "INTRODUCTION In nature, plants coexist with a variety of microorganisms, such as bacteria, fungi, archaea, protists, and viruses (collectively termed the plant microbiota), which coevolve with their host plants and form a coherent biological entity referred to as a holobiont ( 1 – 3 ). Plants represent an ideal, resource-rich ecological niche that allows microbial associates to thrive; additionally, many members of the associated microbiota undoubtedly confer fitness advantages to host plants, including nutrient acquisition and uptake, disease resistance, and abiotic/biotic stress tolerance ( 1 , 2 , 4 , 5 ). Among the plant-associated microbiota, fungi (also called mycobiota) are dominant and play key roles in plant functioning and health as mutualists, saprotrophs, or pathogens ( 4 , 6 – 8 ). For instance, mycorrhizal fungi improve nutritional conditions and enhance the stress resistance of host plants ( 9 – 11 ). Some free-living saprotrophic fungi can establish facultative biotrophic interactions with plants that facilitate plant nutrient uptake ( 12 ). Therefore, understanding the community assembly rules of mycobiota and their ecological coassociations with host plants is a crucial topic for research on plant holobionts. Most attention concerning plant holobiont research has been dedicated to the assemblage of bacterial communities at the soil-root interface ( 13 – 18 ), as plant roots represent the primary site for signal transduction and communication between host plants and their associated microbiota ( 19 ). Along the soil-root continuum, plant roots assemble microbiota from the soil microbial species pool into three separate microhabitats: the rhizosphere (microorganisms surrounding the root), the rhizoplane (root epiphytic microorganisms), and the root endosphere (microorganisms living inside the root) ( 1 , 19 , 20 ). Great progress has been made in deciphering the characterization and assembly mechanisms of rhizospheric and root endospheric microbiota over the last few years ( 19 , 21 ). According to the scholarly consensus, a clear differentiation of bacteria exists between rhizosphere and root endosphere microhabitats ( 14 – 16 ). The bacterial microbiota switches from dense and diverse rhizosphere communities to root endosphere communities with less complexity and reduced diversity ( 17 , 22 , 23 ). Moreover, advances in recent years have provided quantitative evidence that bacterial microbiota establishment in the rhizosphere/endosphere is not completely stochastic (e.g., random dispersal and drift events) but rather predominantly driven by deterministic (e.g., selection) processes ( 17 , 24 – 26 ). However, in contrast to the existing knowledge concerning bacterial community assembly at the rhizosphere-root endosphere barrier, the relative contribution of multiple ecological processes that govern the assemblage of rhizospheric and root endospheric mycobiota is still under debate ( 27 – 31 ). Plant functional traits (including morphological, physiological, and phenological features) represent strategies related to plant growth, reproduction, and survival to some extent ( 32 ). A growing awareness has emerged that complex and intimate linkages exist between host plants and their associated mycobiota; these patterns of codependent plant-fungal associations are considered to be profoundly interwoven with plant functional traits ( 33 , 34 ). Host-specific changes in the abundance and diversity of fungi, especially particular functional guilds, are important predictors in the predictive frameworks of observed plant trait-fungal relationships ( 34 – 36 ). For example, a greater degree to which a plant may depend on mycorrhizal symbioses for nutrient acquisition and uptake indicates a lower specific root length and thicker root diameter ( 34 , 36 ). Plant species with higher shoot nitrogen (N) concentrations and finer roots tend to recruit fewer mycorrhizal fungi and attract diverse fungal pathogens and specialist saprotrophs ( 35 ). Thus, fungi are considered vital components of microbial-mediated mechanisms underlying plant functional traits ( 37 ). However, the understanding of the ecological linkages between rhizospheric and root endospheric mycobiota and plant functional traits from the perspective of overall fungal community composition remains limited. Rice ( Oryza sativa L.) is cultivated globally and consumed by more than half of the world’s population. Here, rice was selected as a model plant for investigating host-microbiota associations. The original analysis of both the rhizosphere and root endosphere fungal microbiota was performed for 87 rice varieties ( Table S1 ) under controlled greenhouse conditions to explore the community assemblages and associated assembly processes, and these data were combined with those for rice phenotypic characteristics to evaluate the linkages between rhizospheric and root endospheric fungal community assemblages and plant functional traits. We hypothesized that, similar to bacteria ( 17 , 24 ), the assemblage of rhizospheric and root endospheric fungal communities could be strongly affected by rhizospheric compartmentalization and dominated by deterministic assembly processes. Moreover, we postulated that the rhizosphere rather than the root endosphere fungal microbiota is strongly associated with plant functional trait profiles because microbial-plant interactions usually occur in the rhizosphere ( 3 , 38 ). This work helps to characterize plant-microbiota associations and lays a foundation for engineering beneficial plant microbiomes for sustainable agricultural production.",
"discussion": "DISCUSSION Distinct fungal assemblages in the rice rhizosphere and root endosphere In agreement with the findings of previous studies on other plant species ( 25 , 41 – 43 ), a significant decrease in fungal richness and the Shannon index from the rhizosphere to the root endosphere compartment was observed in the present study ( Fig. 1a ), which indicated strong filtration for the recruitment of particular microorganisms at the rice rhizosphere-root endosphere barrier ( 1 ). Rhizo-compartmentalization was also the primary driving force of rhizospheric and root endospheric fungal community separation and was used to identify dominant fungal species and functional guilds in the rhizosphere and root endosphere ( Fig. 1b and 2 ), corroborating earlier findings that the distinction in fungal microbiota composition was highly dependent on the rhizocompartment ( 29 , 41 , 44 ). Moreover, greater structural variability in the fungal community was observed in the root endosphere than in the rhizosphere ( Fig. 1b ), as the colonization and formation of endophytic communities appear to be more variable processes ( 14 ). The differences in community assemblage between the rhizocompartments were also reflected by the fungal co-occurrence pattern ( Fig. 1c and d ). Compared with the root endosphere, the rhizosphere harbors a larger and more complex fungal coabundance network ( Fig. 1c ), suggesting greater organization of the fungal community in the rhizosphere. Community cohesion, a measure of biotic interaction strength ( 45 ), was stronger in the rhizosphere than in the root endosphere ( Fig. 1d ), which further supported the view that the rhizosphere contained a more cohesive and stable fungal community. The abundant resources in the rhizosphere may enhance extensive interspecies cooperation and symbiotic interactions and allow more species to maintain free-living populations ( 42 , 46 ). In this case, there are more opportunities for different species to directly or indirectly interact with each other, which might be responsible for increasing network complexity ( 47 ). Moreover, ecologically similar species can coexist by partitioning resources or habitats, and species do not interact with each other. In other words, the strong and intense correlation between taxa in the rhizosphere can also be interpreted as the result of convergent microbial adaptation to microhabitats surrounding the roots ( 48 ). Conversely, the root endosphere represents a relatively separate, divided, and crowded fungal microhabitat, which not only reduces fungal community diversity but also hampers interactions among fungal members ( 42 ). In addition, the presence of more isolated species in the root endosphere indicates that only particular fungal taxa can adapt to an endophytic lifestyle and fill these specific fragmented niche spaces ( 1 , 6 ). Stochastic processes govern the assembly of rhizospheric and root endospheric fungal microbiota at the early stage of rice growth As conceptualized by community ecology theory, four fundamental ecological processes (i.e., selection, dispersal, diversification, and ecological drift) shape the community assembly of plant-associated microbiota ( 49 – 51 ). In this investigation, both stochastic and deterministic components that are embedded in four fundamental ecological processes were concurrently present and determined the establishment of rhizospheric and root endospheric fungal microbiota ( Fig. 3 ). However, contrary to expectations, the variations in the composition of fungal communities inhabiting the rhizosphere and root endosphere were chiefly introduced by stochastic processes ( Fig. 3 ). Studies of plant-associated fungal microbiota commonly report host and environmental dependence, suggesting a certain degree of determinism during the assembly process ( 29 , 52 ); however, a high level of stochasticity was also observed ( 27 , 28 , 31 , 53 ). The assembly processes of plant-associated mycobiota alternate between deterministic and stochastic processes as plants develop ( 24 , 27 , 28 , 31 ). In the early stages of plant growth, fungi tend to be rare and dominated by stochastic processes ( 27 , 31 ), which supports our findings. Moreover, the provision of sufficient nutrients is believed to increase the stochasticity of ecological processes, which subsequently results in the dominance of stochastic process-driven fungal assembly in the rice rhizosphere and root endosphere ( 31 , 53 ). Given that all the plants belong to the same species, the low contribution of deterministic processes may also be attributed to limited sources of variable host selection. Additionally, to our knowledge, community assembly process partitioning may be influenced by primer selection ( 54 ). The finding of stochasticity could partially result from potential primer bias. The specific stochastic-based processes controlling fungal community development varied between rhizocompartments ( Fig. 3b ). Poor spore dispersal ability may result in dispersal limitation of the rhizosphere fungal community, despite the large numbers of spores produced by fungi ( 55 , 56 ). In contrast, endophytic fungal communities inhabiting roots may be prone to ecological drift due to their low overall diversity and abundance ( 27 , 49 , 50 ). Rice rhizosphere mycobiota exhibited more associations with plant functional traits In recent studies, the potential relationships between rhizosphere fungal community composition and host plant morphological and physiological traits have been reasonably evaluated ( 33 , 34 ). Similarly, the association analysis of plant functional traits revealed that plant traits were strongly correlated with both the taxonomic and functional compositions of rhizosphere fungal microbiota ( Fig. 4 to 6 ; Fig. S1 and S3; Tables S2 and S3). The close plant trait‒microbial relationships confirmed that the fungal microbiota inhabiting the rhizosphere is a key player in plant fitness ( 1 ). However, compared with those in the rhizosphere, the strong associations between fungal microbiota profiles and plant functional traits were weakened in the root endosphere ( Fig. 4 ; Fig. S1 to S3; Tables S2 and S3). In general, plants invest carbon (i.e., rhizodeposits) in building a belowground niche, which strengthens plant fitness in a changing environment ( 46 ). Therefore, the high covariation between the rhizosphere fungal community and plant functional traits may be related to the size of the rhizosphere effect created by a given plant. Greater functional complementarity (i.e., greater niche complementarity) and/or redundancy among species are more likely to occur in rhizosphere communities with higher diversity than in root endosphere communities ( 57 , 58 ), which results in more linkages between the rhizosphere fungal community composition and plant functional characteristics. The observed trait-fungal associations could be driven by specific species or trophic guilds of fungi ( 34 ). In the present study, most rhizosphere-specific species were classified as Ascomycota , Chytridiomycota , Basidiomycota , or Mortierellomycota ; were mainly saprotrophs or pathotrophs in terms of trophic guild; and exhibited a close association with plant N content and N accumulation (Fig. S4 and S5). This implies that the specific species residing in the rhizosphere may primarily participate in nitrogen acquisition by plants, thereby influencing the interaction between plant traits and fungal communities ( 59 ). In addition, the diverse fungal species inhabiting the rhizosphere may not only necessarily harbor direct beneficial functions but also contribute to making the rhizosphere a nutrient hotspot for plants through the degradation of organic matter ( 46 ). For instance, a saprophytic fungus (i.e., OTU29) with phylogenetic associations with the genus Mortierella was enriched in the rhizosphere and acted as a hub node in the coabundance network (Fig. S4 and S5). The genus Mortierella , which is among the most abundant and diverse saprophytic fungi, has been proven to be a reliable indicator for predicting plant agronomic traits ( 60 , 61 ). In contrast, most fungi live within roots as commensal endophytes; they have no apparent effects on plant performance in most cases and become functional only under specific conditions ( 6 ). We emphasize that the use of universal fungal primers to characterize the entire fungal community potentially introduces bias and results in a low relative abundance of some taxa, such as arbuscular mycorrhizal fungi ( 62 , 63 ). This study is also limited by the low completeness of fungal trophic guild assignments, which poses challenges for conducting functional assessments ( 64 ). Despite these limitations, the findings presented herein provide evidence that supports the existence of substantial covariation between rhizosphere mycobiota and plant functional traits ( 34 ). Conclusions Stochastic processes dominate the assembly of both rhizosphere (dispersal limitation) and root endosphere (ecological drift) fungal communities at the soil-root interface during the early stage of plant growth. The degree of association between complex and diverse rhizosphere fungal communities and plant functional traits was greater than that between the simplified root endosphere fungal community and plant functional traits. Our work goes beyond simply describing the diversity patterns of rhizospheric and root endospheric fungal communities to decipher the assembly mechanisms structuring rhizospheric and root endospheric mycobiota and to highlight the stronger links between rhizosphere fungal microbiota and plant functional traits. These results could inform future efforts to engineer beneficial root-associated microbiomes for improving plant performance."
} | 4,706 |
36983499 | PMC10057815 | pmc | 5,721 | {
"abstract": "Plants cope with abiotic stress in several ways, including by collaborating with microorganisms. Epichloë, an endophytic fungus, has been shown to improve plant tolerance to extreme external environments. Hordeum bogdanii is a known salt-tolerant plant with the potential to improve alkaline lands. NHX1 plays a key role in the transport of ions in the cell and is overexpressed in plants with increased salt tolerance. However, the expression levels of HbNHX1 in Epichloë endophytic fungal symbionts in H. bogdanii have not been elucidated. We used Hordeum bogdanii (E+) with the endophytic fungi Epichloë bromicola and H. bogdanii (E−) without the endophytic fungi and compared the differences in the ion content and HbNHX1 expression between the shoots and roots of E+ and E− plants under alkaline stress. The absorption capacity of both K + and Na + of H. bogdanii with endophytic fungi was higher than that without endophytic fungi. In the absence of alkaline stress, endophytic fungi significantly reduced the Cl − content in the host H. bogdanii . Alkaline stress reduced SO 4 2− content in H. bogdanii ; however, compared with E−, endophytic fungi increased the content of SO 4 2− in E+ plants. With an increase in the alkaline concentration, the expression of HbNHX1 in the roots of H. bogdanii with endophytic fungus exhibited an upward trend, whereas the expression in the shoots exhibited a downward trend first and then an upward trend. Under 100 mmol·L −1 mixed alkaline stress, the expression of HbNHX1 in E+ was significantly higher than that in E−, indicating that endophytic fungi could increase the Na + region in vacuoles. The external environment affects the regulation of endophytic fungi in H. bogdanii and that endophytic fungi can play a key role in soil salinization. Therefore, the findings of this study will provide technical support and a theoretical basis for better utilization of endophytic fungi from H. bogdanii in saline land improvement.",
"conclusion": "5. Conclusions The results showed that Epichloë endophytic fungi promoted the upregulation of HbNHX1 in the host H. bogdanii in an alkaline environment and participated in the regulation of anion and cation transport in the host plant. This study found that in an alkaline environment, Epichloë endophytic fungi reduced Na + toxicity in H. bogdanii by increasing HbNHX1 gene expression, thereby regionalizing Na + in the vacuole membrane and contributing to the increase in K + and related anions to regulate the effect of alkaline stress. This conclusion provides basic data for the innovation of germplasm resources with endophytic fungi from H. bogdanii , provides a scientific basis and strong guarantee for the breeding of new varieties and the healthy and stable development of grassland animal husbandry, and improves the stress tolerance of new varieties in alkaline environments.",
"introduction": "1. Introduction Soil salinization has severely endangered the environment and the production of economic crops in China [ 1 ]. High salt levels can lead to ion toxicity, osmotic stress, oxidative damage, and other secondary stresses in plants [ 2 ], making it necessary for the plants to maintain the balance between ions in their cells to ensure normal growth and development. A large area of the inland saline-alkaline land in China mainly contains three cations: Na + , K + , Mg 2+ , and four anions: CO 3 2− , HCO 3 − , Cl − , and SO 4 2− [ 3 ]. Excess accumulation of Na + and Cl − will result in ion toxicity, thus reducing the absorption of essential nutrients such as calcium (Ca), potassium (K), phosphorus (P), and nitrogen (N) [ 4 , 5 ]. As soil salinization involves not only salt stress but also alkaline stress, the same stress factors apply to alkaline stress as they do to salt stress, which also involves high pH stress [ 6 ], which leads to a significant amount of metal ions to precipitate, inhibits the absorption of anions, and breaks the ion balance inside and outside cells [ 7 ]. Therefore, to slow down the accumulation of cations such as Na + and K + under alkaline stress, plants maintain the balance of anions and cations by accumulating inorganic anions such as Cl − , NO 3 − , and SO 4 2− . The effects of alkaline stress on the transport of Na + and K + in plants have been extensively studied. Plants cope with a large amount of Na + accumulation under saline-alkaline stress mainly in two ways: SOS1 gene involved ion efflux and the NHX1 gene participation in intracellular ion regionalization. SOS1 is located in the plasma membrane, expressed in the roots of epidermal cells and wood parenchyma cells, and is mainly involved in the efflux of Na + from the cytoplasm to the soil and the transport of Na + to leaves through transpiration [ 1 , 8 ]. NHX1 is located in the vacuole membrane and plays an important role in the transport of Na + in plants by exchanging Na + into vacuoles and H + into the cytoplasm. This reduces the excessive accumulation of Na + in the cytoplasm and maintains the osmotic potential in cells so that they can absorb water normally [ 9 ]. In Arabidopsis and rice, plants overexpressing NHXs can retain K + in the cytoplasm under saline conditions, thereby improving salt tolerance [ 10 , 11 ]. Pérez-Martín et al. [ 12 ] found that high expression of NHX1 in Arabidopsis leaves under saline and alkaline environment can enhance salt tolerance by promoting Na + partition and osmotic regulation. Plants can improve their tolerance to Na + by collaborating with microorganisms [ 13 ]. For example, in tomatoes and lettuce, inoculation of plant roots with fungal endophytes resulted in an improvement in biomass, photosynthetic capacity, and survival rate under salt stress. At the same time, it enhanced the expression of the NHX1 gene and promoted Na + regionalization [ 14 ]. However, many molecular studies have mainly focused on the interaction between root endophytic fungi and plants, whereas there are few studies on the interaction of endophytic fungi in plant stems and leaves. The endophytic fungus of grass spends part or all its life cycle affecting the growth process of grass and does not cause obvious symptoms in plants [ 15 ]. Under saline-alkaline stress, infection with endophytic fungi of Gramineae can improve the tolerance and adaptability of host plants to the external environment [ 16 , 17 ]. Recent research on endophytic fungi of Gramineae mainly focused on the genus Epichloë of Clavicipitaceae, Ascomycota [ 18 ], which affects the resource allocation and mineral absorption capacity of host plants [ 19 ]. H. bogdanii is a salt-tolerant plant with strong ecological adaptability and competitive advantages. It has broad prospects for development as a forage grass and to improve the ecological environments of desert, saline, and alkaline lands [ 20 ]. Recently, studies on H. bogdanii -endophytic fungi symbionts have mainly focused on the classification and identification of endophytic fungi [ 21 , 22 , 23 ] and the effects of endophytic fungi on the growth and development [ 23 , 24 ], physiology, and biochemistry [ 25 , 26 ] of H. bogdanii under abiotic stress. However, HbNHX1 expression and ion balance of Epichloë endophytic fungal symbionts in H. bogdanii under saline-alkaline stress have not yet been reported. In this study, we aimed to explore the regulation mechanism of endophytic fungi on the expression of the HbNHX1 gene of H. bogdanii and the balance of anions and cations in plants under alkali concentration stress. To achieve this, we measured HbNHX1 expression and Na + , K + , Cl − , SO 4 2− and NO 3 − content in the shoots and roots of E+ and E− H. bogdanii plants under alkaline stress, and analyzed the effects of endophytic fungi on HbNHX1 gene expression and ion balance regulation of the host H. bogdanii under alkaline stress.",
"discussion": "4. Discussion 4.1. Effect of Alkali Stress on Na + , K + Content, and K + /Na + Ratio in H. bogdanii Na + and K + have similar radii of hydrated ions, so it is difficult to distinguish them, leading to a competitive relationship between the absorption of Na + and K + by roots [ 5 ]. Excessive salt absorption by plants causes ion toxicity. Plants transport Na + in cells to vacuoles under salt stress to reduce water loss. In most plants living in saline-alkaline environments, Na + accumulates in vacuoles in large quantities and K + absorption is inhibited [ 31 ]. To cope with the excessive accumulation of Na + in the cytoplasm of leaves under salt stress, Hordeum brevisubulatum transported it to vacuoles for Na + localization to reduce Na + toxicity [ 32 ]. Many studies have shown that endophytic infection can improve abiotic and biological resistance of host plants [ 33 , 34 , 35 ]. However, there have also been reports showing no significant differences between plants with and without endophytic fungi [ 36 ]. Our results showed that endophytic fungi and alkaline stress significantly affected the content of Na + , K + , and K + /Na + in plants. Under alkaline stress, the content of Na + in the shoots and roots of E+ and E− H. bogdanii exhibited an increasing trend, which is similar to the results published by Song [ 37 ]. However, this study found that alkaline stress led to Na + content in E+ being significantly higher than that in E-, which may be because endophytic fungi promote the accumulation of Na + in vacuoles during alkaline stress, leading to an increase in the content of Na + . K + is an important mineral nutrient that can protect plants from damage. We found that the K + content in the shoots and roots of H. bogdanii decreased significantly under alkaline stress, indicating that alkaline stress inhibits the absorption of K + by plants. In addition, the K + content of E+ was significantly higher than that of E−, indicating that Epichloë endophytic fungi can increase the K + content in host plants. K + content was higher in the shoots than that in the roots under alkaline stress, which is consistent with the results of Chen et al. [ 38 ], indicating that plants will accelerate K + upward transport when subjected to alkaline stress to alleviate Na + toxicity and maintain ionic balance. Alkaline stress increased the plant Na + content and continuously decreased the K + content, leading to a decrease in the K + /Na + ratio, which was consistent with the trend of K + /Na + ratio decline in Peucedanum littoralis and Hordeum vulgare [ 39 ] after salt stress. 4.2. Effect of Alkali Stress on Cl − , SO 4 2− and NO 3 − Content in H. bogdanii Alkaline stress forces a large amount of Na + in plants to increase, leading to an ion imbalance [ 40 , 41 ]. Plants usually accumulate inorganic anions, such as Cl − [ 42 , 43 ], SO 4 2− , and NO 3 − , or synthetic organic anions [ 44 ] to regulate ion imbalance. In this study, we found that when subjected to alkaline stress, Cl − content was significantly higher than that of the other anions. Additionally, under alkaline stress, the Cl − content of E+ was lower than that of E−, and the Cl − content of E− plants decreased continuously but E+ increased, indicating that endophytic fungi can regulate the Cl − content to regulate the ion balance in plants and reduce the ionic damage in host plants. These results were consistent with studies that reported similar trends in Cl − content in citrus ( Carrizo citrange ) [ 45 ], spring wheat ( Triticum aestivum ), and winter barley ( Hordeum vulgare ) [ 46 ] caused by mycorrhiza. We also found that alkaline stress reduced the content of SO 4 2− in H. bogdanii , which is consistent with the findings of Yang et al. [ 47 ]. The SO 4 2− content of E+ was higher than that of E− when endophytic fungi infected H. bogdanii , indicating that endophytic fungi increased the accumulation of SO 4 2− in host plants. In plants, the increase in Cl − content in the roots is related to a decrease in NO 3 − content [ 48 ]. High pH caused by alkali stress may inhibit the absorption of nitrate [ 49 ], thus weakening the positive role of NO 3 − in maintaining plant alkaline tolerance [ 50 ]. In addition, we found that when subjected to mixed alkali concentrations of 50 mmol·L −1 and 100 mmol·L −1 , the Cl − content in the roots of E+ increased then decreased, and the content of NO 3 − decreased and then increased, which also confirms that the increase in Cl − content is related to the decrease in NO 3 − content. After alkaline stress, the content of SO 4 2− and NO 3 − in the roots decreased. Alkaline stress significantly affected the content of NO 3 − in E+ and E− roots. At 50 mmol·L −1 , NO 3 − content in E+ was significantly lower than that in E−, whereas, at 100 mmol·L −1 , NO 3 − content in E+ was significantly higher than that in E−, indicating that the presence of endophytic fungi can regulate the change in NO 3 − content in host plants to maintain the ionic balance after alkaline stress. 4.3. Effect of Alkali Stress on HbNHX1 Expression in H. bogdanii Roots and Shoots Plants maintain ionic stability by controlling the expression of ion transporters. For example, many plants can help reduce Na + toxicity and affect the expression of Na + -related genes by symbiosis with fungi [ 51 ]. NHX1 is a key gene involved in plant alkaline tolerance. Overexpression of NHX1 has been shown to improve salt tolerance in many plants. The existence of beneficial fungus mycorrhizae affected the gene expression of host plants of Pakchoi ( Brassica campestris ssp. Chinensis ) under salt stress. Moreover, the expression level of NHX1 was higher in root tissues in inoculated plants under both saline and non-saline stress [ 52 ]. In this study, it was found that the expression of HbNHX1 was highest in the stems and leaves of E+ H. bogdanii treated with 100 mmol·L −1 alkaline, while the expression of HbNHX1 was lower in the stems and leaves treated with 50 mmol·L −1 , indicating that under high salt, the endophytic fungi promoted the host H. bogdanii to regionalize more Na + in the vacuoles, promoting the upregulation of HbNHX1 . Diao [ 53 ] found that Suaeda salsa inoculated with Arbuscular mycorrhizal downregulated the expression of SsNHX1 in roots under 100 mmol·L −1 NaCl. However, this study found that under alkaline stress, HbNHX1 expression increased in the roots of H. bogdanii with endophytic fungi, and the expression of HbNHX1 in E+ was significantly higher than that in E− under alkaline stress. The results demonstrated that endophytic fungi increased the expression of HbNHX1 in the roots of H. bogdanii , and that different fungi had different regulatory effects on plants."
} | 3,693 |
38481089 | PMC10938030 | pmc | 5,723 | {
"abstract": "Abstract Ascidians, known for their color variation, host species‐specific microbial symbiont communities. Some ascidians can also transition into a nonfiltering (resting) physiological state. Recent studies suggest that the microbial symbiont communities may vary across different physiological states and color morphs of the host. The colonial ascidian, Polyclinum constellatum , which exhibits several color morphs in the Caribbean Sea, periodically ceases its filtering activity. To investigate if color variation in P. constellatum is indicative of sibling speciation, we sequenced fragments of the ribosomal 18S rRNA and the mitochondrial cytochrome oxidase subunit I genes. Additionally, we sequenced a fragment of the 16S rRNA gene to characterize the microbial communities of two common color morphs (red and green) in colonies that were either actively filtering (active) or nonfiltering (resting). Phylogenetic analyses of both ascidian genes resulted in well‐supported monophyletic clades encompassing all color variants of P. constellatum . Interestingly, no significant differences were observed among the microbial communities of the green and red morphs, suggesting that color variation in this species is a result of intraspecific variation. However, the host's physiological state significantly influenced the microbial community structure. Nonfiltering (resting) colonies hosted higher relative abundances of Kiloniella (Alphaproteobacteria) and Fangia (Gammaproteobacteria), while filtering colonies hosted more Reugeria (Alphaproteobacteria) and Endozoicomonas (Gammaproteobacteria). This study demonstrates that microbial symbiont communities serve as reliable indicators of the taxonomic state of their host and are strongly influenced by the host's feeding condition.",
"introduction": "1 INTRODUCTION Ascidians or sea‐squirts (Chordata; Tunicata) are sessile, benthic invertebrates characterized by a husk‐like protective casing or tunic consisting of secreted protein–cellulose complexes and sulfated polysaccharides. Considerable variation in tunic coloration exists between ascidians, even among conspecific members (Evans, Erwin, et al., 2021 ; Hirose et al., 2009 ; López‐Legentil & Turon, 2005 ; Tarjuelo et al., 2004 ). Color plasticity has been attributed to the presence of different secondary metabolites or pigments (Hirabayashi et al., 2006 ; Turon et al., 2005 ), symbiotic bacteria (Hirose et al., 2009 ), and physical structures present within the tunic (spicules; Monniot et al., 1991 ). Studies have also shown that intraspecific color variation had a genetic basis for some species (Evans, Erwin, et al., 2021 ; Tarjuelo et al., 2004 ) but not others (López‐Legentil & Turon, 2005 ). Furthermore, even within species, some colors were unambiguously linked to particular lineages, while others were not (Evans, Erwin, et al., 2021 ; López‐Legentil & Turon, 2005 ; Tarjuelo et al., 2004 ). For instance, López‐Legentil & Turon, ( 2005 , 2006 ) found that some of the color plasticity described for the colonial ascidian Cystodytes dellechiajei corresponded to sibling species (i.e., blue and purple morphs), while others colors corresponded to intraspecies variation (e.g., blue, green, and white morphs). Similarly, Evans, Erwin, et al. ( 2021 ) described five color groupings for the colonial ascidian Distaplia bermudensis and found two distinct genetic lineages (A and B), each including 2 and 3 color groupings, respectively. Interestingly, Evans, Erwin, et al. ( 2021 ) also reported that each genetic lineage harbored a unique symbiont community. Like all animals, ascidians form symbiotic associations with a wide range of microbes (Erwin et al., 2014 ; Evans et al., 2017 ; Tianero et al., 2015 ). These microbial symbionts may aid in many biological processes, including adaptation to new habitats (Casso et al., 2020 ; Dror et al., 2019 ; Evans et al., 2017 ), secondary metabolite production for chemical defense (Tianero et al., 2015 ), and protection from ultraviolet solar radiation (Hirose et al., 2004 , 2006 ). Similar to sponges and other filter‐feeding invertebrates, ascidians acquire microbial symbionts both vertically and horizontally. Thus, some microbial symbionts are obtained directly from the progenitors (vertical transmission; Hirose, 2000 , 2015 ; Hirose & Nozawa, 2020 ; Hirose et al., 2005 ; López‐Legentil et al., 2011 , 2015 ; Martínez‐García et al., 2007 ) while others are obtained from the surrounding environment (horizontal transmission; Casso et al., 2020 ; Dror et al., 2019 ; Erwin et al., 2013 ; Goddard‐Dwyer et al., 2021 ). Despite the ability to accumulate environmentally sourced symbionts, adult ascidians harbor microbial communities that are highly host‐specific (Erwin et al., 2014 ; Evans et al., 2017 ; López‐Legentil et al., 2023 ) and at least in colonial ascidians, temporally stable (López‐Legentil et al., 2015 ). In addition, some colonial ascidians are known to alternate between actively filtering (active) and nonfiltering (resting, dormant, or topor) phases (Turon, 1992 ). In colonial stolidobranch (botryllids), feeding cessation occurs regularly (e.g. weekly) and consists of resorption of the parental generation and maturation of asexually derived buds (reviewed in Manni et al., 2019 ). In aplousobranchs, the resting phase involves the formation of an external glassy cuticle (a thin layer of superficial tunic tissue) covering the siphonal apertures (López‐Legentil et al., 2016 ; Turon, 1992 ). Internally, the filtering apparatus (branchial sac) is reabsorbed and colonies stop filtering for a few weeks (López‐Legentil et al., 2013 , 2016 ; Turon, 1992 ). This phenomenon allows for the animal's rejuvenation (Turon, 1992 ), the generation of new zooids after reproduction (Molin et al., 2003 ), or survival during periods of adverse conditions (López‐Legentil et al., 2013 ; Nakauchi, 1982 ; Pérez‐Portela et al., 2007 ; Turon, 1988 ; Turon & Becerro, 1992 ). Morphological and metabolic changes between active and resting forms also appeared to impact the composition of the symbiotic microbial communities. López‐Legentil et al. ( 2016 ) showed that although both filtering and resting colonies of the aplousobranch Pseudodistoma crucigaster maintained their core microbial symbionts, shifts in rare bacteria were detected in resting colonies, including the appearance of strictly anaerobic lineages, nitrifying bacterial guilds, and additional environmental microorganisms. Similarly, Hyams et al. ( 2023 ) found that the stolidobranch Botrylloides leachii harbored a resting‐specific microbiota. The colonial ascidian Polyclinum constellatum (Savigny 1816) was first described from the Bermudas and is commonly observed in the Caribbean Sea (Aydin‐Onen, 2018 ; Lambert, 2019 ; Rocha & Costa, 2005 ; Rocha et al., 2010 ; Streit et al., 2021 ). P. constellatum has also been observed in the Pacific, West Indian, and Atlantic oceans (from Canada to Brazil), South Africa, and the Mediterranean Sea (reviewed in Aydin‐Onen, 2018 , Virgili et al., 2022 ) and appears to be spreading rapidly, likely through anthropogenic vectors. Although a few barcode sequences of the cytochrome oxidase I gene (COI) are now available in GenBank (Montesanto et al., 2022 ; Streit et al., 2021 , Virgili et al., 2022 ), the species has been widely misidentified in the past (Montesanto et al., 2022 ), presumably due to multiple color morphs occurring in sympatry (Lambert, 2019 ; Montesanto et al., 2022 ; Rocha et al., 2010 ; Streit et al., 2021 , Virgili et al., 2022 ). In this study, we used ribosomal (18 S rRNA) and mitochondrial (COI) gene fragments to determine the phylogenetic relationships between common color morphs of P. constellatum collected in Puerto Rico (Caribbean Sea). We also characterized the genetic profile and microbial assemblages of the two common color morphs (red and green) and two physiological states of the host (actively filtering and resting). Our goal was to determine whether (1) the observed color morphs corresponded to different genetic lineages, (2) differences in microbial communities occurred between color morphs, and (3) differences in the physiological state of the host (filtering vs. resting) altered the microbial symbiont communities in P. constellatum .",
"discussion": "4 DISCUSSION Color variation in the colonial ascidian P. constellatum did not reveal any genetic lineages. Color has been an unreliable character for species identifications in some ascidians (Bancroft, 1903 ; Van Name, 1945 ), yet, in other cases, different colorations have been linked to sibling speciation (Evans, Erwin, et al., 2021 ; López‐Legentil & Turon, 2005 , 2006 ; Tarjuelo et al., 2004 ). Thus, the reliability of coloration in ascidian identification appears to be species‐specific. Ascidian taxonomists have few available characters to identify species, besides careful observation of the animal, its zooid morphology, and when possible, the larva. Current efforts focus on barcoding unambiguously identified species using mostly the mitochondrial COI gene (e.g., see GenBank, the Barcode of Life Data Systems). However, alternative methods are needed when gene amplifications and DNA sequencing fail. Chemotyping has rarely been employed in ascidians, although it was shown to be a reliable method to discern between sibling species (López‐Legentil & Turon, 2005 ). More recently, the characterization of microbial symbiont communities in species complexes has accurately resolved the taxonomic status of color variable species in ascidians (Evans, Erwin, et al., 2021 ) and other marine invertebrates (Evans, López‐Legentil, et al., 2021 ; Thacker & Starnes, 2003 ). Research herein corroborated these findings, as the red and green morphs of P. constellatum were phylogenetically indistinguishable and hosted similar microbial communities. The physiological state of the host (actively filtering or resting) did alter the composition of the microbial symbiont communities in P. constellatum . To our knowledge, only one previous study has characterized microbial symbiont communities in filtering versus resting ascidians (López‐Legentil et al., 2016 ). Similar to observations herein, microbial symbiont communities in the temperate colonial ascidian P. crucigaster shifted when the animal stopped filtering. Notably, López‐Legentil et al. ( 2016 ) reported the appearance of strictly anaerobic lineages, nitrifying bacterial guilds, and environmental bacteria associated exclusively with resting ascidian colonies. In our study, dissimilarity was driven by an increased relative abundance of the proteobacteria Kiloniella and Fangia in the resting forms and of Ruegeria and Endozoicomonas in the actively filtering colonies. The genus Kiloniella is formed by mesophilic, chemoheterotrophic marine bacteria with an aerobic or facultatively anaerobic (nitrate as electron acceptor) metabolism (Wiese et al., 2009 ). The observed shift in percent mean abundance of Kiloniella (OTU 2) from 2.3% in filtering colonies to 21.4% in resting colonies supports its putative ability to grow at variable oxygen concentrations resulting from filtering cessation. In addition, the ability of Kiloniella species to reduce nitrate to nitrite suggests an important role in nitrogen metabolism (Wiese et al., 2009 ). OTU 10 occurred exclusively in resting colonies and the sequences obtained herein were 100% identical to Fangia hongkongenesis , an aerobic Gammaproteobacteria isolated from coastal seawater in Hong Kong (Lau et al., 2007 ). Thus, similar to López‐Legentil et al. ( 2016 ) observations, when feeding cessation occurs in P. constellatum , facultative anaerobes related to nitrogen metabolism thrive, and environmental bacteria not observed in filtering colonies can colonize the animal. In actively filtering colonies, the relative abundance of the alphaproteobacterium Ruegeria (OTU 1) and gammaproteobacterium Endozoicomonas (OTU 8) were higher than in resting colonies. Except for one species, all members of the genus Ruegeria are marine, and several species have been isolated from marine invertebrates (Menezes et al., 2010 ). All species of Ruegeria are chemoorganotrophic aerobes and thus a decrease in the relative abundance of this symbiont could be related to decreasing oxygen concentrations in the ascidian host. Endozoicomonas are conspicuous chemoorganoheterotrophic Gammaproteobacteria found in a wide range of animal hosts, including coral, sponges, and fish (reviewed in Neave et al., 2016 ). In ascidians, Endozoicomonas are also prevalent (Galià‐Camps et al., 2023 ; Hyams et al., 2023 ; Schreiber et al., 2016 ) and phylogenetic analyses of 16S rRNA genes have revealed an ascidian‐specific clade (Schreiber et al., 2016 ). Members of the same ascidian‐specific Endozoicomonas clade were also detected in seawater, albeit at much lower abundances, suggesting a facultative symbiosis (Schreiber et al., 2016 ). Further analysis revealed the presence of Endozoicomonas microcolonies on the branchial sac of most of the ascidian species investigated (Schreiber et al., 2016 ). In addition, resting colonies of B. leachii were enriched with a novel lineage of Endozoicomonas that appear to occupy specific hemocytes found exclusively in resting animals (Hyams et al., 2023 ). Here, we observed a decrease in the relative abundance of Endozoicomonas in the resting forms of P. constellatum . This decrease was likely due to the observed reduction of the host branchial sac that results from feeding cessation and where Endozoicomonas are typically found since no resting‐specific Endozoicomonas lineage was detected. All in all, the physiological state of the host appears to play an important role in determining microbial symbiont composition, with the appearance of a cuticle over the resting colonies and modified oxygen concentrations appearing to be the main factors driving the observed shifts. In conclusion, our study revealed that color variation observed across colonies of P. constellatum did not correspond to distinct genetic lineages and that different color morphs hosted similar microbial symbiont communities. Thus, data to date indicate that conspecific ascidians harbor similar microbial communities where color variants represent the same species (e.g., P. constellatum , herein) and divergent symbiont communities where they represent distinct host lineages (e.g., D. bermudensis , Evans, Erwin, et al., 2021 ). Furthermore, shifts in microbiome structure were detected in actively filtering versus resting colonies of P. constellatum , indicating that changes in the microenvironment of the host occurred with filtering cessation and altered symbiont composition, with possible functional consequences for host–symbiont interactions (e.g., nitrogen cycling). Understanding the microbiome in relation to the holobiont physiological state is key to unlocking the largely untapped potential of microbes to aid their hosts with unique metabolic products and activities."
} | 3,795 |
39705366 | PMC11661455 | pmc | 5,724 | {
"abstract": "Numerous organisms exploit asymmetrical capillary forces generated by unique fiber or asymmetrical tapered structures to rapidly eliminate undesired liquid for survival in moist or rainy habitats. Human eyelashes, the primary protector of eyes, use a yet-to-be-fully-understood mechanism to efficiently transfer incoming liquid for vision safeguarding. Here, we elucidate that human eyelashes featuring a hydrophobic curved flexible fiber array with surface micro-ratchet and macro-curvature approximating the Brachistochrone is adept at directionally and rapidly expelling incoming liquid to maintain clear vision. These structural attributes are sequentially used for liquid drainage, starting from anisotropic retention via micro-ratchet, followed by the elastic expulsion among deflected hydrophobic flexible fiber arrays and culminating in the fastest sliding off along a Brachistochrone path, which together reduce the contact time by about 20% of that on rigid linear slopes. Investigating the intricate relationship between multistructure and draining efficiency of human eyelashes may inspire the design of advanced liquid-repelling edges on outdoor devices to maintain dryness.",
"introduction": "INTRODUCTION The need for efficient liquid drainage is evident across a wide range of applications, including surface cleaning ( 1 – 3 ), anti-adhesion ( 4 – 7 ), heat transfer enhancement ( 8 , 9 ), wearable electronics ( 10 , 11 ), moisture management ( 12 ), energy generation ( 13 ), as well as the survival of natural organisms ( 14 – 18 ). For instance, water striders effortlessly remain atop the water surface under humid conditions by using their legs, which are covered by microsetae arrays with nanogrooves ( 16 ), for self-removal of condensed water droplets ( 17 ). Similarly, the drain fly maintains dryness in moist environments through the directional movement of condensed droplets along its flexible tentacles, equipped with periodic micro-ratchet ( 15 ). Certain plants enhance water shedding by adopting leaf structures with concave-convex curvatures ( 14 ), while dogs dry themselves by shaking to cluster their wet fur ( 18 ). Drawing inspiration from these natural systems, a variety of artificial devices has been devised, providing substantial advantages in applications such as floating robotics ( 19 ), anticondensation ( 20 ), atomization ( 21 ), underwater aerobic reaction ( 12 ), etc. Nevertheless, research has predominantly concentrated on the dynamics of liquid expelling, breaking, shedding, transferring, and sliding on an individual fiber ( 22 , 23 ), an apex ( 14 ), a cone ( 24 ), within two cones ( 16 , 25 ), a bunch of fibers ( 18 , 26 , 27 ), continuous planar geometries ( 28 ), etc. So far, the sliding and expelling dynamics of liquid on a parallel discontinuous flexible fiber array and the effect on draining direction or time have been scarcely investigated. As human evolution progresses, our body and facial hair have notably reduced, yet the eyelashes remain a distinguishing feature ( 29 ). The physiological or functional rationale behind the presence of eyelashes, traditionally speculated to serve roles like catching dust ( 30 ) or filtering air ( 31 ), has long been debated. The hydrodynamic advantages of eyelashes, particularly how they expel undesired liquid away from the eye to preserve vision clarity, have been seldom explored. For instance, during facial washing or intense physical activity, the eyes are exposed to significant water or sweat without being compromised. Here, we demonstrate that human eyelashes composed of a hydrophobic flexible fiber array with multifaceted integration of structures from surface micro-rachet to macro-curvature favor directionally and rapidly expelling undesired incoming liquid. The hydrophobic flexible fibers deflect outward to construct a wedge that expels droplets away, significantly reducing contact time compared to rigid surfaces. Furthermore, experiments demonstrate that droplets slide off more rapidly along the curved Brachistochrone ( Brach .) than along linear paths. A deeper understanding of the interaction between structural and functional aspects of eyelashes could guide the thoughtful design of innovative drainage solutions, including aesthetically pleasing and protective false eyelashes, waterproof imaging, and ventilated devices.",
"discussion": "DISCUSSION The historical use of eyelash adornments dates back to 4000 BCE in ancient Egypt ( 45 ). Nowadays, people, especially women, pay increasing attention to using eyelash adornments, reflecting a long-standing interest in enhancing personal attractiveness through such means. In 2023, mascara sales in the United States nearly reached $1008.9 million ( 46 ). Previous experiments reveal that the hydrophobicity and the downward-inclined curved flexible fiber array structure of natural eyelashes endow them with functional superiority in water drainage. Despite this, modern beauty standards often encourage practice such as clipping, rolling, or even perming eyelashes to obtain an upward curvature ( Fig. 4A , left, and movie S4) and using hydrophilic mascara ( Fig. 4A , left) to extend and fix eyelashes. It compromises the protective functions due to covering or damaging the scale-like cuticle ( Fig. 4A , left) on eyelashes. As a result, frequent makeup smudging occurs because of the hydrophilic properties of mascara and the improper curvature of eyelashes when exposed to sweat, rain, or tears during blinking. The downward inclined and naturally curved infants’ eyelashes ( Fig. 4A , middle) suggest an inherent design optimized for both protection and aesthetic appeal. So as a tip, for people with sparse eyelashes, hydrophobic curved false eyelashes ( Fig. 4A , right) could offer a practical solution for enhancing appearance while preserving eye protection. Fig. 4. Application assumptions of human eyelash-mimetic drainage edges. ( A ) Image shows a female volunteer using mascara to comb her eyelashes from downward to upward inclined. A contact angle image reveals the hydrophilicity of the mascara. SEM images reveal that the coated mascara covers the micro-ratchet structure of the eyelash (left). Image showing the downward inclined and naturally curved eyelash of an infant volunteer, whose curvature approximates the Brach. (middle). Composite snapshots show the effective water drainage on the hydrophobic Brach. false eyelash (right). ( B ) Schematics show the design of eyelash-mimetic drainage edges for waterproofing in the lens of unmanned flight. ( C ) Images show the drainage process of continuous water flow by the eyelash-mimetic fiber array, where the imaging is misty and clear without and with fibers, respectively. Inset SEM image reveals the surface structure of the 3D-printed eyelash-mimetic fiber array. ( D ) Schematics show the design of eyelash-mimetic ventilated and waterproof windows. ( E ) Images show air passes through, but water is blocked. This understanding of eyelash structure has broader implications, inspiring the design of innovative drainage solutions across various applications. For instance, the principles observed could inform the creation of waterproof lenses ( Fig. 4, B and C ; figs. S19 and S20; and Materials and Methods) for unmanned flights, ensuring clear vision and imaging capabilities, or the development of ventilative, waterproof filter screens for outdoor electrical boxes ( Fig. 4, D and E ), enhancing heat transfer and protecting against moisture. As shown in Fig. 4 (C and E) , the water jet, angled at approximately 45°, is directed toward the lens or the electrical box. Without the eyelash-mimetic fiber array as a shield, the jet would directly affect and wet the lens, resulting in misty imaging. With the eyelash-mimetic curved fiber array in place, the direction of the liquid is altered first, and then it is expelled in the fastest manner. As shown in fig. S19, the pupil plays a role in an eye similar to the aperture in a camera. Therefore, the length of the eyelashes should be such that neither obstruct the light entry nor compromise drainage performance. In addition, as reported in a previous work, a fiber whose length is one-third the width of the lens provides the best protection against dust ( 31 ). Therefore, to optimize performance by considering both waterproofing and dust prevention, we can design a fiber array with a length equal to one-third of the lens width and a curvature corresponding to the Brach . under the condition that the fiber array does not obstruct light."
} | 2,138 |
38373845 | PMC10913062 | pmc | 5,725 | {
"abstract": "Abstract Community assembly is influenced by environmental niche processes as well as stochastic processes that can be spatially dependent (e.g. dispersal limitation) or independent (e.g. priority effects). Here, we sampled senesced tree leaves as unit habitats to investigate fungal community assembly at two spatial scales: (i) small neighborhoods of overlapping leaves from differing tree species and (ii) forest stands of differing ecosystem types. Among forest stands, ecosystem type explained the most variation in community composition. Among adjacent leaves within stands, variability in fungal composition was surprisingly high. Leaf type was more important in stands with high soil fertility and dominated by differing tree mycorrhizal types (sugar maple vs. basswood or red oak), whereas distance decay was more important in oak-dominated forest stands with low soil fertility. Abundance of functional groups was explained by environmental factors, but predictors of taxonomic composition within differing functional groups were highly variable. These results suggest that fungal community assembly processes are clearest for functional group abundances and large spatial scales. Understanding fungal community assembly at smaller spatial scales will benefit from further study focusing on differences in drivers for different ecosystems and functional groups, as well as the importance of spatially independent factors such as priority effects.",
"conclusion": "Conclusions The feasibility of sampling entire, discrete habitat patches with their relative spatial positions has led us to understand that there can be enormous spatial heterogeneity in leaf litter fungal communities over small scales of a few centimeters, as well as at previously recognized regional scales spanning hundreds of kilometers. This heterogeneity is influenced by a variety of processes, both environmental and stochastic. We suggest that further progress in understanding fungal community assembly processes will benefit from hierarchical explorations of fungal functional groups, because mixing functional groups can lead to confounding patterns. In addition, further exploration of priority effects in the context of classic environmental niche-based and dispersal-based metacommunity mechanisms will be critical to explore. Importantly, insights into leaf litter decay are often gained at an ecosystem-level spatial scale, which aggregates across the fine-scale variability in fungal community composition and function that occurs at the scale of adjacent leaves. We suggest that a fuller understanding of the factors structuring fungal communities at fine spatial scales can bring a better understanding to leaf litter decay at an ecosystem-level spatial scale.",
"introduction": "Introduction Processes that drive community assembly range from selection of species by local environmental conditions to stochastic variation in species colonization (Hanson et al. 2012 ). Although these broad groups of hypothesized mechanisms are often presented as alternatives, most communities will not be influenced by only environmental or stochastic factors, leaving community ecologists with the challenge of understanding where a community falls in this spectrum (Chase and Myers 2011 , Zhou and Ning 2017 ). Despite their many key roles in ecosystem processes, understanding mechanisms of fungal community assembly has proven particularly challenging due to high levels of stochasticity (Powell et al. 2015 , Bahram et al. 2016 , Peguero et al. 2022 ). Previous observations of fungal communities have indeed supported both environmental selection and stochastic processes (Peay et al. 2016 ), indicating that they may operate simultaneously or that their relative strengths may depend on other factors such as dispersal frequency and ecosystem characteristics. Saprotrophic fungal communities vary between forest stands dominated by differing tree species (Urbanova et al. 2015 ), in part due to contrasting senesced leaf litter chemistry (Schneider et al. 2012 ). This may also occur at the level of individual leaves, with fungal community composition depending upon leaf types (Aneja et al. 2006 , Kurbatova et al. 2009 , Zhang et al. 2020 ). However, stochastic mechanisms, that are independent of environmental factors, may also play an important role in fungal community assembly. Priority effects occur when initial colonists affect the performance of subsequent colonizers (Debray et al. 2022 ), and has been demonstrated in fungal community assembly in several environments (Kennedy et al. 2009 , Fukami et al. 2010 ). If dispersal ranges are not limited (i.e. proximity to source populations does not affect the probability of dispersal), strong priority effects could result in simultaneous independence from environmental conditions and lack of spatial patterns. However, if dispersal ranges are limited, patchy spatial patterns can emerge that are independent of environmental conditions. For example, Feinstein and Blackwood ( 2013 ) examined fungal communities of individual leaves found together in 20 cm × 20 cm patches on the forest floor and discovered a distance–decay pattern independent of leaf type. Spatial patterns consistent with this scenario have also been found, at various spatial scales, in bulk soil fungal communities (Green et al. 2004 , Zhao et al. 2019 , Peguero et al. 2022 ) and in ectomycorrhizal and leaf endophytic communities (Peay et al. 2010 , Bowman and Arnold 2021 , Cook et al. 2022 ). Although it is clear that fungal communities are affected simultaneously by environmental niche relationships and stochastic processes, the drivers of this mixed community assembly process require further investigation. One explanation for patterns in fungal community composition being consistent with multiple community assembly processes is that fungal communities are comprised of multiple functional groups. These groups are responsible for different biological interactions and ecosystem functions [i.e. necrotrophic and biotrophic plant pathogens, mycoparasites, mycorrhizae, endophytes, yeasts, wood degraders, and primary saprotrophs (nonwood plant tissue degraders)] (van der Heijden et al. 2008 , Zanne et al. 2020 ). The different functional groups have distinct environmental requirements for growth and survival, which should result in strong environmental niche selection at the levels of whole functional groups (i.e. comparisons among functional groups) (Bahram et al. 2016 , Schröter et al. 2019 , Masumoto et al. 2023 ). For instance, fungi that can break down recalcitrant compounds (e.g. wood degraders) are expected to be more prevalent on plant material with a higher lignin content (Rajala et al. 2012 ). Yeasts are thought to be tolerant of desiccation and nutrient depletion and should, therefore, have higher relative abundance in environments characterized by these conditions (Treseder and Lennon 2015 ). In contrast, stochastic factors may be particularly strong within functional groups because species in the same functional group should have similar niche requirements. This functional equivalency may allow species within a functional group to vary independently from the environment, depending on their modes of dispersal, even while the overall abundance of the group is constrained (Schröter et al. 2019 , Masumoto et al. 2023 ). We reasoned that groups that disperse and spread slowly (i.e. wood rot fungi and yeasts) or that have patchier distributions (mycorrhizal fungi) should be more influenced by dispersal limitation (Belisle et al. 2012 , Horn et al. 2014 , Peay and Bruns 2014 ). Necrotrophic plant pathogens and primary saprotrophs are prolific spore producers and have low environmental specificity (Brown and Hovmoller 2002 , van Kan 2006 , Halbwachs et al. 2015 ), and thus may largely be influenced by priority effects without a strong spatial pattern. On the other hand, endophytes and biotrophs should be structured by biological environmental factors, such as leaf type or host, due to their high degree of environmental specificity (Spanu and Kamper 2010 , Wearn et al. 2012 ). The importance of environmental selection, dispersal limitation, or priority effects in community assembly may also be influenced by spatial scale (Green and Bohannan 2006 , Martiny et al. 2011 , Horn et al. 2015 ). Some mechanisms may not be recognized if they are occurring at a larger or smaller scale than the one under investigation, although they still may play a significant role in assembly processes. Scale dependence in community assembly has been demonstrated several times in soil fungal communities (Green et al. 2004 , Pellissier et al. 2014 ). At regional and continental scales, soil fungal community similarity is usually associated with environmental differences (Zhao et al. 2019 , Zheng et al. 2021 ). For soil fungal communities within individual sites, distance decay analysis often shows spatial autocorrelation up to a few meters in bulk soil, whereas at larger distances community similarity appears primarily stochastic or weakly correlated with environmental variables (Bahram et al. 2016 , Zhao et al. 2019 , Peguero et al. 2022 ). Likewise, Feinstein and Blackwood ( 2013 ) found that, at the scale of individual leaves, fungal community similarity decreased as distance between leaves increased, but at the scale of ecosystems, environmental factors were more important. This study was performed to better understand how deterministic and stochastic factors influence saprotrophic fungal communities at both individual leaf and forest stand spatial scales. We focused on individual senesced leaves as natural sampling units due to their discrete nature as distinct resource patches with independent histories. We tested the following hypotheses: H1—At small scales (between adjacent leaves), overall fungal community composition is influenced by both environmental selection (through leaf type) and dispersal limitation/priority effects. H2—In contrast, at larger spatial scales (between forest stands), community composition is more influenced by environmental selection (through ecosystem type). H3—Relative abundances of fungal functional groups will be strongly structured by environmental factors, including ecosystem and leaf type, but not by spatial effects. H4—In contrast, the importance of factors influencing taxonomic composition within each functional group may include dispersal limitation, depending on the dominant dispersal mechanisms in each functional group. To test these hypotheses, leaves were gathered from well-characterized ecosystems in the northern lower peninsula of Michigan. Individual leaves with differing chemical properties were collected after mapping their locations on the forest floor. Fungal communities were characterized using two strategies: terminal restriction fragment length polymorphism (T-RFLP) analysis was used to achieve high-replication of individual leaf communities, and pyrosequencing was used to obtain high-resolution taxonomic profiles and functional characterization of a more limited number of communities.",
"discussion": "Discussion The dominant assembly mechanisms influencing fungal communities are often difficult to discern from spatial patterns because both stochastic and environmental factors are known to play a role (Powell et al. 2015 , Peguero et al. 2022 ). Despite sampling individual leaves as distinct habitat patches, we found that stochastic variation independent from distance decay (i.e. unexplained variation) was consistently strong, as noted by others for fungi in other habitats (Cook et al. 2022 , Bahram et al. 2016 ). Surprisingly, even pairs of leaves that were physically touching did not share notably similar fungal communities, perhaps resulting from priority effects, leaf history, or other unmeasured environmental variability. However, we did find changes in the importance of distance decay and environmental factors according to ecosystem type, spatial scale, and how fungi are treated with respect to functional groups. Environmental factors were particularly important at large spatial scales and for functional group abundances, whereas distance decay had significant effects on taxonomic composition in particular ecosystems and for particular functional groups. Thus, our results point to the importance of considering functional redundancy, environmental heterogeneity, and priority effects in understanding variation in mechanisms of fungal community assembly. Deterministic and stochastic mechanisms influence community assembly of fungal taxa Fungal communities residing in decomposing leaves are often expected to respond strongly to the plant species identity of the leaves because of differences in available resources, secondary chemistry, and the immediate physical environment for growing mycelia (Osono 2007 , Prescott and Grayston 2013 ). However, we were surprised to find that leaf type had a relatively small and inconsistent effect on the taxonomic composition of resident fungal communities. This may imply that many fungi in forest ecosystems are adapted to efficiently utilize any leaf litter that is available every year, as would be the case from multiple dominant tree species within a forest stand. Ecosystem type, on the other hand, had a strong effect on fungal community composition, even though the impacts of ecosystem type on leaf litter fungi could be expected to be more diffuse than the impacts of a particular leaf. The ecosystem types we studied differ on many environmental axes, including contrasting soil types, moisture availability, and plant community members (Host et al. 1988 ). These environmental factors structure fungal communities in other studies (Brockett et al. 2012 , Prescott and Grayston 2013 ), and may physiologically limit fungal communities more than does leaf chemistry, constraining the pool of fungi available to colonize new senesced leaves. The study of metacommunities has begun to focus on the conditions that cause shifts community assembly processes between stochastic and deterministic factors (Chase and Myers 2011 , Zhou and Ning 2017 ). Here, we found that, at the scale of adjacent leaves, differences among ecosystem types could help explain the relative importance of dispersal limitation and niche effects on fungal community composition. Although the effect of leaf type was not as strong as expected overall, the effect was strongest in the SMBW stands, which has the highest soil fertility and most labile leaves. More abundant resources may lead to stronger environmental filtering through both increased production of propagules, which should reduce dispersal limitation, and rapid fungal growth and competition, increasing sensitivity to the leaf litter environment. In addition, sugar maple leaves are likely more biochemically distinct from basswood and red oak leaves than black and white oak leaves are from each other. Sugar maple leaves are known to be biochemically less recalcitrant than red oak leaves (Gallo et al. 2004 ), and sugar maple is an arbuscular mycorrhizal species, rather than mostly ectomycorrhizal like all the other focal trees (Tedersoo and Brundrett 2017 ). Arbuscular mycorrhizal tree leaves are typically less recalcitrant (Cornelissen et al. 2001 ) and lead to fungal communities that are distinct from those associated with ectomycorrhizal species (Bahram et al. 2020 , Eagar et al. 2023 ). Leaf depth within the forest floor was also investigated, but accounted for a negligible amount of variation, possibly because the sites investigated were all upland forests. Although Feinstein and Blackwood ( 2013 ) found that depth in the forest floor was important in explaining fungal community at sites in Ohio, USA, this was especially consistent in forested wetlands, whereas depth was only significant in one of their two upland sites. Distance–decay relationships were frequently found within BOWO or SMRO stands (7 out of 12 stands across April and August, Table 1 ), but not in SMBW stands (one out of six stands). Although we could not test this in the current study, the more recalcitrant nature of oak leaves should result in slower fungal growth rates due to a need for increased investment in extracellular enzyme production (Gallo et al. 2004 , Moorhead and Sinsabaugh 2006 , Osono 2007 ), favoring stronger competitors that are slower to disperse and have patchier species distributions (Kneitel and Chase 2004 ). Colonization between neighboring leaves may also be favored because resources are physically separated in this environment, and cytoplasmic transport through a hyphal network can integrate resource patchiness for many saprotrophic fungi (Bielčik et al. 2019 ) Recent conceptual developments have highlighted the need to more directly incorporate effects of species interactions in metacommunity ecology to fully account for both possible patterns and known mechanisms in community assembly (Leibold et al. 2022 ). Here, despite finding significant effects of ecosystem type, leaf proximity, and leaf type on fungal community composition, a great deal of variation was still left unexplained, implying the presence of strong, spatially unstructured priority effects (Johnson 2015 ). Priority effects have been experimentally documented in wood saprotrophic communities, in which inoculation of specific early colonists can affect future fungal colonizers for prolonged periods of time (Weslien et al. 2011 ) through generation of secondary metabolites, release of nutrients, and niche preemption (Heilmann-Clausen and Boddy 2005 , Fukami 2015 ). In leaf litter, priority effects may begin before leaf senescence even occurs, because endophytic and necrotrophic fungi can shift lifestyles and act as saprotrophs (Osono 2006 , Song et al. 2017 ). Coupled with stochastic movement of individual leaves, these priority effects could lead to the low similarity we observed between fungal communities of leaves even in direct contact with one another. Given the apparent importance of priority effects, and the possibility that outcomes are impacted by conditions such as temperature and plant tissue biochemistry (Hiscox et al. 2016 ), we suggest that this is an important area for further research. Differing community assembly processes among and within functional groups Although species can differ from each other on many niche axes (Rosenfeld 2002 ), simplification of fungal taxa to functional groups has been hypothesized to improve predictions about fungal community assembly (Peay et al. 2016 , Schröter et al. 2019 ). Our study supports this idea, because functional group abundances were better explained overall, and more strongly determined by leaf type and ecosystem than were taxonomic abundances. Moreover, the small-scale spatial factor we investigated (leaf proximity) had no influence on relative abundances of functional groups, despite their importance for taxonomic community composition. This indicates that environmental selection acts to assemble combinations of functional traits based on niches available in the environment, and that the species identity of the organisms filling each niche is less important. Plant pathogens and fungal endophytes were present in large abundances despite leaf senescence 6–12 months before sampling, indicating that these functional groups likely persist in leaf litter as important facultative saprotrophs and reinforcing the idea that priority effects are a key mechanism affecting fungal community assembly. However, relic DNA can also persist in the environment and lead to a snapshot of communities in the past (Lennon et al. 2018 ). Plant pathogens were more prevalent on leaves from the arbuscular mycorrhizal tree sampled (sugar maple) than from the ectomycorrhizal trees (black oak, white oak, and basswood), consistent with observations in other systems (Bahram et al. 2020 , Eagar et al. 2023 ). Unsurprisingly, ectomycorrhizal fungi were found to be most abundant in the ecosystem with the highest abundance of ectomycorrhizal trees, BOWO. Mycoparasites/yeasts and lichens were also notably higher on BOWO leaves, possibly due to water limitation in this ecosystem and their overall resistance to desiccation (Kranner et al. 2008 , Treseder and Lennon 2015 ). Within functional groups, community composition was associated with differing explanatory factors for differing functional groups, in support of H4. Two major categories emerged. Composition within some functional groups was associated with the environmental factors ecosystem and leaf type (mycoparasites/yeasts, necrotrophic plant pathogens, primary saprotrophs, and endophytes), indicating response of specific taxa within these groups to environmental conditions or plant host species. Composition of other functional groups was associated more with forest stand (ectomycorrhizal fungi, lichens, and white rot saprotrophs), possibly consistent with dispersal limitation mechanisms. These findings are consistent with some of our expectations, except that mycoparasites/yeasts were found to be more sensitive to the environment than anticipated, and there was greater dispersal limitation indicated for primary saprotrophs. These results demonstrate that separate groups of fungi are difficult to group together in ecological studies, as they are impacted by different community assembly mechanisms to varying degrees and therefore have confounding patterns (Schröter et al. 2019 , Masumoto et al. 2023 ). Taxonomic composition of fungal communities Ascomycete sequences dominated almost all leaves in all ecosystems tested. This dominance has previously been observed on senesced leaves and can be explained by the presence of Ascomycete endophytes, necrotrophs, and primary saprotrophs and the abundance of labile compounds promoting the growth of fast-growing early colonizing fungi (Schneider et al. 2012 , Urbanova et al. 2015 ). However, some ascomycetes also can produce enzymes capable of breaking down recalcitrant compounds seen in more decomposed litter (Osono 2007 , Gacura et al. 2016 , Fillat et al. 2017 ). Dominance of fungal order Helotiales on most leaves is consistent with a highly diverse fungal order including major groups of necrotrophic plant pathogens, cellulose decomposers, and early leaf litter colonizers (Lindahl et al. 2007 , Purahong et al. 2016 ). Other prevalent groups of Ascomycete fungi found include the orders Hypocreales and Capnodiales, which also include endophytes, plant pathogens, and saprotrophs (Rehner and Samuels 1995 , Crous et al. 2009 ). The highest abundance genus identified was Mycoarthris ( Table S1 , Supporting Information ), which is often found in soil and decomposing plant material (e.g. Baldrian et al. 2012 , Neupane et al. 2021 ), but remains poorly characterized. Basidiomycetes were more prevalent on recalcitrant leaf litter (BOWO leaves). This was expected as they can be more prevalent on leaf litter of lower nutrient quality (Voriskova and Baldrian 2013 ) and include groups that break down recalcitrant plant cell wall components such as cellulose and lignin (Baldrian and Valakova 2008 , Lundell et al. 2010 ). However, we were surprised to find that yeasts from the class Tremellomycetes were the most prevalent group of Basidiomycetes, instead of basidiomycete classes containing species known as specialists in degradation of recalcitrant compounds, such as Agaricomycetes. Soil yeasts have resistant dormant stages and can survive in stressful environments, such as those with frequent desiccation and low productivity (Treseder and Lennon 2015 ). Their prevalence in BOWO ecosystems may, therefore, be due to the very sandy soil and low moisture availability. Conclusions The feasibility of sampling entire, discrete habitat patches with their relative spatial positions has led us to understand that there can be enormous spatial heterogeneity in leaf litter fungal communities over small scales of a few centimeters, as well as at previously recognized regional scales spanning hundreds of kilometers. This heterogeneity is influenced by a variety of processes, both environmental and stochastic. We suggest that further progress in understanding fungal community assembly processes will benefit from hierarchical explorations of fungal functional groups, because mixing functional groups can lead to confounding patterns. In addition, further exploration of priority effects in the context of classic environmental niche-based and dispersal-based metacommunity mechanisms will be critical to explore. Importantly, insights into leaf litter decay are often gained at an ecosystem-level spatial scale, which aggregates across the fine-scale variability in fungal community composition and function that occurs at the scale of adjacent leaves. We suggest that a fuller understanding of the factors structuring fungal communities at fine spatial scales can bring a better understanding to leaf litter decay at an ecosystem-level spatial scale."
} | 6,280 |
29132003 | null | s2 | 5,726 | {
"abstract": "Direct revegetation, or phytostabilization, is a containment strategy for contaminant metals associated with mine tailings in semiarid regions. The weathering of sulfide ore-derived tailings frequently drives acidification that inhibits plant establishment resulting in materials prone to wind and water dispersal. The specific objective of this study was to associate pyritic mine waste acidification, characterized through pore-water chemistry analysis, with dynamic changes in microbial community diversity and phylogenetic composition, and to evaluate the influence of different treatment strategies on the control of acidification dynamics. Samples were collected from a highly instrumented one-year mesocosm study that included the following treatments: 1) unamended tailings control; 2) tailings amended with 15% compost; and 3) the 15% compost-amended tailings planted with Atriplex lentiformis. Tailings samples were collected at 0, 3, 6 and 12months and pore water chemistry was monitored as an indicator of acidification and weathering processes. Results confirmed that the acidification process for pyritic mine tailings is associated with a temporal progression of bacterial and archaeal phylotypes from pH sensitive Thiobacillus and Thiomonas to communities dominated by Leptospirillum and Ferroplasma. Pore-water chemistry indicated that weathering rates were highest when Leptospirillum was most abundant. The planted treatment was most successful in disrupting the successional evolution of the Fe/S-oxidizing community. Plant establishment stimulated growth of plant-growth-promoting heterotrophic phylotypes and controlled the proliferation of lithoautotrophic Fe/S-oxidizers. The results suggest the potential for eco-engineering a microbial inoculum to stimulate plant establishment and inhibit proliferation of the most efficient Fe/S-oxidizing phylotypes."
} | 469 |
29132003 | null | s2 | 5,727 | {
"abstract": "Direct revegetation, or phytostabilization, is a containment strategy for contaminant metals associated with mine tailings in semiarid regions. The weathering of sulfide ore-derived tailings frequently drives acidification that inhibits plant establishment resulting in materials prone to wind and water dispersal. The specific objective of this study was to associate pyritic mine waste acidification, characterized through pore-water chemistry analysis, with dynamic changes in microbial community diversity and phylogenetic composition, and to evaluate the influence of different treatment strategies on the control of acidification dynamics. Samples were collected from a highly instrumented one-year mesocosm study that included the following treatments: 1) unamended tailings control; 2) tailings amended with 15% compost; and 3) the 15% compost-amended tailings planted with Atriplex lentiformis. Tailings samples were collected at 0, 3, 6 and 12months and pore water chemistry was monitored as an indicator of acidification and weathering processes. Results confirmed that the acidification process for pyritic mine tailings is associated with a temporal progression of bacterial and archaeal phylotypes from pH sensitive Thiobacillus and Thiomonas to communities dominated by Leptospirillum and Ferroplasma. Pore-water chemistry indicated that weathering rates were highest when Leptospirillum was most abundant. The planted treatment was most successful in disrupting the successional evolution of the Fe/S-oxidizing community. Plant establishment stimulated growth of plant-growth-promoting heterotrophic phylotypes and controlled the proliferation of lithoautotrophic Fe/S-oxidizers. The results suggest the potential for eco-engineering a microbial inoculum to stimulate plant establishment and inhibit proliferation of the most efficient Fe/S-oxidizing phylotypes."
} | 469 |
33097740 | PMC7584646 | pmc | 5,728 | {
"abstract": "Of the 7–8 silk fibers making up an orb-web only the hierarchical structural organization of semicrystalline radial fibers -composed of major ampullate silk- has been studied in detail, given its fascinating mechanical features. While major ampullate silk’s nanofibrillar morphology is well established, knowhow on mesoscale (> 50–100 nm) assembly and its contribution to mechanical performance is limited. Much less is known on the hierarchical structural organization of other, generally less crystalline fibers contributing to an orb-webs’ function. Here we show by scanning X-ray micro&nanodiffraction that two fully amorphous, fine silk fibers from the center of an orb-web have different mesoscale features. One of the fibers has a fibrillar composite structure resembling stiff egg case silk. The other fiber has a skin–core structure based on a nanofibrillar ribbon wound around a disordered core. A fraction of nanofibrils appears to have assembled into mesoscale fibrils. This fiber becomes readily attached to the coat of major ampullate silk fibers. We observe that a detached fiber has ripped out the glycoprotein skin-layer containing polyglycine II nanocrystallites. The anchoring of the fiber in the coat suggests that it could serve for strengthening the tension and cohesion of major ampullate silk fibers.",
"conclusion": "Conclusions Our results suggest that even highly amorphous spider silk fibers can contain specific mesoscale features suggesting functional differences. Indeed, we have identified two types of amorphous, fine silk fibers by their mesoscale organizations. The fibrillar composite structure of am 1 fibers derived from modeling reciprocal space features in microXRD patterns appears providing enhanced stiffness as for egg case silk fibers although we do not have evidence that both types of fibers have the same glandular origin. The ribbon of weakly interacting nanofibrils wound around a core of disordered protein of am 2 fibers observed in real space by scanning nanoXRD resembles the skin–core structure of MaS fibers 8 suggesting a blend of toughness and flexibility. The results suggest that am 2 fibers can be anchored in the glycoprotein skin-layer of MaS fibers enabling functions related to fine-tuning the tension of the radial threads or increasing the lateral cohesion of bridge-thread fibers. It is probable that am 2 -type fibers and other fibrous surface features have the same glandular origin as MaS fibers protein skin-layer suggesting that care is required when interpreting surface-sensitive spectroscopy and imaging techniques. The evolution of nanoXRD towards sub-100 nm focal spots and enhanced reciprocal space resolution enabled by the upgraded ESRF source 17 , 43 should allow refining the am 1 fiber model by imaging mesofibrils in real space and resolving a possible meridional correlation peak. Probing of fiber sections with smaller focal spots should allow refining the proposed partial assembly of nanofibrils in the am 2 fibers skin-layer into mesofibrils. It will also be necessary developing protocols for transferring delicate orb-web structures on sample supports while maintaining their architecture in order to reduce background scattering and provide a mechanically stable support for scanning nanoXRD. This would allow generating coarse density projections of larger areas followed by high-resolution scanning of specific features. A general conclusion emerging from this work is that practically any orb-web silk fiber has become accessible to scanning nanoXRD.",
"introduction": "Introduction Orb-webs built by Araneoidea spiders are composed of seven to eight functional silk fibers (Fig. 1 A) 1 , 2 . Of these, the hierarchical structural organization of load-bearing radial or dragline fibers, composed principally of major ampullate gland silk (MaS) proteins, has been studied in most detail by scattering, imaging and spectroscopy techniques. Although its blend of strength, extensibility and toughness shows phylogenetic variability attributed to silk protein evolution 3 , 4 , the hierarchical structural organisation of MaS fibers derived by scattering techniques is highly conserved 5 , 6 . Indeed, atomic-scale, wide-angle X-ray and neutron scattering (WAXS/WANS) suggest a two-phase system with 10–15% crystalline, alanine-rich β-sheet nanodomains of a few nm diameters 7 – 10 , randomly dispersed in a disordered, polypeptidic matrix. Diffuse short-range order (SRO) X-ray scattering peaks 8 from the amorphous phase do not allow differentiating specific (e.g. helical 11 ) hydrogen-bonding motifs. Microscopic models for mechanical performance of dry fibers assume that the nanodomains act as reinforcement-nodes of an amorphous network with hydrogen-bonding interactions 12 , 13 . Nanoscale, small-angle X-ray scattering (SAXS) and related techniques agree to self-assembly of nanodomains with less-ordered chain segments into lamellar stacks of nanofibrils of 5–7 nm diameters 8 , 10 , 14 , 15 , backed by bottom-up molecular modelling 16 . Scanning nanobeam X-ray diffraction (nanoXRD) with down to ~ 40 nm focal spots suggest a homogeneous nanofibrillar morphology as volume fractions of nanofibrils and nanodomains appear to be correlated 17 . We note, however, variability of nanofibrillar dimensions and volume concentrations when comparing XRD to atomic force microscopy (AFM) and transmission/scanning electron microscopy (TEM/SEM) results, implying that the nature and contribution of nanofibrils to mechanical properties are not fully resolved 18 . A notable exception are ultrathin MaS ribbons of the genus Loxosceles whose mechanical properties can be directly related to individual nanofibrils 19 , 20 . There is, however, a lack of scattering or imaging evidence of higher-order (mesoscale: > 50–100 nm) assembly of nanofibrils in MaS fibers although hierarchical network models based on nanofibrillar domains allow simulating mechanical data such as strain-hardening 21 . Indirect evidence for nanofibrillar bundles of ~ 150 nm diameter was obtained by X-ray particle size analysis for more crystalline (44%) bagworm silk while a smaller diameter of ~ 40 nm was proposed for MaS fibers 15 . The assumption of a homogeneous hierarchical structural organisation assumed in most experimental and modelling approaches is, however, biased by fiber-specific mesoscale heterogeneities. Indeed, a skin–core structure has been observed by TEM for N. clavipes 22 and nanoXRD for A. bruennichi’s MaS fibers but is absent for B. mori fibers 8 , 17 . Biochemical analysis of N. clavipes fibers suggests a sequence of (from outside) lipidic, glycoprotein and proteneous layers and a core with a heterogeneous distribution of MaSp1 and MaSp2 proteins 23 . Various functions have been attributed to the (mesofibrillar 8 , 24 ) proteneous skin-layer such as providing plasticity and confinement for the core 23 or counterbalancing the stress generated by the nanofibrils in the core 25 . It is, however, fair to say that the phylogenetic origin of the skin–core structure and its contribution to MaS fibers mechanical properties 4 are currently not understood. Figure 1 Optical microscopy of orb-web and hub fragment. ( A ) Adult A. bruennichi spider located in the hub of its orb-web. For the nomenclature of orb-web parts see: 47 . ( B ) Hub-area from A . bruennichi’s orb- web. ( C ) Optical microscopy image of hub-area silk fragment. The rectangular area was probed by scanning microXRD. ( D ) Zoom of rectangular area in ( C ). The twisted fiber indicated by an arrow in ( C , D ) is also visible in the SAXS/WAXS-CIs (Fig. 2 A,B). As compared to MaS fibers, the hierarchical structural organization of less crystalline or amorphous orb-web fibers, such as flagelliform silk (flag) fibers forming the cores of the capture-spiral thread 1 , 2 , is less well explored. Flag fibers appear also to be structurally more diverse, as N. clavipes and A. bruennichi’s flag fibers are amorphous showing strong SRO scattering 26 , 27 while A. trifasciata, E. fuliginea and C. sexcuspidata flag fibers contain a fraction of crystalline polyglycine II (PG-II) nanodomains 26 , 28 of unknown distribution in the matrix. In the absence of network-reinforcing nodes discussed for MaS fibers 12 , 13 , molecular or super-molecular reinforcement motifs (e.g. β-springs 29 ) have been proposed contributing to the blend of toughness and extensibility of flag fibers 30 . Enhancing these functional properties by mesoscale features could provide an evolutionary advantage for stopping larger preys impacting an orb-web while limiting the metabolic cost of making larger diameter fibers. Indeed, a possible enhancement mechanism could be the incorporation of skin–core structures used for improving mechanical performance of synthetic polymers and biomaterials 31 . Silk fiber composites with mesoscale features are, however, not well documented for Araneoidea , as also for other types of spider silks although we note the fibrillar composite structure of A. aurentatia’s cylindrical gland fibers used for constructing egg cases 32 . Probing bulk fibrillar features by TEM 32 , 33 or atomic force microscopy (AFM) 34 requires ultra-thin sections and embedding techniques which are prone to artefacts. In the exceptional case of ultra-thin L. laeta ribbons, single nanofibrils could, however, be directly visualized by AFM 19 . While not reaching the resolution of TEM and AFM, scanning nanoXRD can reveal fibrils down to the 100 nm scale and below as shown for the skin-layer of whole A. bruennichi’s MaS fibers 8 , 17 . Indeed, nanoXRD has confirmed the amorphous nature of A. bruennichi’s flag fibers while A. marmoreus flag fibers show an amorphous core and a mesofibrillar skin-layer containing crystalline PG-II nanodomains 27 . A possible relation of the skin–core structure to the nature of prey caught in the web has been suggested 27 but more flag fibers have to be studied across Araneoidea to establish a trend. Here we e xplore by scanning nanoXRD whether highly amorphous silk fibers with weak chain interactions can contain specific mesoscale features suggesting functional differences. Our aim was detecting such fibers in-situ in the natural environment of orb-web fibers. We focused principally on silks from the hub of A. bruennichi spiders orb-web serving as central foraging site (Fig. 1 A,B). As compared to the geometrically well-defined mesh of the capture-section composed of fibers with known functions such as load-bearing, stiff radial threads and the flexible, sticky capture-spiral, fibers in the centre of the hub are more irregularly arranged (Fig. 1 B) 1 , 2 and functionally less well understood or even differentiated. Indeed, the function of decorating fibers from the stabilimentum attached to the hub is controversial 35 and the nature of fibers used for adjusting the final tensioning of radial fibers in the hub 2 remains to be identified. We used two approaches for analysing nano&mesoscale fiber features: (i) modelling based on microXRD patterns and (ii) imaging by density projections based on scanning nanoXRD combined with modelling. In view of the levels of structural organization (see above) we will assume that single silk fibers are externally homogeneous with diameters down to approximately the micron-range. “Filament” is used for a detached part of a fiber and “fibril” for a fiber sub-structure with an unspecified diameter. The term “nanofibril” is used for diameters up to about 50 nm and “mesofibril” for diameters up to the micron-scale.",
"discussion": "Results and discussion Hub-area density projections Optical images of the hub fragment reveal a conglomerate of multiple fibers (Fig. 1 C,D). We generated composite images (CIs) of the hub fragment by scanning microXRD, corresponding to density projections based on X-ray scattering contrast (see Methods). We identify in the WAXS CI (Fig. 2 A) several orb-web features 1 such as two bunches of four radial threads and the hub-spiral (contrast provided in both cases by Bragg peaks and SRO 8 ) as well as the mesh of decorating silk fibers (contrast provided principally by SRO 17 ). We observe the same features in the SAXS CI (Fig. 2 B). The contrast is provided in this case by fibrillar correlations and shape-transforms 8 . Further microstructural data supporting these identifications are presented in the Supplementary Material. We observe, however, only in the SAXS-CI several fine fibers (Fig. 2 B). The absence or weakness of Bragg peaks and SRO scattering suggests their highly amorphous nature. The scattering contrast for the amorphous fibers is provided by equatorial SAXS streaks, oriented normal to the fiber axis. We identify two different types of amorphous fibers distinguished by narrow (am 1 ) and more diffuse streaks (am 2 ) (Fig. 2 C,D). In the following we will analyse the hierarchical structural organization of am 1 fibers based on a microXRD pattern derived from the SAXS-CI. The mechanically particularly stable support provided by a MaS bridge-thread section for an am 2 -type fiber enabled scanning nanoXRD with subµm step increments. This allowed analysing the am 2 fibers hierarchical structural organization based on real space SAXS&WAXS-CIs and reciprocal space nanoXRD patterns. Figure 2 Scanning microXRD and fibrillar composite structure of am 1 fiber. ( A ) WAXS-CI (angular range of pixels shown in SM Fig. 2A) obtained by scanning microXRD with 2 µm (hxv) step-increments. The twisted fiber is also visible in the optical micrographs (Fig. 1 C,D). ( B ) SAXS-CI (angular range of pixels shown in SM Fig. 2B). Clusters of various size on radial threads are due to lipidic deposits 8 . ( C , D ) SAXS patterns of am 1 /am 2 fibers; Open arrow: fiber axis. ( E ) Intensity profile of am 1 fiber SAXS streak; simulation based on uncorrelated cylinders (SM Eq. 5) with 114.4 nm diameter and smeared by averaging over 5 neighbouring points. Q = 2π/d scale; d: lattice spacing. ( F , G ) Schematic model of am 1 fiber composed of cylinders with about 1/10 of the fibers diameter. Fibrillar composite structure of am 1 fiber The diameter of the projected am 1 fiber was derived from the spatial distribution of equatorial streaks in the SAXS-CI as 2.0 ± 0.4 µm (SM Fig. 2 H,I). The streaks can be related to the transform of fibrillar shapes which can be approximated by cylinders with diameter d c (see Methods). Indeed, the modulated intensity decay of the streak in Fig. 2 D was modeled by cylinders of d c ~ 115 nm diameter where the matrix of (assumed) lower density provides the scattering contrast (Fig. 2 E; SM Eq. 4). The azimuthal width of the streak translates into an orientation distribution of f c = 0.998(1) corresponding to quasi-parallel mesofibrils (SM Fig. 3 C; SM Eq. 1). The homogeneous distribution of streaks across the line-scans of the fiber (SM Fig. 2 I) suggest the model of parallel, cylindrical mesofibrils distributed randomly in the matrix with an undefined volume density (Fig. 2 F,G). The presence of meridional diffuse scattering, which cannot be resolved from the beamstop (Fig. 2 C), suggests that the model of infinite, homogeneous cylinders is an idealization. The model resembles the structure of egg case silk fibers from A. aurantia cylindrical glands 32 , showing stiffness and extensibility comparable to MaS fibers 30 , 36 , 37 . The mesoscale features of am 1 fibers suggest therefore also enhanced stiffness. The lack of an important SRO fraction suggests that molecular contributions to extensibility (e.g. helical motifs) can be excluded. However, while am 1 fibers have a hierarchical structural organization resembling egg case silk it is not established that they are also produced by cylindrical glands. Figure 3 SEM images of MaS and accompanying fibers. ( A ) SEM image of crimped fiber detaching from MaS thread. ( B ) SEM images of bridge-thread fibers with accompanying thin, filamentary fibers. ( C ) Crimped fiber bridging MaS radial thread. ( D ) Zoom into crimped fiber revealing ribbon-like morphology with filamentary substructures, partially fused with the MaS surface. The fibers were obtained from the web fragments studied by micro-/nanoXRD but do not correspond to the features probed (adapted in modified form from 8 ; with permission by the authors). Skin–core structure of am 2 fiber The association of am 2 -type with MaS fibers suggests a functional relation. Indeed, the SAXS-CI obtained by scanning microXRD reveals a crimped am 2 fiber emanating from the upper bunch of radial threads (Fig. 2 B). The separation of a crimped fiber from a MaS thread is also visible in a SEM image (Fig. 3 A). A spectrum of crimped and elongated, flexible fibers with ribbon-like features is covering bridge-thread fibers (Fig. 3 B,C). Partial fusion of crimped fibers with the MaS skin reveals about 300 nm diameter substructures -limited by the SEM resolution- implying surface interactions (Fig. 3 D). The crimped morphology is particularly well visible when the overall am 2 fiber direction is along the MaS fiber axis (Fig. 3 C). The fiber becomes extended when bridging the two fibers of the MaS thread. We generated SAXS&WAXS-CIs based on scanning nanoXRD of an am 2 fiber attached to a bridge-thread section with a gap between the two fibers (Fig. 4 A). The straight, bridging part of the am 2 fiber has remained connected to one of the MaS fibers by a thin filament (Fig. 4 A). This geometry provides stability for scanning nanoXRD and allows observing weak scattering from the am 2 fiber without scattering from the bridge-thread. Indeed, the WAXS-CI reveals the projection of the am 2 fiber and the filament by faint diffuse scattering (Fig. 4 B,C). The fiber is tilted by 7° ± 1° from the horizontal scan axis, corresponding also to the fiber axis direction of the MaS fibers, revealed by the contrast provided by β-sheet peaks and SRO scattering 8 (Fig. 4 B,D). The am 2 fiber density projection is, however, not completely homogeneous as we observe within its contours a several µm long domain defined by two Bragg peaks of 0.418 ± 0.001 nm and 0.378 ± 0.004 nm lattice spacings (Fig. 4 C,E,F). The nature of this domain will be discussed below. The diameter of the am 2 fiber was determined from the intensity distribution of a horizontal scan-line (Fig. 4 C,E) as for the am 1 fiber (SM Fig. 2 I), assuming that the diffuse scattering at each scan-point (Fig. 4 G) is proportional to the amount of protein probed by the nanobeam. Although we masked the strongest (0.418 nm) Bragg peak visible in several patterns (Fig. 4 I) we find a slight asymmetry of the profile suggesting enhanced diffuse scattering from an 2nd, unknown component (Fig. 4 G). We simulated the profile by a sine function with a width of 10 µm fwb (full-width-at-base) (Fig. 4 G) corresponding to a fiber diameter of 1.2 ± 0.2 µm (10 µmxsin7° ± 1°). A sine function is appropriate for the continuous shape-change of a cylindrical cross-section; a hollow cylinder is excluded as the diffuse scattering would increase when probing from the outer skin and decrease towards the center of the cylinder (SM Fig. 4 A,B). Figure 4 Composite images and structural analysis of am 2 fiber. ( A ) Optical microscopy of bridge thread and am 2 fiber. ( B ) NanoXRD WAXS-CI: 0.5 µm (hxv) steps-increments; pixels based on WAXS-range in ( D ), 16 × binned to reduce noise level. Upper intensity level reduced to reveal am 2 fiber scattering. ( C ) Zoom into WAXS-CI revealing am 2 fiber. Contours of crystalline domain (blue rectangle) defined by Bragg peaks. (D) Single pixels with Bragg peaks indexed for the poly(L-alanine) lattice 8 , 48 ; Inset: SAXS range with equatorial streak and meridional peak. ( E ) Zoom into crystalline domain. ( F ) Intensity profile from 11 averaged patterns from crystalline domain (inset) fitted by 3 Gaussians for PG-II Bragg peaks 42 and SRO scattering and a 0-order polynomial for random background (Q = 2π/d scale; d: lattice spacing). ( G ) Integrated diffuse intensity (2 < Q(nm −1 ) < 13.2) of am 2 fiber along violet, dashed arrow in ( D , E ), PG-II (100) peak masked. Red filled circles: am 2 scattering. Bragg peaks were observed at the position of the red open circles. The green open circles correspond to other nanofibrillar features (see text). Red dashed curve: sine function fitted to the am 2 data points, green circles were excluded from the fit. An idealized cylindrical cross-section model for the am 2 fiber and the relative dimensions of the nanobeam is shown as in-set. ( H ) Pattern from position F2 in ( G ) fitted by one Gaussian and a 0-order polynomial). ( I ) Pattern from position F1 in ( G ) fitted by two Gaussians and a 0-order polynomial. The SAXS-CI is based on the contrast provided by the equatorial streaks (Fig. 4 D). The contours of the bridge-thread are revealed by an enhanced contrast of the skin-layer mesofibrils 8 (Fig. 5 A). The banded structure on the bridge-thread will be discussed below. The am 2 fiber is revealed by horizontal groups of streaks defining a skin–core structure (Fig. 5 A,B). The azimuthal width of the strongest streak in a group (e.g. streak 1 in Fig. 6 A) corresponds to a spread in fibrillar orientations of about 8° fwhm or about 15° fwhm for an averaged streak of a whole scan-line (SM Fig. 3 A,B). Averaging across a larger fiber volume is at the origin of the diffuse streak observed by microXRD (Fig. 2 D). Similar groups of streaks define three further fibrous features which are partially overlapping in the am 2 fibers density projection (SM Fig. 6 B). We identify one of these features in the optical micrograph as the filament connecting the am 2 fiber to the lower MaS fibers surface (Fig. 4 A). The other fibrous features in the projection image are optically out of focus. The absence of an equatorial Bragg peak implies the absence of inter-fibrillar correlations. The angular deviation of the fibrillar axis from the direction of the am 2 fiber axis by ~ 8° is attributed to a wound ribbon of fibrils. The intensity variation in a group of streaks (Figs. 6 A, SM Fig. 6 B,C) is due to the geometry of the skin-layer probed. Indeed, for a cylindrical cross-section, the nanobeam will probe more fibrils through the skin-layer at the edge than at the center of the fiber (SM Fig. 4 A,B). The projection of the fiber diameter derived from the spatial extension of the two groups of streaks in a single horizontal line is 1.2 µm ± 0.2 µm as also derived from the diffuse scattering data (Fig. 4 G). The thickness of the skin-layer determined from the extension of the group of streaks is ~ 200 nm. The filament shows also streaks expected for fibrillar morphology (SM Fig. 7A). Albeit the low counting statistics we start seeing a modulation of the intensity profile as for the am 2 fiber profile suggesting mesofibrils (SM Fig. 7B). We assume therefore that the filament and probably the other fibrous features visible in the SAXS-CI (SM Fig. 6 B) have become detached during rupture of the am 2 fiber from the MaS skin-coat. A cylinder diameter of d c = 4.4 ± 0.3 nm diameter was determined by Guinier’s approach (Supplementary Materials) for the strongest streak showing a continuous intensity decay (Fig. 5 C). This diameter corresponds to the scale of nanofibrillar diameters obtained by different techniques. Indeed, diameters of 6–7 nm were obtained Guinier’s approach for nanofibrils in the cores Nephila and Argiope MaS fibers 8 , 14 . A diameter of 4.7 nm was derived from the equatorial SAXS correlation peak for bagworm silk nanofibrils 15 while single nanofibrils with 20 nm × 7 nm cross-section were imaged by AFM for recluse spider silk ribbons 19 . The diffuse scattering profile (Fig. 4 G) suggests that am 2 fiber cores contain disordered protein. For the projection of ribbon-like skin-layer one would expect observing towards the center of the fiber fibrillar scattering at different crossing-angles. Indeed, we observe patterns with multiple weak streaks which we attribute to fibrils at discrete angles (SM Fig. 7C,D). The weakness of these streaks does, however, not provide information on the homogeneity of the skin-layer which might have become locally degraded during the rupturing process. Preliminary evidence suggests at least partial assembly of nano- into mesofibrils in the skin-layer. Indeed, a modulation of the equatorial streak profile is observed for the weaker streaks at the inside of the skin-layer (Figs. 6 B, SM Fig. 8A). We determined the peak positions by fitting Gaussians to the profile (SM Fig. 8B) and derived a cylinder diameter of d c = 86 ± 3 nm from an extrapolation of the order position versus the scattering vector Q (Fig. 6 B) 8 , 38 . A slightly larger value of d c ~ 100 nm is obtained by fitting a 1st order Bessel function to the profile (Figs. 6 B, SM Fig. 8B; SM Eq. 5). These values are in the range of cylindrical diameters for the mesofibrillar skin-layer of MaS fibers 8 . Mesofibrils could, however, only be observed at the inside of the am 2 fibers skin-layer as the modulation of streaks from the center and the outside of the skin-layer is not well pronounced. We prefer therefore the idealized model shown in Fig. 6 C,D. It is possible that aggregation of nanofibrils is related to mechanical compression at the inner skin-layer. We note that mesofibrillar dimensions and braid-like morphology of am 2 -fibers resemble MaS fibers proteneous skin-layer. Figure 5 Skin–core structure of am 2 fiber. ( A ) SAXS-CI based on pixels covering the equatorial streak in Fig. 4 D. The contours of the am 2 fiber in the gap of the MaS thread can be traced by groups of equatorial streaks. ( B ) Zoom of am 2 fiber with skin-layer defined by groups of horizontal streaks. Aspect ratio ~ 3:1 for better visualization of the fiber contours. The X-ray probing points are shown schematically, scaled to the size of the focal spot. Each 0.5 µm horizontal step corresponds to an incremental vertical displacement of the focal spot across the fiber by 61 ± 9 nm (= 500 nmxsin7° ± 1°). Red arrow: averaged local fibrillar axes direction; black arrow: am 2 fiber axis. ( C ) Guinier intensity plot for the streak shown in inset. A linear regression line with slope m = −0.61 (0.7) has been fitted to Q 2 > 0.3 nm −2 14 . Figure 6 Nanofibrillar am 2 fiber skin and related filamentary features. ( A ) Evolution of streaks across skin-layer of am 2 fiber (Fig. 5 B). ( B ) Variation of intensity profiles for two streaks from skin-layer. The profile of streak 2 (blue line, open blue circles) has been fitted by 5 Gaussian (red line, solid red circles. The inset shows a correlation plot of peak positions determined by Gaussian fits to the profile versus Q to determine the cylinder radius (text). A regression line with slope m = 0.073 (2) has been fitted. ( C ) Schematic model of am 2 fiber composed of ribbons of nanofibrils tilted by about 8° against the macroscopic am 2 fiber axis, wound around a disordered core. Only one layer of nanofibrils in a ribbon (not to scale) is shown. ( D ) Projection of model showing assembly into mesoscale nanofibrillar bundles at the inside of the skin-layer. ( E ) Zoom of red, dashed area in Fig. 5 A. Band-like feature indicated by red domain defined by enhanced intensity streaks. ( F ) SEM image of filamentary features covering bridge-thread fibers with schematic fibrillar model for one of the filaments (from same web silk but not from area probed by nanoXRD). Banded pattern on bridge-thread The banded pattern on the surface of the bridge-thread SAXS-CI (Fig. 5 A) is generated by streaks with enhanced intensity along the direction of the bands, overlapping with the equatorial streaks from the core nanofibrils 8 . We exclude scattering from “ordered regions” of a few µm size derived from Raman studies for Nephila and B. mori fibers 39 as no evidence for bulk crystalline domains of this size was obtained by scanning nanoXRD of A. bruennichi’s bridge-threads 8 . We rather attribute the banded pattern to scattering from the mesh of filaments wound around the bridge-thread fibers (Figs. 3 B). The density of these filaments appears to fluctuate locally along the threads or even disappear. We have selected a single band by its enhanced scattering (Fig. 6 E). Selected streaks from the band show the modulated intensity decay of mesofibrils suggesting equatorial-type scattering (SM Fig. 5 A,B). We therefore propose as model layers of mesofibrils which are aligned normal to the axis of the filaments (Fig. 6 F). Given the close association of these band-like features and the am 2 -fibers with the MaS-surface (see also below) we put forward the hypothesis of a common glandular origin. Indeed, the proximal region of the MaS gland has been proposed to be at the origin of the MaS fibers core while protein secreted by the distal region was proposed to be at the origin of Nephila ’s proteneous skin-layer 22 ; also assumed for A. bruennichi ’s MaS fibers 8 . The different fibrous morphologies could therefore be due to different levels of assembly. Interaction of am 2 fiber with MaS fibers coat We discuss here the nature of the domain observed within the contours of the am 2 fiber projection, which is defined by Bragg peaks (Fig. 4 C,E,F). The peaks show an azimuthal spread which can be separated into several Gaussians suggesting a cluster of nanocrystallites (SM Fig. 9A–D). Slight changes in the texture of the peaks suggest clusters differing slightly in orientation along the am 2 fiber. The same peaks corresponding to 60–100 nm nanocrystallites were observed for MaS bridge-threads 8 while we derive 30–40 nm nanocrystallites for the am 2 fiber domain (Supplementary Material). These nanocrystallites, observed initially only together with MaS diffraction patterns (called S,S* peaks), were explained by the non-periodic lattice (NPL) model based on peptide chain side-group ordering of the β-sheet nanocrystalline fraction 40 , 41 . A recent model attributes these peaks, however, to the PG-II lattice as they are also observed for the MaS glycoprotein skin-layer 23 lacking poly(L-alanine) peaks 17 . Peak positions and intensities of the Bragg peaks in the am 2 domain agree also to PG-II (100)/(101) reflections 42 . We assume therefore that a fragment of the glycoprotein shell containing PG-II nanocrystallites has been ruptured together with the am 2 -fiber section from the MaS coat. Indeed, glycoprotein is revealed by its SRO scattering (Fig. 4 H) contributing to the asymmetry of the diffuse scattering profile (Fig. 4 G). The SAXS-CI reveals two rupture zones on the MaS thread and the filament connecting the am 2 fiber to one of the rupture zones (Fig. 5 A, SM Fig. 10A–E). Streaks from the MaS proteneous skin-layer with about 15° azimuthal fwhm (similar to the skin of pristine MaS fibers 8 ) are observed next to the lower rupture zone (SM Fig. 10D) while random SAXS due is observed within the rupture zone (SM Fig. 10E). We attribute the random SAXS to randomly orientated mesofibrils in the proteneous skin-layer below the ruptured glycoprotein layer. Indeed, the radial profile of the streaks shows the modulated intensity decay of the streaks of the MaS mesofibrillar skin-layer 8 . The scattering data support therefore the biochemical analysis of the MaS coat 23 ."
} | 7,885 |
34367082 | PMC8336468 | pmc | 5,729 | {
"abstract": "Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, one of the most promising liquid biofuels. Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n -butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative gel-free proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS) analysis allowed identification and quantification of differentially expressed soluble proteins. The study data are available via ProteomeXchange with the identifier PXD024183. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (HSPs), involved in protein quality control, were up-regulated such as the chaperones GroES (Cpn10), Hsp90, and DnaJ. Globally, our data indicate that protein biosynthesis is reduced, likely not to overload HSPs. Several additional metabolic adaptations were triggered by butanol exposure such as the up-regulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid (e.g., arginine, lysine, methionine, and branched chain amino acids) biosynthesis and proteins involved in cell envelope re-arrangement (e.g., the products of Clocel_4136, Clocel_4137, Clocel_4144, Clocel_4162 and Clocel_4352, involved in the biosynthesis of saturated fatty acids) and a redistribution of carbon flux through fermentative pathways (acetate and formate yields were increased and decreased, respectively). Based on these experimental findings, several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans are suggested. This includes overexpression of HSPs (e.g., GroES, Hsp90, DnaJ, ClpC), RNA chaperone Hfq, V- and F-type ATPases and a number of genes whose function in C. cellulovorans is currently unknown.",
"conclusion": "Conclusion The results obtained by the present investigation indicate that butanol exposure elicits complex responses in C. cellulovorans thus confirming previous observations made in other microorganisms. These responses encompass adaptation mechanisms at the cell wall, cell membrane and cytoplasmic levels: • Butanol-challenged C. cellulovorans dedicated a massive effort in the biosynthesis of proteins involved in protein translation, folding and degradation (28% of the up-regulated proteins). However, lower total cell protein levels measured in butanol-supplemented C. cellulovorans cultures ( Figure 5 ) seems more consistent with the fact that protein translation is inhibited in this condition (possibly by ribosome inactivation through the ribosome hibernation promoting factor, Clocel_3036). This is coherent with observations made on other butanol-stressed clostridia ( Alsaker et al., 2010 ; Venkataramanan et al., 2015 ; Sedlar et al., 2019 ) as well as other butanol-challenged bacteria ( Fu et al., 2013 ; Tian et al., 2013 ). It remains to be determined why a large number of ribosomal subunits were up-regulated in butanol-supplemented C. cellulovorans cultures. • Proteomic data suggest that a re-arrangement of both the cell wall (i.e., peptidoglycan) and membrane composition occurs in butanol-stressed C. cellulovorans . As regards cell membrane, our findings suggest that a higher proportion of saturated fatty acids is incorporated in the cell membrane in this condition thus decreasing membrane fluidity, consistently with observations made in other microorganisms ( Borden and Papoutsakis, 2007 ; Huffer et al., 2011 ; Isar and Rangaswamy, 2012 ; Fu et al., 2013 ). • Many membrane transport proteins, and in particular ion transporters possibly involved in ATP synthesis and/or generation of transmembrane electrochemical gradient were up-regulated in butanol-challenged C. cellulovorans . These proteins include Na + (NatB, Clocel_3460) and Mg 2+ (MgtE, Clocel_1328) transporters and several subunits of a V-type ATPase (V 1 subunits K, Clocel_1656; C, Clocel_1658; F, Clocel_1659; A, Clocel_1660; and B, Clocel_1661) and a F-type ATPase (F 1 subunits γ, Clocel_3050; α, Clocel_3051; and δ, Clocel_3052). • Multiple metabolic pathways were significantly affected by butanol exposure including: (i) up-regulation of enzymes involved in amino acid biosynthesis; (ii) down-regulation of several enzymes involved in pyrimidine and purine biosynthesis; (iii) modulation of the expression of multiple enzymes involved in glycolysis and fermentative pathways. As regards amino acid biosynthesis, the enhancement of enzymes catalyzing biosynthesis of branched-chain amino acids (acetolactate synthase large subunit, Clocel_1324, ketol-acid reductoisomerase, Clocel_1325, and dihydroxy-acid dehydratase, Clocel_0493) could possibly refer to additional mechanisms to modulate cell membrane fluidity ( Mansilla et al., 2004 ). In fact, branched-chain amino acids are used as primers for the synthesis of branched-chain fatty acids ( Mansilla et al., 2004 ; Alsaker et al., 2010 ; Wang et al., 2016 ). Inconsistent with studies reporting butanol inhibition of glucose uptake in other bacteria ( Bowles and Ellefson, 1985 ; Tomas et al., 2004 ; Alsaker et al., 2010 ; Venkataramanan et al., 2015 ; Sedlar et al., 2019 ), butanol caused an increase of the specific glucose consumption in C. cellulovorans . This observation possibly correlates with the up-regulation of a putative glucose PTS transporter (Clocel_2778). In addition, pyruvate fate was significantly perturbed by butanol-stress in C. cellulovorans . Formate production was inhibited (consistent with down-regulation of PFL-activating enzyme, Clocel_1812), and a higher proportion of the acetyl-CoA was driven to acetate instead of butyrate ( Figure 2 ). The latter observation correlates well with the down-regulation of the acetyl-CoA to butyrate pathway (i.e., acetyl-CoA acetyltransferase, Clocel_0192 and Clocel_3058; 3-hydroxybutyryl-CoA dehydrogenase, Clocel_2972; electron transfer flavoprotein α and β subunits, Clocel_2973 and Clocel_2974; and phosphate butyryltransferase, Clocel_3675) ( Figure 8 ). Although the metabolic reason for this re-arrangement is currently unclear, one can hypothesize that down-regulation of the butyrate pathway may also serve to drive more acetyl-CoA toward fatty acid biosynthesis. The present study has highlighted several potential gene targets for improving butanol tolerance of C. cellulovorans through rational metabolic engineering. The complexity of microbial responses to butanol stress makes identification of priority gene modifications aimed at these strategies not trivial. Furthermore, a significant number of previous studies reported involvement of genes with previously unknown/poorly characterized function in butanol tolerance ( Jia et al., 2012 ; Pei et al., 2017 ; Sun et al., 2017 ). Although an exhaustive mechanistic understanding of microbial adaptation to butanol stress currently remains elusive, several studies have obtained significant improvement of butanol tolerance by single/few gene modifications ( Anfelt et al., 2013 ; Si et al., 2016 ; Yang et al., 2020 ) which may serve as general paradigms for these strategies. So far, most examples of targeted gene modification aimed at enhancing butanol tolerance have been based on overexpression of HSPs (e.g., GroES, GroEL, and DnaK) which proved to be effective in very diverse microbial models such E. coli ( Zingaro and Papoutsakis, 2012 ), lactobacilli ( Fiocco et al., 2007 ), cyanobacteria ( Anfelt et al., 2013 ), and clostridia ( Tomas et al., 2003 ). On the basis of the high fold changes measured by the present proteomic analysis, the most interesting C. cellulovorans gene targets include groES (Cpn10, Clocel_2966, FC = 5.05), htpG (Clocel_0510, FC = 3.99), dnaJ (Clocel_1417, FC = 3.32) and clpC (ATPase AAA-2 domain protein, Clocel_3760, FC = 2.80). Recently, an increasing attention has been devoted to the role of sRNAs in response to a variety of stresses including butanol, and studies involving overexpression or suppression of some of them were effective in improving butanol tolerance ( Venkataramanan et al., 2013 ; Jones et al., 2016 ; Sun et al., 2017 ). These data seem to find confirmation in C. cellulovorans since the RNA chaperone Hfq (Clocel_2035) was among the most up-regulated protein under butanol-stress (FC = 3.09). Therefore, overexpression of Hfq is another interesting strategy that would be worth testing. Recently, overexpression of several sRNA regulators was used to improve butanol-tolerance of E. coli ( Xu et al., 2020 ). A further obvious strategy concerns overexpression of proteins involved in maintaining homeostasis of the transmembrane Δψ and of ATP concentration such as the V-type (Clocel_1654-1662) and F-type (Clocel_3048-3055) ATPases identified in this study. It is worth noting that the function of four among the ten most up-regulated proteins in butanol-stressed C. cellulovorans (FC ≥ 3.5) is currently unknown. Our study thus points at interesting gene candidates for engineering butanol-hypertolerant C. cellulovorans and stimulates further studies aimed at characterizing still poorly characterized proteins.",
"introduction": "Introduction The consequences of massive exploitation of fossil fuels on global warming and climate changes have prompted research toward alternative energy sources with lower environmental impact. Biofuels produced by microbial fermentation of plant biomass have attracted substantial interest based on their potential to benefit current environmental, economic, and societal issues ( Lynd, 2017 ). It has been estimated that plant biomass provides 10% of global primary energy and, within this, cellulosic feedstocks are the most abundant and least expensive ( Lynd, 2017 ). Clostridium cellulovorans , an anaerobic, mesophilic, cellulolytic bacterium ( Sleat et al., 1984 ) is among the most promising candidates for industrial production of cellulosic biofuels, with particular reference to n -butanol (hereinafter referred to simply as butanol). Butanol (four carbon chain) has a longer carbon backbone than other established biofuels such as ethanol (two carbons) or methanol (one carbon), which gives it fuel properties more similar to that of gasoline, such as high combustion energy, low volatility and corrosivity ( Liu et al., 2013a ). Pure butanol can be fed to spark ignited engines without any modification, whereas ethanol must be blended with gasoline ( Campos-Fernández et al., 2012 ). C. cellulovorans potential to ferment all the main plant polysaccharides, namely cellulose, hemicelluloses and pectins ( Aburaya et al., 2015 , 2019 ) represents an advantage over other well established plant-degrading microorganisms such as Clostridium thermocellum , Clostridium cellulolyticum , or Thermoanaerobacterium saccharolyticum which show more restricted substrate panel ( Saxena et al., 1995 ; Demain et al., 2005 ). The most abundant C. cellulovorans fermentation products are organic acids (e.g., butyrate, formate, acetate), ethanol, H 2 and CO 2 ( Sleat et al., 1984 ). Although wild type C. cellulovorans cannot biosynthesize butanol, its production in this microorganism has recently been enabled by introducing a single heterologous alcohol/aldehyde dehydrogenase ( Yang et al., 2015 ). In fact, most metabolic reactions leading to conversion of acetyl-CoA to butyrate are in common with butanol biosynthesis. Butyrate production is widespread in solventogenic clostridia such as C. acetobutylicum and C. beijerinckii but is absent in most cellulolytic clostridia such as C. thermocellum and C. cellulolyticum ( Mazzoli and Olson, 2020 ). By using metabolic engineering, recombinant C. cellulovorans strains have been obtained which can produce about 4 g/L and 4.96 g/L of butanol through direct fermentation of crystalline cellulose ( Bao et al., 2019 ) and alkali-extracted corn cobs ( Wen et al., 2020 ), respectively. These butanol titers are the highest reported so far for direct fermentation of plant biomass using a single microorganism ( Mazzoli and Olson, 2020 ). However, butanol displays higher cell toxicity than other biofuels (e.g., ethanol) ( Ingram, 1976 ) which limits fermentation titers. In addition, butanol separation generally requires two distillation columns instead of one ( Vane, 2008 ), which increases capital costs. Native butanol producers (i.e., C. acetobutylicum and C. beijerincki ) typically can attain 15–20 g/L butanol titer ( Tomas et al., 2003 ; Nicolaou et al., 2010 ; Chen and Liao, 2016 ). However, C. cellulovorans cannot grow in media containing more than 8 g/L butanol ( Yang et al., 2015 ). Recently, only moderate improvement of C. cellulovorans tolerance to butanol has been obtained by adaptive evolution ( Wen et al., 2019 ). Although continuous solvent extraction from fermentation medium or two-phase (organic-aqueous) fermentation systems can be used to circumvent solvent toxicity, their cost threatens process viability ( Heipieper et al., 2007 ; Huang et al., 2010 ; Dürre, 2011 ). The development of strains with superior solvent tolerance is highly desirable for sustainable production of biofuels ( Nicolaou et al., 2010 ). Enhancement of microbial solvent tolerance can be achieved through different strategies belonging to two main paradigms: (i) “random” approaches, such as random mutagenesis ( Liu et al., 2012 ), whole genome shuffling ( Mao et al., 2010 ) and adaptive laboratory evolution ( Liu et al., 2013b ) and; (ii) “rational” approaches based on targeted gene modification (e.g., overexpression of genes involved in solvent tolerance) ( Tomas et al., 2003 ; Borden and Papoutsakis, 2007 ; Xu et al., 2020 ). Different experimental approaches have been employed to identify genes involved in solvent resistance such as the construction of genomic or deletion libraries or the use of transcriptomic and proteomic analyses ( Borden and Papoutsakis, 2007 ; Alsaker et al., 2010 ; Mazzoli, 2012 ; Venkataramanan et al., 2015 ). The relatively high butanol cell-toxicity is mainly attributed to its partition coefficient (logP = 1), namely its higher ability to intercalate within the lipid bilayer of biological membranes and increase their fluidity with respect to less hydrophobic biofuels (e.g., ethanol) ( Heipieper et al., 2007 ). From this standpoint, solvent effect on cells is similar to that of temperature upshift (heat shock). Solvents mostly affect the structure and functions of biological membranes, thus compromising vital processes such as energy generation and nutrient transport ( Bowles and Ellefson, 1985 ; Heipieper et al., 2007 ). Butanol was shown to inhibit membrane-bound ATPases, partially or completely abolish the membrane ΔpH ( Bowles and Ellefson, 1985 ; Gottwald and Gottschalk, 1985 ; Terracciano and Kashket, 1986 ) and Δψ ( Terracciano and Kashket, 1986 ), lower intracellular pH and ATP concentration ( Bowles and Ellefson, 1985 ; Huang et al., 1986 ; Terracciano and Kashket, 1986 ), besides interfering with active uptake of glucose and other nutrients ( Bowles and Ellefson, 1985 ). With regards to native butanol producers, most research aimed at understanding mechanisms of butanol tolerance refers to C. acetobutylicum ( Tomas et al., 2004 ; Borden and Papoutsakis, 2007 ; Alsaker et al., 2010 ; Wang et al., 2013b ). It has been reported that butanol-challenged C. acetobutylicum increases the ratio of saturated/unsaturated fatty acids in the cell membrane, likely to balance the fluidity increase caused by solvent (a response also referred as homeoviscous adaptation) ( Borden and Papoutsakis, 2007 ). Furthermore, responses to butanol challenge have been investigated in a number of other microorganisms which have been proposed for recombinant butanol production owing to their native higher tolerance to butanol, e.g., lactic acid bacteria ( Winkler and Kao, 2011 ; Liu et al., 2021 ; Petrov et al., 2021 ) and Pseudomonas putida ( del Cuenca et al., 2016 ), or genetic tractability, e.g., Escherichia coli ( Rutherford et al., 2010 ) and Synechocystis sp. ( Tian et al., 2013 ) with respect to Clostridia. Apart from adaptation mechanisms directly targeted to restore membrane function (which may also include adjustment of the protein content; Weber and De Bont, 1996 ), responses to solvents generally include up-regulation of heat shock proteins (HSPs) and may comprise overexpression of solvent efflux pumps and changes in cell size and shape ( Heipieper et al., 2007 ; Nicolaou et al., 2010 ). However, microbial responses to solvent stress are complex and species-related. The present investigation aimed at digging into C. cellulovorans responses to butanol challenge and identifying genes possibly involved in tolerance to this chemical. A comparative proteomic analysis was performed on C. cellulovorans cultures grown in butanol-supplemented medium and in control medium (i.e., without butanol) leading to identification of 307 differentially expressed proteins. This protein dataset will help future rational metabolic engineering strategies for obtaining butanol-hypertolerant strains to be exploited in large scale production of this biofuel.",
"discussion": "Results and Discussion C. cellulovorans Growth Parameters at Different Butanol Concentrations The effect of supplementing different butanol concentrations (1–8 g/L) on C. cellulovorans growth and metabolism was determined. Growth was only slightly affected by 1 g/L butanol ( Figure 1A ), while higher butanol concentration led to progressive reduction of growth efficiency. At 6 g/L butanol, the maximum biomass reached was about 50% of that obtained in control conditions (no added butanol). Consistently, butanol supplementation negatively affected the specific growth rate (μ) ( Figure 1B ). These results are consistent with those obtained by Yang et al. (2015) . FIGURE 1 Growth kinetics (A) and specific growth rate (μ, B ) of C. cellulovorans grown in media supplemented with different butanol concentration. Bars represent standard errors. Data are the averages of three biological replicates. Asterisks indicate values that significantly (* p < 0.05; ** p < 0.005) differ from that measured in the control condition (no added butanol). Eventually, these analyses were aimed at finding the most suitable butanol concentration for studying butanol-stress response in C. cellulovorans . For this purpose, a butanol concentration that significantly inhibits growth efficiency but allows enough biomass production for proteomic analyses is ideal. Cultures with 4–7 g/L butanol showed comparable growth rate and maximum biomass and were considered suitable for proteomic analyses. Among these, cultures with 6 g/L butanol were finally chosen. Fermentative Metabolism of C. cellulovorans in Medium Supplemented With 6 g/L Butanol Almost a double amount (0.84 ± 0.18 g/L) of glucose was consumed by C. cellulovorans grown in control condition as compared to 6 g/L butanol-enriched cultures (0.46 ± 0.03 g/L) after 4 h ( Figure 2 ). This seems consistent with differences in the specific growth rate measured in the two conditions ( Figure 1B ) and with previous studies reporting butanol inhibition of glucose uptake in other clostridia ( Bowles and Ellefson, 1985 ; Tomas et al., 2004 ; Alsaker et al., 2010 ; Venkataramanan et al., 2015 ; Sedlar et al., 2019 ). However, it is worth noting that butanol inhibition on C. cellulovorans growth is more important than that on glucose consumption ( Figures 1 , 2 ). Butanol-supplemented cultures showed specific glucose consumption about 2-fold higher than control cultures. As regards catabolite production, formate titer significantly decreased (from 0.30 g/L to 0.07 g/L) in butanol-supplemented condition ( Figures 2A,B ) as well as formate yield (from 0.33 g/g glucose consumed to 0.15 g/g glucose consumed) ( Figure 2C ). The opposite pattern was observed for acetate and butyrate yield: 0.29 g/g acetate and 0.62 g/g butyrate were measured in butanol-supplemented condition, while only 0.13 g/g acetate and 0.43 g/g butyrate were produced in the control condition ( Figure 2C ). No significant difference in ethanol yield was measured between the two growth conditions. No significant butanol consumption (<0.35 g/L) was observed in butanol-supplemented cultures (data not shown). In C. cellulovorans , pyruvate may have three metabolic fates: (i) oxidation by pyruvate ferredoxin oxidoreductase (PFOR), leading to production of acetyl-CoA and reduced ferredoxin (possibly used for hydrogen production); (ii) conversion to formate and acetyl-CoA by pyruvate formate lyase (PFL) and; (iii) reduction to lactate by lactate dehydrogenase (LDH) with concomitant consumption of NAD(P)H. According to fermentation end-product determination, LDH reaction accounts for very minor carbon flux, since no detectable lactate amounts were produced, and most pyruvate flux is expectedly taken in charge by PFL and PFOR reactions. The decrease of formate yield in butanol-challenged cultures is indicative of an increase in the carbon flux through the PFOR reaction in this condition. Higher amount of reducing equivalents generated by the PFOR reaction may also have promoted acetyl-CoA-to-butyrate pathway [which requires 3 NAD(P)H per butyrate molecule]. In a number of studies on cellulolytic clostridia, it has been reported that acetate is preferentially accumulated in conditions promoting slower growth ( Riederer et al., 2011 ; Munir et al., 2016 ; Badalato et al., 2017 ). This observation finds confirmation in the present study, in which acetate yield was higher in butanol-supplemented cultures. FIGURE 2 Glucose consumption and catabolite production of C. cellulovorans grown in control condition (no added butanol, A ) or in butanol-supplemented medium (B) . (C) Fermentation end-product yield (g per g of consumed glucose). Data are the mean of triplicate measurements. Bars represent standard errors. Asterisks indicate values that significantly ( ∗ p -value < 0.05; ∗∗ p -value < 0.01) differ between control (green) and butanol-supplemented (red) culture conditions. Proteome Analysis: Identification of Differentially Expressed Proteins in 6 g/L Butanol-Challenged Cultures Biomass samples (four biological replicates for each growth condition) for proteomic analyses were harvested 4 h after culture inoculation, that is during exponential growth phase ( Figure 1A ). A schematic overview of the proteomic workflow used to study C. cellulovorans responses to butanol stress is depicted in Figure 3 . Proteins showing a fold change (FC) ≥ 1.5 or FC ≤ 0.67 ( p-value ≤ 0.05) in butanol-treated cultures versus control ones were considered as differentially expressed. The analysis identified 203 up-regulated and 104 down-regulated proteins ( Table 1 and Supplementary Table 2 ) in butanol-stressed cells. The complete list of identified proteins with protein coverage is reported in Supplementary Tables 3 – 5 . The study data are available via ProteomeXchange with the identifier PXD024183. Differentially expressed proteins were classified according to the Clusters of Orthologous Groups (COGs) ( Galperin et al., 2015 ). It is worth noting that a significant amount of C. cellulovorans genes encode proteins with still unknown function ( Tamaru et al., 2010 ), namely 12% of the up-regulated and 14% of the down-regulated proteins (that is 24 and 15 proteins, respectively) identified in the present investigation ( Figure 4 ). Apart from these proteins, the overexpressed proteins were predominantly mapped to COG categories representing translation (COG category J, 30 proteins), amino acid metabolism (COG category E, 18 proteins), energy production and conservation (COG category C, 18 proteins) and molecular chaperones (COG category O, 11 proteins) which globally account for almost 50% of the up-regulated proteins. More than 50% of the identified down-regulated proteins belongs to nucleotide metabolism (COG category F, 20 proteins), carbohydrate metabolism (COG category G, 13 proteins), transcription (COG category K, 9 proteins) and translation (COG category J, 8 proteins) related groups. Similar findings have previously been reported in butanol-challenged C. acetobutylicum , at least as regards proteins belonging to COG categories C, E, F, and G ( Venkataramanan et al., 2015 ). TABLE 1 List of the most highly differentially expressed proteins quantified in the present study for each COG category. FIGURE 3 Schematic overview of the proteomic workflow used to study C. cellulovorans responses to butanol stress. (1) C. cellulovorans was grown in media supplemented with different concentrations of butanol (0–8 g/L). Whole-cell soluble protein extraction was performed on C. cellulovorans cells grown in control (0 g/L butanol) or butanol-challenged (6 g/L butanol) cultures. (2) Differential proteomics were analyzed through Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS). Phylogenetic protein classification by COGs (Clusters of Orthologous Groups of proteins) was performed and qRT-PCR was used to confirm differential gene expression of a gene pool. (3) Butanol-challenged C. cellulovorans metabolism was described by discussing the differentially expressed proteins (up- and down-regulated in butanol-stressed bacteria). This information led to identification of target genes for rational metabolic engineering strategies to obtain a butanol-hypertolerant C. cellulovorans strain for large scale bio-production. FIGURE 4 COG categories overrepresentation in differentially expressed proteins. The treemap displays the fold enrichment of each COG category in (A) down-regulated and (B) up-regulated proteins. The size and color of the rectangles are proportional to the registered fold enrichment. The proportion of regulated proteins that are annotated to each COG category is shown in brackets. Expression profile of thirteen key genes was validated by means of qRT-PCR analysis ( Table 2 ). Selected genes encode proteins that are representative of eight different COG categories (C, E, F, I, J, KT, O, P). Mostly genes encoding proteins highly up-regulated in butanol-supplemented cultures were considered, but also those coding for slightly (Clocel_1554) and strongly (Clocel_1562) down-regulated proteins were included in the selection. Biomass samples for this analysis were collected at the same time point for which proteomic analysis was carried out. For most of the genes selected (nine), qRT-PCR confirmed the expression profile determined by proteomic analyses ( Table 2 ). However, four genes showed an opposite expression trend, i.e., Heat shock protein Hsp90-like (Clocel_0510), Asparagine synthase (glutamine-hydrolyzing) (Clocel_3893), ATP:guanido phosphotransferase (Clocel_3761) and acyl-ACP thioesterase (Clocel_4352). In general terms, this result is not aberrant, since similar ratio (about 30%) of inconsistencies between proteomic and transcriptomic data has been observed by previous investigations on other microorganisms grown under butanol stress ( Tian et al., 2013 ; Venkataramanan et al., 2015 ). In the study by Venkataramanan et al. (2015) , comparative analysis of proteomic against two sets of transcriptomic data obtained with two different approaches (microarray and RNA-seq) was performed in C. acetobutylicum and opposite patterns between proteomic and transcriptomic results were ascribed to post-transcriptional regulation of gene expression. The present results indicate an overall good quality of our proteomic data but suggest that post-transcriptional regulation mechanisms may also occur in C. cellulovorans . Additional comments to specific genes/proteins will be given in the following sections. TABLE 2 Validation of protein expression profiles obtained by proteomic analysis through quantitative real-time PCR analysis (qRT-PCR). Differentially expressed proteins found in the present investigation will be thoroughly discussed in the next sections. Proteins Involved in Protein Translation, Folding and Degradation Cell exposure to solvents, such as butanol, induces effects which are similar to heat shock, including protein unfolding and aggregation, that is sometimes reported as proteotoxic stress ( Heipieper et al., 2007 ; Schäfer et al., 2020 ). Cell responses to solvent shock generally include modulation of the expression of proteins with housekeeping functions such as protein translation (e.g., to replace denatured proteins), folding, and degradation ( Patakova et al., 2018 ). Proteins belonging to COG category J (translation, including ribosome structure and biogenesis) and O (molecular chaperones and related functions) have generally been found as among the most differentially expressed under butanol stress ( Alsaker et al., 2010 ; Fu et al., 2013 ; Tian et al., 2013 ; Venkataramanan et al., 2015 ; del Cuenca et al., 2016 ; Sedlar et al., 2019 ). In addition, genome sequencing of a butanol-tolerant mutant of C. acetobutylicum detected a high number of point mutations in genes encoding rRNAs which likely affect the structure and function of ribosomes ( Bao et al., 2014 ). Studies on E. coli have indicated that short-chain alcohols inhibit transcription and translation processivity by proning ribosomes to misreading errors and stalling and causing aberrant termination of transcription ( Haft et al., 2014 ). Proteins involved in translation and ribosome structure In the present study, COG category J is one of the most represented (15%) among the proteins overexpressed by butanol-challenged C. cellulovorans . These proteins include 9 aminoacyl-tRNA synthetases, 14 ribosomal proteins, a translation initiation factor and a methionyl-tRNA formyltransferase which are directly involved in protein biosynthesis ( Table 1 and Supplementary Table 2 ). These results appear inconsistent with similar studies performed on a number of other bacterial models such as C. acetobutylicum ( Alsaker et al., 2010 ; Venkataramanan et al., 2015 ), C. beijerinckii ( Sedlar et al., 2019 ), Staphylococcus warneri ( Fu et al., 2013 ), and Synechocystis sp. ( Tian et al., 2013 ) which reported an enrichment of proteins belonging to COG category J among those down-regulated by butanol challenge. As far as we know, the present study is the first reporting extensive up-regulation of proteins belonging to COG category J in microorganisms challenged with butanol. Interestingly, qRT-PCR results, although obtained on a restrained number of genes belonging to COG category J, further support proteomic evidence ( Table 2 ). Down-regulation of genes/proteins involved in translation and ribosome structure has been correlated with growth inhibition observed under butanol stress ( Alsaker et al., 2010 ; Tian et al., 2013 ; Venkataramanan et al., 2015 ; Sedlar et al., 2019 ). Although C. cellulovorans growth was inhibited by butanol supplementation, category J proteins were mostly up-regulated in this microorganism, thus indicating that there is no obvious correlation between the two phenomena. However, protein content of butanol-challenged C. cellulovorans was significantly lower than that measured in control conditions all throughout the growth kinetics, thus indicating that protein translation is less efficient under solvent stress ( Figure 5 ). The latter result seems consistent with up-regulation of (p)ppGpp synthetase I (SpoT/RelA, Clocel_2071) in butanol-supplemented C. cellulovorans cultures, which suggests an increase of cellular levels of (p)ppGpp that mediates the so called stringent response ( Schäfer et al., 2020 ). (p)ppGpp is involved in bacterial response to multiple stresses, including nutrient limitation, heat shock and ethanol ( Harty et al., 2019 ; Schäfer et al., 2020 ). Increased levels of (p)ppGpp have been reported to modulate many aspects of bacterial physiology and metabolism, including inhibition of transcription, translation, GTP biosynthesis, DNA replication and microbial growth ( Potrykus and Cashel, 2008 ; Schäfer et al., 2020 ). Clocel_2071 encodes a bifunctional protein consisting of both (p)ppGpp synthetase and hydrolase domains, however, it is likely that Clocel_2071 up-regulation under butanol stress actually leads to intracellular accumulation of (p)ppGpp. Recently, a link between butanol tolerance and stringent response has also been reported in Lactobacillus mucosae ( Liu et al., 2021 ). A very recent study reported that (p)ppGpp mainly inhibits translation in Bacillus subtilis , for instance by binding to the translation initiation factor IF-2 and other ribosome-associated GTPases ( Schäfer et al., 2020 ). In addition, C. cellulovorans up-regulated proteins under butanol shock include ribosome hibernation promoting factor (Hpf, Clocel_3036) which mediates the formation of functionally inactive 100S ribosomes in bacteria (namely ribosome dimers formed through interactions between their 30S subunits), thus contributing to decrease translation rate ( Yoshida and Wada, 2014 ). Overexpression of Hpf has been considered as a hallmark of the stringent response activation in B. subtilis [its transcription is activated by (p)ppGpp] ( Schäfer et al., 2020 ). Altogether, these data suggest that translation is diminished in butanol-challenged C. cellulovorans , consistent with other investigations indicating that under proteotoxic stress microorganisms decrease translation as a mean to reduce the load on the cellular protein quality control systems (HSPs) ( Schäfer et al., 2020 ). The reason why several ribosomal proteins and other proteins involved in translation were up-regulated in butanol-challenged C. cellulovorans currently remains elusive. FIGURE 5 Protein content of cells grown in control (green) or butanol-supplemented medium (red) in different growth phases. Data are the mean of triplicate measurements. Asterisks indicate values that significantly (** p -value < 0.01) differ between the two growth conditions. The most up-regulated protein in COG class J is the RNA chaperone Hfq (Clocel_2035, FC = 3.09). Hfq is a global modulator of the activity of small regulatory RNAs (sRNAs) ( Cho et al., 2014 ). sRNAs act as major posttranscriptional regulators of gene expression through binding with target mRNAs and Hfq promotes this binding thus acting as an enhancer ( Faigenbaum-Romm et al., 2020 ). In bacteria, sRNAs are involved in regulation of gene expression regulation in response to a variety of stresses, including solvent stress ( Venkataramanan et al., 2013 ; Pei et al., 2017 ). Growth of hfq -deleted Acinetobacter baumannii was dramatically compromised when exposed to stresses including ethanol and temperature ( Kuo et al., 2017 ). In addition, the latter study demonstrated that Hfq is involved in regulation of a number of stress-related genes including groEL . Ethanol has been shown to induce up-regulation of hfq gene in Zymomonas mobilis ( Cho et al., 2017 ). Mutations in hfq gene have been identified in isobutanol hypertolerant E. coli strains obtained by adaptive evolution suggesting the role of this gene in adaptation to solvents ( Minty et al., 2011 ). Butanol stress has been reported to induce Hfq up-regulation and differential expression of 84 sRNAs (several of which were predicted to be Hfq targets) in C. acetobutylicum ( Venkataramanan et al., 2013 ). Molecular chaperones Proteins overexpressed in butanol-supplemented C. cellulovorans cultures encompass 11 members of the COG category O (molecular chaperones and related functions). HSPs comprise two major classes, namely chaperones binding to denatured or misfolded proteins and promoting refolding to their native structure, and ATP-dependent proteases which catalyze hydrolysis of irreversibly damaged proteins ( Schumann, 2016 ). In Gram positive bacteria, HSPs have mainly been studied in B. subtilis , and divided in six classes based on mechanisms that regulate their expression ( Schumann, 2003 ). Some of the proteins identified here, namely the molecular chaperones GroEL (Cpn60, Clocel_2965), GroES (Cpn10, Clocel_2966), DnaJ (Clocel_1417) and DnaK (Clocel_1416), belong to class I (i.e., HrcA-regulated) HSPs which traditionally include the bicistronic groE and the heptacistronic dnaK operons ( Figure 6 ; Schumann, 2003 ). The structure of these operons in C. cellulovorans is highly conserved with respect to B. subtilis . Up-regulation of groE operon and of dnaKJ genes upon butanol exposure has also been observed in C. acetobutylicum and C. beijerinckii ( Tomas et al., 2004 ; Sedlar et al., 2019 ). Consistent with the latter studies, up-regulated HSPs in butanol-challenged C. cellulovorans also include the molecular chaperone Hsp90 (HtpG, Clocel_0510) ( Figure 6 ). Transcription of Class I HSP genes is regulated by CIRCE (controlling inverted repeat of chaperone expression) sequences which are found in the DNA region upstream of the operon ( Schumann, 2003 ). A search for the CIRCE motif (TTAGCACTC-N9-GAGTGCTAA) in the C. cellulovorans genome identified three exact matches, namely upstream of the groE and dnaK operons and hsp90 gene. This confirms previous findings on C. acetobutylicum ( Tomas et al., 2003 ) and indicates that these genes/gene clusters belong to the same regulon even in C. cellulovorans (although hsp90 had previously been located in a different HSP class, at least in B. subtilis ) ( Schumann, 2003 ). Curiously, qRT-PCR analysis confirmed up-regulation of GroES also at the transcript level, but indicates a down-regulation of Hsp90 mRNA ( Table 2 ). C. cellulovorans overexpressed proteins under butanol challenge also include class III (i.e., CtsR-regulated) HSPs, namely ClpP (Clocel_1566) and ClpC (ATPase AAA-2 domain protein, Clocel_3760). ClpC and ClpP form the ClpCP ATP-dependent protease ( Figure 6 ) ( Schumann, 2003 ). In addition, two proteins (Clocel_3761, Clocel_3762) which do not belong to COG category O but are encoded by the same clpC operon, hence belong to the CstR regulon, are differentially expressed in the conditions tested ( Figure 6 ). In particular, the product of Clocel_3761 is up-regulated by butanol stress, while the protein encoded by Clocel_3762 is down-regulated in the same condition. The proteins encoded by Clocel_3761 and Clocel_3762 show high sequence identity with McsB (44%) and McsA (37%) from B. subtilis , respectively. McsAB are modulators of the CtsR activity, however, McsA is thought to stabilize CtsR binding to DNA (thus repressing regulated genes), while McsB phosphorylates CstR making it inactive and promotes its proteolysis by ClpCP (thus enabling transcription of genes under CstR regulation) ( Schumann, 2003 , 2016 ). Opposite expression patterns of these proteins observed in butanol-challenged C. cellulovorans seem therefore consistent with activation of transcription of genes in the CstR regulon. Both peptidase S1 and S6 chymotrypsin/Hap (Clocel_0111, FC 2.22) and HtrA2 peptidase (Clocel_1552, FC 2.18) from C. cellulovorans show some sequence identity with C. acetobutylicum HtrA (40 and 37%, respectively). HtrA belongs to class V (i.e., CssRS-regulated) HSPs and is thought to be a membrane-anchored protease acting on non-native proteins within or on the outer face of the cytoplasmic membrane ( Schumann, 2003 ). These results essentially confirm those previously obtained on butanol-challenged C. acetobutylicum ( Alsaker et al., 2010 ). Furthermore, up-regulation of HSPs has been frequently observed in butanol-challenged microorganisms ( Rutherford et al., 2010 ; Fu et al., 2013 ; Liu et al., 2021 ). FIGURE 6 The expression of several heat shock proteins (HSPs) is affected in butanol-challenged C. cellulovorans . The gene loci encoding the main C. cellulovorans HSPs are indicated in red or green depending on if their protein products were up-regulated or down-regulated in butanol challenged cultures. Products of the genes indicated in blue were not identified in the present study. Amino Acid Metabolism and Transport Almost all (namely 18 out of 21) the differentially expressed proteins involved in amino acid metabolism and transport identified in this study were up-regulated in butanol-stressed C. cellulovorans . COG category E was the second most represented class among overexpressed proteins in butanol-challenged C. cellulovorans . A similar observation was reported on butanol-exposed S. warneri ( Fu et al., 2013 ). Up-regulated enzymes are involved in the biosynthesis of a number of amino acids including proline and arginine (Clocel_2734, Clocel_3150, Clocel_1668), lysine (Clocel_1978, Clocel_3115), methionine (Clocel_1764, Clocel_2896, Clocel_3040), and branched chain amino acids (BCAA) (Clocel_1324, Clocel_1325, Clocel_0493) ( Table 1 and Supplementary Table 2 ). In addition, up-regulated proteins include four aminotransferases (Clocel_1948, Clocel_2059, Clocel_2390, Clocel_3812). The role of amino acids in the cellular stress response is a well-known concern. Increase in pyrroline-5-carboxylate reductase and intracellular levels of proline in response to butanol stress has been observed in B. subtilis ( Mahipant et al., 2017 ). A mutant proline-accumulating Saccharomyces cerevisiae is more tolerant to ethanol stress ( Takagi et al., 2005 ). Experimental evidence suggesting a role of L -proline as inhibitor of protein aggregation and chaperone for protein folding has been reported ( Samuel et al., 2008 ). Additional proline functions include: (i) osmoprotection ( Zaprasis et al., 2015 ); (ii) improvement of stability or solubility of hydrophobic macromolecules and soluble proteins ( Schobert and Tschesche, 1978 ; Samuel et al., 1997 ) and; (iii) reduction of solvent-induced membrane disorder ( Takagi et al., 2005 ). In yeasts, arginine acts both as cryo- ( Morita et al., 2002 ) and osmo-protectant ( Noti et al., 2018 ). A possible role of amino acids as osmoprotectants in butanol stress has been hypothesized ( Wang et al., 2013a ). A study on E. coli indicated that limitations in cellular levels of methionine and methionyl-tRNA could be among the mechanisms of ethanol toxicity and that methionine supplementation could increase ethanol tolerance in this bacterium ( Haft et al., 2014 ). The authors speculated that the main effect of methionine limitation is a reduction of the protein translation efficiency, owing to longer ribosome stalling at non-start AUG codons. Up-regulation of proteins involved in methionine biosynthesis in butanol-challenged C. cellulovorans suggests that higher cellular levels of methionine could be present in these conditions. It is therefore tempting to hypothesize that a phenomenon similar to that observed in E. coli could also occur in C. cellulovorans . In addition, methionine is involved in multiple other functions, e.g., it contributes to oxidative stress response and participates in several methyltransferase reactions ( Rodionov et al., 2004 ; Luo and Levine, 2009 ). Up-regulation of genes involved in BCAA biosynthesis by bacteria exposed to butanol has already been reported in C. acetobutylicum ( Alsaker et al., 2010 ; Janssen et al., 2012 ) and increased intracellular levels of BCAA have actually been measured in this bacterium under butanol stress ( Wang et al., 2016 ). The relationship between the increased levels of BCAA and butanol stress has generally been referred to the role of BCAA as primers for the synthesis of branched-chain fatty acids and the role of the latter in modulating cell membrane fluidity ( Mansilla et al., 2004 ; Alsaker et al., 2010 ; Wang et al., 2016 ). In B. subtilis , incorporation of branched-chain fatty acids in cell membrane has been identified as an alternative strategy to change membrane fluidity with respect to saturating/desaturating fatty acids ( Mansilla et al., 2004 ). It is therefore possible to hypothesize that increased levels of enzymes involved in the biosynthesis of BCAA in C. cellulovorans are involved in strategies to cope with altered membrane fluidity caused by butanol. Differential expression of proteins involved in amino acid biosynthesis and in particular their up-regulation and/or increased intracellular concentration of amino acids has been reported in a number of microorganisms (e.g., C. acetobutylicum , B. subtilis, E. coli ) exposed to butanol and other alcohol stress ( Wang et al., 2013a , 2016 ; Venkataramanan et al., 2015 ; Mahipant et al., 2017 ; Li et al., 2019 ). The present study indicates that C. cellulovorans response to butanol involves at least some elements of the stringent response, which typically includes up-regulation of amino acid biosynthesis mediated by the CodY transcriptional regulator. To understand if CodY could also be involved in up-regulation of amino acid biosynthetic enzymes in butanol-challenged C. cellulovorans , the 15-nucleotide CodY canonical consensus motif AATTTTCWGAAAATT ( Belitsky and Sonenshein, 2013 ) was searched throughout the C. cellulovorans genome. Through this analysis, 1, 9, 126, or 1386 putative CodY binding sites were identified depending on if 0, 1, 2, or 3 mismatches were allowed. It is worth remembering that CodY binding sequences, their location (upstream or within a gene coding sequence), and CodY regulation mechanisms (either negative or positive) may be highly variable. This makes computational approaches for predicting putative CodY-regulated genes hardly conclusive ( Belitsky and Sonenshein, 2013 ) as it was the case for the present study. However, it might be worth testing this hypothesis in future investigations. Cell Envelope Structure and Biogenesis The primary cell target of solvent toxicity is the cell envelope ( Heipieper et al., 2007 ). As other solvents and hydrophobic compounds, butanol compromises cell envelope structure and function, including cell wall and membrane thus requiring activation of repair responses ( Mazzoli et al., 2011 ). Among proteins overexpressed in butanol-challenged C. cellulovorans , eight enzymes were involved in different stages of peptidoglycan biosynthesis or remodeling, that is glucose-1-phosphate thymidylyltransferase (Clocel_3025), UDP- N -acetylglucosamine pyrophosphorylase (Clocel_3808), two D -alanine/ D -alanine ligases (Clocel_3085 and Clocel_0693), mur ligase (domain of unknown function DUF1727, Clocel_2906), peptidoglycan transferase (Clocel_2098), cell wall hydrolase/autolysin (Clocel_2663), and a zinc metalloprotease (Clocel_1781) ( Sangshetti et al., 2017 ). Previous evidence that UDP- N -acetylglucosamine pyrophosphorylase is overexpressed under butanol stress has been reported in S. warneri and Synechocystis sp. PCC 6803 ( Fu et al., 2013 ; Zhu et al., 2013 ). Enzymes involved in cell wall recycling and/or autolysis in clostridia have been associated with butanol tolerance ( Webster et al., 1981 ; Van Der Westhuizen et al., 1982 ; Croux et al., 1992 ). D -alanine/ D -alanine ligase was found as part of the ethanol tolerance response in Oenococcus oeni ( Silveira et al., 2004 ). Studies on Clostridium beijerinckii NRRL B-59 have suggested that increased tolerance to butanol might be associated to peptidoglycan thinning ( Linhová et al., 2010 ). Because of its chaotropic effects, butanol increases biological membrane fluidity ( Heipieper et al., 2007 ). It has been previously reported that Clostridia increase the saturated fatty acid content of cell membrane (thus decreasing its fluidity) when they are exposed to butanol ( Baer et al., 1987 , 1989 ; Huffer et al., 2011 ; Isar and Rangaswamy, 2012 ). In the present study, five proteins involved in the biosynthesis of saturated fatty acids (Clocel_4136, Clocel_4137, Clocel_4144, Clocel_4162, and Clocel_4352) and two enzymes likely involved in phospholipid biosynthesis (Clocel_1331, Clocel_1338) were identified among the proteins up-regulated by butanol. Curiously, one of the two acyl carrier proteins (Clocel_4143) encoded by the C. cellulovorans genome was down-regulated. Globally, these data indicate that fatty acid biosynthesis is improved in butanol-challenged cells, which is likely related to a change in fatty acid composition of the cell membrane. Extensive up-regulation of enzymes involved in fatty acid biosynthesis has been reported in a number of bacteria upon butanol stress, such as S. warneri ( Fu et al., 2013 ). Up-regulated proteins in butanol challenged C. cellulovorans also include two enzymes involved in the synthesis of terpene precursor isopentenyl-PP (Clocel_0126, Clocel_1782). In addition, butanol-challenged C. cellulovorans overexpresses a couple of proteins belonging to the MreB/Mrl family (Clocel_3042 and Clocel_2768) which are involved in cell shape regulation ( Egan et al., 2020 ). Changes in cell shape and/or size have been observed in several bacteria under butanol stress ( Heipieper et al., 2007 ; Fletcher et al., 2016 ). Cell elongation and filamentous growth was reported for E. coli ( Fletcher et al., 2016 ), while an increase of the cell size of P. putida and Enterobacter sp. ( Neumann et al., 2005 ) and a decrease of the cell size of Pseudomonas taiwanensis ( Halan et al., 2017 ) were observed. These modifications likely affect the surface-to-volume ratio of cells. For instance, a reduced surface-to-volume ratio is thought to diminish butanol entry into the cell ( Heipieper et al., 2007 ). Membrane Transport Differentially expressed proteins identified in this study include 16 components of membrane transporters. Most of them (14, namely Clocel_0638, Clocel_0903, Clocel_1272, Clocel_1328, Clocel_1355, Clocel_1356, Clocel_1854, Clocel_2887, Clocel_2598, Clocel_3460, Clocel_3854, Clocel_3857, Clocel_4100, Clocel_4152) were up-regulated in butanol-challenged C. cellulovorans . Currently, the protein product of Clocel_0638 does not have any associated function, however, it shows a very high sequence identity (73–75%) with some ATP-dependent permeases found in other Clostridia (e.g., Uniprot entry U2DAG1, T0N9N5 and A0A1M6LTB1 3 ). Increased levels of membrane transporters have previously been observed in other bacteria under butanol stress or as involved in butanol tolerance ( Gao et al., 2017 ; Yang et al., 2020 ). As mentioned above, butanol negatively alters cell membrane structure and function, including intrinsic proteins involved in nutrient and ion transport ( Bowles and Ellefson, 1985 ). The chaotropic effect of alcohols leads to increased membrane permeability for ions and other small solutes ( Hutkins and Kashket, 1986 ), which partially or completely abolishes transmembrane ΔpH and Δψ ( Bowles and Ellefson, 1985 ; Gottwald and Gottschalk, 1985 ; Terracciano and Kashket, 1986 ; Wang et al., 2005 ). It therefore might be hypothesized that increased levels of transporters could improve ion pumping outside the cell membrane thus compensating solvent effects and restoring homeostasis. Transporters up-regulated in butanol-stressed C. cellulovorans include two cation transporters, namely NatB (Clocel_3460) and MgtE (Clocel_1328). NatB mediates Na + extrusion from cells and was overexpressed by ethanol in B. subtilis ( Cheng et al., 1997 ) and possibly involved in ethanol tolerance in Clostridium phytofermentans ( Tolonen et al., 2015 ). MgtE is considered among the primary Mg 2+ transporters in bacteria and involved in Mg 2+ uptake ( Groisman et al., 2013 ). A mutation in a Mg 2+ transporter has been described among the genetic traits of an ethanol-hypertolerant C. phytofermentans ( Tolonen et al., 2015 ), while Mg 2+ supplementation has been shown to reduce dissipation of membrane ΔpH caused by butanol in C. beijerinckii ( Wang et al., 2005 ). It has been hypothesized that both Na + and Mg 2+ are employed by bacteria to maintain membrane ΔpH by means of Na + /H + antiporters and Mg 2+ -dependent H + -translocating ATPases ( Wang et al., 2005 ). A recent study reported that the overexpression of an ABC transporter (ButTM) belonging to the multidrug resistance (MDR) systems led to substantial increase of C. acetobutylicum tolerance to butanol ( Yang et al., 2020 ). This raised the hypothesis that ButTM acts as a solvent (butanol, ethanol) extruding pump, similar to solvent efflux pumps found in other bacteria ( Patakova et al., 2018 ), although this role was not confirmed by experimental evidence. A btrR-btrT-btrM-btrK -like gene cluster was found in the C. cellulovorans genome (Clocel_4202-5) consistently with other clostridia ( Yang et al., 2020 ) although none of these genes was up-regulated in butanol-supplemented cultures. However, another ABC-related transporter up-regulated in the present study (Clocel_2887) shows significant identity with some MDR transporters (COG category V, i.e., defense mechanisms). Finally, proteins up-regulated by butanol exposure include two components of an ABC transporter putatively involved in dipeptide/oligopeptide transport (Clocel_1355-6). This finding seems consistent with increased requirement of amino acids as suggested by the activation of pathways for amino acid biosynthesis described above. Energy Production and Conversion Five (out of nine) subunits of a V-type ATPase (V 1 subunits K, Clocel_1656; C, Clocel_1658; F, Clocel_1659; A, Clocel_1660; and B, Clocel_1661) and three (out of eight) subunits of a F-type ATPase (F 1 subunits γ, Clocel_3050; α, Clocel_3051; and δ, Clocel_3052) were overexpressed by C. cellulovorans cells grown in butanol-supplemented medium. Expression profile of Clocel_1656 and Clocel_1660 was confirmed by qRT-PCR also ( Table 2 ). Interestingly, one of the subunits of the F-type ATPase was down-regulated (i.e., F 0 subunit B, Clocel_3053). Both enzymes are reversible ATPases/synthetases since they function as molecular motors that hydrolyze or synthesize ATP depending on the physiological conditions ( Nakanishi et al., 2019 ). ATP hydrolysis is used to generate an ion (H + or Na + ) gradient across the cell membrane, while consumption of electrochemical gradient is used to synthesize ATP ( Murata et al., 2005 ; Nakanishi et al., 2018 ). Both V- and F-type ATPases share the same overall architecture consisting of a hydrophilic portion (F 1 /V 1 ) catalyzing ATP synthesis/hydrolysis and a membrane moiety responsible for ion translocation across the membrane (F o /V o ) ( Murata et al., 2005 ; Nakanishi et al., 2018 ). As mentioned above, butanol is known to inhibit membrane-bound ATPases, diminish the membrane ΔpH ( Bowles and Ellefson, 1985 ; Gottwald and Gottschalk, 1985 ; Wang et al., 2005 ) and Δψ ( Terracciano and Kashket, 1986 ), and lower intracellular pH ( Bowles and Ellefson, 1985 ; Huang et al., 1986 ; Terracciano and Kashket, 1986 ) and ATP concentration ( Bowles and Ellefson, 1985 ). Cytoplasm acidification leads to several cell damages such as enzyme denaturation, alteration of nutrient uptake, oxidative stress, depurination and depyrimidination of DNA, and disruption of amino acid pools ( Charoenbhakdi et al., 2016 ; Ju et al., 2016 ). Up-regulation of V- and F-type ATPases is likely a strategy to cope with disruption of membrane ΔpH and/or reduction of intracellular ATP pool and restore cell homeostasis. Interestingly, up- ( Ghiaci et al., 2013 ; Tian et al., 2013 ) or down- ( Mao et al., 2011 ; Fu et al., 2013 ; Liu et al., 2021 ) regulation of ATPase expression has been observed upon butanol stress depending on the microbial model. This may be related to the reversible activity of ATPases/synthetases and different physiology of microorganisms studied. Nucleotide Metabolism The amount of several enzymes involved in purine (10 proteins) and pyrimidine (7 proteins) biosynthesis was significantly lowered by butanol challenge ( Figure 7 ). This concerns nearly all the pathway enzymes from glutamine to UMP (pyrimidine metabolism, Figure 7B ). qRT-PCR analysis seems to further support this evidence since it confirmed down-regulation of the small subunit of carbamoyl-phosphate synthase (Clocel_1562) and dihydroorotase (Clocel_1554) in butanol-stressed C. cellulovorans ( Table 2 ). As regards purine metabolism, almost the entire pathway enzymes from phosphoribosyl pyrophosphate (and glutamine) to IMP and then to XMP and AMP were down-regulated ( Figure 7A ). This picture is similar to that captured by transcriptomic analysis of C. acetobutylicum after butanol challenge ( Alsaker et al., 2010 ), although a more recent proteomic analysis of this strain in the same conditions reported an enrichment of proteins involved in purine biosynthesis among overexpressed proteins ( Venkataramanan et al., 2015 ). Down-regulation of enzymes involved in pyrimidine biosynthesis has been reported also in butanol-challenged Lactiplantibacillus plantarum while effect on purine pathway enzymes is less pronounced ( Petrov et al., 2021 ). Differential regulation of genes involved in nucleotide biosynthesis has been observed after a number of chemical stresses (e.g., acetate, butyrate, NaCl) in other clostridia ( Alsaker et al., 2010 ; Philips et al., 2017 ). However, most frequently genes for pyrimidine biosynthesis were down-regulated, while those involved in purine biosynthesis were up-regulated. The reason for differential expression of genes involved in nucleotide metabolism in stress responses still remains elusive. As regards repression of XMP biosynthesis, this could be part of the stringent response mediated by (p)ppGpp, as previously described in B. subtilis ( Kriel et al., 2012 ). XMP is an intermediate of the biosynthesis of GTP ( Figure 7A ), and repression of the pathway for GTP biosynthesis by (p)ppGpp has been observed in B. subtilis . This is thought to differentially regulate transcription of genes depending on the initiating nucleotide, possibly by simple mass action (thus GTP depletion would inhibit transcription of genes that start with GTP only). It is worth noting that only enzymes involved in de novo biosynthesis of purine and pyrimidine were down-regulated in butanol-challenged C. cellulovorans cultures, while, except for the product of Clocel_3712, enzymes catalyzing nucleotide salvage pathways (i.e., that recycle nucleotide precursors obtained by degradation of polynucleotides) were not identified ( Figure 7 ). De novo nucleotide synthesis requires higher ATP/GTP expenditure than salvage pathways ( Moffatt and Ashihara, 2002 ). Hence, down-regulation of de novo nucleotide synthesis could also be a strategy to reduce energy consumption. FIGURE 7 Schematic representation of purine (A) and pyrimidine (B) metabolic pathways of C. cellulovorans . C. cellulovorans genes encoding each pathway enzyme is indicated. Red and green colors were used for gene loci whose protein product was over- or under-expressed in butanol-challenged cells, respectively. Geni loci whose protein product was identified in this study but was in similar amounts in butanol-supplemented and control cultures were indicated in black, while blue indicates geni loci whose protein product was not identified in the present investigation. Add, adenosine deaminase; Adk, adenylate kinase; AICAR, 1-(5′-Phosphoribosyl)-5-amino-4-imidazolecarboxamide; AIR, Aminoimidazole ribotide; CAIR, 1-(5-Phospho- D -ribosyl)-5-amino-4-imidazolecarboxylate; CarA, carbamoyl-phosphate synthase, small subunit; CarB, carbamoyl-phosphate synthase, large subunit; Cdd, cytidine deaminase; Cmk, cytidylate kinase; FAICAR, 1-(5′-Phosphoribosyl)-5-formamido-4-imidazolecarboxamide; FGAM, 2-(Formamido)- N1 -(5′-phosphoribosyl)acetamidine; FGAR, 5 ′ -Phosphoribosyl- N -formylglycinamide; GAR, 5′-Phosphoribosylglycinamide; GuaA, GMP synthase; GuaB, inosine-5′-monophosphate dehydrogenase; IMP, inosine monophosphate; Ndk, nucleoside-diphosphate kinase; NdrD, anaerobic ribonucleoside-triphosphate reductase; NrdJ, ribonucleoside-diphosphate reductase; P i , inorganic phosphate; Pdp, pyrimidine-nucleoside phosphorylase; PK, pyruvate kinase; PRPP, 5-phosphoribosyl 1-pyrophosphate; PRPS, ribose-phosphate pyrophosphokinase; PurB, adenylosuccinate lyase; PurC, phosphoribosylaminoimidazole-succinocarboxamide synthase; PurD, phosphoribosylamine/glycine ligase; PurE, phosphoribosylaminoimidazole carboxylase; PurF, amidophosphoribosyltransferase; PurH, phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase; PurK, 5-(carboxyamino)imidazole ribonucleotide synthase; PurL, phosphoribosylformylglycinamidine synthase; PurM, phosphoribosylformylglycinamidine cyclo-ligase; PurN, phosphoribosylamine–glycine ligase; PyrB, aspartate carbamoyltransferase; PyrC, dihydroorotase; PyrD, dihydroorotate dehydrogenase; PyrE, orotate phosphoribosyltransferase; PyrF, orotidine 5′-phosphate decarboxylase; PyrG, CTP synthase; PyrH, uridylate kinase; PyrR, uracil phosphoribosyltransferase; SAICAR, 1-(5′-Phosphoribosyl)-5-amino-4- ( N -succinocarboxamide)-imidazole; SurE, 5′-nucleotidase; Trdx, thioredoxin; Udp, uridine phosphorylase; XMP, xanthosine 5′-phosphate. Carbohydrate Transport and Metabolism Butanol challenge induces up-regulation of HPr kinase/phosphorylase (HPrK/P, Clocel_1671), a sensor enzyme involved in the regulation of sugar uptake and carbon metabolism in several bacteria ( Deutscher et al., 2014 ). HPrK/P catalyzes the ATP- and PP i -dependent phosphorylation of Ser46 of HPr, a protein of the PEP-dependent sugar phosphotransferase system (PTS), and also its dephosphorylation ( Fieulaine et al., 2001 ). Conditions leading to increase of fructose 1,6-bisphosphate (F1,6BP) concentration activate kinase activity of HPrK/P which improves the level of phosphorylation of HPr-Ser46 ( Deutscher et al., 2014 ). P-Ser46-HPr acts as a co-regulator (mainly a co-repressor) of gene transcription (e.g., genes involved in carbohydrate transport and catabolism, including glycolysis) ( Shimizu and Matsuoka, 2019 ). On the other hand, increase in intracellular levels of P i inhibits kinase activity of HPrK/P and stimulates its phosphorylase activity ( Fieulaine et al., 2001 ). In addition, a putative glucose PTS transporter (Clocel_2778) is up-regulated in butanol-challenged C. cellulovorans . The latter result seems consistent with increased specific glucose consumption observed in cells grown in butanol-supplemented conditions. However, three glycolytic enzymes, namely phosphoglycerate kinase (Clocel_0720), triose phosphate isomerase (Clocel_0721) and pyruvate phosphate dikinase (Clocel_1454) were down-regulated in butanol-challenged C. cellulovorans . Interestingly, up-regulation of some/most key glycolytic enzymes was reported in other bacteria (e.g., C. acetobutylicum , S. warneri ) in response to butanol stress ( Fu et al., 2013 ; Venkataramanan et al., 2015 ). Central Carbon Metabolism and Fermentative Pathways The amount of some key enzymes involved in C. cellulovorans fermentative pathways (e.g., acetate, butyrate and formate production) was significantly altered by butanol exposure. Acetate kinase (Ack, Clocel_1892, acetyl phosphate + ADP → acetate + ATP) was two-fold overexpressed in butanol-supplemented cultures, which is consistent with increased acetate yield observed in this condition ( Figure 2 ). It is tempting to speculate that improved acetate production could be related to increased ATP requirement under alcohol-stressed conditions. A slight Ack up-regulation was observed also in butanol-stressed C. acetobutylicum ( Alsaker et al., 2010 ). As mentioned above, a statistically significant decrease in formate yield was observed in butanol-challenged C. cellulovorans ( Figure 2 ). Proteomic analysis actually showed 2-fold down-regulation of pyruvate formate lyase (PFL)-activating enzyme (Clocel_1812) in butanol-challenged cells. Down-regulation of PFL after butanol challenge was observed also in C. acetobutylicum ( Tomas et al., 2004 ; Alsaker et al., 2010 ). In addition, a pyruvate ferredoxin oxidoreductase (PFOR, Clocel_1684) and a hydrogenase large subunit (Clocel_3813) were twofold overexpressed in butanol-grown C. cellulovorans . Previous studies have suggested that Clocel_1684 likely encodes the main PFOR of C. cellulovorans ( Ou et al., 2019 ; Usai et al., 2020 ). Its up-regulation also supports the hypothesis that a higher amount of pyruvate is metabolized through PFOR instead of PFL reaction in butanol-exposed cells. Since in the PFOR reaction, pyruvate is oxidized with concomitant reduction of ferredoxin, a higher amount of reduced ferredoxin would be available for hydrogen production in these growth conditions. C. cellulovorans genome encodes four hydrogenases. Interestingly, butanol-exposure induced up-regulation of only the product of Clocel_3813 while that encoded by Clocel_4097 was found to be up-regulated in glucose-grown C. cellulovorans ( Ou et al., 2019 ; Usai et al., 2020 ). Anaerobic bacteria generally have multiple hydrogenases whose specific function is often unclear ( Mazzoli, 2012 ). It is therefore difficult to determine the precise relationship between these particular hydrogenases and changes in their expression depending on environmental conditions. Interestingly, a similar modulation of the pyruvate node after butanol shock was reported in C. acetobutylicum featuring down-regulation of PFL and overexpression of a PFOR and a hydrogenase ( Alsaker et al., 2010 ; Venkataramanan et al., 2015 ). Proteomic analyses indicated that butyrate production pathway is inhibited in butanol-challenged C. cellulovorans . Six gene products (i.e., acetyl-CoA acetyltransferases, Clocel_0192 and Clocel_3058; 3-hydroxybutyryl-CoA dehydrogenase, Clocel_2972; electron transfer flavoprotein α and β subunits, Clocel_2973 and Clocel_2974; and phosphate butyryltransferase, Clocel_3675) out of ten involved in acetyl-CoA conversion to butyrate were down-regulated ( Figure 8 ). In addition, slight down-regulation of enoyl-CoA hydratase/isomerase (Clocel_2976) and butyrate kinase (Clocel_3674), although not statistically significant, provided a further evidence that the butyrate production pathway is repressed by butanol supplementation in C. cellulovorans . These data are inconsistent with increase in butyrate yield observed in butanol-challenged C. cellulovorans ( Figure 2 ). However, carbon flux through a pathway is not solely controlled by the concentration of pathway enzymes. Down-regulation of the butyrate pathway may have contributed to divert a higher proportion of acetyl-CoA toward acetate production and possibly fatty acid biosynthesis. Acetate/butyrate ratio was actually increased by 50% in butanol-challenged cultures ( Figure 2 ). Genes Clocel_2972-2976 form a cluster for butyryl-CoA synthesis which is similar to that found in other clostridia such as C. kluyveri and C. acetobutylicum and was demonstrated to be under the control of the redox-responsive transcriptional regulator Rex ( Hu et al., 2016 ). Actually, upstream of Clocel_2972-2976 is located a gene (Clocel_2977) which encodes a protein that shares 71% sequence identity with C. acetobutylicum Rex. A search for the Clostridium Rex DNA binding element (TTGTTAANNNNTTAACAA) identified two motif instances upstream (–23 and –88 from Clocel_2976 transcription start site) of the Clocel_2972-2976 gene cluster further supporting the hypothesis that Rex regulates its expression. Additional putative Rex binding motif instances were found upstream of acetyl-CoA acetyltransferase encoding genes (Clocel_0192 and Clocel_3058) but not upstream of genes encoding butyrate kinase and phosphate butyryltransferase (Clocel_3674 and Clocel_3675, respectively). These data suggest that Rex may be involved in the regulation of gene expression in butanol-challenged C. cellulovorans . Interestingly, it was recently reported that the Rex regulon was not affected by butanol stress in C. acetobutylicum ( Venkataramanan et al., 2015 ). However, up-regulation of the butyryl-CoA formation operon ( bcd , etfAB , crt ) and thiolase after butanol shock was reported, which corresponds to an opposite trend with respect to what observed in the present study on C. cellulovorans . It is worth remembering that Rex is a gene transcription repressor whose activity depends on low intracellular NADH/NAD + ratio ( Ravcheev et al., 2012 ). These results therefore indicate a different regulation of the butyryl-CoA formation operon between C. cellulovorans and C. acetobutylicum which may reflect different redox states, namely a more oxidized cell status for C. cellulovorans . FIGURE 8 The acetyl-CoA–butyrate pathway is down-regulated in butanol-challenged C. cellulovorans . Gene loci encoding the pathway enzymes are represented in colors indicating their level of relative expression in butanol-supplemented cultures (green, down-regulated; black, identified but not differentially expressed, blue, not identified). Abbreviations: Bcd/EftAB, butyryl-CoA dehydrogenase/electron transfer protein; Buk, butyrate kinase; Crt, crotonase; Fd, ferredoxin; H 2 ase, hydrogenase; Hbd, 3-hydroxybutyryl-CoA dehydrogenase; Pbt, phosphate butyryltransferase; Thl, thiolase. In addition, two alcohol dehydrogenases (ADHs, encoded by Clocel_4197 and Clocel_1949) were two-fold overexpressed in butanol-enriched cultures. The C. cellulovorans genome encodes seven putative ADHs whose function has not been determined in detail yet. Interestingly, the products of Clocel_4197 and Clocel_1949 were not identified in the proteome of glucose- or avicel-grown C. cellulovorans which suggests that they are not biosynthesized in high amounts in physiological conditions ( Usai et al., 2020 ). A number of studies have identified ADHs as involved in conferring alcohol resistance to bacteria since they were overexpressed under alcohol-stress ( Rutherford et al., 2010 ) or their deletion or mutation in cofactor (NADH/NADPH) specificity improved alcohol tolerance ( Brown et al., 2011 ; Dai et al., 2012 ; Tian et al., 2019 ; Mazzoli and Olson, 2020 ). Recently, butanol dehydrogenase activity has been hypothesized for the product of both Clocel_4197 and Clocel_1949 ( Wen et al., 2019 ) although this requires experimental confirmation. However, no significant butanol consumption by C. cellulovorans was detected in the present investigation (data not shown). Currently, an explanation for the up-regulation of these proteins in butanol-challenged C. cellulovorans is therefore elusive. A more detailed characterization of C. cellulovorans ADHs, with particular attention on those encoded by Clocel_4197 and Clocel_1949, aimed at determining their substrate(s), cofactor(s) and metabolic role(s) seems important for improving understanding of C. cellulovorans physiology and strategies to tolerate butanol. Oxidative Stress Response and Redox Balance Overexpression of proteins involved in oxidative stress response under solvent (e.g., ethanol, butanol) stress has been observed in different microorganisms ( Rutherford et al., 2010 ; Zhu et al., 2013 ; Venkataramanan et al., 2015 ; Charoenbhakdi et al., 2016 ). Proteins up-regulated by butanol in C. cellulovorans include a desulfoferrodoxin (Clocel_4154) and a nitroreductase (Clocel_4148). Desulfoferredoxin catalyzes NAD(P)H-dependent reduction of superoxide anion to hydrogen peroxide ( Riebe et al., 2007 ) and was found among up-regulated proteins also in butanol-challenged C. acetobutylicum ( Alsaker et al., 2010 ). Nitroreductase, a flavoprotein that catalyzes NAD(P)H-dependent reduction of substrates, has sometimes been associated with oxidative stress response ( Roldán et al., 2008 ; Rutherford et al., 2010 ; Williams et al., 2015 ). In addition, two enzymes of the tricarboxylic (TCA) cycle, namely citrate synthase (Clocel_3688) and isocitrate dehydrogenase (Clocel_2469) were up-regulated in butanol-challenged C. cellulovorans . Up-regulation of isocitrate dehydrogenase was reported as part of the butanol response of other gram positive bacteria ( Alsaker et al., 2010 ; Fu et al., 2013 ). The function of the TCA cycle in Clostridia has mainly been associated with production of intermediates for biosynthetic routes and regulation of the redox balance (namely, isocitrate dehydrogenase reaction generates NADH) ( Shinohara et al., 2013 ). Determination of Cellular ATP Content Solvents, including butanol, are generally thought to decrease cellular ATP concentration by a number of mechanisms, namely by: (i) increasing membrane permeability to ATP; (ii) inhibiting membrane-bound ATPases; (iii) increasing membrane permeability to protons and other ions thus leading to dissipation of the proton motive force; (iv) inducing energy-consuming adaptation mechanisms (e.g., efflux systems) ( Bowles and Ellefson, 1985 ; Heipieper et al., 2007 ). In addition, the present study revealed that a number of proteins directly involved in ATP synthesis (acetate kinase and several subunits of both V-type and F-type ATPases) were up-regulated in butanol-challenged C. cellulovorans . However, determination of cellular ATP content showed that ATP levels were generally similar in control- and butanol-supplemented cultures of C. cellulovorans ( Figure 9 ). Interestingly, a transient significant increase of ATP levels was observed in butanol-challenged C. cellulovorans 3 h after inoculum. Although an explanation for this observation currently remains elusive, a similar phenomenon was previously reported in E. coli in response to temperature upshift ( Soini et al., 2005 ). It is worth remembering that transient increase of ATP concentration in E. coli did not correspond to improvement of energy charge but rather to a decrease of this parameter, since a higher increase of ADP level occurred concomitantly. It has been speculated that transient ATP level increase in E. coli could be a strategy to cope with higher ATP consumption for protein- and DNA-repair mechanisms ( Soini et al., 2005 ). Although solvent effects on the activity or expression of proteins involved in ATP synthesis/consumption have been reported by several studies ( Heipieper et al., 2007 ; Cao et al., 2017 ), direct determination of ATP content of cells exposed to solvents have seldom been determined. In ethanol-challenged Arthrobacter simplex , a decrease of ATP content with respect to control conditions was observed for ethanol concentrations higher than 4% (v/v) ( Luo et al., 2018 ). However, in the solvent-tolerant bacterium Pseudomonas putida DOT-T1E, no significant difference in ATP content was detected during fermentation with or without 1-decanol ( Neumann et al., 2006 ). It has been hypothesized that maintenance of unchanged levels of ATP and metabolic energy in P. putida in the presence of solvents was likely obtained at the expense of reduced growth efficiency. This explanation could also apply to the present observations on C. cellulovorans . FIGURE 9 Growth curve and intracellular ATP content of C. cellulovorans grown in control condition (green) or in butanol-supplemented medium (red). Bars represent standard deviations ( n = 3). Asterisks indicate values that significantly ( ∗ p -value < 0.01) differ between the two growth conditions."
} | 19,032 |
32206719 | PMC7080450 | pmc | 5,730 | {
"abstract": "We review recent advances and prospects of interfacing functional nanomaterials with biological systems for nanobiohybrids.",
"introduction": "INTRODUCTION For billions of years, life has been constantly evolving to adapt to everchanging environmental niches on Earth. Changes in nutrient level, geographic location, temperature, pressure, moisture, salinity, or pH can alter biological functions and cause mutations to genetic information, which leads to the evolution of life ( 1 ). A fascinating yet challenging-to-overcome example is the antibiotic resistance developed by different strains of deadly bacteria in response to prolonged exposure to antibiotics ( 2 ). In the 1970s, genetic engineering was first introduced as a man-made, highly potent strategy for modifying life at the molecular level. Recent developments in precision genome engineering, such as zinc finger proteins ( 3 ) and CRISPRs ( 4 ), have substantially enhanced the versatility of gene editing. Nevertheless, these techniques still face several challenges including high levels of complexity, noise, epigenetics, and mutations; difficulties in characterization, standardization, and modularity; and a risk of accidental release into the wild, among other things ( 5 ). Moreover, ethical boundaries ( 6 ) have not yet been agreed on, which recently led to the first embryonic genetic modification of humans ( 7 ). In addition, genetic modification is currently irreversible, meaning that genetically modified organisms (GMOs) cannot return to their native state, which has raised notable social concerns, such as the recent debate about GMOs in agriculture ( 8 ). The rapid explosion of materials research and nanotechnology in the past few decades has recently allowed us to explore alternative strategies for enhancing existing, or enabling completely new, functions within biological systems. With careful material design and construction, many synthetic materials have been successfully coupled with biological systems, demonstrating new extrinsic functional properties that surpass many existing natural capabilities of biosystems, such as the adaptation to fatal environments ( 9 – 11 ), the ability to prolong life cycles ( 12 ), and photosynthesis in nonphotosynthetic species ( 13 ). Biological species have gone through millions of years of evolution to achieve many of their biofunctionalities; however, given the amazing compositional and structural diversity of advanced synthetic materials, it is expected that strategies for the integration of functional synthetic materials with biological systems for the design and engineering of nanobiohybrids are a more rapid, powerful, and cost-effective alternative than natural evolution or genetic engineering. Notable effort in the past decade has been devoted to the design of nanobiohybrid systems, which broadly encompasses composite materials that have both a biologically derived component and a synthetic component. The biological component can be anything from purified biomolecules ( 14 ) (e.g., DNA and proteins) to complex biological systems ( 11 , 15 ) (e.g., living cells, tissues, and organisms), while the synthetic component can be inorganic materials ( 16 , 17 ) (e.g., carbon materials, CaCO 3 , SiO 2 , Au, and iron oxide), organic materials ( 18 , 19 ) (e.g., polymers and lipids), or hybrid materials ( 14 , 20 ) [e.g., metal-organic frameworks (MOFs) and metal-phenolic networks (MPNs)]. In these nanobiohybrid systems, the choice of both biological and synthetic components has an impact on different aspects of the final biofunctionality ( Fig. 1 ). Fig. 1 Interfacing functional nanomaterials with biological systems for enabling new biological functions or augmenting existing biological functions. Here, we investigate the recent synthetic materials and engineering efforts surrounding the construction of advanced nanobiohybrid systems for augmenting biofunctionality. Although many functional materials have been widely exploited for biotechnological applications such as biosensing and in vivo drug delivery (e.g., inorganic or hydrogel particles hosting bioactive macromolecules), in this review, we instead primarily focus on materials that introduce novel functionalities to endow or augment the properties of the biological component. Construction of nanobiohybrids generally follows two routes: (i) endogenous (internal) bioaugmentation: engineering synthetic materials inside biosystems, and (ii) exogenous (external) bioaugmentation: engineering synthetic materials outside biosystems ( Fig. 1 ). In the first section, we focus on the materials used for constructing nanobiohybrid systems, ranging from inorganic materials to polymers to hybrid materials, while in the second section, we focus on the enhanced/novel biological functionality arising from the nanobiohybrid systems (see table S1). In the last section, we provide an overview and outlook of the possible synthetic methods and technologies that could be adapted for future bioaugmentation applications."
} | 1,254 |
39429883 | PMC11487568 | pmc | 5,731 | {
"abstract": "Abstract The ruminal microbiota generates biogenic methane in ruminants. However, the role of host genetics in modifying ruminal microbiota‐mediated methane emissions remains mysterious, which has severely hindered the emission control of this notorious greenhouse gas. Here, we uncover the host genetic basis of rumen microorganisms by genome‐ and transcriptome‐wide association studies with matched genome, rumen transcriptome, and microbiome data from a cohort of 574 Holstein cattle. Heritability estimation revealed that approximately 70% of microbial taxa had significant heritability, but only 43 genetic variants with significant association with 22 microbial taxa were identified through a genome‐wide association study (GWAS). In contrast, the transcriptome‐wide association study (TWAS) of rumen microbiota detected 28,260 significant gene–microbe associations, involving 210 taxa and 4652 unique genes. On average, host genetic factors explained approximately 28% of the microbial abundance variance, while rumen gene expression explained 43%. In addition, we highlighted that TWAS exhibits a strong advantage in detecting gene expression and phenotypic trait associations in direct effector organs. For methanogenic archaea, only one significant signal was detected by GWAS, whereas the TWAS obtained 1703 significant associated host genes. By combining multiple correlation analyses based on these host TWAS genes, rumen microbiota, and volatile fatty acids, we observed that substrate hydrogen metabolism is an essential factor linking host–microbe interactions in methanogenesis. Overall, these findings provide valuable guidelines for mitigating methane emissions through genetic regulation and microbial management strategies in ruminants.",
"conclusion": "CONCLUSION We systematically evaluated the effect of host genetic variants and rumen gene expressions on bovine rumen microbial abundance variation. We found that a more direct relationship between gene expression in rumen effector organs and rumen microbiota abundance. Our results highlight that TWAS is a promising method for determining the host and microbiota associations at gene expression level. By combining multiple relationship networks (genes–taxa–VFAs), we observed that host–microbe interactions in the rumen methanogenesis are primarily involved in substrate hydrogen metabolism and transport. Overall, these findings provide novel insights into the host–microbiome interactions in methanogenesis and offer valuable guidelines for genetic regulation and microbial management strategies to mitigate methane emissions in ruminants.",
"introduction": "INTRODUCTION Methane is one of the six greenhouse gases that is second only to carbon dioxide in its performance for global warming [ 1 ]. Mitigating methane emissions from livestock production is crucial to achieve carbon neutrality in China [ 2 ]. The rumen microbiota of ruminants is responsible for the production of methane and contributes about 18% of its total anthropogenic emissions [ 3 ]. Cattle, as crucial domestic ruminant, contribute the majority of livestock production emissions of methane [ 4 , 5 ], which is attributed to their strong fermentation function of rumen microorganisms [ 6 ]. Methane production is closely related to the abundance of methanogenic archaea in the rumen, which are mainly from the Methanobrevibacter genus [ 7 ]. The abundances of methanogens belonging to the Methanobrevibacter genus (e.g., M. gottschalkii , M. smithii , M. boviskoreani , M. millerae , and M. thaurei ) were positively correlated with methane emissions [ 8 , 9 , 10 , 11 , 12 , 13 ], which are common hydrogenotrophic methanogenic archaea usually utilizes hydrogen (H 2 ) and carbon dioxide (CO 2 ) produced from microbial fermentation as substrates to produce methane (CH 4 ) [ 10 , 14 ]. In addition to its negative environmental impact, enteric methane emissions result in a 2%–12% loss of gross energy intake for the host [ 15 , 16 ]. Therefore, it has extremely been desirable to find methods to modulate the rumen microbiome to reduce methane emissions of ruminants all the time. The gut microbiota is shaped by diet, host, environment, and other factors. The regulation of the gut microbiota by host‐derived molecules provides a new perspective for understanding host–microbe interactions [ 17 ]. An increasing number of studies have highlighted the important role of host genetics on gut microbiota in human and animals [ 18 , 19 ]. Heritability estimation of gut microbiota helps to understand the proportion of host genetic factors that explain changes in microbial abundance. According to TwinsUK population studies, 5.3%–8.8% of bacterial taxa have heritability estimates greater than 0.2 in stool samples [ 20 , 21 ]. The host genetic factors of ruminants seem to have a greater impact on the rumen microbiota. According to previous studies, approximately 34% of microbial taxa and 64% core genera had significant heritability from a cohort of 709 beef cattle and 1150 male sheep lambs [ 22 , 23 ]. In addition, the rumen Methanobrevibacter genus of dairy cows also has been reported to have moderate heritability ( h \n 2 = 0.22) [ 24 ]. In recent years, numerous studies have focused on identifying the associations between host genetics variants and microbial abundance variation via genome‐wide association study (GWAS) using the microbiome as complex traits [ 25 , 26 , 27 , 28 ]. Using GWAS, researchers have identified many single nucleotide polymorphisms (SNPs) in cattle, which are related to microbiota composition, feed efficiency, host immunity, and metabolism [ 22 , 29 , 30 ]. However, although methane emissions and methanogen abundance have been reported to have moderate heritability, no major loci have been identified in multiple studies through GWAS [ 22 , 29 , 31 ]. Compared to GWAS, transcriptome‐wide association studies (TWAS) have been developed to interpret the relationship between gene expression and phenotype, which is of great value for explaining the genetic basis of complex phenotypes as well as providing gene‐level associations and have been conducted across various traits and tissues [ 32 ]. Interestingly, establishing the correlations between host genes and rumen methanogens via the TWAS approach is a promising strategy for host regulation of methane emission. Notably, in most studies, TWAS tests usually involve genetically predicted expression using summary data rather than dynamic correlations between gene expression of the effector organs and phenotypes from paired samples, which may cause false hits and bias [ 33 ]. At present, a number of studies have integrated GWAS and TWAS using paired samples to identify molecular markers related to agronomic traits in plants [ 34 , 35 , 36 ]. However, to the best of our knowledge, the integrated GWAS and TWAS analyses of large‐scale paired samples have not been used in animal studies thus far, especially in gastrointestinal microorganisms. In this study, we aim to bridge the host and microbiome in rumen methanogenesis. We hypothesized that the relationship between rumen genes and rumen microbes is more direct than host genetic factors, and the host influences microbial changes by driving rumen gene expression, thereby regulating methane emissions. To address these hypotheses, we conducted matched genome, transcriptome, and microbiome sequencing of the rumen through a single large‐scale cohort of 574 Holstein cattle and performed genome‐wide association study of microbiota (mbGWAS) and transcriptome‐wide association study of microbiota (mbTWAS) to identify the genetic variants and rumen genes influencing the rumen microbiota (Figure 1A ). These works will explore the possibility of genetically regulating rumen microbiota to mitigate methane emissions in cattle and broaden our insights into the potential mechanisms of host–microbe interactions in rumen methanogenesis. Figure 1 Study design and the composition and community structure of rumen microbiota. (A) Workflow of the integrated rumen genome, transcriptome and microbiome to uncover the host genetic basis of rumen microbiota. The composition and abundance of bacterial taxa at the phylum (B) and genus (C) levels. The composition and abundance of archaeal taxa at the phylum (D) and species (E) levels. (F, G) The ⍺ diversity was determined using chao1 and Shannon indices. (H, I) The β diversity principal coordinates analysis plot based on Bray–Curtis distances for bacteria and archaea at genus level. (J) Interaction networks of taxa at the genus level. Only correlation coefficients <− 0.5 or > 0.5 and adjusted p values <0.05 are displayed. The node size represents the average relative abundance. mbGWAS, genome‐wide association study of microbiota; mbTWAS, transcriptome‐wide association study of microbiota; rRNA, ribosomal RNA; SNP, single nucleotide polymorphism.",
"discussion": "DISCUSSION Methane emissions from ruminant livestock contribute a large amount of agricultural greenhouse gas, so a great deal of research has been devoted to finding mitigation strategies. In the past few years, although an increasing number of studies have been conducted to assess the associations among host genetics, rumen microbiota, and methane emission, whether methane emissions could be modulated through the genetic effects of cattle on the ruminal microbiota is still disputed [ 24 , 29 , 31 ]. Moreover, previous studies have not elucidated the relationship between microbes and gene expression in rumen effector organs. Therefore, the host‐microbiome interactions, especially in methanogenesis, remain largely unknown. To better understand this issue, for the first time, we integrated GWAS and TWAS analysis of rumen microbiota in large‐scale population to examine the regulation relationship between host and methanogens. We identified several candidate rumen genes and microbes involved in methanogenesis by combining multiple correlation analyses among host rumen genes, rumen microbes, and VFAs with methanogenic archaea. These preliminary results will provide crucial guidelines for regulating methane emissions through genetic and microbial management strategies. Heritability estimation helps to understand the extent to which host genetics contributes to phenotypic variation. In this study, we found that the heritability of the rumen core microbiota may be nearly universal, with approximately 70% of rumen taxa exhibiting significant heritability, which is consistent with a recent study in sheep [ 23 ]. Previous studies confirmed that CH 4 production is influenced by host genetics and indicated that methane emissions are moderately heritable, with host genetics able to explain 19%–33% of the phenotypic variation [ 24 , 29 , 31 ]. Our results of heritability estimation shown that the rumen archaea also have significant heritability and the average heritability was 0.41, such as M. gottschalkii ( h \n 2 = 0.47), M. boviskoreani ( h \n 2 = 0.33), and M. millerae ( h \n 2 = 0.27) (Figure 2E,H ), which provides important clues for us to search for host genetic markers regulating methanogenic archaeal abundance without directly measuring methane. However, no SNPs passed the significance threshold in the GWAS analysis for methanogens belonging to Methanobrevibacter genus. This conclusion is consistent with the findings of several large‐scale cohorts about host genetic influences on the rumen microbiota in cattle [ 22 , 29 , 43 ]. Given that in multiple studies all over the world, no major signal sites associated with methane emissions and methanogenic archaea abundance have been identified. Hence, we speculate that methane production may be a complex phenotype controlled by host microeffect polygenes, only the GWAS method may not be able to find effective genetic markers. Due to the collected matched transcriptome and microbiome data, this limitation could be addressed by using TWAS, which directly identifies the genes significantly associated with complex traits [ 32 ]. In our study, we are the first to construct TWAS on rumen microbiota, and our TWAS detected 28,260 significant gene‐microbe associations ( p < 3 × 10 −6 ), which involved 4652 unique genes associated with 210 microbes. These host rumen epithelial genes provide us with an unprecedented opportunity to study host–microbe interactions in methanogenesis. We further integrated a triple relationship network among host rumen genes, rumen microbiota, and VFAs with methanogens to explore the valuable genes and microbes participating in the methanogenesis pathway. We found that the four archaea species belong to Methanobrevibacter genus (e.g., M. gottschalkii , M. boviskoreani , M. millerae , and M. thaueri ) were positively correlated with acetic acid and A/P ratio but negatively correlated with propionic acid, which are mainly hydrogenotrophic methanogens [ 10 ]. During the hydrogenotrophic methanogenesis pathway, methanogens generally convert the fermentation products H 2 and CO 2 as substrates to methane [ 14 ]. Therefore, to some extent, a promising strategy is to modulate the supply of substrate H 2 to reduce methane production. In our study, we discovered that some taxa (e.g., Oscillospirales_UCG‐011 , Christensenellaceae_R‐7_group , and Oscillospiraceae_NK4A214_group ) were positively correlated with four methanogens, acetic acid, and acetic to propionic ratio (A/P), which may be potential acetic acid‐producing bacteria. These acetic acid fermentation microbes produced a large amount of hydrogen (H 2 ), which could be used by methanogenic archaea to produce methane. On the contrary, other taxa (e.g., Prevotella , Prevotellaceae_UCG‐003 , and Anaeroplasma ) were negatively correlated with four methanogens but positively correlated with propionic acid (Figures 5A and 6 ). Numerous studies have manifested a strongly negative correlation between the abundance of Prevotella and methane emissions and suggest that members of the Prevotella genus have the ability to utilize hydrogen toward propionic acid production and away from methanogenesis [ 42 , 44 , 45 ]. Our TWAS and correlation analysis results showed that there were significantly related relationships between host rumen epithelial genes and rumen microbiota. The rumen epithelium plays an important role in VFA absorption, metabolism, and H + transport [ 46 , 47 ]. Generally, the metabolic hydrogen ([H]) in the rumen undergoes reoxidation of reduced cofactors by hydrogenases and also transfers electrons to H + to form H 2 (molecular hydrogen), which is intercepted by methanogens for the production of methane [ 40 , 48 , 49 ]. Therefore, we speculated that host–microbe interactions in energy metabolism and methane production primarily occur through H + exchange and transport. In our current study, we identified 252 genes associated with rumen fermentation bacteria and methanogenic archaea that were significantly enriched in metabolic pathways. Among these, we observed several positively related genes with acetic acid‐producing bacteria and four methanogens, which enriched in starch and sucrose metabolism pathways (e.g., AMY2B , PYGB , PYGM , GYG1 , and MGAT4A ). A previous study found that the starch‐rich diet enriched for amylolytic bacteria, enhanced propionate production through the acrylate pathway with lactate as an intermediate, helping to maintain a healthy rumen and decrease the production of H 2 available for methanogenesis [ 50 ]. We thereby speculate host amylase and glycogen phosphorylase‐related genes could competitively degrade starch and glycogen, which reduces the niche advantage of starch‐utilizing bacteria and increases opportunities for acetate‐producing microorganisms to acquire nutrients. This promotes a shift in rumen fermentation toward acetate‐type fermentation, producing more available hydrogen being used by methanogens to produce methane. In addition, rumen carbohydrates fermentation is accompanied by the metabolism and transport of hydrogen, these H 2 occurred reversible oxidation under the action of microbial hydrogenases via the reaction H 2 → 2H + + 2e − [ 50 , 51 ]. Subsequently, these H + enter the mitochondrial electron respiratory chain of host rumen epithelial cells through the proton gradient and participate in energy metabolism. Furthermore, we discovered a large number of protease genes, such as peroxidase (e.g., GPX3 and PRDX6 ), coenzyme (e.g., COQ2 ), cytochrome c oxidase (e.g., COX5A and COX6B1 ), and ATPase (e.g., ATP5ME , ATP6V0A4 , ATP5F1E , and ATP6V1B1 ), which were positively correlated propionic acid‐producing bacteria and negatively correlated with four methanogens (Figure 6 ). These genes are involved in the mitochondrial respiratory chain for ATP synthesis by mediating electron transfer and proton transport across respiratory chain complexes. The respiratory chain complexes I and III generate superoxide (O 2 \n − ) and hydrogen peroxide (H 2 O 2 ) from molecular oxygen (O 2 ), which is the chief reactive oxygen species (ROS) in mitochondria [ 52 ]. The GPX3 gene and PRDX6 gene are important peroxisomal enzymes that catalyze the degradation of hydrogen peroxide into water, thereby controlling mitochondrial ROS levels. This process is accompanied by redox reactions and electron transport of NADH/NAD + in complex I [ 53 ]. COX is the terminal enzyme of the mitochondrial respiratory chain, reduces oxygen (O 2 ) to water, thus contributing to the generation of the electrochemical proton gradient to drive ATP synthesis [ 54 ]. COX5A and COX6B1 are the subunits of cytochrome oxidase involved in mitochondrial electron transport and plays a vital protective role in mitochondrial dysfunction, oxidative stress, and cell apoptosis [ 55 , 56 ]. ATP5ME , ATP5F1E , ATP6V0A4 , and ATP6V1B1 genes are mitochondrial ATP synthase that predominantly utilizes the H + proton gradient for ATP synthesis from ADP and phosphate ions [ 57 ]. These above results indicated that host epithelial cells will competitively consume H + to participate in the mitochondrial respiratory chain for energy metabolism, resulting in decreased the availability of substrate hydrogen for methane production (Figure 6 ). This possible mechanism consistent with the conclusion previously reported that cows with high feed efficiency have a lower abundance of the Methanobrevibacter genus [ 58 ]. In summary, these findings suggest that host–microbiome interactions in methanogenesis are influenced mainly by hydrogen metabolism. More research will be needed to elucidate these complex regulatory mechanisms in future. However, there were some limitations of our study. First, the microbiome data were obtained from 16S rRNA gene amplicon sequencing, and microbial genes and functions were not elucidated. Second, the relationship between methanogens and methane emissions is not clear, so the real methane emission data of some individuals should be determined as a prior in future studies. Nevertheless, as the most abundant methanogens, the Methanobrevibacter genus was widely suggested to contribute to methane emissions [ 10 ]. Notably, previous studies have suggested that propionate‐type fermentation was correlated with low methane emissions, while the acetate‐type fermentation was correlated with high methane emissions [ 59 ]. We found that the four methanogens (members of the Methanobrevibacter genus) were positively correlated with acetate and negatively correlated with propionate, which suggested a potential link between methane emission and methanogenic archaea abundance. Last but not least, our study used a single cohort, and whether these findings are generally applicable to ruminants need to be verified with different cattle populations and more other ruminant species."
} | 4,970 |
32313819 | null | s2 | 5,734 | {
"abstract": "A biofilm is a multicellular consortium of surface associated microbes surrounded by a hydrated, extracellular polymer matrix. The biofilm matrix plays a critical role in preventing desiccation, acquiring nutrients, and provides community protection from environmental assaults. Importantly, biofilms are significantly more resistant to antimicrobials relative to their free-swimming counterparts. The level of antimicrobial tolerance is influenced by a number of factors, including genetic/adaptive resistance mechanisms, stage of biofilm development, and pharmacokinetics of the antibiotic. Here, we describe an "
} | 153 |
35165204 | PMC8892325 | pmc | 5,735 | {
"abstract": "Significance Seagrass meadows colonize shallow coastlines around the world and represent sites of intense carbon cycling. Due to their capacity to produce methane, seagrass ecosystems constitute net sources of methane to the atmosphere. Here, we identify key processes and microorganisms responsible for methane formation in seagrass-covered sediments in the Mediterranean Sea. Our work shows that methane is solely formed from methylated compounds that are produced and released by the plant itself. Due to the persistence of these compounds in buried plant material, microbial methane production continues long after the death of the living plant. These results provide a comprehensive understanding of methane production in seagrass habitats, thereby contributing to our knowledge on these important blue carbon ecosystems.",
"conclusion": "Conclusions Our work shows that the high methane fluxes from seagrass sediments are a result of high rates of methylotrophic methanogenesis and a fast advective and plant-mediated transport, combined with an inefficient microbial methane filter in these sediments. High rates of methane production observed in dead seagrass sediments might be explained by a persistent input of various simple as well as larger methylated compounds through the buried plant rhizomes and deposition of fresh leaf debris. As seagrass habitats are declining around the world due to increased eutrophication and physical disturbances of their habitats, these ecosystems lose their ability to sequester carbon dioxide from the atmosphere. However, the capacity of these sediments to produce methane may persist long after the meadow die-off, thus continuing to offset the blue carbon function of these ecosystems in the long run.",
"discussion": "Discussion Methane Fluxes from P. oceanica Seagrass Meadows. P. oceanica is the dominant seagrass species in the Mediterranean Sea and covers at least 11,687 km 2 of its coastline ( 38 ) ( SI Appendix , Fig. S2 A ). We show that the P. oceanica –covered sediments off the island of Elba were a net source of methane to the water column. The measured net flux of methane from whole-core incubations was comparable to the flux from depth-integrated methane production rate measurements in nonamended sediment incubations; however, the fluxes varied strongly between the investigated cores (4.6 to 490 µmol ⋅ m −2 ⋅ d −1 ). It is notable that the methane emissions into the water column and the methane-producing potential of the dead seagrass sediment remained largely comparable to that of the living seagrass meadow (median of 142 µmol ⋅ m −2 ⋅ d −1 versus 106 µmol ⋅ m −2 ⋅ d −1 , respectively; SI Appendix , Fig. S7 ). This suggests that methane production can be sustained in seagrass sediments long after the living plant’s disappearance. We propose that this observation can be explained by the persistence of methylated compounds in the plant’s tissue, as for example choline, betaines and DMSP were found in high concentrations also in partially degraded rhizomes and leaves buried in or lying on top of the dead seagrass sediments ( Fig. 2 and SI Appendix , Table S3 ). It should be noted that our measured methane fluxes represent dark fluxes and do not account for potential changes associated with the plant’s diel cycle. However, current reports suggest that methane emissions from seagrass-covered sediments might be independent of light or dark conditions ( 30 , 31 ). Although these fluxes represent net fluxes that account for methane oxidation, the gross methane fluxes are likely not substantially different because the rates of methane oxidation in these sediments were lower than the methanogenesis rates. This indicates that the microbial methane filter in these sediments is rather inefficient and cannot substantially mitigate methane emissions into the water column. Our measured methane fluxes from P. oceanica meadows (upscaled 0.0003 to 0.033 Tg CH 4 ⋅ yr −1 for the Mediterranean Sea) are among the highest reported to date from seagrass meadows ( 30 , 31 , 39 ). Methane appears to be efficiently transported out of the sediment through advective and/or plant-mediated transport ( SI Appendix , Texts S1 and S3 ) as indicated by the low methane concentrations in the vegetated sediment (despite high methane production rates) and the lack of structure in the methane concentration profile. Our flux measurements captured the diffusive transport of methane from the sediment but did not account for advective and plant-mediated transport processes or the effects of waves and water movement on methane exchange between the sediment and the water column. Therefore, our measurements may underestimate the total methane emissions from Posidonia -covered sediments. Due to the shallow water depths of the seagrass beds (as a consequence of their dependence on light) and the generally low methane oxidation rates in the mixed water column ( 40 ), the emitted methane will likely efficiently exchange between the water column and the atmosphere, similar to other settings ( 41 ). It has been pointed out previously that the methane emissions from seagrass-covered sediments into the water column partially offset the effect of CO 2 uptake by the plant and therefore affect the blue carbon function of these ecosystems (see discussion in SI Appendix , Text S2 ). Microbial Processes Underlying Methane Emissions from P. oceanica Meadows. The ability of seagrass ecosystems to produce methane is typically assigned to their capacity to release high amounts of labile organic carbon into the underlying sediments ( 31 ). Through their degradation, methanogenic substrates such as hydrogen and acetate can be produced. Posidonia seagrasses additionally bury large amounts of plant material in the form of massive underground peat deposits ( 33 ) ( SI Appendix , Fig. S3 A ) a feature analogous to terrestrial peatlands, another recognized source of methane to the atmosphere ( 42 ). In terrestrial peats, the predominant modes of methane production are acetoclastic and hydrogenotrophic methanogenesis ( 43 ). On the contrary, our substrate-addition experiments with P. oceanica –covered sediments showed that no methane was produced from competitive substrates, such as hydrogen or acetate, as these substrates were likely used up by the abundant sulfate-reducing Deltaproteobacteria ( Fig. 3 ). This highlights an interesting difference between microbial methane production in marine and terrestrial peat ecosystems. Methylotrophic methanogenesis was the sole detected pathway of methane production in vegetated as well as in dead seagrass sediments ( Fig. 2 ). This agrees with observations from other vegetated marine sediments, such as salt marshes ( 20 ) and intertidal sediment containing algal detritus ( 18 , 44 ), that methanogenesis is mainly fueled by noncompetitive substrates, which are largely inaccessible to sulfate reducers. Highest rates of methane production were consistently measured in the uppermost sediment horizon, and the rates appeared to decrease in deeper depths. However, it should be noted that all rates represent turnover rates of the added tracer and not total turnover rates (i.e., do not account for nonlabeled substrates present in the sediment; SI Appendix , Text S4 ). Importantly, all measured activity could clearly be attributed to archaeal methanogenesis as the addition of BES, a specific inhibitor of the archaeal methanogenic enzyme Mcr, consistently inhibited methane production ( Fig. 2 ). Our analyses showed that the plant rhizomes in the uppermost surface layer contained the highest concentrations of methylated compounds; additionally, the surface layer likely receives a continuous supply of these methane precursors from leaf debris. At the same time, the surface sediments also experience periodic events of oxygenation ( Fig. 1 C ), which can potentially interfere with the strictly anaerobic process of archaeal methanogenesis. However, the phenomenon of oxygen-tolerant methane production via methanogenesis has previously been observed in, for example, soils where methanogens seem to be able to sustain their activity through the expression of genes controlling oxygen toxicity (e.g., catalases) ( 45 ) or by occupying anoxic microniches of the oxygenated soil or sediment layer ( 46 ). Seagrasses are a rich source of small, methylated compounds that can act as direct or indirect methane precursors. For example, methanol may be formed during bacterial degradation of lignin ( 43 , 47 ), and compounds such as methylamines can be readily produced as degradation products of, for example, choline or betaines ( 48 ). Phosphatidylcholines and betaines are abundant in plant tissue as membrane components and osmolytes, respectively ( 26 ) and are excreted or leaked into the surrounding sediment through the plant rhizomes and roots. We successfully detected choline (degradation product of phosphatidylcholine), betaines of glycine and proline, and DMSP in seagrass leaves as well as in their rhizomes ( Fig. 2 and SI Appendix , Table S3 ). Highest concentrations of choline, betaines, and DMSP were detected in fresh rhizomes from the surface layer of Posidonia -covered sediments, and the concentrations decreased in older plant pieces (i.e., recovered from deeper parts of the sediment). This distribution mirrors the distribution of our measured methane production rates, which followed the same trend ( Figs. 1 F and 2 ). It is feasible that glycinebetaine, the main plant osmolyte, might act as a direct methane precursor, as methane production from added glycinebetaine was immediate and proceeded linearly over time ( SI Appendix , Fig. S5 B ). However, betaines as well as cholines might further be fermented to trimethylamine (and other methylamines) ( 44 , 49 ) that are suitable substrates for methanogens ( 50 ). Many fermentative bacteria have a documented capacity to degrade glycinebetaine to form methylamines ( 51 ), and our investigated seagrass sediments hosted numerous populations of typical fermenters, including Clostridiales and Vibrionales ( Fig. 3 ), which may be potential candidates for providing methylamines to the methanogens. The abundance of betaines, choline, and DMSP in rhizomes collected from dead seagrass sediments was largely comparable to fresh rhizomes, with the exception of rhizomes collected from the surface layer in which the analyzed compounds were markedly depleted, presumably due to methanogenesis. In addition to rhizomes, seagrass leaves were also found to contain high amounts of all investigated methylated compounds ( SI Appendix , Table S3 ). As Posidonia plants shed their leaves year-round, we speculate that the resulting leaf debris deposited on the surrounding unvegetated sediment may act as an additional and persistent source of plant-derived methane precursors ( Fig. 5 ). Fig. 5. Methane production in vegetated sediments covered by Posidonia seagrasses. 1) Posidonia seagrasses produce a variety of methylated compounds that can act as methanogenic substrates. For example, betaines are stored in the plant's rhizomes and are released into the surrounding sediment, thereby becoming accessible for degradation. 2) Plant pieces buried in the dead seagrass sediments remain a source of methylated compounds for long periods of time. 3) Detached seagrass leaves that get deposited onto the adjacent dead seagrass or bare sediments may act as an additional source of methylated compounds in this ecosystem. 4) In the sediment, a diverse community of methanogenic archaea produces methane either directly from methylated compounds (e.g., betaines) or their degradation products (e.g., methylamines), mainly in the surface sediment layer. Diverse Methylotrophic Methanogens in P. oceanica Sediment. Methylotrophic methanogenesis is generally performed by members of the Methanosarcinaceae family (formerly phylum Euryarchaeota, now Halobacteriota). We recovered 16S rRNA gene sequences belonging to this family from our vegetated sediments, albeit at a relatively low abundance (<2% of the archaeal community). Members of the genus Methanococcoides were the most abundant euryarchaeal methanogens in all sediments based on 16S rRNA gene abundances. Importantly, we also recovered a full McrA protein sequence from the oxic surface sediment that clustered together with McrA sequences of Methanococcoides , with closest phylogenetic affiliation to M. methylutens . M. methylutens is a marine methanogen originally isolated from a sediment underlying a mat of algae and seagrass debris ( 52 ). While it can grow on methylated compounds, it cannot use H 2 or acetate ( 52 ). This fits well to our substrate-addition experiments, where only the addition of methylated compounds resulted in methane production. Other closely related McrA sequences belonged to, for example, a choline-utilizing methanogen isolated from a marine sediment (97.9% amino acid identity to WP_135613124.1) ( 53 ). These methanogens have been observed to form syntrophic associations with fermentative bacteria (e.g., Clostridia sp., Pelobacter sp.), which provide (tri)methylamines from the degradation of glycinebetaine and/or choline ( 51 , 54 ). However, Methanococcoides have also been reported to be capable of using choline and glycinebetaine directly ( 55 , 56 ) and do not necessarily require a bacterial partner for methane production. In a recent study, Methanococcoides , Methanosarcina , and Methanolobus species were successfully enriched on methylated compounds from Zostera seagrass sediments ( 57 ). Based on our combined results, we thus propose that members of these cosmopolitan and ubiquitous methylotrophic methanogens were likely responsible for methane production in P. oceanica seagrass meadows. Interestingly, the P. oceanica –covered sediments also contained high abundances of other putatively methylotrophic methanogenic archaeal groups—such as Ca. Bathyarchaeota ( 9 ), Asgard archaea ( 13 ), and Thermoplasmata ( 11 ). It should be noted that for each of these phyla, only a handful of species is presumably capable of methane metabolism; most Bathyarchaeota, Asgard archaea, and Thermoplasmata are metabolically versatile and are often involved in, for example, lignin and peptide degradation, acetogenesis, and exogenous protein mineralization ( 58 ). Additionally, the Mcr proteins from Bathyarchaea and Helarchaea (subgroup of Asgard archaea) belong to the “divergent” Mcr proteins, which typically share very low amino acid sequence homology to the Mcr from classical methanogens. It has thus been proposed that they may not be involved in methane metabolism ( 59 ). Instead, the divergent Mcr of Ca. Syntrophoarchaeum, for example, has been shown to be involved in butane oxidation ( 12 ), and a similar role has been proposed for the divergent bathyarchaeal ( 59 ) and helarchaeal Mcr ( 13 ). We retrieved seven McrA protein sequences from vegetated sediments that were affiliated with the clade of “divergent” Mcr sequences ( Fig. 4 A ), more specifically, with the McrA sequences from Ca. Helarchaea ( Fig. 4 C ). Given the lack of evidence for the presence of butane in the investigated sediments, it does not seem immediately obvious that the seagrass-associated Helarchaea should make a living off the oxidation of butane. Instead, given the high potential for methane production from a variety of methylated compounds, we suggest that a possible involvement of this Mcr in methane metabolism should be reexamined. Alternatively, the possibility of Mcr-containing Helarchaea being involved in the conversion of methane precursor molecules as a part of a microbial consortium should be considered. In any case, our combined data show that a diverse community of both traditional and putatively novel methanogenic archaea in these sediments is involved in the production of methane from a variety of plant-derived methylated compounds."
} | 3,999 |
19454530 | PMC2843956 | pmc | 5,736 | {
"abstract": "To move our economy onto a sustainable basis, it is essential that we find a replacement for fossil carbon as a source of liquid fuels and chemical industry feedstocks. Lignocellulosic biomass, available in enormous quantities, is the only feasible replacement. Many micro-organisms are capable of rapid and efficient degradation of biomass, employing a battery of specialized enzymes, but do not produce useful products. Attempts to transfer biomass-degrading capability to industrially useful organisms by heterologous expression of one or a few biomass-degrading enzymes have met with limited success. It seems probable that an effective biomass-degradation system requires the synergistic action of a large number of enzymes, the individual and collective actions of which are poorly understood. By offering the ability to combine any number of transgenes in a modular, combinatorial way, synthetic biology offers a new approach to elucidating the synergistic action of combinations of biomass-degrading enzymes in vivo and may ultimately lead to a transferable biomass-degradation system. Also, synthetic biology offers the potential for assembly of novel product-formation pathways, as well as mechanisms for increased solvent tolerance. Thus, synthetic biology may finally lead to cheap and effective processes for conversion of biomass to useful products.",
"conclusion": "5. Conclusions Economical conversion of cheap, abundant, renewable biomass to valuable products will require the combination of a range of different characteristics that do not naturally occur in any one organism. We have seen that metabolic pathways for product formation can be transferred from one host to another, and that synthetic biology offers improved methods for investigating synergy between the many gene products involved in biomass degradation and solvent tolerance. Synthetic biology also offers a suite of rapidly improving tools for the combination of multiple genetic modules in a single chassis (host) organism. For example, different combinations of biomass degradation genes could be generated to provide a set of biomass-degradation modules suited to different types of cellulosic substrate materials. Similarly, product-formation modules could be generated allowing the production of different products suitable for use as biofuels or chemical industry feedstocks, and these could be combined with solvent-tolerance modules suitable for protecting the organism against each particular product. For any desired combination of substrate and product, one could simply choose the appropriate modules for substrate degradation, product formation and product tolerance, and combine them in a robust, high performance chassis to generate an IBPM for each application. Thus, the fortuitous combination of the rapidly developing discipline of synthetic biology and the increasingly urgent requirement for improved biomass conversion processes offers the potential for a sustainable, oil-free future.",
"introduction": "1. Introduction Owing to the twin factors of declining oil reserves and increasing concern about rising carbon dioxide levels, our society must urgently seek a replacement for fossil fuels. The enormous quantities of coal, oil and gas that are used for the generation of electricity may ultimately be partially or completely replaced by other sources such as nuclear, solar-electric, solar-thermal, hydroelectric, geothermal, tidal, wave and ocean-thermal power systems. Considerable attention has also been directed to the development of electric vehicles, which may replace petrol-driven cars for short journeys. However, barring enormous improvements in battery technology, it seems unlikely that liquid fuels can be replaced in the near future for long-distance road transport, sea transport or, most particularly, aviation, which are essential for the efficient global movement of goods and people, and thus critical to the functioning of the modern world. Also, quite apart from energy uses, around 6–10 per cent of fossil fuels recovered are currently directed to the petrochemical industry ( Waltz 2008 ), leading to a wide range of products essential to our way of life. Alternative sources of suitable molecules for these applications are urgently required. Plant-derived material is the only feasible renewable source. A great deal of work has gone into the development of biofuels ( Antoni et al . 2007 ; Dale 2008 ). The majority of effort is associated with bioethanol and biodiesel. Ethanol for biofuel use is produced on a large scale by fermentation using the yeast Saccharomyces cerevisiae . The major producers of bioethanol are Brazil and the USA. In Brazil, ethanol is derived from sucrose in sugarcane; in the USA, it is produced mainly from glucose syrup derived from maize (corn) starch, a procedure that is generally held to be less environmentally favourable than the sugarcane-based process owing to the high-energy inputs involved ( Goldemberg 2007 ; Granda et al . 2007 ). Biodiesel is produced by esterification of fatty acids derived from vegetable oils ( Hill et al . 2006 ; Granda et al . 2007 ). It has been suggested that the CO 2 emissions generated by land clearance for growing biofuel crops may outweigh the reduction in emissions associated with reduced use of fossil fuels ( Fargione et al . 2008 ; Gallagher et al . 2008 ; Searchinger et al . 2008 ). It also seems clear that production of biofuels from maize and vegetable oils is creating unacceptable competition with food, both by diverting food-grade materials and by absorbing land that could be used for growing food crops. This is considered to be one factor in the major rises in staple food prices seen in early 2008, leading to food riots in many parts of the world ( Sachs 2008 ), though the extent to which biofuel production competes with food production has been questioned (e.g. Dale 2008 ). Nevertheless, it seems clear that there is limited scope for increased production of biofuels by these methods. This has led to great interest in ‘second generation’ biofuels, derived from non-food materials, particularly lignocellulosic biomass, the non-edible parts of plants ( Lynd et al . 2005 ; Chang 2007 ; Kumar et al . 2008 ; Wackett 2008 ; Yuan et al . 2008 ). Potential sources of such material include agricultural and timber industry waste, waste paper and purpose-grown plant material derived from rapidly growing non-food plants such as scrub willow, switchgrass and Miscanthus , which can be grown on land unsuited to food production ( Heaton et al . 2008 ). Since all plants consist largely of this material, in principle any plant material could be used, including mixed grasses harvested sustainably from prairie-style systems, thus avoiding the environmental issues associated with intensive monocultures. Processes must therefore be developed for the conversion of lignocellulosic biomass into useful products. The Ideal Biofuel Producing Micro-organism (IBPM) must possess a number of independent characteristics:\n it must be able to hydrolyse cellulosic material effectively, with minimal requirement for pre-processing; it must be able to convert the sugars released into molecules useful as liquid fuels and/or chemical industry feedstocks; it must be able to produce these molecules at a high concentration without poisoning itself, in order to minimize downstream processing costs; and it must be capable of rapid growth in a bioreactor and suitable in other respects for use in an industrial context. Naturally occurring micro-organisms do not fulfil these criteria. Here we will consider how the emerging discipline of synthetic biology might be applied to develop a new organism, which can be applied in such a process. Synthetic biology aims to construct novel biological systems from smaller components. This programme is exemplified in the concept of BioBricks ( Knight 2003 ; Registry of Standard Biological Parts 2009 ): modular, interchangeable DNA components, which, due to the use of a combination of restriction sites generating compatible and incompatible sticky ends, can be assembled in any order and in any desired number to generate complex multi-gene systems, which can be further combined to any desired degree ( figure 1 ). The original BioBrick standard is now under revision (BioBricks Foundation; www.biobricks.org ), but any successor standards will certainly maintain the key principles of modularity and interchangeability. Ultimately, the decreasing cost of synthetic DNA should further simplify the assembly of complex multi-gene systems. The unprecedented flexibility offered by BioBricks and other synthetic biology approaches has tremendous potential for allowing the generation of new micro-organisms combining useful phenotypes from different sources. It is this technology that may finally allow us to generate the IBPM and make conversion of biomass to fuels and chemical feedstocks an economically competitive process. Figure 1. The BioBrick 1.0 assembly standard ( Knight 2003 ; Registry of Standard Biological Parts 2009 ). ( a ) Each BioBrick is a length of DNA bearing a genetic component such as an open reading frame, ribosome-binding site, promoter, transcription termination sequence or any combination of these. Each BioBrick possesses EcoRI and XbaI restriction sites at the 5′ end, and SpeI and PstI sites at the 3′ end. ( b ) Standard prefix and suffix sequences for BioBricks. The six-base pair recognition sites for each restriction endonuclease are shown in bold and dashed lines indicate the staggered cuts made by each enzyme. ( c ) Ligation of an SpeI-cut end to an XbaI-cut end generates a six-base pair ‘scar’, which is not recognized by either XbaI or SpeI. ( d ) By appropriate choice of restriction enzymes, any BioBrick can be inserted either upstream or downstream of any other BioBrick. ( e ) In either case, the product, bearing both components, is also a BioBrick, bearing the same four restriction sites as the original component BioBricks. It can thus be added either upstream or downstream of any other BioBrick. In this way large and complex constructs can be built up quickly and easily from a library of standard parts. In synthetic biology, the characteristic of robust growth in a bioreactor context is ensured by choice of an appropriate host or ‘chassis’ organism, such as Escherichia coli , Bacillus subtilis or S. cerevisiae , which provides background processes to support the new pathways to be introduced. The problem then becomes one of generating the new biological modules: biomass degradation, product formation and solvent tolerance. In the remainder of this paper, we will consider how synthetic biology can contribute to each of these aspects and, finally, can help to bring them all together in one organism, the IBPM."
} | 2,709 |
29938188 | PMC6010905 | pmc | 5,737 | {
"abstract": "Abstract Higher memory density and faster computational performance of resistive switching cells require reliable array‐accessible architecture. However, selecting a designated cell within a crossbar array without interference from sneak path currents through neighboring cells is a general problem. Here, a highly doped n ++ Si as the bottom electrode with Ni‐electrode/HfO x /SiO 2 asymmetric self‐rectifying resistive switching device is fabricated. The interfacial defects in the HfO x /SiO 2 junction and n ++ Si substrate result in the reproducible rectifying behavior. In situ transmission electron microscopy is used to quantitatively study the properties of the morphology, chemistry, and dynamic nucleation–dissolution evolution of the chains of defects at the atomic scale. The spatial and temporal correlation between the concentration of oxygen vacancies and Ni‐rich conductive filament modifies the resistive switching effect. This study has important implications at the array‐level performance of high density resistive switching memories.",
"conclusion": "3 Conclusions In summary, a high‐performance diode‐free resistive switching cell was fabricated with a high on/off ratio. The real‐time in situ TEM analysis of asymmetric MIS structures allowed the study of the dynamics, evolution, and underlying physics governing the switching mechanism, which was previously speculated at best from electrical measurements without much direct evidence. Multiple conductive path formation and rupture events were temporally and spatially uncorrelated. The use of an asymmetric MIS structure clearly assisted in identifying the presence and source of the metal‐rich conductive filament, which originated from the anode electrode while preventing sneak path issues. This fundamental atomic study on the chemistry, morphology, and time‐dependent correlated/uncorrelated switching behavior of filaments provides a strong support to the feasibility of further scaling of future resistive switching technologies, paving the way for very high‐density data storage and future neuromorphic computing.",
"introduction": "1 Introduction Nanocrossbar arrays comprise a set of parallel bottom electrodes and perpendicular top electrodes with a thin layer of resistive switching material in between. Switching materials can be classified into different groups according to their physical mechanisms. 1 , 2 , 3 Materials showing electrochemical metallization or valence change (VCM) effects have been extensively investigated. 4 , 5 Each crosspoint in a memory cell stores logic information as a high resistance state (HRS) or a low resistance state (LRS). 6 , 7 , 8 A selection device, such as a transistor or a diode, is usually necessary in addition to the memory element for the scaling limit of the memory device. Therefore, the simplicity of the geometrical structure and the absence of transistors make the concept extremely interesting for low‐power nonvolatile memory circuitry and high integration. Conductive filament (CF) has been recognized as the key structural element that contributed to resistive switching performance. 6 , 7 , 9 , 10 , 11 Recently, a number of approaches, such as electrochemical redox reaction, 12 , 13 metal nanodots doping, 14 micrsostructural transitions, 15 , 16 and current compliance capping 17 have been explored to address the controlled formation and rupture mechanism of CFs. Here in, solving the disadvantage of sneak paths by simply embedding a diode structure inside the memristor cell is proposed. This paper attempts to close the gap in the understanding of the fundamental mechanism through the real‐time observation of resistive switching at an atomic scale. In situ transmission electron microscopy (TEM) technique is a powerful and versatile tool to examine the interfacial properties of the nanodevices. 18 , 19 , 20 , 21 , 22 The formation of CF under different compliance current levels controls the growth of the filament. Subsequently, the entire device is switched on and in a cyclic mode to investigate the primary mechanism that leads to a self‐rectifying behavior.",
"discussion": "2 Results and Discussion The device used in this study was a unipolar resistive switching device based on an asymmetric metal–insulator–semiconductor (MIS) structure: Ni as the top electrode, HfO 2 as the insulator, and Si substrate as the bottom electrode. Ni has been used extensively in the mainstream complementary metal oxide semiconductor technology as a source/drain contact material. 23 The bottom metal electrode was replaced with a highly doped n ++ ‐type Si ( N \n D ≈10 −19 cm 3 ) to study the role of asymmetric electrode in switching. It comprised of no additional diode and only one resistive switching element (0D1R), which allowed for the construction of highly dense passive crossbar arrays by solving the sneak path problem combined with the drastic reduction of power consumption and area. \n Figure \n \n 1 \n A shows that the resistive switching device demonstrates well‐behaved unipolar switching current–voltage ( I–V ) characteristics under DC sweep. The SET voltage was 2.4 V ( V \n SET ) with a compliance current level of 10 µA. Subsequently, the current level reached the 100 µA range with no compliance capping when the voltage was swept again from 0 V with the same polarity. In the RESET sweep, a current drop was observed (three orders of magnitude) at a threshold voltage of 1.8 V (RESET voltage, V \n RESET ). The HfO 2 ‐based resistive switching demonstrated a high ON/OFF resistance ratio of ≈10 3 with 500 cycles of endurance, as shown in Figure S1 (Supporting Information). Figure 1 B shows the device‐to‐device resistance distribution in a Gaussian cumulative density function plot for 10 devices at 500 DC cycles for each device. The LRS had less spread in its resistance value possibly due to the lower variation in the number of defects for the “harder” stages of dielectric breakdown (BD). In contrast, HRS intrinsically had a more extensive spread in its resistance state of ≈2–3 orders of magnitude. This was expected because the CF rupture driven by the local Joule heating effect was difficult to control and occurred within a time range of a few nanoseconds. Therefore, the extent of rupture varied considerably from cycle to cycle. However, die‐to‐die (device‐to‐device) variations become smaller as the distribution tends to overlap very closely with each other. The devices used in this study exhibited good switching parameter uniformity and high‐temperature operating stability. In the crossbar structure‐based memory circuit, all resistive switching devices in a row were connected to each other by the top electrode, and all resistive switching devices in a column were connected to each other by the bottom electrode. The worst case scenario for reading an HRS was assumed, that is, all unselected devices were set to LRS, as illustrated in Figure 1 C. Sneak leakage paths 24 were reduced to a minimum because the reverse current in the LRS of the resistive switching cell in this study under negative voltage sweep was very low (Figure 1 A). The low reverse currents can be attributed to the n‐type heavily doped Si bottom electrode that functions as a “diode” in itself, as indicated in Figure 1 D. This self‐rectifying effect significantly improved the size of the crossbar array (maximum numbers of rows and columns) because the current flow and voltage drop over the addressed element was strongly dependent on the current flowing through the multiple parallel sneak leakage paths. Furthermore, the overall resistance of the crossbar array depended on the number of “0”s and “1”s stored in the array. Thus, the power consumption decreased when the resistive cells were mainly in the LRS. The integration of a self‐rectifying element addressed the sneak leakage path problem, and this structure is sufficiently scalable. Such devices can also find application in true random number generators, neuromorphic computing and so on. Figure 1 Device electrical performance: A) I–V characteristic of a device with MIS structure: Ni/HfO 2 /SiO 2 /Si‐diode with 100 nm Ni, 3.1 nm HfO 2 , and 0.7 nm SiO x . Note that the reverse current in LRS is very low. The schematic in the inset shows the equivalent circuit after a SET process. B) Device to device resistance distribution in the ON and OFF states for 10 different devices each with 500 DC switching cycles. C) Schematic of an example of a sneak path of a “ON” resistive switching device in a normal circuit. D) Enerrgy band diagram of an “ON” state resistive switching device with negative sweep voltage. In situ TEM experiments were performed to understand the kinetics of the dynamic nucleation–dissolution behavior of CFs for the I–V characteristics shown in Figure 1 A. First, the evolution of the formation and rupture of a single nanofilament was studied and a multistage constant voltage stress algorithm developed in ref. 25 was used. This involved a repetitive sequence of stressing the dielectric from low to high stress voltage conditions at small increments of ≈0.2 V. 25 This algorithm aims to observe the morphological change of the nanofilament at different stress conditions and identify the critical current levels for the HRS → LRS and LRS → HRS transition. The device was stressed using the proposed test scheme at room temperature 26 by using an in situ TEM system. In the test, the forming process was initiated with a voltage sweep from 0 to 4.0 V and a compliance setting of 1 µA. The process began with an initial voltage of 2.7 V. It was selected based on a previous study of the gate dielectric BD mechanism. 27 The in situ stressing voltage was notably higher than the ex situ SET voltage because of the additional parasitic resistance of the in situ TEM tip R \n tip (Figure S2, Supporting Information). The complete current evolution with stress time and the corresponding high‐resolution TEM micrographs are shown in Figure \n \n 2 \n A–E. Heavy atoms from the top metal electrode migrated into the HfO 2 dielectric and Si‐substrate, forming a unique geometrical defect, which resembled an inverted pyramid when viewed from the Si substrate. 28 A “depleted” region with a lower contrast in the top Ni electrode can be seen on top of the pyramidal (triangle in Figure 2 D) defects in the HfO 2 /SiO x dielectrics, indicating that the Ni atoms from the anode migrated downward. The filament inside the dielectric was bowl‐shaped with a wider top of ≈20 nm near the anode and a shorter bottom of ≈10 nm. 28 The shape and size of the filaments in the dielectric are consistent with previous observation in both VCM and conductive‐bridge resistive random access memory (CBRAM) reported devices, as reported by pioneering works from authors of refs. 29 , 30 . Figure 2 E shows a RESET stage, where the dark contrast in the dielectric can barely be seen. The device experienced a significant “switch‐off,” which led to a very low current level with an ON/OFF ratio >1000 as shown in Figure 1 A,B. The nanofilament was disconnected in the dielectric, and hardly any contrast was detectable inside the HfO 2 or SiO x layer. One of the major driving forces that caused the “switch‐off”/rupture of the nanofilament was the Joule heating effect, 31 which was repeatedly observed in numerous devices, as shown in Figure S3 and Video S3 (Supporting Information). The formation of a nanofilament is associated with the formation of a hillock‐like defects in the Si and SiO 2 interface. These findings are in agreement with the previous study of dielectric breakdown‐induced epitaxy (DBIE) in an SiO 2 /Si gate stack. 26 , 27 \n Figure 2 A–J) Evolution of the nanofilament formation and rupture under various compliance currents. Heavy atoms from the top metal electrode migrated into the HfO 2 dielectric and Si‐substrate, forming a unique geometrical defect which resembles an inverted pyramid if viewed from the Si substrate. Scale bar is 5 nm. Multiple nanofilament formation and rupture events in a single device were also observed in the experiment, as shown in Figure \n \n 3 \n . Figure 3 A–C shows the dynamic observation of a sequence of morphological changes during the real‐time localized electrical stressing of an MIS resistive switching device in TEM. The first two Ni residuals (1st and 2nd CFs) in the Si substrate (i.e., Figure 3 B) indicated the locations of the nanofilaments in two initial cycles of SET and RESET. When the device was switched off for the second time (Figure 3 B) at around 9 s for the third SET cycle, a new uncorrelated nanofilament (3rd CF) formed ≈100 nm away from the two previously ruptured filamentary regions (Figure 3 C). Figure 3 D–F shows the high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) images (contrast dominated by the atomic number) of the 1st, 2nd, and 3rd nanofilaments, in which the bright contrast in the HfO 2 and SiO x layers of the SET filament again indicates that heavy metal atoms from the top metal electrode diffused into the oxide layer. The insets in Figure 3 D,F show the Ni and O element electron energy loss spectroscopy (EELS) mapping results. The EELS mapping results confirmed that Ni atoms from the top electrode diffused into the dielectric layers. Ni atoms continued to diffuse along the 〈111〉 direction of the silicon substrate because of the largest inner‐plane distance. 32 The contrast of each filament can be compared because multiple filaments at the three different locations were observed in a single device. The Ni concentration in the RESET region of the dielectrics was much lower than that in the newly nucleated filament site, which further confirmed the dissolution of a Ni metal filament inside the oxide layer at the nearby SET location. 33 Video S1 (Supporting Information) shows the dynamic evolution of the process. Figure 3 Uncorrelated formation and rupture of multiple nanofilaments under constant voltage stress of 2.7 V. Experiment times: A) 0 s, B) 16 s, the first SET/RESET occurred at ≈1 and ≈2 s and the second SET/RESET happened at ≈7 and ≈9 s. C) After third SET/RESET at ≈38 s in (B). D–F) Corresponding HAADF‐STEM images of the three nanofilaments shown in (B) and (C), respectively. Insets in (D) and (F) are the corresponding EELS mapping results of area of interest of the SET/RESET sites. Cyan represents nickel signals, and red represents oxygen signals, respectively. Correlated nanofilament formation and rupture events were also observed. Figure \n \n 4 \n A–D shows that the second SET induced two other metal filaments (2nd and 3rd CFs) very near the first one (1st CF) after the first RESET. Even though the first filament experienced substantial rupture of the nanofilament (evident in the significant drop in the switching current), the second nucleation occurred very close to the location of the first one, which can be attributed to the residual metal fragments that remained in the original site after rupture. These Ni residuals served as a weakest link location for subsequent “more localized” switching. Figure 4 E,F shows the corresponding high‐resolution TEM and STEM images of the correlated CFs, respectively. Figure 4 Correlated formation and rupture of multiple nanofilaments. Experiment times: A) 0 s, B) 1 s, C) 3 s, and D) 8 s. E,F) High resolution TEM and HAADF‐STEM micrograph of the formation of two close nanofilaments near the first CF during second SET. Note that there is no heavy atom contrast left in the oxide in the position of the first nanofiament. This verifies that the first nanofilament ruptured quite extensively during the RESET process. Both single and multiple filaments were typically observed in the in situ TEM experiments. As shown in Figure 4 , spatially correlated CFs were regularly observed in some cases of the formation and rupture of multiple filaments. Figure \n \n 5 \n presents the schematic of the scaling limitations based on the observed multiple filaments model. The new uncorrelated filaments ( A \n 2 ) may occur elsewhere in the device if the resistive switching device area ( A ) is much larger than the observed nanofilament size of 20 nm due to the logically higher probability of having multiple weakest link spots in a larger area structure. In this example, the first nanofilament ( A 1 ) is responsible for the SET and RESET which undergoes a very significant formation and rupture of Ni‐filament. Based on Poisson area scaling theory, any new nanofilament could nucleate at other locations for large area devices, as shown in Figure 5 . Further experiments, such as a statistical and spatial study of CF distribution, are required to reveal and better understand these underlying mechanisms clearly. Uncorrelated filament formation within a device can lead to unstable state at the LRS and is a challenge for resistive switching application. However, if the reset process is carefully tuned to ensure that the switching occurs at the same place (i.e., energy consumed to switch the same CF is smaller than that for creating a new CF), the variability issue can be better managed. The chances of forming a new filament that is uncorrelated are very low (which is attributed to the area scaling effect in the region of the dielectric that is free of filaments) when the resistive switching area is small. Repeated nucleation at the same nanofilament location or the nucleation of another nanofilament very close to the site of a previous one (“correlated”) could occur. In this scenario, the unit size can be downscaled to the area of a single CF (i.e., about 20 nm, as determined in this study), extensively increasing the cell density and decreasing the energy dissipation for storing a single bit (Video S2, Supporting Information). Figure 5 Schematic illustrating the area scaling dependence of multiple filament nucleation probability. The overall area of the resistive random access memory (RRAM) device is denoted as A , the first filament area is A \n 1 , and the second filament area is A \n 2 , respectively."
} | 4,526 |
34556173 | PMC8461902 | pmc | 5,738 | {
"abstract": "Background Consolidated bioprocessing (CBP) technique is a promising strategy for biorefinery construction, producing bulk chemicals directly from plant biomass without extra hydrolysis steps. Fixing and channeling CO 2 into carbon metabolism for increased carbon efficiency in producing value-added compounds is another strategy for cost-effective bio-manufacturing. It has not been reported whether these two strategies can be combined in one microbial platform. Results In this study, using the cellulolytic thermophilic fungus Myceliophthora thermophila , we designed and constructed a novel biorefinery system DMCC ( D irect m icrobial c onversion of biomass with C O 2 fixation) through incorporating two CO 2 fixation modules, PYC module and Calvin–Benson–Bassham (CBB) pathway. Harboring the both modules, the average rate of fixing and channeling 13 CO 2 into malic acid in strain CP51 achieved 44.4, 90.7, and 80.7 mg/L/h, on xylose, glucose, and cellulose, respectively. The corresponding titers of malic acid were up to 42.1, 70.4, and 70.1 g/L, respectively, representing the increases of 40%, 10%, and 7%, respectively, compared to the parental strain possessing only PYC module. The DMCC system was further improved by enhancing the pentose uptake ability. Using raw plant biomass as the feedstock, yield of malic acid produced by the DMCC system was up to 0.53 g/g, with 13 C content of 0.44 mol/mol malic acid, suggesting DMCC system can produce 1 t of malic acid from 1.89 t of biomass and fix 0.14 t CO 2 accordingly. Conclusions This study designed and constructed a novel biorefinery system named DMCC, which can convert raw plant biomass and CO 2 into organic acid efficiently, presenting a promising strategy for cost-effective production of value-added compounds in biorefinery. The DMCC system is one of great options for realization of carbon neutral economy. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-021-02042-5.",
"discussion": "Discussion Plant biomass and CO 2 have many desirable features as industrial raw material to decrease reliance on fossil fuels. Direct conversion of plant lignocellulose and fixing CO 2 into the central carbon metabolism of industrial microbes are promising for cost-effective production of value-add compounds in biorefinery. The thermophilic and cellulolytic M. thermophila has been engineered to produce malic acid using raw plant biomass as the feedstock without addition of hydrolytic enzyme [ 10 ]. In this study, using M. thermophila , the DMCC system (direct microbial conversion of biomass with CO 2 fixation), a novel strategy for biorefinery from plant cell wall and CO 2 , was constructed to convert plant biomass and CO 2 into malic acid efficiently. For fixing and channeling CO 2 into carbon metabolism for producing value-added compounds, there are two different strategies. One approach is the integration of exogenous biosynthetic production pathways into naturally existing carbon-fixing organisms, such as cyanobacteria and algae. Autotrophic microbes have been engineered to produce chemicals and biofuels from CO 2 , such as 2,3-butanediol, lactic acid and malic acid [ 49 – 51 ]. However, production performance remains far below industrial feasibility. The other option is to equip heterotrophic fermentation strains with efficient CO 2 -fixation pathway. Recently, Calvin–Benson–Bassham (CBB) pathway was constructed in heterotrophic microbes to recycle released CO 2 into central metabolic pathway for improved carbon efficiency [ 20 , 22 , 28 ] and even synthesize sugars and other major biomass components from CO 2 [ 29 ]. An efficient energy supply is required for biological CO 2 fixation. Engineered E. coli and P. pastoris could incorporate CO 2 into cell components via heterologous CBB pathway, requiring reducing power and energy from the supplements, such as pyruvate, formate, and methanol [ 18 , 29 , 33 ]. Using sustainable plant biomass as energy source and actuate CO 2 -fixation would be a promising strategy for cost-effective bio-manufacturing. In addition, the main components of lignocellulose comprise glucose, xylose, and arabinose. Intermediate ribulose-5-phosphate of pentose catabolism via pentose phosphate pathway can serve as the substrate of PRK. In E. coli and S. cerevisiae , xylose and arabinose were used to drive CO 2 -fixation of the CBB cycle [ 17 , 20 , 32 ]. In this work, the CBB cycle enzymes were successfully introduced into the cellulohydrolytic fungus M. thermophila to producing CO 2 fixation system, combined with native PYC module to produce malic acid using plant biomass and CO 2 as the carbon sources. The proportion of carbon atoms from fixed CO 2 in the total carbon of malic acid was significantly increased and the average rates of fixing and channeling 13 CO 2 into malic acid achieved 44.4 mg/L/h, 90.7 mg/L/h and 80.7 mg/L/h in strain CP-51, when grown on xylose, glucose and Avicel, respectively (Fig. 4 ). With raw corncob as the feedstock, the yield of malic acid produced by the final engineered strain Gal-1 achieved 0.53 g/g total plant biomass, representing a 10.4% increase over the highest reported yield (0.48 g/g) [ 9 ] and the content of 13 C atom in malic acid was up to 0.44 mol/mol malic acid. Furthermore, this strategy of synergistic conversion of plant biomass and CO 2 can be utilized for the production of other chemicals, such as fumaric and succinic acid. Rapid utilization of all components of the hydrolysate of plant biomass is the prerequisite for efficient production of biochemical. However, due to the preference of microbes for glucose, pentose utilization is inhibited by glucose presented in the culture, which led to two-stage utilization of sugar mixture and low productivity of target products. It was suggested that d -glucose impairs the simultaneous utilization of pentose mainly by inhibition of pentose uptake [ 43 ]. Enhancement of pentose uptake can alleviate this inhibition and facilitate improved pentose utilization and elevated co-fermentation rates of hexose and pentose [ 44 , 52 ]. Recently, transporter engineering and directed evolution have been used for rewiring substrate specificity to obtain glucose-insensitive xylose transporters [ 45 , 53 ]. Herein, a glucose inhibition-free xylose transporter was integrated into engineered strain CP-51 for facilitating pentose utilization to actuate CO 2 -fixation of heterologous CBB pathway and improving co-fermentation rates of hexoses and pentoses for production of malic acid. Titer and yield of malic acid were increased to 76.1 g/L and 1.01 g/g carbon source, respectively, by conversion of a mixture of sugars derived from plant biomass. In addition, although xylose and arabinose are both pentose, there are dramatic differences in transcriptomic profiles in filamentous fungus when exposed to them in a previous study [ 9 ], indicating that that regulation network of xylose catabolism is different from that of arabinose catabolism. Here, it was also observed that titer of malic acid on xylose was obviously below that on arabinose. The exact molecular basis of the two pentose metabolism needs more investigation in the future. In this study, CBB cycle enzymes, RuBisCO and PRK, were integrated into the metabolic network of the thermophilic fungus M. thermophila for enhanced fixation efficiency of CO 2. A novel biorefinery system named DMCC was designed and constructed, which can produce 1 t of bulk chemicals (such as malic acid) using less than 2 t of plant biomass, accompanied by the fixation of 0.14 t CO 2 . This study provides a novel strategy for producing biochemicals and operating carbon neutral."
} | 1,939 |
35424313 | PMC8694127 | pmc | 5,739 | {
"abstract": "In the past decades, drag-reduction surfaces have attracted more and more attention due to their potentiality and wide applications in various fields such as traffic, energy transportation, agriculture, textile industry, and military. However, there are still some drag-reduction materials that need to be deeply explored. Fortunately, natural creatures always have the best properties after long-term evolution; aquatic organisms have diversified surface microstructures and drag-reducing materials, which provide design templates for the development of thriving artificial underwater drag-reduction materials. Aquatic animals are tamed by the current while fighting against the water, and thus have excellent drag reduction that is unparalleled in water. Inspired by biological principles, using aquatic animals as a bionic object to develop and reduce frictional resistance in fluids has attracted more attention in the past few years. More and more aquatic animals bring new inspiration for drag-reduction surfaces and a tremendous amount of research effort has been put into the study of surface drag-reduction, with an aim to seek the surface structure with the best drag-reduction effect and explore the drag-reduction mechanism. This present paper reviews the research on drag-reduction surfaces inspired by aquatic animals, including sharks, dolphins, and other aquatic animals. Aquatic animals as bionic objects are described in detail, with a discussion on the drag-reduction mechanism and drag-reduction effect to understand the development of underwater drag-reduction fully. In bionic manufacturing, the effective combination of various preparation methods is summarized. Moreover, bionic surfaces are briefly explained in terms of traffic, energy sources, sports, and agriculture. In the end, both existing problems in bionic research and future research prospects are proposed. This paper may provide a better and more comprehensive understanding of the current research status of aquatic animals-inspired drag reduction.",
"conclusion": "5. Conclusions and perspectives Drag reduction is a very desirable feature in many applications. In this review, taking aquatic animals as the main research object, the development of related aquatic animals-inspired drag-reduction technology research is introduced, from which some questions and conjectures are discussed next. Bionic drag reduction extends the limitation of traditional drag reduction in many aspects and its effect is obvious. For the mechanism explanation of theoretical research, drag reduction under laminar flow has many mechanism explanations supports, such as the protruding height theory. However, in turbulent flow, fluid motion is disordered and complex, and most researchers focus on the effect of drag reduction, while the mechanism of drag reduction is rarely discussed. So far, drag reduction under turbulence is still a hot and difficult topic. The “water-trapping” effect and the ‘secondary vortex’ effect from the perspective of the vortex are utilized to explain surface drag reduction. Furthermore, boundary layer theory and separation control under reverse pressure conditions have been extensively studied to explore the reasons for drag reduction. The results are now mainstream view aquatic animals in water and low resistance non-smooth surface and mucus stealth effect with combined effects. The preparation of bionic drag reduction materials utilizes this point and the composite drag reduction technology also implies the same feature. The preparation of drag-reduction materials combined with matching technology or methods seems to be the trend of future development. For example, lubricant injection method and drag-reducing porous material that can store lubrication for a long time, 206 the storage of the porous surface, and the lubrication of the lubricant are superimposed without affecting each other, making the lubricant more durable, and hydrophobic materials combined with artificial micro/nano-engine technology to improve the efficiency and speed of the micro device. 209,210 Moreover, the application of bionic drag-reduction materials is inseparable from the development of drag reduction technology and can better compensate for each other's defects. Therefore, the two complement each other, gradually becoming the future trend in the field of underwater bionic drag reduction. The drag-reduction effect of the bionic shark scales is only significant under certain conditions. If the flow field environment, arrangement spacing, and shield scale size are changed, the drag-reduction effect will be weakened or even not. Regardless of whether the shark scale is actively controlled or passively controlled, it can be concluded that the shark scale can swing at an angle of 50° and the dolphin's corrugated skin during high-speed movement. 27 Compared with the fixed shark scales in the experiment, it seems that the shark scales can be regarded as continuously adjusting to the flow field and the imitation sample fixed in the experiment seems to be a moment during the movement of the shark scales. Thus, the common features of drag-reduction bionic surface need to be further explored. The extent to which the scales are regarded as dynamic on skin hydrodynamic function is unclear but it is a promising field to link skin structure with locomotor hydrodynamics. 2 Aquatic animals, as a research object of bionic drag-reduction, have different morphological characteristics, such as the volume, surface texture, and posture of aquatic animals, and their adaptability to the flow field. The color pattern seems to have a potential mechanism for underwater creatures to resist water flow. 108 During the fight between underwater creatures and water flow, a more organized skin pattern has evolved, which vibrates slightly during swimming. 92 For the experimental setup, more abundant measurement techniques and visualization methods for later treatment of the flow field structure can be adopted. The visual treatment of vortices is conducive for the analysis of the generation, development, and extinction of vortices in the boundary layer. Color is introduced into PIV measurement of the boundary layer microbubbles and tomographic particle image velocimetry (TPIV) measurement. 81,211 Also, the measurement of aquatic animals hydrodynamic parameters is jointly verified by experiment and computer simulation. The surface function of aquatic animals in the body during movement is rarely replicated with experimental models at present. Bionic manufacturing is more inclined to replicate the surface of bionic microstructures, which is limited by processing materials and manufacturing processes. Large-scale manufacturing of drag-reduction surfaces is still the key point to be explored in the future. Combining new materials and advanced manufacturing processes will be a breakthrough in drag reduction preparation. For example, the combination of coatings and grooves forms a self-cleaning surface, the surface of the wear-resistant structure is combined with the hydrophobic to form a wear-resistant hydrophobic surface, and micro-device process manufacturing with drag-reduction materials to drive the application of targeted drugs, etc. In addition, bionic drag-reduction materials have broad prospects in anti-fouling, anti-fog, water collection, wear resistance, micro-engine drag reduction, heat dissipation, catalysis, etc. To sum up, although aquatic organisms have developed a variety of materials, surfaces, and structures ranging from macroscopic to nanoscale, using which as imitation objects to solve engineering problems has brought us advanced templates, the close integration of multiple disciplines is still one of the future development trends. Thus, underwater bionic drag-reduction materials still urgently need further exploration and research in the future.",
"introduction": "1. Introduction With the aggravation of the global energy crisis, the research on underwater drag reduction and performance improvement technology is becoming increasingly urgent. Oceans are home to countless aquatic organisms, which are considered as the origin of life. Furthermore, after 3.7 billion years of natural selection and evolution of aquatic species, creatures living underwater are characterized by efficient energy use and low-drag activities. Therefore, they have the best answers when we seek to improve or optimize underwater drag-reduction materials. Fortunately, the application of bionics allows us to imitate biological materials in order to manufacture and process drag-reducing materials. Aquatic organisms survive in water as they have super adaptability to resist the impact and friction of water currents. In the long biological evolution, sophisticated body structure and a characteristic way of motion have been developed to combat the complex environment. There are many types of aquatic organisms, including aquatic bacteria, aquatic fungi, aquatic plants, and aquatic animals. Among them, aquatic animals represented by vertebrates have taken the lead in this long-term evolution. For example, fish are the oldest vertebrates that inhabit almost all marine environments on Earth from freshwater lakes and rivers to saltwater seas and oceans. They have a wide variety of complex species (more than 36 000 living fish species in the world) and their powerful ability to adapt to the environment is widely known. As we know, the living environment of most aquatic animals is liquid water medium for physiological activities. The interface between the water environment and the body surface is crucial for both the physiology and hydrodynamic functions of aquatic animals in water. Oxygen, ions, and carbon dioxide exchange occurs around the surface of the aquatic animals' body tissues to maintain the basic physiological activities. 1 For the hydrodynamic functions of aquatic animals, surface texture and body structure have potential mechanisms for drag reduction. 1,2 Aquatic animals need to consume the most energy to overcome surface resistance during underwater navigation and transportation, and have the function of drag reduction in long evolution and natural selection owing to the physical challenge of motion in water. The countermeasures are rough as follows: (1) friction drag and viscous drag caused by direct contact of the skin surface with fluids. Different kinds of aquatic animals have different special responses to overcome these obstacles, such as shark's scales and dolphin's flexible compliant skin. 3 Furthermore, mucus covering the surface texture of aquatic animals gives a slippery feeling, which has always been considered as an indispensable factor in reducing the frictional drag and viscous drag of aquatic animals. Tian et al. utilized the DSD/SST method to simulate and propose that mucus also has the effect of enhancing thrust and reducing noise. 4 The combination of scales and mucus on aquatic animals' skin protects them from being scratched by sharp objects underwater and swims and wears in the water for a long time, resists parasites, and plays a clean and antifouling effect. (2) The pressure drag exists between the head and the tail. In general, the streamline of the aquatic animals' body greatly reduces the fluid pressure drag due to the weakening of the negative vortex effect or avoiding the formation of negative vortices. Research has indicated that shark scales and flexible skin seem to be able to reduce pressure drag through passive flow control mechanisms. The skin and streamlined body control the vortex to increase the fluid velocity in the boundary layer, thereby reducing separation and thus reducing pressure drag. 3,5 Aquatic animals not only passively reduce drag in the streamline and interface performance but also have an amazing cruise strategy during active swimming. With the expansion of marine operations and exploration, the technical improvement of underwater drag reduction is necessary. Aquatic animals have been used as a source of inspiration for many vehicles, such as ships, aviation, submarines, and underwater operating equipment. 6 The main reasons are their adaptability to environmental changes underwater and the ability to self-heal/correct the system. 7 Cruising aquatic animals can utilize the surrounding environment to save energy. Also, the energy consumption of fish organisms in water is different from common sense. 8 During the active motion of aquatic animals, they accelerate intermittently, then constantly maintain the cruise speed, and alternately climb and sink forward. 9,10 Moreover, biological aquatic animals are “stingy” with their energy while adapting to underwater activities and their cruising strategy is to spend the least energy to reach the maximum distance of sailing from an evolutionary perspective. Compared to large-sized sharks and dolphins, small-sized aquatic animals seem to have insufficient energy to resist turbulent water currents and are eliminated by the environment. On the contrary, Fletcher et al. explained why some smaller fish (flatfish) could maintain stable motion in turbulent river water. Small-sized fish utilize a non-smooth riverbed base with a low-velocity thin layer cruising strategy, which is close to the river bottom, to reduce their energy consumption. 11 In the experiment, aquatic animals utilized less oxygen in high-turbulence compared with low-turbulence flow at medium and high swimming speeds, i.e. , energy consumption is weakened. 8 Aquatic animals not only utilize the turbulent flow state but also borrow the disturbance generated by other large moving objects, which changes their body motion by staying behind the hull and ‘synchronize’ with the current vortex, thereby reducing energy consumption. This behavior is called Karman gaiting. 1,12,13 Passive drag reduction and adaptive active movement provide survival advantages for aquatic animals, also bringing new ideas and inspiration to different fields of research. In recent years, with the discovery of other biomorphic phenomena at different scales, the traditional view of drag reduction on smooth surfaces has been questioned, while the bionic surface of drag reduction inspired by aquatic animals has been extensively studied and experimented. Surface drag reduction emphasizes the actual value in applications, including traffic, energy transportation, agriculture, textile industry, military, sport, and industry. So far, there have been many research results of bionic drag reduction from aquatic animals as bionic prototypes, which can be roughly divided into three categories: sharks, dolphins, and other aquatic animals. Until now, some review articles on bio-inspired drag-reducing surface have been published. 14–17 However, the discussions focusing on the development of the aquatic animals-inspired drag-reduction surfaces are relatively rare. In this review, drag-reduction biological prototypes represented by sharks and dolphins, the latest signs of progress related to biology, drag-reduction surfaces, drag-reduction mechanisms, manufacturing, and applications are summarized. In this review, we aim to discuss the recent developments in the thriving artificial drag-reduction materials inspired by aquatic animals. This review is mainly composed of five sections. Section 2 presents the drag-reduction surfaces inspired by aquatic animals in detail and summarized their drag-reduction mechanism. In the following Section 3, some typical bionic drag-reduction surface processing and manufacturing methods are described. Then, in Section 4, the applications of bionic drag-reduction surfaces in practical engineering are briefly described. In the final Section 5, the problems and development direction of future research on bionic drag-reduction surfaces are proposed and addressed. This review may provide a better comprehension of the current research status of aquatic animals-inspired drag reduction surfaces."
} | 3,998 |
29746457 | PMC5944914 | pmc | 5,740 | {
"abstract": "To cooperatively carry large food items to the nest, individual ants conform their efforts and coordinate their motion. Throughout this expedition, collective motion is driven both by internal interactions between the carrying ants and a response to newly arrived informed ants that orient the cargo towards the nest. During the transport process, the carrying group must overcome obstacles that block their path to the nest. Here, we investigate the dynamics of cooperative transport, when the motion of the ants is frustrated by a linear obstacle that obstructs the motion of the cargo. The obstacle contains a narrow opening that serves as the only available passage to the nest, and through which single ants can pass but not with the cargo. We provide an analytical model for the ant-cargo system in the constrained environment that predicts a bi-stable dynamic behavior between an oscillatory mode of motion along the obstacle and a convergent mode of motion near the opening. Using both experiments and simulations, we show how for small cargo sizes, the system exhibits spontaneous transitions between these two modes of motion due to fluctuations in the applied force on the cargo. The bi-stability provides two possible problem solving strategies for overcoming the obstacle, either by attempting to pass through the opening, or take large excursions to circumvent the obstacle.",
"introduction": "Introduction Many living groups exhibit collective modes of motion [ 1 ]. Among these, groups such as cell clusters [ 2 , 3 ], locust [ 4 ] and fish [ 5 ], have been found to display spontaneous transitions between co-existing collective dynamical phases. Among these collective phases are disordered modes of motion, in which the group swarms whilst remaining cohesive, and ordered modes of motion, where the individuals orient along a single polarized direction, or rotate around the group center of mass. The spontaneous transitions between these collective modes of motion have been attributed to noise [ 2 , 4 ], or interactions with external constraints [ 3 , 5 ]. However, there is currently no theoretical description that defines the necessary conditions for the emergence of co-existing dynamical phases, or a precise mechanism that explains the transitions between the different modes of motion. In this study, we investigate dynamical bi-stability, and its theoretical underpinnings during cooperative transport by a group of ants. Cooperative transport by ants, also known as group retrieval, is the process by which individual ants join efforts to retrieve large items of food [ 6 – 10 ]. Cooperative transport is known to exist in at least forty different ant species [ 11 – 20 ]. Among these species, the longhorn crazy ants Paratrechina longicornis are well known for displaying highly coordinated retrieval, of items, that can reach orders of magnitude larger than their own size and weight [ 13 , 21 , 22 ]. After a recruitment phase [ 22 ], the longhorn crazy ants lift the load above the surface to reduce friction, and pull towards chemical depositions they leave [ 22 ], which mark the pheromone scent trail that leads to the nest [ 23 ]. Cooperative transport by P. longicornis ants was shown to exhibit a rich variety of collective modes which range from random motions to ballistic movement [ 21 ], that is either directed towards the nest [ 21 ] or exhibits oscillatory modes [ 24 ] and direction changes when the motion was externally constrained [ 22 , 25 ] by semi-natural obstacles ( Fig 1A ). In addition, cooperative transport by this species has allowed for a comprehensive theoretical description [ 21 , 24 ]. The existence of multiple modes of motion, along with the theoretical understanding, suggest the potential of this system for investigating the origins and dynamics of co-existing collective modes. 10.1371/journal.pcbi.1006068.g001 Fig 1 The simplified model. (A) Ants encounter an obstacle during cooperative transport. Dashed line indicates the pheromone scent trail that passes beneath the leaf. Solid line indicates an alternative route. The leaf obstructs the direct route to the nest, and the cargo can not be passed along the original scent trail. Bottom left inset shows a previously studied obstacle [ 21 , 22 , 24 ] that resemble this natural scenario. (B) Ants carrying a circular cargo along a confined linear obstacle with an opening. Blue line is a 16 minute trajectory. The sketched part of the obstacle indicates the true proportions of the enclosed frame. (C) An illustration of the simplified one dimensional model. G is the number of informed ants, that are modeled as a restoring force and v is the velocity the cargo. Ant color index: Blue—lifter, red—puller, informed ants are represented by the force G → . (D) The restoring force due to the informed ants in the 2D case. Left inset compares between the projection of the restoring force for a two dimensional cargo along the x axis, and the restoring force of the continuous function we used in the simplified model for ϵ = 1 and r = 1 ( Eq (9) ). The restoring force is normalized by f 0 . Ant color index: Blue—lifter, red—puller, black—informed. To test this possibility, we have used a simple experimental system, in which the ants interact with a rigid obstacle. Under this constraint, the paths to the nest are blocked from all directions, except for a single narrow opening, that allows passage for single ants, but not for the cargo ( Fig 1B and Methods section). In the presence of such an obstacle, ants that accompany the transport of a large cargo were observed to locally mark pheromone trails that lead from the cargo to the narrow opening [ 22 ]. Even though these trails lead to a dead end (the cargo can not fit through the opening), the markings serve as the only available source of information, as new ants that join the carrying effort pull and direct the cargo towards the narrow opening [ 22 ]. Furthermore, the simple geometry of the obstacle makes the motion of the ant group amenable to analytic theoretical description, which allows us to expose the conditions for bi-stability, and the mechanisms governing the transitions between the co-existing dynamical modes of motion. Here, we present a theory of cooperative transport by ants near rigid obstacles, which predicts bi-stability between two dynamical modes of motion: a convergent mode of motion that keeps the cargo near the opening, and large quasi periodic excursions that can circumvent an obstacle. We demonstrate how noise in the discrete decision making process of the ants induces mechanical fluctuations that can stochastically switch the group between these two modes. The predictions of the model are then verified in experiments, where ants carrying cargoes with the size of natural prey [ 21 ] exhibited spontaneous transitions between the bi-stable collective dynamical modes. In addition to the agreement between observations and simulations, our analytical model allows us to explain the conditions for bi-stability, and propose a physical mechanism for how groups of ants overcome obstacles when carrying food to the nest.",
"discussion": "Discussion During cooperative transport of food, ants often encounter obstacles, such as barriers with small openings that allow single ants to pass, but are too narrow for the food to be retrieved [ 21 , 22 ]. These encounters raise a conflict within the carrying group: While the group attempt to maintain a persistent motion that will allow them to bypass the obstacle, informed individuals attempt to direct the load towards the opening, which is identified as the direct route to the nest. In this work, we explain the behavior of ant groups near obstacles, using a physical mechanism that originates from the mechanical interactions between the individuals in the group. We show that when a group of ants encounters such an obstacle during cooperative transport ( Fig 7A ), two modes of motion spontaneously emerge. These two modes of motion can assist the group in overcoming the obstacle: Either by dwelling near an opening, which may allow flexible loads to be squeezed through the direct route to the nest ( Fig 7B ), or, when carrying large items of food, perform persistent excursions ( Fig 7C ), which may lead to obstacle circumvention. ( Fig 7D ). With the use of experiments and simulations, we demonstrate how intrinsic noise, in the form of mechanical fluctuations, allows stochastic transitions between the two modes of motion, and keeps the system out of detrimental behaviors, such as remaining stuck near an opening with large loads that cannot be squeezed through, or oscillating for long durations, when carrying loads that can be passed through the opening. 10.1371/journal.pcbi.1006068.g007 Fig 7 The conflict that ants face when encountering an obstacle during food retrieval to the nest. (a) Informed ants direct the carrying group towards the pheromone scent trail that pass through the opening and as a result encounter the obstacle. (b) The ants dwell near the opening and the motion is dominated by informed ants that attempt to squeeze the cargo through. (c) The ants motion is dominated by the uninformed ants that attempt to align their direction of motion and perform sideways oscillations. (d) The ants perform a large amplitude excursion that take them across the barrier to meet other informed individuals that mark a new pheromone scent trail. Previous studies of cooperative transport by ants in the presence of constraints have investigated the behavior of the carrying ant group near obstacles with open boundaries [ 21 , 22 , 25 ], a fully confining obstacle that trap the group [ 25 ], and by confining the motion of the cargo by a tether [ 24 ]. These studies have related the decision making process of the individual ants to problem solving behaviors during obstacle navigation [ 21 , 22 , 24 ], and proposed a strategy in which the ants’ behavior changes over the course of time when facing obstacles [ 25 ]. Here, we further examine the interaction of ant groups with obstacles, using a simple experimental setup that allows a detailed analysis of the cargo’s motion. Our results provide direct evidence for the emergence of dynamical bi-stability in the presence of rigid obstacles, and elaborates further the current physical understanding of cooperative transport by ants [ 21 , 22 , 24 ]. In addition, the analytical framework displayed in this work provides a detailed explanation of the origin and conditions for maintaining co-existence between the two collective dynamical modes. These findings could have implications for other biological ensembles [ 2 – 5 ] and theoretical models [ 31 , 32 ], that have reported co-existence between several collective dynamical modes."
} | 2,698 |
36297988 | PMC9607013 | pmc | 5,741 | {
"abstract": "The development of environmentally friendly antifouling strategies for marine applications is of paramount importance, and the fabrication of innovative nanocomposite coatings is a promising approach. Moreover, since Optical Coherence Tomography (OCT) is a powerful imaging technique in biofilm science, the improvement of its analytical power is required to better evaluate the biofilm structure under different scenarios. In this study, the effect of carbon nanotube (CNT)-modified surfaces in cyanobacterial biofilm development was assessed over a long-term assay under controlled hydrodynamic conditions. Their impact on the cyanobacterial biofilm architecture was evaluated by novel parameters obtained from three-dimensional (3D) OCT analysis, such as the contour coefficient, total biofilm volume, biovolume, volume of non-connected pores, and the average size of non-connected pores. The results showed that CNTs incorporated into a commercially used epoxy resin (CNT composite) had a higher antifouling effect at the biofilm maturation stage compared to pristine epoxy resin. Along with a delay in biofilm development, a decrease in biofilm wet weight, thickness, and biovolume was also achieved with the CNT composite compared to epoxy resin and glass (control surfaces). Additionally, biofilms developed on the CNT composite were smoother and presented a lower porosity and a strictly packed structure when compared with those formed on the control surfaces. The novel biofilm parameters obtained from 3D OCT imaging are extremely important when evaluating the biofilm architecture and behavior under different scenarios beyond marine applications.",
"conclusion": "4. Conclusions A set of novel structural parameters obtained from OCT imaging was developed to quantify the marine biofilm structure over time and on three different surface materials, one of them with recognized antifouling activity. CNT-modified surfaces delayed cyanobacterial biofilm development in the maturation stage of the biofilm. Biofilms developed on the composite had reduced wet weight, thickness, and biovolume and were smoother and less porous than those formed on epoxy resin and glass (control surfaces). Analysis of novel parameters obtained by OCT imaging enables a deeper understanding of the biofilm development process in different settings, including the marine environment.",
"introduction": "1. Introduction Marine biofouling causes severe economic and energetic losses, along with critical environmental and ecological consequences. Carbon nanomaterials, including carbon nanotubes (CNTs), graphene, fullerenes, and diamond-like carbon, have been recognized for their antimicrobial and anti-adhesive properties [ 1 ]. Due to their remarkable mechanical strength, high thermal conductivity, and structural stability, CNTs are promising nanomaterials for several applications, namely in the industrial, environmental, and medical fields [ 2 , 3 ]. CNTs have already been tested in antifouling formulations to prevent biofouling, mainly to protect ship hulls, as well as in composite materials that come into contact with seawater. Likewise, CNTs have been reported to impact the composition of marine biofilms, as well as the settlement of macrofouling organisms [ 4 , 5 ]. CNTs can be represented as a single rolled-up graphene sheet (single-walled carbon nanotubes, SWCNTs) or a series of concentric graphene sheets (multi-walled carbon nanotubes, MWCNTs) [ 6 ]. These carbon nanomaterials exhibit a concentric cylindrical structure with a diameter in the order of nanometers, varying according to the number of walls, and a length of several microns that is extendable by up to a few millimeters (around 4 mm). CNTs are generally incorporated into a polymeric matrix for application in protective coatings, such as polydimethylsiloxane (PDMS) [ 7 ], to improve their mechanical strength [ 8 ]. The main antibacterial and antifouling mechanisms of CNTs include the disruption of membrane integrity by electrostatic forces between the microbial outer surface and CNTs, leading to membrane oxidation. Moreover, reactive oxygen species generation may directly harm biological molecules of bacteria and/or indirectly prompt DNA destruction [ 1 , 9 , 10 ]. Although the mechanism behind the antifouling properties of CNTs is still not clear, their length, diameter, surface area, concentration, and treatment time play a significant role in their antifouling and antimicrobial activity [ 9 , 10 , 11 ]. Regarding CNT concentration, a loss of cell viability was shown to be correlated with increasing SWCNT loading. Remarkably, 5% SWCNTs may reduce biofilm development from Bacillus anthracis spores by 81%, while SWCNT concentrations ≥ 20% can inhibit biofilm formation [ 12 ]. Likewise, 0.1 wt% SWCNTs can decrease Escherichia coli biofilms by 18%, while, with 1 wt% SWCNTs, the biofilm reduction may reach 76% [ 13 ]. On the other hand, a study in which different CNT concentrations (0.1, 1, 2, 3, 4, and 5 wt%) were tested to inhibit E. coli biofilm development reported an increase in E. coli culturability for surfaces with CNT concentrations between 0.1 and 2 wt% compared to bare surfaces, while a decrease was observed for the remaining CNT concentrations [ 11 ]. Among these higher CNT concentrations, 3 wt% was the most promising surface for the inhibition of E. coli biofilms. Since CNTs’ antimicrobial activity also depends on their dispersion state [ 14 ], a lower dispersion may occur at higher loadings (4 and 5 wt%), leading to a lower antimicrobial effect on CNT-based surfaces [ 11 ]. Moreover, it has also been reported that introducing small quantities of CNTs into a polymer network can result in a considerable increase in the antibacterial performance of that polymeric matrix [ 13 ]. In fact, polyvinyl-N-carbazole (97 wt%) with 3% of SWCNTs demonstrated similar or stronger bactericidal performance than the surfaces consisting of 100% SWCNTs [ 15 ]. The attachment by macrofoulers, such as calcareous hard-fouling organisms (barnacles, mussels, and tubeworms) and soft-fouling organisms (non-calcareous algae, sponges, anemones, tunicates, and hydroids), is responsible for the main consequences of marine biofouling. However, the prevention of adhesion and biofilm development by microfoulers such as bacteria, cyanobacteria, and diatoms reduces the progression of biofouling to the next stages. A deeper knowledge of biofilm behavior and how it interplays with the surrounding environment will enable the development of efficient methodologies to control biofouling and mitigate its negative impacts. New imaging technologies, biochemical methods, and molecular biology tools have contributed to the technological development of biofilm science. Optical Coherence Tomography (OCT) is an exciting modality that overcomes the time-consuming and destructive methodologies of biofilm analysis, such as some microscopic techniques. In addition to the tedious sample preparation, most of the relevant microscopic techniques applied to the study of biofilms require the staining of the sample or the use of fluorochromes, which are expensive and can interfere with the local properties of the biofilm [ 16 ]. Moreover, some of them provide low-resolution images only covering a small field of view (FOV). OCT presents several advantages over the common microscopic methods since it is a simple and inexpensive technique, does not require sample preparation and/or staining procedures, and allows for the reconstruction of 3D images by in situ , non-invasive, and real-time imaging without affecting the biofilm structure [ 17 ]. Moreover, OCT can provide images at the mesoscale relatively quickly, and it allows for a great penetration depth, revealing several details of the biofilm structure [ 18 ]. Despite all of the advantages of this optical technique, only a limited set of image processing scripts have been specifically developed for processing OCT biofilm images. Furthermore, the analysis of structural parameters obtained from OCT is not advanced when compared, for instance, to microscopy techniques such as Confocal Laser Scanning Microscopy (CLSM). Indeed, there are several software tools and libraries for biofilm image processing from microscopy, including Image Structure Analyzer (ISA) [ 19 ], COMSTAT [ 20 ], PHobia Laser scanning microscopy Imaging Processor (PHLIP) [ 21 ], bio Image_L [ 22 ], and DAIME [ 23 ]. Studies focused on assessing the marine biofouling mitigation effect of CNT coatings can greatly contribute to improving the knowledge regarding the antifouling properties of these promising materials. Moreover, most marine studies focus on unicellular bacteria, and it is pertinent to address additional microfouler organisms such as filamentous cyanobacteria due to their improved stress and predation resistance [ 24 ]. Therefore, the main goal of this study was to analyze the potential of CNT-modified surfaces to delay cyanobacterial biofilms, as well as to evaluate their impact on biofilm architecture using an in-depth OCT analysis. CNTs were incorporated in a commercially available polymer, epoxy resin, since it is commonly used to coat the hulls of small recreational vessels [ 25 , 26 ] due to its unique physical, chemical, and mechanical properties, no safety issues, and low cost [ 27 ]. Additionally, epoxy composites have demonstrated high durability and resistance to fatigue and UV irradiation [ 28 ]. CNT loading (3 wt%) was chosen according to results obtained in previous studies, in which these carbon-based surfaces were tested to inhibit E. coli biofilm development in the medical field [ 11 ]. Additionally, textural modifications of CNTs were performed by ball milling (BM) treatment over 4 h to enhance its antimicrobial performance [ 29 ] by adjusting the CNT length and opening their closed ends to increase the specific surface area [ 30 ]. The textural modifications induced by ball-milling treatment proved to be effective in the inhibition of biofilm formation, reducing the amount of biofilm per surface area, biofilm thickness and surface coverage by 31, 47 and 27%, respectively, when compared to surfaces where CNTs were not ball-milled [ 11 ]. The specific aim of this study comprises the development of novel analysis parameters obtained from 3D OCT imaging to evaluate the biofilm structure. Since OCT is an in situ , non-destructive technique that can be applied to different fields (e.g., marine, medical, and industrial), and the knowledge of biofilm architecture is important to understand all phenomena related to this complex lifestyle, the analysis carried out in this work is extremely relevant. To the best of our knowledge, this is the first study evaluating the impact of CNT-modified surfaces on cyanobacterial biofilm behavior using an in vitro platform that mimics the hydrodynamic conditions prevailing in real marine environments.",
"discussion": "3. Results and Discussion 3.1. Surface Characterization It is recognized that surface properties such as topography and physicochemistry affect their antiadhesive and/or antimicrobial behavior [ 45 , 46 ]. Thus, all tested surfaces were first investigated regarding (i) wettability by water contact angle measurements, (ii) topography and roughness by Atomic Force Microscopy (AFM), and (iii) morphology and structure by Scanning Electron Microscopy (SEM). The results obtained from the water contact angle measurements and roughness analysis are shown in Table 2 . Given that substrates with a water contact angle ( θ w ) of <90° are considered to be hydrophilic [ 47 ], glass is the most hydrophilic surface ( θ w = 40.9° ± 7.4°), followed by the CNT composite ( θ w = 68.9° ± 4.9°), and the epoxy resin-coated glass ( θ w = 76.3° ± 2.5°), which is significantly more hydrophobic ( p < 0.05) than the resin composite. Lower hydrophobic properties caused by the incorporation of 3 wt% ball-milled CNTs were also observed in previous work in another polymeric matrix (PDMS) [ 11 ]. Regarding the average roughness ( R a ) value determined by AFM ( Table 2 ), glass and epoxy resin appeared to be smoother surfaces ( R a of 6.3 and 13 nm, respectively) than the CNT composite, which registered a R a value of about 70 times higher than the remaining surfaces. Figure 2 reveals the topography and morphology of the tested surfaces obtained from AFM and SEM imaging, respectively. Glass and epoxy resin-coated glass were the most homogeneous and smooth materials ( Figure 2 a,b,d,e). In opposition, the CNT composite ( Figure 2 c,f) was the roughest surface, presenting CNT agglomerates that form small elevations on the material ( Figure 2 f). 3.2. Biofilm Formation The structure of biofilms can be numerically quantified with imaging tools to investigate and monitor the effect of different compounds, surfaces and/or environmental factors on biofilm architecture. In the present work, cyanobacterial biofilm development on different surfaces was monitored over 7 weeks and the quantitative results obtained from 3D OCT analysis are shown in Figure 3 . The values indicated in Figure 3 b–f are only presented from day 14 since, for the first sampling day (day 7), the biofilm thickness was below the OCT range. In general, a gradual temporal increase in biofilm wet weight ( Figure 3 a), thickness ( Figure 3 b), and biovolume ( Figure 3 d) were observed, showing that this filamentous cyanobacterium is a good biofilm former. However, growth was more evident on glass and epoxy resin than on the CNT composite. In fact, for biofilm wet weight ( Figure 3 a), from day 7 to day 49, increases of 71%, 64%, and 49% were observed for glass, epoxy resin, and the CNT composite, respectively, and for biofilm thickness ( Figure 3 b), increases of 87%, 82%, and 72% were registered over time for the same surfaces. Regarding biovolume, increases of 77%, 64%, and 64% were observed for glass, epoxy resin and the CNT composite, which indicates that these CNT-modified surfaces can delay biofilm development when compared to pristine epoxy resin and glass. Moreover, on days 42 and 49, a reduction in cyanobacterial biofilm development was observed on the CNT composite surface. Regarding the biofilm thickness, on day 42, the values obtained on glass and epoxy surfaces were 58% and 47% higher than those obtained on the CNT composite surface, respectively, while on day 49, the biofilm thickness was 58% and 23% higher than those attained on the composite ( Figure 3 b). Additionally, the biovolume obtained on the glass and epoxy surface was 45% and 43% higher when compared to the values obtained on the CNT composite surface, respectively. Moreover, on day 49, these values were 46% and 6% higher as compared to the modified epoxy resin ( Figure 3 b). Since, in the early stages of biofilm formation (days 7, 14, and 21), all parameters were similar between the surfaces, the results suggest that the CNT composite surface may have a greater antifouling effect on the maturation stage of these cyanobacterial biofilms as compared to the other two types of substrates. Surface topography plays a considerable role in the way in which marine fouling organisms adhere to surfaces, settle on them, and interact with them [ 48 ]. In the nanoregime, the topography can have a robust impact on the wettability of the surface, as well as a direct effect on the contact area available for fouler adhesion. Once more surface contact area is available, more settlement may take place and, consequently, the organisms may be more difficult to remove [ 49 ]. Moreover, the surface roughness may also promote an increase in the settlement and adhesion strength of biomolecules, including the proteinaceous adhesives used by many marine organisms [ 50 ]. Therefore, rough surfaces present an opportunity for fouling organisms to settle within and between the topographic features, protecting them from hydrodynamic forces [ 51 ]. Highly textured surfaces also provide greater surface area for adhesive cements to adhere [ 52 ]. Even though topography does not act as a unique mechanism, it is still a key feature that must be considered in the design of new materials. Since, in the early stages of biofilm formation, the values attained were similar between the surfaces ( Figure 3 ), it appears that differences in surface properties, namely in average roughness ( Table 2 , Figure 2 ), may have been responsible for the differences registered in the later stages of biofilm development. Therefore, an antimicrobial effect rather than an anti-adhesive effect may explain the impact of CNT-modified surfaces in long-term cyanobacterial biofilm development. CNT composites may lead to unstable and weak biofilm development due to the piercing effect in the cell’s membrane of the first biofilm layers [ 11 ]. However, this viability effect may be reflected only in the subsequent biofilm layers, as cell-to-cell adhesion will be hampered if cells in the initial layers are damaged [ 53 ]. Moreover, biological processes such as cell reproduction and extracellular polymeric substance (EPS) production by these first-adhered impaired cells may affect biofilm development. Biofilms formed on the CNT composite were also more homogenous on days 42 and 49 than those developed on glass and epoxy resin ( Figure 3 c), since the values of the contour coefficients were closer to 1, which reflects a homogeneous and flatter biofilm. Indeed, on day 42, the contour coefficient was around 46% and 32% lower for the CNT composite surface when compared to glass and epoxy resin, respectively. On the other hand, on day 49, these differences reached 53% and 15% when compared to the values obtained on glass and epoxy resin. Since the top of the biofilm grown on the carbon-based surface became flattened, without heterogeneous top structures (such as streamer structures), the superficial area in contact with the surrounding environment decreased, as well as the ability of nutrients and oxygen to penetrate within the biofilm. Likewise, in the maturation stage of biofilm development, a lower percentage of porosity ( Figure 3 e) and a smaller average size of non-connected pores ( Figure 3 f) were determined for biofilms formed on the CNT composite surface when compared with control surfaces. The lower porosity and average size of non-connected pores may contribute to the lower viability of the cells located on the deepest biofilm layers since the internal mass transfer of nutrients to the inner layers of the biofilm may be hindered, contributing to the antimicrobial effect of these surfaces. Figure 4 , Figure 5 and Figure 6 show representative 2D cross-sectional and 3D OCT images of Nodosilinea cf. nodulosa LEGE 10377 biofilms on glass, epoxy resin, and CNT composite after 49 days. Both 2D and 3D OCT images illustrate quantitative data on biofilm biomass and porosity ( Figure 3 ). In fact, on the last day of the experiment, a higher percentage of biofilm biomass and porosity was observed on the glass surface when compared with the epoxy resin and the CNT composite surface. The biofilm top structure can also be observed as flatter for biofilms formed on the CNT composite than biofilms developed on glass and epoxy resin ( Figure 4 and Figure 5 ), as it was also indicated by quantitative data ( Figure 3 c). Moreover, in the biofilm formed on the glass, it was possible to observe long streamers, which can reach around 500 µm ( Figure 4 ). On the other hand, according to representative 3D OCT images, biofilms developed on the CNT composite only present structures that reach around 250 µm. Through representative images of the size of non-connected pores, it was also possible to observe that these values can reach around 98,000 µm 3 in biofilms developed on glass, while values around 35,000 µm 3 and 19,000 µm 3 were achieved on epoxy resin and the CNT composite, respectively. Mature biofilms consist of multidimensional heterogeneous structures with interstitial pores, which ensure the water and nutrient flow and that influence their resistance to mechanical or chemical challenges [ 54 , 55 , 56 , 57 ]. Biofilm biomass, thickness, and structure have a strong effect on the performance of underwater marine devices [ 58 ]. Consequently, the knowledge of these biofouling parameters is essential for the design and maintenance of submerged marine equipment. In this work, CNT composite surfaces were tested for the prevention of cyanobacterial biofilms. Biofilms formed on this carbon-modified surface were more homogeneous, flatter, less porous, and had a tightly packed structure compared with the control surfaces, as it can be proved by the quantitative ( Figure 3 c,e,f) and qualitative ( Figure 4 , Figure 5 and Figure 6 ) data. Although biofilm growth is reduced on these surfaces, which may be beneficial for the performance of nautical equipment, it may have an impact on the efficacy of methods to eradicate the biofilms formed on this surface material. Firstly, due to their lower porosity and a smaller average size of non-connected pores ( Figure 3 e,f), the internal mass transfer may be hampered, and chemical compounds used for biofouling control may not reach the inner layers of the biofilm [ 59 ]. Moreover, a homogeneous structure ( Figure 3 c) can be associated with greater cohesion across the whole biofilm structure. In this scenario, the detachment of biofilm components either by increasing hydrodynamic forces or by applying mechanical cleaning methods would be facilitated if a heterogeneous structure was present (such as the presence of some streamers). As a flatter biofilm was present, the detachment or removal phenomena may be hindered. Although there are several biofilm structural parameters described in the literature [ 60 , 61 ], there has been a tendency to use only a limited number of them [ 62 , 63 , 64 ]. This evidence makes cross-comparisons difficult and can be justified by the fact that only some of these parameters computed from biofilm images can be intuitively associated with identifiable biological processes. The analysis of biofilms based on biofilm weight, thickness, and biovolume alone, which are typically used for the description of biofilm structure, does not provide complete information on biofilm development. Only the combination of all evaluated structural parameters leads to a complete overview of biofilm behavior on the different surfaces. Moreover, the contour coefficient reported in this work may be a relevant parameter to replace or complement the analysis of biofilm roughness since more reliable structural biofilm information is provided by this novel parameter. Analysis by SEM was also performed to assess the morphological differences shown by the cyanobacterial biofilms grown on the distinct surfaces ( Figure 7 ). As observed by OCT ( Figure 3 , Figure 4 and Figure 5 ), the SEM analysis reveals differences in the cyanobacterial biofilm growth patterns on different surfaces. The highest and lowest cyanobacterial biofilm amounts were observed on glass and the CNT composite surfaces, respectively. Indeed, SEM observations showed that while the biofilm formed on the control surfaces (glass and epoxy resin) looks like a dense filamentous network that covers practically the entire surface area, the biofilm grown on the composite surface presented lower-density cell aggregates. OCT is a relevant tool for assessing the spatial organization and heterogeneity of biofilm since 2D and 3D datasets contain a representative description of the overall biofilm structure at the mesoscale with µm-resolution [ 17 ]. Moreover, by OCT analysis, the laborious nature, as well as the costs entailed with microscopic techniques, can be reduced. Few studies focus on longer assays for the assessment of CNTs on marine biofilm formation [ 65 ]. In fact, some in vitro studies have been performed between 6 [ 66 ] and 10 days [ 4 ], but only a study performed by Xie et al. [ 67 ] achieved the 20 days necessary to evaluate the antifouling effect of CNT-based antifouling coatings. Moreover, these studies focus on other marine bacteria, diatoms, algae, and macrofoulers [ 4 , 66 , 67 ]. For instance, an in situ study performed by Sun et al. [ 68 ] with pioneer biofilm bacteria over 24 days showed that all CNT/PDMS composites decreased Proteobacteria biofilm formation but increased cyanobacterial biofilm development. Other studies showed a promising reduction in biofilms formed by different microfoulers at different times on distinct CNT-based antifouling coatings [ 5 , 45 , 67 , 69 , 70 , 71 , 72 ]. In this study, biofilm was evaluated as a whole structure, including cells, water, and the compounds excreted by the cyanobacterial cells. Considering that a greater number of active cells in a biofilm can lead to greater surface colonization and biofilm development potential, future studies should include complementary techniques to assess the viability and/or the metabolic state of biofilm cyanobacterial cells [ 73 , 74 ]. Since the mechanisms behind the antifouling properties of CNTs are still not clear [ 2 ], it is relevant to conduct further assays in marine conditions with CNT-/epoxy resin surfaces, incorporating different CNT concentrations, lengths, diameters, and surface areas, as well as functionalized CNTs, with different polymeric matrices and different fouling organisms [ 2 , 9 , 10 , 11 ]. The generation of oxidative stress and mechanical damage through the direct perforation of the microorganisms’ outer membranes and the release of intracellular content are some of the mechanisms that may be involved in CNT action [ 10 ]. Indeed, the representative SEM images of the morphology and structure of CNTs in the composite reveal the presence of CNT agglomerates ( Figure 2 f; Figure S1 in Supplementary Material ), which can interact with the membrane of cyanobacterial cells by the known piercing phenomenon and be one of the strategies associated with the antifouling properties of these surfaces. In situ assays are also particularly recommended to assess the impact of these surfaces on the adhesion and biofilm formation of multiple microfouling organisms, as well as on the subsequent attachment of macrofoulers. Moreover, additional concerns with these nanostructured surfaces may be related to the fact that they may be damaged in harsh marine environments, leading to a reduction in their antifouling ability and lifespan. Hence, the development of robust and mechanically stabilized surfaces is critical [ 75 ]. Different biofilm techniques provide valuable and complementary information about different aspects of the complex biofilm structure. Therefore, a multidisciplinary approach that integrates different methodologies is recommended to obtain a more realistic biofilm representation and to better understand the complex phenomenon of marine biofilm development."
} | 6,736 |
35515466 | PMC9054073 | pmc | 5,742 | {
"abstract": "Aerobic oxidation of native soft wood lignin in an aqueous solution of Bu 4 NOH facilitates efficient production of vanillin (4-hydroxy-3-methoxybenzaldehyde), which is one of the platform chemicals in industry. Oxidation of Japanese cedar ( Cryptomeria japonica ) wood flour at 120 °C for 4 h under O 2 in Bu 4 NOH-based aqueous solutions produced vanillin in 23.2 wt% yield based on the Klason lignin content of the starting material. This yield was comparable to that in alkaline nitrobenzene oxidation of the same material (27.2%), which indicated that our aerobic oxidation exploited the full potential of the wood flour for vanillin production. Further mechanical investigation with lignin model compounds suggested that the vanillin formation occurred mainly through following successive reactions: alkaline-catalyzed degradation of β-ether linkages in middle units of lignin polymer to form a glycerol end group, oxidation of the glycerol end group by O 2 to a HC α \n \n\n<svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>\nCreated by potrace 1.16, written by Peter Selinger 2001-2019\n</metadata><g transform=\"translate(1.000000,15.000000) scale(0.017500,-0.017500)\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>\n\n O moiety, and release of vanillin from the HC α O end. One of the reasons for the high performance of Bu 4 NOH for the vanillin production was explained by the general understanding in organic chemistry that Bu 4 OH is a stronger base than simple alkali, e.g. NaOH. The other more fundamental mechanical aspect was that Bu 4 N + suppressed disproportionation of the vanillin precursor (the C α HO end group) probably due to strong interaction between the cation and the HC α O end group.",
"conclusion": "Conclusions The degradation of Japanese cedar wood flour under O 2 produced vanillin and vanillic acid in 23.2 and 1.2% yields on the basis of the Klason lignin amount, respectively, at 120 °C for 4 h in 1.25 mol L −1 Bu 4 NOH aq., where the OH − concentration was enhanced to 3.75 mol L −1 by the addition of NaOH. These yields have been significantly improved, compared to those obtained under air reported in our previous studies. 8 Also, the reaction time required for achieving the maximum vanillin and vanillic acid yields was drastically shortened to 4 h by the O 2 introduction. It should be noted that the yields of the compounds were comparable to those obtained in the AN oxidation, which is a benchmark for evaluating selectivity of lignin depolymerization methods. The mechanical investigation with the lignin model compounds suggested that the major vanillin formation pathway involved the alkaline-catalyzed β-ether cleavage on the non-phenolic middle units of lignin polymer to produce the glycerol end group, followed by the oxidation of the end group by O 2 to aldehyde and subsequent releasement of free vanillin. The positive effect of Bu 4 NOH over NaOH with regard to the vanillin formation was partially attributed to the basicity of Bu 4 NOH stronger than that of NaOH. More fundamentally, Bu 4 N + significantly suppressed the disproportionation of the aldehyde end so that the releasement of vanillin became to play more roles in entire degradation.",
"introduction": "Introduction Vanillin (3-methoxy-4-hydroxybenzaldehyde) is a major target compound to be produced from soft wood lignin. Quite a few lignin depolymerization methods such as degradation with various oxidants, 1–14 catalytic degradation, 6,12–15 and pyrolytic processes 16 make it clear that vanillin is one of the major minima in the potential energy map in those lignin degradation reactions. Also, vanillin is a platform compound in the chemical industry, and is a source of several medicines, polymer materials, etc. 17,18 Until the 1990s industrial vanillin production was based mainly on aerobic oxidation of lignosulfonate, a major component of waste water from sulfite pulping. 3,6,19–21 This vanillin production was carried out in alkaline media under compressed air with the yield of vanillin being ∼5% based on the original lignosulfonate. 3,6,7 However, management of waste water containing sulfur compounds is a major problem of this biomass-based vanillin production process. Due to this drawback, recent vanillin production is almost completely substituted with phenol-based ones, where phenol is manufactured from fossil resources. Considering recent environmental requirements, however, the biomass-based vanillin production is worth being re-investigated and re-developed. Our previous studies have reported that selective vanillin production can be made by aerobic oxidation of several soft wood lignin samples, e.g. soft wood flour, milled wood lignin, and several types of technical lignins including lignosulfonate, at 120 °C in the presence of tetrabutylammonium ion Bu 4 N + and OH − . 5,8 When Japanese cedar ( Cryptomeria japonica ) wood flour is degraded in 1.25 mol L −1 Bu 4 NOH aq. at 120 °C under air, vanillin and vanillic acid are produced in 15.4 and 3.9%, respectively, based on the Klason lignin content of the wood. 8 These yields are considerably higher than those obtained in NaOH aq. with the same OH − concertation, 5,8 indicating that the Bu 4 NOH solution exhibits much better performance for vanillin production than the corresponding NaOH aq. The Bu 4 NOH-based oxidation method has several advantages over the process with lignosulfonate: this method employs reaction temperature (120 °C) significantly lower than that of the lignosulfonate process (∼170 °C); 3 the process can utilize non-sulfur containing low materials such as wood flour and soda-lignin as well as lignosulfonate. Also, the use of molecular oxygen as an ideal oxidation reagent in this process should be highly emphasized. 4 b On the other hand, there are several issues to be further managed: considerably long reaction times (up to 72 h) and unclear mechanical aspects of the reaction involved in the aerobic oxidation, especially the roles of Bu 4 N + in overall reactions. It is also noted that the highest yield of the target compound obtained with the Bu 4 NOH-based method is still lower than those obtained in the alkaline nitrobenzene (AN) oxidation, 8 which is used as a benchmark to evaluate the performance of lignin oxidation processes. The first half of this article handles the above issues of reaction time and the yields of vanillin and vanillic acid. We will report the reaction time required to reach the maximum vanillin and vanillic acid yields is significantly shortened along with improved yields of the compounds, when the reaction was carried out with the introduction of pure O 2 to the reaction system. In the second haft of this article, we will report results obtained with lignin model compounds (see Scheme 1 for their structures) and discuss the reasons for the high performance of Bu 4 NOH, including the roles of Bu 4 + in the oxidation process. Scheme 1 β- O -4 type dimeric lignin model compounds employed in this study.\n\nVanillin production from wood flour with introduction of O 2 Our previous study reported that aerobic oxidation of the wood flour (14 mg) in 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) under air for 43 h produced vanillin and vanillic acid in 15.4 and 3.9 wt% yields, respectively, based on the Klason lignin amount of the wood flour. 8 The 1.25 mol L −1 aqueous solution of Bu 4 NOH is the molten form of the Bu 4 NOH·30H 2 O crystal. To further enhance the degradation, we carried out similar degradation experiments after flushing the reaction system (sealed 10 mL test tube) with pure O 2 . We will hereafter call this “degradation under O 2 ” for convenience, although the atmosphere of the reaction system is not fully substituted with O 2 . Note that 1.25 mol L −1 is the best concentration of Bu 4 N + in terms of vanillin production according to our previous study: 5 increase in [Bu 4 N + ] to more than 1.25 mol L −1 scarcely boosts the vanillin yield and also results in degradation of Bu 4 N + probably into BuOH and Bu 3 N. \n Fig. 1A shows changes in the yield of vanillin during the degradation in the Bu 4 NOH aq. under O 2 along with our previous results obtained under air in the same reaction medium ( Fig. 1C ). It becomes clear that the vanillin formation is significantly faster under O 2 : the time required for reaching the maximum vanillin yield was 8 h under O 2 whereas the reaction under air needed 43 h. The maximum yield of vanillin was also increased to 19.8 wt% (8 h) from 15.4 wt% (43 h) by the O 2 substitution. After reaching the maximum yield in 8 h, the yield of vanillin was decreased to 17.0 wt% at 20 h and this degradation of vanillin was more significant under O 2 than under air. This indicates that vanillin is somewhat reactive toward O 2 and reaction time should be carefully controlled when the vanillin production is carried out under O 2 instead of air. Fig. 1 Changes with time in yields of vanillin (●), vanillic acid (■), and their total yields (△) during degradation of Japanese cedar wood flour (14 mg) in 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) under O 2 (A), under O 2 with the addition of NaOH(s) (200 mg) (B), and under air 8 (C) at 120 °C. The total amount of Klason lignin and acid-soluble lignin of the wood flour was 34.3 wt%. \n Fig. 1 also shows the yield of vanillic acid (3-methoxy-4-hydroxybenzoic acid) along with that of vanillin, as vanillic acid is the second major product, as reported previously. 5,8 The reaction under O 2 tends to form more vanillin than vanillic acid, when it is compared to the one under air ( Fig. 1A and C ). It is thus stated that O 2 introduction made the reaction more vanillin selective. One of the possible reasons for this selectivity change is that vanillic acid is more reactive toward O 2 than vanillin and readily release CO 2 . As shown in Fig. 1A , the maximum yield of vanillic acid was achieved at 4 h in the Bu 4 NOH aq. under O 2 and then the yield gradually decreased. Note that increase in the yield of vanillic acid was not observed during the degradation of vanillin from 8 to 20 h, which suggested oxidation of vanillin to vanillic acid was not important under the employed conditions. We then carried out the degradation of the wood flour (14.0 mg) in 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) under O 2 with the addition of NaOH(s) (200 mg) to increase the OH − concentration of the reaction solution to 3.75 mol L −1 , as vanillin formation is significantly enhanced in stronger alkaline media. 5 In this alkaline-enhanced solution, as shown in Fig. 1B , the reaction time required for the maximum yield of vanillin was further shortened to 4 h. The yield of vanillin was also improved to 23.2 wt% by the addition of NaOH. We thus tried further addition of NaOH to the solution, but when the concentration of OH − was set at 5.0 mol L −1 by the addition of 300 mg of NaOH, significant decrease in the vanillin yield was observed. \n Table 1 summarizes the maximum yield of vanillin obtained from the wood flour in the above Bu 4 NOH-based reaction solutions along with that obtained in alkaline nitrobenzene (AN) oxidation of the same wood flour. AN oxidation is one of the most selective methods to convert various lignin samples to the corresponding monomeric benzaldehydes (vanillin in the case of soft wood lignins) and used as a benchmark to measure the performance of lignin conversion methods. The maximum vanillin yield (23.0 wt%) and the shortest reaction time (4 h) were both achieved when the reaction was carried out under O 2 in the Bu 4 NOH aq. with the addition of NaOH. Considering that our previous study about the reaction under air required 43 h reaction time to achieve much lower maximum vanillin yield (15.4 wt%), the O 2 introduction investigated in this study had drastic effects for improving the performance of our vanillin production method. Also, the 23.0 wt% vanillin yield obtained in the NaOH-added Bu 4 NOH aq. under O 2 was comparable to those in AN oxidation ( Table 1 ). This also indicates that our Bu 4 NOH-based reaction system well exploits vanillin forming potential of the wood flour. Yields (wt%, based on the Klason lignin amount) of vanillin and vanillic acid from the Japanese cedar wood flour (14.0 mg) degraded in the Bu 4 NOH aq.-based reaction media (2.0 mL) at 120 °C and their yields from alkaline nitrobenzene (AN) oxidation Medium Atmosphere Reaction time a (h) Yield (wt%) Vanillin Vanillic acid Total Bu 4 NOH aq. + NaOH(s) b O 2 4.0 23.2 1.2 24.4 Bu 4 NOH aq. 8 c Air 43 15.4 3.9 19.3 AN oxidation 8 Air 2.5 27.2 1.2 28.4 a The reaction times that result in the maximum vanillin yield are selected for each column. For AN oxidation, the reaction time was 2.5 h according to the standard procedure. b Reaction solution was prepared by the addition of NaOH(s) (200 mg) to 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL). c These are the results presented in our previous study, 8 where 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) was used without the NaOH addition.",
"discussion": "Results and discussion Vanillin production from wood flour with introduction of O 2 Our previous study reported that aerobic oxidation of the wood flour (14 mg) in 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) under air for 43 h produced vanillin and vanillic acid in 15.4 and 3.9 wt% yields, respectively, based on the Klason lignin amount of the wood flour. 8 The 1.25 mol L −1 aqueous solution of Bu 4 NOH is the molten form of the Bu 4 NOH·30H 2 O crystal. To further enhance the degradation, we carried out similar degradation experiments after flushing the reaction system (sealed 10 mL test tube) with pure O 2 . We will hereafter call this “degradation under O 2 ” for convenience, although the atmosphere of the reaction system is not fully substituted with O 2 . Note that 1.25 mol L −1 is the best concentration of Bu 4 N + in terms of vanillin production according to our previous study: 5 increase in [Bu 4 N + ] to more than 1.25 mol L −1 scarcely boosts the vanillin yield and also results in degradation of Bu 4 N + probably into BuOH and Bu 3 N. \n Fig. 1A shows changes in the yield of vanillin during the degradation in the Bu 4 NOH aq. under O 2 along with our previous results obtained under air in the same reaction medium ( Fig. 1C ). It becomes clear that the vanillin formation is significantly faster under O 2 : the time required for reaching the maximum vanillin yield was 8 h under O 2 whereas the reaction under air needed 43 h. The maximum yield of vanillin was also increased to 19.8 wt% (8 h) from 15.4 wt% (43 h) by the O 2 substitution. After reaching the maximum yield in 8 h, the yield of vanillin was decreased to 17.0 wt% at 20 h and this degradation of vanillin was more significant under O 2 than under air. This indicates that vanillin is somewhat reactive toward O 2 and reaction time should be carefully controlled when the vanillin production is carried out under O 2 instead of air. Fig. 1 Changes with time in yields of vanillin (●), vanillic acid (■), and their total yields (△) during degradation of Japanese cedar wood flour (14 mg) in 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) under O 2 (A), under O 2 with the addition of NaOH(s) (200 mg) (B), and under air 8 (C) at 120 °C. The total amount of Klason lignin and acid-soluble lignin of the wood flour was 34.3 wt%. \n Fig. 1 also shows the yield of vanillic acid (3-methoxy-4-hydroxybenzoic acid) along with that of vanillin, as vanillic acid is the second major product, as reported previously. 5,8 The reaction under O 2 tends to form more vanillin than vanillic acid, when it is compared to the one under air ( Fig. 1A and C ). It is thus stated that O 2 introduction made the reaction more vanillin selective. One of the possible reasons for this selectivity change is that vanillic acid is more reactive toward O 2 than vanillin and readily release CO 2 . As shown in Fig. 1A , the maximum yield of vanillic acid was achieved at 4 h in the Bu 4 NOH aq. under O 2 and then the yield gradually decreased. Note that increase in the yield of vanillic acid was not observed during the degradation of vanillin from 8 to 20 h, which suggested oxidation of vanillin to vanillic acid was not important under the employed conditions. We then carried out the degradation of the wood flour (14.0 mg) in 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) under O 2 with the addition of NaOH(s) (200 mg) to increase the OH − concentration of the reaction solution to 3.75 mol L −1 , as vanillin formation is significantly enhanced in stronger alkaline media. 5 In this alkaline-enhanced solution, as shown in Fig. 1B , the reaction time required for the maximum yield of vanillin was further shortened to 4 h. The yield of vanillin was also improved to 23.2 wt% by the addition of NaOH. We thus tried further addition of NaOH to the solution, but when the concentration of OH − was set at 5.0 mol L −1 by the addition of 300 mg of NaOH, significant decrease in the vanillin yield was observed. \n Table 1 summarizes the maximum yield of vanillin obtained from the wood flour in the above Bu 4 NOH-based reaction solutions along with that obtained in alkaline nitrobenzene (AN) oxidation of the same wood flour. AN oxidation is one of the most selective methods to convert various lignin samples to the corresponding monomeric benzaldehydes (vanillin in the case of soft wood lignins) and used as a benchmark to measure the performance of lignin conversion methods. The maximum vanillin yield (23.0 wt%) and the shortest reaction time (4 h) were both achieved when the reaction was carried out under O 2 in the Bu 4 NOH aq. with the addition of NaOH. Considering that our previous study about the reaction under air required 43 h reaction time to achieve much lower maximum vanillin yield (15.4 wt%), the O 2 introduction investigated in this study had drastic effects for improving the performance of our vanillin production method. Also, the 23.0 wt% vanillin yield obtained in the NaOH-added Bu 4 NOH aq. under O 2 was comparable to those in AN oxidation ( Table 1 ). This also indicates that our Bu 4 NOH-based reaction system well exploits vanillin forming potential of the wood flour. Yields (wt%, based on the Klason lignin amount) of vanillin and vanillic acid from the Japanese cedar wood flour (14.0 mg) degraded in the Bu 4 NOH aq.-based reaction media (2.0 mL) at 120 °C and their yields from alkaline nitrobenzene (AN) oxidation Medium Atmosphere Reaction time a (h) Yield (wt%) Vanillin Vanillic acid Total Bu 4 NOH aq. + NaOH(s) b O 2 4.0 23.2 1.2 24.4 Bu 4 NOH aq. 8 c Air 43 15.4 3.9 19.3 AN oxidation 8 Air 2.5 27.2 1.2 28.4 a The reaction times that result in the maximum vanillin yield are selected for each column. For AN oxidation, the reaction time was 2.5 h according to the standard procedure. b Reaction solution was prepared by the addition of NaOH(s) (200 mg) to 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL). c These are the results presented in our previous study, 8 where 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) was used without the NaOH addition. Mechanical investigation with lignin model compounds We have so far shown that vanillin is effectively produced by aerobic oxidation of the wood flour in the Bu 4 NOH-based solutions. Since such effective vanillin production cannot be achieved in a simple alkali solution ( e.g. NaOH aq.) with the same OH − concentration as that of the Bu 4 N + -based one, 5,8 it is clear that chemical nature specific for Bu 4 NOH plays pivotal roles in the vanillin formation. To investigate the mechanisms underlying the vanillin production in the Bu 4 NOH solution, degradation of several types of lignin model compounds (mainly LM1 and LM2, see Scheme 1 ) was carried out in 1.25 mol L −1 Bu 4 NOH and NaOH solutions and the reaction behaviors were carefully compared. In these model experiments, we carried out the degradation under air (not under O 2 ) to minimize degradation of the products. \n Fig. 2 presents a HPLC chromatogram of the reaction mixture obtained from the degradation of LM1 (20 mg) in the Bu 4 NOH aq. (2.0 mL) under air, where β- O -4 is the most abundant linkage type in native lignin. LM1 formed vanillin along with veratraldehyde (3,4-dimethoxybenzaldehyde), veratryl alcohol (3,4-dimethoxybenzyl alcohol), veratric acid (3,4-dimethoxybenzoic acid), and guaiacol. The compounds with the veratryl moiety and vanillin are most likely to be formed from the A ring of LM1 and guaiacol is derived from the B ring (see Scheme 1 for the definition of the A- and B-rings). Fig. 2 HPLC chromatogram of the reaction mixture obtained after the degradation of LM1 (20 mg) in 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) under air for 4 h. As shown in Fig. 3A , degradation of LM1 for short reaction times gave guaiacol and small amount of the A-ring-derived compounds. At the 1 h reaction time (the second plot from the left), for instance, guaiacol was formed in 12.3 mol% yield and the A-ring-derived compounds, mainly veratraldehyde, were produced in 7.2 mol% yield, with 86.4% of LM2 remaining intact. This suggests that the initial step of the degradation is alkaline-catalyzed β-ether cleavage shown as pathway A in Scheme 2 , as reported in many studies. 22–25 This β-ether cleavage results in the formation of veratryl glycerol from the A-ring along with the releasement of guaiacol from the B-ring. Veratryl glycerol was not detected in the HPLC analysis probably due to its very high polarity, but the presence of significant amount of the compound in the reaction mixture was strongly suggested from the GC/MS analysis of the acetylated reaction mixture (see Fig. S1 in ESI † ). The yields of the A-ring-derived compounds were gradually increased at prolonged reaction times ( Fig. 3A ). Veratraldehyde was predominant in the A-ring derived products until 6 h and vanillin then became the major product with almost full consumption of LM1. At 43 h vanillin and veratraldehyde were finally formed in 15.7 mol% and 2.2 mol% yields, respectively, with the LM1 recovery being 3.7%. Note that the required 43 h reaction time is in good agreement with the experimental fact that vanillin yield became almost constant after 43 h in the degradation of the wood flour in the Bu 4 NOH aq. under air (see Fig. 1C ). 8 Fig. 3 Changes with time in recovery of LM1 ( ), yields of guaiacol ( ), veratraldehyde ( ), vanillin ( ), veratric acid ( ), veratryl alcohol ( ), and the total yield of the A-ring-derived products ( ) during degradation of LM1 (20 mg) at 120 °C under air in 1.25 mol L −1 Bu 4 NOH aq. (2.0 mL) (A) and in 1.25 mol L −1 NaOH aq. (2.0 mL) (B). Scheme 2 Reaction pathways from LM1 to major products detected in this study. The above results suggest that, as presented in Scheme 2 , veratryl glycerol formed by the β-ether cleavage is gradually oxidized into veratraldehyde by O 2 via pathway B. However, our attempts to detect any intermediates in this aerobic oxidation step were not successful. Veratraldehyde then undergoes a demethylation reaction on the 4-OMe group to finally form vanillin via pathway C. The detailed mechanism of this demethylation is not clear at the moment. One of the possibilities is a simple S N 2 reaction on the methyl group by OH − , but a mechanism involving a nucleophilic addition of OH − on the 4-position of the benzene ring followed by the elimination of MeO − cannot be ruled out due to the presence of a strong electron-withdrawing CHO group at the 1-position. It is also noted that the demethylation of the 3-OMe group is disfavored because of the lack of the influence of the CHO group at the 3-position. \n Fig. 2 and 3 indicate the reaction mixture also contains considerable amount of veratric acid and veratryl alcohol. It is likely that these two compounds are produced through disproportionation of veratraldehyde via Cannizzaro reaction shown in pathway D in Scheme 2 . This consideration is supported by the fact that the amount of these three compounds increased with the gradual decrease in the amount of veratraldehyde ( Fig. 3A ). There also might be another pathway where LM1 directly gave veratraldehyde, pathway E in Scheme 2 , as suggested from the result that the sum of the guaiacol yield and the LM1 recovery was less than 100% at 43 h reaction time. However, this pathway must be minor compared to the veratraldehyde formation via the pathways A and B, since the guaiacol yield is quantitative at least at the initial stage of the reaction, as discussed above. It is also noted that guaiacyl moieties including guaiacol degrade in the presence of O 2 ( ref. 26–30 ) and this is probably one of the reasons for the small yield of the compound at the prolonged reaction times. We then performed the degradation of LM1 in 1.25 mol L −1 NaOH aq., a simple alkaline solution with the same OH − concentration as that of the Bu 4 NOH aq. As shown in Fig. 3B , the degradation velocity of LM1 was lower in the NaOH aq. than in the Bu 4 NOH aq. The maximum yield of vanillin in the NaOH aq. (0.8 mol%, 6 h) was also much lower in the NaOH solution. One of the plausible reasons for this is that the activity of OH − is greater in the Bu 4 NOH aq. than in the NaOH aq. even when their OH − concentrations are the same. In other words, OH − in the Bu 4 NOH aq. is less influenced by the counter cation Bu 4 N + , which renders the Bu 4 NOH solution more basic over the NaOH aq. The fast degradation of LM1 will result in smooth formation of the veratryl glycerol, which is the major origin of vanillin ( Scheme 2 ). There is another mechanical aspect that accounts for the better performance of Bu 4 NOH for vanillin production. Table 2 summarizes the yields of the major products quantified after the degradation of LM1 for 43 h. In the Bu 4 NOH aq. LM1 formed the aldehyde products (veratraldehyde and vanillin) in 17.9 mol% (18.6 mol%) yield, where the yield in the parenthesis is the one based on degraded LM1. The other non-aldehyde A-ring derived products (veratric acid and veratryl alcohol) were formed in 15.8 mol% yield (16.5 mol%) in total, which was similar to that of the aldehyde products. These results suggest that the reactions via the pathways C and D almost evenly occur in the Bu 4 NOH aq. It is also noted that total yield of the four compounds was 33.7 mol% (35.1 mol% based on degraded LM1) and the rest (64.9 mol%) undetected products probably involve considerable amount of veratryl glycerol (see above discussion and Fig. S1 in ESI † ). Yields (mol%) of major A-ring-derived products after the degradation of LM1 (20 mg) at 120 °C for 43 h under air in 1.25 mol L −1 Bu4NOH aq. and 1.25 mol L −1 NaOH aq. (2.0 mL) Aldehydes Non-aldehydes Total Vanillin Veratraldehyde Total Veratryl alcohol Veratric acid Total Bu 4 NOH aq. 15.7 (16.3) a 2.2 (2.3) 17.9 (18.6) 9.1 (9.5) 6.7 (7.0) 15.8 (16.5) 33.7 (35.1) NaOH aq. 0.4 (0.9) 5.2 (12.1) 5.6 (13.0) 3.9 (9.1) 5.4 (12.6) 9.3 (21.7) 14.9 (34.7) a The value in the parenthesis shows the yield based on the amount of degraded LM1: 96.3% and 42.9% in the Bu 4 NOH aq. and the NaOH aq. based on the initial amount of LM1, respectively. The degradation of LM1 in the NaOH aq., on the other hand, formed relatively small amounts of the four A-ring-derived products (the total yield: 14.9 mol% after 43 h degradation), as summarized in Table 2 . This is because complete degradation of LM1 was not achieved in the NaOH aq. even after 43 h degradation (see Fig. 3B ). The total yield in the NaOH aq. eventually became comparable to that in Bu 4 NOH when it was calculated based on the degraded LM1: 35.1 mol% in the Bu 4 NOH aq. and 34.7 mol% in the NaOH aq. It is therefore likely that reactivity of veratryl glycerol toward O 2 to form veratraldehyde – in other words the velocity of the reactions involved in the pathway B – is almost constant no matter whether the reaction was carried out in the Bu 4 NOH aq. or the NaOH aq. However, as shown in Table 2 , the total yield of the aldehyde products in NaOH aq. based on degraded LM1 (13.0 mol%) was smaller than that in Bu 4 NOH (18.6 mol%) with the total yields of the non-aldehyde products in NaOH aq. (21.7 mol%) being greater than that in Bu 4 NOH (16.5 mol%). These different yields of the aldehyde and the non-aldehyde products suggest that the pathway C is more favored over the pathway D in the Bu 4 NOH aq., which results in the increased yield of vanillin. To check this idea, we degraded veratraldehyde (5.0 mg) at 120 °C for 4 h under N 2 either in the Bu 4 NOH aq. or the NaOH aq. (1.0 mL) and compared the product distribution between the degradations in the two reaction media. As summarized in Table 3 , the degradation of veratraldehyde in Bu 4 NOH resulted in the formation of vanillin, veratryl alcohol, and veratric acid in very similar yields. On the other hand, the degradation in NaOH aq. gave much smaller amount of vanillin and more amount of veratryl alcohol and veratric acid. This is in complete agreement with the above consideration that the demethylation pathway C ( Scheme 2 ) is more favored over the disproportionation pathway D in the Bu 4 NOH aq. We also carried out similar degradation in Bu 4 NCl-added NaOH aq., where the 1.25 mol L −1 Bu 4 NOH aq. was mimicked by the addition of the corresponding amount of Bu 4 NCl to 1.25 mol L −1 NaOH aq. The addition of Bu 4 NCl also increased the selectivity toward vanillin as indicated from the higher vanillin yield (20.2 mol%) based on the degraded veratraldehyde and the lower yield of veratryl alcohol and veratric acid (29.5 and 32.3 mol%, respectively) than those obtained in the simple NaOH aq. Interestingly, in this Bu 4 NCl-added system, the degradation of veratraldehyde was considerably suppressed: much more starting material (57.0%) was recovered in the Bu 4 NCl-added NaOH aq. These results suggest that Bu 4 N + slows down both pathways C and D, and along with that, makes the degradation more vanillin-oriented. Similar recoveries of the starting material obtained between the Bu 4 NOH and the NaOH aq. – they are 11.5 and 8.6%, respectively – would be explained by the idea that the slowing-down effect by Bu 4 N + was compensated by the strong basicity of Bu 4 NOH (see above). Yields (mol%) of vanillin, veratryl alcohol, and veratric acid along with recovery (%) of veratraldehyde after degradation of veratraldehyde (5.0 mg) at 120 °C for 4 h under N 2 in 1.25 mol L −1 Bu 4 NOH aq. and NaOH aq. (1.0 mL) Yield Recovery Vanillin Veratryl alcohol Veratric acid Bu 4 NOH aq. 30.6 (33.5) a 28.1 (30.7) 31.2 (34.1) 8.6 NaOH aq. 5.9 (6.7) 38.3 (43.3) 41.6 (47.0) 11.5 NaOH aq. + Bu 4 NCl(s) b 8.7 (20.2) 12.7 (29.5) 13.9 (32.3) 57.0 a The value in the parenthesis shows the yield based on the amount of degraded veratraldehyde. b Solid Bu 4 NCl was added to the NaOH solution so that the concentration of Bu 4 N + in the reaction solution became 1.25 mol L −1 . To obtain deeper insights into the effect of Bu 4 N + , we carried out 1 H NMR analyses of veratraldehyde in 1.25 mol L −1 NaOD/D 2 O-based solutions. As summarized in Table 4 , the signal of all protons of veratraldehyde considerably shifted to lower magnetic fields by 20 to 112 Hz, when Bu 4 NCl was added to the NaOD/D 2 O. This shift scarcely occurred when the same molar amount of NaCl was added to the solution, which strongly suggested that the shift was caused by Bu 4 N + and not by Cl − . It is therefore likely that veratraldehyde is in a close contact with Bu 4 N + in the solutions containing Bu 4 N + . The largest shift observed for the aldehyde proton (112 Hz) suggests strong interaction between Bu 4 N + and the aldehyde group. The disproportionation reaction is eventually suppressed when both reactants are in the “cage” of Bu 4 N + . On the other hand, in the case of the demethylation (pathway C in Scheme 2 ), one of the reactants is OH − , which is a small molecule and hence can more easily squeeze itself into the “cage”. Although further investigation is necessary to elucidate more detailed mechanism, this idea can reasonably explain the above experimental facts. According to these mechanisms, it can be stated that Bu 4 N + does not have a specific catalytic site, but the interaction between the aldehyde group and Bu 4 N + is a major origin of the good performance of Bu 4 NOH for the vanillin production. Also, as shown in Fig. 3 , the yield of guaiacol in the degradation in the Bu 4 NOH aq. is extremely higher than that in the NaOH aq. and this result may be also explained with the idea that guaiacol was protected from the O 2 attack by the cage effect caused by the presence of Bu 4 N + . Chemical shifts (ppm) in 1 H NMR analyses of veratraldehyde (0.5 mg) in 1.25 mol L −1 NaOD/D 2 O solution (0.75 mL) and those with the additions a of Bu 4 NCl and NaCl \n \n C 2 –H (d) C 5 –H (d) C 6 –H (dd) C α –H (s) C 3 –OCH 3 (s) C 4 –OCH 3 (s) 1.25 mol L −1 NaOD/D 2 O 7.16 6.89 7.32 9.33 3.64 3.69 1.25 mol L −1 NaOD/D 2 O + Bu 4 NCl 7.29 (+52) b 7.09 (+80) 7.50 (+72) 9.61 (+112) 3.69 (+20) 3.75 (+24) 1.25 mol L −1 NaOD/D 2 O + NaCl 7.18 (+8) 6.93 (+40) 7.36 (+16) 9.38 (+20) 3.67 (+12) 3.73 (+16) a Bu 4 NCl and NaCl were added to the NaOD solution with the concentration of the additives being set at 1.25 mol L −1 . b Changes in the chemical shift (Hz) caused by the additive were shown in the parenthesis. \n Fig. 4 shows a typical HPLC chromatogram of the reaction mixture obtained after the degradation of the phenolic lignin model LM2 (5.0 mg) in 1.25 mol L −1 Bu 4 NOH aq. (1.0 mL) at 120 °C under air. In the case of LM2, vanillin and vanillic acid were detected as major A-ring-derived products along with guaiacol as B-ring-derived one. We also found two large peaks at 37.6 and 39.0 min, which were assigned as cis - and trans -isomers of the enol ether EE with 1 H NMR analysis after their isolation from the reaction mixture with column chromatography (see experimental section for details). The formation of the trans -isomer was favored in the degradation. Note that EE are reported as major products in degradation of phenolic lignin models under alkaline conditions in many previous studies. 22,31–33 Fig. 4 HPLC chromatogram of the reaction mixture obtained after the degradation of LM2 (5 mg) in 1.25 mol L −1 Bu 4 NOH aq. (1.0 mL) under air for 1 h. In the degradation of LM2, as shown in Fig. 5A , vanillin, guaiacol, and EEs were produced from the initial stage of the degradation in the Bu 4 NOH aq. After 2 h reaction time, the yield of EEs started to decrease with increase in the yield of vanillin and guaiacol. At 4 h reaction time, vanillin and guaiacol were produced in 59.5 and 61.9 mol% yields, respectively (see also Table 5 ). Vanillic acid started to form after 15 min and the yield reached 12.7 mol% at 4 h, although the compound was not detected in the reaction mixture before 15 min. These results suggest that LM2 is first degraded to EE as presented in pathway F in Scheme 3 and EE is then degraded into vanillin and vanillic acid along with the releasement of guaiacol (pathway G). The pathway F is a simple alkaline degradation process via quinone methide formation followed by succeeding elimination of C γ as formaldehyde, as extensively investigated previously. 22,31–33 The pathway G requires O 2 according to the previous studies reporting that EE is substantially stable under N 2 even at elevated temperatures (∼170 °C) but readily to be oxidized into vanillin and guaiacol in the presence of O 2 . 26,28 Note that, according to comparison between the results in Fig. 3 and 5 , the degradation of LM2 was much faster than that of LM1 and this will be discussed in the next section. Fig. 5 Changes with time in recovery of LM2 ( ), yields of guaiacol ( ), vanillin ( ), vanillic acid ( ), and enol ethers EEs a ( ) during degradation of LM2 (5.0 mg) at 120 °C under air in 1.25 mol L −1 Bu 4 NOH aq. (1.0 mL) (A) and in 1.25 mol L −1 NaOH aq. (1.0 mL) (B). a The yields of EEs are shown as the sum of the cis - and trans -isomers. Yields (mol%) of vanillin, vanillic acid, and guaiacol and the recovery (%) of the starting material after the degradation of cis / tarns mixture of EE (3.0 mg) at 120 °C for 4 h under air in 1.25 mol L −1 Bu 4 NOH aq. and 1.25 mol L −1 NaOH aq. (2.0 mL) Staring material Reaction medium Yield (mol%) Recovery (%) Vanillin Vanillic acid Guaiacol EE LM2 Bu 4 NOH aq. 59.5 12.7 61.9 7.0 1.0 LM2 NaOH aq. 83.8 5.9 65.5 6.0 7.4 EE Bu 4 NOH aq. 43.6 22.2 41.4 — 29.2 EE NaOH aq. 51.8 6.5 49.7 — 17.7 Scheme 3 Reaction pathways from LM2 to the products detected in this study. The formation of vanillic acid via pathway H is less significant than that via pathway G. We also carried out degradation of cis / trans mixture of isolated EE (3.0 mg) in the Bu 4 NOH aq. (1.0 mL) under air. As summarized in Table 5 , the degradation for 4 h resulted in the formation of 43.6, 22.2, and 41.4 mol% of vanillin, vanillic acid, and guaiacol, respectively. Thus, the yields of vanillin and guaiacol from EE are moderately smaller than those from LM2 obtained under the same reaction conditions (vanillin: 59.5 mol% and guaiacol: 61.9 mol%, see Fig. 5 and Table 5 ). These results mildly suggest that there is a reaction pathway forming vanillin and guaiacol directly from LM2 (pathway H in Scheme 3 ). For vanillic acid, on the other hand, the yield from EE (22.2 mol%, Table 5 ) was greater than that from LM2 (12.7 mol%, Fig. 5 and Table 5 ), which suggests that vanillic acid is formed mostly via EE in the degradation of LM2. The degradation of LM2 in the NaOH aq. was then carried out under the same reaction conditions. As shown in Fig. 5B , we observed reaction behaviors qualitatively the same as those observed in the case of the Bu 4 NOH aq. This indicates that the degradation follows the pathways in Scheme 3 both in the Bu 4 NOH and the NaOH aq. Through quantitative comparison of the reaction behaviors between the Bu 4 NOH and the NaOH aq. ( Fig. 5 ), the degradation in the NaOH aq. exhibited (1) slower degradation of LM2, (2) lower yields and faster degradation of EE, (3) larger yields of vanillin and guaiacol, and (4) lower yields of vanillic acid. We will discuss these four differences below. The relatively slow degradation of the starting material in the NaOH aq., the first difference mentioned above, can be explained again with the stronger basicity of Bu 4 OH over NaOH. The reactions in the pathway G ( Scheme 3 ) requires the deprotonation of the phenolic OH of LM2 and C γ –OH of the intermediate quinone methide. These deprotonation steps are considered to be less feasible in the NaOH aq., ending up the slow degradation of LM2. The second and third differences are closely linked together, as the faster degradation of EE is the greater the yields of the products (vanillin and guaiacol) should become. It is possible that, as an analogy to the stabilization of veratraldehyde by Bu 4 N + (see previous section), EE is stabilized by the presence of Bu 4 N + . The larger recovery of EE (29.2%) after the degradation for 4 h in the Bu 4 NOH aq. than that in the NaOH aq. (17.7%, Table 5 ) also supports this idea. As summarized in Table 6 , 1 H NMR analyses of trans -EE in the 1.25 mol L −1 NaOD/D 2 O with and without the addition of Bu 4 NCl indicated that C 2 –H and C 6 –H on the benzene ring and the protons on the C α C β group significantly shifted to upper magnetic fields when Bu 4 NCl was added to the NaOD/D 2 O solution, whereas the shift observed for C 5 –H was relatively small. The addition of the same molar amount of NaCl again did not cause those shifts. These results suggest that Bu 4 N + is located mainly around the C α C β bond of EE. It has been reported that the initial step of the aerobic oxidation of EE to vanillin is addition of O 2 to the C α C β group. 26,28 Binding of the C α C β group to Bu 4 N + will render the O 2 addition more demanding, by which the degradation of EE to vanillin is inhibited. Note that the changes in the chemical shifts of EE by the addition of Bu 4 N + ( Table 6 ) were opposite from those observed for the veratraldehyde–NaOD/D 2 O system ( Table 4 ), that is, upper-magnetic field shift and downer one were observed in the EE and in the veratraldehyde solutions, respectively. However, the reasons for this difference are not clear at the moment. Chemical shifts (ppm) of several protons of trans -EE a (1.0 mg) in its 1 H NMR analyses in 1.25 mol L −1 NaOD/D 2 O (0.75 mL) and those with the additions b of Bu 4 NCl and NaCl \n \n C 2 –H (d) C 5 –H (d) C 6 –H (dd) C α –H (s) C β –H (s) 1.25 mol L −1 NaOD/D 2 O 6.65 6.33 6.54 6.83 6.00 1.25 mol L −1 NaOD/D 2 O + Bu 4 NCl 6.46 (−76) c 6.27 (−24) 6.38 (−64) 6.71 (−48) 5.89 (−44) 1.25 mol L −1 NaOD/D 2 O + NaCl 6.65 (±0) 6.36 (+12) 6.56 (+8) 6.85 (+8) 6.02 (+8) a The results only for the trans -isomer are shown as most of the cis -isomer signals were overlapped with those of the trans -isomer. b Bu 4 NCl and NaCl were added to the NaOD solution with the concentration of the additives being set at 1.25 mol L −1 . c Changes in the chemical shift (Hz) caused by the additive were shown in the parenthesis. The reasons for the low yield of vanillic acid in the NaOH aq. (the fourth difference between the Bu 4 NOH aq. and the NaOH aq. mentioned above) are not clear at the moment. As presented in Table 5 , this vanillic acid-oriented degradation behavior was re-produced also in the degradation of EE in the Bu 4 NOH aq.: EE formed vanillic acid in 22.2 mol% on the basis of degraded starting material in the Bu 4 NOH aq., whereas the yield of the compound was much smaller in the NaOH aq. (6.5 mol%). This suggests that the degradation of EE (pathway G in Scheme 3 ) is modified by the presence of Bu 4 N + to be more vanillic acid-selective. The interaction between EE and Bu 4 N + discussed above would play some roles in this selectivity change, but more systematic investigation is necessary to elucidate the underlying mechanism. Effects of Bu 4 N + on aerobic oxidation of lignin polymer We have so far discussed the degradation behavior of the lignin model compounds processing β- O -4 linkages, which consist around 50% of the inter-unit linkages of native lignin polymer. In this section we wish to discuss aerobic oxidation of native lignin polymer focusing mainly on vanillin production, on the basis of the results obtained above. In the discussion below, we assume that the non-phenolic model LM1 represents the reactivity of the monomeric unit in the middle of the polymer and the phenolic model LM2 corresponds to that of the phenolic end of the polymer. The initial step of the degradation of lignin polymer is simple alkaline degradation of the phenolic end, which is the fastest reaction observed in this study ( Fig. 5 ). When the phenolic end has a β- O -4 linkage, the degradation results in the formation of an enol ether end group. The enol ether end formed – if it is in contact with O 2 – is further oxidized into vanillin or vanillic acid and forms a new phenolic end in the adjacent monomeric unit. When these stepwise reactions continued, lignin polymers could degrade completely into the low molecular weight compounds. However, this type of “unzipping” reaction seems not to play major roles in the degradation. The reaction time required for completing the degradation of LM2 was ∼8 h, which was far from sufficient for vanillin production from the wood flour under the same conditions: at least 43 h is necessary to fully produce vanillin from the wood flour at 120 °C under air, 8 although the introduction of pure O 2 shortens the reaction time (see above). On one hand, the unzipping vanillin/vanillic acid production may be stopped when the reaction “bumps” into a less reactive linkage such as β–5, β–β, 4- O -5 and 5–5′ linkages. On the other hand, other types of reactions such as condensations are possible to prevent the vanillin/vanillic acid production from the phenolic end. Our results obtained for LM2 revealed that Bu 4 N + did no good for the vanillin production after all: Bu 4 N + stabilizes the enol ether to make the vanillin formation slow and changes the selectivity to vanillic acid-oriented. However, these negative effects of Bu 4 N + are not very serious in the vanillin production from native lignin in Bu 4 NOH, since the vanillin production from the phenolic end is fundamentally of little importance in entire vanillin production reactions. Besides a series of the reactions on the phenolic end, alkaline degradation of non-phenolic middle units gradually takes place. As shown in Scheme 4 , the initial step of the degradation on a non-phenolic β- O -4 type middle unit is alkaline-catalyzed ether link cleavage to form a glycerol end group, which corresponds to the pathway A in Scheme 2 . This step is significantly enhanced in Bu 4 NOH due to its strong basicity and this is one of the benefits in the utilization of Bu 4 NOH instead of NaOH. Scheme 4 Vanillin formation from a β- O -4 middle unit of lignin polymer. The pathways with bold arrows are positively influenced by Bu 4 N + . The oxidation of the glycerol group by O 2 , pathway B in Scheme 2 in the case of the model compound LM1, then occurs to form a benzaldehyde end group (vanillin end group when the lignin sample is derived from soft wood lignin). There are two possibilities for the fate of the benzaldehyde end group. One is the free benzaldehyde formation by the cleavage of the ether link connecting the benzaldehyde end to the adjacent monomeric unit, which corresponds to the pathway C in Scheme 2 . The other is the disproportionation of the aldehyde group resulting in the formation of benzoic acid and benzyl alcohol ends (pathway D in Scheme 2 ). The degradation in Bu 4 NOH facilitates the former reaction pathway to increase the benzaldehyde yield. These proposed reactions suggest that the reaction mixture from the wood flour should involve some amount of vanillyl alcohol. However, the compound was not detected in the degradation of the wood flour under any conditions, although vanillic acid was detected as a major product ( Fig. 1 ). This is probably because vanillyl alcohol end group is unstable due to the presence of benzyl hydroxy group which is readily subjected to condensation reactions. 22 It is also noted that vanillic acid, unlike vanillyl alcohol, can also be formed from the oxidation of the enol ether end, as indicated from Fig. 5 , and some of the origins of vanillic acid produced from the wood flour is considered to be phenolic end groups of lignin polymer."
} | 11,756 |
18533821 | null | s2 | 5,743 | {
"abstract": "The scale of large neuronal network simulations is memory limited due to the need to store connectivity information: connectivity storage grows as the square of neuron number up to anatomically relevant limits. Using the NEURON simulator as a discrete-event simulator (no integration), we explored the consequences of avoiding the space costs of connectivity through regenerating connectivity parameters when needed: just in time after a presynaptic cell fires. We explored various strategies for automated generation of one or more of the basic static connectivity parameters: delays, postsynaptic cell identities, and weights, as well as run-time connectivity state: the event queue. Comparison of the JitCon implementation to NEURON's standard NetCon connectivity method showed substantial space savings, with associated run-time penalty. Although JitCon saved space by eliminating connectivity parameters, larger simulations were still memory limited due to growth of the synaptic event queue. We therefore designed a JitEvent algorithm that added items to the queue only when required: instead of alerting multiple postsynaptic cells, a spiking presynaptic cell posted a callback event at the shortest synaptic delay time. At the time of the callback, this same presynaptic cell directly notified the first postsynaptic cell and generated another self-callback for the next delay time. The JitEvent implementation yielded substantial additional time and space savings. We conclude that just-in-time strategies are necessary for very large network simulations but that a variety of alternative strategies should be considered whose optimality will depend on the characteristics of the simulation to be run."
} | 427 |
36385980 | PMC9659854 | pmc | 5,744 | {
"abstract": "Underwater adhesives hold great promises in our daily life, biomedical fields and industrial engineering. Appropriate underwater bonding can reduce the huge cost from removing the target substance from water, and greatly lift working efficiency. However, different from bonding in air, underwater bonding is quite challenging. The existence of interfacial water prevents the intimate contact between the adhesives and the submerged surfaces, and water environment makes it difficult to achieve high cohesiveness. Even so, in recent years, various underwater adhesives with macroscopic adhesion abilities were emerged. These smart adhesives can ingeniously remove the interfacial water, and enhance cohesion by utilizing their special physicochemical properties or functional groups. In this mini review, we first give a detail introduction of the difficulties in underwater bonding. Further, we overview the recent strategies that are used to construct underwater adhesives, with the emphasis on how to overcome the difficulties of interfacial water and achieve high cohesiveness underwater. In addition, future perspectives of underwater adhesives from the view of practical applications are also discussed. We believe the review will provide inspirations for the discovery of new strategies to overcome the obstacles in underwater bonding, and therefore may contribute to designing effective underwater adhesives.",
"conclusion": "Conclusion and perspectives Realizing efficient underwater bonding is challenging due to the obstacles of interfacial water and difficulties in enhancing cohesiveness underwater. In this review, we summarized and overviewed the proposed strategies for overcoming these obstacles and difficulties. Despite that much progress has been made in underwater bonding, current underwater adhesives still have much space to made from the view of practical use. First, many adhesives require complex synthesis process or rigorous synthesis conditions ( Pan et al., 2020 ). Although some adhesives showed high underwater bonding performance, the large production is also urgently needed ( Li et al., 2022 ) for the commercialization. Second, the storability and usability of the adhesives should be carefully considered. Finally, current adhesives mainly focus on the underwater adhesive performance in static water environment, but the practical waters, such as lakes, rivers, and ocean are mainly dynamic. In dynamic water, the diffusion of the adhesive molecules accelerates, and the efficient bonding is more difficult to realize. Future works can focus on the dynamic environment of the water, thus developing underwater adhesives suitable for practical dynamic water environment.",
"introduction": "Introduction Underwater bonding is highly demand in wide range of areas ( Cui et al., 2017 ; Fan and Gong, 2021 ; Wang Z. M., et al, 2021 ; Wu J. et al., 2022 ). For example, in our daily life and industrial field, it often requires to directly repair the water pipeline leakage, attach the underwater sensor, or even repair the broken hull in water. In medical applications, doctors usually need to seal the wounds in moisture environment or even under blood. Efficient underwater bonding can greatly simplify the working procedures with no need for creating dry surfaces, thereby lifting working efficiency and reducing cost ( Xia et al., 2021 ). However, achieving underwater bonding is commonly challenging ( Ahn et al., 2015 ; Narayanan et al., 2021 ). And commercial man-made adhesives, such as cyanoacrylate (Super glue), vinyl acetate (Elmer’s glue), as well as most epoxy and polyurethane glue, cannot perform well in underwater adhesion ( Li et al., 2017 ; White and Wilker, 2011 ; North et al., 2017 ; Cheng et al., 2022 ). The challenges in underwater bonding mainly result from the interfacial water on the submerged surfaces, and the difficulties in achieving high cohesiveness underwater ( Kamino, 2008 ; Kamino, 2013 ; Waite, 2017 ; Fan and Gong, 2021 ; Narayanan et al., 2021 ; Cheng et al., 2022 ). In recent years, researchers have proposed various strategies to overcome these obstacles, and therefore developed new types of adhesives that were capable of underwater bonding. In this mini review, we first introduced the difficulties in underwater bonding, and then overviewed the main discovered strategies that are used to realize underwater macroscopical adhesion. In this part, we mainly focused on the strategies of creating underwater adhesives with macroscopical underwater bonding capacities. In the last part, the future perspectives of adhesives according to practical applications were discussed."
} | 1,155 |
29142826 | PMC5678828 | pmc | 5,746 | {
"abstract": "Processing of lignocellulosic biomass or organic wastes produces a plethora of chemicals such as short, linear carboxylic acids, known as carboxylates, derived from anaerobic digestion. While these carboxylates have low values and are inhibitory to microbes during fermentation, they can be biologically upgraded to high-value products. In this study, we expanded our general framework for biological upgrading of carboxylates to branched-chain esters by using three highly active alcohol acyltransferases (AATs) for alcohol and acyl CoA condensation and modulating the alcohol moiety from ethanol to isobutanol in the modular chassis cell. With this framework, we demonstrated the production of an ester library comprised of 16 out of all 18 potential esters, including acetate, propionate, butanoate, pentanoate, and hexanoate esters, from the 5 linear, saturated C 2 -C 6 carboxylic acids. Among these esters, 5 new branched-chain esters, including isobutyl acetate, isobutyl propionate, isobutyl butyrate, isobutyl pentanoate, and isobutyl hexanoate were synthesized in vivo . During 24 h in situ fermentation and extraction, one of the engineered strains, EcDL208 harnessing the SAAT of Fragaria ananassa produced ~63 mg/L of a mixture of butyl and isobutyl butyrates from glucose and butyrate co-fermentation and ~127 mg/L of a mixture of isobutyl and pentyl pentanoates from glucose and pentanoate co-fermentation, with high specificity. These butyrate and pentanoate esters are potential drop-in liquid fuels. This study provides better understanding of functional roles of AATs for microbial biosynthesis of branched-chain esters and expands the potential use of these esters as drop-in biofuels beyond their conventional flavor, fragrance, and solvent applications.",
"conclusion": "4 Conclusion Biological upgrading low-value carboxylates, derived from lignocellulosic biomass or organic wastes, to high-value esters has significant potential. In this study, we expanded our general, flexible framework for this biological upgrading. By deploying the ester production strains harnessing the acid-to-ester modules with various AATs, we demonstrated the microbial biosynthesis of 16 out of the total 18 potential esters including 5 new branched-chained esters – isobutyl acetate, isobutyl propionate, isobutyl butyrate, isobutyl pentanoate, and isobutyl hexanoate from the carboxylates. Not only did we confirm the substrate preferences of ATF1 (EcDL207) towards long-chain acetate esters, SAAT (EcDL208) towards acyl acylates, and VAAT (EcDL209) towards ethyl C 2 -C 4 acylates, but also demonstrated their activities towards branched-chain esters. Since our study aimed to expand the carboxylate to ester platforms, there is much room to enhance ester production that is currently low in future studies ( Supplementary Tables 1–3 ). Many promising strategies can be employed to improve the ester production such as pathway optimization ( e.g. , modulating promoter, ribosome binding site, gene orthologs to balance and optimize pathway fluxes) and process conditions ( e.g. temperature, medium, pH, substrate feeding, in situ extraction and fermentation).",
"introduction": "1 Introduction The natural, efficient consolidated bioprocessing of lignocellulosic biomass or organic wastes is anaerobic digestion ( Agler et al., 2011 , Jonsson and Martin, 2016 ). In this process, a consortium of mixed microbes ( e.g. anaerobic digesters) can degrade organic wastes directly into carboxylates ( e.g. , linear and saturated C 2 -C 6 organic acids) without the stipulation of any pretreatment ( Batstone and Virdis, 2014 , Thanakoses et al., 2003 ). While these carboxylates have low values and are inhibitory to microbes, they can be biologically upgraded to a large space of high-value chemicals such as esters that are widely used in flavor, fragrance, and solvent industries. Certain carboxylate-derived esters have high hydrophobicity for easy separation from fermentation and encompass high combustion properties that can be used as biodiesels or jet fuels ( Chuck and Donnelly, 2014 , Contino et al., 2011 , Kallio et al., 2014 ). Biologically upgrading the carboxylate to ester platforms has recently been demonstrated ( Layton and Trinh, 2016 ). This conversion was achieved by a modular cell ( Trinh et al., 2015 ) tightly integrated with an engineered acid-to-ester production module − a modular heterologous pathway comprised of an alcohol production submodule, an acid to acyl CoA synthesis submodule, and alcohol and an acyl CoA condensation submodule. The flexible design of these modules served several purposes: (i) expanding the biosynthesis of the ester platform in a plug-and-play fashion using a pure culture or a consortium of mixed cultures and (ii) screening alcohol acyl transferases (AATs) for their novel in vivo activities. Understanding the catalysis of the AAT condensation reaction is critical for efficient ester biosynthesis but is currently limited. Some recent studies have aimed at understanding AAT specificities using various techniques, from whole-cell in vivo approaches using the carboxylates as substrates ( Layton and Trinh, 2016 ) or acid additions from the 2-keto acid synthesis pathway ( Rodriguez et al., 2014 ) to in vitro enzymatic assays ( Lin et al., 2016 ) and in silico protein modeling ( Morales-Quintana et al., 2011 , Morales-Quintana et al., 2015 , Morales-Quintana et al., 2012 , Morales-Quintana et al., 2013 ). To date, the biological upgrading of the carboxylate to ester platforms has only been demonstrated using the ethanol production module, and understanding of whether the targeted AATs have activity towards other alcohols has not yet been investigated. In this study, we biologically upgraded the carboxylate to branched-chain ester platforms by modulating the alcohol submodule from ethanol to isobutanol. Using the engineered Escherichia coli modular cell, we explored the functional roles of three AATs of the acid-to-ester module for the potential synthesis of 18 unique esters from the 5 linear, saturated C 2 -C 6 carboxylic acids commonly found in the carboxylate platform. Microbial biosynthesis of the ester platform with longer- and branched-chain alcohols beyond ethanol modulates the ester flavor and fragrance properties as well as improves the energy density of these esters that can potentially be used as pure or blended biodiesels and jet fuels.",
"discussion": "3 Results and discussion 3.1 Design of microbial biosynthesis of branched-chain ester platform The general framework for biological upgrading of the carboxylate to branched-chain ester platforms utilizes the acid-to-ester pathway ( Fig. 1 A). This framework was built upon our previously established foundation ( Layton and Trinh, 2016 ) by modulating the alcohol submodule from ethanol to isobutanol. In brief, the designed acid-to-ester pathway contained the isobutanol submodule for conversion of pyruvate to isobutanol and the PCT plus AAT submodule that converts carboxylates to acyl CoAs and condenses them with alcohols to produce esters ( Fig. 1 B). As the isobutanol submodule contains the overexpression of an E. coli alcohol/aldehyde dehydrogenase (AdhE), it can reduce acyl CoAs from carboxylates to alcohols that can be used for ester biosynthesis by the PCT plus AAT submodule. In our design, we used the PCT of C. propionicum because it exhibits broad substrate specificity towards C 2 -C 6 carboxylates to produce their respective CoA counterparts ( Layton and Trinh, 2016 , Schweiger and Buckel, 1984 ). We also used three highly active AATs, including ATF1 of Saccharomyces cerevisiae , SAAT of Fragaria ananassa, and VAAT of F. vesca, that encompass various substrate preferences to test for the branched-chain ester biosynthesis ( Layton and Trinh, 2016 ). Specifically, ATF1 exhibits substrate preference towards longer-chain acetate esters, while SAAT and VAAT have substrate preferences towards C 4 -C 6 ethyl acylates and C 2 -C 4 ethyl acylates, respectively. In addition, SAAT has specificity towards acyl acylates. Fig. 1 (A) Microbial biosynthesis of branched-chain esters from carboxylates. (B) Genetic design of the acid-to-ester module. Fig. 1 By modulating the alcohol submodule from ethanol to isobutanol, the designed framework can expand the ester production library from 13 to 18 potential esters by co-fermentation of glucose and five linear, saturated C 2 -C 6 carboxylates ( Fig. 1 A). The five new branched-chain esters that can be synthesized microbially from the carboxylate platform include isobutyl acetate, propionate, butyrate, pentanoate, and hexanoate ( Fig. 1 B). Ester synthesis depends on the availability of precursor metabolites, acyl CoAs and alcohols, and the broad substrate activities of AATs. In this study, we characterized three strains harnessing the acid-to-ester pathways with various AATs while other heterologous genes and their constructs were identical. These engineered strains are EcDL207, 208, and 209 and carry ATF1, SAAT and VAAT, respectively. 3.2 Expanding combinatorial biosynthesis of ester platforms 3.2.1 Microbial biosynthesis of an acetate ester platform A total of 5 targeted acetate esters including ethyl, propyl, butyl, pentyl, and hexyl acetates could be potentially synthesized from 5 carboxylates. Our results show that (i) a mixture of ethyl and isobutyl acetates could be produced from co-fermentation of glucose and acetate ( Fig. 2 A); (ii) a mixture of ethyl, propyl, and isobutyl acetates from co-fermentation of glucose and propionate ( Fig. 2 B); (iii) a mixture of ethyl, butyl, and isobutyl acetates from co-fermentation of glucose and butyrate ( Fig. 2 C); (iv) a mixture of ethyl, isobutyl, and pentyl acetates from co-fermentation of glucose and pentanoate ( Fig. 2 D); and (v) a mixture of ethyl, isobutyl, and hexyl acetates from co-fermentation of glucose and hexanoate ( Fig. 2 E). Due to the various AAT specificities and precursor availability, it is anticipated that each characterized strain might not be able to produce all expected acetate esters for each co-fermentation of glucose and a targeted carboxylate as previously observed ( Layton and Trinh, 2016 ). Fig. 2 Ester production of EcDL207, 208, and 209 after 24 h from co-fermentation of (A) glucose and acetic acid, (B) glucose and propionic acid, (C) glucose and butyric acid, (D) glucose and pentanoic acid, and (E) glucose and hexanoic acid. Fig. 2 Among the characterized strains and acetate esters, EcDL207 produced pentyl acetate at the highest level of 66.30±13.44 mg/L after 24 h from the co-fermentation of glucose and pentanoate ( Fig. 2 D). EcDL207 also synthesized 31.17±0.32 mg/L hexyl acetate ( Fig. 2 E), 20.26±2.82 mg/L isobutyl acetate ( Fig. 2 A) and 10.22±0.85 mg/L butyl acetate ( Fig. 2 C) at much higher titers than EcDL208 and EcDL209. The observed phenotypes of EcDL207 were consistent with our previous study ( Layton and Trinh, 2016 ) where ATF1 exhibited high specificity towards acetate esters. If the application were tailored for production of an acetate ester platform, ATF1 would be a strong candidate to use for the acid-to-ester pathway. Both carboxylates and acetate esters have distinct physical properties. The fruity smell of esters makes them unique presenting broad applications in flavor, fragrance, and solvent industries. While acetate and most of the carboxylates are very soluble in water, causing toxicity to microbes during fermentation, the acetate esters have significant reduction in water solubility ( Fig. 3 A) and can be easily extracted during fermentation as implemented in our study. In addition, biological upgrading of acetate to acetate esters resulted in the improved ONMED values making them suitable for biofuel applications ( Fig. 3 B). For instance, isobutyl acetate (0.639) has a higher ONMED value than ethanol (0.615) and acetic acid (0.312), and has been tested as a biofuel blend ( Olson et al., 2003 ). Lower solubility of isobutyl acetate in water (~7 g/L) in comparison to acetic acid (complete solubility) and isobutanol (~88 g/L) is very advantageous for in situ fermentation and extraction. Fig. 3 Physical properties of carboxylates, alcohols, acetate esters, propionate esters, butyrate esters, pentanoate esters, and hexanoate esters. (A) Water solubility (g/L). inf: complete solubility. (B) Octane normalized mass energy density (ONMED). Fig. 3 3.2.2 Microbial biosynthesis of a propionate ester platform Co-fermentation of glucose and propionate could generate a propionate ester library comprised of ethyl, propyl, and isobutyl propionates ( Fig. 2 B). Among the characterized strains, only EcDL209 could produce all three propionate esters while EcDL207 and EcDL208 could synthesize only isobutyl propionate. Isobutyl propionate was produced at the highest titer of 3.60±1.96 (mg/L) by EcDL209 among propionate esters and characterized strains. It is interesting to notice that EcDL209 harnessing VAAT produced little ethyl propionate. In our previous study, however, we observed that the VAAT exhibited a relatively high activity towards ethyl propionate production with a titer up to 67.24±10.41 mg/L when the ethanol module was used instead of the isobutanol module ( Layton and Trinh, 2016 ). Altogether, these results suggest that the insufficient generation of ethanol in EcDL207, 208 and 209 might have resulted in low ethyl propionate production. Among acylate esters, the production of propionate esters was the lowest ( Fig. 2 ). Propyl acetate, propyl propionate, and isobutyl propionate, each have unique intrinsic physical properties including ONMED values 0.594, 0.639 and 0.675, respectively as well as fruity odors and relatively low water solubility ( Fig. 3 ). The ONMED values of these propionate esters are all slightly lower than propanol (0.697) but higher than ethanol (0.615), which allow them to be blended in biofuels for increasing their octane. 3.2.3 Microbial biosynthesis of a butyrate ester platform From co-fermentation of glucose and butyrate, all characterized strains EcDL207-209 produced the targeted butyrate ester library, consisting of ethyl, butyl, and isobutyl butyrates. EcDL208 produced 21.34±13.76 mg/L butyl butyrate and 41.25±18.76 mg/L isobutyl butyrate at the highest levels among the characterized strains while EcDL209 produced ethyl butyrate at the highest titer of 20.76±0.00 (mg/L). Consistent with the previous study ( Layton and Trinh, 2016 ), SAAT of EcDL208 shows substrate preferences towards C 4 -C 6 acyl CoAs and short-chain alcohols while VAAT of EcDL209 exhibits substrate preferences toward C 2 -C 4 acyl CoAs and ethanol. As expected, ATF1 of EcDL207 produced some amount of butyl and isobutyl acetates because it has substrate preferences towards acetyl CoA and short-chain alcohols. Butyric acid is completely soluble in water and is very toxic to microbes ( Fig. 3 ). However, biological upgrading of butyrate can generate a butyrate ester library with unique properties. The most distinct feature is the odor difference between rancid butyrate and its derived pleasant butyrate esters. Both butyl and isobutyl butyrate have very low water solubility (<0.7 g/L), which is advantageous for simultaneous fermentation, separation, and extraction process development ( Fig. 3 ). These butyrate esters also have higher ONMED value than ethanol and propanol, which make them suitable for biofuel application. For instance, butyl butyrate has been recently tested as a potential jet fuel alternative ( Chuck and Donnelly, 2014 ). 3.2.4 Microbial biosynthesis of a pentanoate ester platform Biological upgrading of pentanoate could expand the pentanoate ester platform to include ethyl, pentyl, and isobutyl pentanoates. Among the characterized strains, EcDL208 could produce all three targeted pentanoate esters at the highest titers, including 3.90±0.50 mg/L ethyl pentanoate, 64.71±10.19 mg/L isobutyl pentanoate, and 62.63±0.75 mg/L pentyl pentanoate. The high production of the targeted pentanoate ester library conferred the substrate preference of SAAT used in EcDL208 ( Layton and Trinh, 2016 ). Like butyric acid, pentanoic acid exhibits a rancid odor. However, biologically-upgraded pentanoate esters have pleasant smells and tastes, and hence are known for their wide use in flavor and fragrance industries. Unlike pentanoic acid, its derived pentanoate esters are mostly insoluble and are advantageous for in situ fermentation and extraction. Since ethyl, isobutyl, and pentyl pentanoates have ONMED values of 0.675, 0.727, and 0.747, respectively, which are close to the isobutanol ONMED (0.749), these esters can be potentially used as drop-in biofuels beyond their conventional flavor, fragrance, and solvent applications. For instance, ethyl pentanoate has undergone road trials and demonstrated stable performance when blended (10%) with gasoline ( Lange et al., 2010 ). 3.2.5 Microbial biosynthesis of a hexanoate ester platform Co-fermentation of hexanoate and glucose could potentially yield a hexanoate ester library including ethyl, isobutyl, and hexyl hexanoates. The characterized strains, however, could only synthesize isobutyl hexanoate, neither ethyl hexanoate nor hexyl hexanoate. EcDL208 produced isobutyl hexanoate with the highest titer of 3.21±0.15 mg/L. SAAT of EcDL208 was also first shown to have the substrate specificity for isobutanol and hexanoyl CoA to produce isobutyl hexanoate. Different from the previous study where the ethanol submodule was used instead of the isobutanol submodule ( Layton and Trinh, 2016 ), both SAAT and VAAT exhibited activities towards ethyl hexanoate production despite low titers. These results suggest that inefficient supply of precursor metabolites in EcDL208 and EcDL209 might have limited ethyl hexanoate biosynthesis investigated in this study. Currently, we have not been able to synthesize hexyl hexanoate likely due to low activity of the characterized AATs towards this ester. Not only does isobutyl hexanoate have one of the highest ONMED values at 0.747 (comparable with pentyl pentanoate) among the biologically upgraded esters, but it also exhibits little to no solubility in aqueous solutions ( Fig. 3 ). Interestingly, ethyl octanoate that also has the same ONMED as isobutyl hexanoate has recently been tested and demonstrated for its use in A-1 jet fuel ( Chuck and Donnelly, 2014 ). The physical property of isobutyl hexanoate makes it a potential candidate for jet fuel application. Table 2 A list of primers for plasmid construction. Table 2 Primers Sequences DL_0001 5′-CATCATCACCACCATCACTAA-3′ DL_0002 5′-ATGTATATCTCCTTCTTATAGTTAAAC-3′ DL_0012 5′-GGCGGCCGCTCTATTAGTGATGGTGGTGATGATGTTAAATTAAGGTCTTTGGAG-3′ DL_0018 5′-GGCGGCCGCTCTATTAGTGATGGTGGTGATGATGCGGATAACATACGTAGACCG-3′ DL_0020 5′-GCCGCTCTATTAGTGATGGTGGTGATGATGCTAAGGGCCTAAAAGGAGAG-3′ DL_0023 5′-AAATAATTTTGTTTAACTATAAGAAGGAGATATACATATG AGAAAGGTTCCCATTATTAC-3′ DL_0024 5′-TCAGGACTTCATTTCCTTCAG-3′ DL_0025 5′-CTGAAGGAAATGAAGTCCTGAAAGGAGATATACATATGAATGAAATCGATGAGAAAAATC-3′ DL_0027 5′-TGGGTCTGAAGGAAATGAAGTCCTGAAAGGAGATATACATATGGAGAAAATTGAGGTCAG-3′ DL_0028 5′-TGGGTCTGAAGGAAATGAAGTCCTGAAAGGAGATATACATATGGAGAAAATTGAGGTCAG-3′"
} | 4,806 |
29725058 | PMC5934359 | pmc | 5,747 | {
"abstract": "Endophytic microbes isolated from plants growing in contaminated habitats possess specialized properties that help their host detoxify the contaminant/s. The possibility of using microbe-assisted phytoremediation for the clean-up of Arsenic (As) contaminated soils of the Ganga-Brahmaputra delta of India, was explored using As-tolerant endophytic microbes from an As-tolerant plant Lantana camara collected from the contaminated site and an intermediate As-accumulator plant Solanum nigrum . Endophytes from L . camara established within S . nigrum as a surrogate host. The microbes most effectively improved plant growth besides increasing bioaccumulation and root-to-shoot transport of As when applied as a consortium. Better phosphate nutrition, photosynthetic performance, and elevated glutathione levels were observed in consortium-treated plants particularly under As-stress. The consortium maintained heightened ROS levels in the plant without any deleterious effect and concomitantly boosted distinct antioxidant defense mechanisms in the shoot and root of As-treated plants. Increased consortium-mediated As(V) to As(III) conversion appeared to be a crucial step in As-detoxification/translocation. Four aquaporins were differentially regulated by the endophytes and/or As. The most interesting finding was the strong upregulation of an MRP transporter in the root by the As + endophytes, which suggested a major alteration of As-detoxification/accumulation pattern upon endophyte treatment that improved As-phytoremediation.",
"conclusion": "Conclusion The success of microbe-assisted phytoremediation is dictated both by the plant as well as the microbes under question. Both the partners, therefore, need to be carefully chosen for an efficient outcome. Our work shows that an endophytic consortium isolated from a plant collected from an As-contaminated site could be used in conjunction with an accumulator plant to improve its As-phytoremediation ability. The consortium but not the individual microbes effectively enhanced both plant growth and root to shoot transport of the metalloid- a tightly controlled property of a plant. As a plant model, S . nigrum was found suitable for removing low concentration of As present in the soil of many highly populated areas. The consortium improved P-nutrition and enhanced ROS/oxidative defense in the plant indirectly affecting plant fitness under stress. Simultaneous in planta increase in As(V)-As(III) conversion was also observed that could have a direct influence on As long-distance transport and detoxification. SnMRP2, an MRP gene from S . nigrum , was specifically upregulated upon endophyte treatment in an As-dependent manner. Metal hyperaccumulator plants are often slow growing. Their use in fields is also constrained by their adaptation to particular geographical areas. This work demonstrates that a suitable plant model used as surrogate host together with endophytes with the appropriate properties can widen the use of endophyte-assisted phytoremediation in the clean-up of soil without such restrictions.",
"introduction": "Introduction Arsenic (As) contamination in groundwater is a major health hazard in widely distributed areas of Eastern India (West Bengal) and Bangladesh 1 where the As-levels in potable water exceed the limit approved (10 ppb) by World Health Organization (WHO) 2 . Arsenic content in soil increases from constant irrigation with contaminated groundwater or through anthropogenic activities 3 ; wherefrom it accumulates in food crops and eventually enters the food chain 4 . Even though As in drinking water is considered as the major cause of As-related diseases, excess As-content in the soil cannot also be overlooked. Although Arsenicosis is widespread among the residents of West Bengal and Bangladesh, contamination levels in the soil in these areas appear to be often ≤25 ppm. Such soil is graded as ‘low’ As-contaminated soil 1 . Highly contaminated areas in the world contain >500 ppm of As like the Zarshuran mining area in Iran where the contamination levels reach 6000 ppm. However, these areas are sparsely populated in contrast to the highly populated areas of the Gangetic delta. This makes the development of suitable strategies for clean-up of low contaminated areas extremely important. Metal contamination in soil is particularly difficult and extremely expensive to remove. The use of plants and their associated microbes to remediate contaminated soil, called phytoremediation, is an extremely relevant strategy in this context. Plant and its associated microbes participate in complex interactions that shape the community structure of these microbes and also the plant’s interaction with its environment. Endophytes are microbes that reside asymptomatically within the plant endosphere. Properties of the endophytes are dictated by the edaphic factors wherein the host plant survives. While one of the selection forces determining the endophytic colonization is the nutritional status of the host 5 , the necessity to detoxify a xenobiotic appears to be an additional driving force. This is exemplified by the fact that the endophytic microbes isolated from plants surviving in contaminated habitats often contain properties useful for the detoxification of the related xenobiotic/s 6 . Endophytes isolated from plants growing in metal-contaminated habitats have been reported to harbor genome or plasmid coded strategies to detoxify metals 7 and contribute to the metal tolerance and accumulation property of the host plant. An endophyte related to Achromobacter piechaudii from the metallophyte Sedum plumbizincicola demonstrated increased uptake of different metals in the root 8 . Enterobacter sp. PDN3, from poplar growing in areas polluted with organic xenobiotics, was able to degrade trichloroethylene (TCE) 6 . Another Enterobacter sp. from water hyacinth contained a plasmid responsible for conferring metal tolerance to the bacteria. Endophytes exert their plant growth promoting (PGP) properties on their hosts in a way analogous to the gut microflora of animals 9 . Although host genotype impacts their properties to a certain degree, a number of studies have shown that this influence is limited indicating that the soil condition (also determining the plant’s nutritional status) predominately drives their entry into a plant 10 , 11 . Endophytes from Pteris vittata - an As hyperaccumulator were found to transform As(V) to As(III) and produced IAA, solubilized phosphate, and siderophores as mechanisms of growth promotion 12 . In contrast, endophytes isolated from S . nigrum growing at a mining site contaminated with different heavy metals were mostly siderophore and ACC deaminase producing 13 . Recent studies have indicated that endophytes can also have beneficial effects on distantly related plant genera besides their natural host 14 , 15 . This is extremely significant because this opens up the possibility that the endophytes can be used to transmit their properties to surrogate plants of agricultural value and also to plants relevant for phytoremediation. For example, nitrogen-fixing endophytes isolated from Typha angustifolia collected from a nutrient-deficient Uranium mine improved nitrogen metabolism in rice 16 . We examined the possibility of using microbe-assisted phytoremediation in the clean-up of soil containing As-contamination levels relevant to highly populated areas of Gangetic West Bengal. As-resistant microbes were isolated from an As-tolerant excluder Lantana camara collected from a contaminated site in Nadia, West Bengal 17 and was applied to a perennial weed Solanum nigrum found in abundance in this area for the purpose of As-phytoremediation. S . nigrum is an established Cadmium (Cd)-hyperaccumulator that has been reported to be an intermediate As-accumulator able to store up to 500 ppm of As 18 . A significant amount of this As is accumulated in the shoot 19 making this plant suitable for As-phytoremediation 18 . Ferns of genus Pteris especially Pteris vittata 20 are widely studied for their unique ability to accumulate over 4000 ppm of As in the above-ground biomass. However, the rhizome and rhizoids of these ferns penetrate only the surface layers of soil restricting their use in in situ phytoremediation. Use of hyperaccumulators in soil clean-up is often limited by their slow and stunted growth. Higher biomass increases the effectiveness at which a soil pollutant can be removed. Brassica juncea 21 , 22 and Brassica carinata are reported to accumulate As 23 . Although not hyperaccumulators these are fast growing plants. However, their leaves and seeds are widely consumed making them undesirable as phytoremediation models. Naser Karimi and his colleagues (2009) reported Isatis cappadocica 24 and Hesperis persica 25 as As-accumulator terrestrial angiosperms. Although both these plants are highly tolerant to As, the bioaccumulation factor reported (shoot: soil concentration) was low; approximately 1 and 0.89 respectively 24 , 25 . Further, Isatis sp . and Hesperis sp. are not endemic to As-contaminated regions of the Gangetic plains of India neither has their As-accumulation ability been tested at low concentrations of As. This prompted us to search for suitable local plant/s. S . nigrum appeared to be a suitable plant host for microbe-assisted As-phytoremediation of low As-contaminated soil. It is a weed well adapted to the As-contaminated region under study showing a much wider geographic distribution. The As-tolerant endophytic consortium from L . camara was found to improve the As-phytoremediation efficiency of S . nigrum without the necessity of a transgenic approach, influencing multiple processes known from previous studies to improve As-tolerance. While individually, the microbes had a variable effect, some showing growth promotion but having little or no role in the improvement of As-bioaccumulation or vice versa, when used as a consortium, they efficiently improved As-accumulation in S . nigrum . Our work demonstrated the suitability of using endophytes possessing desired properties coupled with an appropriate plant model in As-phytoremediation even when the natural host of the microbes was unsuitable for the application.",
"discussion": "Results and Discussions Solanum nigrum - a suitable model for microbe-assisted Arsenic remediation To identify an As-accumulator terrestrial plant (i) a non-biased, as well as (ii) a targeted approach was taken. For the non-biased approach, non-edible plants with As-bioaccumulation factor (shoot/rhizospheric soil As concentration) >1 were searched from an As-contaminated site in Nadia, previously reported for groundwater contamination and As-related diseases. The concentration of As in plant rhizosphere measured by VGA-AAS was found to be 19.3(±3.77) ppm. None of the plants from 14 different species studied from the site were As-accumulators (data not shown). This was not surprising as the exclusion of metals is a predominant strategy adopted by many metallicolous plants adapting to contaminated soils 26 . Lantana camara - a perennial weed was one of the highest accumulators of As among the collected plants, [12.5(±0.25) ppm in shoot] and was therefore selected for isolation of As-tolerant endophytes. Arsenic was supplied as arsenate (sodium arsenate, As hereafter)− the main form of As in aerobic soil. Albeit tolerant to 25 ppm of As under laboratory conditions, L . camara showed a bioaccumulation factor <1. Metal-bioaccumulation is a property largely dictated by the plant and is regulated by complex strategies controlling uptake, root to shoot transport and detoxification of the metal. We envisaged that a successful microbe-assisted phytoremediation model would require an As-accumulator plant model. A targeted approach was therefore adopted and Solanum nigrum , an already known As-accumulator plant 19 , 27 was used as the plant model. We hypothesized that S . nigrum , being an As-accumulator is equipped with strategies to take up, transport and store As at high amounts- a prerequisite to As-phytoremediation. Although not found at the time of collection, S . nigrum is a non-edible short-lived perennial shrub widely found in this region. It is not an As-hyperaccumulator but has a significant biomass and a robust root system. S . nigrum (tetraploid Red Makoi variety) accumulated 152.3 (±12.4)ppm of As in its shoot under laboratory conditions when supplied with 25 ppm of As, which was the upper limit of the As found in the soil of the studied area. Although this As-level was much lower than that reported in Iran by Karimi et al . 24 or in the mine tailings of Mexico 28 the amount was still 2500-fold higher than the acceptable limits and was much more relevant to the contamination level that poses threat to the human populace. S . nigrum has been earlier reported to restrict root to shoot translocation of As 18 , 19 . We found that although the As-concentration in root was higher compared to shoot, the bioaccumulation factor was still >1. S . nigrum was therefore chosen as the As-accumulator plant model in this study. Arsenic tolerant endophytes from Lantana camara contained plant growth promoting properties Seven bacterial endophytes from L . camara tolerant to 4000 ppm of As were isolated based on their distinct colony morphology (Fig. S1A ). The 16S rDNA sequences were compared against EZBiocloud and NCBI database and the generic nomenclature was assigned according to EZBiocloud database (Table 1 ). Their evolutionary relationship was ascertained by a phylogenetic tree (Fig. S2 ). Three out of seven isolates were of Kocuria sp (LC2, LC3, and LC5). The rest belonged to Enterobacteriaceae viz . Enterobacter sp. (LC1, LC4, and LC6) and Kosakonia sp. (LC7) (Table 1 ). The 16S rDNA sequences of the different isolates of Enterobacter and Kocuria showed significant variations indicating considerable diversity amongst them. Enterobacter cloacae is already reported as endophyte of corn 29 , pine 30 and Capsicum 31 . Genome sequence of an As-resistant E . cloacae LSJC7 shows a chromosomally coded arsenic resistance (ars) operon required for detoxification of arsenate, arsenite and antimonite 32 . Recently Kocuria arsenatis , a novel strain of As-tolerant endophytic bacteria, was isolated from Prosopis laegivata - a plant collected from an As-contaminated mine tailings 28 . Notably, K . arsenatis was the closest homolog of one of our isolates, Kocuria sp. LC5. Kocuria palustris was reported as another As-tolerant bacterium containing As-detoxifying genes indicating that such mechanisms may be common in genus Kocuria . Ars operon has been widely studied in E . coli 33 , 34 . Despite such mechanistic studies of As-tolerance in bacteria, its influence on As-phytoremediation in plants has not been reported. Bacterial PGP activities could result in increased plant biomass essentially increasing storage space within the plant for the accumulated As. The ability to produce growth hormone auxin (Fig. 1A), and to solubilize inorganic phosphate (P) appeared to be predominant among the PGP activities (Fig. 1B,C ) both under −As and +As conditions. Interaction effects of As and endophytes on auxin production and P-solubilization were observed [F(6,238) = 5.996 (P < 0.0001) and F(12,105) = 34.75 (P < 0.0001) respectively] suggesting that these PGP activities of the endophytes were influenced by As and that the microbes were able to use these strategies to promote plant growth under As-challenged conditions. Enterobacter sp. LC1, Kocuria sp. LC5 and Enterobacter sp. LC6 were among the highest auxin producers (Two-way ANOVA with Tukey’s post-hoc analysis). LC7 had the highest P-solubilization potential, [14.37(±0.32) ppm in the presence of 1000 ppm As] which was increased by As-treatment (Two-way ANOVA with Tukey’s post-hoc analysis; Fig. 1B ) in agreement with the qualitative assay (Fig. 1C ). All the endophytes solubilized P indicating that their colonization within L . camara was likely driven by induced P-deficiency as a consequence of As-contamination in the soil since arsenate enters plants through the opportunistic use of P-transporters 35 . Table 1 Identification of arsenic-tolerant endophytes from Lantana camara shoot. Endophyte name GenBank Accession No. Maximum similarity with (In EZBioCloud) and similarity score Maximum similarity with (in NCBI BLAST) and similarity score Bacterial Phylum Enterobacter sp. LC1 KT873248 Enterobacter cancerogenus LMG 2693 (Z96078), 99.52% Enterobacter cloacae S20504 (KF956588), 99.3% Proteobacteria Kocuria sp. LC2 KU821101 Kocuria rhizophila DSM 11926 (Y16264), 99.36% Kocuria rhizophila F2 (KM577162), 99.5% Actinobacteria Kocuria sp. LC3 KT873249 Kocuria rhizophila DSM 11926 (Y16264), 99.72% Kocuria rhizophila F2 (KM577162), 98.2% Actinobacteria Enterobacter sp. LC4 KT873250 Enterobacter hormaechei subsp. oharae DSM 16687 (CP017180), 99.38% Enterobacter cloacae S20504 (KF956588), 99.5% Proteobacteria Kocuria sp. LC5 KT873251 Kocuria arsenatis CM1E1 (KM874399), 97.16% Kocuria rhizophila F2 (KM577162), 96.0% Actinobacteria Enterobacter sp. LC6 KU051718 Enterobacter ludwigii EN-119 (JTLO01000001), 99.24% Enterobacter cloacae 34983 (CP010377), 99% Proteobacteria Kosakonia sp. LC7 KT873252 Kosakonia cowanii JCM 10956 (BBEU01000098), 97.91% Escherichia sp. CZBRD4 (KJ184949), 97.6% Proteobacteria Figure 1 Lantana camara endophytes were auxin producing and phosphate solubilizing. ( A ) The endophytes were grown in Luria- tryptophan broth in the presence of 0 and 1000 ppm As. Auxin production was quantified in culture supernatants at A 540 against a standard curve of IAA; n = 18 (Df for As:1, for endophytes:6, for interaction:6, error:238). ( B ) & ( C ) The endophytes were grown in Pikovskaya media in the presence of 0 and 1000 ppm As and phosphate solubilization potential was measured both quantitatively at A 880 against a standard curve of K 2 HPO 4 ; n = 12 (Df for As:2, for endophytes:6, for interaction:12, error:105), ( B ) and qualitatively by halo formation around the spotted cells ( C ). LC1.: Enterobacter sp. LC1, LC2.: Kocuria sp. LC2, LC3.: Kocuria sp. LC3, LC4.: Enterobacter sp. LC4, LC5.: Kocuria sp. LC5, LC6.: Enterobacter sp. LC6, LC7.: Kosakonia sp. LC7, U = Uninoculated control. Data are represented as mean ± SEM. Bars with different letters indicate significant differences amongst different endophytic isolates at a particular As-level (bold italics for +As) obtained from two-way ANOVA with Tukey’s post-hoc test. Significant differences for an individual isolate between −As and +As treatments have been marked by ***(P < 0.001); ns = no significance. Lantana camara endophytes colonized Solanum nigrum endosphere and promoted its growth in the presence of arsenic Unlike other plant-microbe interactions viz , plant-pathogen and legume-rhizobia interactions, endophytes show a broader host range due to relatively less influence of the host genotype on their colonization. For example, Azoarcus BH72, isolated from Kallar grass 36 and nitrogen-fixing endophytes from Typha angustifolia 16 were also found to colonize rice. Rhodotorula graminis , a basidiomycotan endophytic yeast isolated from poplar, similarly showed a broad host range 37 . Likewise, LacZ-labeled L . camara endophytes (Fig. S3A ) were tested for their ability to colonize the endosphere of S . nigrum . Representative figures following X-gal staining showed blue staining in the infected plant (Fig. 2A ) and bacterial colonization in the apoplastic spaces in the root (Fig. 2B ). Our results confirmed that the L . camara endophytes established themselves within S . nigrum endosphere as a surrogate host (Fig. 2A,B ). The endophytes also translocated systemically in the stem, node, and leaf (Figs 2A , S3B ) possibly through the xylem vessels as also reported for other beneficial endophytes 38 , 39 . Next, the effect of the L . camara endophytes on the growth of S . nigrum seedlings was studied in the presence and absence of As. Significant interaction effects were observed between As and endophyte treatments on all the growth parameters measured by total plant biomass, root length, shoot length, leaf number and area (Two-way ANOVA; Table 2 ). Individually, the endophytes had differential effects on plant growth. Some had growth promoting effects both under +As and −As conditions (LC2, LC3) while some showed growth retardation (LC5, LC6; Figs 2C–G , S4 ). LC4 showed growth promotion in absence of As but compromised growth under +As (Two-way ANOVA with Tukey’s post-hoc test). The bacteria were also used as a consortium which ensured that the interspecies interactions that occurred in their natural environment were maintained while interacting with the plant. Multi-strain consortiums are often reported to be more effective in their PGP activity than a single bacterium 40 . Inoculation with the consortium resulted in an increase in plant height and biomass 4-weeks post infection (wpi). The growth promotion was visible even in the absence of As. Nonetheless, the difference in growth between uninoculated vs. inoculated plants was more pronounced when the plants were treated with As (Fig. 2C–G ; Table 2 ) indicating that the consortium was most potent in increasing the vigor of S . nigrum . Figure 2 L . camara endophytes colonized S . nigrum endosphere and promoted plant growth. ( A , B ) One week old S . nigrum seedlings were infected with LacZ-labeled endophytes and stained with 100 µg/mL X-gal. Stained regions of plants are shown with arrows indicating colonization. LC1.: Enterobacter sp. LC1, LC2.: Kocuria sp. LC2, LC3.: Kocuria sp. LC3, LC4.: Enterobacter sp. LC4, LC5.: Kocuria sp. LC5, LC6.: Enterobacter sp. LC6, LC7.: Kosakonia sp. LC7. U: uninoculated control plant, Scale bar = 0.5 cm. Infected roots were visualized under a light microscope. Kocuria sp. LC2 colonization in root sections has been shown. Scale bar = 300 µm. Bacterial chains have been shown in arrowheads ( B ). ( C-G ) One week old S . nigrum plants were treated with 25 ppm Na 3 AsO 4 or 1 × MS in presence or absence of the endophytes added individually or as a consortium. The biomass ( C ), root length ( D ), shoot length ( E ), leaf number ( F ) and leaf area ( G ) were determined 4wpi; n = 12. Data are represented as mean ± SEM. Bars with different letters indicate significant differences amongst different endophytic isolates at a particular As-level (bold italics for +As) obtained from two-way ANOVA with Tukey’s post-hoc test. Significant differences for an individual isolate between −As and +As treatments have been marked by *P < 0.05, **P < 0.01 ***(P < 0.001); ns = no significance. (Df for As:1, for endophytes:8, for interaction:8, error:198). Table 2 F-values for different plant properties due to As or endophyte (main effects) or their interaction. Plant Properties As-effect Endophyte-effect Interaction effect Phenotypic effects Biomass F(1,198) = 797.2; P < 0.0001 F(8,198) = 1040; P < 0.0001 F(8,198) = 239.7; P < 0.0001 Root length F(1,198) = 134.9; P < 0.0001 F(8,198) = 115; < 0.0001 F(8,198) = 53.19; P < 0.0001 Shoot length F(1,198) = 118.8; P < 0.0001 F(8,198) = 194.7; P < 0.0001 F(8,198) = 64.18; P < 0.0001 Leaf number F(1,198) = 281.3; P < 0.0001 F(8,198) = 60.17; P < 0.0001 F(8,198) = 28.74; P < 0.0001 Leaf area F(1,198) = 93.25; P < 0.0001 F(8,198) = 112.3; P < 0.0001 F(8,198) = 29.43; P < 0.0001 Total chlorophyll content F(1,32) = 300.9; P < 0.0001 F(1,32) = 258.6; P < 0.0001 F(1,32) = 24.30; P < 0.0001 Enzyme activities \n As-effect \n \n Endophyte-effect \n \n Interaction effect \n \n Root \n \n Shoot \n \n Root \n \n Shoot \n \n Root \n \n Shoot \n GR activity F(1,36) = 39.35; P < 0.0001 F(1,36) = 157.9; P < 0.0001 F(1,36) = 6.627; P = 0.0143 F(1,36) = 1.894; P = 0.1772 (ns) F(1,36) = 0.01235; P = 0.9121 (ns) F(1,36) = 43.95; P < 0.0001 GST activity F(1,36) = 2.212; P = 0.1457 (ns) F(1,36) = 1.198; P = 0.281 (ns) F(1,36) = 2.435; P = 0.1274 (ns) F(1,36) = 21.46; P < 0.0001 F(1,36) = 0.06844; P = 0.7951 (ns) F(1,36) = 51.66; P < 0.0001 Glutaredoxin activity F(1,36) = 38.97; P < 0.0001 F(1,36) = 21.64; P < 0.0001 F(1,36) = 23.24; P < 0.0001 F(1,36) = 16.91; P = 0.0002 F(1,36) = 79.74; P < 0.0001 F(1,36) = 45.77; P < 0.0001 AsPOX activity F(1,36) = 67.44; P < 0.0001 F(1,36) = 0.1590; P = 0.6924 (ns) F(1,36) = 27.85; P < 0.0001 F(1,36) = 0.2627; P = 0.6114 (ns) F(1,36) = 152.0; P < 0.0001 F(1,36) = 1.329; P = 0.2566 (ns) POX activity F(1,36) = 4.360; P = 0.0439 F(1,36) = 0.6863; P = 0.4129 (ns) F(1,36) = 3.986; P = 0.0535 (ns) F(1,36) = 9.178; P = 0.0045 F(1,36) = 7.454; P = 0.0097 F(1,36) = 20.62; P < 0.0001 Arsenate reductase activity F(1,36) = 106.4; P < 0.0001 F(1,36) = 97.34; P < 0.0001 F(1,36) = 2.22; P = 0.1449 (ns) F(1,36) = 34.02; P < 0.0001 F(1,36) = 26.57; P < 0.0001 F(1,36) = 31.43; P < 0.0001 The endophytic consortium increased arsenic phytoremediation potential of S. nigrum We measured the As-concentration in plants treated with the individual microbe and the consortium under +As condition by ICP-OES. Without the endophytes, S . nigrum retained most of the As (1267 ± 56.88 ppm) in the root. LC2 increased the root retention of As. LC4, LC5, LC7 and the consortium decreased As-concentration in root (One-way ANOVA with Tukey’s HSD test) concomitantly increasing shoot As suggesting that these microbes regulated the root-to-shoot transport of As that is extremely vital for As-phytoremediation. LC4 and LC5 increased the shoot As-concentration to >2000 ppm and negatively affected plant growth which inversely correlated with shoot As-concentration. The consortium-treated plants were the only exception where both growth and root-to-shoot translocation of As increased in +As plants (Fig. 3 ; Table 2 ). The consortium also reduced As in the rhizosphere soil (Fig. S5 ). The ratio of shoot-As to soil-As was approximately 16 after endophyte treatment compared to Isatis cappadocica and Hesperis persica where this ratio was reported to be around 1. A 6.74-fold increase in plant biomass (Fig. 2C ) suggested a significant increase in As-cleanup from the soil by endophyte-treated plants. Since the consortium appeared to be most effective in improving the potency of As-phytoremediation of S . nigrum , the endophytes were used as a consortium in the subsequent studies. Figure 3 L . camara endophytes increased As accumulation and translocation in S . nigrum . S . nigrum plants were grown in presence or absence of 25 ppm As, treated with or without the endophytes. ( A – C ) As concentration was measured in root and shoot by ICP-OES. As concentration in plant root ( A ) and shoot ( B ), and translocation factor (shoot As/root As) ( C ) have been plotted. n = 6. Data are represented as mean ± SEM. Bars with different letters indicate significant differences amongst different endophytic isolates obtained from one-way ANOVA with Tukey’s post-hoc test. The endophytic consortium increased photosynthetic efficiency in S . nigrum in the presence of arsenic This increased fitness of the consortium-treated plants was further confirmed by measuring their photosynthetic efficiency. As-stress is known to decrease the photosynthetic rate in plants 41 – 44 . As-treated S . nigrum plants also showed leaf chlorosis and a 26.02(±0.0057)% decrease in total chlorophyll content (Figs 4A , S6 ). This reduction was largely restored when plants were supplemented with the consortium. The consortium increased the chlorophyll content even without As, the increase was, however, significantly greater in the presence of As [24.55(±0.0057)%] (Two-way ANOVA with Tukey’s post-hoc analysis, Table 2 ; Fig. 4A ). The quantum efficiency (Fv/Fm) of photosystem II (PSII) increased by 1.42-times on consortium treatment (unpaired t-test; Fig. 4B ). Several derived parameters were measured and are shown by a radar plot (Fig. 4C , Table S 3 ). The results suggested that the number of photosynthetically active reaction centers was enriched by 25% in endophyte-treated plants compared to untreated control. The consortium-treated plants showed a greater PSII performance with enhanced energy flux towards electron transport, reduced energy loss by dissipation, reduction in the number of inactive reaction centers and an increase in Q A turnover rate. (Fig. 4C , Table S 3 ). This is in contrast to the reported effect of As on non-hyperaccumulator Ceratophyllum demersum L, where higher concentrations of As disrupted PSII performance completely 45 . Since photosynthetic performance is a direct reflection of plant growth and yield, the consortium seemed to circumvent the growth inhibition caused by As-stress imparting a fitness advantage to the plants leading to better survival in the presence of the metalloid. Figure 4 Endophyte consortium treatment improved photosynthetic efficiency and phosphate nutrition in S . nigrum . ( A ) Total chlorophyll content in plant leaves was compared between endophyte consortium-treated plants and uninoculated plants grown in the presence or absence of 25 ppm As. ***P < 0.0001 (endophyte-treated vs uninoculated; two-way ANOVA with Tukey’s post-hoc test); n = 9 (3 leaves/plant). (Df for As:1, for endophytes:1, for interaction:1, error:32). ( B and C ) Photosynthetic efficiency of endophyte consortium-treated plants was compared with untreated plants grown in the presence of As, represented by Fv/Fm ratio ( B ) and the radar plot ( C ). ***P < 0.0001 (unpaired two-tailed t-test); n = 9 (3 leaves/plant). ( D ) The total phosphate content of endophyte consortium treated and uninoculated plants grown in presence of 25 ppm As was measured by ICP-OES 4wpi. n = 9 (each in 3 experimental replicates). ***P < 0.0001(unpaired two-tailed t-test). Data are represented as mean ± SEM. The endophytic consortium influenced several mechanisms, directly and indirectly, influencing As tolerance and/or detoxification The endophytic consortium improved phosphate nutrition in S. nigrum in the presence of arsenic Arsenate competes with phosphate for P-transporters to gain entry into the plant and thus interferes with P-sensing and responses 46 . We investigated the effect of the consortium on the total P-level of the plants. Recently, it was shown that colonization of endophytic fungus Colletotrichum tofieldiae in Arabidopsis thaliana was controlled by P-deficiency response of the plant. Phosphate was supplemented by the fungus 5 which helped it bypass plant innate immunity. The observation that all the L . camara endophytes solubilized phosphate possibly indicated a similar nutritional requirement of the host plant under As-stress. Endophytes isolated from Typha angustifolia growing in the marginal wetland of a Uranium mine appeared to be primarily N-fixing 16 and improved N-nutrition in the plant again highlighted that the endophyte properties are driven by plant nutritional status (controlled by the soil). In the presence of As, treatment with the consortium increased the total P-content per plant by about 8.05-times (unpaired t-test; Fig. 4D ). Downregulation of P-transporters is a common strategy adopted by tolerant plants to bypass As-uptake 47 . However, this is likely to obstruct P-acquisition by plants affecting plant growth. The consortium improved P-nutrition of S . nigrum under As-stress and restored plant fitness in the presence of As without such a compromise. Elevated P has been reported to completely change the uptake and intracellular dynamics of As in plants 45 . Nonetheless, this improvement of P-nutrition could further increase the As-load in S . nigrum . Tu 48 reported that supplementation of P in the growth medium indeed increased As-phytoremediation by P . vittata . The endophytes, through their effect on P-nutrition, had a two-fold effect. On the one hand, it improved plant health in presence of As, on the other hand, it increased As-phytoremediation potential. The endophytic consortium enhanced reactive oxygen species generation in S. nigrum A major cause of As-induced cell damage is believed to be oxidative stress 49 , 50 . Several detoxification strategies in plants are known to contribute towards As-tolerance. These processes were investigated in consortium-treated plants to understand the mechanism of consortium-mediated As-detoxification. Leaves of 4-week old S . nigrum plants grown in the presence and absence of As, with and without the consortium, were stained with Nitroblue tetrazolium (NBT) that stains superoxide radicals 51 . The consortium-treated plants showed intense staining even in absence of As (Fig. 5A ). Staining was noted around the veins in plants treated with only As or the consortium (Figs 5A , S7A left panel). ROS generated due to the accumulation of uncomplexed As(III) is reported to accumulate in plant veins 52 . The pattern of superoxide accumulation noticeably changed upon As + consortium treatment. The staining became most intense and occurred throughout the leaf (Figs 5A , S7A right panel). Whether this change in the ROS-staining was due to change in As-distribution is difficult to predict, however, microscopic studies showed that the staining occurred in the chloroplasts (Fig. 5B ). Plant ROS responses occur in distinct organelles like chloroplast, mitochondria, peroxisome, apoplast and the nucleus. Depending on the site of production, ROS has specific signatures and distinct biological readouts. For example, ROS production in chloroplast is implicated in signaling response and appears to be distinct from peroxisome where the ROS production triggers repair 53 . The ROS production was also highest in roots of As + consortium-treated plants, where the staining pattern appeared to be distinctly different from As-treated plants; probably indicating that different organelles participate in ROS production (Fig. S7B ). The endophytes, therefore, appeared to contribute to sustained ROS generation in plants without having any deleterious effect on the plant health. This was clearly different from the ROS generation in plants that occur as an immediate response to its encounter with microbe-associated-molecular-patterns (pathogenic or symbiotic) 54 . Recently it has been suggested that ROS is not necessarily indiscriminately cytotoxic as believed earlier, but rather functions in cellular surveillance and signaling 55 , 56 . A balance between ROS production and induction of anti-oxidative defense is central to acclimation to abiotic stress. Beneficial interaction of plants with arbuscular mycorrhiza and Piriformospora indica is known to confer cross-tolerance to a variety of abiotic and biotic stressesand requires the generation of ROS 57 . P . indica reduced As induced root damage and As translocation in rice 58 . Total glutathione level was increased 1.8-times in root and 3.4-fold in the shoot (Fig. 5C ) in the presence of As + consortium compared to As-treated plants (unpaired t-test). Although the total glutathione content increased, the reduced (GSH) to oxidized glutathione (GSSG) ratio was decreased (Fig. 5D ) further confirming more ROS generation in As + endophyte plants. Figure 5 L . camara endophyte consortium augmented ROS production and anti-oxidative defense in S . nigrum . ( A ) Young leaves of endophyte consortium-treated and uninoculated plants grown in presence or absence of 25 ppm As were stained with 0.05% NBT 4wpi and visualized under a stereo microscope M205FA. Scale bar = 5 mm. ( B ) ROS production in chloroplasts of plants treated with As and endophytes. Scale bar = 50 µm. ( C and D ) Total glutathione content ( C ) and GSH/GSSG ratio ( D ) of endophyte consortium-treated plants were compared with untreated plants grown in presence of As. n = 14. *P < 0.05, ***P < 0.0001 (unpaired two-tailed t-test). ( E – I ) Antioxidant enzyme activities were measured in root and shoot of endophyte consortium-treated and uninoculated plants grown in presence or absence of As. Glutathione Reductase (GR) ( E ), glutathione S-transferase (GST) ( F ), glutaredoxin ( G ), peroxidase (POX) ( H ) and ascorbate peroxidase (APX) activities ( I ) were plotted using GraphPad Prism5. n = 10 (each in 2 experimental replicates). *P < 0.05, **P < 0.01, ***P < 0.0001, ns = no significance (endophyte-treated vs uninoculated; Two-way ANOVA with Tukey’s post-hoc test). Data are represented as mean ± SEM. (Df for As:1, for endophytes:1, for interaction:1, error:36). The endophytic consortium elicits distinctly different oxidative defense response in root and aerial tissues Enhanced glutathione biosynthesis and metabolism is an accepted strategy that improves As tolerance in plants 59 . Glutathione influences the activity of many enzymes involved in detoxification, sequestration, and transport of As directly or indirectly 60 . Activities of enzymes that regulate the reduced glutathione pool or indirectly depend on the glutathione pool were tested. These were glutathione reductase (GR) that catalyzes the formation of GSH from GSSG, glutaredoxin and glutathione S-transferase (GST). Glutaredoxins receive electrons from glutathione and donate them to oxidized substrates. Overexpression of glutaredoxins has been reported to increase As-tolerance in plants 61 , 62 . GSTs transfer glutathione moieties to xenobiotics for detoxification. Activities of all three ‘glutathione-related’ enzymes were increased in shoots of As-treated plants specifically upon consortium treatment (Fig. 5E–G ) with significant interaction effects between As and endophytes (Two-way ANOVA, Table 2 ). The consortium appeared to expand the glutathione pool and influenced glutathione-related detoxification mechanisms without the requirement of any transgenic approach long sorted to improve As-resistance in plants. Activities of these enzymes were however not significantly altered in roots. In contrast, activities of ascorbate peroxidase (APX) and peroxidase (POX) were enhanced in As-treated plant roots (Fig. 5H,I ) rather than shoots upon endophyte treatment with significant interaction effects between As and endophytes (Two-way ANOVA, Table 2 ) indicating that As-mediated upregulation of anti-oxidative defense was affected by the consortium. Peroxidases are components of the ascorbate-glutathione cycle that constitute the most prominent ROS detoxification system in plants. Peroxidase activity scavenges H 2 O 2 and is almost always associated with a concomitant increase in GR activity. Interestingly, it was not so in our case. Peroxidase and GR activities were antagonistic in root and shoot indicating additional layers of tissue-specific regulation. In the shoot, increased As-content as a consequence of endophyte treatment probably triggered glutathione-mediated detoxification. In contrast, more peroxidase activity implied more H 2 O 2 generation in roots which were scavenged or potentially participated in signaling responses. In summary, the endophytes boosted both the non-enzymatic and enzymatic antioxidant defense in an As-dependent manner in the plant and this together with heightened ROS-levels provided enhanced protection against As-stress. The endophytic consortium increases As(V) to As(III) conversion in plants As(V) to As(III) conversion is an important mechanism for As-detoxification. As(III) forms stable complexes with thiol-containing compounds, glutathione, and glutathione-derived peptides− phytochelatins (PCs) 63 and are immobilized in vacuoles by MRP transporters. Arsenic is transported to the shoot via aquaporin proteins as As(III), which is the preferred form of As for phytoremediation 64 . As(III) can also be extruded out of plant cell 64 – 67 . The activity of arsenate reductase catalyzing As(V) to As(III) conversion was increased both in shoot and root of consortium-treated plants in an As-dependent manner (Two-way ANOVA, Table 2 ; Fig. 6A ). Overexpression of bacterial arsenate reductase and gamma-glutamylcysteine synthetase conferred greater As-tolerance 59 . The endophytic consortium expanded the glutathione pool and simultaneously increased arsenate reductase activity, mimicking an analogous physiological state in S . nigrum . The endophytes were individually tested for their ability to convert As(V) to As(III) using the ability of AgNO 3 to provide specific color (yellow or orange) to As(V) and As(III) mixed in a given ratio 68 . All the endophytes (but not the dead bacteria) individually or as a consortium changed the color of AgNO 3 in a way suggesting a 25–40% conversion of As(V) to As(III) (Fig. 6B ). Arsenic speciation in S . nigrum shoot extracts was performed by separating As(V) from As(III) utilizing the selective ability of As(V) to bind Fe-doped Ca-alginate beads at pH3 followed by ICP-OES 69 . pH had negligible effect on As(V) to As(III) conversion (data not shown). Our results revealed that S . nigrum had an innate ability to convert As(V) to As(III). 51% of As(V) was converted to As(III) in untreated plants. The As(III) increased to 68% when the plants were treated with the consortium (Fig. 6C ). This confirmed that the endophytes reduced As and potentially contributed to the long-distance transport and detoxification of As. Figure 6 L . camara endophyte consortium reduced arsenate, upregulated arsenate reductase activity in As- dependent manner and differentially regulated aquaporin and MRP genes. S . nigrum plants were grown in presence or absence of 25 ppm As, treated with or without the endophytes used as a consortium. ( A ) Arsenate reductase activity was measured in the root and shoot 4wpi. n = 10 (each in 2 experimental replicates). *P < 0.05, ***P < 0.001 (endophyte- treated vs uninoculated; Two-way ANOVA with Tukey’s post-hoc test). (Df for As:1, for endophytes:1, for interaction:1, error:36). ( B ) AgNO 3 was added to bacterial cells (live & dead) incubated in Tris-Cl buffer with arsenate for 48 h and the colour was compared to standards having varying As(V)/As(III) ratio. Viability of live cells was measured. LC1.: Enterobacter sp. LC1, LC2.: Kocuria sp. LC2, LC3.: Kocuria sp. LC3, LC4.: Enterobacter sp. LC4, LC5.: Kocuria sp. LC5, LC6.: Enterobacter sp. LC6, LC7.: Kosakonia sp. LC7., Mix: Consortium, U: uninoculated control. ( C ) As(V) and As(III) speciation was performed in S . nigrum shoot extracts grown in the presence or absence of the consortium. As(V) and As(III) was separated using calcium alginate beads followed by ICP-OES. Results have been represented as pie charts showing the percentage of each species in the total As. n = 6. ( D ) cDNA was prepared from root and shoot of 4-week infected plants and expression of 4 aquaporins and 3 MRP transporters in root and shoot were measured by real-time PCR. The fold change of expression has been normalized to that of control. SnTIP2-1, SnATIP and SnTIP2-2 = Tonoplast intrinsic proteins; SnPIP1 = Plasma membrane intrinsic protein; SnMRP1, SnMRP2 and SnMRP3 = Multidrug resistance-associated proteins. *P < 0.05,**P < 0.01,***P < 0.001. Data are represented as mean ± SEM. This results led us to investigate the expression of aquaporin-like proteins which play important role in the transport of As(III) 70 , 71 and H 2 O 2 across membrane 72 . Expression of three S . nigrum tonoplast intrinsic proteins (TIPs) and one plasma membrane intrinsic protein (PIP) was checked by real-time PCR. In roots, SnTIP2-2 (GU594261) and SnPIP1 (GU575314) were upregulated by 2.1 and 1.4-fold respectively, and SnTIP2-1 (GU594266) and SnATIP (GU594268) were downregulated by 3.3 and 1.3-fold respectively upon endophyte treatment. In the presence of As + consortium, the downregulation of SnTIP2-1 and SnATIP was further pronounced (4.4 and 3.9-fold respectively) (Fig. 6D ). Although upregulated on consortium treatment, SnPIP1 was strongly downregulated (3.7-fold) when As was together with the consortium. The endophytes upregulated all four aquaporins in the shoot. Arsenic treatment alone also led to upregulation of the aquaporins in shoots, but at a much lower level compared to the consortium -treated plants. Further, expression of MRP transporters was studied in As and/endophyte treated plants. MRP transporters are known for sequestration of As-PC complexes in plant vacuoles and are reported to be upregulated in response to As-stress 73 which make them important candidates for studying As detoxification. Unlike the aquaporins, no sequences for S . nigrum MRPs were available in the nucleotide database. Therefore three partial MRP sequences SnMRP1 (1015 bp, KY448289), SnMRP2 (959 bp, KY448290) and SnMRP3 (634 bp, KY448291) (Fig. S8A–C ) homologous to Arabidopsis thaliana AtABCC1 and AtABCC2 73 were cloned from S . nigrum . The putative SnMRPs belonged to three phylogenetically distinct classes. Among these, in the root, SnMRP1 was upregulated upon consortium treatment but downregulated by 3-fold upon As-treatment. SnMRP2 showed the most interesting regulation. It was strongly upregulated (5-fold) in root upon endophyte treatment exclusively in presence of As. In the shoot, SnMRP2 was upregulated (1.5-fold) only in presence of As. Endophyte consortium treatment downregulated the expression of all the other MRPs in the shoot (Fig. 6D ). Differential regulation of these genes may indicate potentially important roles played by them in controlling plant’s response towards As and/or the consortium."
} | 11,433 |
33972407 | PMC8166140 | pmc | 5,748 | {
"abstract": "Significance The growth of coral reefs is threatened by the dual stressors of ocean warming and acidification. Despite a wealth of studies assessing the impacts of climate change on individual taxa, projections of their impacts on coral reef net carbonate production are limited. By projecting impacts across 233 different locations, we demonstrate that the majority of coral reefs will be unable to maintain positive net carbonate production globally by the year 2100 under representative concentration pathways RCP4.5 and 8.5, while even under RCP2.6, coral reefs will suffer reduced accretion rates. Our results provide quantitative projections of how different climate change stressors will influence whole ecosystem carbonate production across coral reefs in all major ocean basins.",
"discussion": "Results and Discussion Net carbonate production in every coral reef at every site included in our analysis was reduced by each of the projected scenarios, with the extent of declines being dependent on scenario and location ( Figs. 2 – 4 and SI Appendix , Figs. S1–S3 ). We project declines in net carbonate production so severe that reef accretion will cease globally by 2100 under RCP4.5 and 8.5 ( Fig. 2 ) even under our optimistic estimates that do not factor for physical erosion. Even under RCP2.6, we project mean declines in global net carbonate production of 71% by 2050 and 77% by 2100. These declines are largely the result of reduced coral cover from bleaching events rather than from the direct impacts of ocean warming or acidification on calcification or bioerosion ( SI Appendix , Figs. S1–S4 ). We further predict that median reef net carbonate production will switch from production to erosion under higher emissions scenarios. Specifically, we project 119% mean declines in global net carbonate production by 2050 and 148% by 2100 under RCP4.5, while we estimate declines in net carbonate production of 149% by 2050 and 155% by 2100 under RCP8.5. Fig. 2. ( A ) Net carbonate production rates (kg CaCO 3 m −2 ⋅ y −1 ) and ( B ) potential vertical accretion rates (mm ⋅ y −1 ), presently and under the interactive effects of ocean acidification and ocean warming. These data account for reduced future coral cover due to mass bleaching events across three ocean basins for the mean of each of 183 reefs. Scenarios shown are three RCP scenarios (2.6, 4.5, and 8.5) by 2050 and 2100. Medians, 75% quartiles, 95% whiskers, and outliers are presented. See SI Appendix , Figs. S1 and S3 for the accretion and carbonate production rates projected under each stressor singularly. For accretion without sediment dissolution, see SI Appendix , Fig. S2 . Fig. 3. Location of study regions and their net carbonate production (kg CaCO 3 m −2 ⋅ y −1 ) under the following scenarios: ( A ) present-day and projections of the interactive effects of ocean acidification, warming, and mass coral bleaching by 2050 at ( B ) RCP2.6, ( C ) RCP4.5, and ( D ) RCP8.5 and by 2100 under ( E ) RCP2.6, ( F ) RCP4.5, and ( G ) RCP8.5 occurring at each of 183 reefs. Present-day Palmyra reef is higher than 15 kg CaCO 3 m −2 ⋅ y −1 . Fig. 4. Examples of how the effects of ocean acidification, ocean warming, and mass coral bleaching are projected to impact net carbonate production through changes in bioerosion and net calcification of corals and of coralline algae. Displayed here are regions in the Atlantic ( A and B ), Indian ( C and D ), and Pacific Oceans ( E and F ) with high ( A , C , and E ) and low ( B , D , and F ) present-day net carbonate production. Scenarios are the same as in Fig. 1 : present-day and RCP2.6, 4.5, and 8.5 in 2050 and 2100. Photo credits: A , B , and C were taken by Chris Perry; D was taken by Nicholas Graham; E was taken by Gareth Williams; and F was taken by Christopher Cornwall. We project much lower decreases in net carbonate production when the impacts of coral bleaching are excluded. For example, we estimate net carbonate production will only be reduced 4% by 2050 and 3% by 2100 under RCP2.6. If CO 2 emissions are kept to within RCP2.6, our meta-analysis of past experimental work forecasts relatively small declines (1 to 6%) in individual coral and coralline algal calcification ( Table 1 ) and only small changes in bioerosion rates (4% declines to 6% increases). However, we estimate 15% declines in net carbonate production by 2100 under RCP4.5, while under RCP8.5, we estimate 58% declines in global net carbonate production by 2100. The scenarios without coral beaching would only manifest if coral thermal tolerances increased dramatically to the point where they are no longer impacted by coral bleaching events, which is a highly unlikely scenario. Nevertheless, these scenarios demonstrate the impacts that ocean warming and acidification will have on metabolic rates of remaining thermally tolerant corals and coralline algae. The declines in net carbonate production caused solely by the impacts of climate change on the processes of calcification and bioerosion could be construed as being relatively small (e.g., SI Appendix , Figs. S1 and S4 ). However, there is considerable natural variability in net carbonate production presently, and there are many heavily degraded reefs that already have low net carbonate production ( 33 ). To put these declines in net carbonate production rates into perspective, mean global declines in net carbonate production (e.g., SI Appendix , Fig. S4 ) predicted under RCP8.5 by ocean acidification alone outweighs the present-day net carbonate production rates for 31% of these reefs. Table 1. Mean percentage change on individual components of the carbonate budget by 2050 and 2100 relative to today, caused by ocean warming, acidification and their interactive effects. Values were calculated using multiple linear regressions of responses measured in the laboratory against region-specific increases in temperature, and decreases in pH. See Table S1 for a full study list. The 100% increase in sediment dissolution indicates full removal of all sediment within those scenarios Values were calculated using multiple linear regressions of responses measured in the laboratory against region-specific increases in temperature and decreases in pH. See SI Appendix , Table S1 for a full study list. The 100% increase in sediment dissolution indicates full removal of all sediment within those scenarios. Different colors represent the different RCP scenarios (white 2.6, light blue 4.5, red 8.5). We project that many presently degraded reefs will logically continue to have low levels of net carbonate production in the future (see examples in Fig. 4 B , D , and F ) but that reefs with higher present-day production rates will follow trajectories that are largely related to three factors. These factors are 1) their present-day net carbonate production rates; 2) the biotic composition of the reef presently, where reefs with higher bioerosion fare worse and those with higher coralline algal carbonate production fare slightly better under scenarios with reduced coral cover; and 3) their geographic location, as projections of ocean warming and the associated timing and magnitude of loss of coral cover due to bleaching events varies by region ( SI Appendix , Fig. S5–6 ). Reefs in the Pacific Ocean tend to fare better than others under most future scenarios, and those in the Atlantic fare the worst under RCP2.6 scenarios ( Figs. 2 – 4 ). This is reflective of the generally higher present-day rates of net carbonate production in the Pacific Ocean and lower rates in the more heavily degraded coral reefs in the Atlantic Ocean ( 34 , 35 ). Additionally, higher bioerosion rates in the Indian Ocean reefs (mean 2.90 kg CaCO 3 m −2 ⋅ y −1 compared to 1.93 kg CaCO 3 m −2 in the Atlantic Ocean and 1.52 kg CaCO 3 m −2 in the Pacific Ocean) and higher contributions of coralline algal carbonate production in Pacific Ocean reefs (mean gross production = 1.08 kg CaCO3 m −2 ⋅ y −1 compared to 0.06 kg CaCO 3 m −2 in the Atlantic and 0.31 kg CaCO 3 m −2 in the Indian Oceans) alter the trajectories of these reefs. The contribution of corals to gross carbonate production and its trajectory under climate change plays the largest role in dictating net carbonate production and accretion rates on reefs. Present-day rates of bioerosion are also important in determining net carbonate production under RCP8.5 once coral cover is severely reduced in our model ( R 2 = 0.85). However, our estimated changes in total bioerosion rates in situ are relatively small. This is because parrotfish and sea urchins—which erode mainly by biophysical means—contribute the greatest to total bioerosion at most of our sites (mean: 1.74 kg CaCO 3 m −2 ⋅ y −1 ) as opposed to micro- or macrobioeroders living in/on the carbonate framework (mean 0.65 kg CaCO 3 m −2 ⋅ y −1 ), which also can bioerode by chemical means. Coralline algal gross carbonate production plays a limited role on many reefs (mean gross production = 0.28 kg CaCO 3 m −2 ⋅ y −1 ). However, our data illustrate that Pacific Ocean reefs with higher coralline algal carbonate production rates are more robust under scenarios where coral gross production is reduced by bleaching, but these reefs are comparatively more susceptible to the ocean acidification scenarios. This is due to the larger adverse effects of ocean acidification on the net calcification rates of coralline algae compared to corals (up to 12% in 2100 under RCP 8.5; Table 1 ). Coralline algae appear more robust to the impacts of marine heatwaves than corals ( 17 ), and thus we did not decrease their abundance here under ocean warming scenarios. All reefs with positive net carbonate production under RCP8.5 by 2100 had present-day coralline algal net carbonate production rates ≥1.8 kg CaCO 3 m −2 ⋅ y −1 . Together, this indicates that coralline algal calcification could initially act as a short-term substitute to provide carbonate in reefs heavily influenced by mass coral bleaching. However, coralline algal–dominated reefs will offer very different (or reduced) ecological services and structural complexity compared to coral-dominated reefs ( 11 ), and their capacity to produce carbonate will also be limited once ocean acidification intensifies. Conversely, their ability to support parrotfish and sea urchin bioeroders will also be reduced ( 36 – 38 ), which could therefore support slightly higher net carbonate production than equivalent coral-dominated reefs with equal gross carbonate production ( 33 ). A major consequence of declining net carbonate production rates on reefs relates to their capacity to accrete at the same rate as rising sea levels. Median global coral reef accretion potential is estimated as 2.80 mm ⋅ y −1 in our present-day scenario (range: −1.77 to 13.20), but we project this will fall to −1.11 mm ⋅ y −1 (−3.51 to 1.51) under the interactive effects of coral bleaching, ocean warming, and acidification in RCP8.5. Global sea level rises of up to 15 mm ⋅ y −1 (range: 10 to 15) are projected by 2100 under RCP8.5 ( 39 ). While rates vary between regions, no reefs here maintain accretion rates that will match the projected global mean rates of sea level rise. However, rates of sea level rise are much lower under RCP2.6 (mean: 4 mm ⋅ y −1 ) ( 39 ), and, here, accretion rates only drop to 0.47 mm ⋅ y −1 (−2.75 to 6.31) ( Fig. 2 B ). However, only four reefs still maintain rates of accretion that match mean increases in sea level rise by 2100 under RCP2.6. These aforementioned accretion rates assume sediment dissolution rates on reefs will equate to those measured on lagoon sediments ( 20 ). However, the link between the two processes is currently unknown. If sediment dissolution rates measured in lagoons are not accounted for here, mean accretion rates are likely to decline less in all future scenarios: −0.74 and 0.89 mm ⋅ y −1 under RCP8.5 and 2.6, respectively ( SI Appendix , Fig. S2 ). While previous global-scale assessments have demonstrated the likelihood of lagoon sediments becoming sites of net sediment dissolution under future ocean acidification scenarios ( 20 ), we demonstrate here that this negative trajectory is likely to extend to whole reef–scale net carbonate production and that most reefs will likely suffer net erosion by 2100 under business-as-usual scenarios. However, it is possible that some shallower reefs may actually benefit from rising sea levels, with increasing accommodation space allowing for increased coral vertical growth ( 40 ). Indeed, this has been observed in the past ( 41 ) during much slower rates of sea level rise. It is unknown whether these possible gains in accretion will outweigh losses due to ocean warming and acidification, but determining when and where this could occur should be an urgent focus for future research. We note that the capacity for reef-building taxa to gain tolerance to marine heatwaves, and ongoing ocean warming and acidification over the coming decades, is largely unknown ( 30 , 31 , 42 – 45 ). The trajectories of reef accretion projected here will be highly sensitive to changes in coral community thermal tolerance. The fast rate of environmental change relative to the time required for adaptation suggests it will be difficult for corals to maintain their current role, especially those with longer generation times. The only remaining corals after repeated mass bleaching events could be heat-sensitive species or phenotypes ( 30 ). However, it is unlikely that these heat-tolerant corals would maintain similar rates of gross carbonate production and cover as the current assemblages. If so, these possibilities represent the only real avenues for future reef persistence and slowing of rates of reef surface submergence for the majority of reefs under RCP4.5 and 8.5 scenarios as coral cover continues to decline under these emissions scenarios. In an analysis such as this, it is also not feasible to include other moderating effects, such as the effects of pH/temperature variability, light, nutrients, and water velocity in modifying responses at a site level ( 46 – 49 ). These could further modify trajectories of individual reefs. However, on a global scale, we would assume that the individual studies included here would encompass a range of environmental conditions, both the estimates of net carbonate production and the effects of climate change stressors on key ecological processes across sites. Our results indicate that the net carbonate production and accretion of most the world’s coral reefs will be fundamentally reduced by ongoing climate change. Increasingly negative impacts are associated with higher levels of emissions and environmental change, and thus the two most contrasting futures for coral reef carbonate production are highlighted by this analysis: one in which the RCP2.6 stabilization scenario is achieved and the second in which emissions continue to rise under conditions similar to those predicted under RCP4.5 through to 8.5. Under RCP2.6, most coral reefs could maintain positive net carbonate production, with a small subset even having accretion rates that match sea level rise. If rapid action is taken to reduce CO 2 emissions, there is a higher potential for coral reefs to maintain their many key functional roles in the future ( 50 ). In alternate scenarios where emissions are not curbed sufficiently, almost all coral reefs will suffer losses in net carbonate production so severe that it will halt their capacity to accrete vertically and no reefs will match sea level rise (given the caveats above). This will progressively limit their capacity to provide important services such as habitat for reef-associated taxa, protection of shorelines from wave action, and serving as centers of tourism and fisheries. Our projections here are likely optimistic given that we do not account for increasing storm frequency, which could further remove reef framework via physical erosion, nor do we include some other factors that reduce coral cover or calcification rates, such as disease, pollution, and frequent outbreaks of Crown of Thorns. Given the increased risk of globally declining coral cover and the mean global net decline in carbonate production predicted under current emissions trajectories, we must now markedly reduce CO 2 emissions to have any possibility of sustaining positive carbonate production and reef accretion rates, thus maintaining the critical ecological and societal services that reefs provide."
} | 4,145 |
25643398 | PMC4348357 | pmc | 5,749 | {
"abstract": "The perception and response to cellular death is an important aspect of multicellular eukaryotic life. For example, damage-associated molecular patterns activate an inflammatory cascade that leads to removal of cellular debris and promotion of healing. We demonstrate that lysis of Pseudomonas aeruginosa cells triggers a program in the remaining population that confers fitness in interspecies co-culture. We find that this program, termed P. aeruginosa response to antagonism (PARA), involves rapid deployment of antibacterial factors and is mediated by the Gac/Rsm global regulatory pathway. Type VI secretion, and, unexpectedly, conjugative type IV secretion within competing bacteria, induce P. aeruginosa lysis and activate PARA, thus providing a mechanism for the enhanced capacity of P. aeruginosa to target bacteria that elaborate these factors. Our finding that bacteria sense damaged kin and respond via a widely distributed pathway to mount a complex response raises the possibility that danger sensing is an evolutionarily conserved process. DOI: \n http://dx.doi.org/10.7554/eLife.05701.001",
"introduction": "Introduction Bacteria can occupy highly dynamic environments, where survival is linked to the ability to sense and respond to an assortment of threats ( Cornforth and Foster, 2013 ). It is increasingly clear that in addition to well-understood environmental and nutritive stresses, antagonistic factors elaborated by other bacteria are a common threat that bacteria must cope with ( Little et al., 2008 ; Hibbing et al., 2010 ). A number of antagonistic strategies have been identified, including the production of diffusible factors such as small molecule antibiotics. It has been suggested that sub-inhibitory concentrations of these molecules induce specific changes in bacteria, including the production and distribution of resistance mechanisms ( Hoffman et al., 2005 ; Linares et al., 2006 ; Andersson and Hughes, 2014 ). Other antagonistic pathways, such as contact-dependent inhibition (CDI) and the type VI secretion system (T6SS), require cell contact ( Hayes et al., 2010 ; Konovalova and Sogaard-Andersen, 2011 ). Kin cells are protected from the toxic proteins delivered by these pathways by specific cognate immunity proteins; however, escape from or defense against these pathways by non-kin is not well understood ( Hood et al., 2010 ; Russell et al., 2011 ). The T6SS is a versatile export machinery that can deliver a wide range of proteinaceous effector molecules from donor to recipient Gram-negative bacterial cells ( Hood et al., 2010 ; Coulthurst, 2013 ; Russell et al., 2014a ). One of the best characterized bacterial targeting T6SSs is the Hcp Secretion Island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa ( Hood et al., 2010 ). The H1-T6SS transports a cargo of at least seven effectors, termed type VI secretion exported 1–7 (Tse1–7) ( Hachani et al., 2014 ; Whitney et al., 2014 ). The outcome of intoxication by these proteins can be lysis or cessation of growth ( LeRoux et al., 2012 ; Li et al., 2012 ). Like other species with interbacterial T6SSs, P. aeruginosa has the capacity to target cells of its own genotype with the H1 pathway. To inhibit self-intoxication, cognate type VI secretion immunity proteins (Tsi) are produced and localized to the cellular compartment that contains the target of the corresponding effector ( Russell et al., 2013 ; Benz and Meinhart, 2014 ; Durand et al., 2014 ; Russell et al., 2014a ). T6SSs are found in at least three genetically distinct configurations present among multiple bacterial phyla, making it one of the most widespread pathways mediating interbacterial antagonism known ( Russell et al., 2014b ). Expression and activity of the H1-T6SS is tightly regulated at multiple levels ( Silverman et al., 2012 ). Stringent post-transcriptional regulation of the H1-T6SS is achieved through the g lobal ac tivation of antibiotic and cyanide synthesis/ r egulator of s econdary m etabolism (Gac/Rsm) pathway ( Goodman et al., 2004 ; Mougous et al., 2006b ). This global regulatory system impacts protein production via RsmA, a CsrA-type protein that binds to target mRNA molecules and generally acts to repress their translation ( Lapouge et al., 2008 ). RsmA is modulated by levels of the small RNA (sRNA) molecules rsmY and rsmZ , which bind to and sequester it from its targets. Transcription of the sRNAs is promoted by phosphorylated GacA, the cognate response regulator of the sensor kinase GacS. Finally, two hybrid sensor kinases, RetS and LadS, acting through GacS, repress or stimulate GacA phosphorylation, respectively ( Ventre et al., 2006 ; Goodman et al., 2009 ). Consistent with regulation of T6S by the Gac/Rsm pathway, many of its targets in P. aeruginosa and related γ-proteobacteria are involved in the production of social or antagonist factors ( Lapouge et al., 2008 ). This theme of Gac/Rsm-dependent modulation of antibiotic activity is exemplified by the defect of P. fluorescens gac mutants in bacterial and fungal growth inhibition on plants ( Laville et al., 1992 ). Though the precise cues that activate the Gac/Rsm pathway are unknown, Haas et al. have found that one or more signals accumulate in spent bacterial culture supernatants deriving from both self and non-self organisms ( Dubuis and Haas, 2007 ). Posttranslational regulation by the threonine phosphorylation pathway (TPP) constitutes a second level of control over H1-T6SS activity. In this pathway, phosphorylation of a fork head-associated domain-containing protein, Fha1, triggers apparatus assembly and effector secretion ( Mougous et al., 2007 ). PppA, a phosphatase, opposes the activity of PpkA on Fha1, returning the system to the inactive state. Additional components of the TPP, encoded by type VI secretion associated genes Q-T ( tagQ-T ), act upstream of these proteins and are thought to be involved in signal transduction ( Hsu et al., 2009 ; Casabona et al., 2013 ). Two signals of the TPP have been proposed: surface-associated growth and membrane perturbation ( Silverman et al., 2011 ; Basler et al., 2013 ). The latter signal is thought to underlie the observation that organisms with active T6S or type IV secretion (T4S) are more efficiently targeted by the H1-T6SS than those without ( Ho et al., 2013 ). It was proposed that the activity of these apparatuses induces local membrane perturbations in P. aeruginosa that are sensed by the TPP, leading to posttranslational activation of the H1-T6SS and enhanced recipient cell death ( Basler et al., 2013 ). A caveat of these studies is that the P. aeruginosa strain used bears an inactivating mutation in retS , which constitutively activates the Gac/Rsm pathway, potentially masking the contribution of this major regulatory mechanism to the defense mounted by P. aeruginosa against the antagonistic pathways of competing bacteria. Here, we show that lysed kin cells act as a danger signal that is sensed by the Gac/Rsm pathway of wild-type P. aeruginosa . Our experiments provide a mechanism for T6S-dependent killing of competitor bacteria possessing either the T6 or T4 secretion pathways, as both induce P. aeruginosa lysis, stimulate the Gac/Rsm pathway, and lead to posttranscriptional de-repression of the H1-T6SS. These findings provide a rationale for the regulation of promiscuous antibiotic mechanisms by a pathway that can respond to self-derived signals.",
"discussion": "Discussion We have shown that self-derived signal(s) generated as a consequence of cell lysis activate the Gac/Rsm pathway of P. aeruginosa , and thus stimulate the production of antibiotic factors under its control. In growth competition experiments, the capacity to mount this multifaceted response grants P. aeruginosa a significant fitness benefit. Our results suggest that similar to multicellular organisms, injury to a bacterial colony can trigger the release of danger signals that lead to a coordinated response against the threat ( Matzinger, 1994 ; Kaczmarek et al., 2013 ). In this study, we focused on offensive factors under control of the Gac/Rsm pathway; however, defensive factors are also likely to be elaborated. For example, a consequence of Gac/Rsm activation in P. aeruginosa is the production of c-di-GMP, which activates exopolysaccharide production of P. aeruginosa ( Lee et al., 2007 ; Irie et al., 2012 ). This increases cellular adhesiveness, which facilitates multicellular aggregates that are more resistant than planktonic cells to an assortment of antibacterial molecules and environmental stresses ( Colvin et al., 2011 ; Billings et al., 2013 ). In the context of an infection such as the chronic lung infections that occur in cystic fibrosis patients, host-induced cellular lysis could activate Gac/Rsm and inadvertently convert cells to a state that is more resistant to killing by antibiotics and the immune system. If PARA was the sole mechanism contributing to enhanced T6S-dependent killing of bacteria with T6 or T4 systems, it would follow that P. aeruginosa cells lacking the sensor kinase RetS should target bacteria irrespective of these pathways. However, previous studies have shown that P. aeruginosa ∆ retS retains some ability to differentially target T6S- and T4S-positive vs -negative cells ( LeRoux et al., 2012 ; Basler et al., 2013 ). Thus, the response of P. aeruginosa to antagonism is comprised of a global response mediated by the Gac/Rsm pathway and a secondary T6S-specific element that is not fully understood. We speculate that this ability of a ∆ retS strain is related to coordinated spatiotemporal localization of the apparatus among adjacent cells, and that these two mechanisms operate in concert to hone the offensive response of P. aeruginosa . PARA may constitute an initial adaptation in which cells perceive a threat in their proximity and increase expression of the H1-T6SS, followed by the orientation of effector translocation specifically toward aggressor cells. We find that RP4-containing E. coli cells induce lysis in P. aeruginosa , trigger PARA, and in turn are subject to increased antagonism by the H1-T6SS. This mechanism differs from the model put forth by the Mekalanos laboratory, which suggested that the TPP is required for the response of wild-type P. aeruginosa to an incoming conjugative apparatus ( Ho et al., 2013 ). A key finding in the prior study was that polymyxin B, an outer membrane-disrupting antibiotic, induces clpV1 foci formation in wild-type P. aeruginosa , but not in a strain lacking tagT. This finding, among other data involving strains in the ∆ retS background of P. aeruginosa , led the authors to propose that membrane perturbations caused by an incoming T4 apparatus are sensed by the TPP. A tagT deletion strain intrinsically lacks H1-T6SS activity; therefore, interpreting its inability to respond to the antibiotic as evidence of TPP involvement is problematic ( Basler et al., 2013 ; Casabona et al., 2013 ; Ho et al., 2013 ). We found that H1-T6SS-active strains that lack the TPP (∆TPP ∆ tagF ) display a generalized targeting defect, but retain the ability to discriminate T4S + and T4S − \n E. coli . An alternative explanation for the findings of Ho et al. is that the application of polymyxin B promotes cell lysis, leading to PARA induction ( Barrow and Kwon, 2009 ; Ho et al., 2013 ). Attempts to test the validity of this explanation were confounded by pervasive cell death at the antibiotic concentration reported by the authors (20 μg/ml, ∼40-fold the minimum inhibitory concentration against P. aeruginosa PAO1) ( Barrow and Kwon, 2009 ). The adaptive significance of RP4-induced lysis and its mechanistic basis remain to be resolved. This process could be an altruistic behavior of P. aeruginosa that both aborts the T4S-dependent transfer event and alerts surrounding kin, thus decreasing the probability of foreign DNA acquisition by the colony. However, a second possibility is that plasmids such as RP4 carry interbacterial antagonistic factors that provide fitness to their hosts under certain conditions. It is interesting to note that Ho et al. identified an RP4 transposon mutant that was not targeted by P. aeruginosa but retained a functional T4SS ( Ho et al., 2013 ). This insertion resides in trbN , a gene encoding a periplasmic transglycosylase. It is plausible given the requirement for both T4 structural genes and this peptidoglycan-degrading accessory factor, that the T4S apparatus facilitates the transfer of this protein to recipient cells, where it induces lysis. Alternatively, upon plasmid transfer, the product of trbN may stochastically trigger lysis in a small portion of recipient cells. Efforts to characterize Gac/Rsm-stimulating signal(s) have been performed primarily in P. fluorescens ( Lapouge et al., 2008 ). This organism is a close relative of P. aeruginosa , and the Gac/Rsm pathways of the two species share a number of characteristics including regulation of hydrogen cyanide production and an H1-T6SS-like pathway. Haas et al. have made a number of intriguing observations pertaining to the production and sensing of Gac/Rsm-stimulating signals in P. fluorescens that are consistent with our findings in P. aeruginosa . Most notably, they found that conditioned media extracts derived from dense cultures of P. fluorescens , and, to a lesser extent, P. aeruginosa and Vibrio cholerae , are sufficient to activate the Gac/Rsm pathway ( Dubuis and Haas, 2007 ). In agreement with our results, this indicates that the signal can be self-produced; however, it also raises the intriguing possibility that the lysis of non-self bacteria may activate PARA. While we found that B. thai -derived lysate was not sufficient to stimulate PARA, it is possible that other organisms produce the signal. This could lead to a positive feedback loop by which killed competitor cells further stimulate P. aeruginosa to produce antibacterial factors. Despite decades of research, the chemical structure of the molecule(s) that stimulate the Gac/Rsm pathway of P. aeruginosa , presumably via interaction with the periplasmic ligand binding domains of its associated sensor kinases, remain unknown. Our own efforts to identify the signaling molecule(s) contained within P. aeruginosa lysate, which included assorted enzymatic treatments, have so far been unsuccessful. Structural studies of RetS revealed its periplasmic region bears a fold resembling known carbohydrate interaction domains. A similar domain is predicted in the ecto domain of LadS ( Vincent et al., 2010 ). Given that RetS is required for PARA transduction, it is tempting to speculate that cell-associated carbohydrate(s) are released upon lysis and serve as a signal that activates Gac/Rsm in P. aeruginosa ( Jing et al., 2010 ). The additional observation that extracts derived from multiple bacterial species can stimulate Gac/Rsm in P. fluorescens suggests two non-mutually exclusive hypotheses: that the molecule is broadly conserved or that the pathway has evolved to respond to a number of inputs ( Dubuis and Haas, 2007 ). The existence of three sensor kinases that operate upstream in the Gac/Rsm pathway of P. aeruginosa supports the latter hypothesis. In what they referred to as ‘competition sensing’, Cornforth and Foster recently proposed that bacterial stress responses include antagonistic components, and that these pathways have evolved to respond to threats posed by other bacteria ( Cornforth and Foster, 2013 ). For example, in response to DNA damage, the SOS pathway stimulates colicin production in E. coli , and in P. aeruginosa , exogenous peptidoglycan fragments have been shown to stimulate quorum-regulated toxins ( Cascales et al., 2007 ; Korgaonkar et al., 2013 ). Our study demonstrates that ‘competition sensing’ includes an antibacterial response to cellular damage in kin cells. While we found that T6S-dependent killing by P. aeruginosa is part of an antagonistic response to lytic threats, Borgeaud et al. reported that in V. cholerae , T6S is co-regulated with competence machinery and utilized for obtaining access to exogenous DNA ( Borgeaud et al., 2015 ). Together, these studies demonstrate how functionally conserved machinery can be incorporated into diverse cellular programs exhibited by bacteria. The ‘danger theory’ of eukaryotic immunity proposes that in addition to the foreign substances that they introduce, threats can be sensed by virtue of cellular damage and ensuing mislocalization of host factors ( Matzinger, 1994 ; Kono and Rock, 2008 ). For instance, uric acid microcrystals, which form upon release of the molecule to the sodium-rich extracellular milieu, stimulate dendritic cell maturation ( Shi et al., 2003 ). Our study shows that bacteria can also recognize threats by sensing self-derived signals associated with cell damage ( Figure 9 ). Moreover, we find that the response to such signals includes the activation of factors that combat the threat—akin to the stimulation of inflammation in eukaryotes. It remains to be determined whether danger sensing is common among bacteria. The Gac/Rsm pathway is conserved widely among Gram-negative γ-proteobacteria; however, the variability of genes under its control confounds a prediction of its general involvement in danger sensing ( Lapouge et al., 2008 ). An intriguing possibility is that bacteria can utilize a diversity of signaling systems to sense and respond to kin cell damage. 10.7554/eLife.05701.028 Figure 9. Bacterial danger sensing. The model depicts antagonism between two species of bacteria, represented in green and brown. The green cells possess a danger sensing pathway; specifics of PARA are provided in parentheses. DOI: \n http://dx.doi.org/10.7554/eLife.05701.028"
} | 4,464 |
40108116 | PMC11923276 | pmc | 5,752 | {
"abstract": "Superamphiphobic and flame-retardant fabrics offer effective protection for firefighters and industrial workers operating under hazardous conditions. However, limitations in deformation resistance, wear comfort, and environmental adaptability hinder their practical applications. Here, a monolithic hierarchical macro-/micro-/nanostructure is constructed to achieve durable repellency against water and oils, even under significant deformations. This coating integrates fluorinated nanoparticles, flame retardant microparticles, and a cross-linking adhesive. Hydrogen bonding and the adhesive define the coating’s morphology, robustness, and adaptability. The coated surface exhibits an ultralow water adhesion force (0.002 mN) and excellent anti-fouling performance against extreme temperatures (100 °C, −196 °C) and corrosive liquids, including aqua regia and concentrated H 2 SO 4 . Upon fire exposure, the coating enables self-extinguishing behavior on cotton fabrics. The coated fabrics also demonstrate remarkable mechanical and UV resistance while preserving wear comfort. Overall, we achieve a balance between desirable properties and wear comfort in superamphiphobic, flame-retardant fabrics, enabling protective clothing applications previously unattainable.",
"introduction": "Introduction Clothing serves as a crucial second skin, offering both comfort and protection across diverse environments. Fabrics possessing flexibility and breathability are one of the most pivotal clothing materials, providing great convenience in industrial production and daily life 1 . These fabrics primarily comprise natural fibers such as cotton, silk, and linen, as well as synthetic fibers like polyester, nylon, and polypropylene 2 . However, when used in personal protective textiles for industrial workers and firefighters in hazardous conditions, several challenges arise: (1) the vast majority of fabrics are highly susceptible to rapid ignition upon exposure to fire, increasing the risks of fire hazards during use 3 , 4 ; (2) fabrics are prone to contamination from water and oil, leading to appearance defects and performance degradation; (3) the growing demand for diverse applications requires fabrics to withstand harsh environments, including UV radiation, chemical corrosion, high temperatures, and freezing conditions 5 , 6 . To address these issues, many efforts, such as physical blending 7 , chemical grafting 8 , organic/inorganic hybrid coating 9 – 12 , and sol-gel reactions 13 , have been explored to enhance fabrics’ flame retardancy, hydrophobicity, and oleophobicity. The incorporation of flame-retardant elements, including halogens, phosphorus, nitrogen, silicon, and boron, has been shown to be effective in enhancing fire safety. However, the high sensitivity of these components to moisture often leads to their migration and failure, especially in humid environments or after repeated washing, which significantly limits their practical application 14 . To fight against the leaching of flame retardants, integrating superhydrophobicity with flame retardancy provides a promising solution. Various materials, such as polydimethylsiloxane-silica/ammonium polyphosphate (APP) hybrid coatings 15 – 18 , phytic acid metal complex aggregations/dimethyloctadecyl [3-(trimethoxysilyl) propyl] ammonium chloride hybrid coatings 19 , hydroxyethyl acrylate/sodium vinylsulfonate copolymers 20 , and ethylene-vinyl acetate/aluminum trihydroxide composite 21 , have demonstrated the feasibility of this strategy. However, superhydrophobicity alone is insufficient to prevent appearance defects or reduce the increased fire risk caused by oil contamination (e.g., edible oil, diesel) on flame-retardant materials, particularly under hazardous conditions. Compared to superhydrophobic materials, superamphiphobic surfaces provide superior oil repellency and enhanced anti-fouling performance 22 . Over the past decade, many methods, such as laser-etching 23 , template-assisted electrochemical deposition, 3D-printing technologies, and inorganic/organic hybrid coating 24 – 30 , have been proposed to create superamphiphobic surfaces. However, sophisticated fabrication processes often hinder their practical application, making scalable, easy-to-manufacture coatings the preferred solution 31 , 32 . By optimizing the composition, size, and parameters of the inorganic/organic components, the properties of coated fabrics can be significantly improved 33 , 34 . Despite these advancements, it remains a daunting challenge to maintain superamphiphobicity under harsh external environments such as long-term mechanical abrasion or large deformations 35 – 43 . In addition, personal protective clothing requires resistance to extreme acid/base corrosion, UV irradiation, and hot/cold liquids, while also ensuring wear comfort (e.g., water moisture/air permeability and bending rigidity) 44 . Unfortunately, so far, there exists a glaring gap in achieving the integration of these functions within one material with well-balanced performance. In this work, a distinctive scalable coating with integrated superamphiphobicity and flame retardancy for functional fabrics used in personal protective clothing is presented. Attributed to the well-arranged fluorinated silica (F-SiO 2 ) nanoparticles, micro-sized aluminum diethylphosphinate (ADP) particles and cross-linking polydimethylsiloxane (PDMS) adhesive, the obtained coating exhibits super-repellency to both water and oils, even under significant deformations. The inorganic functional micro/nanoparticles provide optimal multi-scale roughness and generate a monolithic hierarchical macro-/micro-/nanostructure with substrates, endowing the surfaces with impressive air trap capability. The trapped air cushion imparts durable repellency to hot (100 °C), cold (−196 °C), and corrosive liquids (even aqua regia and concentrated H 2 SO 4 ). The coated surface achieves an ultralow water adhesion force as low as 0.002 mN. The combined efforts of hydrogen bonding interactions and cross-linking elastic adhesive enhance the coating’s mechanical robustness, and environmental adaptability, such as UV resistance, and corrosion resistance, while maintaining excellent water moisture permeability, bending rigidity, and tensile strength. More importantly, applying the coating to cotton fabric imparts self-extinguishing behavior in the vertical burning test. This multifunctional, superamphiphobic, and flame-retardant material demonstrates significant potential for advanced functional textiles and personal protective clothing applications."
} | 1,645 |
24924356 | PMC5381534 | pmc | 5,753 | {
"abstract": "Reaching a comprehensive understanding of how nature solves the problem of degrading recalcitrant biomass may eventually allow development of more efficient biorefining processes. Here we interpret genomic and proteomic information generated from a cellulolytic microbial consortium (termed F1RT) enriched from soil. Analyses of reconstructed bacterial draft genomes from all seven uncultured phylotypes in F1RT indicate that its constituent microbes cooperate in both cellulose-degrading and other important metabolic processes. Support for cellulolytic inter-species cooperation came from the discovery of F1RT microbes that encode and express complimentary enzymatic inventories that include both extracellular cellulosomes and secreted free-enzyme systems. Metabolic reconstruction of the seven F1RT phylotypes predicted a wider genomic rationale as to how this particular community functions as well as possible reasons as to why biomass conversion in nature relies on a structured and cooperative microbial community.",
"discussion": "Discussion Our cumulative understanding regarding microbial plant biomass degradation is built from laboratory performance-based assessment of singular cellulolytic isolates and their enzymes. However this does not reflect the true capabilities of naturally occurring cellulolytic ecosystems, which utilize syntropic interactions within a consortium to envelop both polysaccharide hydrolysis and downstream metabolism. Here we have utilized the recent progression in DNA sequencing and computational biology technologies to describe both the genomes and proteomes for all the as-yet uncultured members within a cellulolytic consortium. Predictive metabolic “blue-print” analysis of all F1RT phylotypes presented a genomic rationale as to how the F1RT consortium functions, why it resisted initial aerobic culturing efforts and why it displayed structural similarity to the independently generated SF356 enrichment. In addition, our “omics” analyses allowed for the first time, the reconstruction of cellulosomes from uncultured bacteria and indications for the presence of extracellular (poly)cellulosomes, a poorly understood facet of the cellulosome “paradigm”. Moreover, our analysis demonstrated the putative operation of both cellulosome and free-enzyme systems by different phylotypes within a consortium. Indeed, recent in vitro studies indicate synergism between recombinant cellulosomes and free-enzymes, due to the two enzyme systems using different mechanisms for interacting with microcrystalline surfaces 19 23 . Our findings suggest that such a multi-system strategy for cellulose-degradation conceivably exists within naturally occurring microbial consortia. The functional capabilities of F1RT combined with the observed structural similarities with the previously described SF356 consortium would suggest that F1RT-like populations play crucial roles in natural cellulose-degrading soil ecosystems. Verification of this hypothesis would create significant insights towards understanding the impact of such populations on a broader scale."
} | 769 |
35391826 | PMC8980759 | pmc | 5,754 | {
"abstract": "Summary Compared with conventional von Neumann’s architecture-based processors, neuromorphic systems provide energy-saving in-memory computing. We present here a 3D neuromorphic humanoid hand designed for providing an artificial unconscious response based on training. The neuromorphic humanoid hand system mimics the reflex arc for a quick response by managing complex spatiotemporal information. A 3D structural humanoid hand is first integrated with 3D-printed pressure sensors and a portable neuromorphic device that was fabricated by the multi-axis robot 3D printing technology. The 3D neuromorphic robot hand provides bioinspired signal perception, including detection, signal transmission, and signal processing, together with the biomimetic reflex arc function, allowing it to hold an unknown object with an automatically increased gripping force without a conventional controlling processor. The proposed system offers a new approach for realizing an unconscious response with an artificially intelligent robot.",
"introduction": "Introduction Neuromorphic systems are used to mimic the biological signal perception process of the biological system because of the ultrahigh speed of parallel operation for spatial and temporal information ( Fu et al., 2018 ; Han et al., 2015 ; Mead, 1990 ; Zhu et al., 2014 ). Several generations of neuromorphic systems have been developed including algorithm-based neuromorphic computing and hardware-based neuromorphic circuit for emulating parallel calculations ( Tuchman et al., 2020 ). Because both of them depended on central processing units, graphics processing units and tensor processing units still faced the high energy consumption concern for the traditional von Neumann’s architecture and limitations of Moore’s law ( Berggren et al., 2020 ; Moore, 1965 ; Xia and Yang, 2019 ). Recently, third generation of “in-memory computing” based neuromorphic sensing systems were reported by mimicking the biological signal perception function ( Berggren et al., 2020 ; Xia and Yang, 2019 ). The biological signal perception function includes environmental sensing, signal transmission, and signal processing. Different types of neuromorphic sensing systems with signal perceptions functions have been developed to mimic the sensing functions, such as vision, touch, and taste sensing ( Bao et al., 2021 ; Berridge et al., 2000 ; Kyung et al., 2015 ; Lee and Lee, 2019 ). An ideal neuromorphic sensing system not only demonstrates signal perception but also controls artificial effectors such as humanoid hands by refining a control procedure ( Tuchman et al., 2020 ). An artificial afferent nerve with a pressure sensor was demonstrated for the control of a cockroach leg ( Kim et al., 2018 ). Also, the vision-based optoelectronic sensorimotor can manipulate artificial muscle based on light cognition ( Lee et al., 2018 ). In the biological system, there are two different control mechanisms; a brain processed conscious control and a spinal cord processed unconscious control. In some biological systems, not all environmental stimuli are processed by the brain ( He et al., 2020 ). Instead, the spinal cord produces a faster unconscious response as a simple reflex arc, thereby reducing the brain’s tasks. Recently, the concept of unconscious controlling was replicated through an artificial somatic reflex arc system ( He et al., 2020 ). Moreover, a conscious response has been demonstrated using an artificial stimulus-response system, and the response time was reduced through training (S. Kim et al., 2021a ). However, the effect of training on the unconscious response of neuromorphic systems has not been studied yet. In the field of pressure sensing, artificial skin has been highlighted as a type of biomimetic touch sensors. Different types of pressure sensors have been proposed for the sensor parts, including capacitance-based and resistance-based sensors ( Chortos et al., 2016 ). A force-sensitive resistor (FSR)-based sensor has been used as a pressure sensor, because of its low cost, small size, and high sensitivity to force ( Gupta et al., 2011 ). However, the sensing mechanism of FSR, deformation of active layer or diaphragm depending on the force, generates concerns such as its non-linearity and non-repeatability. Such concerns are considered as evaluation criteria of the sensors ( Abdul Razak et al., 2012 ; Kumar and Pant, 2014 ). Therefore, there is a need for a force-sensitive resistor without deformation of the active layer. Utilizing the sensing mechanism of a potentiometer is a possible solution, as it shows a variable resistance change without physical deformation of the active layer by changing the length of the conducting path. The remaining challenge is to develop a class of variable resistors that change axial forces into rotational forces. The 3D origami structures have been demonstrated with their rotational multifunctionalities ( Li et al., 2019 , 2020 ). In particular, the Kresling origami, formed by a series of tessellated triangles, shows rotating characteristics when compressed ( Zhai et al., 2018 ). In addition, its rotating behavior can be tuned by modifying the angular design of the tessellated triangle(s), allowing for customization of the force sensing ( Novelino et al., 2020 ; Zhai et al., 2018 ). Applying the origami structure to the pressure sensor allows for force conversion without requiring other components, such as gears or axial shifters. Indeed, employing such a structure simplifies the entire pressure sensing system for various applications ( Kaur et al., 2021 ). For the fabrication of three-dimensional (3D) structural components, 3D printing has been widely adopted for prototyping because of its advantages in regard to custom and facile fabrication. Various 3D printing technologies have been developed, such as fused filament fabrication (FFF), digital projection lithography, direct ink writing (DIW), and selective laser sintering. Among these, the DIW method for paste printing has attracted significant attention for the preparation of functional materials ( Dong et al., 2018 ; Kim et al., 2019 ; Lewis, 2006 ; Valentine et al., 2017 ; Zhang et al., 2019 ; Zhou et al., 2017 ). DIW-printed structures are mainly dependent on the properties of the printed materials, whereas in-situ sintering with a laser facilitates the fabrication of 3D structures. For example, freestanding structures have been achieved using laser-assisted methods ( Skylar-Scott et al., 2016 ). Circuits made from copper have been demonstrated on a 3D surface using a laser-assisted DIW method ( Jo et al., 2020 ). However, 3D printing on curvilinear surfaces while keeping the nozzle vertical to the surface is difficult in the typical DIW because of the limited freedom of current three-axis DIW printers. One unique solution is to add more freedom of motion to the printers by using a multi-axis robot arm. Robot arms are widely used in the industry for more complex applications, such as welding for automobiles. Nonetheless, to the best of our knowledge, this work is the first DIW printing method with a six-axis industrial robot arm. We used a DIW approach using a multi-axis robot arm, as shown in Figure 1 A. The six-axis robot arm enables the seamless fabrication of conductive traces on vertical and tilted surfaces in a single printing process. Moreover, the 3D-printed conductor can be sintered in situ using an infrared laser (IR) laser with optimized parameters, as shown in Figure 1 B. Unlike conventional three-axis 3D printing systems, the demonstrated six-axis robot 3D printing can fabricate 3D electronics on curvilinear surfaces. Figure 1 3D printing through the six-axis robot (A) Schematic diagram of robot 3D printing for three different types of surfaces. (B) Schematic diagram of printing procedure with in situ laser sintering. (C) The six-axis robot 3D printing setup. (D) Image of the laser spot and sintering (top: in-situ scanning; bottom left: as-printed conductive trace; bottom right: sintered conductive trace). (E) Relation between pulse width and induced temperature at a frequency of 100 Hz. Data are represented as mean ± SEM. (F) Resistance changes with scanning at a speed of 4 mm/s. (G) Relation between scanning speed and sintering efficiency. Data are represented as mean ± SEM. (H) Left: sample printed by six-axis robot on the vertical wall of a 3D-shaped oscillator. Right: design of 3D-shaped oscillator. (I) Left: the kirigami conductive path on a 3D origami finger using six-axis robot 3D printing. Right: design of the kirigami conductive path on the tilted surfaces. Here, we demonstrated a neuromorphic humanoid hand, fabricated by six-axis robot-based DIW method and integrated with origami-inspired pressure sensors. A portable neuromorphic system is added for the bioinspired signal transmission and processing without conventional spike-based control algorithm. The 3D structural neuromorphic humanoid hand is trained to demonstrate a reflex arc by gripping an object with an unconsciously increased force, based on the learning and non-volatile memory functions of the neuromorphic system.",
"discussion": "Discussion A sensory humanoid hand with a proposed origami pressure sensor can detect variable resistance and connect with a portable neuromorphic system integrated by a 3D oscillator and a synaptic transistor for the transmission and processing of the signals. The 3D structural neuromorphic robot hand is created to provide a perception function, including the detection, transmission, and processing of signals. For the fabrication of such a complicated 3D neuromorphic system, we introduced a multi-axis robot DIW method. The six-axis robot-based DIW method printed conductive traces on curvilinear surfaces such as vertical and tilted surfaces seamlessly. We demonstrated a unique DIW method for the fabrication of 3D conductive traces by optimizing parameters such as the speed of robot motion to improve the efficiency of the 3D printing process. The neuromorphic humanoid hand can also control its motion unconsciously, as the artificially intelligent robot mimics the reflex arc performance. The robot hand is trained to replicate the learning progress of gripping a ball, similar to the gripping motion of human hands. Different gripping forces are monitored through the compressed depths to the soft ball with different steps, such as before training, during training, and after training. The neuromorphic humanoid hand demonstrates an increased gripping force sufficient to hold an unknown object. The force output from our neuromorphic system after training is controllable by modulating the initial training force. Because the non-volatile effect of the neuromorphic device is dependent on the bias applied on the gate electrode of synaptic transistors, it is possible to modulate by control of the amplitude and duration of training force. So, different training effect is possible to control for grasping of various unknown objects. When the system grasps flexible objects, smaller training force needs to produce weak grasping force by reducing the enhancement during the training process, while stronger training force requires to produce a higher grasping force for a rigid object. Therefore, an artificially intelligent robot hand is exhibited with the demonstration of an unconscious response through training. Limitations of the study Integration of the 3D-printed neuromorphic system with a humanoid hand is the first attempt for the realization of an artificially intelligent robot using non-volatile effect. So, this research still needs to find the way to improve the efficiency of training. Our demonstration here can be the first step toward combination of multiple input signals to mimic the biological signal perception functions in future."
} | 2,960 |
37047567 | PMC10094943 | pmc | 5,756 | {
"abstract": "The development of polymeric materials as antifouling coatings for aquaculture nets is elaborated in the present work. In this context, cross-linked polymeric systems based on quaternary ammonium compounds (immobilized or releasable) prepared under mild aqueous conditions were introduced as a more environmentally friendly methodology for coating nets on a large scale. To optimize the duration of action of the coatings, a multilayer coating method was applied by combining the antimicrobial organo-soluble copolymer poly(cetyltrimethylammonium 4-styrenesulfonate-co-glycidyl methacrylate) [P(SSAmC 16 -co-GMA20)] as the first layer with either the water-soluble copolymer poly(vinylbenzyl trimethylammonium chloride-co-acrylic acid) [P(VBCTMAM-co-AA20)] or the water-soluble polymers poly(acrylic acid) (PAA) and poly(hexamethylene guanidine), PHMG, as the second layer. The above-mentioned approach, followed by thermal cross-linking of the polymeric coatings, resulted in stable materials with controlled release of the biocidal species. The coated nets were studied in terms of their antifouling efficiency under accelerated biofouling conditions as well as under real conditions in an aquaculture field. Resistance to biofouling after three water-nutrient replenishments was observed under laboratory accelerated biofouling conditions. In addition, at the end of the field test (day 23) the uncoated nets were completely covered by marine contaminants, while the coated nets remained intact over most of their extent.",
"conclusion": "4. Conclusions In the present work a new greener methodology was developed for the preparation of anti-fouling coatings, based on aqueous polymeric systems which could be applied to aquaculture industry in a large scale. Active polymeric systems were prepared using the copolymer P(SSAmC 16 -co-GMA20), which had given encouraging results of antimicrobial activity with controlled release of biocidal species in a previous study [ 19 ]. Intending to prolong aquaculture coated nets’ activity, a multilayer-coating method was used to encapsulate the AmC 16 biocidal groups in the inner layer of the net leading to their slow release into the marine environment. It is worth noting that for coating preparation, the solvent mixture H 2 O/DMSO was used, instead of other typical organic solvents (CHCl 3 , etc.) leading to a more environmentally friendly system, thus combining the use of organo-soluble P(SSAmC 16 -co-GMAx) copolymers (first layer) with water-soluble P(VBCTMAM-co-AAx) copolymers or water-soluble PAA and PAA/PHMG polymers (second layer). The release study of the coated nets through gravimetric and TOC/TN analysis showed generally good behavior of all coatings under aqueous conditions, especially under simulated seawater environment (NaCl 0.6 M). When tested under laboratory accelerated biofouling conditions, the coated nets showed resistance to biofouling successfully up to day 20 and after three water-nutrient replenishments. The most significant finding was in the final test of the coatings under real field test applications, where the coated nets remained clear up to day 16, compared to the uncoated nets. The difference was more striking at the end of the test (day 23), where the uncoated nets were completely covered by marine contaminants, while the coated nets remained intact over most of their extent. While this time is quite sufficient for some cases (for instance, fry production), the versatility of the present methodology offers several possibilities of optimization to attain longer effective periods, suitable for typical aquaculture applications. Though the proof-of-concept was demonstrated here using DMSO as the solvent of the first layer, in important aspect could be to avoid the use of any organic solvent and developing wholly water-based systems. Such a design could be feasible and offer possibilities to prolong the effectiveness of the coated nets through the control of interlayer depth and crosslinking density. In conclusion, the combination of the organo-soluble P(SSAmC 16 -co-GMAx) copolymers with the water-soluble P(VBCTMAM-co-AAx) copolymers or water-soluble PAA/PHMG polymers in a two- (or four-) layer structure resulted in coatings with high antifouling activity. This may be owed to the presence of the cationic releasable groups (SSAmC 16 , PHMG) and immobilized cationic groups (VBCTMAM) in the polymeric material, imparting a biocide-release and contact-kill action, respectively, while the outer layer consisting of PAA acts as a fouling-resistant surface for contaminants. According to the above results, the new multi-functional and environmentally friendly polymeric coatings can be promising alternatives to address marine biofouling in aquaculture.",
"introduction": "1. Introduction Marine biofouling is an undesirable process in which micro- and macro-organisms attach to any object submerged in the sea and its adverse effects are reflected in the economy, ecology and shipping industries [ 1 ]. In the aquaculture industry, biofouling is one of the main obstacles to efficient and sustainable production [ 2 ]. The growth of aquatic species, affects shellfish, fish and seaweed farming worldwide, with the direct economic costs of managing biofouling in the aquaculture industry estimated at 5–10% of production costs [ 3 ]. The growth of algae, barnacles and bivalves can cover up to 100% of the mesh openings in cage nets and lead to net deformation and volume reduction. In addition, health of farmed fish is directly affected by biofouling, as they are exposed to pathogens associated with the fouling organisms, while water exchange is also impeded thereby reducing oxygen levels [ 4 ]. Every natural and artificial substrate in the marine environment is rapidly colonized by various species of micro- and macro-organisms. In general, researchers have defined four phases of colonization of marine biological contaminants: adsorption of dissolved organic molecules (molecular biofouling), colonization by prokaryotes, colonization by unicellular eukaryotes (diatoms, flagellates, amoebae and ciliates) and recruitment of invertebrate larvae and algal spores [ 5 , 6 ]. These phases may occur sequentially, overlap or take place simultaneously [ 7 ]. Nevertheless, some correlation may be shown, since the early phases, which are the adsorption of molecules and the colonization by prokaryotes, are crucial for the later phases, which are the settlement of larvae and spores. Biofouling control in aquaculture comprises: the prevention of the primary establishment of contaminants by repelling or killing them, the inhibition of the development of established organisms by lowering their adhesion ability or by removing them when small and immature and the removal or elimination of established growth of biological foulants. The main strategies used to date for tackling marine biofouling are divided in two broad categories: antifouling coatings (AF) and fouling release coatings (FR) [ 8 ]. The performance of FR coatings is based mostly on hydrophobic surfaces which prevent the adhesion of biofoulants to the surface due to their low surface energy [ 9 ]. The most used materials for such surfaces are based on polydimethylsiloxane (PDMS) [ 10 , 11 ] and fluorinated materials [ 12 ] which are generally proven to be highly effective against biofouling. On the other hand, AF coatings are based on the impregnation and subsequent release of active biocides such as copper and organotin compounds which despite their high efficiency, they have raised concern worldwide due to their high toxicity levels [ 13 , 14 ]. For example, Kartal et al. [ 15 ] encapsulated and impregnated an eco-friendly antifouling chemical, econea active agent, to the ultra-high-molecular-weight polyethylene (UHMWPE) fishing nets using polyurethane and acrylic as binders. The antifouling activities of encapsulated econea showed high durability in the fish farm environment, open pores of the fishing nets and no viable proliferation when polyurethane was used as binder. In another work [ 16 ], nano-ZnO, Cu 2 O, zinc borate, and Econea multilayer films were applied to UHMWPE fishing nets through the Layer by Layer technique to enhance their antifouling properties. The nets covered with the Econea/PDDA 16 (diallyldimethylammonium chloride) nanoparticles prevented biofouling and mesh occlusion on the nets compared to the other coatings. Frydenberg et al. [ 17 ] encapsulated copper pyrithione (CuPT) crystals by silica aerogel to obtain loadings of 50−80 % wt. CuPT. The results showed that the spatial confinement of CuPT crystals and the strong attachment to the coating enable the air gel to maintain a controlled release of dissolved CuPT for a prolonged time period. Cao et al. [ 18 ] fabricated an antifouling surface via coating Urgency BMox2 (TB) onto dopamine-modified 304 stainless steel (304 SS) and found that the adhesion rates of Vibrio natriegens and Phaeodactylum tricornutum on the antifouling surface were reduced by 99.85% and 67.93%, respectively, from those of untreated samples. To improve the fouling resistance of AF coatings, other research approaches are focusing recently on the exploration of amphiphilic systems by introducing hydrophilic polymers, zwitterionic polymers [ 19 , 20 , 21 ] or quaternary ammonium compounds (QACs) [ 22 , 23 , 24 , 25 , 26 ]. Within this frame, in our laboratory we are focusing on the development of antifouling coatings based on cross-linked polymeric networks which bear QACs [ 27 , 28 , 29 ]. More specifically, synthesized antimicrobial copolymers bearing immobilized 4-vinylbenzyldimethylhexadecylammonium chloride (VBCHAM) biocidal groups and 4-styrenesulfonate cetyltrimethylammonium (SSAmC 16 ) units with releasable cetyltrimethylammonium (AmC 16 ) biocidal groups, were mixed and cross-linked in organic solvents, providing stable coatings with long-lasting antifouling efficiency. Furthermore, towards to a greener approach, some first attempts to prepare water-based antifouling coatings were recently presented [ 30 ], where immobilized quaternary ammonium groups with a short aliphatic chain acted effectively against biofouling. However, these coatings did not show long duration of action when tested under accelerated laboratory conditions. The aim of the present work was to develop a methodology for the preparation of antifouling coatings with increased long-lasting activity, based on the use of water mostly as solvent, which could lead to a facile fabrication approach for marine industries, while maintaining green characteristics. Hence, having in mind the L-b-L (layer-by-layer) concept, we herein present a multilayer coating method of fishing nets, taking advantage of our previous knowledge on thermal cross-linking reaction of complementary carboxyl and epoxy groups of acrylic acid (AA)-containing copolymers and glycidyl methacrylate (GMA)-containing copolymers, respectively. For the first layer, the antimicrobial copolymer poly(cetyltrimethylammonium 4-styrenesulfonate-co-glycidyl methacrylate) [P(SSAmC 16 -co-GMA20)] was used, dissolved in dimethyl sulfoxide (DMSO). DMSO is an organic solvent widely used in several applications due to its less toxic nature, compared to other organic solvents [ 31 ]. The water-soluble copolymer poly(vinylbenzyl trimethylammonium chloride-co-acrylic acid) [P(VBCTMAM-co-AA20)], which was synthesized in a previous work [ 30 ], was used as the second layer, and it was stabilized through the thermal cross-linking reaction. Alternatively, an aqueous solution of poly(acrylic acid) (PAA) or a mixture of PAA with the biocidal poly(hexamethylene guanidine hydrochloride) (PHMG) was used as the second layer of the coated nets. PHMG was chosen for this study since it is well known for its wide range of action against bacteria, fungi and viruses, combined with antifouling activity against macro-pollutants in the aquatic environment [ 32 , 33 , 34 ]. The application of this protocol once or twice results in two-layer or four-layer coatings, respectively. The prepared coatings were studied in terms of their release ratio in aqueous conditions as well as their antifouling effectiveness in accelerated and real conditions, using a scale up process for coating aquaculture nets.",
"discussion": "2. Results and Discussion 2.1. Characterization of Polymers Poly(hexamethylene guanidine hydrochloride), PHMG, was prepared via condensation polymerization of hexamethylenediamine and guanidine hydrochloride. The structural characterization of PHMG oligomer, was conducted through 1 H-NMR spectroscopy in deuterated DMSO-d6. The respective spectrum and the attribution of the chemical shifts of the protons derived from PHMG are shown in Figure 1 . In particular, the protons of the -CH 2 - methyl groups (c) appear at the 1.2–1.5 ppm region, the two protons of the -CH 2 -NH- group (b) at 3.1 ppm, the guanidine -NH 2 - amino groups (a) at 7.0–8.1 ppm and the two protons of the terminal amino group (d) appear at 2.7 ppm. The success of the polycondensation of guanidine salt with hexamethylene diamine was verified by ATR-FTIR spectroscopy, as shown in Figure 2 . In the PHMG spectrum, the presence of the guanidine group is evidenced through the main peak at 1626 cm −1 , attributed to the C=N double bond vibrations, as well as the broad peaks with maxima at about 3150 and 3280 cm −1 , corresponding to the N-H stretching vibrations of guanidine bonds, as well as the methylene groups. The presence of a hexamethylene unit is attested by the two peaks at 2924 and 2861 cm −1 , attributed to the vibrations of the methylene -CH 2 - group. The molecular weight of the oligomer was found to be Μ η : 646, using the Mark–Houwink–Sakurada equation: [ η ] = K × M a , where the values of K and α are known from the literature [ 35 ]. Therefore, given that the molecular weight of the structural unit of PHMG is 158, the oligomer appears to be composed of four structural units of hexamethylene guanidine hydrochloride. The results are shown in Table 1 . 2.2. Preparation of Coated Nets The multilayer coating concept was applied for the present polymeric systems because it offers several advantages. On the one hand, the combination of the desired organo-soluble copolymer with the water-soluble polymers in two (or four) consecutive coatings is easily achieved, thus avoiding the case of poor miscibility of the polymer solutions. On the other hand, in this way the biocide AmC 16 is expected to be trapped more effectively in the inner polymeric layer(s), resulting in a slower release rate in the marine environment, thus, providing possibly a long-term antifouling performance. In this line, the net was first immersed in a solution of P(SSAmC 16 -co-GMA20) providing the first coating. Regarding the second layer, two alternative options of water-soluble cationic polymeric materials were used. The first one was the P(VBCTMAM-co-AA20) copolymer which combines immobilized trimethylammonium groups with possible antimicrobial activity [ 36 ], with carboxyl groups of acrylic acid which can be used for further cross-linking reaction with the first polymeric layer, P(SSAmC 16 -co-GMA20), after heat treatment at 120 °C, as shown in Figure 3 . The second option consisted of the water-soluble homopolymer PAA, which was used either alone, as a hydrophilic outer coating that would stabilize the whole system through cross-linking with GMA and potentially act as a fouling-resistant surface, or synergistically with the cationic oligomer PHMG, which provides strong antimicrobial activity. The mol/mol ratio of PAA/PHMG polymers in the mixture was 1/2. Table 2 shows the three different cases of two-layer coated nets, the w / w ratios of the two layers and the total percentage of polymeric loading on each net, as determined gravimetrically. SEM characterization reveals that the nets were quite uniformly coated ( Figure S1 ), while the presence of atoms characteristic of the two layers was verified through EDS ( Figure S2 ). Subsequently, characterization of the coated nets was carried out by ATR-FTIR spectroscopy, through which the successful modification of the nets was certified. More specifically, Figure 4 A shows the spectra of the nets modified with the CN1 system: P(SSAmC 16 -co-GMA20)/P(VBCTMAM-co-AA20) 55/45 % wt. The graph also shows the spectra of P(SSAmC 16 -co-GMA20), P(VBCTMAM-co-AA20) copolymers and the uncoated net for comparison. As can be seen, the characteristic peaks of the individual copolymers are also observed in the spectra of the net after each coating. Indicatively, in the first layer the peaks of the methyl groups of SSAmC 16 are observed at 2920 and 2840 cm −1 , while in the second layer a peak at 1703 cm −1 owed to the carbonyl group of acrylic acid is observed. Similarly, in the spectra of CN2: (P(SSAmC 16 -co-GMA20)/PAA20 70/30 % wt. ( Figure 4 B), the same peaks at 2920 and 2840 cm −1 of the first layer are shown, while in the second layer a peak at 1703 cm −1 attributed to the carbonyl group of PAA is also shown. In the spectra of the net CN3: P(SSAmC 16 -co-GMA20)/PAA/PHMG 70/30 % wt. ( Figure 4 C), the same characteristic peaks of the polymers P(SSAmC 16 -co-GMA20) and PAA are observed (2920, 2840 and 1703 cm −1 ). Furthermore, a new peak at 1626 cm −1 is shown, which is attributed to the C=N vibrations of PHMG on the outer layer. 2.3. Release Study of Polymer Coatings For each cycle the soluble fraction of the polymeric material % wt. and the solvent uptake % wt. were determined. The results are shown in Table 3 . From the results of Table 3 we may draw the conclusion that all systems appear more stable in salt solution than in water, as they show very low soluble fraction values (from 2 to 9 % wt.), with CN1 net (P(SSAmC 16 -co-GMA20)/P(VBCTMAM-co-AA20)) showing significantly higher mass loss in pure water (32–35 % wt.) than the other systems (5–15 % wt.). In terms of solvent uptake, CN1 net also showed higher values in pure water in comparison to CN2 and CN3. This observation, associated with the high mass loss, probably indicates that the system is loosely cross-linked (high solvent uptake) and a high percentage of uncross-linked P(VBCTMAM-co-AA20) polymer chains are, in fact, present (high mass loss) in the outer layer (55/45 % wt.). A general observation is that the solvent uptake in salt solution is similar or even higher than that observed in pure water. In particular, the CN2 (P(SSAmC 16 -co-GMA20)/PAA) net also shows a high solvent uptake in the salt solution (190 % wt.) while its mass loss was very low (4 % wt.). This observation is encouraging for the application of the coating in real seawater conditions, as the increased solvent uptake of the PAA layer suggests that it could possibly act as a highly hydrated antifouling surface. In an attempt to better understand the behavior of coated nets, the release of structural groups from the coatings was evaluated using Total Organic Carbon (TOC) and Total Nitrogen (TN) measurements. For this study, the nets in Table 3 were immersed only in aqueous 0.6 M NaCl solution, as the final application will be in seawater of a similar salinity. As shown in Figure 5 A–C, regarding the first release cycle lasting 10 days, the TOC and TN values of the three coatings CN1, CN2 and CN3 remained at low levels until the end of the experiment, as compared with the TOC, TN values calculated assuming that the entire polymer coating could be dissolved. The low TOC, TN values observed in the first ten days are qualitatively in agreement with the relatively low mass loss observed in saline solution. Figure 5 C shows the atomic C/N ratio values calculated from the TOC, TN measurements for each coating, as well as the theoretical values obtained for each releasable group or polymer that could potentially be dissolved. According to the overall results, we can draw a more confident conclusion about the type of released material. For the coated net CN1, a low release of the outer coating P(VBCTMAM-co-AA20) is observed as indicated by the atomic C/N ratio (~11–14). In the case of CN2 net, a very low release is also observed. Though the atomic C/N ratio (~14–16) is somewhat underestimated, due possibly to the low TN values, the release of AmC 16 groups (C/N ratio = 19) is most probable in this case, since much higher values (>27) should be observed if the whole inner layer ((P(SSAmC 16 -co-GMA20)) or the whole coating was releasable. In the case of the CN3 net, on the other hand, a higher release is observed in this first cycle, as the TOC and TN values are higher, while, according to the C/N value (~2), PHMG is immediately released. These values are due to the excess of PHMG contained in the PAA/PHMG outer layer mixture, which apparently is not retained through some special interaction with the other polymers and is quickly released. After 10 days the nets were transferred to a new aqueous 0.6 M NaCl solution, where they remained for a second 15-day cycle and the new TOC, TN and C/N measurements (after 25 days) are shown in Figure 6 A–C. During the second cycle of the release study, even lower TOC and TN values were observed in all coatings, demonstrating the continued slow release of biocidal materials, leading to controlled release systems. Though determination uncertainties are important as a consequence of the marginal TN values measured, it is clear that only the P(VBCTMAM-co-AA20) copolymer is released from the CN1 in the first 50 h, and then the biocidal bottom layer copolymer, P(SSAmC 16 -co-GMA20) (C/N~27), begins to be released as well. Moreover, the CN2 net shows very low release of the biocide AmC 16 (C/N~19), while the CN3 net this time shows low release which is probably due to both biocides, PHMG and AmC 16 . In conclusion, the release study through gravimetry and TOC/TN measurements revealed a promising behavior of the coated nets under aqueous conditions, especially under simulated seawater conditions (NaCl 0.6 M). These results are a guide for the expected antifouling effect of the coatings when applied to fish farming nets and immersed in seawater. 2.4. Action of New Polymeric Coatings against Biofouling 2.4.1. Testing under Accelerated Biofouling Conditions The next step was to test the coated nets with the above-mentioned stable polymeric systems for their anti-fouling performance as a function of time. Initially, tests were carried out on a laboratory scale and under accelerated biofouling conditions. In this line, the nets presented in Table 2 were immersed in aquariums with seawater to which nutrients were added to enhance the growth of bio-foulants (algae, etc.). Knowing that the growth cycle of biofouling takes six to seven days to be completed, the seawater-nutrient solution was replenished three times over a 24-day period, when the experiment was terminated. As shown in Figure 7 , the uncoated net presented biofouling from the very first days. Specifically, on day 11, the net was full of fouling, while the surrounding water exhibited a dark green color and high turbidity. In contrast, nets CN1 (P(SSAmC 16 -co-GMA20)/P(VBCTMAM-co-AA20)), CN2 (P(SSAmC 16 -co-GMA20)/PAA) and CN3 (P(SSAmC 16 -co-GMA20)/PAA/PHMG) showed successful resistance to biofouling up to day 20 despite the three water-nutrient replenishments. At the end of the experiment (day 24), a dark green alga was observed on the surface of the CN1 net coated with P(SSAmC 16 -co-GMA20)/P(VBCTMAM-co-AA20), suggesting that the fouling organisms may still be active on the surface of the net, probably due to the high percentage of immobilized biocide groups on the coating that do not allow detachment of the inactive organisms. In this way, the accumulation of dead organisms on the surface acts as a secondary pollution-source which maintains the biofilm formation. On the other hand, in the cases of CN2 (P(SSAmC 16 -co-GMA20)/PAA) and CN3 (P(SSAmC 16 -co-GMA20)/PAA/PHMG) nets, the brownish color of alga indicates that the action of the hydrophilic PAA coating as a resistant-surface for the organisms, in synergy with the gradual release of the biocidal AmC 16 into the water, exhibits a more effective activity against biofouling. The same phenomenon is also observed for the PAA/PHMG coating, but without showing any improvement in the overall activity. To avoid the harmful effects of biofouling development, the nets used in aquaculture are periodically cleaned with high-pressure washers. Following this line, the nets used in this study after their removal from the aquariums were washed with high-pressure water in the laboratory. Photographs of the nets before and after washing are shown in Figure 8 . As can be seen, the cleaning efficiency of the uncoated net and the coated CN1 (P(SSAmC 16 -co-GMA20)/P(VBCTMAM-co-AA20)) net was not sufficient, in contrast to the other two coated nets CN2 (P(SSAmC 16 -co-GMA20)/PAA) and CN3 (P(SSAmC 16 -co-GMA20)/PAA/PHMG). More specifically, contaminants were easily removed from these nets, which showed significant difference before and after washing. From these results we can conclude that the coated nets CN2 and CN3 showed very weak fouling adhesion making them good candidates for possible use as a coating in aquaculture nets that will improve their life expectancy in marine applications. A crucial question for the potential application is whether the nets can be repeatedly coated after use. To check this point, the net CN1, after application under accelerated laboratory conditions and washing with high pressure water, was re-coated using the same procedure. From the weight change a significant loading was verified, while the presence of the materials of both layers was revealed through ATR-FTIR characterization ( Figure S3 ). 2.4.2. Testing under Real Field Conditions The final aim was to test the effectiveness of the new materials under real conditions, in the field. Therefore, the aforementioned polymeric coatings were applied to larger nets (30 × 20 cm) applying the methodology described earlier. To achieve a longer duration of action, replicate nets were coated for each system were coated twice (four-layer coatings) to attain a higher percentage of total loading ( Table 4 ). Thus, for each system studied, two nets with different percentages of coating (two-layer and four-layer coating) and one uncoated net were used, which were fitted in a special circular arrangement placed in an aquaculture cage and immersed in the sea for 23 days. As shown in Figure 9 , the uncoated nets started to exhibit fouling from day 7 of immersion, while the coated nets appeared to be more resistant. However, the difference between coated and uncoated nets was clear at the end of the field test (day 23), where the uncoated nets were completely covered by fouling organisms, while the coated nets still had intact, clean areas. Regarding the activity of the studied coatings, as can be observed, the coated nets showed equally high performance in all cases until the end of the test. More specifically, the nets were free of contaminants until day 16, while on day 23 they were partially coated, with nets S13b and S14a showing a slightly higher clean surface area. This observation is consistent with the results of the accelerated experiment conducted in the laboratory ( Figure 7 ). Furthermore, it is clear that the increased loading percentage enhanced the efficiency of the respective polymer system. In conclusion, the combination of organo-soluble P(SSAmC 16 -co-GMAx) copolymers with the water-soluble P(VBCTMAM-co-AAx) copolymers or water-soluble PAA/PHMG polymers by using the L-b-L coating concept, resulted in coatings with high antifouling activity, which may be owed to three reasons. First, the cationic releasable compounds (SSAmC 16 , PHMG) in the polymeric material impart a killing activity via biocide-release. Second, the cationic immobilized compounds (VBCTMAM) contribute through a contact-kill action. Third, the outer layer consisting of PAA acts as a fouling-resistant surface for fouling organisms due to its increased hydrophilicity and low surface charge [ 37 ]. According to the above results, the present study broadens the research on multiple polyelectrolyte coatings towards the formation of highly active anti-fouling materials [ 38 , 39 , 40 ]. Finally, as it may be seen from the photographs of Figure 9 , the growth of the pollutant microorganisms started from the cages and transferred to the nets due to the very close distance between them. Bearing in mind that the experiment was conducted during the summer season where there is high bioaccumulation, and that the growth of microorganisms is a dynamic phenomenon strongly influenced by environmental conditions, the movement of cage and nets as well as the presence of fish in the cage, we can conclude that the observed activity of the coated nets in this study is quite encouraging."
} | 7,246 |
37102922 | PMC10137362 | pmc | 5,757 | {
"abstract": "Bio-based polymers are attracting great interest due to their potential for several applications in place of conventional polymers. In the field of electrochemical devices, the electrolyte is a fundamental element that determines their performance, and polymers represent good candidates for developing solid-state and gel-based electrolytes toward the development of full-solid-state devices. In this context, the fabrication and characterization of uncrosslinked and physically cross-linked collagen membranes are reported to test their potential as a polymeric matrix for the development of a gel electrolyte. The evaluation of the membrane’s stability in water and aqueous electrolyte and the mechanical characterization demonstrated that cross-linked samples showed a good compromise in terms of water absorption capability and resistance. The optical characteristics and the ionic conductivity of the cross-linked membrane, after overnight dipping in sulfuric acid solution, demonstrated the potential of the reported membrane as an electrolyte for electrochromic devices. As proof of concept, an electrochromic device was fabricated by sandwiching the membrane (after sulfuric acid dipping) between a glass/ITO/PEDOT:PSS substrate and a glass/ITO/SnO 2 substrate. The results in terms of optical modulation and kinetic performance of such a device demonstrated that the reported cross-linked collagen membrane could represent a valid candidate as a water-based gel and bio-based electrolyte for full-solid-state electrochromic devices.",
"conclusion": "3. Conclusions Bio-based polymers represent a more sustainable and eco-friendly alternative to conventional polymers in various fields of applications. In this work, the possibility of employing collagen membranes as hydrogel-polymeric electrolytes in full-solid-state electrochemical devices has been investigated. Indeed, collagen can absorb large amounts of water, and thus it could be employed as a polymeric matrix that can retain large quantities of aqueous electrolyte and can be used as a self-standing electrolyte membrane. Uncrosslinked and cross-linked collagen membranes were obtained by air-drying, and a detailed FTIR characterization was performed to evaluate the effectiveness of the cross-linking and the preservation of typical triple-helical conformation of collagen after cross-linking. The stability and the mechanical properties of the membranes after impregnation in water and aqueous electrolyte were tested; the resulting measures demonstrated that cross-linked membranes could absorb the aqueous electrolyte and preserve their integrity and mechanical characteristics. The ionic conductivity of the cross-linked membranes after impregnation with the aqueous electrolyte (1%, 2%, and 5% v / v H 2 SO 4 ) was measured by EIS spectroscopy, showing a high ionic-conductivity value, close to 10 −4 S/cm for all the tested concentrations. Considering these results and the good optical characteristic of the collagen membranes in terms of transparency and low haze, we tested the H 2 SO 4 -impregnated membrane as an electrolyte in an electrochromic device. The glass/ITO/PEDOT:PSS/impregnated collagen/SnO 2 /ITO/glass device showed a maximum ΔT of about 35% at about 650 nm (at −3 V) and a very fast kinetic with τc equal to 3 s and τb equal to 2 s. The good performances of the fabricated electrochromic device demonstrated that collagen represents a good candidate for the future development of greener full-solid-state electrochemical devices.",
"introduction": "1. Introduction One of the most important components in an electrochemical device is the electrolyte since it is responsible for ionic transportation within the device, thus influencing its performance. Traditional liquid electrolytes are not suitable for the evolution of electrochemical devices since they show several disadvantages, such as the employment of corrosive and inflammable solvents, leakage and evaporation problems, and unfit for flexible devices [ 1 ]. For these reasons, there is a growing interest in the development of new electrolytes suitable for full-solid-state devices since they allow at the same time to improve device safety, simplify the fabrication process, and achieve flexible devices [ 1 ]. In this context, polymers represent promising candidates [ 2 ]. In particular, gel polymer electrolytes contain liquid electrolytes immobilized in a host made up of one or more polymer matrices [ 2 , 3 , 4 ]. Their features are excellent mechanical integrity, film-forming ability, easy processability, and higher ionic conductivity compared to solid-state polymer electrolytes [ 2 , 3 , 5 ]. Among the liquid electrolytes that can be immobilized into a polymeric matrix, aqueous solutions represent an interesting alternative for the development of safe and non-toxic gel polymer electrolytes. Hydrogels are three-dimensional (3D) polymeric networks composed of hydrophilic-functional groups allowing them to absorb and retain a large amount of water without dissolving [ 6 ]. Therefore, hydrogels can hold a large amount of aqueous ionic solution providing good ionic conductivity, keeping at the same time good mechanical stability originating from the polymeric network [ 6 ]. Different synthetic polymers have already been employed to obtain hydrogels, but natural polymers have gained attention as promising substitutes for traditional synthetic polymers thanks to their advantageous properties, such as biodegradability, biocompatibility, non-toxicity, sustainability, etc. [ 7 , 8 ]. Indeed, global warming, price fluctuations, reduced oil resources, and pollution are just some of the factors that are pushing toward increasing use of eco-friendly biomaterials. Natural materials directly extracted from biomass resources, such as polysaccharides, proteins, and lipids, represent one of the most relevant categories. In particular, polysaccharides are the most studied bio-polymeric electrolytes for their large availability, abundance, and accessibility. For all these reasons, they were adopted in the fabrication of eco-friendly devices [ 9 , 10 ]. Among proteins, soybean protein, gelatin, and collagen were used as matrices for gel polymeric electrolytes aiming for more sustainable solid-state electrochemical devices [ 11 , 12 , 13 , 14 ]. In particular, gel polymeric electrolytes were obtained by saturating a membrane of soybean protein with an aqueous solution of Li 2 SO 4, and such electrolytes were employed for the fabrication of solid-state electric double-layer capacitors [ 11 ]. Similarly, a gelatine-based gel electrolyte consisting of porcine skin-derived gelatine and sodium chloride was employed in screen-printed and stencil-printed supercapacitors [ 12 ]. In this context, type I collagen is the most abundant structural protein of vertebrates’ connective tissue, where it provides strength and structural stability, performing regulatory functions [ 15 , 16 ]. Its unique fingerprint consists of three left-handed polyproline-II helices of about 1000 amino acid residues that assemble in a right-handed triple helix [ 16 , 17 , 18 ]. Each polyproline-II chain is characterized by the replication of the (Gly-X-Y) n triplet, where Gly is glycine, and the X and Y positions are usually occupied by proline and hydroxyproline, respectively [ 19 ]. In this recurrence, glycine plays a fundamental role in the three α helices packing, while proline and hydroxyproline are crucial elements in the triple-helical-structure stabilization [ 20 ]. Thanks to its advantageous intrinsic properties, such as biodegradability, biocompatibility, bioactivity, easy manufacturing, and customizable properties, collagen is one of the most used biomaterials for healthcare applications [ 20 , 21 , 22 , 23 , 24 , 25 ]. Collagen harvesting mostly relies on its extraction from animal tissues by-products of the food industry (i.e., skin, tendon, scales, cartilage, bone, and so on) [ 21 , 26 ]. The recovery and valorization of waste materials make collagen eco-friendlier and more cost-effective than other approaches [ 27 ]. Recently, aside from applications in the biomedical, pharmaceutical, cosmetic, and food fields, collagen started to be investigated in the field of energy devices as a sustainable source for nanoporous carbon materials exploitable as batteries anode [ 28 ]. Regarding the application of collagen as a polymeric network for hydrogel-based electrolytes, a collagen fiber membrane infiltrated with Na 2 SO 4 aqueous solution showed an ionic conductivity of about 9 × 10 −3 S/cm and it was used as an electrolyte in an electrical double-layer capacitor [ 13 ]. Collagen was also employed as an electrolyte of fuel cells in humidified conditions [ 14 ]. Indeed, Matsuo et al. demonstrated that the proton conductivity of the collagen membrane goes from 1 × 10 −5 to 4 × 10 −3 S/cm when the relative humidity is increased from 53% to 100%. Such an effect is due to the formation of water bridges bonded with the collagen peptide chains, inducing an increase in proton conductivity compared to a dry-collagen membrane. Starting from such a background, in this work, uncrosslinked and cross-linked collagen membranes derived from equine tendons have been investigated as alternative gel-like polymeric electrolytes for the fabrication of electrochromic devices. Because of produced uncrosslinked collagen membranes’ low degradation resistance in acid environments, a physical cross-linking method was chosen and applied among cross-linking strategies to enhance collagen substrate properties without reducing their eco-friendly character and their biocompatibility profile. In particular, a heat-mediated cross-linking method was selected to induce the formation of amide and ester bonds via condensation reactions [ 29 ]. The swelling capability and the mechanical properties of the two kinds of membranes were evaluated, and the cross-linked collagen proved to be the best candidate for the preparation of the gel electrolyte. The ionic conductivity and the optical properties of the membrane after impregnation with H 2 SO 4 aqueous solution were measured, demonstrating its suitability for the fabrication of electrochromic devices thanks to its good transparency and excellent conductivity. An electrochromic device was fabricated exploiting such a polymeric gel electrolyte, showing good modulation properties and excellent kinetic properties. These results demonstrate the potential of eco-friendly and biodegradable collagen-based membranes as new and effective hydrogel-like electrolytes for the fabrication of full-solid-state electrochemical devices and, in particular, electrochromic devices.",
"discussion": "2. Results and Discussion Uncrosslinked (UN) and cross-linked (DHT) collagen membranes were prepared by air-drying; their IR spectra are reported in Figure 1 . The peaks of amide I, amide II, and amide III of type I collagen were detected, as well as amide A and B contributions [ 30 , 31 ]. The amide I (1621–1635 cm −1 ) band is associated with C=O hydrogen-bonded stretching, the amide II (1535–1548 cm −1 ) peak is associated with C-N stretching and N-H in-plane bending from amide linkages, and the amide III (1220–1240 cm −1 ) to the N-H bending [ 32 , 33 , 34 , 35 , 36 ]. The peaks at 1400 and 1340 cm −1 were assigned to the wagging and deformation modes of -CH 3 and -CH 2 of the glycine backbone, besides the proline and hydroxyproline sides [ 32 ]. The contributions at approximately 1080 cm −1 and 1030 cm −1 were attributed to the stretching vibration of C-O-C and C-O, respectively [ 34 ]. Lastly, contributions at about 3400–3500 cm −1 and 3000–3080 cm −1 could be observed and attributable to the amide A and amide B, which are ascribed to the N-H stretching coupled with intramolecular H-bond and N-H bend, respectively [ 37 ]. The presence of contributions attributable to type I collagen confirmed that the process employed for collagen-membranes production did not significantly affect the material structural conformation. The peaks of amide I, II, II, A, and B were found to be almost the same in uncrosslinked and cross-linked samples, with a slight shift to lower frequencies for cross-linked samples. In particular, the shift to lower frequencies in the amide I and III of cross-linked samples prompted the involvement of the -C=O and -NH groups in new bonding interactions and, thus, the effectiveness of the applied physical cross-linking treatment [ 31 ]. In Figure 1 B, a representation of the chemical structure after the cross-linking process is shown, and the new bonds are highlighted in red. To investigate in-depth collagen secondary structure in the matrices after the production process, five contributions were identified from amide I deconvolution ( Figure 2 ). In particular, β-sheet (peak center: 1610–1642 cm −1 ), random coil (peak center: 1642–1650 cm −1 ), α-helix (peak center: 1650–1660 cm −1 ), β-turn (peak center 1660–1680 cm −1 ), and β-antiparallel (peak center 1680–1700 cm –1 ) components were detected ( Table 1 ) [ 38 , 39 , 40 ]. The increase of the β-sheets component in cross-linked samples could be explained by assuming the formation of bonds among collagen molecules that are laterally associated that spectroscopically mimic β-sheet structures [ 41 , 42 ]. Moreover, the integrity of the collagen triple-helical unit was evaluated by the amide III/A 1450 ratio [ 43 ]. A ratio equal to or higher than unity confirmed its conformational structure preservation after the production process (uncrosslinked amide III/A 1450 = 2.1; cross-linked amide III/A 1450 = 1.9). Additionally, the –OH stretching band (range 4000–3000 cm −1 ) corresponding to the amide A was analyzed and deconvoluted into four components ( Figure 3 ) whose frequencies were related to different O-H bond lengths ( Table 2 ), which in turn were correlated to the hydrogen bond network around the protein. Differences in hydrogen bond distances could provide information about cross-linking due to protein modifications. Thus, following the procedure of the second derivative analysis, four Gaussian components were identified, corresponding to the four classes of water molecules that can be bound to the protein. Each of them has different vibrational energies and a single average H-bond distance (H···OH length) of 0.31, 0.29, 0.2,8, and 0.25 nm, respectively [ 41 , 44 ]. The defined peaks were found at about 3670, 3460, 3250, and 3110 cm −1 , according to the literature [ 41 , 45 ]. The sub-band peaking at about 3590–3690 cm −1 region corresponds to H-bond distances, characteristic of a vapor-like state, attributed to protein non-H-bonded or weakly H-bonded O–H groups [ 41 , 46 ]. As collagen films were in a dehydrated state, about 4.4% of non-H-bonded O–H groups were found in UN films, while no non-H-bonded O–H groups contributions were found in DHT films. The two-component bands peaking at 3480–3490 cm −1 and 3240–3250 cm −1 attributed to water molecules coordinated by two or three H-bonds corresponding to water molecules that form inter- or intra-molecular bridges. Indeed, they were found to constitute about 18% (for ν2) and 50% (for ν3) of the total water content for both UN and DHT. As suggested by Bridelli et al., the corresponding H-bond distances suggested that they could be attributed to the hydrogen bonding of –C=O groups belonging to glycine (d(C=O···W) = 0.295 nm) and hydroxyproline (Hyp) (d(C=O···W) = 0.284 nm) hydroxyl residues [ 41 ]. Lastly, the component at about 3110 cm −1 , which was attributed to water molecules hydrogen-bonded to polar and charged groups exposed to the macromolecule surface, was found to be higher for DHT (about 33%) than UN (26%), suggesting the presence of a high number of hydrogen bonds in DHT films. Moreover, a variation was noticed in the band-intensities ratio of –CH 2 and –CH 3 (uncrosslinked CH 2 /CH 3 ratio: 1.6; cross-linked CH 2 /CH 3 ratio: 6.0); the increase in the band intensity assigned to the methylene groups (2925 cm −1 ) compared to the methyl groups one (2950 cm −1 ) suggested that cross-linking reaction occurred in cross-linked collagen matrices [ 47 ]. Considering the application of the collagen film as a hydrogel-based electrolyte, their stability in water and aqueous electrolytes and their water/aqueous electrolyte-absorbing capacity was evaluated. Sulfuric acid was selected as an aqueous electrolyte because of its high ionic conductivity, and three different concentrations were tested. Cross-linked and uncrosslinked collagen membranes were soaked overnight in water and sulfuric acid (1%, 2%, 5% v / v ). Uncrosslinked collagen membrane in water showed a very high water-absorption capability, clearly visible from the significant swelling-degree ratio (close to 10) and thickness increase (dry uncrosslinked film thickness: 55 ± 6 µm; uncrosslinked film thickness in H 2 SO 4 : 856 ± 24 µm) ( Figure 4 A). Non-significant differences in thickness variation related to sulfuric acid concentration were detected ( p > 0.5). However, the uncrosslinked matrix turned out not to be stable in sulfuric acid solutions since it lost its film-like structures and almost completely melted in 1–5% v / v sulfuric acid. Vice versa, a lower water-absorption capability and a reduced thickness increase were observed for cross-linked membranes (dry cross-linked film thickness: 38 ± 5 µm; cross-linked film thickness in H 2 SO 4 : 242 ± 12 µm; swelling-degree ratio close to 3). Again, non-significant differences in thickness variation related to sulfuric acid concentration were detected ( p > 0.5). Despite the reduced ability to retain H 2 SO 4 , cross-linked films, unlike non-crosslinked ones, turned out to be able to withstand their structure even after soaking in 5% ( v / v ) sulfuric acid. The swelling ratio of the different membranes soaked in different solutions is reported in Figure 4 B. Tensile tests were performed to characterize uncrosslinked and cross-linked collagen matrices, in terms of mechanical properties, after overnight incubation in water and 1–5% ( v / v ) H 2 SO 4 . The mechanical properties of uncrosslinked films in H 2 SO 4 were not assessed because of its almost complete dissolution in sulfuric acid that did not allow for handling and clamping it in a tensile-test-machine tool. As expected, the stress-strain curves of matrices were all characterized by a linear elastic region, followed by a non-elastic region and a rupture region ( Figure 5 ) [ 31 , 40 ]. The constitutive bond of the uncrosslinked matrix was found to be statistically different from that of the cross-linked matrices in terms of E and εr ( Table 3 ). In particular, the elastic modulus of the uncrosslinked matrix (1.2 ± 0.3 MPa) proved to be lower than that of the cross-linked matrices (2.7 ± 0.5 MPa), suggesting a matrix stiffening due to the applied physical cross-linking treatment ( p = 0.01). Moreover, while σ max was found to be almost the same for both uncrosslinked and cross-linked matrices ( p = 0.2), the εr value was significantly reduced by the cross-linking treatment (UN-H 2 O treatment: εr = 131 ± 7%; DHT-H 2 O treatment: εr = 67 ± 7%; p = 0.0003). As regards the presence of the aqueous electrolyte, it was verified to significantly influence the E value as well as the εr value. Indeed, the sulfuric acid increased cross-linked matrices’ E values and reduced their εr value. In particular, the sulfuric acid concentration was proven to be inversely proportional to E values and directly proportional to εr values ( p < 0.01). In other words, the increase in sulfuric acid concentration was responsible for the cross-linked matrix mechanical properties lost. Representative snapshots of uncrosslinked and cross-linked collagen matrices at ε% = 0% and at their maximum deformation before the break in the presence of water and aqueous electrolytes are reported in Figure 6 . Considering the reported results, cross-linked membrane proved to be a good candidate to obtain a self-standing hydrogel-like electrolyte that can be easily integrated into a multilayer device; for such reasons, it was further investigated. In particular, to integrate the reported hydrogel electrolyte in an electrochromic device, optical properties and ionic conductivity should be evaluated. Therefore, the ionic conductivity of the cross-linked membrane impregnated with water at different H 2 SO 4 concentrations (1%, 2%, and 5% v / v ) was evaluated through electrochemical impedance spectroscopy. Nyquist plots of electrochemical impedance spectra of the different membranes, measured at 0 V, have been reported in Figure 7 A. The electrolyte ohmic resistance is obtained by the intercept at the real impedance axis of high frequency. The ionic conductivity (see equation in experimental details) of M-Water, M-H 2 SO 4 (1%), M-H 2 SO 4 (2%), and M-H 2 SO 4 (5%) hydrogel electrolytes was found to be 0.004, 0.12, 0.16, and 0.15 mS/cm, respectively. All H 2 SO 4 -impregnated membranes showed higher ionic conductivity than water-based hydrogel. The ionic conductivity of the membrane impregnated with pure water can be attributed to the intrinsic conductivity of the wet collagen membrane. In particular, it has been largely reported that ionic conductivity in collagen is strongly affected by its hydration state [ 48 ]. The water molecules that are absorbed by the membrane interact with the N-H, O-H, and C=O groups of the peptide chains through hydrogen bonds. When a small quantity of water is absorbed, the water molecules are isolated from each other, but when the number of absorbed water molecules increases, firstly, intra-helix bridges and after, inter-helix bridges form, thus allowing the formation of a water-bridge network into the collagen matrix. The water-bridge network allows the proton conduction into the film through the Grotthuss mechanism. Therefore, the ionic conductivity of the pure water-impregnated membrane is due to the transfer of protons which are intrinsically present in the polymeric matrix. After impregnation with H 2 SO 4 solutions, a strong improvement of proton conductivity is visible, which is imputable to the increase in the number of membrane protons that can be transferred. As a result, the obtained ionic-conductivity values for the H 2 SO 4 -impregnated membranes are suitable for application as electrolytes in electrochemical devices. In particular, considering the ionic conductivity and the mechanical characterization, we evaluated that the cross-linked membrane impregnated with H 2 SO 4 1% ( v / v ) represents the best option. Regarding optical properties, the total transmittance and the haze of the wetted membrane (with H 2 SO 4 1% v / v ) were measured in the visible range of the electromagnetic spectrum; the results have been reported in Figure 7 B. An excellent global transmittance was measured, with a mean transmittance value higher than 90% in the range of 400–800 nm. Concerning haze, it is used to measure the milky or cloudy appearance that is due to the scattering of light in transparent material. The lower the haze, the higher the clarity of the film, a desirable feature for application in electrochromic devices, where high transparency/clarity is required in the bleached state. The mean haze value of the cross-linked membrane is about 15% in the range of 400–800 nm, which is suitable for the mentioned application. Considering the promising characteristics of the cross-linked membrane, it was tested as an electrolyte in an electrochromic device. The cross-linked collagen membrane impregnated in H 2 SO 4 1% ( v / v ) was sandwiched between glass/ITO/PEDOT:PSS and glass/ITO/SnO 2 ( Figure 8 A). In this structure, PEDOT:PSS acts as an electrochromic material while SnO 2 is the ion storage layer; both materials were deposited by solution processes [ 4 ]. In Figure 8 D, the electromagnetic spectra in the visible range of this device at different applied voltages have been reported. In the bleached state, the device shows good transparency with a mean transmittance higher than 70% in the 400–800 nm range. When a negative voltage is applied, a reduction of the transmittance is visible, and an absorption band, peaked at about 650 nm, appears [ 49 ]. Such absorption band is typical of PEDOT:PSS, and it is imputable to the intercalation of H + into the PEDOT:PSS thin film. The maximum ΔT is about 35% at about 650 nm (at −3V), and such transmittance variation can also be appreciated in Figure 8 B,C, where the device is shown in the bleached and colored state. The kinetics of the device was also evaluated by measuring the transmittance at 650 nm, applying a square-wave potential signal from −3V to +1V and back. The results are reported in Figure 8 E. The device shows very fast coloration and bleaching kinetics with τc (coloration time) equal to 3 s and τb (bleaching time) equal to 2 s, which confirm the good performance of the selected electrochromic material but also the excellent ionic-conduction properties of the collagen membrane electrolyte."
} | 6,280 |
32703952 | PMC7378083 | pmc | 5,758 | {
"abstract": "Empirical evidence for the response of soil carbon cycling to the combined effects of warming, drought and diversity loss is scarce. Microbial carbon use efficiency (CUE) plays a central role in regulating the flow of carbon through soil, yet how biotic and abiotic factors interact to drive it remains unclear. Here, we combine distinct community inocula (a biotic factor) with different temperature and moisture conditions (abiotic factors) to manipulate microbial diversity and community structure within a model soil. While community composition and diversity are the strongest predictors of CUE, abiotic factors modulated the relationship between diversity and CUE, with CUE being positively correlated with bacterial diversity only under high moisture. Altogether these results indicate that the diversity × ecosystem-function relationship can be impaired under non-favorable conditions in soils, and that to understand changes in soil C cycling we need to account for the multiple facets of global changes.",
"conclusion": "Conclusion To face climate change we must understand how global environmental changes will impact soil C cycling. To our knowledge, this is the first study that actively manipulated microbial communities to explore how biotic and abiotic components interact to drive CUE in a soil system. Our results highlight that shifts in microbial communities can change CUE, and that the positive effect of microbial diversity on CUE is neutralized under dry conditions.",
"introduction": "Introduction The provision of ecosystem functions is dually threatened by human-induced climate change 1 – 3 and biodiversity loss 4 , 5 . One such function threatened by these factors is the storage of organic carbon (C) in soils 6 – 9 , which is crucial for climate regulation 3 . This C stock is regulated in part by the rate and efficiency with which the microbes living within soil incorporate fresh plant inputs into their biomass and more stable components of soil organic matter 10 , 11 . Indeed, predictions of soil carbon stocks under warming are highly sensitive to the assumptions made about microbial carbon use efficiency (CUE) 7 , 12 – 14 , which is the fraction of C taken up by microbial cells and retained in biomass as opposed to being respired. CUE can be directly affected by global changes such as climate warming and shifts in soil moisture due to modifications in precipitation regimes 3 , 15 . Meanwhile, global changes are also driving shifts in the diversity and structure of microbial communities 16 , 17 . Understanding the drivers of CUE is crucial to determine the fate of C in the soil. However, it is uncertain how these direct and indirect impacts of global changes are driving CUE in soils. Factors such as temperature, moisture, microbial community structure, substrate quality, substrate availability, and soil physico-chemical properties are all likely to affect CUE 11 , 15 , 18 – 20 , but parsing out their relative importance in natural ecosystems remains a challenge 21 . Climate change is impacting soil temperature and water availability, which are known to directly influence microbial metabolism and can therefore impact CUE. Generally, elevated temperatures increase respiration more than growth, and therefore CUE tends to decrease with increasing temperature 18 . However, this decrease in CUE with temperature is not ubiquitous 22 , and has been observed to vary with substrate quality 20 . Our knowledge of the impact of soil water content specifically on CUE is limited to two studies 19 , 23 . Normally, soil microbial communities living in drier soils are expected to have higher metabolic costs due to osmoregulatory mechanisms 24 such as production of intracellular solutes 24 . Another response to drought is the production of extracellular polysaccharide (EPS), which might also imply in further costs 15 . In addition, low water availability can decrease substrate supply to microbial cells due to slow diffusion rates 25 resulting in a greater proportion of substrate allocated for maintenance metabolism and less available to growth. In either case, moisture limitation is expected to reduce CUE 15 . In addition to these direct effects of abiotic factors on CUE, temperature and moisture can also drive changes in microbial diversity 17 and community structure 26 , thus indirectly impacting CUE. The impact of diversity and community structure for microbial CUE remains unexplored. Positive relationships between diversity and soil functions have been observed, for example, for denitrification 27 – 29 and methanogenesis 30 , 31 , which are soil functions attributed to relatively restricted groups of microorganisms. Broader soil processes, such as C cycling, are considered to show extensive functional redundancy and be less subject to changes in diversity 32 , 33 . However, some studies have demonstrated that even broad processes within C cycling can show a positive relationship with diversity, as has been shown for respiration 8 , 34 and decomposition 35 . Moreover, community composition, rather than richness, can have a large impact on C cycling in soils 32 , 36 . CUE is known to differ between bacterial strains grown under identical conditions 22 . This suggests that communities with distinct members could have different community CUE. Moreover, it has been shown that abiotic factors (e.g., temperature) can modulate the relationship between diversity and growth in liquid cultures 2 , but it remains unclear how temperature and moisture could alter the relationship between diversity and growth efficiency in soils. In the context of global change, it is crucial to better understand how CUE is subject to changes in microbial diversity and community composition. The overall aim of this study is to provide empirical evidence for the response of CUE to the combined effects of temperature, moisture, diversity loss and distinct community compositions. We hypothesize that: (1) more diverse soil communities have higher CUE compared to less diverse soil communities; (2) an increase in temperature or decrease in soil moisture both reduce CUE and (3) abiotic conditions modulate the relationship between diversity and CUE. To overcome the challenges in determining the response of CUE to environmental factors that co-vary across space and time in natural soils, we develop a model soil (described in “Methods”) to control and manipulate the desired variables. We extract microbial communities from field soil collected from a temperate deciduous forest at the Harvard Forest Long-term Ecological Research (LTER) site. We manipulate the diversity of the extracted microbial community in one of three ways prior to inoculation: (1) diversity removal approach 37 with three diversity levels (non-diluted “D0,” 1000× diluted “D1,” and 100,000× diluted “D2”); (2) filter to 0.8 μm to exclude fungi and have predominantly bacteria (“bacteria only treatment,” or “B o n l y ”); and (3) enrichment for spore-forming organisms 38 (“SF”). These communities are inoculated into the model soil and incubated for 120 days under two different temperatures (15 and 25 °C), and two soil moistures (30 and 60% water holding capacity (WHC)), in a full factorial design totaling 200 samples (Fig. 1 ). At the end of the incubation, we measure CUE using the 18 O–H 2 O method 39 and assess bacterial and fungal diversity. We also measure three additional parameters that are proposed to affect CUE. Potential activity of the extracellular enzymatic pool is measured as a proxy for enzyme production 40 . The ratio of ITS to 16S rRNA is used to estimate the fungal:bacterial ratio 41 . Soil aggregation is measured as a proxy for substrate supply. For example, under low water content connectivity is greater within than between aggregates while under higher water content connectivity is increased more between aggregates than within an aggregate 42 . We find that bacterial phylogenetic diversity is positively correlated with CUE under high but not low soil moisture. Using path analysis to distinguish between direct and indirect drivers of CUE, we find that temperature and moisture indirectly influence CUE by altering microbial community structure, but it is the microbial components that directly explain CUE. Our work shows that the impact of diversity on CUE depends on soil moisture, indicating a dynamic interplay between the abiotic and biotic drivers of CUE. Fig. 1 Experimental design for manipulation of microbial diversity. The microbial diversity of a soil inoculum obtained from a temperate deciduous forest was manipulated by (1) sequential dilutions; (2) excluding fungi (“B only ”); and (3) selecting for spore-forming microorganisms (SF) ( a ). These inocula were added to artificial soil incubated for 120 days under two moisture (30 and 60% water holding capacity) and two temperature (15 and 25 °C) regimes ( b ). Images of model soils at the end of incubation ( c ). Average bacterial (gray) and fungal (white) richness (operational taxonomic units) for each diversity treatment ( d ). Significant differences between treatments within a microbial group (bacteria or fungi) are indicated by different letters (one-way ANOVA followed by Tukey HSD test, P < 0.05, df = 171, n = 176 for bacteria and for fungi df = 156, n = 161). In the boxplots, whiskers denote the minimum value or 1.5× interquartile range (whichever is more extreme), and box denotes interquartile range. The horizontal line denotes the median. Points indicate biological replicates, n = 40 and 40 for D0 and D1, 35 and 21 for D2, 38 and 40 for B only and 23 and 20 for SF for bacteria and fungi, respectively.",
"discussion": "Results and discussion Microbial community assembly in model soils Representatives of four bacterial and three fungal phyla grew in the model soil, with 1036 bacterial operational taxonomic units (OTUs) (100% identity), and 270 fungal OTUs (97% identity). The experimental manipulations successfully altered microbial diversity, with higher bacterial and fungal richness in the communities derived from the least diluted inocula (D0) compared to all the others (Fig. 1 and Supplementary Figs. 1 and 2 ). However, as previously observed 27 , the decrease in diversity was not commensurate with the degree of dilution: similar reductions in diversity were observed for dilutions of three (D1) and five (D2) orders of magnitude for both bacteria 77.4% (CI 95% = [63.4–90.6%]) vs. 80.9% (CI 95% = [71.6–106.2%]), and fungi 57.9% CI 95% = [45.4–70.5%]) vs. 43.9% (CI 95% = [19.5– 68.3%]). Filtering the D0 inoculum at 0.8 μm was overall successful at removing eukaryotic cells (“B only ”), as most samples showed zero fungal richness in this treatment (Fig. 1 and Supplementary Fig. 2 ). The presence of fungal sequences in some B only samples could suggest that some spores present in the initial soil resisted sterilization, although none of our uninoculated controls indicated growth (described in “Methods”). Finally, we successfully enriched communities in spore-formers (SF) by subjecting the same amount of inoculum soil as was used in D0 to dry heat (120 °C for 30 min) and phenol (1.5% for 1 h), as evidenced by the significantly higher relative abundance of Firmicutes in relation to all other treatments ( F = 338.3, df = 172, P < 0.0001). This vigorous pretreatment reduced the size of the inoculum to such a degree that no growth was observed in the low moisture treatment at 15 °C, and only four replicates showed growth at the high moisture treatment at 15 °C (Supplementary Figs. 1 and 2 ). We evaluated fungal and bacterial abundance at the end of the 120 day incubation by real-time quantitative PCR (qPCR) of a bacterial and a fungal house keeping gene. Bacterial and fungal gene copy number did not statistically differ between treatments except for the B only treatment which showed a significantly higher number of bacterial cells and lower fungal numbers than all other treatments (Supplementary Figs. 3 and 4 , respectively). We were able to generate communities with distinct diversity and community structure within the model soil. Richness was predominantly driven by the diversity manipulations, while community structure was responsive to soil moisture and temperature manipulations (Table 1 and Supplementary Figs. 5 – 8 ). The microbial richness in the model soil was lower than in natural soils 43 , but greater than previous studies aiming to evaluate the relationship between diversity and ecosystem processes 2 , 8 , 32 . While caution is needed when interpreting our findings in relation to natural soils, by using a soil-mimicking matrix we were able to begin to address how biotic and abiotic factors interact to drive microbial CUE in a spatially structured soil environment 2 , 8 . Empirical link between diversity and CUE We measured community CUE with the substrate independent 18 O–H 2 O method 39 under the same temperature and moisture conditions the samples had been incubated at for the previous four months. CUE varied across the range of values measured in other studies 18 (Supplementary Fig. 9 ). We hypothesized that CUE is positively correlated with diversity. Overall, we observed higher community CUE in the most diverse treatment (D0) compared with communities derived from the first (D1) and second dilutions (D2) (Supplementary Fig. 9 ), and lower CUE in B only compared to all other treatments. CUE represents the allocation of C to growth versus respiration, and to understand how it is affected by diversity, we separately evaluated growth and respiration responses. We observed no significant relationship between fungal diversity and CUE. Regarding bacteria, under high moisture conditions, growth rate increased faster with phylogenetic diversity (PD) (Fig. 2 a) than did respiration (Fig. 2 b), leading to a significant positive relationship between bacterial phylogenetic diversity and CUE (Fig. 2 c) in moist but not dry soils. Interestingly, CUE appeared to be constrained to high values in soils with high bacterial diversity (50–80%). This was confirmed by a break point analysis which showed a threshold at a PD value of 4.48 after which only high CUE values were observed ( t = 4.51, df = 86, R 2 = 0.28, P < 0.001). By contrast, the lower diversity samples showed the full range of CUE values suggesting that other factors such as community composition 22 and environmental factors are important in determining community CUE. While we report a positive relationship between diversity and CUE, other studies have evaluated the relationship between respiration and diversity, and found it to be positive 8 , 44 , neutral 44 , 45 , or negative 46 . Fewer studies have evaluated the relationship between diversity and growth rate and/or CUE 11 , 47 . A previous study found no relationship between microbial community composition based on phospholipid fatty acid (PLFA) analysis and CUE 47 , though PLFA has much lower resolution compared to sequencing for measuring community composition. The disparities in responses between diversity × C-cycle functions in different studies may be due to the distinct levels of diversity within different experiments as suggested in a recent review 32 , where it was concluded that a positive relationship between diversity and C-cycle functions is only consistently observed for low diversity communities (<10 species) 32 . However, we only observed no significant relationship between diversity and CUE after a PD value of 4.48. This indicates that in our simplified soil-mimicking system a moderate level of diversity was required to ensure high CUE. While the level of diversity observed in natural soils is higher than in our simplified system, the complexity of natural soils is also higher. For example, microorganisms living in natural soils experience a multitude of different substrates while in our simplified system we used a single substrate (cellobiose). This suggests that natural soils may require higher levels of diversity before the relationship between diversity-CUE saturates. Overall, these results suggest that by evaluating the diversity–function relationship in a soil-mimicking system, the level of diversity needed to saturate this relationship was higher compared to less complex environments such as found in liquid cultures 2 , 32 . Moreover, because we used a solid-matrix system, we were able to evaluate how the interplay between biotic and abiotic conditions shapes this component of C cycling. Fig. 2 Relationship between bacterial diversity and growth, respiration and CUE. Relationship between bacterial phylogenetic diversity (PD) and growth ( a ), respiration ( b ), and CUE ( c ). Microcosms incubated under 30 and 60% WHC are shown on the left and right panels, respectively. Monotonic relationships between the diversity metric and growth, respiration or CUE are evaluated with Spearman correlation and when significant are indicated with a blue line. We fit linear curves for growth and respiration. Biologically, CUE cannot be >100%, thus we fit a saturating curve to the CUE data. The vertical dashed line indicates the threshold at which there is no more significant relationship between bacterial diversity (PD) and CUE. Shaded area denotes 95% confidence intervals. There were 84 and 92 replicates for 30% and 60% WHC, respectively. A complementarity effect may explain why we observed a positive relationship between diversity and CUE under moist and not dry conditions. Complementarity effects arise from facilitation and niche differentiation that resulted from inter-species interactions increasing overall community productivity 48 . The influence of complementarity effect on function has been previously shown to vary with abiotic conditions 2 . In our study, we propose that the aqueous phase acted as a “gatekeeper” of microbial interactions 49 , 50 allowing species interactions and a complementary effect to emerge in the high moisture but not low moisture treatment. A mechanism that could explain complementarity interactions between species is sharing resources via cross-feeding. This could positively influence growth, if for example some microorganisms are producing amino acids from gluconeogenic substrates while others produce them from glycolytic substrates 51 . In this example, microorganisms could obtain amino acids produced by one of their neighbors under high moisture, resulting in a more efficient (less expensive) community growth. Moreover, as CUE is a compilation of growth and respiration, CUE is only positively impacted by diversity if diversity influences growth more than respiration (Fig. 2 and Supplementary Fig. 10 ). Changes in abiotic factors could also alter the nature of species interactions by changing resource uptake rates 25 and/or requirements 52 . We observed a positive relationship between soil aggregation and growth, respiration and CUE within the microcosms incubated at low water content (Supplementary Fig. 11 ). This could indicate that under low moisture conditions, microbial community growth was more limited to the resources present at the aggregate level and therefore correlated to aggregate size. On the contrary, in moist soils no relationship was observed between soil aggregation and growth, respiration or CUE suggesting that in these soils microorganisms were not limited to the resources present at the aggregate level. Thus, low water content may have limited the extent of possible inter-species complementarity interactions which could explain the absence of positive relationship between diversity and CUE in these soils. Alternatively, another possible mechanism is the additional costs due to desiccation stress 15 , which could have impaired the positive relationship found in high moisture samples. Thus, the impact of microbial diversity on CUE is contingent upon abiotic conditions. Temperature and moisture effects on CUE Temperature 15 , 18 and water content 19 , 23 drive CUE in soil, and in accordance with previous studies 18 , we measured lower CUE in microcosms incubated at higher temperatures ( t = 10.75, df = 172, P < 0.0001). This decrease in CUE for communities incubated at higher temperature was associated with an increase in estimated rrN copy number (Supplementary Fig. 12 ), a high rrN copy number has been related to a lower growth efficiency 53 , although this has not always been observed 22 . Moisture treatment showed no significant impact on either CUE ( t = −1.81, df = 161, P = 0.070) or rrN (Supplementary Fig. 12 ). Given that microbial communities differed among the long-term temperature and moisture conditions (Table 1 and Supplementary Figs. 5 – 8 ) we simultaneously ran an additional incubation to evaluate the direct physiological response to short-term changes in temperature and moisture. Table 1 Percentage of variance explained by the diversity manipulations (Div), moisture (Mois) and temperature (Temp) treatments, and their interactions for bacterial and fungal alpha diversity metrics and bacterial and fungal community structure. Parameter Div Mois Temp Div: Mois Div: Temp Mois: Temp Div:Mois: Temp Residuals Bacterial diversity (PD) 49.81*** 0.04 0.39 1.73 2.46 . 1.61* 1.88 42.08 (0.001) (0.714) (0.266) (0.239) (0.093) (0.025) (0.117) N/A Fungal diversity (Shannon) 33.37*** 0.11 0.05 2.68 2.27 1.12 0.95 59.44 (0.001) (0.596) (0.735) (0.192) (0.26) (0.116) (0.54) N/A Bacterial community structure 29.74*** 10.57*** 2.80*** 5.96*** 2.78* 0.53 1.41 46.22 (0.001) (0.001) (0.001) (0.001) (0.036) (0.163) (0.166) N/A Fungal community structure 21.81*** 2.92*** 1.52* 1.91 1.86 0.24 1.11 68.62 (0.001) (0.001) (0.015) (0.503) (0.49) (0.863) (0.741) N/A Bacterial and fungal community structures correspond to the first axis of a non-metric multidimensional scaling analysis (NMDS). The percentage of explained variance is obtained by dividing the group sum of squares by the total. Significant variables are indicated ( . P < 0.01, * P < 0.05, *** P < 0.0001), and the exact P values are shown below each explained variance. We measured CUE under all different abiotic combinations in a subset of microcosms (Fig. 3 a). For this we selected all D0 microcosms grown under low water content. While long-term abiotic conditions are known to alter microbial community structure 26 the short-term shift in these conditions should induce physiological changes independent of community shifts 15 . We hypothesized CUE would decrease with increasing temperature 15 , 18 . However, the short-term increase of 10 °C in temperature significantly increased mass-specific respiration (Fig. 3 b) and growth (Fig. 3 c) to a similar degree (188% and 176%, respectively), and did not resulted in a significant CUE response (Fig. 3 d) ( t = 0.36, df = 51, ns ). Garcia et al. showed that greater species richness is required to cope with warm temperatures to maintain growth 2 . Our D0 treatment was the most diverse, and these results suggest that its community was able to cope with the 10° increase in temperature without changing the respiration:growth relationship, and consequently without altering CUE. Fig. 3 Effect of short-term changes in temperature and moisture on respiration, growth and CUE. Microbial communities from the less diluted treatment (D0) grown at both temperatures (15 and 25 °C) and at 30% water holding capacity (WHC) were incubated under all combinations of water content and temperatures (experimental outline; a ). Influence of moisture and temperature shifts on respiratory quotient (RQ; b ), growth ( c ), and CUE ( d ) in the model soils. We used linear mixed effect models to evaluate the impact of short-term changes in abiotic conditions on respiration, growth and CUE with microcosm as the random effect ( n = 72, df = 51). Dashed boxplots represent the long-term soil incubation conditions. In the boxplots, whiskers denote the minimum value or 1.5× interquartile range (whichever is more extreme), and box denotes interquartile range. The horizontal line denotes the median. Points represent individual biological samples ( n = 8 for each incubation condition). Short-term changes in moisture had a stronger impact on CUE than temperature. An increase from 30 to 60% WHC elevated respiration and growth by 146% and 169%, respectively. The higher increase in growth compared to respiration after wetting the soil resulted in an 8% absolute increase in CUE (Fig. 3 d). We hypothesize that higher growth was possibly due to higher nutrient availability when increasing the water content. Another possible explanation for moisture being a strong driver of CUE is the difference in water potential experienced by the microbial community at low compared to high water content 54 (Supplementary Fig. 13 ). A previous study showed that CUE decreased with drought duration 23 , which could be associated with extra costs due to desiccation and the production of intracellular solutes or EPS, neither of which are captured by the DNA-based method of growth measurement used here. CUE as a function of interactions between biotic and abiotic drivers Model soil systems provide a unique platform for controlling specific biotic and abiotic components that play a major role governing soil processes, allowing the isolation of specific components from other confounding variables compared to natural soils. Thus, they can be used to increase understanding of major microbial ecology questions. We used structural equation modeling (SEM) to determine the degree to which the biotic components (fungal and bacterial diversity, community structure, fungal:bacterial ratio, potential extracellular enzyme activity and microbial-derived soil aggregation) mediate the influence of abiotic factors on CUE (Fig. 4 and Supplementary Figs. 14 – 16 ). The model path structure was based on the supposition that abiotic drivers (water content and temperature) drive CUE directly, but also indirectly by impacting the biotic drivers of CUE (Supplementary Fig. 14 ). We used the SEM to test the following hypotheses: (1) distinct community structure will result in different community CUE; (2) bacterial diversity positively impacts CUE; (3) the extracelullar enzymatic pool represents a cost to microbial growth efficiency and therefore has a negative effect on CUE; (4) the presence of fungi increases CUE, and (5) microbial-driven soil aggregation has a positive effect on CUE a proxy for substrate supply to cells. Overall, our model explained 30% of variance in CUE (Fig. 4 ). Fig. 4 Structural equation model showing the relative influence of soil abiotic and biotic factors on CUE. Significant paths are shown in blue if positive or in red if negative. Path width corresponds to degree of significance as shown in the lower left. The amount of variance explained by the model ( R 2 ) is shown for each response variable, and measures of overall model fit are shown in the lower right. Bacterial community structure: axis 1 of NMDS; Bacterial alpha diversity: bacterial phylogenetic diversity index; Fungi presence: presence/absence of fungi; F:B ratio: 16S rRNA gene copy number g −1 soil: ITS gene copy number g −1 soil; Enzyme activity/Biomass: maximum activity recorded for Betaglucosidase/microbial biomass carbon. CUE: carbon use efficiency; Global goodness-of-fit: Fisher's C. Exact P values for each path coefficient are reported in Supplementary Table 1 . Although temperature is commonly considered as a controlling variable for CUE 18 , our structural equation model indicates that temperature and moisture influenced CUE only indirectly, and instead acted through the biotic components (Fig. 4 ). Bacterial community structure and diversity were the strongest drivers of CUE. Bacterial diversity positively influenced CUE. However, we cannot make conclusions from the signal of the path coefficient between bacterial community structure and CUE because community structure is represented by the first axis of the non-metric multidimensional scaling (NMDS), which has an arbitrary direction. The other direct drivers of CUE were the presence of fungi, the extracelullar enzymatic activity, and the soil aggregation. The potential extracellular enzymatic activity/MBC was negatively related to CUE, supporting the idea that the enzyme poll represents a cost hindering growth efficiency as previously suggested 15 , 40 , 55 . We found fungal:bacterial ratio did not impact CUE in contrast to a previous study 41 . This difference might be due to the lower fungal:bacterial ratio in our artificial soil compared to natural soils 41 (Supplementary Fig. 17 ). Nonetheless, we observed a higher CUE in microcosms in which fungi were growing (“Fungi presence” component in the model). Accordingly, the B only treatment showed the lowest CUE values (Supplementary Fig. 9 ). However, fungal richness and community structure were not drivers of CUE (Supplementary Figs. 15 and 16 ), and we hypothesize that the 24 h of incubation for CUE measurements captured mainly bacterial growth as bacteria grow faster than fungi 56 . The positive effect of fungi presence on CUE indicates that some general fungal function is important for community growth efficiency (Fig. 4 and Supplementary Fig. 16 ). For instance, fungi could have provided sources of organic nitrogen to bacteria as evidenced by little to no N-acetylglucosaminidase (NAG) activity in B only microcosms (Supplementary Fig. 18 ). Thus, the impact of a microbial community on CUE can play out through a variety of mechanisms. CUE is a composite variable of respiration and growth, which will depend on microorganisms physiology and environmental conditions. It is important to highlight that a substantial fraction of CUE variation remains unexplained in the model, meaning that other factors are important and were not captured here. Altogether these results highlight how changes in the abiotic environment (e.g., temperature and moisture) interact with community composition and diversity loss to impact community CUE."
} | 7,531 |
39370430 | PMC11456600 | pmc | 5,759 | {
"abstract": "The mesophilic methanogenic archaeal model organism Methanosarcina mazei strain Gö1 is crucial for climate and environmental research due to its ability to produce methane. Here, we establish a Ribo-seq protocol for M. mazei strain Gö1 under two growth conditions (nitrogen sufficiency and limitation). The translation of 93 previously annotated and 314 unannotated small ORFs, coding for proteins ≤ 70 amino acids, is predicted with high confidence based on Ribo-seq data. LC-MS analysis validates the translation for 62 annotated small ORFs and 26 unannotated small ORFs. Epitope tagging followed by immunoblotting analysis confirms the translation of 13 out of 16 selected unannotated small ORFs. A comprehensive differential transcription and translation analysis reveals that 29 of 314 unannotated small ORFs are differentially regulated in response to nitrogen availability at the transcriptional and 49 at the translational level. A high number of reported small RNAs are emerging as dual-function RNAs, including sRNA 154 , the central regulatory small RNA of nitrogen metabolism. Several unannotated small ORFs are conserved in Methanosarcina species and overproducing several (small ORF encoded) small proteins suggests key physiological functions. Overall, the comprehensive analysis opens an avenue to elucidate the function(s) of multitudinous small proteins and dual-function RNAs in M. mazei .",
"introduction": "Introduction An unexpected complexity and density of genes within microbial genomes have been revealed by the steadily growing number of sequenced prokaryotic genomes in combination with high throughput OMICS profiling technologies such as next generation sequencing of DNA and RNA and optimized proteomics 1 , 2 . In addition, discovering genes encoding longer proteins or non-coding RNAs, genome-wide studies have also revealed a potential wealth of small proteins in all kingdoms of life. Small proteins are defined here as ribosomal synthesized proteins of ≤ 70 amino acids (aa) in length that are translated from small open reading frames (sORFs). They have been frequently overlooked in the past due to various technical and methodological difficulties e.g., in mass spectrometry 3 , 4 . Recent emerging tools and evidences demonstrated that small proteins in eukarya, bacteria, and viruses are implicated in important and diverse cellular functions, such as transport, sporulation, signal transduction, virulence, symbiosis or antiCRISPR activity 4 – 12 . Small proteins in the domain of archaea are underrepresented in recent studies, and only few small proteins are characterized until now 13 , 14 . This occurs to the smaller number of identified and cultivable archaea. Many archaea live in extreme habitats or in symbiosis with multicellular organisms. Consequently, due to the challenging experimental work and prediction tools optimized for bacteria, less is known about small proteins in archaea 14 . Methanosarcina mazei strain Gö1 is an archaeal model organism and represents a methylotrophic methanogenic archaeon 15 . It produces methane from carbon sources like CO 2 plus H 2 , methanol, methylamine or acetate 16 – 19 and can fix molecular nitrogen under nitrogen (N) limitation 20 . The molecular mechanisms of regulating nitrogen fixation in response to environmental conditions is well studied on transcriptional and post-transcriptional level 21 – 27 and the established genetic system allows to perform functional studies in vivo 28 – 30 . Using a differential RNA-seq approach to identify regulatory small (s)RNAs of M. mazei under N sufficient ( + N) and N limited (-N) condition, 44 sRNAs encoding for a putative small protein were already discovered in 2009 27 . Following genome-wide RNAseq studies under different growth conditions analysed by manual inspection or automated bioinformatics tools predicted a total of 1442 sRNAs encoding for small proteins ≤ 70 aa 14 . The first three identified small proteins of M. mazei were experimentally verified and quantified by LC-MS in 2015, demonstrating an increased amount of the small proteins sP36 and sP41 in the mid exponential phase under -N 31 . The first functional characterized small proteins from M. mazei are, sP26 and sp36. sP26 has been shown to interact with glutamine synthetase (GlnA 1 ) to stabilize its dodecameric structure under -N resulting in increased activity 32 ; sP36 is required for ammonium transporter (AmtB 1 ) regulation 33 . RNA-seq technologies have been applied to study prokaryotic transcriptomes and have revolutionized the discovery of novel transcripts, including regulatory sRNAs. While RNA-seq can provide evidence for sORF transcription and can greatly facilitate gene prediction, it cannot be used to directly distinguish between coding and non-coding transcripts, to provide sORF coordinates, or to predict protein abundance since mRNA expression does not necessarily correlate with protein levels due to post-transcriptional regulation. The development of Ribo-seq, which is based on deep-sequencing of ribosome protected footprints to determine genome-wide ribosome occupancy, is more amenable to detection of sORFs, and has provided direct evidence for translation of a wealth of novel unannotated small proteins in diverse organisms 34 – 37 , viruses 38 and more recently also in haloarchaea 39 , 40 . Consequently, Ribo-seq can be used to investigate whether some of the recently-revealed sRNAs in diverse prokaryotes may in fact be either short mRNAs encoding small proteins or act as dual-function RNAs, having both regulatory and coding potentials 41 – 43 . In this study, we developed and applied Ribo-seq coupled with RNA-seq on M. mazei strain Gö1 to map its global translatome with a particular focus on the small proteins. The use of MNase in our Ribo-seq protocol effectively cleaved mRNA regions that were not shielded by ribosomes, like untranslated regions, enabling distinction between translated and untranslated regions. Besides detecting the translation of 93 annotated sORFs, some of which are included in recent genome annotation updates, we also discovered 314 translated unannotated sORFs. The translation of 62 annotated and 26 unannotated sORF encoded small proteins was further validated via re-assessment of previous proteomics datasets (both top-down and bottom-up) using a protein database incorporating the unannotated small proteins. Importantly, the re-analysis of top-down datasets further validated many small proteins as being translated as full-length proteoforms, with several identified small proteins present as proteoforms with and without excision of the first methionine. Further targeted in vivo validation by western blot analysis confirmed translation of 13 out of 16 small proteins, thereby validating predictions derived from Ribo-seq data. Interestingly, differential analysis (-N vs. +N) inferred regulation for some of the identified translated sORFs, strongly suggesting important physiological functions. Overall, this Ribo-seq analysis provides a large catalogue of unannotated M. mazei sORFs (314), many of which are conserved in (methano)archaea, serving as a comprehensive resource to further investigate their functions in M. mazei e.g., their role in N metabolism or confirm and study their potential role as dual-function sRNAs.",
"discussion": "Discussion The intricate world of the small proteome ( < 71 aa), a largely uncharted component of the archaeal cellular apparatus, is brought to light in this study for the archaeon M. mazei Gö1. We present an integrated approach that combines Ribo-seq, computational predictions, filtering, and manual curation under two different growth conditions. This is complemented by LC-MS-based validation and additional in vivo protein validation, together providing an exhaustive portrayal of the small protein repertoire of M. mazei . Conducting comparative translatomics with Ribo-seq under two conditions ( + N and -N) in archaea is rare and has only recently been carried out in the first bacterium, E. coli , where the focus however was on the large proteome 61 . Ribo-seq has emerged as a potent method to study protein synthesis, and certainly boasts several advantages over proteomics 62 , 63 . However, it is not advisable to rely solely on Ribo-seq due to the inherent challenges, including complex ribosome behaviours, biases in library preparation, and differential ribosome occupancy 63 , 64 . Consequently, to enhance the reliability of Ribo-seq data, it’s essential to combine it with manual curation and filtration of both Ribo-seq and RNA-seq datasets, as well as with validation methods like MS based proteomics and in vivo validation of proteins, as used in recent studies 39 , 40 , 45 , 63 . For bioinformatic analysis, the HRIBO workflow 48 , was successfully used for the downstream analysis of Ribo-seq data. To identify robust unannotated sORF candidates we used DeepRibo prediction results instead of REPARATION, because DeepRibo predicted 37% more annotated sORFs than REPARATION (Fig. 3A ). A benchmarking study by Clauwaert, et al. 49 found that DeepRibo surpassed REPARATION and other methods in terms of accuracy and sensitivity in performance across seven datasets. This more robust performance aligns with our choice of using DeepRibo for predictions. As reported by Gelhausen, et al. 65 , DeepRibo is prone to a high rate of false positives, which we detect in our results as well, where DeepRibo predicted eight false positive out of 63 translated annotated sORFs and 57 false positives out of 255 predicted unannotated sORFs (Supplementary Data 4 ). Consequently, manual curation of the DeepRibo predictions with stringent cut-off filters based on control proteins (e.g. MM_RS08540) on prediction outputs was performed and provided more confident results in our study. The manual confirmation of translation status for the filtered DeepRibo predicted outputs for annotated ORFs, annotated sORFs and unannotated sORFs. One major challenge in the data processing is that the effectiveness of bioinformatic workflows like DeepRibo, REPARATION, deltaTE (for differential expression analysis), which are all optimized for bacterial genomes, might be limited on archaeal datasets. However, our optimized workflow using two conditions generated a dataset which provides an important resource for training these algorithms for archaeal data, suggesting a larger and more comprehensive M. mazei small proteome than currently understood. One of the common sources of gene misannotation is the incorrect determination of translation initiation sites. This issue can be specifically addressed with Ribo-seq 66 , since e.g. unconventional translation events are illuminated, enhancing the accuracy of gene annotations 67 . Ribo-seq revealed that 51 ORFs initiate translation downstream of the previously annotated start codons in M. mazei , suggesting N-terminal truncations in comparison to earlier annotations (Fig. 2B depicting one example). The remaining 61 ORFs out of 112 misannotated genes that were either divided into two smaller ORFs or showed a shift in their reading frames, which further emphasizes the role of Ribo-seq in accurately determining the coding potential of a genome. Moreover, the November 2022 Refseq annotation update of the M. mazei strain Gö1 genome validated our findings by incorporating corrections for 55% (61 out of 112) of the ORFs we identified as misannotated. This further reinforces the reliability and significance of our findings, emphasizing the value of our study in improving the accuracy of the genome annotation for M. mazei Gö1. Ribo-seq effectively detects global translation with high sensitivity, yet unlike its performance in eukaryotic systems it falls short of achieving codon resolution in prokaryotes (bacteria or archaea). This represents a major challenge in achieving codon resolution in prokaryotic genomes 40 , 45 , 47 , 68 , 69 . Our optimized Ribo-seq workflow for M. mazei (see Methods), resulted in the accurate MNase restriction of 5’ and 3’ regions of translated mRNAs (Figs. 1 C, D , S 9 ). Our Ribo-seq libraries, obtained under both +N and -N conditions, exhibit a wide range of footprint lengths spanning from 15 to 40 nucleotides, with a prevalent occurrence of footprints measuring 23-25 nucleotides (Supplementary Fig. 8 ). Higher TE values of annotated CDS in comparison to non-coding sRNAs and tRNAs (Fig. 3C ) and pronounced ribosome protection of up to 16 nt upstream and downstream of start and stop codons (Fig. 1 C, D ) clearly demonstrated the successful establishment of Ribo-seq for M. mazei . Similar validation metrics have recently been reported in the successful establishment of Ribo-seq for Sinorhizobium meliloti in one standard growth condition 45 . LC-MS analysis confirmed the translation of 62 annotated sORFs predicted by Ribo-seq (Supplementary Data 6 and 7 ). While Ribo-seq, complemented by manual curation and gene neighborhood filtering, identified 314 unannotated sORFs, 26 of which were substantiated at the protein level by LC-MS, suggesting that relying exclusively on Ribo-seq or LC-MS approach likely underestimates the total number of sORFs, again highlighting the importance of in vivo validation to reinforce predictions from either method (Fig. 8 ). Furthermore, we studied in vivo expression of sORFs by epitope-tagging the respective sORFs under the control of their native promoter. Here we obtained eight out of ten tested sORFs, from which three showed strong nitrogen regulation (see Fig. 6A ). Particularly the membrane-bound unannotated small proteins were not detected by LC-MS but validated via epitope tagging and immunoblotting analysis, e.g., sORF_05 (Fig. 6B ), which illustrates the analytical strength of our combinatorial approach for detecting small proteins. Considering that current genomic annotation algorithms exhibit bias towards longer ORFs, consequently leading to the systematic underrepresentation of sORFs in genomic databases 70 , this experimentally validated Ribo-seq dataset now allows for refining the small proteome of M. mazei as previously described in E. coli and Staphylococcus aureus 7 , 71 , 72 . Additionally, the M. mazei strain Gö1 Refseq annotation from November 2022 subsequently incorporated 34 out of the 314 unannotated sORFs predicted in our study using the Genbank2014 annotation. This further emphasizes the high quality of our data (Supplementary Data 5 ). Fig. 8 Overview of detection methods and validation for 314 unannotated sORFs after manual curation. 22 sORFs were predicted with NCBI ORF finder, 74 sORFs had a positive prediction score from Deepribo prediction, 34 sORFs are included in the actual Ref seq annotation (November 2022), 26 sORFs were validated by MS analysis and 13 out of 16 sORFs were confirmed by western blot analysis and two of them are not included in the unannotated sORFs list (internal to the ORFs). In addition to providing comprehensive genome-wide archaeal Ribo-seq analysis for a strictly anaerobically growing archaeon it is important to point out that this is a ribosome-binding map under two growth conditions ( + N and -N). Our Ribo-seq analysis demonstrated that, across +N and -N conditions, 1556 and 1430 ORFs were actively translated, representing 45% and 42% of the 3440 annotated ORFs, respectively. Overall, 1633 ORFs showed translation in either or both conditions, with 1353 ORFs common to both, 77 unique to -N, and 203 exclusive to +N conditions (Fig. 3A ). Moreover, the Ribo-seq analysis identified 93 and 95 translated annotated sORFs under +N and -N conditions, respectively, accounting for 51% and 52% of the total 184 sORFs coding for small proteins ( < 71 aa), with a combined total of 96 sORFs translated across conditions, 92 shared between both, three specific to -N, and one exclusive to +N condition (Supplementary Data 3 ). Although this study examines two conditions, the detection of 37% annotated sORFs in M. mazei aligns with previously observed ranges in S. meliloti (33%, Hadjeras, et al. 45 ), and in E. coli (40%, Weaver, et al. 73 , yet falls substantially below those found in Haloferax volcanii (65%, Hadjeras, et al. 40 ), and Salmonella (76%, Venturini, et al. 69 ) The discovery of 314 unannotated sORFs in M. mazei dramatically outnumbers those identified in other prokaryotes using exclusively Ribo-seq alone or combined with LC-MS, such as Salmonella (42, Venturini, et al. 69 ), E. coli , (68, Weaver, et al. 73 ), H. volcanii (48, Hadjeras, et al. 40 ), and S. meliloti (48, Hadjeras, et al. 45 ) However, the number of previously annotated sORFs in M. mazei (184), of which 93 were confirmed by manual curation, is much smaller than in other organisms. Together, we can now provide a list of 407 small proteins with high confidence which represent approximately 12% of total ORFs. In bacterial genomes, a recent study showed that 16% ± 9% of total ORFs are sORFs 74 , indicating that a similar ratio can be expected for archaea. Overall, the variation in the detection of unannotated sORFs across the different studies and reports is likely a product of unique experimental designs, computational methodologies, and inherent biological differences between organisms (including the genome size). Genes involved in N metabolism in M. mazei have been shown to be transcriptionally regulated in response to N availability by a global repressor NrpR 23 , 24 . Those include the structural genes of nitrogenase ( nifHDK genes), glnA 1 encoding glutamine synthetase, the glnK 1 \n amtB 1 operon encoding the nitrogen sensing P-II like protein (GlnK 1 ) and the ammonium transporter B 1 , and the nrpA encoding the nif specific activator 75 . The reported differential expressions are mainly in agreement with the results of the current study (Fig. 3 E, F ) summarized and compared in Table 1 . In addition to transcriptional regulation, we identified one regulatory RNA (sRNA 154 ) as a central post-transcriptional regulator in the nitrogen metabolism in M. mazei 26 . Under nitrogen limitation loop 2 of sRNA 154 activates translation of the nrpA mRNA, whereas in case of glnA 2 mRNA loop2 is masking the ribosome binding site and thus inhibits translation initiation 26 . In agreement, our current study confirms high up-regulation of sRNA 154 under -N (Supplementary Figs. 9 E and 10A ) and demonstrates a notable decrease in the translational efficiency of glnA 2 under -N (TE of 10.41) compared to +N (TE of 24.25) as shown in Supplementary Fig. 10B , and Table 1 . Moreover, the effect of post-transcriptional regulation of nrpA -mRNA by sRNA 154 , enhancing translation efficiency as proposed by Prasse, et al. 26 is as well confirmed by the current data set (see Table 1 and Fig. 3 E, F ). Of particular note is that sRNA 154 is one of the sRNAs where the current analysis identified a leaderless sORF (Supplementary Fig. 11 ). This sORF_154 encodes a small protein of 20 aa (nct 4-66), which is highly conserved in Methanosarcina (Supplementary Fig. 6 ). Consequently, we propose that sRNA 154 represents a dual-function sRNA, which will be validated in the future. Table 1 Validation of previously published results for genes involved in nitrogen metabolism Published 75 This Study Genes Transcription FC ± SD -N/ + N Transcription FC ± SD -N/ + N Translation FC ± SD -N/ + N nifH 197.1 ± 89.7 143.43 ± 3.62 153.69 ± 4 glnK 1 15.6 209.39 ± 3.71 49.62 ± 3.39 amtB 1 167.4 ± 37.5 146.34 ± 3.75 53.58 ± 3.74 glnA 1 26.5 ± 9.3 14.3 ± 3.53 11.18 ± 3.34 glnA 2 0.96 1.61 ± 3.53 0.62 ± 3.32 nrpA 39.6 ± 9.6 1.24 ± 4.39 35.7 ± 3.84 nifD 213.7 ± 107.2 74.39 ± 3.46 65.8 ± 3.96 nifK 202.5 ± 77.7 58.64 ± 3.24 34.92 3.95 Investigating the start codon distribution of annotated and unannotated sORFs, we observed that the usage of the canonical start codon ATG is reduced from 83% (annotated sORFs) to 56% (unannotated sORFs). In addition, annotated sORFs (with 60 aa or more in length) are in general longer than the unannotated sORFs, of which more than 50% are 40 aa or shorter. The identification of the very small sORFs and the sORFs with non-canonical start codons proves the importance of Ribo-seq, because other methods for ORF identification like proteomics and many pipelines for ORF detection are optimized for longer ORFs and have difficulties to identify those small sORFs (reviewed in ref. 76 ). Nevertheless, whether those very small proteins have a cellular function has yet to be shown. The genomic context of sORFs might give a hint to their putative function. sORFs located in 5’UTR of longer genes often act as so called uORFs, influencing the translation of the downstream longer ORF 73 (see Supplementary Data 8 ). Other hints to a function can be the cellular localization of the encoded small protein. Membrane associated small proteins can act as toxins by forming pores in the membrane or interact with protein complexes in the membrane (reviewed in ref. 76 ). We found 64 out of 314 (21%) of the unannotated sORFs on previously published sRNAs (Table S5 from 27 ), out of which 31 were already identified as so-called spRNA (small protein encoding sRNA), and others (33) were classified as regulatory sRNA. These translated sRNAs (see Supplementary Data 9 ) might be either unidentified dual-functional sRNA, regulating cellular processes based on a non-coding regulatory RNA part and an encoded small protein 41 , 43 , 77 , 5 , 78 , or they are in fact spRNA (small mRNAs) and the putative sORF was overlooked by in silico prediction tools or manual inspection in previous studies. In this respect, Ribo-seq is a powerful tool to uncover these sORFs as it can show whether a sRNA is in fact a non-translated sRNA or a small mRNA. However, the Ribo-seq method faces limitations in identifying alternative sORFs internal to the longer ORFs, necessitating TIS-profiling, which has to be performed to stall ribosomes at initiation sites for accurate detection and correction of misannotated genes 47 , 73 . In conclusion, this study reveals a more extensive and dynamic small proteome than previously appreciated, underscoring the nuanced regulatory mechanisms at play in the cellular machinery of M. mazei . Our approach exemplifies the strength of combining multi-omic strategies, including Ribo-seq, to correct and enhance genome annotations, particularly for sORFs, which are often overlooked e.g., due to biases towards longer ORFs. This dual-condition translational landscape illuminates the underexplored small proteome of M. mazei Gö1 and demonstrates its plasticity across N-rich and N-depleted conditions, contributing to a refined understanding of archaeal proteomics. Our findings underscore the effectiveness of multi-omic analyses in overcoming biases against small ORFs, enhancing the precision of genomic annotations and providing a critical resource for future research into archaeal biology. By providing a more accurate representation of small proteins, which constitute around 12% of total ORFs in M. mazei , our study sets a latest standard for genomic annotation in archaeal organisms and offers a valuable resource for further biological and evolutionary studies."
} | 5,829 |
34984249 | PMC8717399 | pmc | 5,761 | {
"abstract": "Furfural (FF) and\n5-(hydroxymethyl)furfural (HMF) are well-recognized\nbiomass-derived chemical building blocks with established applications\nand markets for several of their derivatives. Attaining a wide spectrum\nof petrochemicals is the primary target of a biorefinery that employs\nFF and HMF as the chemical feedstock. In this regard, cyclopentanone\n(CPN) is a crucial petrochemical intermediate used for synthesizing\na diverse range of compounds with immense commercial prospects. The\nhydrogenative ring rearrangement of FF to CPN in an aqueous medium\nunder catalytic hydrogenation conditions was first reported in 2012,\nwhereas the first report on the catalytic conversion of HMF to 3-(hydroxymethyl)cyclopentanone\n(HCPN) was published in 2014. Over the past decade, several investigations\nhave been undertaken in converting FF and HMF to CPN and HCPN, respectively.\nThe research studies aimed to improve the scalability, selectivity,\nenvironmental footprint, and cost competitiveness of the process.\nA blend of theoretical and experimental studies has helped to develop\nefficient, inexpensive, and recyclable heterogeneous catalysts that\nwork under mild reaction conditions while providing excellent yields\nof CPN and HCPN. The time is ripe to consolidate the data in this\narea of research and analyze them rigorously in a review article.\nThis work will assist both beginners and experts of this field in\nacknowledging the accomplishments to date, recognize the challenges,\nand strategize the way forward.",
"conclusion": "12 Conclusions Biomass-derived FF and HMF can be used as renewable\nchemical feedstocks\nfor synthesizing CPN and HCPN, respectively, which are otherwise sourced\nfrom petroleum. A major motivation behind this research is the commercial\nprospect of CPN, HCPN, and many of their derivatives. The conversion\nof FFs to CPNs involves a combination of hydrogenation and rearrangement\nreactions. The process is routinely performed in an aqueous medium\nat elevated temperatures in the presence of a metal-based heterogeneous\ncatalyst and a source of hydrogen. Molecular hydrogen is routinely\nemployed for the hydrogenation reaction, even though hydrogen donors\nlike methanol and 2-propanol have also been examined. Both noble metal\nand non-noble metal-based heterogeneous catalysts have been studied,\nand few of them afforded near-quantitative (>95%) yields of CPN\nand\nHCPN under optimized reaction parameters. Nanocatalysts have shown\nremarkable activity compared to traditional heterogeneous catalysts.\nBoth monometallic and bimetallic NPs have been employed as efficient\ncatalysts for transforming FF and HMF into CPN and HCPN, respectively.\nThe supporting materials for the active metal catalyst play multifaceted\nroles. Apart from playing conventional roles like increasing the surface\narea, uniformly dispersing the metal particles, and giving favorable\nmechanical properties, the supporting materials often establish electronic\ncommunications between the metal sites modifying their hydrogenating\nactivity, provide acidic sites for the rearrangement reaction, and\nalso provide adsorption sites for substrate and hydrogen molecules.\nWater was found to be the best solvent for producing CPN by molecular\nrearrangement of the furan ring, whereas the use of organic solvents\n(e.g., alcohol, ether) promoted hydrogenation. In some cases, the\nmixture of two solvents like 2-propanol/water and toluene/water provided\npromising results. The reaction required elevated temperatures in\nthe range of 140–170 °C for the best selectivity and yield\nof CPN and HCPN. High activation energy is needed for the ring-opening\nmolecular rearrangement reaction involving hydration and dehydration\nsteps. An overpressure of hydrogen gas in the range of 1–5\nMPa is typically used during the hydrogenation step. The acidic sites\non the catalyst promote hydration and rearrangement reactions. Lewis\nacidic sites are very efficient for the reaction, whereas the Brønsted\nacidic sites lead to side products and decomposition reactions. Many\ncatalysts have been reported that contained both metal sites and acid\nsites for hydrogenation and rearrangement reactions. Interestingly,\na physical mixture of the metal catalyst and Lewis acid catalyst also\nshowed comparable results. Many studies have reported excellent yields\nof CPN and HCPN using recyclable catalysts. The reaction is believed\nto proceed via the Piancatelli mechanism, although\nalternative mechanisms have also been proposed and substantiated with\nexperimental data. As a petrochemical, CPN has several applications\nand established markets for its derivatives. Novel applications of\nbiorenewable CPN, such as jet-fuel-range cycloalkanes and green solvents,\nhave been intended. For example, the high-density, jet-fuel-range,\ncyclic hydrocarbon fuels have been synthesized from lignocellulosic\nbiomass via CPN in three catalytic steps. It may\nbe concluded that remarkable advances have been made toward the renewable\nsynthesis of CPNs from FFs since their discovery in the past decade,\nand exciting discoveries are awaited in the coming years.",
"introduction": "1 Introduction Chemical industries are\nundergoing an exhilarating transformation\nwhere the manufacturing processes, both revamped and newly developed,\nemphasize not only the process economics but also environmental perspectives. 1 Since the advent of green chemistry in the early 1990s, ecological aspects of the industrial processes\nare thoroughly evaluated from time to time. 2 , 3 The\nprimary obstacle for the chemical industries to become truly sustainable\nis their unyielding dependence on exhaustible fossilized resources\nlike petroleum. With the demand for petrofuels and petrochemicals\nreaching newer heights, concerns over the fast depleting petroleum\nreserves and the seemingly irreversible environmental degradations\nhave intensified. 4 The full potential of\ngreen chemistry can be comprehended by utilizing a renewable carbon-based\nfeedstock in the chemical industries. In this aspect, biomass is the\nonly organic carbon in nature with the commercial prospect to replace\npetroleum, at least partially, in many futuristic scenarios. 5 , 6 Cellulosic biomasses (e.g., terrestrial lignocellulosic, freshwater\nand marine algae) are of particular interest since they are available\nin large excess, not considered food for humans, often regarded as\nwastes, geographically diverse, and inexpensive. 7 Lignocellulosic biomass primarily consists of biopolymers,\nsuch as cellulose (35–50%), hemicellulose (15–25%),\nand lignin (20–30%). 8 Hemicellulose\nis a nonlinear polymer consisting of hexose (e.g., glucose) and pentose\n(e.g., xylose) sugars, whereas cellulose is a linear, crystalline\npolymer of glucose connected by the β-1,4-glycosidic bond. Lignin\nis a complex polymer of substituted phenolic compounds connected by\nether and ester linkages. The chemical-catalytic pathways for biomass\nvalue addition are fast, selective, and biomass-independent and work\nunder reagent-economic and energy-efficient conditions. 9 , 10 A frequently used strategy initially hydrolyzes the hemicellulose\nand cellulose fractions into the constituent sugars, which are subsequently\ndehydrated into furanic compounds under acid catalysis for downstream\nsynthetic upgrading. 11 , 12 The lignin fraction is typically\nseparated and combusted to generate process heat and electricity.\nThe catalytic deconstruction of lignin to phenolic fuels and chemicals\nhas gained traction in recent years. 13 , 14 The\nacid-catalyzed dehydration of pentose sugars in hemicellulose\nforms furfural (FF), whereas dehydration of hexoses in cellulose and\nhemicellulose produces 5-(hydroxymethyl)furfural (HMF). 15 The acid-catalyzed dehydration process sequentially\neliminates water molecules from the sugar moieties without forming\nany significant waste streams. Both FF and HMF are known for over\na century and have significant literature presence as renewable chemical\nplatforms. 16 The production and derivative\nchemistry of FFs have been explored and perfected over the past several\nyears. Many of the derivatives of FF, such as furfuryl alcohol (FAL),\nhave well-known applications and established markets. 17 Many of the synthetic transformations of FF and HMF to\nbulk and fine chemicals employ chemocatalytic pathways. 18 Catalysis that grew as a separate field in its\ninitial years of development has found widespread applications in\norganic synthesis and the chemistry of renewables. 19 A new generation of inexpensive, eco-friendly, and recyclable\ncatalysts (homogeneous and heterogeneous) are being developed that\ncan produce the targeted compound(s) in desirable selectivity and\nyield. 20 , 21 A wide range of transportation fuels, chemicals,\nand polymers have been synthesized by the structural modifications\nof FF and HMF in the presence of a suitable catalyst. 22 − 25 Interestingly, FF can be converted into HMF by formylation at the\nC5-position, whereas HMF can be converted to FAL by the decarbonylation\nreaction. 26 , 27 Therefore, the derivatives of HMF can potentially\nbe accessed from FF and vice versa. Two major categories of\nproducts are sourced from biomass. The\nfirst category of bio-based products is structurally relatable to\nthe existing products of the petrochemical origin, has analogous properties,\nand functions as their renewable replacement of the latter. 28 , 29 In contrast, the second category of bio-based products is the drop-in\nequivalent of the petrochemicals that can be seamlessly integrated\ninto the existing infrastructure and markets. 30 Novel synthetic strategies must be developed that allow transforming\nFF and HMF into drop-in petrochemicals, widen their derivative chemistry,\nand strengthen their value chains in a biorefinery setup. In\nthis regard, cyclopentanone (CPN) is a chemical intermediate\nfor synthesizing several compounds of commercial significance, such\nas solvents, fragrances, cosmetic products, and agrochemicals. 31 , 32 CPN is commercially produced by the intramolecular decarboxylative\nketonization of adipic acid. 33 The process\nhas been optimized over the years, and Ba(OH) 2 was found\nto be an efficient and recyclable base catalyst. 33 The diesters of adipic acid, such as dimethyl adipate,\nhave also been used to synthesize CPN in the vapor phase. 34 However, the feedstock (i.e., adipic acid) is\nrelatively expensive, requires a multistep synthesis, and produces\nsignificant waste streams during its preparation. 35 CPN can also be produced by the hydration of cyclopentene\n(obtained by the steam cracking of naphtha) and dehydrogenation of\nthe cyclopentanol (CPL) intermediate. Cyclopentene can be reacted\nwith acetic acid by addition–esterification and then transesterified\nwith methanol to form CPL. However, the processes require an expensive\ncatalyst and harsh reaction conditions (280–300 °C, 25–40\nMPa) and produce significant waste streams, adversely affecting the\nscalability and process economy. Besides, both the processes described\nabove typically use starting materials from exhaustible fossilized\nresources and have questionable sustainability. In this regard,\nFF has attracted attention as a plausible renewable\nchemical feedstock for CPN since the former is a C5 compound of renewable\norigin ( Figure 1 ). Figure 1 Synthetic\nroute of CPN from petroleum and its prospective preparation\nfrom biomass. FF has an oxidation level of 6\n(three π-bonds, one ring,\nand two oxygen atoms), whereas CPN has 3 (one π-bond, one ring,\nand one oxygen atom). 36 The chemical equation\nfor transforming FF into CPN reveals that 3 moles of hydrogen are\nrequired per mole of FF, forming 1 mole of water as the byproduct\n( Scheme 1 A). Analogously,\nHMF has an oxidation level of 7 (three π-bonds, one ring, and\nthree oxygen atoms) compared to 4 in 3-(hydroxymethyl)cyclopentanone\n(HCPN) (one π-bond, one ring, and two oxygen atoms) ( Scheme 1 B). Scheme 1 Catalytic\nConversion of (A) Xylose to CPN via FF\nand (B) Glucose to HCPN via HMF CPL is generally formed as a minor side product by the\nover-reduction\nof CPN during its preparation from FF. CPL has potential applications\nas dyes, pharmaceutical products, fragrance agents, and solvents.\nBesides, it is a promising feedstock for various biofuels, including\njet and aviation fuels. Targeted production of CPL from FF has also\nbeen attempted and is discussed in detail in Section 7 ."
} | 3,097 |
32523595 | PMC7261841 | pmc | 5,762 | {
"abstract": "A plant growing under natural conditions is always associated with a substantial, diverse, and well-orchestrated community of microbes—the phytomicrobiome. The phytomicrobiome genome is larger and more fluid than that of the plant. The microbes of the phytomicrobiome assist the plant in nutrient uptake, pathogen control, stress management, and overall growth and development. At least some of this is facilitated by the production of signal compounds, both plant-to-microbe and microbe back to the plant. This is best characterized in the legume nitrogen fixing and mycorrhizal symbioses. More recently lipo-chitooligosaccharide (LCO) and thuricin 17, two microbe-to-plant signals, have been shown to regulate stress responses in a wide range of plant species. While thuricin 17 production is constitutive, LCO signals are only produced in response to a signal from the plant. We discuss how some signal compounds will only be discovered when root-associated microbes are exposed to appropriate plant-to-microbe signals (positive regulation), and this might only happen under specific conditions, such as abiotic stress, while others may only be produced in the absence of a particular plant-to-microbe signal molecule (negative regulation). Some phytomicrobiome members only elicit effects in a specific crop species (specialists), while other phytomicrobiome members elicit effects in a wide range of crop species (generalists). We propose that some specialists could exhibit generalist activity when exposed to signals from the correct plant species. The use of microbe-to-plant signals can enhance crop stress tolerance and could result in more climate change resilient agricultural systems.",
"introduction": "Introduction Plants in nature are always in relationships ( Raina et al., 2018 ) with a microbial community (the phytomicrobiome); some members of the soil microbial community assist plant growth and development ( Prithiviraj et al., 2003 ; Smith et al., 2015a , b ). The phytomicrobiome plus the plant constitute the holobiont—the holobiont is the entity that evolution acts upon, and that produces crop yield ( Smith et al., 2017 ; Cordovez et al., 2019 ). When adaptation to environmental stressors is needed, the plant: (1) alters its own gene expression and resulting physiology, and also (2) adjusts the diversity, composition, and activity of its phytomicrobiome ( Smith et al., 2015b ; Gopal and Gupta, 2016 ). The latter allows for very short-term adjustments, including evolution of the phytomicrobiome; the plant genome evolves much more slowly ( Mueller and Sachs, 2015 ). The genome of the phytomicrobiome (much larger than the plant genome) plus the plant genome comprises the hologenome or the pan-genome (the host plus the microbial metagenome) ( Berendsen et al., 2012 ; Guerrero et al., 2013 ; Turner et al., 2013 ; Bordenstein and Theis, 2015 ). It seems that evolution of more complex eukaryotic cells (Phylum Lokiarchaeota— Turner et al., 2013 ; Spang et al., 2015 ) from simpler prokaryotes, allowed development of the holobiont ( Embley and Martin, 2006 ; Douglas, 2014 ; Koonin and Yutin, 2014 ; Graham et al., 2018 ). Beneficial relationships between terrestrial plants and microbes have existed since plants moved into the terrestrial environment, almost half a billion years ago ( Knack et al., 2015 ). For about a billion years prior to this, algae had relationships with compatible microbial species, sometimes leading to new organisms. For example, Ascophyllum nodosum appears to be a fusion of a macroalga and a fungus ( Deckert and Garbary, 2005 ). The phytomicrobiome is tissue-specific and relationships vary in intimacy all the way to complete incorporation/fusion, as is the case with mitochondria and chloroplasts ( Backer et al., 2018 ). The most abundant and diverse element of the phytomicrobiome is the rhizomicrobiome where microbes live around or within the root tissues, often in the spaces between cells of the cortex (the root is the niche space of these microbes), and use root exudates as a source of energy/reduced carbon ( Schlaeppi and Bulgarelli, 2015 ). Rhizomicrobiome members can stimulate root growth and so improve plant water and nutrient uptake."
} | 1,053 |
38909236 | PMC11193907 | pmc | 5,765 | {
"abstract": "Heimdallarchaeia is a class of the Asgardarchaeota , are the most probable candidates for the archaeal protoeukaryote ancestor that have been identified to date. However, little is known about their life habits regardless of their ubiquitous distribution in diverse habitats, which is especially true for Heimdallarchaeia from deep-sea environments. In this study, we obtained 13 metagenome-assembled genomes (MAGs) of Heimdallarchaeia from the deep-sea cold seep and hydrothermal vent. These MAGs belonged to orders o _ Heimdallarchaeales and o _ JABLTI01 , and most of them (9 MAGs) come from the family f _ Heimdallarchaeaceae according to genome taxonomy database (GTDB). These are enriched for common eukaryote-specific signatures. Our results show that these Heimdallarchaeia have the metabolic potential to reduce sulfate (assimilatory) and nitrate (dissimilatory) to sulfide and ammonia, respectively, suggesting a previously unappreciated role in biogeochemical cycling. Furthermore, we find that they could perform both TCA and rTCA pathways coupled with pyruvate metabolism for energy conservation, fix CO 2 and generate organic compounds through an atypical Wood-Ljungdahl pathway. In addition, many genes closely associated with bacteriochlorophyll and carotenoid biosynthesis, and oxygen-dependent metabolic pathways are identified in these Heimdallarchaeia MAGs, suggesting a potential light-utilization by pigments and microoxic lifestyle. Taken together, our results indicate that Heimdallarchaeia possess a mixotrophic lifestyle, which may give them more flexibility to adapt to the harsh deep-sea conditions. Supplementary Information The online version contains supplementary material available at 10.1186/s40793-024-00585-2.",
"conclusion": "Conclusion We analyze 13 MAGs of Heimdallarchaeia from the deep-sea cold seep and hydrothermal vent. We show that these Heimdallarchaeia clades enable reduce sulfate and nitrate to sulfide and ammonia, respectively, revealing their undiscovered roles in biogeochemical cycling of deep sea. Of note, we demonstrate that Heimdallarchaeia clades could synthesize bacteriochlorophyll and carotenoid, and might utilize light through these light-sensing pigments. In addition, we further found that Heimdallarchaeia clades could fix CO 2 through an atypical Wood-Ljungdahl process, and a novel multi-function MetH enzyme might play a key role in this process. Lastly, we thus propose that Heimdallarchaeia possess a mixotrophic lifestyle, which may give them more flexibility to adapt to harsh deep-sea conditions, and make them contribute to the biogeochemical cycling in deep biosphere.",
"introduction": "Introduction Archaea are important microorganisms that play important roles in the biogeochemical cycle of Earth [ 1 ], and are indispensable for the study of evolution [ 2 ]. To date, four supergroups of archaea have been described: Euryarchaeota, TACK, Asgard, and DPANN [ 3 ]. Breakthroughs in metagenomic sequencing technology are rapidly transforming our understanding of microbial evolution, particularly with the discovery of the Asgardarchaeota phylum and the prediction of their position at the base of the eukaryotic tree of life [ 4 ]. The Heimdallarchaeia as well as their newly derived orders (including o_Hodarchaeales , o_Heimdallarchaeales , and o_JABLTI01 according to genome taxonomy database, GTDB) [ 5 ] currently represent the closest predicted archaeal relatives of eukaryotes [ 6 ]. Although they have been given a new name to o _ JABLTI01 as Gerdarchaeales in recent literature [ 7 ], the original names are still used here in order to be consistent with the GTDB (R220 taxonomy from v2.4.0). Compared with other members of the Asgardarchaeota (e.g. Lokiarchaeia [ 8 ] and Thorarchaeia [ 9 ]), studies of Heimdallarchaeia are lagging due to a lack of metagenomic data and cultured strains. Nonetheless, the available genomic information supports the hypothesis that Heimdallarchaeia could survive in strictly anaerobic habitats, as well as in a sunlit microoxic niche [ 10 ]. The aerobic respiration would allow Heimdallarchaeia to use a wide range of organic substrates [ 8 ], and enable them to oxidize organic substrates by using oxygen as an electron acceptor, and allow them to conserve the energy by coupling ferredoxin reoxidation to respiratory proton reduction [ 11 ]. Metagenome-assembled genomes (MAGs) of Heimdallarchaeia class as well as their new derived clades were obtained from both marine and fresh water environments [ 4 , 10 , 12 ]. But the number of Heimdallarchaeia MAGs was much lower than Lokiarchaeia and Thorarchaeia in the database, which greatly limits the understanding of their metabolism, lifestyle, and contributions to biogeochemical cycling. Previous studies have mentioned that Heimdallarchaeia clades, including o _ JABLTI01 and o _ Hodarchaeales possessed citrate cycle (TCA), succinate dehydrogenase and NADH-quinone oxidoreductase for aerobic respiration, and rhodopsins for light sensing [ 2 , 10 ]. These unique archaea may have existed in microoxic and light-exposed habitats during their evolutionary history [ 4 , 10 , 13 , 14 ]. However, many key metabolism pathways were indicated to be lacking in MAGs of Heimdallarchaeia , such as sulfur metabolism, the Wood-Ljungdahl (WL) pathway, and the reductive citrate cycle (rTCA) [ 2 , 6 , 15 ]. These findings suggest that the metabolism and lifestyle of Heimdallarchaeia remains to be explored. Cold seeps and hydrothermal vents are two special deep-sea environments rich in methane, carbon dioxide (CO 2 ), and sulfur. These are ideal locations for the study of biogeochemical cycling, novel metabolic pathways, and the biological origins and evolution of life [ 16 – 19 ]. Sulfur metabolism and CO 2 fixation are thought to be important metabolic pathways for microorganisms in these environments [ 20 – 24 ]. It is generally believed that there is no light in the deep-sea environment below 1,000 m. However, the geothermal light has been mentioned in hydrothermal vents to provide a selective advantage for the evolution of photosynthesis from a chemotrophic microbial ancestor [ 25 ]. In addition, some bacteria have been reported to use light-sensing molecules for phototaxis toward light associated with the geothermal light in deep-sea vents [ 25 – 28 ]. For archaea, key enzymes involved in photosynthetic pigment synthesis have been reported in Thermoproteota (former Crenarchaeota and Bathyarchaeota ) [ 29 , 30 ]. Therefore, we are interested in whether some photosynthetic pigments are also present in Asgardarchaeota from deep-sea vents. In the present study, we first analyzed the community structure of archaea in deep-sea cold seep and vent sediment, and then obtained 13 MAGs of Heimdallarchaeia , belonging to order o _ Heimdallarchaeales ( o _ UBA460 ) and o _ JABLTI01 , respectively. Based on these MAGs, we confirmed the eukaryotic signatures of deep-sea Heimdallarchaeia ; identified indications for their involvement in sulfur, nitrogen, and carbon cycling; and discovered their potential aerobic light sensing lifestyle. This work could pave the way for the future explorations of unexpected light utilization mechanisms and other special environmental adaptations possessed by Heimdallarchaeia in the deep biosphere.",
"discussion": "Results and discussion Phylogenetic status and eukaryotic signatures of deep-sea Heimdallarchaeia To investigate the metabolic characteristics of deep-sea Heimdallarchaeia , we sampled four sediments (C1, C2, C4, and C5) from a deep-sea cold seep (depth greater than 1,100 m) in the South China Sea, and one sample (H2) from a deep-sea hydrothermal vent (depth 2,194 m, outside of the black chimney, environmental temperature 5.5 °C) in the Western Pacific Ocean (Table S1 ). These environments are rich in CH 4 , sulfur, and different metal ions (Figure S1 ). Metagenomic DNA from these five samples was extracted and sequenced. Sequence statistics indicated that proportion of annotated genes belong to Heimdallarchaeia were relatively abundant among Asgardarchaeota in both cold seep and vent environments (Figure S2 ). To explore the metagenomic characteristics of these Heimdallarchaeia , 13 MAGs were obtained using a hybrid binning strategy combined with manual inspection and data curation. Seven of 13 MAGs (> 80% completeness, < 5% contamination) were considered as high-quality genomes according to the reported standards (Table S2 ) [ 31 ]. Other MAGs were of medium-quality (> 50% completeness, < 5% contamination) [ 32 ], except for C2.bin.3, C5.bin.12 and H2.bin.2. However, since the completeness of these three MAGs was higher than 70%, we also used them for subsequent functional gene annotation and analysis. The maximum-likelihood phylogenetic tree was generated based on concatenation of 53 marker proteins for Archaea from GTDB database (Release 220). Both the MAGs from the present study and other published Heimdallarchaeia MAGs clustered with Asgardarchaeota members, and displayed an obvious evolutionary distance from other archaeal phyla (Fig. 1 A, Supplementary Dataset 1 ). These 13 MAGs fallen in two orders ( o _ Heimdallarchaeales , and o _ JABLT101 ) based on the phylogenomic tree were further used for the amino acid identity (AAI) analysis (Fig. 1 B) [ 6 ]. Among them, 11 MAGs mainly belonged to three families in o _ Heimdallarchaeales , including f _ Heimdallarchaeaceae (C1.bin.1, Ci.bin.2, C1.bin.21, Ci.bin.76, C2.bin.3, C4.bin.14, C4.bin.22, C4.bin.51 and C5.bin.5), f _ Kariarchaeaceae (H2.bin.2) and f _ DAOWED01 (C1.bin.20). Of note, f _ Kariarchaeaceae , f _ DAOWED01 and f _ JAJRWK01 were displayed a more similar identity distinguishing from f _ Heimdallarchaeaceae MAGs in o _ Heimdallarchaeales according the results both of phylogenetic tree and AAI analysis (Fig. 1 ). However, according to the analysis results of AAI, we found that the corresponding values of other three families ( f _ Kariarchaeaceae , f _ DAOWED01 and f _ JAJRWK01 ) and f _ Heimdallarchaeaceae ranged from 43 to 45%, but the values of o _ JABLTI01 and f _ Heimdallarchaeaceae ranged from 46 to 47%, and the values of o _ Hodarchaeales and f _ Heimdallarchaeaceae ranged from 43 to 45% (Supplementary Dataset 1 ). This result might indicate that AAI analysis can only be used to define taxonomic ranks below the family level [ 31 ]. In addition, C1.bin.20 is the second reported MAG in f _ DAOWED01 according to records of the GTDB database. This will further enrich the species composition of f _ DAOWED01 family. \n Fig. 1 Phylogenetic analysis of Heimdallarchaeia clades. ( A ) Maximum-likelihood phylogeny of Heimdallarchaeia MAGs. Phylogenetic analysis was performed based on concatenation of 53 marker proteins for Archaea, which were chosen by Phylosift (1,000 bootstrap replicates). Detailed sequence information from different species in compressed clades is listed in Supplementary Dataset 1 . ( B ) Amino acid identity correlation matrix of MAGs of Heimdallarchaeia clades was calculated by Compare M \n As the closest archaeal lineage to eukaryotes, eukaryote-specific proteins (ESPs) were indeed identified in the present 13 MAGs of Heimdallarchaeia , which is consistent with other members of the Asgardarchaeota (Figure S3 , Supplementary Dataset 2 ). However, we found that the distribution of these ESPs was different in order levels of Heimdallarchaeia [ 6 ]. For instance, some ESPs, including eukaryotic ribosomal proteins, nucleus related proteins, vacuoles and signal transforming related proteins were found to have an almost complete distribution in all 13 MAGs (Figure S3 , Supplementary Dataset 2 ). These ESPs were mainly involved in the storage of genetic material, gene transcription, protein translation, and material transport processes, which were recognized as the “basic part” of eukaryotic cells [ 10 , 14 , 33 – 35 ]. On the other hand, ESPs of ubiquitin-proteasome system, cytoskeleton, mitochondrion, and chloroplast, viewed as the “functional part” of eukaryotic cells, showed different distribution characteristics between o _ Heimdallarchaeales and o _ JABLTI01 (Figure S3 , Supplementary Dataset 2 ). Despite the lack of hard evidence, we speculate that the differences in order levels of Heimdallarchaeia might be related to the subsequent evolution of cellular complexity and functional differentiation of eukaryotic cells [ 7 ]. Heimdallarchaeia clades could participate in the sulfur biogeochemical cycle Sulfur cycling is believed to be a dominant form of metabolism for microorganisms living in the sampling locations presented in this study [ 36 ]. However, recent studies have shown that many key molecules of sulfur metabolic pathway are absent in the MAGs of Heimdallarchaeia and other Asgardarchaeota , such as dissimilatory sulfite reductase (DsrA), adenylylsulfate reductase (AprA) and anaerobic sulfite reductases (AsrAB) [ 2 , 6 , 11 ]. In this study, although DsrA and AprA proteins were also absent in our obtained MAGs, AprB and AsrAB instead could be identified from Heimdallarchaeia . In addition, other key enzymes in inorganic sulfur metabolism, including sulfate transport proteins (CysUWA), sulfate adenylyltransferase (Sat), adenylylsulfate kinase (ApsK), and phosphoadenosine phosphosulfate reductase (CysH), were widely distributed in the MAGs of the cold seep Heimdallarchaeia (Fig. 2 A and B, Supplementary Dataset 3 ). Sulfate is known to be important environmental factors in the sulfate methane transition zone (SMTZ) of cold seeps [ 16 , 37 , 38 ]. Thus, our results suggest that Heimdallarchaeia inhabiting cold seep sediments have the potential to participate in inorganic sulfur metabolism for energy production through assimilatory and partly dissimilatory sulfate reduction pathways [ 39 , 40 ]. In contrast, mostly enzymes (AprB, ApsK, CysH, and Sat) involved in sulfate and sulfite metabolism were absent in the MAG H2.bin.2 obtained from the vent environment. However, enzymes associated with dimethyl sulfone (DMS) metabolism, such as dimethyl sulfone monooxygenase (SfnG) and alkanesulfonate monooxygenase (SsuD), were identified (Fig. 2 A and B, Supplementary Dataset 3 ), suggesting that some members of Heimdallarchaeia may be able to perform organic sulfur metabolism. Notably, sulfide: quinone oxidoreductase (SQR) is broadly distributed in Heimdallarchaeia MAGs (Fig. 2 A, Supplementary Dataset 3 ). SQR is known as one of the most widespread markers of marine sulfur-oxidizing microorganisms [ 41 , 42 ]. It is a ubiquitous membrane-bound flavoprotein involved in sulfide detoxification via the oxidization of sulfide to zero-valent sulfur, through which electrons are transferred to the membrane quinone pool for energy conservation processes [ 42 ]. Previous studies have reported that Heimdallarchaeia could uniquely metabolize H 2 S [ 8 , 10 ]. Therefore, SQR protein could play an important role in this metabolic process. In the present study, seven out of nine f _ Heimdallarchaeaceae MAGs and H2.bin.2 contained complete amino acid sequences for SQR, most of which belonged to the type III group (Fig. 2 C), consistent with SQRs identified in other archaea [ 41 , 43 , 44 ]. This ratio is significantly higher than the previously reported SQRs in Heimdallarchaeia MAGs [ 8 ]. An SQR present in MAG C1.bin.76 was found to form a distinct clade with SQRs from Streptomyces aidingensis (SFC92959.1), Salegentibacter agarivorans (SFF97228.1) and Heimdallarchaeia LC3 (OLS23614.1). This clade is distinct from the six typical groups (Fig. 2 C), suggesting that a novel type of SQR group may exist. \n Fig. 2 Sulfur metabolic pathway identified in Heimdallarchaeia MAGs. ( A ) Distribution of identified key enzymes involved in the sulfur metabolism of Heimdallarchaeia clades. The presence of enzymes involved in the sulfur metabolic pathway is indicated for each MAG using green colored rectangles. ( B ) Sulfur metabolic pathway identified in Heimdallarchaeia clades. ( C ) Maximum-likelihood phylogeny of sulfide: quinone oxidoreductases (SQRs) identified in Heimdallarchaeia clades (1,000 bootstrap replicates). Nodes indicate bootstrap values greater than 70. Numbers represent the tree scale of each branch. The previously reported SQRs in MAGs of Heimdallarchaeia were labeled by the black star. AprB, adenylylsulfate reductase, subunit B; ApsK (CysC), adenylyl-sulfate kinase; AsrA and AsrB, anaerobic sulfite reductases; CysH, phosphoadenosine phosphosulfate reductase; CysA, sulfate transport system ATP-binding protein; CysU, sulfate transport system permease protein; CysW, sulfate/thiosulfate transport system permease protein; HydA, HydB, HydD and HydG, sulfhydrogenases. Sat, sulfate adenylyltransferase; SfnG, dimethylsulfone monooxygenase; SQR, sulfide: quinone oxidoreductase; SsuD, alkanesulfonate monooxygenase; SoxB, S-sulfosulfanyl-L-cysteine sulfohydrolase; TST, thiosulfate/3-mercaptopyruvate sulfurtransferase. Detailed protein information related to this figure is listed in Supplementary Dataset 3 \n Based on these results, we propose that more complete sulfur metabolism pathways (including assimilatory sulfate reduction and sulfide oxidation) in these deep-sea Heimdallarchaeia MAGs may be due to the higher concentrations of sulfur compounds in cold seep environments than previously reported Asgardarchaeota MAGs [ 2 , 4 , 11 , 45 , 46 ]. Therefore, Heimdallarchaeia clades may be important participants in sulfur cycling in the cold seep environments, particularly a higher concentration of sulfide (reach the greatest > 20 mM in the surface sediments) in deep-sea cold seeps [ 46 ]. Heimdallarchaeia clades use diverse nitrogen compounds for growth Nitrogen is the fourth most abundant element in cellular biomass, and it comprises the majority of Earth’s atmosphere [ 47 , 48 ]. However, nitrogen is a limiting nutrient for biological systems in marine environments [ 49 – 51 ]. Hence, the nitrogen cycle is critical for both the growth of microorganisms and the biogeochemical cycles of the ocean [ 48 – 50 ]. Like sulfur metabolism pathways, the nitrogen metabolism pathways present in Heimdallarchaeia that derived from cold seeps and vents are different. The enzymes responsible for nitrate reduction to nitrite (NarI) [ 48 ], nitrite reduction to ammonium (NirD) [ 48 , 49 ], nitrite reduction to nitric oxide (NirK and NirS) [ 52 – 54 ], hydroxylamine reduction to ammonia (Hcp) [ 48 , 55 ], and ammonia transformation to glutamate (GlnA, GltD and GdhA) were all identified in the MAG H2.bin.2 obtained from vent sediment. However, these enzymes, with the exception of enzymes responsible for the transformation of ammonium to glutamate, were almost not detected in cold seep Heimdallarchaeia MAGs (Figures S4 A and S4B, Supplementary Dataset 4 ). These results suggest that Heimdallarchaeia living in vents may play important roles in nitrate reduction, while those living in cold seeps may be essential participants in the metabolism of ammonia, which is potentially derived from the methylamine in this environment. Methylamine is thought to be an important nitrogen source for marine microorganisms, and is released through the biodegradation of proteins and N-containing osmolytes [ 56 , 57 ]. In cold seep environments, methylamine is also a key substrate of archaeal methanogenesis, a process which may release a large amount of ammonia into the environment [ 58 ]. We consistently found large amounts of methane in cold seep sampling sites (Figure S1 ). Therefore, we infer that the different pathways for sulfur and nitrogen metabolism identified in deep-sea Heimdallarchaeia clades from diverse habitats may be the result of their long-term adaptation to the deep-sea extreme environment. Nitrilase (nitrile aminohydrolase) has also been widely identified in deep-sea MAGs of Heimdallarchaeia clades (Figures S4 A and S4B, Supplementary Dataset 4 ). Nitrilase catalyzes the hydrolysis of nitriles to form a carboxylic acid product with the concomitant release of ammonia [ 59 , 60 ]. The existence of nitrilase in microorganisms endows them with the ability to use nitriles as a source of nitrogen for growth [ 61 , 62 ]. Nitrilases have been divided into six subgroups according to their substrates (e.g. aliphatic and aromatic nitriles [ 59 , 63 ] or amides [ 59 , 64 ]). Phylogenetic analysis revealed that nitrilases in deep-sea MAGs of Heimdallarchaeia clades were mainly clustered in clades induced by aliphatic nitriles and amides (Figure S4 C), suggesting that abundant aliphatic nitriles and amides might exist in the deep sea. We speculate that Heimdallarchaeia clades use diverse nitrogen compounds for growth, and play important roles in nitrogen cycling in deep-sea environments. Heimdallarchaeia sense the light by chlorophyll and carotenoid Previous reports of the existence of rhodopsins in archaeal phyla (e.g. Bathyarchaeia , Lokiarchaeia and Heimdallarchaeia [ 10 , 30 ]) have suggested that archaea can sense the light. In this study, no rhodopsin homologs were identified in the present MAGs of deep-sea Heimdallarchaeia clades. However, many typical chloroplastic proteins (including protochlorophyllide reductase, chlorophyll(ide) b reductases NOL/NYC1, NAD(P)H quinone oxidoreductase, and the photosystem I assembly proteins Ycf3 and phycocyanobilin lyase) were identified (Figure S3 , Supplementary Dataset 2 ). Comparative genomic analysis revealed that a series of enzymes involved in the porphyrin and bacteriochlorophyll synthesis pathways were present in MAGs of deep-sea Heimdallarchaeia clades (Fig. 3 A, Supplementary Dataset 5 ). Notably, almost all of the necessary bacteriochlorophyll synthesis components were widely distributed in Heimdallarchaeia MAGs from vents (including H2.bin.2 and LC2), which suggests that Heimdallarchaeia clades may be able to synthesize bacteriochlorophyll (Fig. 3 A, Supplementary Dataset 5 ). Protochlorophyllide reductase (Por) is a key enzyme in bacteriochlorophyll synthesis that could catalyze the transition between divinyl protochlorophyllide and divinyl chlorophyllide a [ 65 ]. Total four homologs of Por coding gene were found from these 13 Heimdallarchaeia MAGs. Phylogenetic analysis revealed that Por homologs from cold seep and vent were clustered in a sister clade, respectively (Fig. 3 B, Supplementary Dataset 5 ). Then Por proteins from Heimdallarchaeia , Acidobacteria , and Rhodobacterales were located in branch with photosynthetic organisms, including Cyanobacteria , eukaryotic Algae, and Plants ( Streptophytina ). Interestingly, the Por homologs identified from Bathyarchaeia [ 66 ] displayed a closer evolutionary relationship to that of Chloroflexia , Rhodospirillales , and Chromatiales (Fig. 3 B). These might indicate that Por homologs in Heimdallarchaeia are more closely evolutionarily related to eukaryotes compared with Por proteins of bacterial origin. \n Fig. 3 Porphyrin and bacteriochlorophyll biosynthesis pathways identified in Heimdallarchaeia MAGs. ( A ) Analysis of porphyrin and bacteriochlorophyll biosynthesis in different Heimdallarchaeia MAGs. ( B - C ) Phylogenetic analyses of protochlorophyllide reductase (Por) and bacteriochlorophyll synthetase (BCS). A rooted maximum-likelihood tree of Por ( B ) or BCS ( C ) homologs derived from different photosynthetic organisms identified in this work (1,000 bootstrap replicates). The solid arrows indicate the enzymes associated with bacteriochlorophyll biosynthesis present in Heimdallarchaeia MAGs. Dotted arrows indicate the enzymes associated with bacteriochlorophyll biosynthesis absent in MAGs. The gray box highlights MAGs from o_Hodarchaeales . The frame highlights assembled genomes of other Asgard archaea. The red highlights MAGs obtained in this study. EARS, glutamyl-tRNA synthetase; HemA, glutamyl-tRNA reductase; HemL, glutamate-1-semialdehyde 2,1-aminomutase; HemB, porphobilinogen synthase; HemC, hydroxymethylbilane synthase; HemE, uroporphyrinogen decarboxylase; HemN, coproporphyrinogen oxidase; HemG, protoporphyrinogen oxidase; HemH, protoporphyrin/coproporphyrin ferrochelatase; BchM, magnesium-protoporphyrin O-methyltransferase; BchE, anaerobic magnesium-protoporphyrin IX monomethyl ester cyclase. Por, protochlorophyllide reductase; NOL/NCY1, chlorophyll(ide) b reductase NOL/NCY1; BCS, bacteriochlorophyll synthase. The detailed information of key enzymes involved in bacteriochlorophyll biosynthesis and proteins used for phylogenetic analyses is listed in the Supplementary Dataset 5 \n Bacteriochlorophyll synthase (BCS) is capable of synthesizing bacteriochlorophyll a by esterification of bacteriochlorophyllide with phytyl diphosphate or geranylgeranyl diphosphate [ 29 ]. Digeranylgeranylglyceryl phosphate synthase (DGPS) might perform the similar function in archaea. Key enzymes of bacteriochlorophyll biosynthesis, including BCS, have been reported in Thermoproteota ( Crenarchaeota ) [ 29 ] and Bathyarchaeia [ 30 ], suggesting that bacteriochlorophyll may be a common molecule used by archaea to utilize light. We phylogenetically analyzed the evolutionary relationship between BCS and DGPS, and found that archaeal DGPS was located at the outer group of the tree, separated from the clade containing BCS in phototrophic bacteria and chlorophyll synthase in photosynthetic organisms (Fig. 3 C, Supplementary Dataset 5 ). Four homologues of BCS in the present Heimdallarchaeia MAGs clustered in a clade with the DGPS in Heimdallarchaeia LC2, which is located between the DGPS and BCS branches (Fig. 3 C, Supplementary Dataset 5 ). Finally, a previously reported functional bacteriochlorophyll synthase derived from uncultured Thermoproteota ( Crenarchaeota ) [ 29 ] was found to cluster on a branch with the DGPS from Heimdallarchaeia LC3 [ 4 ]. This cluster displayed a close evolutionary relationship with the photosynthetic bacteriochlorophyll and chlorophyll synthase branches (Fig. 3 C). In addition to bacteriochlorophyll, other light-sensing pigments, including carotenoids [ 67 , 68 ] and bacteriophytochrome [ 69 ], are identified to be synthesized in Heimdallarchaeia (Supplementary Dataset 6 ). Carotenoids are ubiquitous and essential pigments for photosynthesis [ 67 ]. Carotenoids function as accessory light-harvesting pigments that transfer absorbed energy to bacteriochlorophylls and thereby expand the range of wavelengths that are able to drive photosynthesis [ 70 , 71 ]. We reconstructed the complete synthesis pathway of lycopene [ 72 ], a biologically important carotenoid derived from acetyl-CoA, using Heimdallarchaeia MAGs from cold seeps (Figure S5 , Supplementary Dataset 6 ). It has been previously observed that light in the 450–550 nm (blue-green light) region of the solar radiation spectrum is not effectively absorbed by chlorophylls in photosynthesis, but is effectively absorbed by carotenoids [ 67 , 68 ]. Moreover, carotenoids protect organisms from photo damage by quenching both singlet and triplet states of bacteriochlorophylls under strong illumination, and function as photosynthetic membrane stabilizers in chloroplasts [ 67 ]. Therefore, the biosynthesis of carotenoids in Heimdallarchaeia could complement bacteriochlorophyll to enable high-efficiency light energy utilization and thus provide a competitive advantage in habitats with light. What remains unclear is the ecological function or benefit of light energy utilization in Heimdallarchaeia clades, which reside predominantly in marine sediments [ 4 ]. A recent study on rhodopsins in Heimdallarchaeia provides evidence for their existence in light-exposed habitats that would provide sufficient energy [ 10 ]. The recovery of Heimdallarchaeia from deeper environments may be due to the high deposition rates characteristic of the sampling locations 10 . There is substantial evidence to demonstrate that both long wavelength (> 650 nm) and short wavelength (< 650 nm) light have been detected in vents [ 73 , 74 ]. The blue-green light (450–550 nm) from the dim sunlight penetrating ocean water or the bioluminescence might exist in deep seafloor about 1,000 m [ 75 , 76 ]. Our sampling site from the cold seep was also around this depth [ 77 ]. Thus, the necessary conditions for light energy utilization may exist in these environments. Heimdallarchaeia have the inferred capability to detect light of different wavelengths in the environment, and thus could utilize photoelectrons for energy conversion and thereby have an advantage in the competition for nutrient resources. However, more works are still needed to prove the functions of these pigments in Heimdallarchaeia for light sensing. The mixotrophic and aerobic lifestyle of Heimdallarchaeia clades According to previous studies, Heimdallarchaeia clades exhibited a mixotrophic lifestyle similar to other members of the Asgardarchaeota MAGs [ 10 , 11 ]. Heimdallarchaeia clades were able to simultaneously use the nearly complete TCA (from 2-oxoglutarate to malate) and transport exogenous organic matter through the metabolic circuitry for coupling catabolism with pyruvate metabolism [ 4 , 11 , 78 ]. They could also utilize a reverse tricarboxylic acid cycle (rTCA) for autotrophic CO 2 assimilation [ 4 , 10 , 78 ]. In the present study, we found that these Heimdallarchaeia have the potential ability to fix CO 2 with an atypical Wood-Ljungdahl pathway (Fig. 4 A, Supplementary Dataset 7 ). In this atypical Wood-Ljungdahl process, the methylenetetrahydrofolate reductase (MetF) and 5-methyltetrahydrofolate corrinoid/iron sulfur protein methyltransferase (AcsE) are missing. However, a kind of protein (annotated as the bifunctional homocysteine S-methyltransferase/5,10-methylenetetrahydrofolate reductase, MetH) was found in deep-sea Heimdallarchaeia clades, which was not clustered with the traditional MetH proteins in the phylogenetic tree but located between the clades of AcsE and MetF. This novel function MetH protein (MetH-N) is thought to perform the functions of MetF and AcsE simultaneously, thereby catalyzing the production of tetrahydrofolate (THF) from 5,10-Methylene-THF (Fig. 4 B, Supplementary Dataset 7 ) [ 79 , 80 ]. In addition, CAZy (Carbohydrate-Active enZYmes database) analysis revealed a variety of polysaccharide-degrading enzymes, including chitinase, xylan/chitin deacetylase, diacetylchitobiose deacetylase, and cellulase, in these Heimdallarchaeia MAGs (Figure S6 , Supplementary Dataset 6 ). These CAZymes and results previously presented suggest that macromolecular organic carbon compounds utilization and (homo)acetogenic fermentation may be the main metabolic strategies used for energy production by Heimdallarchaeia clades in deep-sea sediments, as shown for many other microorganisms [ 81 ]. Together, these versatile carbon metabolism patterns provide evidence that Heimdallarchaeia clades live a mixotrophic lifestyle which may be advantageous in deep-sea conditions [ 10 ]. \n Fig. 4 Wood-Ljungdahl (WL) pathway identified in Heimdallarchaeia clades. ( A ) Distribution of identified key enzymes involved in the WL pathway in Heimdallarchaeia . The presence of the enzymes involved in the WL pathway is indicated for each MAG using green colored rectangles. ( B ) WL pathway identified in Heimdallarchaeia . ( C ) Maximum-likelihood phylogeny of a potential difunctional enzyme (MetH) involved in the WL pathway in Heimdallarchaeia (using the LG + G4 + F model; 1,000 bootstrap replicates). Nodes indicate bootstrap values greater than 70. The numbers represent the tree scale of each branch. MetF, methylenetetrahydrofolate reductase; AcsE, 5-methyltetrahydrofolate corrinoid/iron sulfur protein methyltransferase; MetH, 5-methyltetrahydrofolate-homocystenie methyltransferase; CdhE (AscC), acetyl-CoA decarbonylase/synthase, CODH/ACS complex subunit gamma; CooF, anaerobic carbon-monoxide dehydrogenase iron sulfur subunit; FdhA, formate dehydrogenase (NADP + ) alpha subunit; Fhs, formate-tetrahydrofolate ligase; FolD, methylenetetrahydrofolate dehydrogenase (NADP + )/methenyltetrahydrofolate cyclohydrolase. Detailed protein information related to this figure is listed in Supplementary Dataset 7 \n Some aerobic metabolic pathways, distinguished from the metabolic pathways of anaerobic Lokiarchaeia and Thorarchaeia [ 4 , 9 – 11 ], have been found in Heimdallarchaeia [ 9 , 10 ]. We asked whether deep-sea Heimdallarchaeia clades could have a strict anaerobic lifestyle, given the surrounding environment. Similarly, aerobic respiration pathways such as the TCA and oxidative phosphorylation pathway were also found in deep-sea Heimdallarchaeia (Fig. 5 , Supplementary Dataset 6 ). Moreover, both the aerobic kynurenine pathway and aspartate pathway for NAD + de novo synthesis were reconstructed in the present and previously published Heimdallarchaeia MAGs [ 10 , 82 ]. In addition, other proteins involved in oxygen-dependent metabolism and peroxide removal, such as aerotaxis receptors, bacterioferritin, superoxide dismutase (SOD) and catalase (CAT), were identified in MAGs of deep-sea Heimdallarchaeia clades (Fig. 5 ). According to concentrations of dissolved oxygen (DO) in the South China Sea cold seep, there is more than 3 mg/L DO in the surface reduced sediments [ 77 ]. In the present study, the relative proportion of functional genes from Heimdallarchaeia in surface sediments (C1 and C2) is higher than that in deep sediments (C5), indicating that the distribution of Heimdallarchaeia is positively correlated with the concentration of DO (Figure S2 ). Taken together, this evidence strongly suggests that oxygen is present in the environments inhabited by Heimdallarchaeia , which seems to contradict the strict anaerobic lifestyle of other Asgardarchaeota (e.g. Thorarchaeia and Lokiarchaeia ) that occupy the same habitat [ 4 , 10 ]. We speculate this should be a survival strategy for Heimdallarchaeia to adapt to different environments. When living in the microaerobic environment, these Heimdallarchaeia could resist the toxicity of oxygen and use oxygen for physiological metabolism. However, Heimdallarchaeia could also transfer electrons through anaerobic respiration to obtain energy in the strict anaerobic environments. \n Fig. 5 Reconstruction of the mixotrophic lifestyle of Heimdallarchaeia . Solid arrows indicate the enzymes associated with corresponding metabolic pathways identified in Heimdallarchaeia MAGs. Dotted arrows indicate the enzymes associated with corresponding metabolic pathways not identified in 13 MAGs in this study but in other Heimdallarchaeia MAGs, or some metabolic pathways different from the classical pathways in KEGG. Detailed information of key enzymes related to this figure is listed in Supplementary Dataset 6"
} | 8,796 |
34841159 | PMC8613869 | pmc | 5,766 | {
"abstract": "A high-performance\nelastomer was obtained by a multinetworking\nsystem of a covalent bond, hydrogen bond, and clay plane bond. By\ntaking advantage of the characteristics of each cross-linking, the\nthermoplastic elastomer shows excellent compression set resistance,\ngood flowability, and high tensile properties. The hydrogen bond gives\nflowability due to the bond cleavage under heating. The covalent bond\ncontributes to the low compression set by prevention of polymer chain\nflow. Moreover, the clay plane bond affects the tensile properties\nby de-localization of the entire cross-link and increase of hysteresis\nenergy between the clay plane surface and other cross-linking points.\nFurthermore, the self-healing properties and high heat resistance\nand recyclability were also observed.",
"conclusion": "3 Conclusions A high-performance elastomer\nwas obtained by a multinetworking\nsystem of a covalent bond, hydrogen bond, and clay plane bond. By\nusing each feature of the cross-linking bonds, the thermoplastic elastomer\nshows excellent compression set resistance, good flowability, and\nhigh tensile properties. The hydrogen bond shows flowability due to\nthe bond cleavage under heating. The covalent bond contributes to\nthe low compression set by preventing the polymer chain flow. Moreover,\nthe clay plane bond affects the tensile properties by de-localization\nof the entire cross-link and increase of hysteresis energy between\nthe clay plane surface and hydrogen bond cross-link. Furthermore,\nthe self-healing properties and high heat resistance and recyclability\nwere also observed.",
"introduction": "1 Introduction Cross-linking is one of the most important factors for rubber properties.\nSince Charles Goodyear invented sulfur vulcanization in 1839, the\nrubber industry has been developed so far. Since the interaction between\nrubber chains consisting of hydrocarbons is very weak, the rubber\ncannot show good mechanical properties without cross-linking. Therefore,\nthe rubber goods are cross-linked by covalent bonds such as sulfur\nor peroxide cross-links. However, since the covalent bond, which has\na high bond energy, cannot be cleaved by heating, the rubber cannot\nbe remolded. Hence, more than 50% of the worn-out tires are burned\nas a thermal energy source without material recycling. Reusage of\nthe waste vulcanized rubber as a raw material is an urgent issue for\nenvironmental conservation. On the other hand, the thermoplastic elastomer\nis cross-linked by physical interaction between the polymer chains\ninstead of a covalent bond. Since the physical cross-linking can be\neasily cleaved by heating, the thermoplastic elastomer can be remolded.\nHowever, since the bonding energy of the physical interaction is low,\nthe mechanical properties, especially compression set, are poor. A double-network system using different kinds of cross-links had\nbeen applied to the rubber. For example, double networks consisting\nof sulfur and peroxide cross-links had been reported to increase the\nstrength of the rubber. 1 − 6 Moreover, in the field of gel science, double networks using strong\nand weak cross-links have been reported to increase the strength,\nbecause the weak cross-link is destroyed sacrificially to dissipate\nenergy, prior to breaking of the strong cross-linkage. 7 Furthermore, Haraguchi reported that a high-strength gel\ncan be obtained by clay plane cross-linking 8 because clay plane cross-linking points interact with the polar\ngroups of polymers and dissipate energy under external stresses. Consequently,\nsince the stress concentration to the polymer chain is prevented,\nthe strength of the gel increases. Before we have developed\nthermoreversible cross-linking rubber,\nwhich is recyclable, reformable, and has similar mechanical properties\nto vulcanized rubber at room temperature, by using hydrogen bonds. 9 − 12 However, the compression set of the elastomer was high (poor) owing\nto the cleavage of the hydrogen bonds under heating. Compression set\nis one of the most important properties for rubber goods. If the compression\nset is high, the rubber goods cannot be used due to deformation. In this article, we tried to develop a high-performance elastomer\nby using a multinetwork system comprising a covalent bond, hydrogen\nbond, and clay plane bond. The covalent bond is expected to affect\nthe compression set due to the stable cross-linkage. However, introduction\nof a covalent bond may deteriorate the tensile properties. Therefore,\nwe try to further introduce clay plane cross-linking, which is expected\nto increase the hysteresis energy to improve the tensile properties. 13 Moreover, the hydrogen bond gives flowability\ndue to the bond cleavage under heating. Research on a multi-cross-linking\nsystem using a hydrogen bond, covalent bond, and clay bond has never\nbeen reported so far. If the total energy of the covalent bond,\nhydrogen bond, and clay\nplane bond can be the same as that of normal rubber cross-link, the\nmultinetwork elastomer (MNE) may show the same characteristics as\nthe normal rubber at room temperature, and develop fluidity by cleavage\nof the hydrogen bond under heating.",
"discussion": "2 Results\nand Discussion 2.1 Synthesis and Properties As a method\nof introducing hydrogen bonding and covalent bonding moiety to the\nelastomer, addition reactions of maleic anhydride moiety followed\nby reaction of active hydrogen compounds, in the solid phase, have\nbeen used due to the following reasons: (1) Since the addition reaction\nof active hydrogen compounds with the cyclic acid anhydride moiety\nhas been known to proceed effectively, the solid mixing reaction of\na maleated elastomer with active hydrogen compounds was expected to\nproceed effectively. (2) The addition reaction of cyclic acid anhydride\nand active hydrogen compounds was also expected to generate covalent\ncross-linking and strong hydrogen bonding moieties such as carboxylic\nacid, ester, and amide. (3) Since the addition reaction of maleic\nanhydride to elastomer has been widely used in the industry as the\neasiest method to introduce polar groups, 14 , 15 maleated elastomers are easily available on the industrial market. Maleated ethylene-butene elastomer was selected as a base elastomer,\nbecause of its low hardness, low T g , and\nsufficient mechanical properties. The low hardness of the elastomer\nshould be derived from low crystallinity. The introduction reaction\nof active hydrogen compounds to the maleated ethylene-butene elastomer\nwas performed at 180 °C by using an internal mixer ( Figure 1 ). Figure 1 Synthesis of a multinetwork\nelastomer. The reaction of the hydroxyl and\namine groups of various active\nhydrogen compounds with the acid anhydride ring was confirmed by IR\nand solid-state NMR analyses. The introduction of active hydrogen\ncompounds was identified by the disappearance of the absorption peaks\nat 1790 cm –1 due to acid anhydride, as well as the\nexistence of the peaks due to ester or imide. The properties\nof the obtained elastomer are shown in Table 1 . Table 1 Formulations and\nProperties of Test\nSamples run 1 2 3 4 5 maleated\nethylene-butene rubber 100 100 100 100 100 organic clay 5 5 5 3-amino-1,2,4-triazol\n(ATA) 1.26 tris(2-hydoxyethyl)isocyanurate\n(THI) 1.31 1.31 polyether polyol 3.83 sulfamide 0.72 antioxidant a 0.1 0.1 0.1 0.1 0.1 hardness 57 39 66 62 66 tensile strength (MPa) 4.82 1.34 8.22 6.92 5.45 elongation at break (%) 628 108 181 446 545 compression set (%) 80 16 17 39 96 a 3-(3,5-Ditert-butyl-4-hydroxyphenyl)propanoic\nacid. As a hydrogen bond\ncross-linking, 3-amino 1,2,4-triazole was reacted\nwith the maleated ethylene-butene elastomer. 1 − 4 In this case ( Table 1 run 1), although the tensile\nstrength and the elongation at break are sufficient, the compression\nset is high (poor). Cleavage of the hydrogen bonds under heating may\nresult in the high compression set. Next, as a cross-linker that can\ngenerate covalent and hydrogen bonds simultaneously, tris(2-hydroxyethyl)isocyanurate\n(THI) was selected. THI can generate 3 covalent cross-linking points\nand the isocyanurate ring can form hydrogen bonds with carboxylic\nacid and ester, which is generated by the reaction of the maleic anhydride\nmoiety and the hydroxyl group. Although the tensile strength and the\nelongation at break are low, the compression set is low (good). The\nlow compression set might be caused by the stable covalent cross-linking\nunder heating ( Table 1 run 2). Meanwhile, the low tensile strength and the low elongation\nat break should be caused by the lack of hysteresis energy. Since\nstress may concentrate on specific points due to the lack of hysteresis\nenergy, the elastomer structure may be broken quickly. In order to\nimprove the tensile properties by prevention of the stress concentration,\nclay was added ( Table 1 run 3, Figure 1 ).\nClay is expected to increase the hysteresis energy by interaction\nwith the hydrogen bond cross-link. It has been reported that addition\nof clay can increase the strength of the gel. 8 First, bentonite was selected as a clay. However, untreated bentonite\ncannot be dispersed sufficiently in the elastomer. Therefore, organic\nclay was used in order to improve the dispersion. Addition of the\norganic clay before introduction of hydrogen bond and covalent bond\ngave better properties than addition of organic clay after the introduction\nof hydrogen and covalent bond. Addition of the organic clay before\nthe cross-linking may give high dispersion because the cross-linking\nnetwork in an elastomer is surmised to prevent the sufficient dispersion\nof clay. When the organic clay was added before the formation of cross-linking,\nthe tensile strength and elongation at break were improved without\ndeterioration of the compression set. The dispersion of organic clay\nis discussed in “ Section 2.4 ”. Next, in order to find the best cross-linking\nagent that can generate\nthe hydrogen bond and covalent bond, various chemicals were examined.\nAs representative examples, polyetherpolyol and sulfamide are shown\nin run 4 and 5, respectively. Since polyether polyol has 4 same-length\nlinkers, improvement of the compression set was expected by uniformity\nof the length of cross-linking. 16 Since\nsulfamide has a planar structure, increase of mechanical properties\nwas also expected by formation of two-dimensional hydrogen bonds. 17 Although both compounds gave good tensile properties,\nthe compression sets were not sufficient. The reason for the remarkable\nproperties of THI as a cross-linking agent may be the interaction\nof hydrogen bonds of the isocyanurate ring, ester, and carboxylic\nacid, in addition to the stable covalent bonds. 2.2 Effect of Varying Amounts of Organic Clay Successively,\namounts of the organic clay and THI were investigated\n( Table 2 ). Table 2 Effect of Amounts of THI and Organic\nClay run 1 2 3 4 5 6 maleated ethylene-butene rubber 100 100 100 100 100 100 organic clay 0 1 2.5 2.5 2.5 5 tris(2-hydoxyethyl)isosianurate\n(THI) 1.31 1.31 0.983 1.31 1.64 1.31 antioxidant a 0.1 0.1 0.1 0.1 0.1 0.1 hardness 39 57.5 58 61.5 62.5 65.5 100% modulus 1.35 2.08 2.45 3.14 4.51 5.66 300% modulus 3.77 4.97 6.86 tensile strength (MPa) 1.34 3.88 4.69 6.78 7.78 8.22 elongation\nat break (%) 108 291 296 308 233 181 compression set (%) 16 38 39 2 5 17 a 3-(3,5-Ditert-butyl-4-hydroxyphenyl)propanoic\nacid. Since the addition\nof organic clay could increase the hysteresis\nenergy and improve the tensile properties, the amount of organic clay\nwas investigated. Addition of 0, 1, 2.5, and 5 phr (parts per\nhundred rubber, Table 2 runs 1, 2, 4, 6)\nof the organic clay were investigated ( Figure 2 ). In conclusion, elongation at break, which\nindicates dispersion, was highest around 2 phr of the clay. The tensile\nstrength was saturated to 7–8 MPa more than 2.5 phr of the\nclay. Figure 2 Correlation between amount of clay and stress or strain in the\npresence of 1 equiv of THI. 2.3 Effect of Varying Amounts of THI Next,\nin 2.5 phr addition of clay, 0.75 ( Table 2 run 3), 1 ( Table 2 run 4), and 1.25 ( Table 2 run 5) equiv of the hydroxyl group of THI\nagainst the maleic anhydride moiety were investigated ( Figures 3 and 4 ). Since 0.75 equiv of THI is less than the molar stoichiometry of\nmaleic anhydride moiety, the cross-linking density should decrease\nand the elongation at break may be improved. Figure 3 Correlation between amount\nof THI and stress or strain in the presence\nof 2.5 phr of organic clay. Figure 4 Correlation\nbetween amount of THI and the compression set in the\npresence of 2.5 phr of organic clay. Moreover, since 1.25 ( Table 2 run 5) equiv is more than the molar stoichiometry of the\nmaleic anhydride moiety, the branch of THI increase and the cross-linking\ndensity should decrease, which may also improve the elongation at\nbreak. However, 1 equiv ( Table 2 run 4) of THI gave the best elongation at break and the best\ncompression set. It indicates that 1 equiv gave the most efficient\ncross-linking structure. The properties of the obtained elastomer\n( Table 2 run 4) are\nshown in Table 3 . Table 3 Properties of a Multinetwork Elastomer a glass transition temperature\n(°C) b –41.2 5% weight loss temperature (°C) c 405 recyclability d >10 times self-healing temperature (°C) e 140 a Table 2 , run 4. b Determined by DSC. c Determined\nby TGA. d Time of reforming. e Self-adhesion ability. The glass transition temperature\n( T g ) of the obtained elastomer, which\nis observed by a dynamic viscoelasticity\nanalyzer, was almost the same as that of the starting maleated ethylene-butene\nelastomer ( T g : −40.4 °C).\nIt indicates that cross-linkages do not disturb the micro-Brownian\nmotion of the main chain segment. The obtained elastomer has similar\ngood properties at low temperature ( ex . low brittle\ntemperature) to the starting elastomer. Moreover, the 5% weight\nloss temperature was very high (405 °C),\nwhich is higher than that of the general thermoplastic elastomer.\nThe obtained elastomer had thermoplasticity and can be recycled at\nleast 10 times without deterioration of the properties. It was confirmed\nthat the tensile strength after 10 times reforming was maintained\nat 94% (6.37 MPa) of the initial strength. Moreover, the self-healing\ntemperature, which can adhere itself by heating, was confirmed at\n140 °C. The 1 mm thickness sheet having a 5 mm notch was heated\nby elevating the temperature by 20 °C from 60 °C and was\nkept under each temperature for 10 min. Adhesion of the notch was\nconfirmed at 140 °C, which can be derived from the recombination\nof hydrogen bonds (adhesion strength 4.61 MPa, retention rate 68%).\nIntroduction of a covalent bond can improve the compression set, but\ndeteriorate the self-healing property. This is thought to be due to\nthe difficulty of recombination of the cross-linking. Self-healing\nmaterials are important research subjects from the viewpoint of long\nlife and maintenance free, and it is shown that the MNE can be expected\nto be applied as a self-healing material. 18 , 19 2.4 Analysis of Structure 2.4.1 Transmission\nElectron Microscope (TEM) Analysis Figure 5 shows the\nTEM image of a multinetwork elastomer (MNE, Table 1 run 3). The added clay can be confirmed\nto have a black streak shape. The clay size is 200–500 nm and\nis stacked at intervals of several ten nm. The clay is oriented in\nthe direction of the arrow in Figure 5 . Since the sheet was made by press molding, the orientation\ndoes not occur normally. However, the clay seems to be oriented. It\nis considered that the cross-linking is formed by hydrogen bonding\nbetween the carboxyl group and the clay surface. The orientation may\naffect the improvement of tensile properties. Figure 5 TEM image of a multinetwork\nelastomer (MNE). 2.4.2 Dynamic\nMechanical Analysis (DMA) DMA measurements have also confirmed\nan increase in tan δ,\nwhich indicates hysteresis energy, by adding of organic clay. This\nsupports that tensile properties are improved by energy dissipation\nin the multinetwork system (THI and organic clay), compared to the\nHB + CB system (THI) ( Figure 6 ). Figure 6 DMA graph of the obtained elastomer synthesized with 1 equiv of\nTHI and/or 1 phr of organic clay. MNE: multinetwork elastomer, HB:\nhydrogen bond, CB: covalent bond, PB: clay plane bond. 2.4.3 Atomic Force Microscopy (AFM) Measurement AFM measurement of the rubber surface was performed in viscoelastic\nmode to sense the Young’s modulus of the sample ( Figure 7 ). The yellow part of the image\ncorresponds to the high elasticity and the purple part corresponds\nto the low elasticity. Figure 7 AFM images of the elastomers synthesized from 1 equiv\nof THI and/or\n1 phr of organic clay. MNE: multinetwork elastomer, HB: hydrogen bond,\nCB: covalent bond, PB: clay plane bond. In the PB system (organic clay), the low-elasticity part (purple)\nforms a domain of about 20 to 30 nm, and the high-elastic part (yellow)\nsurrounds the area. The low-elastic purple part can be widely observed,\nand the high-modulus portion is thin like a thread. In the HB\n+ CB system (THI), the high-elastic yellow part spreads\nthroughout the sample, but the domain size of the low-modulus portion\nis the same as about 20–30 nm. On the other hand, in the multinetwork\nsystem (THI and organic clay, HB + CB + PB), the domain size of the\nlow modulus was slightly reduced, an intermediate modulus of elasticity\nwas widely observed overall, and the size of the low-modulus part\nis uneven. That is, the modulus of elasticity is dispersed. 2.4.4 Small-Angle Neutron Scattering (SANS) Analysis The\noverall scattering intensity is high over the entire wave number\nin the case of HB + CB system (THI) and scattering peaks are observed\nnear q = 0.03 Å –1 of wave\nnumber. This is d = 20.9 nm (=2π/0.03) when\nconverted to real space size. This size matches the domain size (about\n20–30 nm) of the low-modulus portion as confirmed in the previous\nAFM image. It may represent the spacing of the high-modulus portion\nby the cross-linking portion. In the case of the PB system (organic\nclay), the peak of scattering is observed near q =\n0.03 Å –1 and the scattering intensity is greatly\nreduced in the region with a large wave number compared to the sample\nof THI. The peak of 0.03 Å –1 may indicate the\naggregate structure of the acid anhydride structure. Since the peak\nis the same as the peak in the HB + CB system, the size of the collective\nstructure may not change even if cross-linked. Since the size of clay\nobserved in TEM is 200–500 nm, it is thought that the clay\ncannot be observed in this range of Figure 8 . Figure 8 SANS profiles of the elastomers synthesized\nfrom 1 equiv of THI\nand/or 1 phr of organic clay. MNE: multinetwork elastomer, HB: hydrogen\nbond, CB: covalent bond, PB: clay plane bond. On the other hand, in the multinetwork system (THI and organic\nclay, HB + CB + PB), the scattering intensity near the peak is greatly\nreduced, and concentration fluctuations of 5 to 50 nm size are reduced.\nThis result is consistent with the results of AFM, and the dispersion\nstate of cross-linking points seems to be more uniform ( Figure 8 ). 2.4.5 Infrared\nSpectroscopy (IR) Measurement By adding organic clay to hydrogen\nbonds + covalent bonds (HB +\nCB), it was confirmed that a new absorption peak appeared in 1716\ncm –1 in addition to the absorption of carboxyl groups\nof 1701 and 1734 cm –1 as shown in Figure 9 . This absorption is thought\nto be derived from the hydrogen bonding of the carboxyl groups and\nclay. Figure 9 IR spectra of the obtained elastomer synthesized with 1 equiv of\nTHI, and 1 equiv of THI with 1 phr of organic clay. MNE: multinetwork\nelastomer, HB: hydrogen bond, CB: covalent bond, PB: clay plane bond. 2.5 Speculation of the Network\nStructure The speculated network structure of the cross-linking\npoint is shown\nin Figure 10 . When\nthe covalent bonds of ester were generated by the reaction of the\nmaleic anhydride moiety and the hydroxyl group of THI, the carboxylic\nacids were generated at the same time. The isocyanurate ring can make\ncomplicated hydrogen bonds with the carboxylic acid and the ester.\nMoreover, the organic clay can also make hydrogen bonds with carboxylic\nacids and the isocyanurate rings. Figure 10 Structure of the cross-linking point. Since the TEM image shows good dispersion and orientation\nof the\norganic clay, the structure of the elastomer is speculated to be as\nshown in Figures 11 and 12 . The polymer chains are cross-linked\nby hydrogen bonds and covalent bonds. Moreover, the carboxylic acids\nand isocyanurate rings of polymer may make hydrogen bonds with the\nclay surface and de-localize the overall cross-linking points. Figure 11 Schematic\nstructure of the multinetwork elastomer. Figure 12 Schematic\nstructure of the multinetwork elastomer. The covalent bond cross-link contributed to improve the compression\nset by preventing the polymer chain flow. The hydrogen bond cross-link\ncontributes to improving the flowability because of the cleavage under\nheating. The clay plane bond seems to nonlocalize the overall cross-linking\npoints and increase the hysteresis energy to improve the tensile properties."
} | 5,283 |
35919591 | PMC9277715 | pmc | 5,772 | {
"abstract": "Flexible molds with micro-nano hierarchical structures on the surface were fabricated by a two-step template process using anodic porous alumina as a starting material. The obtained flexible molds could be used to form micro-nano hierarchical pillar arrays on the surface of glass tubes and convex lenses by photo-nanoimprinting. The contact angle characteristics of the surfaces with hierarchical pillar arrays were measured, and it was confirmed that they exhibit superhydrophobic properties with a water-droplet contact angle exceeding 150°. The flexible molds obtained in this study can be used repeatedly and efficiently to form micro-nano hierarchical surfaces with superhydrophobic properties on the surfaces of substrates with various curvatures.",
"conclusion": "Conclusions Flexible molds with hierarchical patterns were fabricated using a hierarchical pillar array prepared by the anodization of Al as a template. Photo-nanoimprinting using the obtained flexible molds enabled the formation of ordered pillar arrays with micro-nano hierarchical structures on the surfaces of tubular substrates and convex lenses. The flexible molds obtained in this study deform following the shape of the substrate, indicating that the flexible molds are effective for preparing fine patterns on surfaces with various curved shapes. The structure of the hierarchical pillars formed on the substrate could also be controlled by changing the geometrical structures of anodic porous alumina used as the starting material. Contact angle measurements of the surfaces with the hierarchical pillar array obtained by this process confirmed superhydrophobic properties with a water-drop contact angle exceeding 150°. In addition, the molds could be used repeatedly, and the water repellency of the surfaces obtained from the 1st to the 10th photo-nanoimprinting remained almost the same. This method is promising for imparting superhydrophobic properties based on micro-nano hierarchical patterns on curved surfaces.",
"introduction": "Introduction Nanostructured surfaces exhibit various functions depending on their surface morphology. 1–4 Nanoimprinting is an effective method for preparing various functional nanostructured surfaces, such as antireflective, water-repellent, and antibacterial surfaces, because it enables the efficient patterning of the controlled fine structures on the substrate surface. 5–14 Nanoimprinting molds are usually fabricated by a combination of electron beam lithography and dry etching. 15,16 However, these methods have some problems, such as the difficulty in fabricating molds with large-area patterns and high-aspect-ratio structures. We have been investigating the formation of ordered fine patterns by nanoimprinting using an anodic porous alumina mold obtained by the anodization of Al in an appropriate acidic electrolyte. 17–19 Anodic porous alumina is characterized by its ability to effectively form ordered nanohole array structures with high aspect ratios, in addition to large areas. 20 Nanoimprinting using anodic porous alumina as a mold is suitable for preparing structures with high aspect ratios and large sample sizes. In the present work, we have expanded the nanoimprinting process using anodic porous alumina molds to the preparation of micro-nano hierarchical structures on curved surfaces. The preparation of fine patterns on curved surfaces is important for the functionalization of the surfaces. As one possible process for the preparation of the fine patterns on the curved surfaces by nanoimprinting, we have demonstrated the nanoimprinting process by using a mold prepared by the anodization of an Al substrate with a given curved surface. 21,22 The problem with this process is that applicable samples are limited to substrates with curvatures identical to the mold curved surface. To overcome this problem, we have introduced flexible molds originating from the anodic porous alumina used as a starting structure. The use of a flexible mold composed of polydimethylsiloxane (PDMS) has been reported in the preparation of fine patterns of polymers on various curved surfaces. 23–25 Since the flexible mold can deform itself to follow the surface shape of the substrate, it is possible to form fine patterns on various curved surfaces using a single mold. In contrast, the sample areas and aspect ratios of the obtained structures were limited in the previously reported process based on mold preparation processes. In addition, to the best of our knowledge, there are no reports of the formation of micro-nano hierarchical structures, which are effective for superhydrophobic surfaces, 26 by nanoimprinting using flexible molds. Here, we describe the preparation of high-aspect-ratio hierarchical pillar arrays on curved surfaces by nanoimprinting using a flexible mold fabricated from anodic porous alumina and their application to superhydrophobic surfaces for the first time. In the present work, the flexible molds with hierarchical structures were prepared by a two-step template process using anodic porous alumina as a starting material. First, anodic porous alumina with hierarchical structures was prepared by an integrated process composed of anodization and selective patterning. 27,28 Next, polymer negatives of anodic porous alumina with the hierarchical structures were formed, and finally, the flexible mold was obtained by molding using the polymer negative as a mold. This process can effectively form high-aspect-ratio micropillars with nanopillar arrays at their tips on curved surfaces. Although the formation of hierarchical structures by nanoimprinting has been reported, a method that can form hierarchical pillar arrays with aspect ratios exceeding 5 on curved surfaces has not been reported. Surfaces with hierarchical pillar arrays formed with high aspect ratios are considered to trap a layer of air between water droplets and the substrate surface more easily than surfaces with hierarchical structures formed with low aspect ratios. According to the Cassie–Baxter model, a surface with air trapped between water droplets and the substrate surface functions as an excellent water-repellent surface. 29 Therefore, this process is expected to be an efficient method for making curved substrate surfaces superhydrophobic. In addition, this process can form a hierarchical pillar array structure with a high aspect ratio, similar to the hierarchical structure found on the sole of a gecko foot, and is expected to be used for preparing structural adhesive surfaces on curved substrates. 30",
"discussion": "Results and discussion \n Fig. 2 shows surface and cross-sectional SEM images of anodic porous alumina with hierarchical structures. The SEM observation of the specimen was performed after the re-anodization of anodic porous alumina with a microcavity array. The low-magnification image of the surface in Fig. 2(a) shows a square lattice array of microcavities with a diameter of approximately 2 μm and a period of 5 μm. This shows good correspondence with the surface pattern of the stamp used for resist formation. From the cross-sectional SEM image shown in Fig. 2(b) , it can be observed that the porous alumina layer in the resist aperture is completely dissolved to the bottom by selective etching. When anodic porous alumina is immersed in an etchant, the etchant penetrates to the bottom of the holes, causing the uniform dissolution of the hole walls from the top to the bottom of the holes and increasing the hole diameter. As etching continues, the hole walls dissolve and the holes coalesce, further dissolving the residue and exposing the residual Al substrate. If a resist mask is formed on anodic porous alumina prior to etching to cover some of the holes, as in this process, the etchant penetrates only inside the holes of the anodic porous alumina at the resist openings, and the hole walls are dissolved. This makes it possible to dissolve only the alumina layer at the resist opening selectively. This is an anisotropic etching process based on the hole array structure of anodic porous alumina. Any acid can be used for etching the alumina layer, but etching can be stopped when the Al substrate is exposed by using an etchant containing chromic acid. This is because when the Al substrate is exposed by etching, a passive film is formed on the Al surface by the effect of the chromic acid, and no further etching occurs. From Fig. 2(b) , it can also be observed that a nanohole array structure is formed at the bottom of the microcavity by re-anodization. The depths of the microcavities and nanoholes in the obtained sample were 8 μm and 220 nm, respectively. The thickness of the resist mask formed on the stamp surface by dipcoating depends on the concentration of the polychloroprene solution and the pull-up speed. If a thick resist is formed by dip coating on the entire micropattern on the surface of the stamp, the resist pattern transferred to the porous alumina surface has no openings. On the other hand, if the resist film formed on the stamp surface is very thin, the holes of anodic porous alumina are not covered by the resist, and the etchant penetrates into all the holes during etching, resulting in dissolution over the entire sample surface. Therefore, to selectively dissolve anodic porous alumina by this process, it is important to control the thickness of the resist film formed on the stamp surface by dip coating. Fig. 2 (a) Surface and (b) cross-sectional SEM images of anodic porous alumina with hierarchical structures. This process can produce anodic porous alumina with a hierarchical structure even when the interhole distance of anodic porous alumina is varied. Fig. 3 shows the results for micro-nano hierarchical structures fabricated from anodic porous alumina with interhole distances of 100, 200, and 300 nm as starting structures. The SEM images in Fig. 3 show that nanohole arrays with hole periods of 100, 200, and 300 nm are formed at the bottom of microholes with a diameter of 2 μm. Fig. 3 SEM images of micro-nano hierarchical structures fabricated from anodic porous alumina with interhole distances of 100, 200, and 300 nm as starting structures. The anodic porous alumina with an interhole period of 100 nm was formed by the anodization of Al in 0.3 M oxalic acid at 40 V and 17 °C for 60 min. The anodic porous alumina with an interhole period of 200 nm was formed by the anodization of Al in 0.05 M oxalic acid at 80 V and 0 °C for 60 min. The anodic porous alumina with an interhole period of 300 nm was performed by the anodization of Al in 0.05 M oxalic acid at 120 V and 0 °C for 60 min. \n Fig. 4 shows SEM images of a micro-nano hierarchical pillar array formed on a flat substrate using the anodic porous alumina shown in Fig. 2 . The low- and high-magnification SEM images of the cross section in Fig. 4(a) and (b) show vertical hierarchical pillars of uniform diameter and height on the substrate. The height of the obtained hierarchical pillars was 8 μm, which corresponded to the depth of the microcavities in the anodic porous alumina used as the starting structure. The high-magnification SEM image of the upper part of the hierarchical pillars in Fig. 4(c) shows the nanopillar array formed at the tip of a micropillar. When nanoimprinting was performed using the anodic porous alumina master with a releasing layer on its surface, a defect-free hierarchical pillar array was formed over the entire surface of the sample. Fig. 4 (a) and (b) Cross-sectional SEM images of hierarchical pillar arrays prepared from anodic porous alumina. (c) High-magnification SEM image of upper part of hierarchical pillars. \n Fig. 5 shows the observation results of a flexible mold fabricated using the hierarchical pillar array shown in Fig. 4 as a template. The photograph in Fig. 5(a) confirms that a flexible mold with a size of 2 cm square was successfully formed. The surface SEM image in Fig. 5(b) shows a square lattice of microholes with a diameter of approximately 2 μm and a spacing of 5 μm. In addition, a nanohole array with an interhole distance of 500 nm was observed to be transferred at the bottom of the microcavities. The cross-sectional SEM image in Fig. 5(c) shows that the depth of the microcavities is 8 μm, confirming that the structure of porous alumina used as the starting structure is retained even after two transfer processes. Fig. 5 (a) Photograph of flexible mold. (b) Surface and (c) cross-sectional SEM images of flexible mold. \n Fig. 6 shows the results of hierarchical pillar arrays formed on the surface of a glass tube with a diameter of 7 mm by photo-nanoimprinting using the flexible mold. Fig. 6(a) shows photographs of the glass tube after photo-nanoimprinting. It can be seen that the nanoimprinted area has turned white. This is due to light scattering in areas with hierarchical pillar arrays formed by nanoimprinting using the flexible mold. The flexible mold fabricated in this study deforms along with the shape of the substrate, making it possible to form a pattern on the entire surface of the glass tube. The optical microscopy image of the imprinted sample shown in Fig. 6(b) confirms that a square lattice of micropillars is formed over a large area. From the SEM images in Fig. 6(c) and (d) , vertical micropillars with a nanopillar array at the tip were observed to form on the curved surface. The flexible mold could be detached from the substrate while deforming even during peeling, making it possible to form hierarchical pillars that maintained an upright structure on each curved surface. Fig. 6 (a) Photographs of specimen after photo-nanoimprinting using flexible mold. (b) Optical microscopy image of specimen. (c) Low- and (d) high-magnification SEM images of hierarchical pillar array formed by photo-nanoimprinting using flexible mold. \n Fig. 7(a) shows the results of preparing hierarchical pillar arrays of different heights on a curved surface by photo-nanoimprinting using a flexible mold. Similar to the experimental results shown in Fig. 6 , a glass tube with a diameter of 7 mm was used as the substrate for nanoimprinting. The surface structure of the flexible mold was controlled by varying the thickness of the porous alumina film used as the starting material. The SEM images in Fig. 7(a) show that the heights of the obtained hierarchical pillars were 5, 8, and 16 μm. Even in the case of the tallest pillar fabricated in this study (16 μm), it was possible to form pillar arrays on a curved surface while maintaining the upright structure. The SEM images in Fig. 7(b) show the results of controlling the height of the nanopillars at the tip of the micropillar. The height of the nanopillars was varied from 200 nm to 420 nm by varying the anodization time from 1 h to 2 h during the preparation of anodic porous alumina with a hierarchical structure. These results show that the geometrical structure of hierarchical pillar arrays formed on curved surfaces by photo-nanoimprinting using flexible molds can be controlled by changing the starting structure. Fig. 7 (a) SEM images of hierarchical pillar arrays with controlled micropillar height. (b) SEM images of hierarchical pillar arrays with controlled nanopillar height. \n Fig. 8 shows the results of micro-nano hierarchical pillar arrays formed by nanoimprinting using a flexible mold on convex lens surfaces with different curvature radii. From the photographs of the specimens, it can be seen that the entire surface of each spechimen is white, for all curvature radii of 52, 26, and 10 mm, indicating that the fine patterns were transferred. SEM observations of each sample also indicated that hierarchical pillar arrays were formed without defects on all of the curved surfaces of different curvature radii. These results indicate that the flexible molds fabricated in this study can be used to form hierarchical pillar arrays not only on two-dimensional curved surfaces, such as cylinders and tubes, but also on three-dimensional curved surfaces such as lenses. We believe this is because the flexible mold made of PDMS can not only deform flexibly but also stretch to follow the convex lens shape. Fig. 8 Photographs and SEM images of nanoimprinted specimens with different curvature radii. \n Fig. 9(a) shows the observed results of hierarchical pillar arrays obtained by the 1st, 5th, and 10th photo-nanoimprinting using the same flexible mold. In all samples, the formation of a 2 μm-diameter, 4 μm-high micropillar array with a nanopillar array at the tip was confirmed. These results show that flexible molds made of PDMS can be used repeatedly. Fig. 9(b) shows the measured contact angles with water droplets exhibited by the surfaces obtained by the 1st through the 10th nanoimprinting. The wettability of the fine patterns obtained by nanoimprinting was evaluated using patterns formed on a smooth substrate rather than on a curved substrate. All of the samples exhibited a water-drop contact angle of approximately 155° and were confirmed to have high waterrepellency. Although the static contact angle could not be measured because of the difficulty in placing the water droplets on the surface, the curved surfaces with the hierarchical pillar arrays were also observed to exhibit high water repellency. Fig. 9 (a) SEM images of hierarchical pillar arrays formed by 1st, 5th, and 10th photo-nanoimprinting using same flexible mold. (b) Contact angles of water droplet on hierarchical pillar arrays."
} | 4,371 |
37609252 | PMC10441418 | pmc | 5,773 | {
"abstract": "Lateral gene transfer (LGT) is an important mechanism for genome diversification in microbial populations, including the human microbiome. While prior work has surveyed LGT events in human-associated microbial isolate genomes, the scope and dynamics of novel LGT events arising in personal microbiomes are not well understood, as there are no widely adopted computational methods to detect, quantify, and characterize LGT from complex microbial communities. We addressed this by developing, benchmarking, and experimentally validating a computational method (WAAFLE) to profile novel LGT events from assembled metagenomes. Applying WAAFLE to >2K human metagenomes from diverse body sites, we identified >100K putative high-confidence but previously uncharacterized LGT events (~2 per assembled microbial genome-equivalent). These events were enriched for mobile elements (as expected), as well as restriction-modification and transport functions typically associated with the destruction of foreign DNA. LGT frequency was quantifiably influenced by biogeography, the phylogenetic similarity of the involved taxa, and the ecological abundance of the donor taxon. These forces manifest as LGT networks in which hub species abundant in a community type donate unequally with their close phylogenetic neighbors. Our findings suggest that LGT may be a more ubiquitous process in the human microbiome than previously described. The open-source WAAFLE implementation, documentation, and data from this work are available at http://huttenhower.sph.harvard.edu/waafle .",
"introduction": "Introduction Lateral gene transfer (LGT), or the movement of genetic material between organisms through means other than vertical inheritance from parent to offspring, is a major force in the evolution and diversification of microbes 1 – 6 . Indeed, studies estimate that 10–20% of genes in some bacterial clades were acquired by LGT 7 – 9 . Also referred to as horizontal gene transfer (HGT), LGT is thought to be an important process in microbial communities, as it may provide recipients with advantageous traits, such as antibiotic resistance, the ability to degrade certain compounds, or survive in different environmental niches 10 . However, the majority of methods for detecting LGT have focused on isolate genomes, making it difficult to assess the prevalence, modes, and functions of LGT in complex microbial communities. Prior studies of LGT in the human microbiome have applied traditional methods of LGT event detection to isolate genomes, typically using phylogenetic approaches based on gene-species tree construction and reconciliation or relying on composition-based inference to identify candidate acquisitions 11 – 16 . Such studies have revealed enrichments for LGT among microbes native to oral and gut sites and that LGT rates were influenced by host 17 , 18 (e.g. lifestyle and geography) and microbial attributes (e.g. phylogeny and ecology) 19 . For example, comparisons of gut microbiomes from Fijian and North American individuals revealed differences in transferred genes linked to diet and geography 20 . Another study found that LGT events occur more frequently in industrialized and urban gut microbial communities 17 . Moreover, while LGT is more common between phylogenetically related species 21 , 22 , this trend was secondary to LGT enrichment among species associated with specific environments, such as the human body 11 . A recent study also found that LGT from maternal gut bacteria may drive infant development through the sharing of functions associated with immunity and dietary changes 10 . These findings are consistent with a proposed theory that LGT is highly adaptive among niche-sharing microbes facing dynamic environmental pressures 17 , 18 , 23 , an idea further supported by observed LGT-induced enrichments for survival-related pathogenicity factors like antimicrobial-resistance genes and carbohydrate usage pathways 11 , 24 , 25 . However, while LGT acts as an ecological force where acquisition of adaptive genes may benefit recipients and promote community stability 19 , LGT could also negatively impact the host and its microbiome 18 , underscoring the need to rigorously study this phenomenon. While studying microbial community LGT from isolate genomes has thus avoided the challenges of culture-independent methodologies, the strategy suffers from several drawbacks. Foremost is that the set of LGT events involving a recipient species must be gleaned from one or a few reference genomes per organism. Therefore, variation in the occurrence and fixation of LGT events within species may go undetected, resulting in a dramatic underestimation of LGT-based strain personalization. Applying these conventional LGT detection methods on metagenome assemblies may also have practical limitations due to the fact that LGT contigs do not bin well, potentially as a consequence of complex flanking repeat regions that can result in loss of coverage 2 . Additionally, assessing LGT from a single reference genome obscures its evolutionary history within environments. For example, evolutionary trajectories vary for human-associated microbes from ancient (i.e. predating the origin of modern humans) to those arising within the host’s lifetime (herein referred to as “recent” LGT events). While such limitations could in principle be ameliorated by assembly of complete microbial genomes, this process is computationally challenging and limited to the highest-coverage species, particularly when analyzing difficult-to-assemble mobile elements. Relatively few methods have been specifically designed to identify and profile LGT events in microbial communities. Notably, general methods of LGT detection based on variability in sequence composition (e.g. Alien_Hunter 26 and DarkHorse 27 ) can theoretically be applied to metagenomic contigs and/or bins, though their effectiveness in that context is unclear. Daisy 28 was among the first methods to specifically target LGT events in microbial communities, which it accomplished by mapping metagenomic reads to putative donor and recipient genomes. The need to pre-specify these genomes limits the method’s utility in comprehensive community LGT profiling, though the more recent DaisyGPS 29 has been developed to aid in donor and recipient selection. Other methods, such as LEMON 30 , follow a related approach of comparing reference genomes with metagenomic reads to identify potential LGT breakpoints in the underlying community strains. In contrast, MetaCHIP 31 identifies LGT events between microbial community members by inspecting their metagenome-assembled genomes (MAGs) for discordance between species and gene trees (independent of external reference genomes). While this design makes MetaCHIP a highly general community LGT profiler, it is expected to lack sensitivity to LGT events occurring outside of a sample’s higher-quality MAGs or involving genetic material from outside the community. Thus, a method that can profile microbial community LGT both broadly and accurately remains an unmet need. To address these issues, we developed a phylogenetically agnostic computational method for novel LGT detection and profiling from shotgun metagenomic assemblies which we call WAAFLE ( W orkflow to A nnotate A ssemblies and F ind L GT E vents). We benchmarked WAAFLE on highly fragmented synthetic assemblies, identifying the majority of expected spiked-in LGT with <0.5% false-positive detection rate and with improved sensitivity compared to existing community-applicable methods. We then carried out the first comprehensive culture-independent profiling of LGT across diverse human body sites, drawing on >2,000 assembled metagenomes from 264 individuals and 16 body sites from the expanded Human Microbiome Project (HMP1-II) 32 . We identified over 100,000 high-confidence novel LGT events (with “novel” defined here as “not previously observed in microbial isolate genomes”). We also experimentally validated high-confidence candidates in a second independent metagenomic cohort. WAAFLE was thus used to interrogate a wide variety of complex microbial communities, and these results considerably expand our understanding of the network of transferring species, functions, and general determinants of LGT among human-associated microbes.",
"discussion": "Discussion Together, these results provide substantial new insights into the landscape of LGT in the human microbiome, enabled by a novel methodology for culture-independent LGT detection and profiling in complex microbial communities. Unlike previous approaches focused on identifying LGT from sequenced isolate genomes 29 , 30 , WAAFLE’s focus on raw unbinned metagenomic contigs improves sensitivity and avoids the challenge of assembling complete microbial genomes from metagenomes. By applying WAAFLE to metagenomic assemblies from the human microbiome, we uncovered >100K putative LGT events across body sites, all of which were novel relative to a microbial isolate genome catalog. These findings not only highlight the vast network of species transferring genetic material within the human microbiome but also expand our understanding of LGT-based strain-level diversity and personalization. While molecular functions enriched in novel LGT events often represented mobile elements, others were of potential adaptive significance. However, in contrast with previous findings 44 , antibiotic resistance was not a dominant function among our newly detected LGT. Instead, our findings highlight the extent to which ORFs of unknown function are laterally transferred and thus likely of previously unrecognized advantage. Expanding upon known determinants of LGT frequency, we found that not only did LGT rates between species vary inversely with phylogenetic separation, but also that spatial proximity (e.g., co-occurrence in biofilms) could overcome this. In addition, we found that a taxon’s abundance was positively correlated with its rate of LGT donation but not receipt. This trend is consistent with a physical model of LGT in which a recipient encounters the cells or free-floating DNA of abundant donors at a higher frequency. Alternatively, treating abundance as a measure of fitness, this pattern could be interpreted as an increased probability of fixation from more fit donors. These trends manifested as two distinct layers of preferential attachment: species commonly participating in LGT with abundant hub species and hubs favoring repeated LGT with phylogenetically similar partners. Surprisingly, more abundant species were not observed to acquire more genes via LGT. While such species have more opportunities to gain LGT, new events enter their populations at a lower frequency, which may act as an antagonistic force to fixation. These findings suggest that integrated biophysical and evolutionary modeling might be fruitful. As a hybrid of reference- and assembly-based approaches, WAAFLE inherits their respective advantages and limitations. Notably, due to its stringent quality control filters to accommodate fragmented assemblies, WAAFLE is sensitive only to LGT events that are essentially fixed within a community. Loci containing potential LGT with coverage differing from adjacent regions—which could occur during a sweep prior to fixation—cannot be reliably distinguished from assembly errors and are excluded, potentially rejecting LGT events that are specific to one lineage (e.g., a strain) or not fixed in the population. In addition, while WAAFLE does not directly map reads to a reference database, it is still dependent on a reference catalog of microbial pangenomes for taxonomic and functional annotation of metagenomic contigs. Incompleteness within this catalog can potentially lead to spurious LGT calls: for example, if a gene is core to a given genus but deleted from the single reference genome of a species X within that genus, WAAFLE may consider the gene to have been acquired by interspecies LGT when observed in new metagenomic strains of X (WAAFLE’s ambiguity and sister-genome filters conservatively remove such cases where possible). Conversely, because WAAFLE is focused on detecting new instances of genetic material entering a species’ pangenome, by design it will not highlight known (and potentially ancient) LGT events within the pangenome when they are re-detected in novel metagenomic strains of the species. This limitation could be addressed in future versions of the software by adding known LGT events as a new layer of pangenome annotation. More generally, future analyses with WAAFLE will benefit from improved reference genome catalogs, specifically from recent efforts to expand the catalog of human-associated microbial genomes 45 – 50 . Because WAAFLE uses metagenomic contigs as input, it is not sensitive to the general challenge of assembling complete genomes from metagenomes. That said, future versions could also be re-tuned for application to metagenomic assemblies of increasing quality or to utilize explicit pre-defined taxonomic binnings, which may aid in disambiguating recipient and donor taxa among candidate LGT embedded within short contigs. WAAFLE is ultimately most sensitive to the reliability of individual contigs and will benefit from new methods to limit or identify misassembly events, including improvement in metagenomic assemblers themselves 51 , as well as downstream filtering methods expanding the junction-coverage approach implemented here 15 . In the future, these limitations of assembly could similarly be bypassed entirely by applying WAAFLE to long-read sequencing data 52 , as long (multi-kilobase) reads share the multi-gene “scope” of short metagenomic contigs still fall short of lengths required by existing LGT methods, and may avoid misassembly issues. While we acknowledge these limitations inherent to our current implementation of a contig-based LGT profiler, we note that our attempted benchmarking efforts of other potentially suitable classical sequence-based LGT detection methods were either not successful (e.g., Alien_Hunter) or highlighted comparatively poorer LGT detection (e.g., DarkHorse). Additionally, we showed that WAAFLE outperformed MetaCHIP 31 , an explicit phylogenetic LGT profiler for detecting within-community LGT, using synthetic benchmarking data. Still, WAAFLE does not incorporate a phylogenetic validation of candidate LGT, suggesting an ongoing and complementary role for methods like MetaCHIP. While many trends were evident from our initial analyses of novel LGT in the human microbiome, much remains to be uncovered. Further investigation of the mechanisms of transfer and fixation of LGT-enriched functions is warranted, particularly those associated with uncharacterized domains or not obviously attributable to mobile element processes. Finally, while WAAFLE-identified LGT are, by definition, novel, more work is needed to formally establish their age. While some candidate events are likely ancient, low levels of adaptation to the recipient genomes, coupled with evidence of stable personalization of LGT across participants here, suggest that novel LGT events arise and fix within individual human microbiomes as part of their long-term developmental dynamics 3 , 53 , potentially influencing host health and disease."
} | 3,826 |
38719821 | PMC11078967 | pmc | 5,776 | {
"abstract": "Self-powered skin attachable and detachable electronics are under intense development to enable the internet of everything and everyone in new and useful ways. Existing on-demand separation strategies rely on complicated pretreatments and physical properties of the adherends, achieving detachable-on-demand in a facile, rapid, and universal way remains challenging. To overcome this challenge, an ingenious cellulose nanofiber-mediated manifold dynamic synergy strategy is developed to construct a supramolecular hydrogel with both reversible tough adhesion and easy photodetachment. The cellulose nanofiber-reinforced network and the coordination between Fe ions and polymer chains endow the dynamic reconfiguration of supramolecular networks and the adhesion behavior of the hydrogel. This strategy enables the simple and rapid fabrication of strong yet reversible hydrogels with tunable toughness ((Value max -Value min )/Value max of up to 86%), on-demand adhesion energy ((Value max -Value min )/Value max of up to 93%), and stable conductivity up to 12 mS cm −1 . We further extend this strategy to fabricate different cellulose nanofiber/Fe 3+ -based hydrogels from various biomacromolecules and petroleum polymers, and shed light on exploration of fundamental dynamic supramolecular network reconfiguration. Simultaneously, we prepare an adhesive-detachable triboelectric nanogenerator as a human-machine interface for a self-powered wireless monitoring system based on this strategy, which can acquire the real-time, self-powered monitoring, and wireless whole-body movement signal, opening up possibilities for diversifying potential applications in electronic skins and intelligent devices.",
"introduction": "Introduction Existing skin-attachable electronics with autonomous powering ability are desired for obtaining accurate and reliable biological/physical information and can be reversibly attached to arbitrary surfaces and detached without leaving residues 1 , 2 . Thus the utilization of reversible adhesion hydrogels is of great significance for self-powered electronic skins (e-skins) 3 , 4 . In the past few years, great efforts have been devoted to realizing the reversible adhesion of hydrogel with a variety of hard and soft materials 5 , 6 . Reversible adhesion can be achieved using chemical connection consisting of reversible bonds 7 , 8 , including dynamic covalent bonds, noncovalent bonds with specific chemical groups, and physically topological entanglement through external stimuli, such as pH 9 , temperature 10 , current 11 , and rays 12 . Suo’s group 7 developed a smart solution adhesive, where the spreading of an aqueous solution of polymer chains on the surface of two adherends to crosslink them together for topological adhesion. To further shorten the interfacial adhesion time and afford the effective interfacial linking between the bulk hydrogel and adherends to transmit force and elicit energy dissipation, there is a need to design a dynamic adhesion strategy with quick and strong long-term adhesion while leaving a window period for reversible easy detachment. In this work, we successfully constructed a supramolecular hydrogel with both reversible tough adhesion and easy photodetachment via an ingenious cellulose nanofiber (CNF)-mediated manifold dynamic synergy strategy (Fig. 1a , b). The comprehensive mechanical properties and cohesive strength of the supramolecular hydrogel can be facilely tuned through UV light-driven photo-Fenton-like (P.F.) reaction between CNF networks and Fe 3+ in the hydrogel material (Fig. 1c, d ). In this way, the interfacial toughness and viscoelasticity can be controlled facilely in a detachable-on-demand way. Combined with the rapid, reliable, and reproducible regulating method, as well as adhesion-on-demand properties based on supramolecular network engineering, the developed convertible hydrogel holds great potential for practical application in photo-detachable self-powered e-skins. Fig. 1 Design of CNF-DA/PAA@Fe 3+ dynamic hydrogel based on a light-driven supramolecular network engineering strategy via photo-Fenton-like (P.F.) reaction. a Schematic illustration of the design of the strong adhesion and photo-triggered detachment mechanism based on the P.F. reaction. b Schematic diagrams of a reversible structure of the adhesive and photo-detachable dynamic hydrogel with light-driven supramolecular network. c Photographs of the molecular switch being peeled off from human skins under UV irradiation and air oxidation. d Comparison of the adhesive strength of photo-detachable hydrogel before and after UV irradiation. Values in d represent their means ± SDs from n = 6 independent samples. Source data are provided as a Source Data file.",
"discussion": "Discussion In summary, we constructed a supramolecular CNF-DA/PAA@Fe 3+ hydrogel with both reversible tough adhesion and easy photodetachment via a CNF-mediated manifold dynamic synergy strategy. The CNF-reinforced network, the physical entanglement between CNF and PAA chains, as well as the strong coordination between Fe 3+ ions and CNF networks endow the excellent reversible mechanical and adhesive properties of the hydrogels. Additionally, the on-demand easy detachment was achieved through the dissociation of coordination complexes when the Fe 3+ ions was reduced to Fe 2+ ions upon exposure to UV light, allowing the adjustability of the supramolecular network to provide the hydrogel with switchable toughness and adhesion. The dynamic polymer network brings about the tunability of the mechanical properties and interfacial toughness (tunability ratio of up to 86%), and on-demand adhesion energy (tunability ratio of up to 93%). Strong and photo-detachable adhesion of the fabricated hydrogel works with diverse substrates, including skin, glass, PDMS, aluminum, PET, PTFE, polyamide, and hydrogel, demonstrating the attractive broad-range adhesion-detachment properties of our hydrogel. In addition, the photo-detachable adhesion strategy based on the P.F. reaction is applicable to other biomacromolecules and petrochemical polymers, including gelatin, chitosan, alginate, starch, PAAm, and PVA, where CNF/Fe 3+ serves as a Fenton-like reagent and the adhesive groups of DA provide initial adhesion. As a proof-of-concept demonstration, a self-healing and mechanically compliant PdA-TENG is fabricated using the CNF-DA/PAA@Fe 3+ hydrogel as the photo-detachable adhesion substrate and the electrode, and a PDMS elastomer as the electrification layer. The self-powered e-skin based on the single-electrode working mode PdA-TENG achieves complex whole-body physiological wireless signal monitoring including blinking, respiring, walking, and major joint motion detections such as knuckle, elbow, and knee. This study demonstrates the potential application of photo-detachable hydrogel in smart e-skins, self-powered biomechanical monitoring systems, and beyond."
} | 1,732 |
39816185 | PMC11730947 | pmc | 5,778 | {
"abstract": "Microalgae offer\na compelling platform for the production of commodity\nproducts, due to their superior photosynthetic efficiency, adaptability\nto nonarable lands and nonpotable water, and their capacity to produce\na versatile array of bioproducts, including biofuels and biomaterials.\nHowever, the scalability of microalgae as a bioresource has been hindered\nby challenges such as costly biomass production related to vulnerability\nto pond crashes during large-scale cultivation. This study presents\na pipeline for the genetic engineering and pilot-scale production\nof biodiesel and thermoplastic polyurethane precursors in the extremophile\nspecies Chlamydomonas pacifica . This\nextremophile microalga exhibits exceptional resilience to high pH\n(>11.5), high salinity (up to 2% NaCl), and elevated temperatures\n(up to 42 °C). Initially, we evolved this strain to also have\na high tolerance to high light intensity (>2000 μE/m 2 /s) through mutagenesis, breeding, and selection. We subsequently\ngenetically engineered C. pacifica to\nsignificantly enhance lipid production by 28% and starch accumulation\nby 27%, all without affecting its growth rate. We demonstrated the\nscalability of these engineered strains by cultivating them in pilot-scale\nraceway ponds and converting the resulting biomass into biodiesel\nand thermoplastic polyurethanes. This study showcases the complete\ncycle of transforming a newly discovered species into a commercially\nrelevant commodity production strain. This research underscores the\npotential of extremophile algae, including C. pacifica , as a key species for the burgeoning sustainable bioeconomy, offering\na viable path forward in mitigating environmental challenges and supporting\nglobal bioproduct demands.",
"conclusion": "Conclusions This study demonstrates the potential of developing extremophile\nmicroalgae as a commercially viable production platform. Here, we\nhave demonstrated the successful transformation of the novel extremophile\nspecies, C. pacifica , into a robust,\nhigh-yield strain. Additionally, pilot-scale cultivation has shown\nthe feasibility of producing biobased fuel and material, highlighting\nthe potential for large-scale applications. By enhancing its resilience,\nemploying genetic engineering, and achieving large-scale growth, we\nhave demonstrated C. pacifica’s potential to produce valuable bioproducts such as biofuels and polyurethane.\nThe versatility of C. pacifica as a\nbiofactory highlights the promise of extremophile algae in a future\nsustainable bioeconomy, offering potential solutions to address climate\nchange and global resource demands. Future research should focus on\noptimizing downstream extraction processes and conducting comprehensive\ntechno-economic analyses to fully realize the potential of C. pacifica for industrial applications.",
"introduction": "Introduction As the 21st century progresses, humanity\nfaces unprecedented challenges\nin the form of climate change and a rapidly growing global population. 1 , 2 These twin crises threaten food security, strain conventional energy\nsources, and exacerbate environmental degradation. 3 − 5 The quest for\nsustainable solutions has never been more urgent, compelling the exploration\nof innovative strategies that can provide the next generation of food\nand fuel without further harming our planet. 6 − 8 In this context,\ndeveloping renewable resources that can reduce carbon emissions and\noffer a viable alternative to traditional agricultural and energy\nproduction methods is critical. The need for such solutions is underscored\nby the increasing scarcity of arable land, the depletion of freshwater\nresources, and the necessity to reduce greenhouse gas emissions. 9 , 10 As we navigate this pivotal moment in human history, the identification\nand cultivation of sustainable, efficient, and versatile sources of\nfood and fuel emerge not just as a scientific endeavor but as a vital\ncomponent of our collective response to the existential challenges\nof our time. Against this backdrop of environmental and societal\nchallenges,\nmicroalgae emerged as a promising solution with the potential to revolutionize\nour approach to sustainable food, fuel, and material production. 11 − 15 Microalgae, with their exceptional photosynthetic efficiency, can\nconvert sunlight into biomass more effectively than traditional crops,\noffering a high-yield, renewable resource that grows rapidly and requires\nminimal inputs. 16 Their cultivation does\nnot compete for arable land, as they can thrive in nonarable environments,\nincluding deserts and saline waters, utilizing nonpotable water and\neven wastewater, thereby reducing the strain on freshwater resources. 17 Furthermore, microalgae are capable of producing\na wide range of valuable bioproducts, from biofuels that can reduce\nour dependence on fossil fuels to high-protein biomass for food and\nfeed, along with bioplastics, pharmaceuticals, and cosmetics. 18 − 22 This versatility positions microalgae as a potential keystone of\na circular bioeconomy, capable of addressing both energy and food\nsecurity while contributing to carbon mitigation efforts. 23 Harnessing the full capabilities of microalgae\nas a sustainable resource remains fraught with obstacles. Scaling\nup production to meet the burgeoning global needs introduces a set\nof complex technical and financial challenges. 24 , 25 Chief among these is the necessity to sustain stable cultivation\nenvironments on a large scale, a task complicated by the risk of contamination\nand the variability of environmental factors, which can precipitate\nculture crashes and disrupt biomass yield. 26 , 27 For example, although Chlamydomonas reinhardtii , a model green alga, thrives in closed bioreactors where growth\nconditions can be carefully controlled, it is not well-suited for\nlarge-scale cultivation in open systems due to the risk of contamination\nfrom zooplankton grazers and other unpredictable environmental factors. 26 , 28 − 32 A promising strategy to navigate the hurdles associated with\nlarge-scale\nmicroalgae cultivation involves focusing on extremophile strains of\nmicroalgae. 33 − 37 These organisms naturally thrive in extreme conditions, such as\nhigh salinity, extreme temperatures, acidic or alkaline environments,\nwhere conventional microalgae strains would struggle or fail to survive.\nBy leveraging the inherent resilience of these extremophiles, it is\npossible to reduce the risk of pond crashes due to environmental stressors,\nthus ensuring a more stable and reliable biomass production. Furthermore,\nthe genetic engineering of these extremophile strains to enhance their\ncellular productivity opens new avenues for bioproduct production\noptimization. 38 − 40 Through targeted modifications, these strains can\nbe engineered to have increased lipid, carbohydrate, or protein content,\nthereby maximizing the yield of desired bioproducts. This dual approach\nof utilizing extremophile microalgae and enhancing their productivity\nthrough genetic engineering not only mitigates some of the challenges\nof stable large-scale cultivation but also paves the way for economically\nviable and environmentally sustainable production of biofuels, food,\nand other valuable bioproducts. This innovative strategy harnesses\nthe robustness of extremophiles against environmental challenges while\npushing the boundaries of their natural productivity, offering a holistic\nsolution to the sustainability puzzle posed by the increasing demands\nfor food and fuel in the face of climate change. In this study,\nwe showcase a complete comprehensive pipeline from\nthe genetic engineering of extremophile microalgae in the laboratory\nto their pilot-scale production for biodiesel and thermoplastic polyurethane\nsynthesis. Our research centers on the novel extremophile species Chlamydomonas pacifica (402 and 403; Chlamydomonas\nResource Center ID, CC-5697, and CC-5699, respectively), which our\nlaboratory discovered in San Diego, California. 41 This microalga is particularly suited for industrial applications\ndue to its exceptional resilience to extreme environmental conditions,\nsuch as high pH, high salinity, and elevated temperatures, its ability\nfor mating and high throughput selection, and its ability for recombinant\ngene expression. 41 Initially, we enhanced\nthe strain’s tolerance to high light intensity through mutagenesis,\nbreeding, and selection. Following this, we employed genetic engineering\ntechniques to develop C. pacifica strains\nthat produce high levels of lipids and starch without compromising\ntheir growth rates. We successfully demonstrated the scalability of\nthese engineered strains by cultivating them in pilot-scale raceway\nponds and converting the resulting biomass into biodiesel and thermoplastic\npolyurethane (TPU). This study is pioneering in showcasing the complete\ncycle of transforming a newly discovered species into a commercially\nrelevant strain. The research highlights the potential of C. pacifica as a key microalgae species in the sustainable\nbioeconomy, offering a viable solution to environmental challenges\nand supporting global demands for renewable resources.",
"discussion": "Discussion The potential of microalgae as biofactories has already been demonstrated\nby their ability to produce a diverse range of bioproducts, including\nbiofuels, nutraceuticals, and biobased chemicals. 11 − 15 , 39 However, large-scale\ncultivation of microalgae continues to present significant challenges,\nprimarily due to uncontrolled growth conditions, the risk of contamination,\nand pond crashes. 26 Our lab recently discovered\na novel extremophile, C. pacifica ,\ndemonstrating remarkable growth resilience under high pH, high temperature,\nand high salinity conditions. 41 Through\nmutagenesis and mating, we have further evolved this strain to tolerate\nhigh light intensity, making it a robust candidate for open pond cultivation.\nThe mating step during laboratory evolution might have altered the\nploidy of the cells from haploid to diploid. However, we suspect that\nthe subsequent generation of the Evolved C. pacifica strain reverted to haploid, as indicated by the presence of vegetative\nor gamete cells observed in the microscopy images ( Figure 3 ). Nonetheless, future studies\ncould focus on identifying the quantitative trait loci (QTLs) in Evolved C. pacifica that are associated with high light tolerance\nto gain a deeper understanding of the genes involved in this adaptation. C. pacifica , belonging to the same\ngenus as the well-studied C. reinhardtii , allows for the opportunity to leverage existing research on C. reinhardtii to accelerate the development of C. pacifica as an industrial strain. Methods to enhance\nbioproduct productivity in C. reinhardtii can be directly applied to C. pacifica . In this study, we improved lipid and starch content in evolved C. pacifica by overexpressing the soybean Dof transcription factor and the PGM1 gene from C. reinhardtii and demonstrated significant product\nenhancements ( Figures 3 and 4 ). Similar genetic engineering strategies,\nsuch as overexpressing chloroplast-type glyceraldehyde-3-phosphate\ndehydrogenase (cGAPDH) to enhance overall biomass productivity or\nutilizing hybrid breeding approaches with selection-enriched genomic\nloci (SEGL) to boost photosynthetic productivity under diverse conditions\ncould be used to further streamline the development of C. pacifica into a robust, high-yield strain. 83 , 84 Here, we successfully demonstrated that the evolved C. pacifica can be grown in pilot-scale raceway ponds,\nleveraging its pH tolerance to avoid contamination and crashes ( Figure 4 ). While the evolved\nwildtype and evolved Dof strains grew well without\nany crashes in three consecutive rounds, the evolved PGM1 strain experienced\na crash in the first round but recovered in the subsequent rounds.\nWe suspect the initial crash was due to contamination of the inoculum,\nprepared under heterotrophic conditions in a medium with acetate as\nthe carbon source at a nearly neutral pH. To mitigate any future contamination\nrisks, preparing the inoculum at a higher pH and using CO 2 captured through direct air capture technologies could be valuable. 85 This approach may reduce contamination risk\nand potentially make the process a net-positive source for the bioeconomy.\nHowever, further life cycle assessment is required to validate this\ncultivation method. 86 Moreover, given the\ngrowing interest in sustainable aviation fuel (SAF) within the airline\nindustry, it would be highly beneficial to test the conversion of\nlipids extracted from the engineered strain of C. pacifica into SAF. 87 , 88 Additionally, performing a Techno-Economic\nAnalysis (TEA) would provide a comprehensive understanding of C. pacifica’s potential as a viable biofactory\nfor producing SAF. Even though, our experiments were conducted\nin an 80 L raceway\npond, representing a small-scale model of larger production systems.\nScaling up any strain presents new challenges, particularly in large-scale\nopen raceway systems, where longer raceways and varying mixing levels\nimpact light availability, nutrient uptake, and gas exchange. 89 Economic feasibility is also a key factor for\nscalability, which has yet to be demonstrated for this new species.\nBiomass productivity plays a crucial role in determining feasibility,\nbut a more thorough evaluation through a TEA is needed to identify\nareas for optimization. Previous TEAs on other species have highlighted\nthe importance of biomass productivity and lipid content. 90 Another crucial factor along the same lines\nis year-round productivity, which was outside the scope of this study\nbut remains an important consideration. C. pacifica likely exhibits seasonal variations in productivity, suggesting\nthat its cultivation may be more suited to certain seasons. Rotating\nit with other strains could enhance overall farm productivity while\nalso disrupting the buildup of strain-specific pathogens, potentially\nprotecting other algae species in the system. 69 , 91 One further challenge is the use of recombinant strains. While\nour experiments were conducted in a controlled greenhouse environment,\nlarge-scale production would require fully open systems, raising concerns\nabout potential strain escape. However, an environmental study using\ngenetically modified C. reinhardtii showed no spread of the recombinant strain into surrounding environments\nover the study period, suggesting that similar risks for C. pacifica may be manageable, though further investigation\nis warranted. 92 Another key consideration\nis that most studies have primarily focused\non extracting a single valuable feedstock, such as lipids, carbohydrates,\nor proteins, from algal biomass. To enhance the economic viability\nof algae, it would be beneficial to optimize downstream extraction\nprocesses to efficiently extract all proteins, carbohydrates, and\nlipids simultaneously. 93 , 94 These can then be converted into\nvaluable products such as therapeutic proteins, animal feed, biomaterials,\nand biofuels. This comprehensive approach would reduce the burden\nand high cost associated with a single commodity and help integrate\nalgae into the mainstream of biotechnology. C. pacifica is an excellent candidate for such tests, given its compatibility\nwith genetic engineering and large-scale cultivation."
} | 3,836 |
26891056 | PMC4758630 | pmc | 5,779 | {
"abstract": "Methane produced by methanogenic archaea in ruminants contributes significantly to anthropogenic greenhouse gas emissions. The host genetic link controlling microbial methane production is unknown and appropriate genetic selection strategies are not developed. We used sire progeny group differences to estimate the host genetic influence on rumen microbial methane production in a factorial experiment consisting of crossbred breed types and diets. Rumen metagenomic profiling was undertaken to investigate links between microbial genes and methane emissions or feed conversion efficiency. Sire progeny groups differed significantly in their methane emissions measured in respiration chambers. Ranking of the sire progeny groups based on methane emissions or relative archaeal abundance was consistent overall and within diet, suggesting that archaeal abundance in ruminal digesta is under host genetic control and can be used to genetically select animals without measuring methane directly. In the metagenomic analysis of rumen contents, we identified 3970 microbial genes of which 20 and 49 genes were significantly associated with methane emissions and feed conversion efficiency respectively. These explained 81% and 86% of the respective variation and were clustered in distinct functional gene networks. Methanogenesis genes (e.g. mcrA and fmdB ) were associated with methane emissions, whilst host-microbiome cross talk genes (e.g. TSTA3 and FucI ) were associated with feed conversion efficiency. These results strengthen the idea that the host animal controls its own microbiota to a significant extent and open up the implementation of effective breeding strategies using rumen microbial gene abundance as a predictor for difficult-to-measure traits on a large number of hosts. Generally, the results provide a proof of principle to use the relative abundance of microbial genes in the gastrointestinal tract of different species to predict their influence on traits e.g. human metabolism, health and behaviour, as well as to understand the genetic link between host and microbiome.",
"introduction": "Introduction By 2050, the human population will grow to over 9 billion people, and in the same time frame, global meat consumption is projected to increase by 73% [ 1 ]. However, intensive food production puts a strain on the environment, and there is a need to produce more food ethically and in a way that does not harm the environment. Methane is a greenhouse gas with a global warming potential 28-times that of carbon dioxide [ 2 ] and ruminants are the major source of methane emissions from anthropogenic activities. Finding means to mitigate methane emissions is an intractable problem, despite large international research efforts. A fundamental problem is that the ruminal microbiota is able to adapt rapidly to intervention methods that have been tried so far—such as different dietary formulations, chemical and biological feed additives, chemo-genomics and anti-methanogen vaccines [ 3 ]. In this study we show that genetic selection of low methane emitting animals is a viable option. The gut microbial ecosystem is particularly important in ruminants due to its ability to convert indigestible fibrous plant material into absorbable nutrients. From the environmental and energetic efficiency point of view, there is a disadvantage in that the anaerobic microbial fermentation process can result in excess hydrogen that is used by methanogenic archaea to produce methane and then eructed into the atmosphere. The loss of feed gross energy as methane has been estimated at 2 to 12% [ 4 ]. In order to address food security as well as economic and environmental impacts of food production, sustainable intensification has been suggested [ 5 ] with genetic improvement of feed conversion efficiency of highest importance in farm animals. Therefore, the overall aim of our work was to improve the efficiency of the rumen microbial community in converting feed into nutrients with minimal production of methane. The host animal provides the environment for the microbial ecosystem in the rumen and may therefore have an impact on its composition and efficiency. Studies in rodents and humans suggest that there is a host genetic influence on the microbiome [ 6 – 9 ]. In addition, research in bovine and ovine indicates that there is a host genetic influence on methane emissions and feed conversion efficiency without considering and evaluating the impact of the microbiome [ 10 – 13 ]. Our previous study found a phenotypic correlation between the composition of the rumen community and methane emissions [ 14 ]. However, direct evidence for a genetic control of the microbiota by the host in ruminants is rather weak. Therefore, the main aim of this study was to investigate whether there is a genetic influence of the host on the ruminal microbial community which affects methane production. If the genetics of the host animal has a significant role in determining key activities of the microbiota, then breeding would be a cost-effective tool to reduce methane emissions and improve feed conversion efficiency, provided that an accurate selection criterion is available. Therefore, this study also aimed to find the best selection criterion for mitigation or improvement of these traits. Metagenomics allows the identification of the composition of the whole microbial community, as well as the abundance of their genes. It could be used to develop new selection criteria for difficult-to-measure traits or to understand the link between host genetics, the microbiome and its activity. Our study design allowed us to provide an insight into the genetic influence of the host animal on methane production by archaea, the impact of diets on methane emissions and their interactions with the host genetics. We found novel selection criteria related to microbial characteristics of each host which can be used to select for low methane emitting animals. Specifically, the relative abundance of microbial genes, identified in a metagenomic analysis, was highly informative for predicting methane emissions, but also for other traits such as feed conversion efficiency, and is recommended for exploitation in genetic selection of hosts or to understand the additive genetic link between host genetics and microbiome. Host selection based on a functional microbiome microarray containing microbial genes associated with methane emissions, feed conversion efficiency, health and other traits will provide a novel and cost-effective selection opportunity without measuring these difficult and costly to record traits and has the potential to enable large scale breeding for these performances. This study was carried out using cattle but the identified best microbial criterion (microbial composition, genes and pathways) to achieve insight into the host-microbiome interactions should be transferable to other traits and species.",
"discussion": "Results and Discussion The results are based on a 2 × 2 factorial design experiment of crossbred breed types and diets in which methane emissions of individual animals were measured in respiration chambers and the microbial community was determined by qPCR targeting the 16S rRNA gene. The experiment was designed using sire progeny groups to estimate the genetic control of the host on methane emissions due to change in microbial community. Substantial variation in methane emissions and microbial community among hosts The basis for an efficient selection program to mitigate methane emissions due change in microbial community depends on the genetic variation of these characteristics among animals. There were large phenotypic ranges in methane emissions between the extreme low and high emitting animals within breed type and diet groups ( Fig 1 , S1 Table and S1 Dataset ). The differences in daily methane emissions between the extremes within crossbred breed type were similar between diets suggesting that the diet effect represents only a scaling effect. If at least part of the variation is influenced by the host animal, then selection for mitigation of methane emissions is expected to be efficient. Even larger variation was obtained for relative archaeal abundance measured as archaea:bacteria ratios, within breed type and diet, as shown by coefficients of variation in the range of 35% to 50% and 39% to 65% for forage and concentrate-based diets respectively. 10.1371/journal.pgen.1005846.g001 Fig 1 Distribution of methane emissions and archaea:bacteria ratios within breed type and diet. The box plot shows the large variation and range of methane emissions (per day or per kg DMI = dry matter intake) and archaea:bacteria ratios within crossbred breed type (AA = Aberdeen Angus sired, LIM = Limousin sired) and diet (CON = concentrate based diet, FOR = forage based diet). The total number of animals in the 2 × 2 factorial design experiment was 68. Breed types and diets are changing methane emissions and microbial community A first indication for a host genetic influence on methane emissions and on the composition of the microbial community can be derived from breed type differences. The least squares means (LSM) for daily methane emissions were significantly different at 184 g/d and 164 g/d for the Aberdeen Angus (AA) and Limousin (LIM) breed types respectively, but not significantly different for methane emissions per kg dry matter intake (DMI) ( Table 1 ). These results indicate that the significant difference between breed types in daily methane emissions were due to higher feed intake of AA (11.3 ± 0.36 kg and 10.2 ± 0.36 kg DMI for the concentrate and forage based diet, respectively) compared to LIM (9.8 ± 0.37 kg and 8.8 ± 0.36 kg, for the same diets, respectively). Animals offered the forage based diet had significantly higher methane emissions than those offered the concentrate based diet. This difference is due to higher propionate production from fermentation of starch in concentrate diets, which leads to less hydrogen being available for methanogenesis [ 15 – 17 ]. Estimated LSM for archaea:bacteria ratios taken from live or slaughtered animals were significantly different for diet effects, but not for breed type effects. In the interpretation of the breed type results, it has to be considered that this effect represents only an expected 2/3 of the additive genetic contribution of the sire breed and that non-additive genetic effects can also have an impact. Consistent with diet effects on methane emissions, low archaea:bacteria ratios were obtained for animals offered the concentrate- in comparison to forage-based diet. The differences in archaea:bacteria ratios between diets were 3.7 for both rumen contents samples taken from live and slaughtered animals, suggesting that these measurements can be used interchangeably. 10.1371/journal.pgen.1005846.t001 Table 1 Comparison of least squares means (LSM) for the breed type and diet effects on methane emissions and archaea:bacteria ratios. Trait Breed type LSM SE P-value Diet LSM SE P-value Methane g/day Aberdeen Angus sired 183.8 5.63 <0.0001 Forage 205.2 5.72 <0.0001 Limousin sired 164.4 5.83 <0.0001 Concentrate 142.9 5.75 <0.0001 Breed type difference 19.4 0.0196 Diet difference 62.3 <0.0001 Methane g/kg DM Aberdeen Angus sired 17.37 0.555 <0.0001 Forage 21.63 0.566 <0.0001 Limousin sired 17.96 0.575 <0.0001 Concentrate 13.69 0.564 <0.0001 Breed type difference -0.59 0.463 Diet difference 7.94 <0.0001 Archaea:Bacteria ratio in live animals Aberdeen Angus sired 5.53 0.498 <0.0001 Forage 6.86 0.520 <0.0001 Limousin sired 4.41 0.536 <0.0001 Concentrate 3.09 0.519 <0.0001 Breed type difference 1.12 0.132 Diet difference 3.77 <0.0001 Archaea:Bacteria ratio in slaughtered animals Aberdeen Angus sired 4.47 0.347 <0.0001 Forage 6.52 0.347 <0.0001 Limousin sired 4.88 0.351 <0.0001 Concentrate 2.82 0.351 <0.0001 Breed type difference -0.41 0.407 Diet difference 3.70 <0.0001 Host genetics affects methane emissions Sire progeny groups differences were used to identify the host genetic influence on methane emissions. Estimates of LSM for daily methane emissions among sire progeny groups showed significant differences ranging from 136 to 205 g/d ( Fig 2 ). In contrast to the breed type effects, there were also significant differences between LSM for sire progeny group effects on methane emissions relative to the amount of feed consumed ( Fig 2 ). Slightly different rankings of sires based on methane emissions per day in comparison to those based on per kg DMI are likely due to differences in feed intake among sire progeny groups. In some cases, the differences in methane emissions between sire progeny groups were even larger than the differences between the diets, indicating a substantial genetic influence of the host animal. The differences in LSM for methane emissions among sire progeny groups, as well as the similar ranking (r = 0.6) when methane emissions are expressed per day or per DMI, indicate that there is a direct genetic influence of the host on the rumen microbial methane production independent of the amount of feed consumed. Population genetic studies using beef cattle [ 10 ], dairy cattle [ 11 ] and sheep [ 12 , 13 ] lend supporting evidence for a genetic influence of the host on methane production. The advantage of the present study is that methane was measured using the considered “gold standard” measurement technique of respiration chambers and that the genetic and diet effects, as well as their interaction were estimated in a powerful experimental design under standardised conditions. 10.1371/journal.pgen.1005846.g002 Fig 2 Host genetic effects on methane emissions and relative microbial abundance. Host genetic effects were estimated by least squares means (± standard errors, different letters above bars indicate significant different estimates) of sire progeny groups (AA = Aberdeen Angus sired, LIM = Limousin sired) adjusted for diet, respiration chamber and randomized block effects. Relative microbial abundance was calculated as archaea:bacteria ratio. Absence of interactions between host genetics and diets There were no significant interactions between breed type (or sire) and diet effects in the present study. The absence of interactions indicates that the genetic ranking of sires would not change according to diet. This observation is of substantial importance for implementation of this approach within genetic improvement programmes, and should be confirmed in further independent studies. These results provide fundamental insight into the regulation of methane emissions, indicating that there is an additive genetic influence of the host on methane production by the archaea and that the genetic influence of the host on methane emissions does not change with the diet. The scaling effect of the diet on methane production could be adjusted for in genetic models. In contrast, if interactions between host genetics and diet are present, it would be necessary to use more complex selection strategies. Host genetics shape the microbiome The host additive genetic influence on the microbiome was estimated based on differences in the archaea:bacteria ratio in rumen contents among sire progeny groups. Our earlier studies showed that the archaea:bacteria ratio in rumen contents from live animals can be used to predict methane emissions with a reasonable phenotypic correlation of 0.49 [ 14 ]. Other methods have also be investigated to predict methane emissions of animals e.g. the use of laser methane detector in sheep or beef cattle [ 18 ] and milk mid-infrared spectra in dairy cows, however, further discussion are beyond the scope of this study [ 19 ]. The ratio of archaea:bacteria in the rumen contents sample from each animal was more informative than the absolute amount of those microbes, most likely because the ratio is e.g. independent of dilution effects and differences in PCR amplification of 16S rRNA genes between samples. Comparison of sire progeny group estimates for the archaea:bacteria ratio (taken from live animals shortly after they left the respiration chambers) with those of methane emissions measured as g/day and kg/DMI showed similar ranking with correlations of r = 0.8 and 0.65, respectively. In addition, similar rank correlations (r = 0.72 and 0.67, respectively) with methane emissions were found for the archaea:bacteria ratios based on rumen contents samples taken in the abattoir even after a time lag between leaving the respiration chambers and slaughter of up to 15 days ( Fig 2 ). Most of the deviations in ranks were due to two small progeny groups associated with the highest standard error. Therefore, the general consistency in ranking of sire progeny groups based on microbial and methane emission levels provides evidence that there is an additive genetic influence of the host on the rumen microbial community and their metabolic activity to produce methane. Thus, the archaea:bacteria ratio could be used as selection criteria for reduction of methane emissions. In particular, the rumen samples taken in the abattoir could be used to test a large cohort of sire progeny groups to accurately estimate their breeding values for methane emissions. Using the same experimental data, in a previous study we reported similar correlations between methane emissions per kg DMI and archaea:bacteria ratio in rumen contents samples taken in the abattoir as those taken from live animals [ 14 ], which opens up many opportunities to collect rumen microbial information as a basis for mitigating methane emissions and other traits such as feed conversion efficiency. Metagenomic gene abundance and methane emissions To investigate the use of microbial gene abundance as an alternative selection criterion to mitigate methane emissions, the extreme animals for methane emissions within breed type and diet were selected and their rumen microbial genes determined using a metagenomic analysis. A previous study showed that the metagenomic data are highly informative, e.g. that higher abundance of the Proteobacteria Succinovibrionaceae was significantly associated with low emitting animals [ 20 ]. The high methane emission group had 88% higher emissions than the low group ( S1 Fig ). In the metagenomic study, 3970 KEGG genes ( S2 Dataset ) were identified in rumen contents samples taken in the abattoir, of which 1570 genes were used based on the relative abundance of more than 0.001% and the predictability within the univariate GLM analysis. The relative abundance of microbial genes is expected to be more informative than their absolute abundance because it is e.g. independent from dilution effects and the difference in amplifications of the genes between samples. Based on the relative abundance of these 1570 KEGG genes, we carried out a network analysis and found distinct functional clusters of gene networks ( Fig 3A and S2 Table ). In particular, cluster 4 and 6 formed a distinct group compared to all other clusters. Interestingly, these two clusters contained most genes known to be associated with methane metabolism (e.g. KEGG database pathway information). In contrast, in some other clusters, microbial genes directly or indirectly related to methane production occurred only sporadically e.g. cluster 13 ( phosphoenolpyruvate carboxylase ), cluster 21 ( acetate kinase ) and cluster 26 ( tetrahydromethanopterin S-methyltransferase subunit B) . These clusters comprise only on a small number of genes and are well dispersed and distinct from cluster 4 and 6. 10.1371/journal.pgen.1005846.g003 Fig 3 Functional clusters of microbial genes identified using network analysis. (A) Correlation analysis of microbial gene abundance was used to construct networks, where nodes represent microbial genes and edges the correlation in their abundance. Networks were clustered using the MCL algorithm and the profiles of clusters 1 to 6 are shown. Each chart represents the average abundance of genes in a cluster across the animals studied. Animals are ordered alternately being low (red bar beneath plot) and high methane emitter (yellow), whereby the first and last 4 bars represent animals offered concentrate (green) and forage (light blue) diets, respectively. See S2 Table for KEGG genes associated within each cluster. (B) Microbial gene networks of cluster 4 and 6 contained most of the microbial genes associated with methane metabolism; explicitly shown in yellow are the KEGG genes identified by the PLS analysis to be most closely associated with methane emissions (see Fig 4 and S3 Table ). To identify the importance of the different microbial genes to predict methane emissions we performed a partial least squares analysis, firstly on all microbial genes in cluster 4 and 6 and thereafter only on those genes directly stated in the literature or in the KEGG database to be involved in the methane metabolism pathway [ 20 – 24 ]. For further discussion of the microbial genes within metabolic pathways of methane metabolism see our previous study [ 20 ]. Using this information, the relative abundances of 20 microbial genes explained (including the diet effect) 81.7% of the variation in methane emissions ( Fig 4 and S3 Table ). The identified microbial genes are only in cluster 4 and interact closely with each other ( Fig 3B ). Excluding the diet effect from the model reduced the explained variation in methane emissions only slightly to 77.1%. However, inclusion of the diet effects in the prediction equation is recommended because then the influence of the microbial enzyme genes on methane emissions is estimated whilst taking diet effects into account. Based on a regression analysis of methane emission on the relative abundance of different microbial genes within diet we will later show that the slope of the regression lines are similar for the different diets and only shifted to a different level depending on the diet, as we would expect for a fixed effect. In general, the analysis suggests that methane emissions for the large cohort of animals necessary to obtain accurate genetic breeding values could be predicted accurately from the relative abundance of these 20 KEGG genes. 10.1371/journal.pgen.1005846.g004 Fig 4 Heatmap of the relative abundance of microbial genes associated with methane emissions as identified in the partial least squares analysis. The relative abundance of microbial genes (blue = low to yellow = high) changed depending on methane emissions (g/kg DMI) for the animals selected for low and high methane emissions within breed type and diet. The labels on the horizontal axis indicate the crossbred breed type (AA = Aberdeen Angus sired, LIM = Limousin sired), diet (CON = concentrate based diet, FOR = forage based diet) and the amount of methane emissions (g/kg DMI). All analysed genomes of methanogenic archaea carry the methyl-coenzyme M reductase alpha subunit ( mcrA ) gene, which catalyses the last step in the methanogenesis [ 25 , 26 ]. Comparing the mcrA gene abundance between the low and high emission groups (170% increase) resulted in a highly significant difference ( S2 Fig ). An association between mcrA gene abundance and methane emissions has been reported in dairy cattle [ 27 ] and sheep (at the transcriptomic level) [ 28 ], whilst this gene is recommended for monitoring the process performance of anaerobic digesters [ 29 ]. Another identified archaeal gene was formylmethanofuran dehydrogenase subunit B ( fmdB ), which is also involved directly in methanogenesis and catalyses the reversible reduction of CO 2 and methanofuran via N-carboxymethanofuran (carbamate) to N-formylmethanofuran, the first and second steps in the methanogenesis from CO 2 [ 30 , 31 ]. For this gene, the high methane emissions group showed 173% greater relative abundance than the low group ( S3 Fig ). Within each of these genes, similar slopes of the regression lines for the diets provided were obtained which indicates that there were no interactions between microbial gene abundances and diet effects ( S4 and S5 Figs). This is consistent with the absence of interactions between sire and diet effects described earlier. There was only a constant effect relating to the different diets, which can be considered as a fixed effect in the genetic evaluation model. The study of [ 28 ] found a significant association between methane emissions and KEGG genes using data from metatranscriptomic, but not metagenomic, sequencing. In contrast, we obtained significant associations in data from metagenomic sequencing. This may be partly due to the higher statistical power of the present experiment, with a difference between selected high and low methane emitter groups of 11.8 g / kg DMI compared to 4.4 g / kg DMI in the study of [ 28 ]. Metagenomic gene abundance and feed conversion efficiency The relative KEGG gene abundances need to be determined only once in a metagenomic study and can then be further used to investigate their relationship with other potential traits. In this study we also analysed association with feed conversion ratio and found that 49 genes were important to predict this trait and explained 88.3% of the variation (including breed type and diet effects) and 85.5% (excluding these effects) as illustrated in Fig 5 and summarised in S4 Table . Most of those genes were in clusters 2 and 5, indicating a close network of the genes associated with feed conversion efficiency ( S3 Fig and S2 Table ). However, these clusters were much more disperse and closer connected to other clusters than the clusters associated with methane. The reason is most likely that the animals were selected on the basis of extreme methane emissions, which provided more power to distinguish microbial gene networks associated with methane emissions than those related to feed conversion efficiency. The microbial genes associated with feed conversion efficiency encoded enzymes involved in host-microbe interactions (e.g. GDP-L-fucose synthetase ( TSTA3 ), L-fucose isomerase ( FucI )), the synthesis of amino acids and vitamins (e.g. anthranilate phosphoribosyltransferase , uroporphyrinogen III methyltransferase ), degradation of amino acids and proteins (e.g. aminopeptidase ), enzymes associated with genetic information processing (e.g. aspartyl-tRNA synthetase ) and membrane processes ( cobalt/nickel transport system permease protein ). None of the 49 genes associated with feed conversion ratio were associated with methane emissions. Of particular interest is the abundance of TSTA3 and FucI , which may reveal the importance of host-microbe cross talk in ruminants. These two genes related to feed conversion efficiency are involved in fucose metabolism. Fucose is a component of innate immunity glycoproteins (mucins) produced by the intestinal mucosa [ 32 ] and in saliva to help maintain the integrity of the mucosal barrier. ‘Fucose sensing’ has been identified as an important cross-talk between the intestinal microbiome and host tissues in studies with mice [ 33 ] and rabbits [ 34 ]. The degradation of mucins often requires enzymes from a range of bacteria, but some Bacteroides and Ruminococcus spp. are able to degrade mucins completely [ 35 ]. In particular, a cluster of bacterial genes involved in fucose uptake (FucP: L- fucose permease ) and fucose utilisation (FucI: L- fucose isomerase ; FucA: L- fucose aldolase ; and FucK: L- fucose kinase ) are controlled by a transcriptional repressor gene (FucR: L- fucose operon activator ). FucR also controls bacterial signal production affecting host production of fucosylated glycans (i.e. mucins). This provides a mechanism to match bacterial demands for fucose with supply [ 35 ] and so affect the development of the microbiome [ 34 ]. 10.1371/journal.pgen.1005846.g005 Fig 5 Heatmap of the relative abundance of microbial genes associated with feed conversion efficiency as identified in the partial least squares analysis. The relative abundance of microbial genes (blue = low to yellow = high) changed depending on feed conversion ratio (kg feed intake/kg growth) for the animals selected for low and high methane emissions within breed type and diet. The labels on the horizontal axis represent the breed type (AA = Aberdeen Angus sired, LIM = Limousin sired), diet (CON = concentrate based diet, FOR = forage based diet) and the feed conversion ratio (kg daily feed intake / kg daily growth). Ross et al. [ 36 ] used the vector of counts of sequenced reads aligned to each contig in a database to create the metagenomic relationship matrix. We used an alternative approach of aligning the reads to identify the microbial genes first and then using the relative abundance of those genes to predict their influence on the trait of interest. The approach used in this study may have the advantage that the abundance of the microbial genes is highly related to the activity of the microbial ecosystem in the rumen. Possible mechanisms of host control of the gut microbiome The mechanisms behind genetic influences of the host on the microbial community composition are expected to be based on many different biological factors. The pH of ruminal digesta is known to have a substantial effect on the microbial community structure and diversity in the rumen ecosystem. Saliva contains bicarbonate and tends to maintain rumen pH between 6 and 7 [ 37 ]. Adult cattle produce a substantial amount of saliva with an average of 150 L/day [ 38 ], though with substantial variation that is most likely influenced by host genetics as well as diet [ 39 ]. Furthermore, differences in bicarbonate secretion, short chain fatty acid absorption and passage rate of protons out of the rumen, all affect ruminal pH [ 37 ] and may be partly genetically determined. Variation in the physical structure and size of the rumen, as well as the intensity of contractions and rate of passage of digesta are all expected to have an influence on the rumen microbial community. Lower methane emissions were found in sheep with small rumens, most likely as a result of reduced digesta retention time [ 40 ]. Digesta retention time in ruminants has been shown to be heritable [ 41 ]. A recent review illustrates the highly complex interactions between microbiome and host [ 42 ], with the concept of microbiome-gut-brain axis interactions emerging. For example, the host’s central nervous system affects the gut microbiome through satiation signaling peptides affecting nutrient availability, hormones such as cortisol released by the hypothalamus-pituitary-adrenals axis during stress regulating gut contraction and integrity, and the immune system can be activated to alter gut flora—which links to ‘fucose sensing’ as discussed earlier based on our results. Microbial characteristics as selection criteria We showed that there is an additive genetic effect of the host animal on the amount of methane produced by cattle via effects on the rumen microbial community. As a consequence, the characteristics of the rumen microbial community of each host (e.g. archaea:bacteria ratio) can be used as selection criteria to mitigate methane emissions. Even better prediction of methane emissions were obtained by using the relative abundance of microbial genes of each host. The relative abundance of rumen microbial genes can be related to any other trait associated with rumen function. In the present study, we demonstrated this for feed conversion efficiency, but there may also be associations between the relative abundance of microbial genes and animal health, meat quality, animal behaviour, milk composition, fatty acid composition, etc. Deep metagenomic sequencing remains relatively costly; therefore a functional metagenomic microarray to cost-effectively determine the relative abundance of rumen microbial genes would be a useful development. This would provide the opportunity to develop new selection strategies for these difficult to measure traits (e.g. methane emissions, feed conversion efficiency, animal health, and animal behaviour)—similar to the adoption of genome-wide selection in dairy breeding [ 43 ]. Using a reference population in which the traits of interest are measured, the prediction equations could be developed based on the relative abundance of rumen microbial genes and then used to predict e.g. methane emissions of other animals based only on the microbial composition in rumen contents samples without measuring the traits directly. Alternatively, the relative abundance of rumen microbial genes could be used directly for selection using e.g. relative weights equivalent to their effects on methane emissions. In addition, the recommended approach enables us to understand the biological significance of the specific genes in order to add further confidence for the application of the prediction equations. We may go further and hypothesize that selection of the host genetics based on the microbial gene abundances may be more efficient for improvement of feed conversion efficiency than using measured feed intake per unit of weight gained because the true conversion of feed may be more strongly related to the rumen microbial metabolism than to the measured feed conversion ratio, which are influenced by other factors (e.g. errors in measurements of feed intake and weight gain). A further attraction of this approach is that the relative abundance of microbial genes can be based on rumen samples taken from either live or slaughtered animals so that efficient selection strategies can be developed using potential breeding animals as well as their slaughtered relatives. A potential adverse consequence of a successful selection of animals for reduced methane may be the accumulation of the substrate gas, H 2 , which is a product of fermentation by acetate and butyrate producing microorganisms, and that this accumulation would suppress fermentation rates in the rumen [ 44 ]. This result was founded mainly upon pure-culture studies in which H 2 accumulation by a single H 2 -producing bacterial species resulted in thermodynamic inhibition of fermentation and growth [ 44 – 46 ]. Co-culture with a methanogen relieved this inhibition. As the main cellulolytic species are H 2 producers, it was feared that preventing methane emissions would lead to H 2 accumulation which would in turn slow fibre breakdown. The effects of H 2 concentration are in fact much more complex [ 16 ]. Studies in gnotobiotic lambs lacking methanogens [ 47 ] and inhibiting methane emissions in goats and cattle using experimental halogenated compounds [ 48 ] suggested that growth was normal and other effects such as on feed intake were minor. The overall outcome of inhibiting methanogenesis seems to be fairly neutral, neither beneficial nor detrimental [ 49 – 51 ] although further research is necessary to clarify this issue. More generally, the results may open up opportunities to use the relative abundance of microbial genes in the gastrointestinal tract of different species to predict their influence on traits e.g. health and behaviour. There is substantial evidence in humans that individuals harbour different microbial communities in their gut, with implications for host health in areas as diverse as obesity, cognitive function and allergy [ 52 – 54 ]. Experiments in rodents indicate a host-driven regulation of the gut microbiota that is genetically encoded [ 6 – 8 ]. Research in humans also indicates a host genetic influence on the gut microbiota using the abundance of microbial taxa, in particular the family of Christensenellaceae , which formed a co-occurrence network with other bacteria and with methanogenic archaea and impacts metabolism [ 9 ]. Here, we show that the use of the abundance of the microbial genes is much more closely associated with metabolism than the abundance of the microbial community (for which the archaea:bacteria ratio was the best predictor [ 14 ]) and therefore a much better criterion to predict the host genetic influence on those traits. In addition, specific microbial genes, their networks and pathways can be used to better understand the association between host genetics and microbial activity related to the trait of interest. This could provide opportunities for personalized medicine considering the genetic link between host and microbiome and its activity, e.g. for treatment of inflammatory bowel disease in humans, which showed strong host-microbe interactions [ 55 ]."
} | 9,133 |
29115043 | PMC5743825 | pmc | 5,780 | {
"abstract": "Summary A successful bioeconomy depends on the manifestation of biorefineries that entirely convert renewable resources to valuable products and energies. Here, the poorly exploited hemicellulose fraction ( HF ) from beech wood organosolv processing was applied for isobutanol production with Corynebacterium glutamicum . To enable growth of C. glutamicum on HF , we integrated genes required for d ‐xylose and l ‐arabinose metabolization into two of 16 systematically identified and novel chromosomal integration loci. Under aerobic conditions, this engineered strain CA rXy reached growth rates up to 0.34 ± 0.02 h −1 on HF . Based on CA rXy, we developed the isobutanol producer strain CI sArXy, which additionally (over)expresses genes of the native l ‐valine biosynthetic and the heterologous Ehrlich pathway. CI sArXy produced 7.2 ± 0.2 mM (0.53 ± 0.02 g L −1 ) isobutanol on HF at a carbon molar yield of 0.31 ± 0.02 C‐mol isobutanol per C‐mol substrate ( d ‐xylose + l ‐arabinose) in an anaerobic zero‐growth production process.",
"conclusion": "Conclusions In the presented study, we systematically identified 16 landing pads, which represent prominent loci for chromosomal integration of additional genetic information in C. glutamicum . As a proof of concept, we integrated synthetic operons into two CgLPs that enabled growth on d ‐xylose and l ‐arabinose as well as on a so far unexploited hemicellulose fraction derived from beech wood organosolv processing. For the first time, we showed isobutanol production with engineered C. glutamicum based on pentoses within this fraction. The work demonstrates the suitability to microbially convert complex side streams to valuable products, enabling a holistic exploitation of renewable resources in biorefinery approaches. Moreover, the proposed chromosomal integration loci can be prospectively used as basis for metabolic engineering in future studies.",
"introduction": "Introduction The future shortage of fossil oil and energy resources raises the demand for a sustainable bioeconomy which mitigates greenhouse gas emissions, relies on alternative energies and exploits renewable material streams and value chains. Biorefineries play a key role in processing lignocellulosic materials (reviewed in Cherubini, 2010 ; Valdivia et al ., 2016 ; Rabaçal et al ., 2017 ) but require a high efficiency in holistically converting the input biomasses in an economic manner to marketable products and energies. Because of the complexity and variability of the lignocellulosic feed, side streams evoke from conversion technologies such as the organosolv processing or fast pyrolysis, which are tedious to exploit and therefore limit the overall efficiency of the applied biorefinery approach. With respect to their abundance, hemicelluloses, which constitute between 15% and 35% of lignocellulosic biomass (Sauer et al ., 2014 ), have initiated much consideration for biotechnological applications (Álvarez et al ., 2016 ). However, they are still commonly wasted (Gírio et al ., 2010 ) due to their complexity, limited accessibility for microorganisms and potential to form toxic components (e.g. weak acids and furan derivatives). During the organosolv processing, a mixture of lignocellulose, organic solvent (e.g. ethanol), water and catalysts (e.g. sulfuric acid) is heated to 180–210°C, which fractionizes fibres (cellulosic material) and a black liquor (containing lignin and hemicelluloses; reviewed in Brosse et al ., 2017 ; Zhao et al ., 2017 ). After recovery of the organic solvent by distillation, the black liquor is diluted with water to yield precipitated lignin and the remainder liquid HF (Zhao et al ., 2009 ). Cellulosic fibres can be enzymatically saccharified and used for fermentation purposes (Zhao et al ., 2017 ) and high purity lignin fractions for example for functionalized materials, fuels, biodegradable polymers or adhesives (Brosse et al ., 2011 ; Liu et al ., 2015 ). Typically, the HF comprises weak acids, sugars (e.g. d ‐xylose, l ‐arabinose, d ‐glucose, d ‐mannose, d ‐galactose), furan derivatives, phenolic residues and other extractives, and was proposed to be used for fermentation and production of chemicals (e.g. xylitol, furfural) (Zhao et al ., 2009 ). Still, due to its complexity, the HF remains difficult to access. The need for technologies that utilize the HF without further laborious treatments lies therefore at hand. Microorganisms generally possess a versatile metabolism allowing in principle the conversion of such complex substrate mixtures to value‐added products through fermentation processes. In this study, we applied the industrial workhorse Corynebacterium glutamicum , which has a long tradition in biotechnological production of amino acids but is also exploited for the biosynthesis of organic acids, alcohols and specialty chemicals (Liebl, 2005 ; Becker and Wittmann, 2015 ). This Gram‐positive, facultatively anaerobic bacterium (Nishimura et al ., 2007 ) is robust and accepted as suitable candidate for future biorefinery applications (Jojima et al ., 2013 ). Previously, C. glutamicum has been engineered to produce isobutanol, a next‐generation biofuel and precursor for chemical synthesis of rubber and specialty chemicals, from glucose (Smith et al ., 2010 ; Blombach et al ., 2011 ; Yamamoto et al ., 2013 ). Alternative carbon source utilization has been implemented in tailored strains (Leßmeier et al ., 2015 ) and harnessed for production of e.g. l ‐lysine from pretreated hemicellulosic materials (Gopinath et al ., 2011 ). However, hemicelluloses such as the organosolv‐derived HF have not been assayed for isobutanol production so far. Although tools for genetic engineering, omics and systems level analysis of this industrial workhorse are available (Kirchner and Tauch, 2003 ; Eggeling and Bott, 2005 ; Wendisch et al ., 2006 ; Burkovski, 2015 ; Cho et al ., 2017 ; Lee and Wendisch, 2017 ), there is still a need for suitable chromosomal sites to integrate genetic information, such as synthetic operons, to expand the metabolism for enhanced substrate consumption or production purposes. This issue was the moving cause to systematically identify suitable gene integration loci in this study. We inserted synthetic operons for d ‐xylose and l ‐arabinose metabolization into two of these sites to enable aerobic growth and anaerobic isobutanol production on HF with engineered C. glutamicum strains.",
"discussion": "Results and discussion Identification of Corynebacterium glutamicum landing pads (CgLPs) Metabolic engineering aims at enhancing the substrate or product spectrum of microorganisms, which is a crucial prerequisite to fully exploit their biotechnological potential. This essentially requires the integration of additional genetic information into the host chromosome to circumvent the inherent disadvantages of plasmid‐based gene expression. So far, no general strategy to identify suitable spots for insertion was formulated. To propose such gene integration loci (designated as C. glutamicum landing pads, CgLPs), we harnessed the knowledge about transcription units (Pfeifer‐Sancar et al ., 2013 ), non‐essential gene clusters (Unthan et al ., 2014 ) and prophage regions (Kalinowski, 2005 ). First, the three prophage regions of C. glutamicum [CGP1 (cg1507‐cg1524), CGP2 (cg1746‐cg1752) and CGP3 (cg1890‐cg2071)] were excluded from the search for relevant integration sites (Kalinowski, 2005 ). Although they were shown to be non‐relevant for ordinary growth under laboratory conditions, the overall function is to date not clarified in depth and a genetic stability is not guaranteed (Baumgart et al ., 2013 ). Second, we contemplated non‐essential chromosome sections in the published list of deletable regions (Unthan et al ., 2014 ). These provide ideal arrays for the integration of genes and exclude lethal effects that arise from disruption of essential genetic structures. Third, the non‐essential regions were analysed for suitability regarding knowledge about transcription start sites, operon structures and Rho‐independent termination sites (Pfeifer‐Sancar et al ., 2013 ). In total, 16 landing pads were identified throughout the chromosome as suitable spots for integration of additional genetic information (cf. Table 1 , Fig. S1 ). All CgLPs locate after a Rho‐independent terminator of the upstream gene and are succeeded by a downstream gene stop or start codon at > 50 bps spacing (Fig. 1 , Table 1 ). The distance between the CgLP and the upstream gene terminator was chosen between 10 and 40 bps depending on the size of the intergenic region. Integration of synthetic gene constructs should in general provide a strong termination site to minimize downstream effects. Two of the identified integration loci, CgLP4 and CgLP12, were exemplarily used in this study for integration of synthetic operons for d ‐xylose and l ‐arabinose metabolization respectively (cf. Fig. 1 , Table 1 ). Table 1 Compilation of identified C. glutamicum landing pads (CgLPs) for chromosomal integration of additional genetic information \n C. glutamicum Landing Pad Base Position a \n Adjacence b \n Spacer c \n Upstream gene d \n Downstream gene/operon e \n Experimental verification CgLP1 97220 ◁ ⌇ ⋂ ◄ 20 cg0121 cg0120 – CgLP2 287966 ► ⋂ ⌇ ◁ 20 cg0327 cg0328 – CgLP3 f \n 558101 ► ⋂ ⌇ ◁ 40 cg0634 ( rplO ) g \n cg0635 – CgLP4 836158 ► ⋂ ⌇ ▷ 10 cg0901 cg0902 \n xylAB \n CgLP5 837445 ► ⋂ ⌇ ▷ 20 cg0903 cg0904 – CgLP6 f \n 857008 ► ⋂ ⌇ ▷ 20 cg0928 g \n \n rrnB \n – CgLP7 1205320 ◁ ⌇ ⋂ ◄ 20 cg1302 cg1301 ( cydA ) – CgLP8 1427460 ► ⋂ ⌇ ▷ 40 cg1538 ( coaE ) g \n cg1540 – CgLP9 2741407 ► ⋂ ⌇ ◁ 40 cg2880 cg2883 – CgLP10 f \n 2971748 ◁ ⌇ ⋂ ◄ 40 cg3112 ( cysZ ) g \n cg3111 – CgLP11 3077633 ► ⋂ ⌇ ▷ 10 cg3212 cg3213 yes h \n CgLP12 3094266 ► ⋂ ⌇ ▷ 20 cg3227 ( lldD ) cg3228 \n araBAD \n CgLP13 3191992 ► ⋂ ⌇ ▷ 10 cg3344 cg3345 yes h \n CgLP14 f \n 3213531 ▷ ⌇ ⋂ ◄ 10 cg3365 ( rmpC ) cg3364 ( trpA ) g \n – CgLP15 3229705 ◁ ⌇ ⋂ ◄ 10 cg3385 ( rhcD2 ) cg3384 – CgLP16 3248838 ◁ ⌇ ⋂ ◄ 40 cg3397 cg3396 – \n a. Referring to the C. glutamicum ATCC 13032 complete genome NCBI reference sequence: NC_006958.1 . \n b. ⌇ = CgLP; ⋂ = Terminator loop; ◄, ► = upstream gene; ◁, ▷ = downstream gene; arrowheads indicate direction of adjacent genes. \n c. Spacer between the predicted end of terminator site (Pfeifer‐Sancar et al ., 2013 ) and the CgLP position. \n d. Delivers the terminator site. \n e. In succession of the CgLP. \n f. Directly adjacent to the non‐essential gene cluster [outside location CgLP3 (80 bps), CgLP6 (39 bps), CgLP10 (342 bps), CgLP14 (123 bps)]. \n g. Gene outside (up‐ or downstream) the non‐essential gene cluster (Unthan et al ., 2014 ); downstream gene is located inside the non‐essential gene cluster. \n h. Were used in our laboratories and are evidentially feasible (data not shown). John Wiley & Sons, Ltd Figure 1 Schematic chromosomal location of C. glutamicum landing pads (Cg LP ) for chromosomal integration of genetic information. The synthetic operons P tuf ‐ xyl AB ‐T rrnB and P tuf ‐ ara BAD ‐T rrnB for d ‐xylose and l ‐arabinose metabolization were inserted exemplarily into Cg LP 4 and Cg LP 12 respectively. P tuf : promoter of the C. glutamicum elongation factor EF ‐ TU (cg0587); T rrnB : terminator of the E. coli rrnB operon; xyl AB : genes encoding XylA (xylose isomerase) of Xanthomonas campestris and XylB (xylulokinase) of C. glutamicum ; ara BAD : encoding AraB ( l ‐ribulokinase), AraA ( l ‐arabinose isomerase) and AraD ( l ‐ribulose‐5‐phosphate 4‐epimerase) of E. coli \n MG 1655. Arrows indicate gene direction. \n d \n ‐ Xylose and l \n ‐ arabinose metabolization in CArXy To enable growth of C. glutamicum on d ‐xylose and l ‐arabinose as abundant components of the organosolv‐derived hemicellulose fraction, we integrated the synthetic operons P tuf ‐ xylAB ‐T rrnB and P tuf ‐ araBAD ‐T rrnB into CgLP4 and CgLP12 respectively, yielding the strain CArXy ( C. glutamicum Δ pqo Δ ilvE Δ ldhA Δ mdh CgLP4::(P tuf ‐ xylAB ‐T rrnB ) CgLP12::(P tuf ‐ araBAD ‐T rrnB ); cf. Fig. 1 ). Cloning, isolation and purification of plasmids, PCR fragments or genomic DNA, and procedures for strain construction are given in the Appendix S1 , where a detailed list of the applied bacterial strains, plasmids and oligonucleotides (cf. Table S1 ) is also provided. In brief, the integration of both synthetic operons into the chromosome harnessed a previously published method (Schäfer et al ., 1994 ) for plasmid‐based (pK19 mobsacB ) gene disruption and allelic exchange by homologous recombination. We designed homologous flanking regions of > 500 bps to specifically locate the additional genetic information to designated CgLPs. The two synthetic operons express the xylAB genes encoding XylA (xylose isomerase) of Xanthomonas campestris and XylB (xylulokinase) of C. glutamicum and araBAD encoding AraB ( l ‐ribulokinase), AraA ( l ‐arabinose isomerase) and AraD ( l ‐ribulose‐5‐phosphate 4‐epimerase) of E. coli MG1655 under control of the constitutive promoter of the C. glutamicum elongation factor EF‐TU (cg0587, P tuf ) and are terminated by the E. coli rrnB operon terminator (T rrnB ) respectively, following already published operon architectures (Schneider et al ., 2011 ; Meiswinkel et al ., 2013 ). First, we characterized growth of C. glutamicum CArXy in shaking flask cultivations for single and combined metabolization of d ‐glucose, d ‐xylose and l ‐arabinose. CArXy reached a growth rate (μ) of 0.39 ± 0.03 h −1 , a biomass/substrate yield (Y X/S ) of 0.52 ± 0.02 g CDW per g d ‐glucose and showed a biomass‐specific uptake rate (q S ) of 4.18 ± 0.16 mmol d ‐glucose per g CDW per h (cf. Fig. 2 A). All growth parameters were identical to previously described values (Buchholz et al ., 2014 ) for the wild type of C. glutamicum and indicate that integration of both synthetic operons does not negatively interfere with the strain's vitality under standard cultivation conditions. Furthermore, C. glutamicum CArXy grew on d ‐xylose and l ‐arabinose with rates of 0.18 ± 0.02 h −1 and 0.16 ± 0.01 h −1 , respectively (cf. Fig. 2 B, C). Previous studies using plasmid‐based expression of araBAD (Schneider et al ., 2011 ) or xylAB (Meiswinkel et al ., 2013 ) yielded maximal rates of 0.31 h −1 or 0.20 h −1 respectively. In our experiments, a full consumption of the pentoses was not achieved at the end of cultivation (78 ± 7% of d ‐xylose and 14 ± 4% of l ‐arabinose metabolized). Poor l ‐arabinose uptake can be explained by a high Monod constant (Schneider et al ., 2011 ) and could be overcome by additional expression of the transporter araE , which was shown to also improve d ‐xylose consumption (Sasaki et al ., 2009 ). Combined supplementation of d ‐glucose, d ‐xylose and l ‐arabinose showed a clear preference for the consumption of the hexose compared to the pentoses (cf. Fig. 2 D), a fact that has been described previously for C. glutamicum (e.g. Kawaguchi et al ., 2008 ; Radek et al ., 2014 ). In contrast to the isomerase pathway, the Weimberg pathway enables a more carbon efficient utilization of d ‐xylose and allows a parallel consumption of d ‐xylose and d ‐glucose in C. glutamicum (Radek et al ., 2014 , 2016 ). However, the maximal net generated biomass (4.7 ± 0.4 g CDW L −1 ) was doubled with respect to sole d ‐glucose (2.2 ± 0.1 g CDW L −1 ), and the higher cell density allowed a full consumption of d ‐xylose and 80% of l ‐arabinose within the given cultivation time (cf. Fig. 2 D). Figure 2 Shaking flask cultivations of the strain CA rXy ( C. glutamicum Δ pqo Δ ilvE Δ ldhA Δ mdh Cg LP 4::(P tuf ‐ xyl AB ‐T rrnB ) Cg LP 12::(P tuf ‐ ara BAD ‐T rrnB )) in a modified CGXII minimal medium based on the literature (Eikmanns et al ., 1991 ; Keilhauer et al ., 1993 ) with either combined or single supplementation of 25 mM \n d ‐glucose, d ‐xylose and l ‐arabinose. Bacterial growth (cell dry weight, CDW ) and substrate consumption are depicted over time. Cultivations were performed in 50 ml medium in 500 ml baffled shaking flasks on a rotary shaker at 120 rpm and 30 °C. Detailed information concerning strain construction, medium, seed train and cultivation conditions is given in the Appendix S1 . Error bars represent the standard deviation ( SD ) of three independent experiments. In summary, the strain CArXy functionally expresses the synthetic operons in the identified CgLPs enabling d ‐xylose and l ‐arabinose metabolization without negatively influencing the cell's general viability under given conditions. Aerobic growth on the hemicellulose fraction The aqueous hemicellulose fraction (HF) was derived from a beech wood ethanol/water organosolv processing after lignin precipitation (without enzymatic hydrolysis and further purification procedures) as a black liquor with high viscosity (Ludwig et al ., 2014 ). A description of the short pretreatment procedure extracting water‐soluble compounds is given in the Appendix S1 . To investigate aerobic growth of C. glutamicum CArXy (cf. Table S1 ) on the HF, shaking flask cultivations were performed (cf. Fig. 3 A, B, Fig. S2 ). In contrast to previous studies, in which engineered C. glutamicum was shown to proliferate on aci d ‐pretreated lignocelluloses such as rice straw and wheat bran in minimal medium (Gopinath et al ., 2011 ), growth in the presence of organosolv‐derived HF was only manifested upon additional supplementation of 5 g of yeast extract (YE) L −1 (data not shown) and was therefore included in all following experiments. In minimal medium with 5 g YE L −1 and 9.7 g HF L −1 , 19.3 g HF L −1 or 38.7 g HF L −1 combined with 5 g YE L −1 , CArXy showed growth rates of 0.14 ± 0.03 h −1 , 0.34 ± 0.02 h −1 , 0.33 ± 0.01 h −1 and 0.17 ± 0.02 h −1 and maximal net generated biomasses of 0.29 ± 0.06, 1.02 ± 0.15, 1.50 ± 0.11 and 2.19 ± 0.41 g CDW L −1 respectively. A consecutive consumption of acetate and the pentoses d ‐xylose and l ‐arabinose was found, and the depletion of acetate coincided with an arrest of the exponential growth phase (cf. Fig. 3 A, B, Fig. S2 ). Figure 3 Aerobic cultivation (A, B) of the strain CA rXy ( C. glutamicum Δ pqo Δ ilvE Δ ldhA Δ mdh Cg LP 4::(P tuf ‐ xyl AB ‐T rrnB ) Cg LP 12::(P tuf ‐ ara BAD ‐T rrnB )) and anaerobic isobutanol production (C, D) with CI sArXy ( CA rXy harbouring pJC 4 ilv BNCD ‐ pnt AB and pBB 1 kivd ‐ adhA ) using the hemicellulose fraction ( HF ). A. CA rXy was cultivated in CGXII minimal medium supplemented with 5 g YE L −1 as reference (open circles) and variable concentrations of hemicellulose fraction ( HF ) [9.7 g HF L −1 (triangles), 19.3 g HF L −1 (squares) and 38.7 g HF L −1 (diamonds)] + 5 g YE L −1 . B. Consumption of acetate (circles), d ‐xylose (triangles) and l ‐arabinose (squares) is depicted for the respective experiment using 38.7 g HF L −1 . C. Zero‐growth isobutanol production was realized with the strain CI sArXy using 77.3 g HF L −1 + 5 g YE L −1 in sealed 100 ml flasks containing 50 mL CGXII medium. D. Metabolization of d ‐xylose and l ‐arabinose during the incubation is shown. Error bars represent SD of three independent experiments. Detailed information concerning strain construction, medium, seed train and cultivation conditions is given in the Appendix S1 . Although substrate consumption is still improvable, we show the capability of C. glutamicum to grow efficiently on HF which in general opens the opportunity to exploit this biorefinery side stream for microbial production of chemicals and fuels. Two‐stage isobutanol production To prove our concept, we aimed to utilize HF for the production of isobutanol under anaerobic conditions. Therefore, we transformed CArXy with the plasmids pJC4 ilvBNCD ‐ pntAB and pBB1 kivd ‐ adhA , which enabled isobutanol production in C. glutamicum (cf. Table S1 , Blombach et al ., 2011 ). Then, the resulting strain CIsArXy was applied in a zero‐growth production processes (Lange et al ., 2016 ), where an aerobic stage was implemented to generate biomass that is used in a subsequent anaerobic, growth‐arrested phase to produce isobutanol at high cell densities (cf. Fig. 3 C, D). Under anaerobic conditions, we observed a simultaneous metabolization of d ‐xylose and l ‐arabinose (cf. Fig. 3 D, acetate was not consumed cf. Fig. S3 ), which directly served as substrate for isobutanol production (cf. Fig. 3 C). No significant production of lactate or succinate (< 0.4 mM) was found. About 15.5 ± 0.6 mM (46 ± 1%) and 1.7 ± 0.0 mM (43 ± 1%) of d ‐xylose and l ‐arabinose were metabolized respectively, and CIsArXy produced 7.2 ± 0.2 mM of isobutanol within 28 h of cultivation. With respect to the analysed pentoses, a carbon molar product/substrate yield (Y P/S ) of 0.31 ± 0.02 C‐mol isobutanol per C‐mol substrate ( d ‐xylose + l ‐arabinose) was achieved, which is already in the range of d ‐glucose‐based processes with engineered C. glutamicum strains (0.15–0.52 C‐mol C‐mol −1 ; Blombach et al ., 2011 ; Smith et al ., 2010 ; Yamamoto et al ., 2013 ). Isobutanol production based on the pentoses d ‐xylose and l ‐arabinose has so far not been demonstrated and therefore represents a promising example for the valorization of HF within a novel value chain. As a future perspective, a dual‐phase process (Lange et al ., 2016 ) is apparent, where an aerobic growth based on acetate within the HF would be directly followed by an anaerobic isobutanol production phase based on the remaining pentoses."
} | 5,446 |
32630740 | PMC7409166 | pmc | 5,782 | {
"abstract": "Reactive pyritic mine tailings can be populated by chemolithotrophic prokaryotes that enhance the solubilities of many metals, though iron-reducing heterotrophic microorganisms can inhibit the environmental risk posed by tailings by promoting processes that are the reverse of those carried out by pyrite-oxidising autotrophic bacteria. A strain (IT2) of Curtobacterium ammoniigenes , a bacterium not previously identified as being associated with acidic mine wastes, was isolated from pyritic mine tailings and partially characterized. Strain IT2 was able to reduce ferric iron under anaerobic conditions, but was not found to catalyse the oxidation of ferrous iron or elemental (zero-valent) sulfur, and was an obligate heterotrophic. It metabolized monosaccharides and required small amounts of yeast extract for growth. Isolate IT2 is a mesophilic bacterium, with a temperature growth optimum of 30 °C and is moderately acidophilic, growing optimally at pH 4.0 and between pH 2.7 and 5.0. The isolate tolerated elevated concentrations of many transition metals, and was able to grow in the cell-free spent medium of the acidophilic autotroph Acidithiobacillus ferrooxidans , supporting the hypothesis that it can proliferate in acidic mine tailings. Its potential role in mitigating the production of acidic, metal-rich drainage waters from mine wastes is discussed.",
"conclusion": "4. Conclusions This study provides further evidence to explain how C. ammoniigenes , a moderately acidophilic, heterotrophic actinobacterium, can be found and can proliferate in mine pyritic tailings, where primary producers, such as chemolithotrophic acidophiles like A . ferrooxidans , sustain the growth of heterotrophic iron-reducing bacteria, which may contribute to mitigating the formation of acidic, metal-rich waters from mineral tailing dumps.",
"introduction": "1. Introduction Waste materials from metal mining, such as mineral tailings, have little or no economic value, making their exploitation not profitable. In the context of mine management, tailings have the potential to pose a long-term threat to the environment. Mine tailings are one of the major waste products generated during the mining of metal ores, and have variable physical and chemical compositions, dependent on the ore body being processed and the mining operations [ 1 ]. Following the crushing and grinding of ores (comminution), target minerals are segregated from other (gangue) minerals by froth flotation. The fine-grain mineral wastes produced (tailings) may account for up to 99% of the primary ore body [ 2 ]. While the mineralogical composition of tailings is highly variable, they frequently contain significant amounts of potentially acid-generating minerals, such as pyrite (FeS 2 ), though the acidity generated in fresh tailings can be neutralized by basic materials, such as lime (CaO), that are often added to enhance froth flotation [ 3 ]. The dissolution of sulfide minerals requires water and an oxidizing agent, which may be either molecular oxygen or ferric iron, and may occur in either aerobic or anaerobic (micro) environments via mechanisms that have been widely reported [ 4 ]. In many cases, the potential for acid generation greatly exceeds the neutralization potential of tailings, and liquors within and draining from tailing deposits can become highly acidic, and enriched with soluble transition metals derived from the dissolution of residual sulfidic (e.g., chalcopyrite; CuFeS 2 ) and other minerals. In addition, such waters are highly toxic to most life-forms [ 5 ]. The reactivity of pyritic mine tailings derives from their small particle size, and their content of acid-generating and metal-rich sulfidic minerals [ 6 ]. There have been a number of studies on the microbiology of tailing deposits located in different parts of the world [ 7 , 8 , 9 , 10 ]. Indigenous prokaryotes include well-known chemolitho-autotrophic acidophiles, such as Acidithiobacillus and Leptospirillum spp., and chemolitho-heterotrophic species (e.g., Ferrimicrobium and Ferroplasma spp.), which use the ferrous iron and/or reduced sulfur as electron donors. Most species of iron-oxidizing acidophiles that oxidize iron when oxygen is present can also use ferric iron as an alternative electron acceptor in anaerobic environments [ 11 ]. Some species of obligately heterotrophic acidophiles, including Acidiphilium, Acidocella and Acidobacterium which are able to reduce ferric iron but not oxidize ferrous iron, have also been identified in mine tailings [ 8 ]. Interestingly, heterotrophic acidophilic bacteria that reduce iron attach to sulfide minerals and form biofilms. Pyrite particles colonized with Acidiphilium and Acidocella spp. were found to be less susceptible to accelerated oxidation by mineral-oxidizing acidophiles, and a technique based on this observation, referred to as “bioshrouding”, was suggested as a method of partially securing reactive mine wastes [ 12 ]. In a series of mesocosm experiments, set up to examine how engineering the microbial communities of reactive mine tailings could be used to limit the generation of acidity and the release of metals [ 13 ], it was found that those that had either not been inoculated, or had been inoculated only with a mixed culture of iron-oxidizing chemolithotrophic acidophiles, became heavily colonized (~ 3 × 10 6 colony forming units/g), within 12 months, by a bacterium that was identified (from its partial 16S rRNA gene sequence) as a strain of Curtobacterium ammoniigenes (99% gene similarity). This bacterium (and two others that were also isolated from the tailings) was inferred to have originated from the tailings themselves, and had not been completely eradicated by pre-treatment of the tailings, which were washed with strong (3 M) sulfuric acid to remove the residual lime. C. ammoniigenes is a heterotrophic, ammonium-oxidizing actinobacterium, the type strain of that had been isolated from water weeds growing in highly acidic (pH 2–4) swamps adjacent to acid sulfate soils in Vietnam [ 14 ]. There have been no previous reports of this bacterium in acidic mine-impacted environments, such as acid mine drainage, biomining sites, or waste rock and tailings deposits. Curtobacterium ammoniigenes strain IT2 has been shown to be a moderate-acidophile and an obligate heterotroph, which tolerates elevated concentrations of many transition metals, and also catalyses the dissimilatory reduction of ferric iron. These characteristics infer that is has a potential role in mitigating the formation and migration of acidic, metal-rich waters from tailings dumps.",
"discussion": "3. Results and Discussion Pale yellow colonies of strain IT2 were observed, after 7 days of incubation, on yeast extract/fructose solid medium pH 3.5. Cells were non-motile rods of irregular shape, and did not appear to produce endospores. The isolate did not oxidize elemental sulfur or ferrous iron autotrophically or heterotrophically. It grew poorly on defined single carbon sources, but the addition of small amounts of yeast extract promoted growth on sugars, fructose in particular, presumably due to the requirement of one or more growth factors ( Table 1 ). The isolate was able to use a relatively limited range of defined organic substrates, including glucose, fructose and galactose, compared with Acidobacterium capsulatum and Acidiphilium cryptum also found in mine tailings. Its substrate range differed from other acidophilic bacteria found in mine tailings [ 20 ]. All of the amino acids tested inhibited the growth of the bacterium at the concentrations tested (i.e., growth was less than in the presence of yeast extract alone), even though they grew well on tryptone soya broth. It was also noted that some of the low molecular weight organic acids tested, such as acetic and citric acid, inhibited the growth of strain IT2 [ 21 ]. The absence of growth in an organic carbon-free medium suggested that C. ammoniigenes IT2 is an obligate heterotroph. Culture doubling times of isolate IT2 grown at different temperatures and pH values are shown in Figure 1 . Strain IT2 grew between pH 2.7 and 5.0, with a pH growth optimum of 4.0. The optimum temperature for growth was found to be at 30 °C, and the maximum temperature at which growth was observed at 37 °C. Under optimum conditions of temperature and pH, its culture doubling time was 3.8 h, equivalent to a growth rate of 0.18 h −1 . The isolate was unable to grow below pH 2.7 and above pH 5.0, indicating that it is a moderate acidophile. Strain IT2 is therefore more tolerant of extreme acidity than C. ammoniigenes B55 T (pH range 3.5–8.0, with optimal growth at pH 4.0). All other species of Curtobacterium are neutrophiles [ 14 ]. In addition, Curtobacterium isolate IT2 showed similar copper tolerance to that reported for Acidiphilum cryptum [ 22 ], a heterotrophic acidophilic bacteria that has also been found in pyritic mine tailings [ 8 ] and can also grow on a wide range of monosaccharides, dicarboxylic acids and tricarboxylic acids. As is the case with many other acidophilic bacteria, isolate IT2 exhibited a high tolerance to elevated concentrations of ferrous iron, zinc and nickel ( Table 2 ), which helps to explain why it is able to proliferate in pyritic tailings [ 13 ]. Tolerance to transition metals is another major characteristic of heterotrophic bacteria isolated from mineral tailings, though, in general, heterotrophs are less tolerant to dissolved metals than iron-oxidizing chemolithotrophs ( A . ferrooxidans in particular) [ 23 ]. C. ammoniigenes IT2 was able to grow in the spent medium of A . ferrooxidans . Figure 2 shows that numbers of C. ammoniigenes cells increased by over one order of magnitude within 12 days, and that this was accompanied by a decrease in the concentration of total DOC. However, only about 24% of the total DOC was metabolized over this period, and the cessation of growth of C. ammoniigenes isolate suggests that the residual DOC was not metabolized by this strain. No changes in the concentration of DOC were observed in the control cultures containing sterile spent medium of A. ferrooxidans, which were also confirmed to be devoid of bacterial cells (data not shown). This result provides further support that primary-producing chemolitho-autotrophic acidophilic bacteria, such as A . ferrooxidans, can support the growth of heterotrophic bacteria by providing them with electron donors and carbon sources. Diaby et al. [ 8 ] proposed a model using the microbiological and geochemical results to explain how autotrophic acidophiles sustained the growth of heterotrophic iron-reducers present in mine tailings at the Andina mine, CODELCO, Chile. DOC, mainly lysates and exudates from A. ferrooxidans and other primary producers, was proposed to sustain the heterotroph communities dominated by Acidiphilium , Acidocella and Acidobacterium spp. Carbon transfer between acidophilic prokaryotes that either fix or produce CO 2 was demonstrated by Kermer et al. [ 24 ], using protein-based stable isotope probing, to be a two-way process. The syntrophic relationship of the acidophilic species involves organic carbon, derived from autotroph (as exudates or cell lysates), being used as the carbon source by heterotrophic bacteria, and CO 2 generated by heterotrophic species being using as carbon source by autotrophs. The latter is particularly important in low pH environments, where the solubility of CO 2 is very low [ 25 ]. Previously, Schnaitman and Lundgren [ 26 ] had shown that 10% of labelled carbon ( 14 CO 2 ) was leaked by A. ferrooxidans into its growth medium, and pyruvic acid was identified as one the low molecular weight exudates. Besides, low molecular weight carboxylic acids, such as formate, acetate and pyruvate, were detected from two copper mine tailings [ 27 ]. Nancucheo and Johnson [ 28 ] reported that glycolic acid was produced and excreted by mineral-oxidizing bacteria, such as L. ferriphilum , Acidithiobacillus caldus and A. ferrooxidans , and demonstrated that this was used as a carbon and energy source by Sulfobacillus spp. The results of this study confirmed that glycolic acid in the spent medium of A. ferrooxidans, as previously reported by Nancucheo and Johnson [ 28 ], was also used, at least in part (~20%), by the Curtobacterium isolate ( Figure 2 ). To mitigate the risk of reactive mineral tailings generating metal-rich, extremely acidic waste drainage waters, they are usually stored under water to limit contact with oxygen. Even so, ferric iron, generated in the aerobic upper layers, can diffuse into tailings and oxidize sulfide minerals in the absence of oxygen [ 29 ]. Diaby et al. [ 8 ] found that, in pyritic tailings (deposits below the “oxidation front”, the junction between the oxidation and neutralization zones), the dissimilatory reduction of ferric iron was a dominant geochemical process, since ferric iron, produced by iron-oxidizing acidophiles in the aerobic tailings surface and migrating downwards in percolating drainage waters, can act as a terminal electron acceptor, for both heterotrophic and many autotrophic species (including A. ferrooxidans ), when oxygen is limited or absent. Extremely acidic environments usually contain ferrous and ferric iron in much greater concentrations than those typically found in neutral pH water bodies [ 30 ]. The redox potential of the ferrous/ferric couple is relatively high, at pH values less than ~2.0 (~ +680 mV in sulfate-rich liquors; [ 31 ]), due to the enhanced solubility of both ionic species of this metal. This makes ferric iron a thermodynamically attractive alternative electron acceptor to oxygen in acidic environments, both for heterotrophic (coupled to organic carbon) and autotrophic (coupled to reduced sulfur or hydrogen) acidophiles [ 25 ]. Previously, Nancucheo and Johnson [ 13 ] showed that the C. ammoniigenes strain IT2 catalysed the dissimilatory reductive dissolution of amorphous ferric hydroxide (concurrent with a corresponding increase in cell numbers), when incubated under anaerobic conditions in cultures containing glucose as the electron donor. This important trait was not previously described for this genus, and adds another species of mesophilic, acidophilic bacteria to the list of those that can use the dissimilatory reduction of ferric iron to support growth in oxygen-limited cultures in highly acidic environments, such as those found in many pyritic mine tailings. Most currently known iron-reducing heterotrophic acidophiles found in pyritic mine tailings are Proteobacteria [ 32 ]. Interestingly, dissimilatory reduction of ferric iron has also been described for other genera of acidophilic actinobacteria, including Ferrimicrobium acidiphilum , Ferrithrix thermotolerans , Aciditerrimonas ferrireducens and Acidithrix ferrooxidans [ 33 ], and the novel recently-described genus Acidiferrimicrobium australe [ 11 ]. In addition, species of acidophilic actinobacteria, except Aciditerrimonas ferrireducens and the isolate IT2, also oxidize ferrous iron. By lowering concentrations of ferric iron, the prime oxidant of sulfide minerals in low pH environments, C. ammoniigenes strain IT2 (and other iron-reducing acidophiles) can, in theory, help control the production of metal-rich mine waters, especially where mineral wastes are ecologically engineered to stimulate such bacteria by limiting oxygen ingress and (possibly) promoting the influx of organic electron donors (e.g., algal exudates, [ 13 ]). New strategies are required to stabilize the storage of mineral tailings, which represents a long-term engineering and environmental challenge, where, occasionally, catastrophic environmental pollution has occurred due to the failings of the retaining dam of a tailings impoundment [ 6 ]. Preventing the oxidation of metallic sulfides in mineral tailings has been highlighted as a key criterion for the ecological restoration of mine tailings by revegetation, and heterotrophic bacteria such as Curtobacterium spp. may possibly be used as biological indicators for monitoring mineral tailings during the process of restoration, in order to minimize the solubilization of a variety of transition metals associated with sulfide minerals."
} | 4,085 |
35557652 | PMC9088794 | pmc | 5,784 | {
"abstract": "Neuromorphic computing\nis an emerging area with prospects to break\nthe energy efficiency bottleneck of artificial intelligence (AI).\nA crucial challenge for neuromorphic computing is understanding the\nworking principles of artificial synaptic devices. As an emerging\nclass of synaptic devices, organic electrochemical transistors (OECTs)\nhave attracted significant interest due to ultralow voltage operation,\nanalog conductance tuning, mechanical flexibility, and biocompatibility.\nHowever, little work has been focused on the first-principal modeling\nof the synaptic behaviors of OECTs. The simulation of OECT synaptic\nbehaviors is of great importance to understanding the OECT working\nprinciples as neuromorphic devices and optimizing ultralow power consumption\nneuromorphic computing devices. Here, we develop a two-dimensional\ntransient drift–diffusion model based on modified Shockley\nequations for poly(3,4-ethylenedioxythiophene) (PEDOT)-based OECTs.\nWe reproduced the typical transistor characteristics of these OECTs\nincluding the unique non-monotonic transconductance–gate bias\ncurve and frequency dependency of transconductance. Furthermore, typical\nsynaptic phenomena, such as excitatory/inhibitory postsynaptic current\n(EPSC/IPSC), paired-pulse facilitation/depression (PPF/PPD), and short-term\nplasticity (STP), are also demonstrated. This work is crucial in guiding\nthe experimental exploration of neuromorphic computing devices and\nhas the potential to serve as a platform for future OECT device simulation\nbased on a wide range of semiconducting materials.",
"conclusion": "Conclusions We present a robust\nsimulation platform for 2D time-dependent PEDOT:PSS-based\nOECTs. Applying the concept of phase separation in the semiconductor\nmaterial and ion injection physics, we are able to reproduce lots\nof experimental ion transport and charging data of OECTs. Moreover,\nwe demonstrate different typical synaptic phenomena of OECTs under\nboth inhibitory and excitatory modes. Our model is very effective\nfor the simulation of synaptic behaviors of OECTs. At the same time,\nour platform enables the simulation of tailored OECTs with a fast\nresponse speed, high transconductance, and low power consumption,\nopening a new paradigm for energy-efficient neuromorphic computing\nplatforms. High tunability and applicability to a wide range of semiconductor\nmaterials make our platform crucial for developing future organic-based\nneuromorphic devices.",
"introduction": "Introduction The rapidly developing\nartificial intelligence (AI) is pushing\nthe traditional von Neumann computational architecture to its energy\nefficiency limit. 1 In the von Neumann architecture,\nthe dynamic random access memory (DRAM) and the processing units are\nseparated physically, resulting in immense energy consumption associated\nwith data movement. 2 , 3 On the contrary, in human brains,\nmassive information can be processed in parallel in memory at an extremely\nfast speed with a super low power consumption of merely 1–100\nfJ per synapse. 4 , 5 Inspired by human brains, the\nemerging neuromorphic computing has attracted massive research interest.\nA key component for neuromorphic computing and artificial neural networks\nis artificial synapses. 6 Emulating biological\nsynapses, an artificial synapse responds to stimuli of action potential\nspikes with programmed postsynaptic current by modulating the device\nconductance. 7 Recently, different synaptic\nfunctions such as short-term plasticity (STP), 8 long-term potentiation, 9 , 10 and spike-timing-dependent\nplasticity (STDP) 11 have been achieved\nby organic and inorganic artificial synaptic devices. Massive research\neffort are put into the materials selection for synaptic transistors,\nincluding zero-dimensional (0D) quantum dots, 12 − 14 one-dimensional\n(1D) nanostructure, 15 − 18 two-dimensional (2D) nanostructures, 19 − 22 three-dimensional (3D) architectures, 23 − 25 transition-metal oxide, 26 ferroelectric\nmaterials, 27 , 28 alloy, 29 mixed structure, 30 − 32 and organic materials. 33 , 34 Among the\nartificial synaptic devices, synapses based on organic electrochemical\ntransistors (OECTs, Figure 1 A–C) have emerged as attractive alternatives to inorganic\ncounterparts owing to their fast response speed, 35 high transconductance, 36 less\nstochastic writing, 2 continuous conductance\ntuning, 37 and low driving voltage comparable\nto biological synapses. 38 A schematic representation\nof an OECT synapse is shown in Figure 1 D. The phosphate-buffered saline (PBS) electrolyte\ntogether with a gold gate electrode of an OECT transmits a presynaptic\nsignal, while the PEDOT: polystyrene sulfonate (PSS) channel together\nwith the source and drain electrodes transmits a postsynaptic output\nsignal in the form of source–drain current ( I ds ). The experimental work has demonstrated that OECTs\nhave synaptic functionalities like spike-timing-dependent plasticity\nand homeostatic plasticity. 39 , 40 However, theoretical\nunderstanding of the working principles of OECT-type artificial synapses\nis still in its very early stage. Figure 1 Device apparatuses and phase separation.\n(A) Sketch of an electrolyte-gated\nPEDOT:PSS OECT. Adapted from ref ( 19 ). (B) 2D overview of the synaptic OECT. The essential\ncomponents are a PEDOT:PSS channel with gold source and drain contact,\nan electrolyte, and an Ag/AgCl gate electrode. (C) Schematic demonstration\nof phase separation in PEDOT:PSS. The blue part stands for the PEDOT\nphase, while the gray parts stand for the PSS phase. When V gate = 0, PEDOT:PSS is doped, I ds > 0, when V gate ≫\n0, OECT is in depletion mode, PEDOT:PSS is de-doped, carrier density\nin the polymer film decreases, I ds = 0.\nThe polarons in the PEDOT phase are stabilized by immobilized counterions\nin the PSS phase. (D) Schematic representation of the synaptic OECT\nin analogy to a biological synapse. Theoretical modeling of OECT synaptic performances is crucial since\nit does not only allow us to understand the working principles of\nOECT as neuromorphic devices but also guides future experiments. In\nan OECT ( Figure 1 A),\nan applied potential on the gate drives ions from the electrolyte\ninto the polymer channel, changing its redox state and conductivity\nas a result. Typically, there are two types of device modes for OECTs:\nthe depletion mode and the accumulation mode. 41 In the depletion mode, the channel material is fully oxidized (heavily\np-doped) such as PEDOT:PSS (the case illustrated in Figure 1 A,B). When a positive gate\npotential is applied, cations are injected from the electrolyte into\nthe channel; as a result, the holes in the channel are depleted and\nthe conductance of the channel is dropped ( Figure 1 C). In the accumulation mode, the channel\nmaterials are usually nearly intrinsic semiconducting polymers with\na very small number of mobile holes. When a negative gate potential\nis applied, anions are injected into the channel and electrochemical\ndoping is induced. Therefore, the channel conductance increases. The\nchange in channel conductance is typically transient or volatile in\nOECTs, meaning that the conductance returns to its initial value after\nthe applied gate voltage is removed. The volatile conductance tuning\nis essential for short-term synaptic behaviors in OECT-based artificial\nsynapses. The short-term synaptic behaviors are essential for critical\ncomputational functions such as signal transmission, encoding, and\nfiltering of neuronal signals. 3 , 6 Modeling the synaptic\nbehaviors of OECT-based artificial synapses\nhas been a crucial yet long existing challenge for the field of neuromorphic\ncomputing. The fundamental equations used to describe the charge-carrier\nand ion transport process in OECTs include the Poisson equation, the\ndrift–diffusion equation for electronic charge-carrier transport,\nand the drift–diffusion equation for ion transport. These equations\nare analogous to the well-known Shockley equations 42 for modeling electron and hole transport in semiconductor\ndevices such as p–n junction diodes and metal-oxide semiconductor\nfield-effect transistors (MOSFETs). Efforts have been put into modifying\nand solving the Shockley equations, which would provide physical insight\ninto the system. Shirinskaya et al. described the doping–de-doping\ninterface as the moving front, based on which a numerical model for\nthe current–voltage characteristics of OECTs was developed. 43 Tybrandt et al. proposed a time-dependent approach\nbased on the drift–diffusion–Poisson equation and phase\nseparation. Their model successfully describes the experimental data.\nThe model though is limited to one-dimensional (1D) across channel\nand electrolyte and does not reflect neuromorphic behavior. The frequency\ndependency of transconductance and the unique bell-shaped transconductance–gate\nbias curve are also not reproduced by their model. 44 The experimental and modeling results of Volkov et al.\nprovide a solid argument that the major contribution to the capacitance\nof the two-phase PEDOT:PSS originates from electric double layers\n(EDLs) formed along the interfaces between the PEDOT-rich region and\nthe PSS-rich region. 45 However, limited\nwork has been done on modeling the artificial synaptic behaviors of\nOECTs. The key challenge of this task is that there is still a lack\nof 2D dynamic models of the cross section of OECT describing the complex\nelectrochemical processes in OECT synaptic tuning, which provides\na deeper perspective on the working principles of OECTs. At the same\ntime, understanding the electronic structure of semiconducting polymers\nis also essential for modeling key synaptic behaviors of OECTs. To address this key challenge, we adopted the concept of PEDOT\nand PSS phase separation and built a 2D transient model for the prototype\ndepletion-mode OECT and demonstrated OECT transistor characteristics\nand synaptic behaviors with a modified Shockley equation model for\nthe first time. Typical OECT transistor behaviors such as transfer\ncharacteristics, output characteristics, and a small signal transconductance\nare reproduced. In addition, the bell-shaped transconductance–gate\nbias curve is reproduced by assuming a Gaussian-shaped density of\nstates (DOS) in the organic semiconductor. 44 , 46 The frequency dependency of transconductance is also studied using\nour 2D dynamic model, demonstrating the physical validity of our model. 47 − 49 Moreover, synaptic behaviors, such as excitatory/inhibitory postsynaptic\ncurrent (EPSC/IPSC), paired-pulse facilitation/depression (PPF/PPD),\nshort-term plasticity (STP), spike-amplitude-dependent plasticity\n(SADP), spike-duration-dependent plasticity (SDDP), are achieved.\nThis work lays the foundations for the simulation of large-scale programmable\nand functional neuromorphic arrays for energy-efficient computing.\nIn addition, this work will provide a modular platform for the design\nof novel OECT synaptic devices.",
"discussion": "Results and Discussion Model\nDescription In the Bernards model, 50 the OECTs are considered as consisting of two\ncircuits: the ionic circuit, where ions are transported in the polyelectrolyte\nblends, and the electronic circuit, where holes are transported on\nthe conjugated polymer backbone. Based on this idea, Tybrandt et al. 44 treated these two phases distinctively in a\nclassic PEDOT:PSS system: the electronic conjugated polymer (PEDOT,\nCP) phase and the ionic polyelectrolyte (PSS, PE) phase ( Figure 1 C). Typical OECT\ncharacteristics such as transfer characteristics and output characteristics,\nalong with charging characteristics, are reproduced by considering\nthe drift–diffusion for both electronic and ionic carriers\nand the effect of EDL capacitance between these two phases in a 1D\nmodel. Recently, Paulsen et al. 51 brought\nup the concept of organic mixed ionic–electronic conductors\n(OMIEC) for an efficient description of not only ionic and electronic\ntransport but more importantly ionic–electronic coupling. PEDOT:PSS,\nas the OMIEC, and OECTs, as a typical configuration of OMIEC devices,\nallows the adaption of the OMIEC concept in our model. In our\nwork, we extended the prototypical model based on the Shockley equations\nto two dimensions with a focus on transient behaviors. PEDOT:PSS is\na classic two-component OMIEC with anions chemically linked to the\nPSS component. The electronic transport mechanism in the PEDOT phase\nshould contain both thermally activated hopping and band-like transport,\ndepending on its crystallinity. 52 In our\nmodel, it is described by a classical drift–diffusion equation\nmodified by electrochemical potential with the unit of energy (μ p ) ( eq 1 ), where p is the hole concentration, D p is the diffusion coefficient of holes in PEDOT, and f is F / RT , which is the ratio between\nFaraday’s constant and RT according to the\nEinstein relation, and is the flux of holes. By assuming\nGaussian\ndensity of states (DOS), the chemical potential can be modified as eq 2 , where E DOS is the center energy of the Gaussian DOS, σ\nis the standard deviation of the DOS and is a measure of the energetic\ndisorder, p t is the total available hole\ndensity, and B is defined as eq 3 . 1 2 3 Similarly, the ionic transport in the PE phase\nfollows a hopping mechanism, which is described by the classic drift–diffusion\nequation ( eq 4 ), where is the\nflux of cations and anions, respectively. c ± is the concentration of cations and anions,\nrespectively. Because of phase separation, the electrostatic potential\nin these two phases is distinctly labeled as V p for the CP phase and V c for the\nPE phase. 4 At the interface between\nphase separating\nregions, the spatial separation between the electronic and ionic charge\ncarriers causes the formation of EDLs. This process exists throughout\nthe system, which enables us to consider this process as a volumetric\nproperty when viewed from a macroscopic level. This volumetric capacitance\nof EDL is labeled as C V . Continuity equations\n( eqs 5 and 6 ) and Poisson’s equation ( eq 7 ) are implemented to relate charge-carrier\nconcentration to flux densities. It is assumed that holes that compensate\nfor negative ionic charges in EDL do not contribute to Poisson’s\nequation ( eq 8 ). 5 6 7 8 Boundary conditions are adapted from Tybrandts’\nmodel considering the continuity of Fermi level and charge neutrality\nat the PEDOT–electrode interface. Full sets of the drift–diffusion\nequations and boundary conditions are shown in Figure S1 . The presence of net ionic charge in the PE phase\nleads to the presence of electronic charge in an OMIEC of the opposite\nsign. The balancing process of excess ionic charge with electronic\ncharge is called electrochemical doping as it causes an increase in\nthe electrical conductivity in the OMIEC. In PEDOT:PSS, stabilizing\nionic charge is immobilized in the PE phase, thus it is inherently\ndoped. Transistor Characteristics Unless specified otherwise,\nthe parameters used in all of the calculations are shown in Table S1 . One can refer to Figure S3 for the dimensions and mesh in the simulations for\na single transistor with channel length L = 200 μm\nand channel thickness W = 10 μm. The current\ndensity is obtained by integrating all charge-carrier species flux\nthroughout the channel on the cross-sectional area. Similarly, the\ncurrent density in the electrolyte ( I g ) can also be calculated by integrating ionic carriers throughout\nthe cross-sectional area in the electrolyte. As shown in Figure 2 , typical transistor\ncharacteristics of OECTs are qualitatively reproduced. 36 The output characteristics in Figure 2 A are qualitatively in good\nagreement with typical PEDOT:PSS-based OECTs, where I ds initially increases as V drain decreases and then reaches a plateau. Higher V gate requires less negative V drain to reach a plateau and results in a lower drain current. The transfer\ncharacteristics and the associated transconductance ( g m ) in Figure 2 B also align with the typical PEDOT:PSS-based OECTs. 47 I ds reaches a maximum\nplateau as V gate decreases and a minimum\nplateau as V gate increases. The transconductance\nhas a non-monotonic dependence on gate voltage, 53 , 54 which is a unique characteristic for OECTs and agrees with the convex-shaped\ntransconductance curve in Figure 2 B,C. The non-monotonic transconductance is an intrinsic\nproperty of OECTs. This happens because of the behavior of holes filling\nthe DOS in PEDOT as the gate voltage gets lower, assuming a Gaussian\nDOS. When the DOS is much less than half-full, both hole concentration\nand hole mobility increase with increasing holes, thus transconductance\nincreases as gate voltage becomes more negative. When the DOS is nearly\nhalf-full, the rate of increase of hole concentration and hole mobility\nslows with increasing holes. As a result, transconductance decreases\nwith a more negative gate voltage. When DOS is more than half-full,\nadding holes leads to a decrease in hole mobility, resulting in a\nnegative transconductance. 47 Figure 2 Simulation\nresults of transistor characteristics. (A) Output characteristics\nof V gate vary from −0.5 V (top\ncurve) to 0.3 V (bottom curve). (B) Transfer characteristics and the\nassociated transconductance for V drain = −0.5 V. (C) Steady-state transconductance. (D) Frequency\nresponse of the transconductance. The frequency response in Figure 2 D is obtained by measuring the small signal transconductance.\nA 100 mV oscillation is applied on the gate electrode and the transconductance\nis determined by the amplitude ratio between output I ds oscillation and the corresponding gate bias. This behavior\nis in agreement with the fact that typical OECTs have higher transconductances,\nin the range of millisiemens, and can only operate at lower frequencies\ncompared to organic field-effect transistors (OFETs). 41 Synaptic Behavior Synapses are the\nconnections between\nthe neuron circuits that dominate the architecture of animal brains.\nEach neuron has over 1000 synapse connections with other neurons.\nArtificial synapse devices with similar physical properties, such\nas OECTs, would enable board applications to neuromorphic computing. 55 Modeling of OECT synaptic behaviors is a crucial\nstep toward an improved perspective on synaptic behaviors. The phosphate-buffered\nsaline (PBS) electrolyte with an Au electrode receives a presynaptic\ninput signal (in the form of gate voltage) and passes the signal to\nthe channel. The PEDOT:PSS channel responds to the presynaptic signal\nand transmits a postsynaptic output signal in the form of a source–drain\ncurrent ( I ds ). For our simulation, the\namplitude of the presynaptic spike is set to be 0.5 V. V pre = 0, 0.5 V are chosen as input baselines. The choice\nof these two conditions ensures intense initial doped and de-doped\nstates of polymer, respectively, which leads to better comparison. When a positive voltage V pre with a\nduration t d is applied at the gate electrode,\ncations in PBS electrolyte (mostly Na + ) are driven to penetrate\ninto the PEDOT:PSS channel and de-dope PEDOT from PSS, therefore lowering\nthe conductance of the channel. In Figure 3 B, upon the application of a single positive\npresynaptic spike with an amplitude of 0.5 V and a duration of 2 ms,\nthe postsynaptic current (PSC) decreases immediately by around 1/3.\nBecause of the positive spike applied, cations in the electrolyte\nare driven into the polymer channel and compensate for holes in hole/PSS\npairs. As a result, originally positively charged PEDOT is reduced,\nand the channel conductance decreases. This is analogous to IPSC in\nbiological inhibitory synapses. After the removal of the spike, injected\ncations return to the electrolyte, PEDOT:PSS gets reversibly doped,\nand PSC gradually recovers to its original state. Figure 3 Simulation results of\ntypical OECT synaptic behaviors. (A) EPSC\ntriggered by a postsynaptic pulse ( V pre = 0.5 V , V post = 0\nV, t d = 2 ms, V drain = −0.3 V). (B) IPSC triggered by a presynaptic pulse ( V pre = 0 V, V post = 0.5 V, t d = 2 ms, V drain = −0.3 V). (C) PPF triggered by a pair of\npresynaptic pulses ( V pre = 0.5 V, V post = 0 V, t d =\n2 ms, Δ t = 2 ms). (D) PPD triggered by a pair\nof presynaptic pulses ( V pre = 0 V, V post = 0.5 V, t d = 2 ms, Δ t = 2 ms). (E) EPSC respond to a\ntrain of 1 kHz presynaptic pulses ( V pre = 0 V, V post = −0.5 V). (F) IPSC\nrespond to a train of 1 kHz presynaptic pulses ( V pre = 0 V, V post = 0.5 V).\n(G) PPD and PPF ratio ( A 2 / A 1 ) as a function of spike interval time (Δ t ). In contrast, when a negative presynaptic\nspike with the same amplitude\nand duration is applied, PSC is boosted due to cations extracted from\npolymer while also recovering a little after. This process reproduces\nEPSC in biological neurons ( Figure 3 A). Temporally correlated behaviors between presynapse\nand postsynapse are important as it contains short-term memristive\nbehavior. The process of synaptic facilitation and depression both\noccur and decay within a short period of time after being simulated.\nA paired-pulse study is used to analyze the temporal correlation. 56 A pair of pulses with identical amplitude and\nduration is applied successively with a certain time interval as a\npresynaptic signal. The resulting postsynaptic current is recorded\nsimultaneously as a function of time. A typical time interval of 2\nms is used to reproduce paired-pulse facilitation (PPF) and paired-pulse\ndepression (PPD). PPF and PPD are forms of short-term synaptic plasticity\nand are reported to be essential for decoding temporal information\nin biological systems. Such behaviors can be mimicked by synaptic\ntransistors and thus our simulation. 11 When\na pair of negative pulses is applied, since the time interval is short,\nions are not completely transported and PEDOT is still relatively\nhighly doped, which results in a stronger second current compared\nto the first one as in Figure 3 C. The second postsynaptic current is facilitated, which means\nthe maximum drain current difference A ( Figure S4 ) of the second postsynaptic current\n( A 2 ) is greater than that of the first\npulse ( A 2 / A 1 > 1). This behavior is analogous to PPF in biological synapses.\nOn the contrary, with a pair of positive pulses applied, the second\npostsynaptic current is depressed ( A 2 / A 1 < 1) as PPD in biological synapses. 57 The ratio of A 2 / A 1 represents the information processing\nability of the synapse. 56 Figure 3 G shows the PPD and PPF ratio\nas a function of spike interval\ntime (Δ t ). With a longer Δ t , the injected ions have more time to return to the electrolyte and\nthe PPD/PPF values increase/decrease exponentially to approach the\nvalue of 1 with a critical value around 40 ms. For Δ t longer than the critical value, ions have sufficient time\nto return and the channel recovers to its original state after the\nfirst pulse. The information between spikes is lost and the synaptic\nOECT runs in the information nonprocessing mode. The temporal\ncorrelation effect was further validated by synaptic\nfacilitation ( Figure 3 E) and depression ( Figure 3 F). 58 Both results are produced\nby applying a 1 kHz train of pulses of an amplitude of 0.5 V. The\nspike-amplitude-dependent plasticity (SADP) is also a typical postsynaptic\nbehavior of the STP effect. As presented in Figure S6 , the value difference between baseline and result PSC (ΔPSC)\nfor IPSC decreases with V pre in a significant\nlinear manner. However, for EPSC, ΔPSC decreases with V pre nonlinearly while approaching zero, as one\ncan predict with increasing gate voltage. The behavior agrees well\nwith previous experimental results. Postsynaptic behavior is further\ndemonstrated by the spike-duration-dependent plasticity (SDDP) ( Figure S5 ); ΔPSC decreases in the IPSC\nmode but increases in the EPSC mode, both synchronously with duration\ntime t d . It can be explained by the fact\nthat more cations are injected/extracted in/from the polymer for a\nlonger duration of time of the applied gate voltage. At a certain\npoint when cations can no longer keep injecting/extracting, the system\nsaturates, which leads to the plateau at ∼15 ms for IPSC and\n∼28 ms for EPSC."
} | 6,062 |
35985998 | PMC9391474 | pmc | 5,785 | {
"abstract": "Microbially mediated nitrogen cycling in carbon-dominated cold seep environments remains poorly understood. So far anaerobic methanotrophic archaea (ANME-2) and their sulfate-reducing bacterial partners (SEEP-SRB1 clade) have been identified as diazotrophs in deep sea cold seep sediments. However, it is unclear whether other microbial groups can perform nitrogen fixation in such ecosystems. To fill this gap, we analyzed 61 metagenomes, 1428 metagenome-assembled genomes, and six metatranscriptomes derived from 11 globally distributed cold seeps. These sediments contain phylogenetically diverse nitrogenase genes corresponding to an expanded diversity of diazotrophic lineages. Diverse catabolic pathways were predicted to provide ATP for nitrogen fixation, suggesting diazotrophy in cold seeps is not necessarily associated with sulfate-dependent anaerobic oxidation of methane. Nitrogen fixation genes among various diazotrophic groups in cold seeps were inferred to be genetically mobile and subject to purifying selection. Our findings extend the capacity for diazotrophy to five candidate phyla (Altarchaeia, Omnitrophota, FCPU426, Caldatribacteriota and UBA6262), and suggest that cold seep diazotrophs might contribute substantially to the global nitrogen balance.",
"conclusion": "Conclusion In the deep-sea cold seep sediments that are impacted by darkness, low temperatures, and high hydrostatic pressure, growth of microbiomes consuming rich hydrocarbons is also supposed to be nitrogen limited. Biological nitrogen fixation is one main source of bioavailable nitrogen, offsetting localized nitrogen limitation and promoting ecosystem productivity. The present work demonstrates the diversity, abundance, and distribution of diazotrophs at cold seeps, revealing this metabolic guild to be diverse, widespread and probably sufficiently abundant to influence deep-sea benthic nitrogen cycling. To our knowledge, most diazotrophs detected in these cold seeps belong to candidate phyla, including the first known diazotrophs with the Altarchaeia, Omnitrophota, FCPU426, Caldatribacteriota and UBA6262. Of the 35 recovered diazotrophic MAGs, 23 represent microorganisms that are involved directly or indirectly in hydrocarbon metabolisms, including anaerobic methane-oxidizing archaea and anaerobic non-methane alkane-degrading bacteria. The tight correlation between hydrocarbon-derived carbon and nitrogen cycles indicates that nitrogen fixation pathways might be selected for microorganisms making use of the most abundant energy source at cold seeps. Moreover, we show that HGTs and purifying selection mediate cold seep nitrogenase evolution. Overall, the findings in this study highlight the importance of exploring the diversity and activity of diazotrophs in deep-sea benthic ecosystems and suggest cultivation of novel diazotrophs from cold seep sediments should be possible.",
"introduction": "Introduction Cold seeps occur in continental margins worldwide. At these sites, there is discharge of biologically or geologically sourced hydrocarbons, ranging in complexity from methane to the varying constituents of petroleum 1 , 2 . Cold seeps are often classified as slow-flow mineral-prone or high-flux mud-prone systems according to their hydrocarbon fluid regime 1 . They span oil and gas seeps, methane seeps, gas hydrates, asphalt volcanoes, mud volcanoes, brine pools, and brine basins among others. The seeping hydrocarbons support the development of extensive local diversity of archaea and bacteria, dominated by aerobic methane-oxidizing bacteria (MOB, e.g., members of the methanotrophic family Methylococcaceae) mainly at the oxygen-rich sediment-water interface 3 and microbial consortia of anaerobic methane-oxidizing archaea (ANME) with sulfate-reducing bacteria (SRB) within anoxic sediment layers 4 – 6 . Various studies have revealed microorganisms that oxidize non-methane hydrocarbons, such as ethane, butane, propane, liquid alkanes and aromatic hydrocarbons, also inhabit these environments 7 – 12 . In contrast with the rich and expanding knowledge of microbial hydrocarbon oxidation at cold seeps, little is known about how microbiomes in these ecosystems control the cycling of other essential nutrients. Seeping hydrocarbons introduce little nitrogen into these carbon-dominated systems, making cold seep sediments inherently limited by nitrogen supply to support biomass production 1 , 13 . Biological nitrogen fixation (diazotrophy)—the reduction of atmospheric dinitrogen gas (N 2 ) to ammonia (NH 3 ) with concomitant hydrogen gas (H 2 ) production—is a critical source of bioavailable nitrogen for living organisms 14 – 16 . The key enzymes mediating this process are nitrogenases, which include three forms distinguished by their active site metal cofactors: molybdenum-iron nitrogenase Nif (Mo-Fe), vanadium-iron nitrogenase Vnf (V-Fe) and iron-only nitrogenase Anf (Fe-Fe) 17 , 18 . All three nitrogenase forms are structurally and functionally similar, each containing two protein components: a dinitrogenase reductase (NifH, VnfH, or AnfH) and a catalytic component (NifDK, VnfDGK, or AnfDGK). Most biological N 2 fixation is catalyzed by the more efficient Mo-Fe nitrogenase, while Fe-V and Fe-Fe nitrogenases are alternative enzymes used in Mo-limited settings 19 , 20 . A combination of rate measurements, lab cultivation, flow cytometry, molecular analysis, and cellular imaging have revealed that diazotrophs are active throughout the oceans. Multiple cyanobacterial diazotrophs are responsible for a substantial portion of new nitrogen input in the surface ocean 21 – 24 . Various diazotrophs are also active in both surface and deeper waters, including diverse heterotrophic Proteobacteria 25 – 28 . Over the past decade, the deep benthos has been found to host diverse groups of previously unrecognized diazotrophs that actively and significantly contribute to local nitrogen balance, including members of Acidobacteria, Firmicutes, Nitrospirae, Gammaproteobacteria and Deltaproteobacteria 15 , 29 , 30 . Despite these advances, there remains limited knowledge about the distribution and evolution of biological nitrogen fixation in sediments from the deep sea, which covers nearly two-thirds of the Earth. Multiple lines of evidence have demonstrated diazotrophy in different deep-sea cold seep sediments. These include a methane seep in the South China Sea, a mud volcano offshore Costa Rica, gas hydrate mounds in the Gulf of Mexico, and an active methane seep in the Eel River Basin 13 , 31 – 34 . Based on 15 N 2 tracer experiments coupled with nanoSIMS, to date only two cold seep taxa have been identified as diazotrophs, the methanotrophic ANME-2 archaea and their sulfate-reducing bacterial partners of the SEEP-SRB1 clade 31 , 32 , 34 , 35 . However, PCR amplicon surveys targeting the nitrogenase reductase nifH gene suggested greater phylogenetic diversity among diazotrophs in methane seep sediments 36 – 38 . Biological nitrogen fixation requires large amounts of ATP and high-potential electrons, whereas anaerobic methane oxidation associated with ANME and SRB are among the lowest energy-yielding reactions that can sustain life 20 , 31 , 39 . From this point of view, we hypothesize that biological nitrogen fixation in cold seeps does not necessarily rely on sulfate-dependent anaerobic oxidation of methane. Considering the existence of phylogenetically and functionally diverse communities in cold seep sediments 11 , 40 , other catabolic processes are predicted to also drive nitrogen fixation. In this study, we investigate the hidden diversity and distributions of nitrogenases and diazotrophs, and compile evidence for their in situ activities within deep-sea cold seep sediments. To this end, gene- and genome-centric analyses of 61 metagenomes are coupled with six metatranscriptomes derived from 11 globally distributed areas of hydrocarbon seepage (Fig. 1 and Supplementary Data 1 ). Samples originate from five types of cold seeps, namely gas hydrates, mud volcanoes, asphalt volcanoes, oil and gas seeps and methane seeps. Most seep types have previously been shown to have lighter δ 15 N indicative of biological nitrogen fixation, compared to nearby background sediments 13 , 41 , 42 (Supplementary Fig. 1 and Supplementary Notes ). Overall, this study corroborates deep-sea cold seep sediments as overlooked habitats for uncovering diverse diazotrophs from uncultivated lineages supported by diverse energy sources, and emphasizes the importance of nitrogen fixation in a carbon-dominated environment. Fig. 1 Geographic distribution of 11 cold seep sites where metagenomic and metatranscriptomic data were collected. These samples were originated from five types of cold seeps: gas hydrates, mud volcanoes, asphalt volcanoes, oil and gas seeps and methane seeps. Sites with red asterisks denote that both metagenomes and metatranscriptomes were collected, sites with blue asterisks denote that only metatranscriptomes were collected, and sites without asterisks denote that only metagenomes were collected. Also see details in Supplementary Data 1 . The world map was drawn using the ggplot2 package in R v4.0.3.",
"discussion": "Results and discussion Cold seeps harbor canonical and novel nitrogenase gene homologues The nifH marker gene, which encodes a key structural protein of the nitrogenase enzyme, is commonly used to explore the diversity and abundance of diazotrophs in various environments 15 , 43 . Annotations of contigs assembled from 61 metagenomes collected at 11 globally distributed cold seep sampling stations (Fig. 1 and Supplementary Data 1 ) revealed 202 non-redundant nifH homologues falling into the nitrogenase superfamily. The phylogenetic tree (Fig. 2 ) suggested that nifH homologues were classified into distinct bona fide nitrogenase sequences (canonical groups I to III) as well as nitrogenase-like groups (groups IV to VI) 44 – 48 . These include (1) typical Mo-Fe nitrogenases from aerobic and facultative anaerobic bacteria (group I; n = 1); (2) Mo-Fe nitrogenases from anaerobic bacteria and archaea (group II; n = 32); (3) alternative nitrogenases (Mo-independent Anf and Vnf) and some Mo-Fe nitrogenases from Euryarchaeota 48 (group III; n = 11); (4) poorly characterized nif homologues (group IV; n = 123); (5) bacteriochlorophyll and chlorophyll biosynthesis genes 49 (group V; n = 1); and (6) putative tetrapyrrole cofactor biosynthesis genes 44 (group VI; n = 5). Group IV genes include its subclusters B ( n = 19), C ( n = 19) and E ( n = 16) with unknown functions, as well as subcluster D ( n = 69) involved in archaeal methionine biosynthesis 44 – 48 . Subcluster A within group IV also includes functional nitrogenases found in Endomicrobium proavitum that can fix nitrogen 50 , but none of the identified nifH homologues from the cold seep assemblies are affiliated with this subcluster. Despite this diversity, this classification scheme (based on Meheust et al.) 44 still did not sufficiently reflect the variety of nitrogenase genes found in cold seep diazotrophic populations. Following the approaches reported by Dekas et al. 35 , Miyazaki et al. 37 and Al-Shayeb et al. 51 , unclassified sequences formed three distinct lineages (Fig. 2 ) including (1) a clade similar to nifH found in Methanosarcina species but not clearly falling into the canonical groups (i.e., Methanosarcina -like group, MSL; n = 7), (2) a novel clade proposed here as group VII ( n = 15), and (3) a novel clade proposed here as group VIII containing nifH -like genes ( n = 6). Among the three novel lineages, MSL and group VII were considered as bona fide nifH based on the analyses of nitrogenase operon structure and conserved motif detailed below. Fig. 2 Maximum-likelihood phylogenetic tree of non-redundant nitrogenase subunit NifH identified from cold seep metagenomic assemblies. Homologues of nifH were classified into canonical groups I to III, nitrogen fixation-like groups IV to VI, and newly assigned groups including groups of Methanosarcina -like, VII and VIII. NflH denotes NifH-like sequences. Scale bar indicates the mean number of substitutions per site. Read abundance ratios of bona fide nifH ( n = 66) and single-copy ribosomal protein genes were used as a proxy for the relative abundance of putative diazotrophs in the total microbial community 52 , 53 . Diazotrophs were typically abundant within various types of cold seep sediments (24 ± 22% of the total bacterial and archaeal community) (Supplementary Data 2 ), but this varied greatly between samples (from 0.7% at a North Pacific gas hydrate to 93% at Gulf of Mexico oil and gas samples) exhibiting correlation with seep type and sediment depth (Fig. 3a ). Heterotrophic microorganisms must oxidize large amounts of organic carbon to generate sufficient ATP to fix nitrogen under anoxic conditions 43 . Accordingly, cold seeps classified as high-flux mud-prone systems, including oil, gas and methane seeps, hosted the highest densities of diazotrophs (Fig. 3a, b ), suggesting a potential control exerted by hydrocarbon flux rates on cold seep diazotrophs. Interestingly, a positive correlation was also observed between the gene abundance for nifH and the oxidative mcrA gene typical of ANME archaea (Spearman’s ρ = 0.77, P = 2.2e−16; Fig. 3c ). Variants of the oxidative methyl-coenzyme M reductase A (McrA) are used as indicators for estimating the relative abundance of anaerobic methane- and multi-carbon alkane-metabolizing archaea 5 , 54 , 55 . In this context, nifH sequences belonging to the newly discovered clade group VII were the most abundant (Fig. 3a ), highlighting the unique diversity of hydrocarbon seep diazotrophs compared to other studied ecosystems, including the deep-sea background sediments 29 , 56 . Fig. 3 Relative abundance patterns of 202 nifH genes. a Relative abundances of 202 nifH genes from different cold seep sediments, shown as RPKM (reads per kilobase per million mapped reads). b Comparison of nifH gene abundances in different types of cold seep ecosystems. n values refer to the number of biologically independent samples for statistics analysis. Asterisks indicate statistically significant differences between groups of mud volcanoes, oil and gas seeps, and methane seeps (determined by two-sided Wilcoxon Rank Sum test; * for P < 0.05, ** for P < 0.01 and *** for P < 0.001). Boxplot components: center line, median values; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. c Significant Spearman correlation between relative abundances of nifH and the oxidative mcrA gene. Percentages were calculated by dividing the RPKM value of nifH genes by the mean of RPKM values estimated from 14 single-copy marker genes. The gray shadow indicates the 95% confidence interval. The abbreviations of the sites are shown in Fig. 1 . Source data are provided as a Source Data file . Diverse diazotrophs from ten different phyla reside in cold seep sediments Using metagenomic assembly and binning strategies, we recovered 1428 non-redundant bacterial ( n = 1146) and archaeal ( n = 282) population genomes (Supplementary Data 3 ) belonging to 76 phyla based on the Genome Taxonomy Database (GTDB; see Methods). Most genomes were affiliated with the phyla Chloroflexota ( n = 239, namely Chloroflexi in NCBI taxonomy), Desulfobacterota ( n = 185, namely Deltaproteobacteria), Halobacteriota ( n = 114, namely Euryarchaeota), Proteobacteria ( n = 130), Acidobacteriota ( n = 70, namely Acidobacteria), Bacteroidota ( n = 65, namely Bacteroidetes), Planctomycetota ( n = 54, namely Planctomycetes), Thermoplasmatota ( n = 48, namely Thermoplasmata), and Asgardarchaeota ( n = 43, namely Asgard superphylum). Among these genomes, 20 bacterial and 15 archaeal MAGs spanning ten different phyla encoded nitrogenase genes (Fig. 4a and Supplementary Data 4 ), and belong to the Halobacteriota ( n = 14), Desulfobacterota ( n = 11), Chloroflexota ( n = 2), UBA6262 ( n = 2, candidate phylum), Altarchaeota ( n = 1), Caldatribacteriota ( n = 1, namely Atribacteria), Omnitrophota ( n = 1, namely Omnitrophica), FCPU426 ( n = 1, candidate phylum), Verrucomicrobiota ( n = 1, namely Verrucomicrobia), and Firmicutes ( n = 1). Within the phylum Halobacteriota, nitrogenase-encoding MAGs span the lineages ANME-1 ( n = 1), ANME-2 ( n = 7), Methanotrichaceae ( n = 1), and Methanosarcinaceae ( n = 5). Within the Desulfobacterota, nitrogenase-encoding MAGs belonged to the order of “C00003060” (aka SEEP-SRB1c 39 ) along with other non-ANME-associated bacterial groups such as BuS5 (aka Desulfosarcina sp. BuS5 in NCBI taxonomy), Desulfatiglandaceae and Syntrophales known to degrade alkanes or aromatic hydrocarbons coupled with sulfate reduction 7 , 57 . The increased diversity of bacterial and archaeal diazotrophic lineages substantially broadens the genomic database of microbial diazotrophs in deep-sea cold seep sediments 48 , which previously only included ANME-2b and SEEP-SRB1g 34 . Indeed, to our knowledge, this represents the first genomic evidence of nitrogen fixation potential in five different phyla, namely Altarchaeia, Omnitrophota, Caldatribacteriota along with two bacterial candidate phyla FCPU426 and UBA6262 48 , 58 . Among archaea, only lineages related to anaerobic methanogens and closely related anerobic methanotrophs are known or predicted to possess nitrogenases 58 , 59 . Our detection of Altarchaeia and other archaeal lineages (e.g., ANME-1) as potential diazotrophs also expand the known diversity of nitrogen-fixing archaea (Fig. 4a and Supplementary Data 4 ). Fig. 4 Maximum-likelihood phylogenetic trees of nitrogen-fixing MAGs and their NifH protein sequences. a Phylogenomic analysis of 35 MAGs containing nitrogen fixation genes. This maximum-likelihood tree is based on concatenation of 43 conserved protein sequences. MAGs are colored based on their phylogenetic affiliation at the phylum level. b Phylogenetic analysis of identified NifH protein sequences and genomic context of corresponding nif genes in the same 35 MAGs with nitrogen fixation genes. The scale bar represents one amino acid substitution per sequence position. For both trees, bootstrap values of >70% are indicated as black circles at the nodes, and scale bars indicate the mean number of substitutions per site. Phylogenetic analysis of NifH (Fig. 4b ) reveals that nitrogenases encoded by these 35 genomes belong to groups of II, III, MSL, and VII. A large majority of these genomes (32 out of the 35) encode nifHDK gene clusters for synthesis of the complete nitrogenase complex. Pairwise alignments of amino acids with bona fide nitrogenases (Supplementary Fig. 2 ) show that 28 identified NifH sequences contain conserved residues important for ATP hydrolysis and [4Fe4S] cluster coordination (Cys97 and Cys132) 46 , 60 . These NifH sequences also contain conserved residues (Arg100) for ADP-ribosylation, a reversible post-translational modification for nitrogenase activity regulation in the bona fide nitrogenases 45 . All residues required for the coordination of the P-cluster (Cys62, Cys88 and Cys154) with the Fe atom of the FeMo cofactor (Cys275 and His442) are conserved among 30 NifD sequences 45 , 61 (Supplementary Fig. 3 ). Crucial residues of the P cluster (Cys70, Cys95, and Cys153) are also conserved in 31 NifK sequences (Supplementary Fig. 4 ). By contrast, one or more conserved cysteine residues in the molybdenum nitrogenase subunits NifD and NifK for P-cluster coordination are absent in the bacteriochlorophyll oxidoreductase (ChlLNB and BchXYZ) and reductive cyclase of F 430 synthesis (CbfCD) systems (which both ligate a catalytic [4Fe4S] cluster instead) 46 , 62 . Overall, the conserved active sites observed among NifH, NifD and NifK homologues suggest that the newly assigned groups MSL and VII nitrogenases most likely function analogously to their canonical group I-III counterparts. For these 35 MAGs, each nif gene cluster also contained a pair of genes downstream of nifH that are here designated as nifI 1 and nifI 2 (Fig. 4b ). The products of nifI 1 and nifI 2 are both members from the P II family of nitrogen-regulatory proteins, known to switch-off nitrogenase activity at the post-translational level 63 . NifI 1 I 2 regulatory mechanisms are typically present in anaerobes, including all diazotrophic methanogens, as well as anaerobic bacteria including Chlorobium tepidum , Dehalococcoides ethenogenes and some Desulfobacterota 64 , 65 . Based on read mapping, distributions of the 35 diazotrophs were compared across metagenomes obtained in different types of samples from all of the cold seeps analyzed in this study (Supplementary Fig. 5 and Supplementary Data 5 ). Their overall relative abundance is 4 ± 3%, far below the estimated values based on read abundance ratios of nifH genes (Supplementary Data 2 ). Two possible explanations might account for this: (1) diazotrophic MAGs might contain multiple copies of nifH ; (2) there are still some diazotrophs that we did not recover here. When considered individually, Desulfobacterota (comprising up to 2% of the microbial community) and Caldatribacteriota (also up to 2%) represented the major bacterial diazotrophs, and Halobacteriota constituted major archaeal diazotrophs (up to 13%). While members of the Caldatribacteriota phylum are prevalent in cold seep sediments 11 , 40 , no previous studies have inferred that they are diazotrophic. These results highlight that Caldatribacteriota may play biogeochemically and ecologically significant roles within diverse cold seeps besides their role in carbon cycling 66 . Most other diazotrophs are at lower abundance (<1% of the microbial community). Overall, it can be speculated that cold seep diazotrophs are widespread and abundant to substantially contribute to the deep-sea nitrogen balance. Various organic and inorganic energy sources support nitrogen fixation With the consumption of 16 ATP molecules per dinitrogen reduced, the nitrogenase system is energetically costly for microorganisms 28 . To provide a global view of functional capabilities among the 35 diazotrophs, metabolic capabilities were annotated based on marker genes and pathways. Genomic analyses of these 35 MAGs identified four distinct groups regarding carbon cycling (Fig. 5 and Supplementary Data 6 – 8 ): (1) anaerobic methane-oxidizing archaea, including one ANME-1 and six ANME-2 (Fig. 5a ); (2) hydrogenotrophic methanogens, including one Methanotrichaceae and three Methanosarcinaceae (Fig. 5c ); (3) anaerobic non-methane alkane-degrading bacteria, including two Desulfobacteria (one Desulfatiglandaceae and one BuS5), one Syntrophales and one Caldatribacteria (Fig. 5d ); and (4) heterotrophs capable of degrading complex organic matter, such as cellulose, chitin, glucan, pectin, polyphenols, and starch (Supplementary Data 8 ). With respect to electron acceptors, sulfate reduction genes were identified in eight Desulfobacterota and one Caldatribacteriota (Fig. 5b and Supplementary Data 7 ), in agreement with a previous report that sulfate reduction supports diazotrophy in marine sediments 29 . Metal reduction related mtrC was identified in the genome of ETH-SRB1 (Supplementary Data 6 ), suggesting that this organism may also use iron or manganese as a terminal electron acceptor 67 . Three Desulfobulbales may be capable of both nitrate reduction and nitrogen fixation based on the presence of napA and napB (Supplementary Data 6 ). Based on the presence of two structural genes of form I RuBisCO and various genes for the Wood Ljungdahl pathway (Supplementary Data 6 ), some microorganisms represented by these MAGs can function as autotrophs, suggesting potential chemolithoautotrophic diazotrophy. The genomes also exhibit the potential for further assimilation of fixed ammonium into amino acids through the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) enzymes or NADH-glutamate dehydrogenase (GDH) 68 (Fig. 5 ). Fig. 5 Metabolic reconstruction of core pathways for nitrogen-fixing MAGs. a Anaerobic archaeal oxidation of methane; b dissimilatory sulfate reduction; c archaeal methanogenesis; d anaerobic degradation of alkanes by bacteria. Red font indicates that not all MAGs retrieved include the gene (numbers of MAGs with the corresponding gene indicated in parentheses). The percentages between brackets indicate the estimated completeness of the corresponding MAGs. Mtr N 5 -methyltetrahydromethanopterin–coenzyme M–methyltransferase, Mer 5,10-methylenetetrahydromethanopterin reductase, Mtd methylenetetrahydromethanopterin dehydrogenase, Mch methenyltetrahydromethanopterin cyclohydrolase, Ftr formylmethanofuran-tetrahydromethanopterin N-formyltransferase, Fwd formylmethanofuran dehydrogenase, Hdr heterodisulfide reductase, APS adenosine phosphosulfate, Apr adenylylsulfate reductase; Sat sulfate adenylyltransferase, GS glutamine synthetase, GOGAT glutamate synthase, GDH NADH-glutamate dehydrogenase, Ass alkylsuccinate synthase, AssK CoA-ligase, Mcm methylmalonyl-CoA mutase, Pcc propionyl-CoA carboxylase, Acd acetate-CoA ligase (ADP-forming). Detailed enzyme annotation is presented in Supplementary Data 7 . Nitrogenases not only mediate the reduction of molecular nitrogen into ammonia, but also reduce protons into molecular hydrogen during their reaction cycle 69 . Some diazotrophs identified here, including those within Caldatribacteriota, Desulfobacterota and Methanosarcinaceae (Supplementary Data 9 ), have the potential to internally recycle this hydrogen as an energy source, for example by using group 1 [NiFe]-hydrogenases linked to anaerobic respiratory chains 70 . Not all diazotrophs possess hydrogenases, potentially allowing non-diazotrophic bacteria to deploy uptake hydrogenases to consume hydrogen released by hydrogenase-deficient diazotrophs. The latter include various Chloroflexota, Desulfobacterota, Gammaproteobacteria, Campylobacterota, and Planctomycetota in the cold seep sediments. Thus, the nitrogenase reaction is also likely to have diverse consequences for nutrient cycling in cold seep sediments. To infer whether the potential diazotrophs identified in the metagenomes can fix N 2 under in situ conditions, two metatranscriptomes sequenced from Haima cold seep sediments and four metatranscriptomes sequenced from Jiaolong cold seep sediments (Fig. 1 and Supplementary Data 1 ) were mapped against nitrogenase-encoding MAGs. For both seep sites, the nifH genes of ANME-1, ETH-SRB1, and Caldatribacteriota were transcribed at moderate to high levels, up to 60–335 transcripts per million reads (TPM) (Supplementary Data 10 ), whereas fewer transcripts from ANME-2 were detected. Transcript levels were higher in deeper sediments relative to surficial layers, suggesting nitrogen fixation is particularly important when microbial carbon metabolism (e.g., methane oxidation) is prevalent and nitrogen oxides are limited 71 . Transcribed nifH genes in various microbial groups suggest that diverse catabolic processes actively fuel nitrogen fixation in cold seep sediments. This supports the hypothesis that nitrogenases have been acquired by organisms inhabiting nearly every characterized ecological niche, consistent with a selective advantage for organisms able to relieve nitrogen limitation. nif genes are subject to mobile genetic element transfers and purifying selection Microorganisms can acquire genes through horizontal gene transfer (HGT), which enables them to adapt to changing environmental conditions and thus occupy expanded ecological niches 72 . Previous studies have suggested that HGT events have crucially impacted the distribution of nitrogenase genes 45 , 48 , 73 . For example, thermophilic Aquificales acquired the ability to fix N 2 from thermophilic Deferribacteres 56 , and the acquisition of Nif by Firmicutes possibly arose through a HGT event with an ancestral methanogen 58 , 74 . Gene neighborhood analyses of MAGs from cold seeps revealed gene clusters of mobile genetic elements (MGEs) together with nifHDK , nitrogenase regulation and metal cofactor biosynthesis genes, and molybdenum/molybdate and ammonium transporter genes (Fig. 6 ). The MGEs identified here included retrotransposable and transposable elements, with the former transferred via an RNA intermediate 75 between host genomes and the latter serving as mobile DNAs 72 . Five diazotrophic MAGs contained genes for retrotransposable elements, including reverse transcriptase, endonuclease, DEAD/DEAH box helicase and nucleotidyltransferase. A transposon gene, integron integrase, was only found in one ETH-SRB1 genome. MGEs near nif gene clusters have been previously reported in other diazotrophs, indicating HGT 26 , 60 . To integrate into the host genome, MGEs require ATP hydrolysis 76 . Accordingly, AAA-type ATPase genes are interspersed with retrotransposable elements in the nif gene cluster of UBA6262 HMR_13C2018. These proteins have been biochemically demonstrated to control efficient transposition through DNA remodeling and transposase recruitment 77 . Meanwhile, the phylogenies of most NifH sequences were observed to be inconsistent with their corresponding taxonomies (Fig. 4 ). Except for Methanosarcina -like group NifH, sequences from group II, group III and group VII were scattered among diverse bacterial and archaeal phyla (Fig. 4 ). For example, six different bacterial phyla encoded NifH sequences of group II. Combined with the MGEs analysis, these results suggest that HGTs occurred among cold seep communities during their evolution. Nevertheless, vertical transmission of these genes in deep-sea cold seeps, like what has been observed for nitrogenases in the surface ocean, cannot be ruled out 25 . Fig. 6 Genomic context of nitrogen fixation genes. Gene neighborhoods of nifHDK include retrotransposable or transposon elements, regulatory nitrogen fixation genes, nitrogenase metal cofactor biosynthesis genes and transporter genes. Source data are provided as a Source Data file . Intra-population genetic diversity (i.e., microdiversity) may increase the fitness of a genotype in ecosystems with changing conditions. InStrain 78 was used to assess within-sample nif microdiversity based on metagenomic paired reads. Genomic nucleotide diversity (π) was calculated based on all reads, and as the average number of nucleotide differences per base pair for nifHDK genes. The observed nucleotide diversity was low, ranging from zero to 0.04, and mostly varied without significance between five different cold seep types (Fig. 7a ). This indicates that these genes are highly conserved both across and within samples, regardless of sampling location, possibly because few mutations accumulated during sediment burial 79 . Nucleotide diversity was also estimated to be similar among nifH , nifD and nifK genes (Fig. 7a ). The ratios of the two rates of non-synonymous to synonymous polymorphism (pN/pS) in nifHDK were determined (Fig. 7b ) to assess if genes are under purifying (negative) selection which involves the selective removal of deleterious mutations 79 . In general, pN/pS ratios below 1 indicate that a gene is under selective pressure to remove deleterious mutations to maintain protein function 80 . Calculation values were all well below 1 (between 0.02 and 0.5), suggesting that nif H, nif D and nif K genes are under strong purifying selection in cold seep sediments 81 . This is consistent with previous studies, as generally microbial genes encoding key functions will undergo higher purifying selection compared to genes that are dispensable 80 . Fig. 7 Evolutionary metrics of nitrogen fixation genes. a Nucleotide diversity (π) of nifHDK genes at different types of cold seeps; b pN/pS ratio of nifHDK genes at different types of cold seeps. Nucleotide diversity is used to measure genetic diversity within a population (microdiversity), which is calculated using the formula: 1 − [(frequency of A) 2 + (frequency of C) 2 + (frequency of G) 2 + (frequency of T) 2 ]. pN/pS is the ratio of non-synonymous to synonymous polymorphism rates within a population. n values refer to the number of biologically independent samples for statistics analysis. The significances were analyzed by two-sided Kruskal–Wallis Rank Sum test. Boxplot components: center line, median values; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. Source data are provided as a Source Data file ."
} | 8,201 |
32776389 | null | s2 | 5,786 | {
"abstract": "The order Sulfolobales was one of the first named Archaeal lineages, with globally distributed members from terrestrial thermal acid springs (pH < 4; T > 65°C). The Sulfolobales represent broad metabolic capabilities, ranging from lithotrophy, based on inorganic iron and sulfur biotransformations, to autotrophy, to chemoheterotrophy in less acidophilic species. Components of the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle, as well as sulfur oxidation, are nearly universally conserved, although dissimilatory sulfur reduction and disproportionation (Acidianus, Stygiolobus and Sulfurisphaera) and iron oxidation (Acidianus, Metallosphaera, Sulfurisphaera, Sulfuracidifex and Sulfodiicoccus) are limited to fewer lineages. Lithotrophic marker genes appear more often in highly acidophilic lineages. Despite the presence of facultative anaerobes and one confirmed obligate anaerobe, oxidase complexes (fox, sox, dox and a new putative cytochrome bd) are prevalent in many species (even facultative/obligate anaerobes), suggesting a key role for oxygen among the Sulfolobales. The presence of fox genes tracks with a putative antioxidant OsmC family peroxiredoxin, an indicator of oxidative stress derived from mixing reactive metals and oxygen. Extreme acidophily appears to track inversely with heterotrophy but directly with lithotrophy. Recent phylogenetic re-organization efforts are supported by the comparative genomics here, although several changes are proposed, including the expansion of the genus Saccharolobus."
} | 385 |
37448571 | PMC10336218 | pmc | 5,787 | {
"abstract": "Introduction Soil microbial communities are critical in regulating grassland biogeochemical cycles and ecosystem functions, but the mechanisms of how environmental factors affect changes in the structural composition and diversity of soil microbial communities in different grassland soil types is not fully understood in northwest Liaoning, China. Methods We investigated the characteristics and drivers of bacterial and fungal communities in 4 grassland soil types with 11 sites across this region using high-throughput Illumina sequencing. Results and Discussion Actinobacteria and Ascomycota were the dominant phyla of bacterial and fungal communities, respectively, but their relative abundances were not significantly different among different grassland soil types. The abundance, number of OTUs, number of species and diversity of both bacterial and fungal communities in warm and temperate ecotone soil were the highest, while the warm-temperate shrub soil had the lowest microbial diversity. Besides, environmental factors were not significantly correlated with soil bacterial Alpha diversity index. However, there was a highly significant negative correlation between soil pH and Shannon index of fungal communities, and a highly significant positive correlation between plant cover and Chao1 index as well as Observed species of fungal communities. Analysis of similarities showed that the structural composition of microbial communities differed significantly among different grassland soil types. Meanwhile, the microbial community structure of temperate steppe-sandy soil was significantly different from that of other grassland soil types. Redundancy analysis revealed that soil total nitrogen content, pH and conductivity were important influencing factors causing changes in soil bacterial communities, while soil organic carbon, total nitrogen content and conductivity mainly drove the differentiation of soil fungal communities. In addition, the degree of connection in the soil bacterial network of grassland was much higher than that in the fungal network and soil bacterial and fungal communities were inconsistently limited by environmental factors. Our results showed that the microbial community structure, composition and diversity of different grassland soil types in northwest Liaoning differed significantly and were significantly influenced by environmental factors. Microbial community structure and the observation of soil total nitrogen and organic carbon content can predict the health changes of grassland ecosystems to a certain extent.",
"conclusion": "5. Conclusion In summary, our results implied that the structural composition and diversity of both bacterial and fungal communities in different grassland soil types in northwestern Liaoning Province were significantly different, but bacterial and fungal communities differed in their responses to the environment and bacterial community network was more stable than fungal community network. Temperate steppe-sandy soils were the most degraded grasslands. The diversity of bacterial and fungal communities was highest in the warm and temperate ecotone soils, while the warm-temperate shrub soils had the lowest bacterial diversity. The microbial community structure of temperate steppe-sandy soil differed significantly from the others. Our findings revealed the crucial influence of soil pH and plant cover on fungal community diversity and suggested that soil total nitrogen content, pH, electrical conductivity and soil organic carbon content predominantly explained the variability of soil microbial community structures. Our study demonstrated that changes in grassland ecosystem health can be predicted to some extent by microbial community structure and the corresponding observations of soil total nitrogen and organic carbon content.",
"introduction": "1. Introduction Grasslands are one of the most widespread vegetation types in the world, covering about 1/3 of the land area ( Kemp et al., 2013 ), and are an important part of terrestrial ecosystems ( Merbold et al., 2014 ). Grassland types are divided into nine major types ( Wu, 1990 ), temperature and precipitation are often used in the study of grassland type classification ( Del Vecchio et al., 2018 ; Peng et al., 2021 ). The climate of northwestern Liaoning Province is transitional from temperate semi-humid to semi-arid from southeast to northwest, and is characterized by abundant sunshine, rain and heat in the same season, high cumulative temperature, and low precipitation ( Wang et al., 2013 ). The land in this area is barren, with serious soil erosion and desertification ( Wang et al., 2021 ). Under the combined influence, many complex grassland soil types have been formed, among which the temperate steppe and the warm-temperate shrub are the most unique and have great research value. With the in-depth research and practice, many researchers have realized that the degradation of grassland is not only the change of surface vegetation and soil physicochemical properties, but also the change of grassland soil microbial community structure and diversity ( Yu et al., 2021 ; Wang et al., 2022 ). Soil is rich in living microbiota, which are an important part of soil ecosystem. The species, quantity, distribution, life activity pattern of microbial communities and the transformation with the materials and energy in the soil are the values of soil microbial resources. It plays a leading role in ecosystem functions such as soil organic matter decomposition and nutrient cycling ( Wei et al., 2017 ). Compared with soil microorganisms such as fungi, actinomycetes and soil protozoa, soil bacteria are the most important taxon, accounting for about 70 to 90% of the total soil microorganisms, and play a dominant role in biogeochemical cycling processes ( Van der Heijden et al., 2008 ; Bar-On et al., 2018 ). However, fungi have higher relative abundance and diversity in the rhizosphere soil ( Bahram et al., 2021 ). Soil bacteria and fungi together drive and regulate biological processes such as mineralization, degradation and transformation of various nutrient element in the soil, and also have an important impact on plant growth and biological yield ( Kaiser et al., 2016 ). They are directly participate in biochemical processes such as ammonification, nitrification, denitrification and biofixation of nitrogen in the soil ( Huang et al., 2013 ), and play a key role in soil organic matter degradation and soil carbon cycling processes ( Chen and Sinsabaugh, 2020 ). Thus, the composition and biodiversity of soil bacterial and fungal communities in grassland ecosystems have become the focus of attention by many scholars ( Bhattacharyya et al., 2014 ). The construction process of microbial communities is decisively influenced by environmental factors ( Tatari et al., 2017 ). Soil microorganisms are very sensitive to the living microenvironment and can respond rapidly to changes in the soil environment, thereby affecting the species diversity of plant community and the formation of soil structure. Therefore, changes in soil microbial community structure and diversity can be used as important indicator to measure the health changes of grassland ecosystems and the degree of grassland degradation or restoration ( Wu et al., 2014 ). Tardy et al. (2014) showed that the biodiversity of soil bacterial community determined the structure of soil bacterial community and its ecological service functions, while the study by Jia et al. (2020) showed that environmental factors play a very important role in driving fungal community construction. Existing studies have shown that soil microbial diversity differed significantly among different grassland community type ( Qi et al., 2021 ), and the more suitable the habitat conditions (High total nitrogen and organic carbon content and suitable soil pH, etc.), the higher the diversity was Santamaría et al. ( Santamaría et al., 2018 ). Besides, the diversity of different soil bacterial communities was mainly influenced by pH, organic matter, alkaline nitrogen and effective phosphorus content ( Dunbar et al., 2002 ; Roy et al., 2020 ; Jiang et al., 2021 ), while soil pH and organic matter content played a decisive role in the structure of different soil fungal communities ( Guo et al., 2021 ; Shang et al., 2021 ). Therefore, the microbial community structure and diversity could differ under different grassland soil types and may be influenced by distinct environmental factors. Further studies to analyze the bacterial and fungal communities and their diversity in different regional grassland soil types are conducive to better utilization and development of grassland resources. Although many studies have been conducted on the influence of environment on microbial communities, against the background of relatively limited overall knowledge of soil microorganisms in different grassland types in northwest Liaoning, the study on the characteristics of soil microbial communities and their influencing factors in different grassland types in northwest Liaoning can not only enrich theoretically the research content of different grassland types, but also provide scientific basis for maintaining the stability of fragile ecosystems in our province and protecting the diversity of regional biological resources. To understand the characteristics and influencing factors of bacterial and fungal communities in different grassland soil types in northwest Liaoning, we used high-throughput sequencing to answer two questions: (1) Are there differences in the structural composition of bacterial and fungal communities in different grassland soil types? and (2) What are the underlying mechanisms for the effects of environmental factors on soil bacterial and fungal communities? Based on the results of previous studies, we hypothesize that: (i) There could be significant differences in the structural composition of the bacterial and fungal communities in different grassland soil types and (ii) Changes in environmental factors such as soil total nitrogen (TN) and soil organic carbon (SOC) would drive the observed changes in soil bacterial and fungal communities.",
"discussion": "4. Discussion Actinobacteria and Ascomycota were the common dominant phylum of bacterial and fungal communities in different grassland soil types in northwest Liaoning ( Figure 3 ). This is similar to the structural composition of fungal communities in Hulunbeier sand area, Loess Plateau grassland and Inner Mongolia desert grassland ( Huang et al., 2021 ), but there are obvious differences with the structural composition of their bacterial communities, probably because the grassland in northwest Liaoning is in the process of recovery. Actinomycetes have a variety of antibacterial, antioxidant and enzyme inhibitor functions ( Amrita et al., 2012 ; Arumugam et al., 2017 ), and they have positive effects on both soil and plants in grassland, which can change the dominant flora and eliminate harmful microorganisms, while protecting and promoting root growth. And Ascomycota have the function of degrading environmental pollutants and restoring soil ( Germain et al., 2021 ), while most of the grasslands in northwest Liaoning are degraded grasslands, which can explain the dominant role of Ascomycota in this study. Moreover, there may be a close link between the relative abundance of soil bacterial community genera and their functions, soil physicochemical properties may be an important factor influencing the differences in bacterial community composition ( Liang et al., 2021 ). Subgroup 6 plays an important role in the global carbon cycle ( Pan et al., 2019 ), and their relative abundance correlates with soil carbon content. In this study, the relative abundance of Subgroup 6 of temperate steppe-sandy soil was significantly lower than that of other grassland soil types, while the soil organic carbon content was similarly significantly lower than that of other grassland soil types ( Table 2 ). The relative abundance of RB41 is somewhat representative of inter-root soil nutrient changes, and there is a significant positive correlation between them ( Liu et al., 2022 ). The soil nutrient content of temperature step sandy-soil in this study is the lowest ( Table 2 ), which explains why its relative abundance of RB41 is significantly lower than that of other grassland soil types. Rubrobacter is highly resistant to radiation, salinity, heat and even thermophilicity, and soils with higher relative abundance of Rubrobacter are more resistant to stress ( Kouřilová et al., 2021 ). In the study, the relative abundance of Rubrobacter in temperate steppe-sandy soil was significantly lower than in other grassland soil types, indicating that temperate steppe-sandy soil had the least resistant. In this study, most of the soil fungal community genera contained high virulence and pathogenicity, such as Fusarium and Penicillium . The reason may be the degradation of the sampled grassland soils. Pseudogymnoascus belongs to cold-tolerant fungi, which are more suitable for survival in alpine environments ( Shi et al., 2021 ), which explains why he relative abundance of Pseudogymnoascus was the highest in temperate steppe-sandy soil. Meanwhile, the more connections in the soil microbial community network, the higher its stability and the stronger the ability to inhibit the invasion of pathogens ( Shi et al., 2016 ; Wei et al., 2018 ). In this study, the degree of connection in the soil bacterial network of grassland was much higher than that in the fungal network ( Figure 4 ), indicating that the bacterial community had a strong resistance to invasion, which further proved that the bacterial community dominated the grassland in northwest Liaoning. In addition, the extent of grassland degradation or restoration can be predicted to some extent by soil microbial symbiotic networks. Studies have shown that enhanced microbial interactions and higher levels of recovery in grasslands lead to more complex interaction networks ( Gao et al., 2021 ). The microbial symbiotic network in this study was relatively simple and belonged to a degraded grassland ( Figure 4 ). Microbial diversity has an important influence on the stability of grassland ecosystems, and the microbial diversity index can evaluate the abundance and evenness of microbial communities in soils, as well as reflect the different species composition and functions of microbial communities ( Kim et al., 2017 ). Alpha diversity of bacterial and fungal communities in different grassland soil types showed significant differences in the study of Liu (2019) , which is consistent with the findings of this study. The abundance, number of OTUs, number of species and diversity of both soil bacterial and fungal communities in the warm and temperate ecotone were all the highest ( Figure 1 ). The reason may be that the warm and temperate ecotone is simultaneously influenced by both warm and temperate grassland types, with a large environmental gradient and high primary and secondary productivity resulting in a rich diversity of soil bacterial communities in grasslands. Which is reflected in Huang et al. (2016) , where microbial diversity was enriched under the influence of dual environmental factors. However, Araujo et al. (2021) reported that the soil nutrient content and microbial diversity index in the interlacing zone showed intermediate values of adjacent communities. Ecotones are areas where two biomes meet due to sudden changes in soil properties caused by anthropogenic activities or changes in climatic conditions, and are areas of very high species abundance and diversity ( Eddy, 1990 ; Bossuyt et al., 1999 ). Most studies on ecotones have focused on plants, animals and insects, and ecological ecotones are usually identified by obvious changes in above-ground vegetation ( Pinheiro et al., 2015 ; Coelho et al., 2017 ). Nevertheless, there is no clearer understanding of soil microbial properties, so the study of microorganisms in ecotones needs to continue to be explored continuously. In addition, the bacterial diversity index of warm-temperate shrub was almost significantly lower than that of other grassland soil types ( Figure 1 ). The reason may be that the presence of shrubs makes the grass covered by stunted growth and development, which in turn affects the diversity of bacteria. In Idbella’s study ( Idbella et al., 2022 ), it was demonstrated that the abundance of soil oligotrophs under shrub cover decreased significantly, which in turn decreased the soil bacterial diversity. At the same time, there were also obvious differences in microbial community structure among different grassland soil types ( Figure 2 ). The structure of both bacterial and fungal communities in temperate steppe-sandy soil differed significantly from other grassland soil types ( Supplementary Figure S2 ). In the soil environment of the whole Liaoning Province, the sand content is relatively high, which is due to the desertification caused by soil degradation. We divide the temperate steppe into two soil types (sandy soil and loamy soil) according to soil texture and properties, while the warm and temperate ecotone and the warm-temperate shrub are both loamy soil, which makes temperate steppe-sandy soil is absolutely unique. In the study of Sessitsch et al. (2001) , changes in soil particle size lead to changes in soil microbial community structure, which could explain the significant differences of microbial community structure between temperate steppe-sandy soil and other grassland soil types. In addition, studies have shown that there were significant differences in bacterial and fungal community structure during the succession stages of grassland degradation and restoration ( Guo et al., 2019 ), which means that the degree of degradation of grassland soil can be judged by observing the differences in microbial community structure of different grassland soil types. In this study, the microbial community structure was significantly different in the temperate steppe-sandy soil ( Figure 2 ), which proved the highest degree of soil degradation. Environmental factors were strongly associated with soil microbial communities ( Franklin and Mills, 2009 ). The Pearson correlation analysis showed that the diversity of soil fungal communities was positively correlated with plant cover and negatively correlated with soil pH ( Figure 4 ). As the most important characteristic of soil, pH could affect the diversity of soil microorganisms by altering their nutrient utilization efficiency and enzyme activity. In the study by Zhang et al. (2017) , higher soil pH could reduce the nutrient use efficiency of fungi, which in turn reduced the diversity of the fungal community. In addition, there was a correlation between soil pH and soil salinity, and a joint increase in both also inhibited microbial activity ( Yang et al., 2020 ). Besides, there was a positive correlation between plant cover and soil phosphorus content, and microbial assimilation and storage of P was fundamental for its availability for plant colonization ( Canini et al., 2019 ). In the study by Dang et al. (2018) , the increase of plant cover improved soil ecological functions and optimized the nutrient environment, which in turn increased fungal diversity. However, the Alpha diversity of soil bacterial communities was not restricted by the measured environmental factors in spite of the significant differences in bacterial diversity among different types. What’s more, it can be seen that the responses of soil bacterial and fungal communities to environmental changes were inconsistent, this is supported by the different responses of soil bacterial and fungal communities to different nitrogen application levels and pH intervals in the study by Li et al. (2019) . The RDA analysis showed that the environmental factors affected the soil bacterial and fungal communities, which was consistent with the finding of Furtak that microbial communities with higher relative abundance are significantly affected by environmental factors ( Furtak et al., 2020 ), but the dominant influencing factors would be different. In the present study, soil total nitrogen content, organic carbon, pH and electrical conductivity were found to be main factors explaining the changes observed in the microbial community structure in grasslands of northwest Liaoning. Soil total nitrogen content has multiple effects on the growth, composition and function of microorganisms ( Zhang et al., 2018 ). Soil pH could shape microbial community structure by modifying enzymes activity and controlling the accessibility of nutrient and moisture supplements ( Cao et al., 2016 ; Ren et al., 2018 ). Soil electrical conductivity affects the conversion and cycling of phosphorus and carbon in the soil, which in turn influences the microbial communities ( Nan and Petra, 2013 ). The key parameter of soil organic carbon cycling is microbial carbon use efficiency, and its content significantly affects the activity of soil microorganisms ( Wu et al., 2022 ). What’s more, RDA ordination revealed distinct differences in microbial community composition between grassland soil types. Microbial communities of the temperature steppe-sandy soils tended to be distributed in environments with lower soil total nitrogen and organic carbon contents ( Figure 6 ), which illustrated the lowest soil properties in the temperature steppe-sandy soils ( Table 2 ). However, the environmental factors selected in our study were not enough to explain the structural changes in soil bacterial and fungal communities in northwest Liaoning grasslands. Soil microbial communities could also be influenced by soil salinity, climate, season, and geographic location factors ( Liang et al., 2022 ). Furthermore, light may affect carbon cycling and thus influence microbial communities through plant photosynthesis, and microbial communities would be also strongly influenced by temperature ( Ruiz and Azcon, 1996 ), and there may be interaction effects between the various factors ( Yao et al., 2018 ). Therefore, a wider range of environmental factors should be further investigated in the future to obtain more comprehensive information. Soil total nitrogen and organic carbon content are considered important indicators of grassland quality ( Wang et al., 2022 ), and studies have shown that soil quality in degraded grassland is severely deteriorated and soil total nitrogen and organic carbon content was significantly reduced ( Byrnes et al., 2018 ). Yang (2022) also demonstrated that soil carbon and nitrogen levels gradually increased as grasslands were restored. In this study, the temperate grassland sandy soil had the lowest total nitrogen content and significantly lower organic carbon content than other grassland soil types, which also proved that it had the highest degree of grassland degradation."
} | 5,755 |
35622915 | PMC9140962 | pmc | 5,788 | {
"abstract": "Among the earliest consequences of climate change are extreme weather and rising sea levels—two challenges to which coastal environments are particularly vulnerable. Often found in coastal settings are microbial mats—complex, stratified microbial ecosystems that drive massive nutrient fluxes through biogeochemical cycles and have been important constituents of Earth’s biosphere for eons. Little Ambergris Cay, in the Turks and Caicos Islands, supports extensive mats that vary sharply with relative water level. We characterized the microbial communities across this variation to understand better the emerging threat of sea level rise. In September 2017, the eyewall of category 5 Hurricane Irma transited the island. We monitored the impact and recovery from this devastating storm event. New mat growth proceeded rapidly, with patterns suggesting that storm perturbation may facilitate the adaptation of these ecosystems to changing sea level. Sulfur cycling, however, displayed hysteresis, stalling for >10 months after the hurricane and likely altering carbon storage potential.",
"introduction": "INTRODUCTION Coastal environments are uniquely vulnerable to the consequences of climate change; lying at the interface of land, ocean, and atmosphere, they are directly affected by sea level rise, extreme weather, and changes in both air and ocean temperature and chemistry. Some coastal ecosystems, e.g., coral reefs and salt marshes, are already suffering devastating losses in extent and biodiversity ( 1 – 3 ), while others, e.g., mangrove forests, appear to be expanding and even mitigating climate change impacts by enhancing land stabilization and carbon storage ( 4 – 7 ). Here, we examined a predominantly microbial ecosystem facing these challenges: photosynthetic microbial mats, which are often found in close association with mangroves and are thought to play a major role in shallow sediment nutrient availability ( 8 ). Photosynthetic microbial mats are assemblies of microbes that form layered, macroscopic structures. Their fabric is commonly built by filamentous Cyanobacteria ( 9 ), and the communities that inhabit them rank among the most diverse microbial ecosystems known ( 10 – 12 ). Within a mat, steep physicochemical gradients partition a complex network of niche spaces ( 13 , 14 )—sunlight drives phototrophy in the surface layers ( 15 – 17 ); in the subsurface, redox stratification and other chemical gradients support a wide range of anaerobic metabolisms ( 18 – 23 )—and tightly coupled metabolic interactions fuel rapid and dynamic biogeochemical cycling with a diurnal cadence ( 13 , 24 ). These ecosystems have been important components of the biosphere since long before the rise of plants and animals ( 25 – 27 ), a history recorded by their mineralized vestiges preserved in ancient sedimentary rocks ( 28 – 30 ). Little Ambergris Cay is an uninhabited island in the Turks and Caicos with a broad, shallow interior basin widely paved by benthic microbial mats ( Fig. 1, A and B ). This remote environment is an ideal natural laboratory—both for better understanding modern mat ecosystems and as an analog for the ancient mat ecosystems that dominated the Earth through much of its history ( 31 – 35 ). The mats on Little Ambergris Cay exhibit a variety of macroscopic textures ( Fig. 1 ), ranging in thickness from millimeters to decimeters, in consistency from leathery to gelatinous, and in surface character from botryoidal or tufted to smooth. In previous studies, these mats have been categorized into three end-member types ( 32 ), termed blister mats, polygonal [or biscuit ( 31 )] mats, and smooth [or flat ( 31 , 33 )] mats ( Fig. 1, C to H ). The basis for this morphological diversity has been of interest to the geobiological community, as mat textures preserved in the geological record provide clues about ancient microbial ecosystems ( 31 , 32 ). Fig. 1. Maps and context images. ( A ) Satellite image of the Caicos carbonate platform, white arrow pointing out Little Ambergris Cay. ( B ) Drone orthomosaic of Little Ambergris Cay with study areas indicated. Aerial images of these regions documenting changes over time and sample details can be found in fig. S1. ( C to H ) Surface (C to E) and cross-sectional (F to H) photographs of end-member mat types—blister mats, of millimeter-scale thickness characterized by rough, black, or gray surfaces (C and F); polygonal mats, of centimeter- to decimeter-scale thickness with highly cohesive, often fibrous mat fabric and dark green tufted surfaces characterized by desiccation cracks that delineate polygons (D and G); and smooth mats, of generally centimeter-scale thickness and ranging in consistency from moderately cohesive to loose and goopy, often covered in beige exopolysaccharide material (E and H). ( I ) National Oceanic and Atmospheric Administration Geostationary Operational Environmental Satellite network infrared image of Hurricane Irma with the eye directly over Little Ambergris Cay on 7 September 2017, 22:45 UTC. Black traces indicate land masses, and white box indicates the area shown in (A). Previously, we conducted a comprehensive mapping effort of these different mat types across Little Ambergris Cay and showed that the primary factors determining their distribution are water depth and tidal exposure time above water ( 32 ). The mats exist only within a narrow elevation range; areas higher than 30 cm above mean water level host scrubland rather than mat, and areas lower than 20 cm below mean water level strong hydrodynamic forces inhibit mat development. Within this range, blister mats occur in the highest, driest areas (subaerial exposure times of 22 to 24 hours/day), polygonal mats in intermediate areas (subaerial exposure times of 12 to 23 hours/day), and smooth mats in lower, wetter areas (subaerial exposure times of 0 to 12 hours/day). Since small (centimeter-scale) differences in water level exert such a strong control on mat habitat ranges, this is a system that is acutely sensitive to one of the most immediate consequences of global climate change—sea level rise ( 36 ). However, observations of ancient mat ecosystems from the geological record demonstrate that mats have persisted across numerous intervals of rising and falling sea level, with textural changes tracking changes in water depth ( 37 , 38 ). This history suggests the hypothesis that while mat ecosystems are finely tuned to water level, they may also be robustly adaptable. The present study of Little Ambergris Cay microbial mat communities was initiated to better understand the ecological differences among mat types. Our approach combined 16 S ribosomal RNA (rRNA) gene amplicon sequencing and community analysis with physical, geochemical, and biological field observations. Initial field campaigns were conducted in July 2016 and August 2017, surveying the diversity of microbial mats across the island in 2016 and focusing on the ecosystem structure with depth in 2017. In September 2017, Little Ambergris Cay experienced a direct hit by the eyewall of category 5 Hurricane Irma ( Fig. 1I )—one of the strongest hurricanes ever recorded in the Atlantic—with 920-mbar average atmospheric pressure and sustained 170 miles/hour winds accompanied by an estimated 3.2-m storm surge ( 39 , 40 ). Tropical cyclones of increasing intensity are another impending consequence of climate change ( 41 ). While sea level rise, warming, and acidification manifest over time scales of decades, extreme weather events cause marked environmental changes over time scales of hours to minutes and therefore can be much more immediately devastating to vulnerable ecosystems ( 1 , 42 ). In contrast to the adaptability of mat ecosystems to changes in sea level, the geological record demonstrates that sudden blanketing with a sediment layer can terminate mat growth ( 28 ). Having characterized the baseline ecosystem just before Hurricane Irma, we were uniquely well poised to investigate how the mat communities responded to such a catastrophic disturbance. Follow-up studies were conducted in March 2018, July 2018, and June 2019, to document the impact and subsequent recovery.",
"discussion": "DISCUSSION In ecological theory, perturbations are classified as pulses—discrete, relatively instantaneous alterations—or presses—sustained, gradual alterations ( 47 , 48 ). Global climate change is, by definition, a press; however, it also increases the frequency and severity of pulses, including but not limited to extreme storm events such as Hurricane Irma ( 41 , 49 ). These different types of perturbation tend to carry different patterns of microbial community response, and the impacts of multiple perturbations may interact with each other in complex ways ( 50 ). Therefore, understanding the ecological implications of climate change requires understanding how each type of perturbation affects communities, the extent to which communities can recover from them, and how they might influence each other. The dataset presented here has implications for both pulse (Hurricane Irma) and press (sea level rise) perturbations on a coastal microbial mat ecosystem. Our depth profile characterization described an ecosystem governed by carbon cycling through primary producers and decomposers (and secondary and tertiary decomposers) and sulfur cycling through both producers and consumers of sulfide. In many ways, this nutrient cycling is the microbial equivalent of the trophic levels that comprise classical macrofaunal ecosystems; rather than predator/prey relationships, species interactions are based primarily on the production and consumption of chemical substrates. The hurricane severely disrupted the chemical gradients that enabled many of those interactions. The rapid development of new growth in the wake of the hurricane reflected the populations not dependent on those gradients or the buildup of certain substrates—phototrophs, aerobic heterotrophs, and metabolically flexible mixotrophs—but lacked many of the niche spaces available in the climax community, exemplified by the absence of sulfur cycling taxa and a sulfidic chemocline. By analogy to classical ecology, the populations dependent on an intricate food web (or higher trophic levels) lagged behind the initial community. The subsequent return of sulfate-reducing and sulfide-oxidizing bacteria along with a sulfidic chemocline illustrated the recovery of biogeochemical cycling characteristic of a mature mat ecosystem. The sulfur cycle has important connections to the carbon storage potential of mangrove and mat ecosystems. Reactions between dissolved sulfides and organic matter have been implicated in decreasing organic matter lability and thereby increasing its preservation potential. This phenomenon is known to occur in the Little Ambergris mats ( 51 ) and has been suggested to account for as much as half of the organic matter preservation associated with mangrove forests ( 52 )—ecosystems noted for their disproportionately important contributions to global carbon storage and therefore targeted by restoration and conservation efforts aimed at ameliorating anthropogenic carbon emissions ( 53 ). In the absence of sulfides generated by microbial sulfate reduction, these sulfurization reactions are unlikely to occur. Therefore, although the mat sulfur cycle ultimately recovered from the hurricane impact, the interruption that we observed likely carries consequences in the form of lost carbon storage potential. This means that the expected increase in extreme storm events due to climate change may have adverse implications for the carbon sequestration capacities of mangrove and mat ecosystems. Since the sulfur cycle disruption was seen even in mats that remained fully intact, this aspect of the hurricane impact was likely due to the extreme degree of fluid inundation flushing away soluble substrates and overwhelming anaerobic communities with oxic waters rather than physical disruption of mat integrity or burial. That being said, the sediment underlying the Little Ambergris mats comprises primarily ooid sand grains, which approximate close-packed spheres and therefore accommodate substantial pore space that promotes fluid permeability. This means that considerable flushing likely accompanies normal tidal cycles, introducing oxic seawater and moving soluble nutrients ( 34 ), and the gradients powering mat biogeochemical function are robust enough to weather that degree of flushing. Therefore, the flushing induced by Hurricane Irma must have exceeded some critical threshold in their O 2 -buffering capacity. Irma was the strongest hurricane ever to hit Little Ambergris Cay in recorded history, although the island experiences hurricane force winds on average once every 5.5 years ( 54 ), and tropical storms more frequently than that. A better understanding of where this threshold sits on the continuum from normal daily tidal flushing to Hurricane Irma is required to appreciate the severity of these implications for changes going forward. In contrast to the posthurricane rapid colonization of fresh surfaces and reestablishment of gradients in surviving mats, adaptation to changing sea level requires mats in a given location to shift from one type to another as relative water level shifts around them. For the community differences among mat types to persist, taxa that are specific to a given mat type—and therefore likely well adapted to the narrow habitat ranges that distinguish them—will have to migrate into areas that previously hosted a different mat type. However, our transplant experiment demonstrated impressive persistence of a polygonal mat community in the environmental context of a blister mat. This suggests that although mat morphologies will shift with changing sea level, established mat communities that can tolerate the change may exhibit priority effects, inhibiting the immigration of exogenous taxa that would otherwise be better adapted to that specific environment ( 48 , 55 ). Nonetheless, we observed posthurricane new growth analogous to the full range of mat types—with analogous community differences—after the hurricane had scoured out or buried much of the mat area. This new growth occurred at a much higher rate than steady-state mat growth, suggesting that the hurricane perturbation enabled the new growth, perhaps by resetting whatever factors limit growth, creating fresh surfaces for colonization, or aiding in dispersal. It is possible that by disrupting the invasion-resistant established mat communities and promoting the redistribution of taxa, these perturbations could facilitate the development of mat communities most optimized to a given habitat range. Therefore, the occurrence of pulse disturbances such as a hurricane may enable adjustment to the press disturbance of sea level change for this ecosystem, exemplifying the complex effects of multiple simultaneous forcing factors. Together, this study demonstrates the substantial resilience of Little Ambergris Cay microbial mats in the face of both pulse and press disturbances induced by climate change. The mat communities and putative biogeochemical functions largely recovered from Hurricane Irma—a markedly destructive perturbation—within 2 years. In contrast, catastrophic hurricanes threaten extinction for island macrofauna with limited reproduction rates and dispersal abilities ( 56 ). While this robustness in the face of environmental perturbation is consistent with the geological record of microbial mat ecosystems persisting through past intervals of climate change, this study resolved a granularity that can only be observed in the modern and rates of both perturbation and recovery that likely exceed most historical examples."
} | 3,943 |
34346142 | PMC8456820 | pmc | 5,790 | {
"abstract": "Summary Coastal salt marshes are key sites of biogeochemical cycling and ideal systems in which to investigate the community structure of complex microbial communities. Here, we clarify structural–functional relationships among microorganisms and their mineralogical environment, revealing previously undescribed metabolic activity patterns and precise spatial arrangements within salt marsh sediment. Following 3.7‐day in situ incubations with a non‐canonical amino acid that was incorporated into new biomass, samples were resin‐embedded and analysed by correlative fluorescence and electron microscopy to map the microscale arrangements of anabolically active and inactive organisms alongside mineral grains. Parallel sediment samples were examined by fluorescence‐activated cell sorting and 16S rRNA gene sequencing to link anabolic activity to taxonomic identity. Both approaches demonstrated a rapid decline in the proportion of anabolically active cells with depth into salt marsh sediment, from ~60% in the top centimetre to 9.4%–22.4% between 2 and 10 cm. From the top to the bottom, the most prominent active community members shifted from sulfur cycling phototrophic consortia, to putative sulfate‐reducing bacteria likely oxidizing organic compounds, to fermentative lineages. Correlative microscopy revealed more abundant (and more anabolically active) organisms around non‐quartz minerals including rutile, orthoclase and plagioclase. Microbe–mineral relationships appear to be dynamic and context‐dependent arbiters of biogeochemical cycling.",
"conclusion": "Conclusions The biological community of Little Sippewissett salt marsh sediment demonstrated notable differences in its composition, anabolic activity patterns, spatial arrangements and mineralogical associations at the three distinct horizons analysed in this study. Following incorporation of HPG into new biomass during a 3.7‐day in situ incubation experiment, correlative microscopy, BONCAT‐FACS and 16S rRNA gene amplicon sequencing demonstrated that the most prevalent active constituents shifted from sulfur cycling phototrophic consortia in the surficial horizon, to putative sulfate‐reducing bacteria likely oxidizing a range of organic compounds, to a range of fermentative lineages in the lower horizons. We observed a rapid decay in the proportion of active organisms from ~60% in the top centimetre to between 9.4%–22.4% in the horizons between 2–10 cm depth, offering a quantifiable reflection of the shift to the dark, anoxic environment. By embedding sediment cores in resin, we mapped biomass and mineral grains with microscale resolution and found that, on average, organisms were most likely to be found inside mineral grains in the lowermost horizon. Plagioclase, orthoclase and rutile minerals recruited more abundant communities that contained a higher proportion of anabolically active organisms compared with quartz grains. Taken together, these findings give the impression of a more spatially and metabolically expansive community in surface sediments, fuelled by sunlight and a range of available niches, that is streamlined by burial and mineralogical weathering. This benchmark study presents a promising new approach for exploring the anabolic activity of a complex microbial community by mapping the precise spatial configuration of anabolically active organisms within mineralogically heterogeneous salt marsh sediment through correlative fluorescence and electron microscopy, while simultaneously identifying active organisms in neighbouring sediment with BONCAT‐FACS and 16S rRNA gene sequencing. The structure, activity and evolutionary trajectory of complex microbial communities are determined by the interactions between biotic and abiotic components of an ecosystem. Spatial relationships are a powerful indication of these interactions, particularly in concert with the identification of metabolically active organisms. Looking forward, the incorporation of rRNA‐targeted FISH into this workflow would enable a more direct connection between microbe‐mineral spatial arrangements and taxonomically constrained activity patterns. Improved approaches for understanding microscale ecosystems in a new light, such as those presented here, reveal environmental parameters that promote or constrain metabolic activity and clarify the impact that microbial communities have on our world.",
"introduction": "Introduction Salt marshes are vibrant microbial habitats that play important roles in the biogeochemical cycling of intertidal ecosystems (Tobias and Neubauer, 2019 ). The confluence of high organic input and seawater‐derived sulfate fuel a wide range of carbon, nitrogen, phosphorous and sulfur transformations over compressed spatial scales, leading to abundant, redox‐specific niches and microbial communities with high phylogenetic diversity (Lozupone and Knight, 2007 ; Bowen et al ., 2012 ). Because of this, salt marshes represent ideal sites to explore the intricacies of microbial community structure from the microscale to the ecosystem scale. Within complex microbial communities, spatial relationships are increasingly seen as central determinants of key ecological parameters. In salt marshes, metabolic activity within specific sediment horizons ultimately shapes emergent properties such as carbon sequestration or greenhouse gas emissions (Abdul‐Aziz et al ., 2018 ; LaRowe et al ., 2020 ). More generally, microbe–microbe and microbe–mineral interactions establish evolutionary trajectories (Cordero et al ., 2012 ; Andersen et al ., 2015 ), niche development (Morton et al ., 2017 ), and community structure, function, and stability (Boetius et al ., 2000 ; Wright et al ., 2012 ; Coyte et al ., 2015 ). Furthermore, inter‐organism arrangements govern chemical communication (West et al ., 2007 ), metabolite exchange (Romine et al ., 2017 ) and competition for resources (Mitri et al ., 2016 ). Nonetheless, these critical spatial relationships are neglected by the most commonly used methods in microbial ecology such as bulk meta‐omics and geochemical approaches. As a result, important metabolic activities may be obscured, including inter‐species nutrient cycling (Wilbanks et al ., 2014 ; Cordero and Datta, 2016 ) and electron transfer to (Lovley and Phillips, 1988 ; Myers and Nealson, 1988 ) or from (Shelobolina et al ., 2012 ) specific minerals. Recent efforts have made progress in analysing microbial communities at the microscale. Nanoscale secondary ion mass spectrometry (nanoSIMS) coupled with stable isotope probing (SIP) and fluorescence in situ hybridization (FISH) can resolve anabolic patterns and taxonomically identify individual cells. However, this method typically separates microbial assemblages from their broader environmental context (McGlynn et al ., 2015 ; Musat et al ., 2016 ; Gyngard and Steinhauser, 2019 ). By combining energy‐dispersive X‐ray spectroscopy (EDS) with X‐ray computed tomography images, Hapca et al . extended chemical analyses into a third dimension with resin‐embedded soil, but no cellular information was attained (Hapca et al ., 2015 ). Correlative imaging with nanoSIMS and electron and fluorescence microscopy enabled Schlüter et al . to pinpoint the position of a subset of the microbial community in relation to leaf fragments, but metabolic activity and microbial identities were not considered (Schlüter et al ., 2018 ). A promising addition to this emerging field is SIP combined with non‐destructive Confocal Raman microspectroscopy, which was recently used to measure the in situ activity and substrate uptake of microbes in transparent soil microcosms (Sharma et al ., 2020 ). The work presented here advances this line of microbial ecology research. The methods herein not only preserve spatial arrangements and link cell positions to mineralogy through correlative microscopy, but also establish the presence, location and mineralogical associations of anabolically active cells. Anabolic activity was assessed with bioorthogonal non‐canonical amino acid tagging (BONCAT), a next‐generation physiology approach (Hatzenpichler et al ., 2020 ) that uses substrate analogue probing to visualize protein synthesis in active cells. A non‐canonical amino acid, such as l ‐homopropargylglycine (HPG) or l ‐azidohomoalanine, is incorporated into growing peptides by native methionyl‐tRNA synthetases. Subsequent azide‐alkyne click chemistry allows fluorescent detection of newly synthesized proteins (Sletten and Bertozzi, 2009 ). BONCAT was initially developed in neuron (Dieterich et al ., 2006 ), eukaryote (Hinz et al ., 2011 ) and cultured bacteria (Hatzenpichler et al ., 2014 ) systems; more recently, it was optimized for environmental microbial communities and shown to have no measurable effect on community composition or metabolic activity (Hatzenpichler et al ., 2014 , 2016 ). The approach has been proven effective in a diverse range of bacterial and archaeal cultures (Hatzenpichler et al ., 2014 ; Hatzenpichler and Orphan, 2015 ); ocean water (Samo et al ., 2014 ; Leizeaga et al ., 2017 ; Sebastián et al ., 2019 ), marine sediment (Hatzenpichler et al ., 2016 ), hot spring (Reichart et al ., 2020 ), and soil microbiomes (Couradeau et al ., 2019 ); as well as marine viruses and bacteriophages (Pasulka et al ., 2018 ). BONCAT appears to be a taxonomically agnostic measure of anabolic activity that correlates well with other metrics of activity (Bagert et al ., 2014 ; Hatzenpichler et al ., 2014 , 2020 ; Samo et al ., 2014 ) with only small effects on metabolism (Steward et al ., 2020 ) and protein chemistry (Bagert et al ., 2014 ; Lehner et al ., 2017 ). In this study, we mapped the anabolic activity of microorganisms in sediments from Little Sippewissett salt marsh in Falmouth, MA, where terrestrial freshwater runoff, seawater, high organic input, and abundant light and chemical energy lead to dramatic redox stratifications within the top few centimetres of sediment and a wide range of metabolic niches (Wilbanks et al ., 2014 , 2017 ; Larsen et al ., 2015 ). Using purpose‐built equipment (Fig. 1 ), we incubated a series of sediment cores with HPG in situ for 3.7 days. One core was processed for horizon‐specific fluorescence‐activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. A parallel core was used for correlative microscopy; samples were embedded in resin to maintain precise spatial arrangements, sectioned, stained, and analysed using fluorescence and electron microscopy to map active and inactive biomass as well as identifiable mineral grains. Fig. 1 An overview of the experimental and sample processing approach deployed in this study (components are not to scale; please refer to the text for the correct dimensions). The PETG tube is cut to the appropriate dimensions and the lower edge is bevelled (1). Cut‐off 50 ml Falcon tube tops are secured to the PETG tube with epoxy (2), and sediment is collected from the marsh by pressing the tube downward into the sediment (3). A sterile plug of glass wool is added to the bottom to keep the material in place, and the full tube is pulled back out of the sediment. Tube lids are secured; the top lid has a perforated top to allow contact with an oxic atmosphere (4). In an anoxic chamber, lids are removed and fluid is replaced drop‐wise by pipette with 50 μM HPG in 0.22 μm‐filtered Berry Pool water (5). (Not all cycles of fluid replacement are shown; see text for full protocol.) PDMS membranes are secured to top and bottom of tube with twist‐on lids (6). Sample tubes are returned to the marsh; immediately prior to emplacement in the Berry Pool sediment, the bottom lid is perforated to allow gaseous continuity with the environment (7). The sample is placed back in the sediment at the initial collection location for the duration of the incubation period (8); upon recovery, lid perforations are immediately covered with electrical tape to minimize gas exchange during transport back to the lab (9). In the anoxic chamber, incubation fluid is replaced with fixative and incubated for 4 h at room temperature (10). Correlative microscopy cores are processed according to steps 11a‐15a. The fixed core is removed from the anoxic chamber and infiltrated with an ethanol dehydration series (11a) followed by LR White resin (12a), which is allowed to cure during a 36 h incubation at 60 °C. The embedded core is then sectioned by a sterile water‐cooled diamond saw (13a), and sectioned surfaces are incubated in the click solution for 60 min in the dark in an anoxic chamber (14a). Sample sections are now ready for SYBR green counterstaining and fluorescence and electron microscopy (15a). Cores for cell sorting and sequencing are processed according to steps 11b‐16b. Following fixation, the core is rinsed with 0.22 μm‐filtered Berry Pool water (11b). The overlying liquid and top 1.0 cm of sediment are removed and replaced by a plug of sterile glass wool for transport (12b). A sterile plunger was used to extrude the core in 1 cm increments (13b). Cells were extracted from these subsamples and then incubated in the click solution for 30 min in the dark (14b). Cells were then washed (15b) and introduced to the cell sorter, which separated BONCAT positive and BONCAT negative cells (16b) for downstream sequencing. [Color figure can be viewed at wileyonlinelibrary.com ] Mineralogical identities of individual grains were assessed in order to determine whether different mineral types corresponded with notable differences in organism abundance, configuration, or anabolic activity. Like the vocabulary used to describe microbe–microbe interactions, microbe–mineral interactions can be harmful, neutral, or beneficial for the organism. Microbial sorption to quartz grains – which are dominant in coastal settings – has been demonstrated, but repellent electrical charges make the interaction less favourable than those with other mineral types (Mills et al ., 1994 ; Gong et al ., 2018 ). The best‐studied beneficial interactions are the microbial reduction of iron or manganese oxides (Thamdrup, 2000 ), which enable bacteria to off‐load reducing power, altering mineral structure and chemistry in the process (Kawano and Tomita, 2002 ; Welch and Banfield, 2002 ). A number of factors influence the nature of these interactions, including accessible surface area, mineral lattice structure, co‐occurrence of organic matter, and other environmental conditions such as temperature and pH (Dong et al ., 2009 ). Beyond iron and manganese, microbes have been shown to associate with other cations, acquiring potassium from silicates (Valsami‐Jones et al ., 1998 ), releasing organic ligands that adhere to aluminium (Rogers and Bennett, 2004 ), and using reducing power from photo‐catalytically activated titanium oxide (Lu et al ., 2012 ). By identifying the mineral grains with which the native microbial community associates, the nature of previously undetected microbe–mineral relationships can be examined in further detail. The novel approach presented here allowed us to map active and inactive organisms in their native microscale configuration and identify the active and inactive microbial communities in adjacent sediment horizons. Our results indicate that the proportion of anabolically active organisms decreased dramatically below the photic zone and that mineralogy likely has an impact on the relative abundance and anabolic activity of mineral grain‐associated organisms. High‐throughput 16S rRNA gene sequencing of active and inactive microbial communities in adjacent sediment cores revealed a continuous progression of community structure with depth, oriented around shifting metabolisms of photosynthesis, sulfur cycling and fermentation. Notably, with correlative fluorescence and electron microscopy, we observed differential cell association with distinct mineral types and a greater proportion of organisms inside mineral grains in lower (6–7 cm) sediment horizons compared with shallower zones. While the full potential of microbiome mapping remains to be realized, this benchmarking study unveils a new experimental approach to (i) evaluate how metabolic activity relates to microscale environmental factors, and (ii) develop testable hypotheses regarding metabolic interactions among members of complex microbial communities.",
"discussion": "Results and discussion This study reveals how microbial presence and anabolic activity in salt marsh sediment relate to sediment depth and mineralogical distributions at the microscale with a new level of realism. In this section, we first present results of quality assurance tests to demonstrate the reliability of our newly developed methods before sharing insights on community structure derived from BONCAT‐FACS sequencing and findings of spatial arrangements and putative microbe–mineral interactions via correlative microscopy. We close with key insights made possible through the integrated combination of the two newly developed techniques. Experimental treatments and depth‐based sampling details for the samples discussed below are provided in Table S1 and Fig. S1 respectively. Control experiments constrain false positives and cell entrainment To validate our protocols, control samples were analysed and targeted procedural experiments were performed. The contribution of false‐positive ‘organism’ designations via fluorescence microscopy was quantified by comparing samples BM and AM; both sediment cores were incubated in the presence of 50 μM HPG, but the latter had been autoclave‐sterilized, eliminating anabolic activity and the incorporation of HPG into new biomass (Table S1 ). Across five fields of view from sample BM's top horizon (7.6 mm depth) and an arbitrary horizon in the homogenized AM core, sample AM exhibited 3.5% as many SYBR‐active objects and 3% as many Cy3‐active objects as sample BM (Fig. S2 ). When corrected for the pre‐sterilization biomass abundance in sample AM (9.81 × 10 8 cells/cm 3 ), these apparent false‐positive rates increased to 6.9% for SYBR signal and 5.9% for BONCAT signal. Analysis areas contained roughly equivalent proportions of mineral surface area and pore space. Although the comparison between the two samples was not exact – autoclaving could have both denatured photosynthetic pigments and incompletely degraded cells – this analysis suggests that our efforts to constrain background fluorescence or non‐specific binding of HPG or the dyes could benefit from future optimization, but false‐positive signal did not dramatically influence our results. When comparing the top horizons of samples BM and CM (a live environmental incubation that did not receive HPG), we found that CM had 23% more SYBR‐active objects but just 2.9% as many Cy3‐active objects as sample BM (or 2.3% as many when normalized by SYBR‐active biomass, Table 1 ; Fig. S2 ). These data strongly suggest that HPG did not affect SYBR signal but was required for BONCAT signal and that Cy3‐active features were not attributable to cellular autofluorescence. Table 1 Cell abundance and percentage of anabolically active cells as determined through fluorescence microscopy and BONCAT‐FACS analyses. Fluorescence microscopy analysis FACS analysis Sediment depth (mm) Cell abundance (cells/cm 3 ) % Active % Active Sediment depth (mm) Sample CM Sample BM Sample BS 7.6 2.4 × 10 9 \n 1.95 × 10 9 \n 51.3 70.0 0–10 12 2.46 × 10 9 \n 2.86 × 10 9 \n 22.3 22.4 10–20 21.8 20–30 35.5 3.66 × 10 8 \n 10.5 30–40 17.4 40–50 13.0 50–60 60.7 5.8 × 10 8 \n 6.85 × 10 8 \n 12.1 14.5 60–70 11.5 70–80 12.8 80–90 9.4 90–100 Where % active values are available for both fluorescence microscopy and FACS datasets, the linear correlation coefficient was 0.99. Cells with gray shading indicate the absence of data. Clarifying the role that our experimental treatment had on the microbial community and the empirical biases that may result was a key priority. Daily fluctuations of the Berry Pool water level, which ranges from ~5 to 30 cm water depth over the course of a tidal cycle, consistently introduce and remove transient organisms that may not be physically associated with sediment particles. Nonetheless, it is possible that the percolation of fluids through the incubation chambers associated with our protocol might transport microbial cells outside of their naturally occurring habitats. To test this possibility, we introduced 1 ml of 1 × 10 9 ml −1 1 μm diameter YG carboxylate fluorescent microspheres (Polysciences, Warrington, PA) to the overlying water of a salt marsh sediment core (Sample MM, Table S1 ). These microspheres are commonly used to simulate microorganism transport and constrain contamination in sediments, soils and subsurface environments (Smith et al ., 2000 ; House et al ., 2003 ; Goeppert and Goldscheider, 2011 ; Bang‐Andreasen et al ., 2017 ; Labonté et al ., 2017 ; Daly et al ., 2018 ). Following microsphere addition, the core was treated identically to the BM sample through the embedding step (see Experimental procedures section). Throughout the process, flow‐through liquid fractions were collected and concentrated on 0.22 μm polycarbonate filters. After embedding, multiple horizons were sectioned and examined with fluorescence microscopy. Bead counts over five representative fields of view at 10.7 mm above the sediment–water interface, 2.0 mm depth, 5.3 mm depth, 9.8 mm depth, and 23.3 mm depth (Fig. S3 ), as well as 16 liquid fractions, were averaged and scaled by the overall cross‐sectional area of the core. Z ‐axis transmission of bead fluorescence under confocal microscopy examination was 8.75 μm. Linear interpolation of data points indicated that 99.3% of beads remained above the 7.6 mm horizon, which was the shallowest horizon used for microscopy analysis. Assuming a cell density of 10 6 ml −1 in the overlying water and 30 ml of overlying water in the initial core sample, we calculate that 6 × 10 −3 % and 8 × 10 −4 % of cells detected in the 7.6 and 12 mm horizons respectively, are attributable to entrained surface water cells. Because a horizon lower than 60.7 mm was not examined with the bead test, an analogous figure is not attainable for the 60.7 mm horizon. However, given the trends observed here, we believe the contribution from surface‐entrained organisms is negligible. This analysis gave us confidence in interpreting mineral‐associated organisms as native to the observed sediment horizons. BONCAT‐FACS illuminates community structure and prominent metabolic functions Several previous studies have elucidated key aspects of the Little Sippewissett salt marsh microbiological system and its role in biogeochemical cycling (Shapiro et al ., 2011 ; Wilbanks et al ., 2014 , 2017 ; Larsen et al ., 2015 ; Salman et al ., 2015 ; Mackey et al ., 2017 ). Because geochemical measurements were not conducted as a part of this study, we leverage this heritage to convey prevailing conditions within different sediment horizons and infer physiological traits based on the 16S rRNA gene data we collected. By incorporating BONCAT‐FACS into community analyses of centimetre‐thick sediment horizons, we can focus specifically on the anabolically active subset of the community and detect shifting metabolic priorities. Sequence data and relative abundances of assigned lineages for bulk, active and inactive microbial communities across all horizons are reported in Supporting Information Dataset 1 . The top horizon (0–10 mm) of Little Sippewissett salt marsh sediment exhibits dramatic redox gradients as oxygen concentrations fall below detection by 5 mm, sulfide rises from 0 to between 0.5–1.5 mM, and pH drops 1–2 units immediately below the surface before stabilizing at ~7.0–7.3 at night and ~6.0 during the day below 4 mm (Salman et al ., 2015 ). Our results showed that the majority of microbes recovered from this horizon via FACS were anabolically active during the incubation (70%, Table 1 ) and that the community was dominated by the phyla Proteobacteria (48% relative abundance) and Bacteroidetes (30%), whose metabolically diverse members are suggestive of a range of redox conditions and substantial heterotrophic cycling in the upper sediment layer (Spain et al ., 2009 ; Gómez‐Pereira et al ., 2012 ). The prevalence of the purple sulfur bacteria Thiohalocapsa (14.4%) and putative sulfate‐reducing bacteria Desulfobulbaceae (6.5%) in the bulk community were reflective of the abundant ‘pink berries’ found at the sediment surface (Fig. S4 ) (Seitz et al ., 1993 ; Wilbanks et al ., 2014 ). Thiohalocapsa were substantially less abundant in both the active (3.8%) and inactive sorted cells (2.7%), likely due to incomplete disaggregation during cell extraction (see Experimental procedures section below). Desulfobulbaceae were more prevalent in the active community (14.0%) than in the bulk cells (6.5%), suggesting that not all of these sulfate‐reducing organisms were syntrophically associated with purple sulfur bacteria ( Dataset 1 ). Among organisms potentially involved with sulfur‐cycling consortia, we observed a more diverse distribution of putative sulfate‐reducing bacteria lineages (65 genus‐level Desulfobacterales amplicon sequence variants, or ASVs) compared with a more streamlined set of purple sulfur bacteria with a single dominant representative (19 genus‐level Chromatiales ASVs, with Thiohalocapsa accounting for 83% of the recovered sequences). Sequencing of active and inactive communities in the 0–10 mm range revealed eight lineages that each represented >1% of the overall relative abundance that was significantly more abundant in the anabolically active subset ( Dataset 1 ). Of these, six were putative members of the pink berry consortia ( Chromatiales or Desulfobacterales orders), one was a photoheterotroph ( Halieaceae ) that may encode multiple light‐harvesting complexes (Spring et al ., 2015 ), and one was a member of the metabolically diverse Rhodobacteraceae family (Pujalte et al ., 2014 ; Pohlner et al ., 2019 ). Many of the other abundant inactive lineages – including three putative sulfate reducers and three putative purple sulfur bacteria – were among the most abundant ASVs in both the active and inactive fractions ( Dataset 1 ). This overlap may indicate stochastic activity of particular consortia or a metabolic dependence upon physicochemical traits on a sub‐cm scale, such as pore connectivity or identity of neighbouring organisms. Alternatively, our conservative gating approach may have captured some active cells with low fluorescence in the inactive gate (Fig. S5 ). In the sediment horizons below 10 mm, oxygen is absent and sulfide concentrations range from ~0.5 to 2.5 mM. Day or night pH values remain largely consistent with depth to 30 mm, producing values of ~6.9–7.2 at night, and ~6.0–6.3 during the day (Salman et al ., 2015 ), typical of carbonate‐buffered anoxic sediments (Soetaert et al ., 2007 ). Under these more energetically constrained conditions, the proportion of active organisms detected by FACS decreased substantially from 70% in the top layer to values between 9.4% and 22.4% in the subsurface horizons (Table 1 ), signifying a shift from primary production to burial and degradation. The most prominent sulfur‐metabolizing orders, Desulfobacterales and Desulfarculales , accounted for a higher proportion of the anabolically active community – 34.6% ± 3.3% SD and 5.7% ± 1.6% SD respectively, across the nine horizons between 10 and 100 mm – than in the top sediment horizon (25% and 3.6% respectively; Fig. 2 ). The prominence of these lineages is consistent with previous observations that sulfate reduction is the main metabolism responsible for remineralization of organic matter in salt marsh sediments, accounting for 67%–80% of total respiration in various salt marshes (Howarth and Teal, 1979 ; Howarth and Giblin, 1983 ; Howarth and Merkel, 1984 ) and more than 90% of primary production degradation at neighbouring Great Sippewissett Marsh (Howarth and Teal, 1979 ). Members of the most abundant order across all subsurface horizons, Desulfobacterales , were more prevalent among anabolically active than inactive organisms, while the Desulfarculales frequently exhibited the opposite relationship (Fig. 2 ). The latter order consisted of the Desulfatiglans genus, whose abundance in subseafloor environments has been attributed to its metabolic versatility in the degradation of aromatics (Jochum et al ., 2018 ). In the context of the salt marsh, this versatility has seemingly enabled the genus to persist throughout the core, but the cost of a diverse metabolic portfolio could be substantial lag times in metabolic re‐routing or extended periods of quiescence for organisms whose metabolic substrate is not present at a given time. Fig. 2 Trends of the relative abundances of active and inactive subsets of the eight most prevalent orders with sediment depth, as detected by BONCAT‐FACS combined with 16S rRNA gene sequencing for sample BS. At each horizon, the relative abundance contribution for each order was determined in both the anabolically active sorted cells and the inactive sorted cells. Values to the right of the axis indicate the relative abundance of that order in the active fraction; values to the left indicate the relative abundance in the inactive fraction. The coloured bars reveal if the order was enriched in the active fraction (yellow bars) or the inactive fraction (blue bars) in a given horizon. The length of bars shows fold‐enrichment, as indicated by the x ‐axis, calculated by dividing the larger relative abundance value by the smaller relative abundance value for each order in each sediment horizon. [Color figure can be viewed at wileyonlinelibrary.com ] Purple sulfur bacteria of the order Chromatiales , as anticipated, comprised a decreasing proportion of active cells down‐core in the absence of light. Nonetheless, one Chromatiaceae ASV (of the Halochromatium genus) was the second most abundant lineage among active organisms between 10 and 20 mm, suggesting that bioturbation late in the incubation contributed to in‐mixing from more photosynthetically active surface layers, or that chemotrophic growth occurred in situ in microoxic microenvironments (Hell et al ., 2008 ; Hunter et al ., 2008 ). More broadly, Chromatiales was the most abundant order in several inactive fractions, suggesting that purple sulfur bacteria may be among the larger microbial contributors of organic matter to deeper sediments. Cellvibrionales and Rhodobacterales were found at higher relative abundance in active than inactive fractions at the top of the core, but the opposite was true below 20 mm depth. Cellvibrionales have traditionally been considered oligotrophs, but some members of the order contain sulfur oxidation pathways and others can grow photoheterotrophically (Spring et al ., 2014 , 2015 ); this diversity of environments may explain their relatively consistent presence among both active and inactive sequences throughout the core. Rhodobacterales are noted early colonizers of particles (Dang et al ., 2008 ); one of the most prominent genera detected throughout the core was Rubribacterium , a non‐sulfur purple bacterium that is a facultative aerobe (Boldareva et al ., 2009 ). These traits help explain the order's presence at all horizons and its decrease in the active fraction with depth. The observed vertical profile of Pirellulales sequences is consistent with aerobic chemoorganotrophs (Schlesner et al ., 2004 ) which may have been deposited onto the sediment surface, metabolically inactivated quickly upon burial and the onset of anoxic conditions, and potentially scavenged by the anoxic heterotrophs. Fermentation may be a prominent metabolism in the anoxic horizons we sampled. Sphingobacteriales are typically associated with carbon remineralization in oxic soils (Fierer et al ., 2007 ), but they do retain fermentation‐associated genes (Hester et al ., 2018 ) that may explain their presence among the active cell fraction we recovered from below 10 mm depth (Fig. 2 ). Members of the fermentative Clostridia class (Mead, 1971 ; O'Brien and Ljungdahl, 1972 ; Winter et al ., 1987 ) increased in relative abundance downcore (Pearson's r = 0.76, p < 0.05), as did their enrichment in the active fraction ( r = 0.79, p < 0.01). In salt marshes, fermentation produces organic acids such as acetate (Gandy and Yoch, 1988 ; Kostka et al ., 2002 ) that may promote syntrophic relationships with the abundant sulfate‐reducing bacteria we observe (Ford, 1993 ; Bahr et al ., 2005 ). Few sequences from putative methanogens were observed, potentially due to primer bias (Bahram et al ., 2019 ), seasonality (Buckley et al ., 2008 ), and the presence of abundant sulfate‐reducing bacteria and a range of homoacetogens that may be more successful at attaining hydrogen (Oremland and Polcin, 1982 ; Ye et al ., 2014 ). Correlative microscopy reveals precise spatial relationships between microbes and minerals Correlative microscopy analyses at three distinct horizons revealed changes in organism abundance from 1.95 × 10 9 cm −3 at 7.6 mm depth to 2.86 × 10 9 cm −3 at 12 mm depth and 6.85 × 10 8 cm −3 at 60.7 mm depth. Moving downward along these three horizons, the proportion of anabolically active organisms decreased from 51.3% (7.6 mm) to 22.3% (12 mm) to 12.1% (60.7 mm), a trend that correlated well with BONCAT‐FACS data ( R \n 2 = 0.99; Table 1 ). The uppermost section examined by correlative microscopy was located within the top sequenced horizon, at a depth of 7.6 mm (Fig. 3 ). In the analysed area, 15 of the 20 mineral grains were quartz (SiO 2 ), while albite (NaAlSi 3 O 8 ), orthoclase (KAlSi 3 O 8 ), rutile (TiO 2 ), plagioclase (a solid solution range from NaAlSi 3 O 8 to CaAl 2 Si 2 O 8 ) and Ca/K/Mg/Fe silicate grains of indeterminant mineralogy were also observed. 73.4% of cells were associated with (located inside or around) quartz grains (Table 2 ). However, when cell biomass abundances were normalized by proxies for mineral surface area and volume, non‐quartz grains exhibited 33% greater biomass per unit surface area and 43% greater biomass density per unit volume. Fig. 3 Correlative fluorescence and electron microscopy from the uppermost section (7.6 mm sediment depth). A. Overlain on the base SEM image are two fluorescence channels showing SYBR‐active features in blue, and BONCAT‐active features in yellow. The dark zonation indicates the fluorescence microscopy footprint. B. Composite elemental maps derived from EDS analysis show the mineral grains that were analysed, labelled by mineral type. 1 = Quartz; 2 = Plagioclase; 3 = Orthoclase; 4 = Rutile; 5 = Albite. Yellow arrows indicate particularly large mineral grain pore spaces for illustrative purposes. C–E show three mineralogically distinct sites in detail. (i) SYBR green, (ii) BONCAT and (iii) merged channels, as well as (iv) EDS elemental abundance maps (in which dark blue background represents the resin). Brightness and contrast of all fluorescence microscopy views have been increased by 40% to enhance signal visibility; original images as presented in the methods can be found at the following Dropbox link: tinyurl.com/4mcw39xu [Color figure can be viewed at wileyonlinelibrary.com ] Table 2 Proportions of cells, and the anabolically active subsets, associated with mineral exteriors and interiors at the three horizons examined by correlative microscopy. % of associated biomass % active Associated biomass per unit surface area Associated biomass per unit volume % Outside % of Outside biomass that was active % Inside % of Inside biomass that was active 7.6 mm horizon All minerals 100 51.3 77.5 51.7 22.5 52.8 Quartz 73.4 49.7 0.97 0.93 80.6 48.9 19.4 50.1 Plagioclase 10 62.6 1.3 1.44 75.9 63.6 24.1 59.5 Orthoclase 6.7 61.2 1.31 1.11 90 60.2 10 64.9 Rutile 5.3 66 1.26 1.5 42 78 58 57.3 12 mm horizon All minerals 100 22.3 80.2 20 19.8 24.8 Quartz 85.6 21.7 0.99 0.94 79.1 19.1 20.9 24.3 Orthoclase 9.5 29.8 1.24 1.68 89.6 30 10.4 29.8 60.7 mm horizon All minerals 100 12.1 62.2 10.3 37.8 14.9 Quartz 62.1 10.3 0.86 0.82 69.5 9.3 30.5 12.5 Plagioclase 21 12 1.52 1.6 45.6 7.6 54.4 15.6 Orthoclase 13.5 15 1.39 1.6 55.7 12.9 44.3 17.6 For the biomass per surface area and volume, the relative proportion of biomass associated with a given mineral type was divided by the relative proportion of surface area or volume accounted for by that mineral type. Values less than 1 indicate fewer associated cells than would be expected given an even distribution of biomass across mineral perimeters or surfaces. Only mineral types that accounted for at least 5% of the observed biomass in a given horizon are included in this analysis. Color shadings are intended to clarify different data categories. Orange cells refer to all observed biomass, blue cells refer to all biomass located outside mineral grains, and green cells refer to all biomass located inside mineral grains. Overall, 77.5% of observed cells were outside their associated mineral grains while 22.5% were found inside, frequently along fractures or pores up to several hundred μm 2 in cross‐sectional area visible by SEM (see e.g. Fig. 3B ). We found that the degree of anabolic activity was higher around non‐quartz minerals when compared with quartz‐associated cells (Table 2 ). Although low abundances of these mineral types make generalizations difficult, it is possible that metal cations in the mineral structures facilitate a wider range of metabolic reactions than the more chemically inert quartz (Shi et al ., 2016 ). The electrical semi‐conductivity of titanium oxide can promote extracellular electron transfer (Zhou et al ., 2018 ) and, via photo‐catalysis, stimulate the growth of non‐phototrophic microbes (Lu et al ., 2012 ); these mechanisms may account for the elevated proportion (78%, compared with a mean of 51.7% for this horizon) of active cells associated with the exterior of the titanium oxide rutile grain. Within the 10–20 mm depth zone, a post‐BONCAT embedded section from a depth of 12 mm was examined by correlative microscopy (Fig. 4 ). Twenty‐two mineral grains were analysed; as above, the vast majority of grains were quartz, and the microbes associated with non‐quartz grains (in particular, orthoclase) had a higher proportion of anabolically active constituents (Table 2 ). Some of the highest concentrations of active cells were associated not with well‐defined minerals, but rather with heterogeneous patches that include small particles of quartz, sodium and iron (Fig. 4C ). In comparison with larger mineral grain interfaces, these particle assemblages offer greater chemical diversity and more potentially reactive surface area, factors that may facilitate interactions among microbes. The higher surface roughness associated with particle assemblages may also influence fluid flow through the column, reducing drag and providing more stable microenvironments (Taylor et al ., 2006 ). Fig. 4 Correlative fluorescence and electron microscopy from the sediment section at 12 mm sediment depth). A. Overlain on the base SEM image are two fluorescence channels showing SYBR‐active features in blue, and BONCAT‐active features in yellow. The dark zonation indicates the fluorescence microscopy footprint. B. Composite elemental maps derived from EDS analysis show the mineral grains that were analysed, labelled by mineral type. 1 = Quartz; 2 = Plagioclase; 3 = Orthoclase. Yellow arrows indicate particularly large mineral grain pore spaces for illustrative purposes. C–E show three mineralogically distinct sites in detail. (i) SYBR green, (ii) BONCAT and (iii) merged channels, as well as (iv) EDS elemental abundance maps (in which dark blue background represents the resin). In (D), zones containing BONCAT‐positive biomass in (i), (ii) and (iii) are marked with white arrows. Brightness and contrast of all fluorescence microscopy views have been increased by 40% to enhance signal visibility; original images as presented in the methods can be found at the following Dropbox link: tinyurl.com/4mcw39xu [Color figure can be viewed at wileyonlinelibrary.com ] The deepest section used for correlative microscopy analysis was at a depth of 60.7 mm. Forty‐three mineral grains were observed in the fluorescence microscopy field of view, which also contained the highest abundance of small mineral particles and heterogenous patches of the three sections (Fig. 5 ), potentially due to the degradation processes that accompany burial and longer residence times within the sediment column (Curtis, 1987 ). Despite the high abundance of associated mineral interfaces across a range of spatial scales, this horizon exhibited the lowest microbial abundance and the lowest proportion of anabolically active organisms. This observation is consistent with commonly observed trends in sediments, where electron acceptor depletion and the progressive loss of labile carbon with depth can lead to energetically constrained conditions (Blume et al ., 2002 ; Jörgensen et al ., 2002 ; Stone et al ., 2014 ). It is also possible that longer incubation times would have resulted in a higher proportion of anabolically active organisms, as our incubation duration was determined by lab‐based experiments using the more active upper sediment horizons (0–5 cm; see Experimental procedures ). Fig. 5 Correlative fluorescence and electron microscopy from the sediment section at 60.7 mm sediment depth. A. Overlain on the base SEM image are two fluorescence channels showing SYBR‐active features in blue, and BONCAT‐active features in yellow. The dark zonation indicates the fluorescence microscopy footprint. B. Composite elemental maps derived from EDS analysis show the mineral grains that were analysed, labelled by mineral type. 1 = Quartz; 2 = Plagioclase; 3 = Orthoclase; 4 = Rutile; 5 = Albite; 6 = Ca, K, Mg, Fe silicate; 7 = Hornblende. Yellow arrows indicate particularly large mineral grain pore spaces for illustrative purposes. C–E show three mineralogically distinct sites in additional detail in (i) SYBR green, (ii) BONCAT and (iii) merged channels, as well as (iv) EDS elemental abundance maps (in which dark blue background represents the resin). In (C) and (D), zones containing BONCAT‐positive biomass in (i), (ii) and (iii) are marked with white arrows. No such zones were detected in E. Brightness and contrast of all fluorescence microscopy views have been increased by 40% to enhance signal visibility; original images as presented in the methods can be found at the following Dropbox link: tinyurl.com/4mcw39xu [Color figure can be viewed at wileyonlinelibrary.com ] In the 60.7 mm horizon, quartz grains had the lowest cell abundances per unit surface area and volume of the three examined sections, while orthoclase and plagioclase had higher‐than‐average biomass densities (Table 2 ). The proportion of anabolically active organisms, however, was not substantially different among distinct mineral types, suggesting that cells adhere more strongly to plagioclase and orthoclase grains, and/or that quartz is more readily degraded during weathering and burial, disrupting surficial microbial association. This horizon also exhibited the highest proportion of cells located inside mineral grains (37.8%), an observation that could reflect the extensive remineralization of external biomass with burial (Mackin and Swider, 1989 ). Compiling findings across horizons When integrating sequencing and microscopy data across all horizons, intriguing trends of anabolic activity, diversity and spatial arrangement emerged. With increasing depth into the sediment, where geochemical and thermal conditions were more stable, alpha diversity metrics of bulk pre‐extraction communities revealed a decrease in richness but increase in evenness (Fig. S6 ). Among the anabolically active and inactive communities, no substantial change in the number of distinct ASVs with depth was observed, but the evenness of their distribution increased down‐core for the active constituents. This pattern may reflect a wider range of available niches with fewer dominant lineages below the photic zone, as organic matter is remineralized through a range of metabolic routes, making these deeper communities' emergent effects more resistant to environmental changes (Wittebolle et al ., 2009 ). Beta diversity analysis revealed a clear separation between both bulk and extracted communities as well as between active and inactive communities (Fig. S7 ). The distinction between the bulk, sediment‐associated community and the extractable microbes (Fig. S7 A) reveals an opportunity for improved cell extraction procedures, and may be attributable to differences in DNA collection protocols, strongly adherent non‐extractable cells and relic DNA not contained in cells (Carini et al ., 2016 ). The community composition differences between active and inactive communities (Fig. S7 B) confirms that organisms respond to environmental cues in a taxonomically differentiated manner and that anabolic activity is not a random process. Furthermore, for both active and inactive communities, the closer two sediment horizons were in depth, the more similar their community compositions. This trend likely reflects depth‐based gradients that form the energetic basis for metabolic activity, as well as the burial process in which a given horizon's community represents the confluence of local selective pressures operating on an assemblage of organisms ‘imported’ from above or from below due to tidal pumping. At each of the three horizons examined through correlative microscopy, cells appeared to be distributed not as evenly spaced individual cells, but rather as aggregations suggestive of inter‐organism interactions consistent with a ‘sphere of influence’ on the order of a few microns (Dal Co et al ., 2020 ; Steinberg et al ., 2021 ) (Figs 3 , 4 , 5 ). Throughout the sediment column, quartz was the dominant mineral type, yet microbial communities associated with quartz grains had the lowest proportion of anabolically active members. Other mineral types – such as orthoclase, plagioclase and rutile – had a broader set of cations (Al, Ti, K) that may have offered additional electron transfer or nutrient acquisition opportunities for active cells. With increasing sediment depth, organisms were more likely to be located inside mineral grains, and these ‘internal’ cells were increasingly likely to be anabolically active compared with their ‘external’ counterparts (Table 2 ): at 7.6, 12 and 60.7 mm depth, internal organisms were 2.1%, 24% and 45% more likely to be active than those outside minerals, respectively. These observations are consistent with a more stable intra‐mineral environment that may be less susceptible to predation, particularly in the more energetically constrained anoxic sediment horizons."
} | 11,818 |
38716125 | PMC10989591 | pmc | 5,791 | {
"abstract": "Abstract Optimization of dye decolourization for wastewater and power production are explored in dual‐chamber microbial fuel cells (MFCs) with TiO 2 /CdS photocathodes. The rapid reduction of azo dye methylene blue (MB) and power production were enhanced with TiO 2 /CdS photocathode under illumination. The analysis of electrochemical impedance spectra indicated that the photocatalysis of TiO 2 /CdS accelerated the electron transfer process of photoelectrode reduction. Moreover, the UV‐visible light spectrophotometer showed that the maximum degradation of the MFCs was 98.25%, which illustrated that MB may be cleaved by photoelectrons generated by light irradiation on the illuminated TiO 2 /CdS photocathode. Finally, the power production of MFCs in this work promoted reductive decolourization of the dye MB solution.",
"conclusion": "4 CONCLUSION In this article, a photocathode doped with CdS was prepared, and the performance of the electrode in generating electricity and degrading MB in MFCs was studied. First, the surface morphology of the synthesized photocathode was characterized by SEM and EDS, and the results showed that CdS was uniformly distributed on the electrode surface. Second, CV showed that the CdS electrode has high electrochemical activity, which further improved the oxidation‐reduction performance of the reactor. Electrical impedance spectroscopy showed that the CdS electrode has low ohmic impedance and high conductivity, which could increase the electron transfer rate. The LSV curve showed that the CdS electrode has high ORR activity. The diffuse reflectance spectrum showed that the CdS photocathode has extended the spectral response range and has a clear light response under visible light. The degradation experiment of MB solution showed that the best degradation condition is that the closed‐circuit MFC is under light conditions. The reason for the high degradation rate of the composite material was that CdS broadens the electrode spectral response range, and the photogenerated electrons and the electrons transferred from the anode participate in the organic reduction. During the process, the bond breakage and decolourization of MB were realized. In short, photoelectric MFCs provide new ideas for the degradation of dye wastewater.",
"introduction": "1 INTRODUCTION In recent years, the production and use of printing and dyeing have been rapidly developed, resulting in a large amount of dye wastewater containing organic dyes. \n 1 \n , \n 2 \n , \n 3 \n It is well known that dye wastewater can seriously affect the local ecological environment, especially the healthy growth of aquatic organisms due to its wide variety, high concentration, complex organic components, poor bioavailability, strong toxicity and emissions. \n 4 \n Therefore, developing a low cost, high efficiency and reliable dye wastewater treatment technology has been particularly important for industrial dye wastewater degradation. \n 5 \n , \n 6 \n , \n 7 \n \n Recently, the photoelectric coordinated system for wastewater treatment has attracted more and more attention. \n 8 \n , \n 9 \n The microbial fuel cell with photoelectrodes, as a new technology with biological power generation and wastewater treatment, provides a promising option for the treatment of dye wastewater. \n 10 \n , \n 11 \n , \n 12 \n In the microbial fuel cells (MFCs) with photoelectrodes, organic substances (such as acetate) are oxidized by the electricity‐generating microorganisms in the anode chamber. The electrons released to the anode are transferred to the cathode by the external circuit and then combine with an electron acceptor to form water. \n 13 \n , \n 14 \n , \n 15 \n , \n 16 \n , \n 17 \n Meanwhile, the process in which photo‐generated holes are left as electron acceptors and combined with electrons transferred from the anode increases the reaction rate of the cathode. \n 18 \n Photoelectrodes have an important impact on the capacity and degradation of MFCs, and therefore many researchers have focused on developing efficient photoelectrodes. \n 19 \n , \n 20 \n Li et al. \n 21 \n prepared photocatalytic electrodes by coating polydopamine and horseradish peroxidase on titanium dioxide (TiO 2 ) nanotube arrays and exhibited good sensitivity and selectivity for hydrogen peroxide biosensing. Chen et al. \n 22 \n designed a new type of microbial fuel cell with a nano‐structured TiO 2 semiconductor photocathode. Its proton exchange membrane can separate the anode and cathode compartments, thereby overcoming the thermodynamic barriers of producing hydrogen from acetic acid without the help of a power source. Lu et al. \n 11 \n introduced rutile coated cathode in MFCs, which can achieve maximum power density and improve conversion efficiency under light irradiation. However, TiO 2 can only perform photocatalysis under UV irradiation, and the wide band gap seriously hinders its wide application. \n 23 \n On the one hand, the photocatalytic reaction of electrodes is considered as a surface reaction, and the specific surface area of nanostructured catalysts affects the catalytic efficiency. On the other hand, the specific surface area of untreated TiO 2 materials is limited, and most of them cannot directly participate in the photocatalysis process. However, the composite of other narrow band gap semiconductor materials and TiO 2 can solve the above problems, and further improve the photocatalytic performance. It has huge application potential in the application of photocatalytic wastewater treatment due to low photogenerated electron‐hole pair separation efficiency, carrier recombination ultrafast and cadmium sulfide (CdS) extremely aggregated nanoparticles. \n 24 \n \n Therefore, this work mainly prepared CdS‐doped photocathodes to study the electrode's performance in generating electricity and degrading methylene blue (MB) in MFCs. In order to explore the performance of photocathode and CdS, several test methods were used. First, the surface morphology of the synthesized photocathode was characterized by a scanning electron microscope (SEM) and energy dispersive spectrometer (EDS). X‐ray photoelectron spectroscopy (XPS) and X‐ray diffraction analysis (XRD) were used for characterization in order to observe the morphology and crystal structure of the electrode. Second, the photoelectrochemical method was used to detect the influence of light on the electrochemical activity of the photocathode MFC in MB. Finally, a UV‐visible light spectrometer (UV‐vis) was used to study the dye degradation ability of MFC. This work further revealed the potential possibility of TiO 2 /CdS with the photocatalytic activity on improving composite for photocatalytic degradation of dye wastewater in MFCs and provided a novel idea for the enhancing utilization of photocatalysts. We hope that this research could contribute to the MFCs in wastewater treatment and their development in the future.",
"discussion": "3 RESULTS AND DISCUSSION 3.1 Characterization of the photocathode The microscopic surface morphology and structure of the electrode were characterized by SEM. \n 4 \n It could be seen that Figure 1A,B show SEM images of TiO 2 /CdS electrodes with high magnification, which mean that after anodization, the smooth surface of the titanium plate becomes a rough surface, and CdS nanometers were electrodeposited in situ on the TiO 2 electrode. After the particles, the surface of the electrode was overlapped and wrapped by a large number of CdS quantum dot nanoparticles, which enhanced the light utilization performance and the electron transfer rate. To this end, an energy spectrum SEM‐EDS was also carried out to verify the distribution of CdS on the electrode surface. As shown in Figure 2B,C , TiO 2 was uniformly distributed on the electrode surface. As shown in Figure 2D,E , the CdS quantum dots were widely distributed on the electrode surface, obviously more than titanium and oxygen. This showed that CdS is successfully synthesized on the surface of the electrode, which could provide more quantum dots. FIGURE 1 Scanning electron microscope (SEM) micrograph of (A) TiO 2 /CdS electrode (10 K). (B) TiO 2 /CdS electrode (40 K) FIGURE 2 (A) Scanning electron microscope (SEM) image of the TiO 2 /CdS electrode. (B–E) Elemental maps of as‐prepared electrode In order to study the chemical state and surface composition of the electrode, we used XPS to study the XPS characteristics of the TiO 2 /CdS electrode. The standard binding energy of C 1S of 284.6 eV was used to correct the binding energy positions of all peaks in Figure 3 . As shown in Figure 3A , in the survey spectrum of the electrode, the characteristic peaks of the three elements were detected, corresponding to the three elements Ti, S and Cd. The peak of C1s came from the XPS test instrument itself. In Figure 3B , the Ti 2p peak is higher in the range of 450–475 eV. The double peaks at 458.3 and 464.5 eV correspond to Ti 4+ 2p 2/3 and Ti 4+ 2p 1/2 , respectively, which were mainly Ti 4+ (TiO 2 ). In Figure 3C,D are the XPS spectra of CdS. The band intensities of Cd 3 d 3 , Cd 3 d 5 and S 2p were 411.89, 405.17, 161.47, and 162.59 eV, respectively, which also correspond to the Cd 2+ and S 2– ions in CdS. Among them, the presence of a weak peak at 168.52 eV indicated that S 2– was slightly oxidized to SO 4 \n 2– . The results further indicated that TiO 2 and CdS were completely present in the electrode. FIGURE 3 (A) X‐ray photoelectron spectroscopy (XPS) survey spectra CdS electrode. (B) High‐resolution Ti 2p XPS spectra of CdS electrode. (C) High‐resolution S 2p XPS spectra of CdS electrode. (D) High‐resolution Cd 3d XPS spectra of CdS electrode In order to study the crystal phase of the prepared electrode, we used XRD to analyze the crystal structure of the electrode material. As shown in Figure 4 , the peaks observed at 25.331°, 35.174°, 38.516°, 40.239°, 48.093°, 53.096°, 53.875°, 54.962°, and 70.691° correspond to TiO 2 . Two distinct reflections of anatase TiO 2 appeared at 25.331°, 38.516°, 53.875°, 54.962°, 70.691°, and 48.093°, which corresponded to (101, (112), (105), (211), (220) And (200) crystal plane (PDF#21‐1272). Others were at 35.174°, 40.239°, 53.096°, corresponding to the (101), (200) and (211) crystal planes of rutile TiO 2 . According to the hexagonal wurtzite structure Greenockite CdS standard card, the peak values of 26.803 and 69.707 corresponded to (002)s and (210) crystal planes, respectively. According to the cubic CdS standard card, 31.948 and 71.883 corresponded to the (200) and (331) crystal planes, respectively, which verified the cubic morphology of CdS in Figure 1B of the SEM. The size of the crystal grain size was calculated according to the Scherer formula:\n \n (2) \n D = 0.9 λ / β c o s θ \n \n FIGURE 4 X‐ray diffraction (XRD) pattern of different electrodes In the formula, λ is 0.154 nm, which is the wavelength of the CuKα line. β is the half‐width corresponding to a peak with a certain radian, and θ is the Bragg angle. From this calculation, the crystal grain sizes of TiO 2 and CdS were 62.543 and 42.24 nm, respectively. According to related literature, this increase in specific surface area by reducing the size of the crystal size was beneficial to promote the Photoelectrocatalytic (PEC) reaction process. 3.2 Electrochemical characterization of photoelectrodes The peak height and symmetry of the oxidation wave and the reduction wave obtained from the CV could be used to judge the reversibility of the reaction of the electroactive material on the electrode surface. \n 26 \n As shown in Figure 5A , the heights of the oxidation wave and the reduction wave of CdS and TiO 2 were different, the symmetry of the curve was poor, which showed that the reaction of the two electrodes is irreversible. Compared to TiO 2 , CdS had an obvious oxidation peak current, which promoted the electrochemical reaction activity. As shown in Figure 5B , under light conditions, the redox peak of CdS was significantly higher than the height under dark conditions, resulting in different electrochemical activities of the electrode. The CV curve showed that the CdS electrode has higher electrochemical reaction activity under light conditions, which was beneficial to speed up the electron transfer rate of the MFCs reactor. FIGURE 5 (A) Cyclic voltammetry (CV) curves of TiO 2 and CdS electrode electrolyte in three‐electrode mode. (B) CV curve of CdS electrode in the dark and light In order to further determine the electrochemical parameters of the anode, an EIS experiment was carried out. \n 27 \n Figure 6A,B is the measured Nyquist diagram of the electrode obtained under different conditions. As shown in Figure 7A , the fitted equivalent circuit under dark conditions was R 1 (CPE 1 R 2 )O, R 1 represents the ohmic resistance of the electrolyte, CPE 1 represents the electric double‐layer capacitance, and R 2 represents the charge transfer resistance, which reflected the intensity of the redox reaction. O stands for finite Warburg. The fitted equivalent circuit under CdS illumination was R 1 (CPE 1 R 2 ) (CPE 2 R 3 )O, CPE 2 and R 3 represents the oxide film electrical impedance and the oxide film capacitor, respectively (Figure 7B ). Under the light, the charge transfer resistance of TiO 2 in the control group was 7.673×1014 Ω, and the minimum charge transfer resistance of CdS was 1509 Ω. The charge transfer resistance was determined by the electrode reaction kinetics. The smaller the value, the faster the electron transfer process on the electrode. The data fitting results of the Z‐SimpWin software showed that the charge transfer resistance of the CdS electrode under light conditions is the smallest (Table 1 ). The reason may be that it provides more electrochemically active sites and high electron transfer during light irradiation. FIGURE 6 (A) Nyquist plots of different electrodes. (B) Nyquist plots of CdS electrode in the dark and light. (C) Tafel polarization curves of different electrodes. (D) Linear sweep voltammetry (LSV) curves of CdS under dark and light conditions FIGURE 7 Equivalent circuit models were used in the analysis of electrodes in microbial fuel cells (MFCs). (A) Under the dark conditions. (B) Under illumination TABLE 1 The fitting results of different cathodes electrodes from Nyquist plots \n (A) Dark conditions \n \n (B) Under illumination \n \n Cathodes \n \n R 2 (Ω cm –2 ) \n \n CPE (Ω cm –2 S n ) \n \n R 2 (Ω cm –2 ) \n \n CPE (Ω cm –2 S n ) \n \n R 3 (Ω cm –2 ) \n \n CPE (Ω cm –2 S n ) \n TiO 2 \n 1.027 × 10 \n 4 \n \n 1.209 × 10 –4 \n 3.062 × 10 \n 13 \n \n 4.936 × 10 –4 \n 7.673 × 10 \n 14 \n \n 3.211 × 10 –2 \n CdS 1.745 × 10 –2 \n 4.435 × 10 –14 \n 1.02 × 10 \n 4 \n \n 9.994 × 10 –5 \n 1.509 × 10 \n 3 \n \n 2.14 × 10 –3 \n John Wiley & Sons, Ltd. The Tafel curve test was to change the electrode potential as a function of its own variable in a certain linear relationship during the whole process and measured the tracking of the electrode potential change by the current flowing through the electrode. The higher the corrosion potential of the electrode, the slower the corrosion rate, and the corrosion rate was related to the phenomenon of metal polarization. The higher the self‐corrosion current density, the faster the corrosion rate and the worse the corrosion resistance. Figure 6C was the Tafel curve of different electrodes. The electrochemical corrosion parameters were calculated in Table 2 , and the results showed that the corrosion potential of CdS is the lowest and the corrosion resistance is the best, which provided a good foundation for the long‐term operation of MFCs. TABLE 2 Corrosion parameters of different electrodes \n Ecorr (vs. SCE) (V) \n \n Icorr (μA cm –2 ) \n Ti 0.059 11.69 TiO 2 \n 0.067 7.175 CdS 0.103 5.725 John Wiley & Sons, Ltd. The LSV curve showed that CdS has superior ORR catalytic activity under the light. It could be seen the CdS electrode under light has a smaller overpotential, a large current density, and the best catalytic performance compared to dark conditions (Figure 6D ). The main reason may be that more electrochemically active sites and higher conductivity are exposed, which greatly increased the possibility of the prepared electrode being in contact with electrons, thereby further speeding up the ORR process. At the same potential, the current density and the catalytic performance of the electrode exhibited a proportional relationship. 3.3 The optical absorption characteristics of electrodes Figure 8 shows the UV‐vis diffuse reflectance absorption spectrum of the electrode. It could be seen that nano‐TiO 2 has a strong absorption peak at 200–380 nm in the UV region, while the CdS‐doped composite electrode had strong absorption at 200–800 nm in the UV and visible regions (Figure 8a ). The absorption of the CdS‐doped composite electrode in the visible light region was higher than that of the TiO 2 electrode, which was caused by the electron transfer on the conjugated compound produced by polymer recombination. Electrons flowed from substances with a higher conduction band (CB) energy level to TiO 2 with a lower CB energy level, facilitating photogenerated carrier separation. \n 28 \n CdS could effectively extend the absorbance range of TiO 2 to the visible region. \n 29 \n The electrons could migrate to TiO 2 more effectively, and the cavities were oxidized and consumed by transfer, which reduced the recombination of carriers and enhanced the photocatalytic activity. \n 30 \n According to Equation ( 2 ), the forbidden bandwidth of the electrode material was estimated, and the calculation results were shown in Figure 8 b and c.\n \n (3) \n α h v = β h v − E g m / 2 \n \n FIGURE 8 (A) UV‐Visible light absorption diffuse reflectance spectroscopy (DRS) patterns. (B) Calculated energy band gap of TiO 2 . (C) calculated energy bandgap of TiO 2 /CdS Among them, α is the absorption coefficient, β is the proportional constant related to the band tailing parameter, hv is the incident photon energy related to the Planck constant, and Eg is the indirect optical energy gap. The energy (hv) and (αhv) 2 could be used as the x and Y coordinate axes to draw the band gap diagram of TiO 2 and CdS doped electrodes. The arctangent was made along the intersection of the calculated curve and the x‐axis, and the value obtained at the intersection was the band gap value of the TiO 2 and CdS‐doped electrode. Compared to the TiO 2 electrode, the absorption of CdS for light was significantly enhanced, and the range of absorption was expanded. According to Figure b,c, the band gap of TiO 2 was about 3.22 eV and the band gap of the TiO 2 /CdS composite electrode was about 3.16 eV. The positions of the valence band (VB) and the CB of CdS and TiO 2 can be calculated by empirical equations, \n 30 \n , \n 31 \n the CB positions are ‐0.48 eV and−0.30 eV and the VB positions are 1.86 and 2.92 eV, respectively. Based on the results above, there are differences in the band gap between CdS and TiO 2 , resulting in band overlap. This structure leads to the transfer and separation of photogenerated carriers between catalysts of different energy levels, which effectively suppresses photogenerated electron‐hole recombination, improves the photogenerated electron yield and catalytic efficiency of TiO 2 , and effectively expands the light response range. 3.4 The degradation performance of photoelectrodes The instantaneous photocurrent response curve of the prepared photoelectrode under visible light irradiation was shown in Figure 9 . The curve showed that the photocurrent response of the CdS electrode is obvious, and the test time interval between the change of light and dark conditions is 20 s. Generally, the separation ability of electron and hole pairs was proportional to the photocurrent density, which had a significant effect on improving photocatalytic activity. It could be seen from the response curve that the photocurrent response intensity of the photoelectrode is relatively strong, possibly due to the doping of CdS, which had a positive effect on the separation/transport of electrons and holes, and further improved the production capacity of MFCs. FIGURE 9 Transient photocurrent responses of microbial fuel cells (MFCs) with CdS photocathode In order to study the degradation performance of the photoelectrode of the microbial fuel cell to the dye, we used a UV‐vis spectrophotometer to evaluate the MB solution. Under visible light irradiation, 100 ml of 10 mg/L MB solution was added to the MFC with a CdS photocathode. The result of the spectral scan was shown in Figure 10A . The intensity of the characteristic peak at 663 nm gradually decreased with time and disappears after 2 h. This showed that CdS photoelectrodes can effectively degrade MB in MFCs. Formula (1) was used to calculate the concentration of MB in MFC under different reaction conditions. Figure 10B showed that photocathodes under different conditions have different photocatalytic effects. The degradation curve showed that the closed‐circuit MFC has the lowest degradation rate under dark conditions, and the open‐circuit MFC has the highest degradation rate under light conditions. Among them, the degradation rates of open‐circuit MFC under dark and light conditions reached 32.67% and 98.25%, respectively, and the degradation rates of closed‐circuit MFC under dark and light conditions reached 0.20% and 79.86%, respectively. Under light conditions, the degradation rate of open circuit MFC was 18.69% higher than that of a closed circuit. Under dark conditions, the degradation rate of open circuit MFC was 32.47% higher than that of a closed circuit. It showed that the photoelectric synergy of MFCs can further promote the degradation of dyes. FIGURE 10 (A) Photo images of methylene blue (MB) in the photodegradation. (B) Degradation efficiency curve during open/closed circuit/light‐dark conversion"
} | 5,477 |
31354694 | PMC6636552 | pmc | 5,792 | {
"abstract": "Mining of mineral resources substantially alters both the above and below-ground soil ecosystem, which then requires rehabilitation back to a pre-mining state. For belowground rehabilitation, recovery of the soil microbiome to a state which can support key biogeochemical cycles, and effective plant colonization is usually required. One solution proposed has been to translate microbial inocula from agricultural systems to mine rehabilitation scenarios, as a means of reconditioning the soil microbiome for planting. Here, we experimentally determine both the aboveground plant fitness outcomes and belowground soil microbiome effects of a commercially available soil microbial inocula (SMI). We analyzed treatment effects at four levels of complexity; no SMI addition control, Nitrogen addition alone, SMI addition and SMI plus Nitrogen addition over a 12-week period. Our culture independent analyses indicated that SMIs had a differential response over the 12-week incubation period, where only a small number of the consortium members persisted in the semi-arid ecosystem, and generated variable plant fitness responses, likely due to plant-microbiome physiological mismatching and low survival rates of many of the SMI constituents. We suggest that new developments in custom-made SMIs to increase rehabilitation success in mine site restoration are required, primarily based upon the need for SMIs to be ecologically adapted to both the prevailing edaphic conditions and a wide range of plant species likely to be encountered.",
"conclusion": "Conclusion In this study, we analyzed changes occurring within mine site topsoil microbiomes and chemical parameters after a 12-week incubation experiments using agriculture-based SMI and/or ammonium sulfate. This is the first study to assess the efficacy of protocol translation from agriculture practices into a semi-arid restoration context, shedding light into an establishing, but poorly studied restoration practice. Our results revealed that an important depletion of semi-arid microbiome diversity and evenness occurred when SMIs were added and further exacerbated when ammonium sulfate was also added in conjunction. Such a loss in native microbial diversity, along with incompatible interactions from exogenous microbes, likely explained the loss in A. ancistrocarpa fitness proxies (seedling emergence and shoot to root ratio). Therefore, future mine site restoration protocols must carefully consider preservation of native microbiome diversity through appropriate topsoil handling and storage as well as careful selection of any exogenous taxa that may be added to maximize a protocol’s potential success. For the latter, key taxa which deliver primary ecosystem functions (such as N fixation) and can survive the semi-arid conditions are a priority. Here, we demonstrate that α-proteobacterial nitrogen fixing organisms are likely to be of particular interest and suitability to speed-up nitrogen cycle restoration in mining affected areas.",
"introduction": "Introduction The mining of ores and minerals results in deleterious environmental outcomes ( Bradshaw, 2000 ) such as clearance of landscape biota and the production of large amount of by-products. Central to the amelioration of mining impacts is the rehabilitation of mined landscapes, with the final goal of the establishment of aboveground flora and fauna of adequate composition, and diversity ( Maestre et al., 2005 ; Baldwin et al., 2006 ; Brooker, 2006 ). In parallel, the belowground microbiome also needs to be rehabilitated, but is often overlooked, a key goal being the generation of high microbial diversity which can produce stable ecosystem services. These services are critical since they have central roles in the supply to the plant of key nutrients, pathogen protection, and water access. Therefore, in order to maximize the outcomes from aboveground restoration approaches we need to develop equivalent belowground microbiome “reconditioning” strategies which provide an optimum soil microbiome, one that will sustain aboveground biomass, and provision long term seed emergence and survival. During mining operations, initial vegetation clearance is followed by removal, and storage of the topsoil to expose the deeper mineral containing substrates. This topsoil is usually stored in a non-planted state for extended periods of time, often years. Over time, there are significant declines in key traits such as carbon content, seed banks of locally adapted native plants, and the composition of the microbiome ( Golos and Dixon, 2014 ; Muñoz-Rojas et al., 2016a ), including community diversity, and function ( Kumaresan et al., 2017 ). This decline in the soil microbiome is a critical factor within this stored topsoil, not only due to potential loss of key nutrient cycling pathways ( Wagg et al., 2014 ) but growing evidence also suggests that plant diversity, and fitness can be determined by the surrounding microbiome composition ( Lau and Lennon, 2011 , 2012 ; Panke-Buisse et al., 2015 ; Wubs et al., 2016 ). To this end, the addition of exogenous microbiomes in the form of soil microbial inocula (SMIs), such as those traditionally used in agricultural practices, is becoming increasingly mainstream in mine site restoration practices. Due to the economic importance of agriculture ( Johnston and Mellor, 1961 ; Knowler and Bradshaw, 2007 ) SMIs have been developed over decades for addition to the soil to maximize plant establishment, growth and productivity. These include plant growth promoting rhizobacteria (PGPR) developed and deployed across a range of agricultural soil types ( Çakmakçi et al., 2006 ; Singh et al., 2011 ; Chaparro et al., 2012 ; Upadhyay et al., 2012 ). Broadly, PGPRs encompass Nitrogen fixation and P solubilizing microorganisms such as Rhizobium , Pseudomonas , Azotobacter , and Azospirillum which directly, or indirectly, promote plant growth ( Singh et al., 2011 ). Additionally, inoculation with microorganisms such as Trichoderma , Pseudomonas , Glomus , Bacillus , and Agrobacterium can alter the plant’s physiological state and enhance plant growth ( Conrath et al., 2006 ; Rodrigues et al., 2008 ) and response to environmental stress ( Jung et al., 2012 ). The addition of microorganisms to trigger such processes is termed “bio-priming” and has been successfully applied to increase production in wheat ( Meena et al., 2016 ), rice ( Rodrigues et al., 2008 ), maize ( Akladious and Abbas, 2012 ), and soybean ( Entesari et al., 2013 ). Finally, the supplementation of SMIs to mineral fertilizers to allow slow and controlled release of nutrients has gained increased attention as a means of efficient nutrient use and has shown significant promise in improving crop growth ( Wu et al., 2005 ; Chang and Yang, 2009 ; Pereira et al., 2009 ; Leaungvutiviroj et al., 2010 ). Whilst important and clearly beneficial in agricultural systems, the ecological outcomes of deploying SMIs to mine site systems have been little studied. One recent example has shown the successful increase in germination and seedling growth of two plant species native to Western Australia, using indigenous cyanobacteria isolates ( Muñoz-Rojas et al., 2018 ). However, there is significant interest in using existing commercially produced SMIs to directly apply the technology within a mine site restoration setting. Theoretically, several barriers may exist to their effective deployment. These include much lower nascent nutrient levels in mine soil substrates, which will dictate whether copiotroph or oligotrophic adapted microorganisms survive and drive the ecosystem functionality ( Fierer et al., 2007 ; Carbonetto et al., 2014 ) and the ratio of bacteria to fungi, an important ecosystem property ( Güsewell and Gessner, 2009 ). Further, natural ecosystems tend to harbor microbiome diversity which has co-evolved with the native plants, including taxa that promote germination ( Muñoz-Rojas et al., 2018 ), or those that can solubilize recalcitrant macronutrients ( Lambers et al., 2009 ). Here, in order to resolve the efficacy of agricultural derived SMIs within mine site rehabilitation strategies, we assess the influence of an agricultural derived SMI upon the fitness of an Australian native plant Acacia ancistrocarpa , dominant in arid lands in Western Australia and commonly used for restoring semi-arid mine sites. We determined seedling emergence, a critical life stage transition in arid plants, and shoot to root ratio in tandem with emergent properties of the soil microbiomes in the first effort to assess the potential rehabilitation implication of an SMI consortium that is used in agriculture. We conclude that agricultural derived SMIs can be compromised in habitats such as semi-arid ecosystems and SMIs derived from cognate environments are likely have a higher chance of efficacy and plant growth promotion.",
"discussion": "Discussion We studied the effect of a commercially available soil microbial inoculum (SMI) within a mine site, semi-arid ecosystem context. We assessed soil microbiome and plant parameters as a first approach to gauge whether currently available SMIs can be applied to mine site restoration practices to enhance aboveground outcomes and overcome the reduced microbiome capacity within soils which have been subjected to mineral extraction ( Kumaresan et al., 2017 ). We further tracked the most abundant OTUs from the SMI to understand if such microorganisms can survive the relatively harsh conditions within mine waste soil for subsequent planting of native flora. Treatment selection in this experiment (Control, Nitrogen, Microbes, and Microbes+Nitrogen) can be viewed as an increasing intervention scale within the topsoil’s native microbiome and the soil’s chemical properties. The nitrogen addition treatment did not contain any allochthonous microorganisms and the differences in microbial diversity, evenness, or soil respiration shown in this study were similar to the experimental controls. However, the addition of the SMI, principally under the Microbes+Nitrogen treatment, substantially altered the plant response, reducing seedling emergence, microbial activity, and overall taxonomic diversity. Since the SMI was derived from agricultural soils and used in a more depauperate ecosystems we conclude that reduction in seedling emergence is likely due to a mismatch between plant and the introduced microbiome and that key drivers of emergence are likely present in the native microbiome associated with a given species of plant. These observations are in line with previous suggestions that native microbial communities drive aboveground diversity ( Wagg et al., 2014 ; Wubs et al., 2016 ; van der Putten, 2017 ), improve plant overall fitness ( Lau and Lennon, 2012 ), dictate seed bank persistence [reviewed in Long et al. (2015) ], or even affecting flowering time ( Panke-Buisse et al., 2015 ). Similar to our results, Batten et al. (2008) demonstrated that changes in the soil indigenous microbiome (in this case, mediated by invasive plants) negatively affected the performance of the American native plant Lasthenia californica . The net effects of soil microbial symbionts upon plant fitness can fluctuate along the mutualism-parasitism continuum depending on their origin, genetics, and environmental conditions ( Klironomos, 2003 ; Denison and Kiers, 2004 ). Hence, the reduced seedling emergence in the presence of the SMI is likely associated with the (a) reduced abundance and diversity of the indigenous microbes that presumably promote seed germination/emergence, and/or (b) negative/incompatible interactions between the native plant A. ancistrocarpa and the microorganisms introduced via addition of the microbial consortium. Clearly, further work investigating native plant and microbe signaling will be crucial to unveil such key constraints to seed emergence within these semi-arid ecosystems. SMI With or Without Nitrogen Addition Does Not Improve Plant Fitness The proposed key linkages between the native microbial diversity and plant fitness (germination, biomass) were evident when considering a decline of 33% in seedling emergence was observed when SMIs were added with Nitrogen, when compared to the Control. This implied that a severe loss in the topsoils’ native microbiome (as observed for SMI addition treatments) could significantly impact plant recruitment. Currently, we cannot fully explain the exact mechanism, other than highlight there must be key linkages for this species to the native microbiome, as our knowledge is limited on both biological entities (i.e., A. ancistrocarpa seedlings and native microbiome). However, it is widely accepted that the soil microbiome is integral for seed germination, as it can mediate seed coat break down ( van Leeuwen, 1981 ; Delgado-Sánchez et al., 2011 ), degrade germination inhibitors ( Zhu et al., 2011 ), and/or protect the seeds from pathogenic attack ( Dalling et al., 2011 ). As discussed above, a fraction of the reduced plant fitness could be linked with incompatible/pathogenic interactions between the exogenous microbes and the native plant. Furthermore, it has been shown that seed exudates can drive microbiome composition in the immediate surroundings, either by encouraging microbial growth via the supply of carbon compounds ( Roberts et al., 2009 ) or discouraging them by the production of defense proteins ( Rose et al., 2006 ) such as peroxidases ( Fuerst et al., 2011 ). We await the resolution of the detailed mechanisms in this case, but in general, a dense beneficial microbial assemblage is often formed around seeds ( Chee-Sanford et al., 2006 ) and clearly altering such an assemblage for native semi-arid species, as here, has significant repercussions for seed success. Depletion of the initial native microbiome was seen throughout the incubation experiment when comparing the characteristics of the parental topsoil (Basal) and the Control incubation treatment. This loss in both diversity and evenness over time within the control soil is a parallel for topsoil storage scenarios and is in line with previous studies documenting decreases in archaeal and bacterial microbiome diversity, functional capacity ( Kumaresan et al., 2017 ), earthworm communities ( Boyer et al., 2011 ), and seedling recruitment ( Rokich et al., 2000 ; Golos and Dixon, 2014 ) when fresh topsoil is stored unplanted for later use. We observed significant decreases in microbiome diversity and evenness (up to 20%) even in a relatively short 12-week time period. The impact of the loss of microbiome diversity during storage/experimental incubation must be assessed with caution, however, as higher microbiome diversity does not always correlate with healthier ecosystems ( Fernandez et al., 2000 ; Hashsham et al., 2000 ). Shade (2017) argue that comparing microbiomes by their diversity indices and assuming that higher diversity is better, oversimplifies the underlying mechanisms that set such diversities values. Nevertheless, more diverse microbial communities signify a higher potential for metabolic redundancy and, by extension, community resilience and plasticity. Despite clear changes within the native microbiomes during storage/incubation, stockpiling is a common practice in open cut mines where topsoil is removed from its original site, and stored elsewhere ( Muñoz-Rojas et al., 2016a ) in conditions that do not resemble its natural planted state. While this practice seems to be a logical procedure for restoration, a priority must be the resolution of whether better storage protocols can improve the conservation of the native belowground microbial communities ( Shade, 2017 ) and, by extension, improve the success of above and belowground outcomes when restoration commences. SMI OTUs Have Differential Response to Semi-Arid Conditions Many component taxa within the SMI clearly found the semi-arid conditions challenging for establishment. Despite the ease of access to commercial SMIs, they tend to be derived from high nutrient agricultural systems, systems which are vastly different from the edaphic conditions within semi-arid soils. The last can be explained by the differential interest from both the academia and the industry to develop novel ways to enhance soil quality ( O’Callaghan, 2016 ; Muñoz-Rojas, 2018 ). Several criteria include substantial differences in water and nutrient availability, metal concentrations and physical structure. Here, from the 40 most abundant OTUs within the SMI, 16 could not be detected reliably within the treatments, and 12 OTUs were present across all the treatments. Amongst these, four fell within the order Rhizobiales ( Microvirga , Bradyrhizobium , an unknown family and an unknown genus within Hypomicrobiaceae ). Such microorganisms may play a fundamental role in the early stages of primary succession ( Van Der Heijden et al., 2008 ; Wubs et al., 2016 ), as they can be the only source of nitrogen in such nutrient-limited soils. Specifically, such organisms have been demonstrated within early ecological succession of glacier forefronts ( Knelman et al., 2012 ), where a strong correlation between similar OTUs with vegetation development has been observed ( King et al., 2010 ). Addition of symbiotic or free-living nitrogen fixing microorganisms to semi-arid environment can be a significant step to potentiate nitrogen cycle in mine-impacted soils, especially addition of free-living diazotrophs, which has been shown to be active N 2 fixers ( Buckley et al., 2007 ) in many terrestrial systems ( Cleveland et al., 1999 ). Hypomicrobiaceae members are of great interest as they are ubiquitous soil microorganisms ( Oren and Xu, 2014 ) known to be abundant in both mining impacted environments ( Reis et al., 2013 ) and in agricultural soils ( Zhalnina et al., 2013 ), which opens the possibility for future approaches using them to initiate N cycling dynamics. An abundant OTU falling within the Arthrobacter genus was also found across all treatments, where Arthrobacter spp. are among the most ubiquitous indigenous soil bacteria that harbor broad metabolic and ecological functions to cope with harsh conditions ( Mongodin et al., 2006 ), and have been detected as plant endophytes ( Hardoim et al., 2015 ), within soda lakes ( Duckworth et al., 1996 ; Chakkiath et al., 2013 ), and mine tailings ( Bondici et al., 2013 ). Microorganisms with such ubiquity and functional plasticity can be suitable components for early colonization of nutrient-limited, semi-arid environments and post-mining vegetation rehabilitation."
} | 4,659 |
21946161 | null | s2 | 5,796 | {
"abstract": "Cell-free synthetic biology is emerging as a powerful approach aimed to understand, harness, and expand the capabilities of natural biological systems without using intact cells. Cell-free systems bypass cell walls and remove genetic regulation to enable direct access to the inner workings of the cell. The unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the rapid development of engineering foundations for cell-free systems in recent years. These efforts have led to programmed circuits, spatially organized pathways, co-activated catalytic ensembles, rational optimization of synthetic multi-enzyme pathways, and linear scalability from the micro-liter to the 100-liter scale. It is now clear that cell-free systems offer a versatile test-bed for understanding why nature's designs work the way they do and also for enabling biosynthetic routes to novel chemicals, sustainable fuels, and new classes of tunable materials. While challenges remain, the emergence of cell-free systems is poised to open the way to novel products that until now have been impractical, if not impossible, to produce by other means."
} | 290 |
35962867 | PMC9375759 | pmc | 5,798 | {
"abstract": "A novel methanogenic strain, CaP3V-MF-L2A T , was isolated from an exploratory oil well from Cahuita National Park, Costa Rica. The cells were irregular cocci, 0.8–1.8 μm in diameter, stained Gram-negative and were motile. The strain utilized H 2 /CO 2 , formate and the primary and secondary alcohols 1-propanol and 2-propanol for methanogenesis, but not acetate, methanol, ethanol, 1-butanol or 2-butanol. Acetate was required as carbon source. The novel isolate grew at 25–40 °C, pH 6.0–7.5 and 0–2.5% (w/v) NaCl. 16S rRNA gene sequence analysis revealed that the strain is affiliated to the genus Methanofollis . It shows 98.8% sequence similarity to its closest relative Methanofollis ethanolicus . The G + C content is 60.1 mol%. Based on the data presented here type strain CaP3V-MF-L2A T (= DSM 113321 T = JCM 39176 T ) represents a novel species, Methanofollis propanolicus sp. nov. Supplementary Information The online version contains supplementary material available at 10.1007/s00203-022-03152-w.",
"conclusion": "Taxonomic conclusion Based on phylogenetic, morphological, and physiological characteristics, strain CaP3V-MF-L2A T is considered to display a novel species within the genus Methanofollis (Table 1 ). Description of Methanofollis propanolicus sp. nov. Methanofollis propanolicus sp. nov. (pro.pa.no´li.cus. N.L. n. propanol; L. suf. -icus -a -um suffix used with various meanings; N.L. masc. adj. propanolicus) regarding to propanol, based on the substrate propanol, which can be metabolized by this species. Cells are irregular cocci, motile, 0.8–1.8 μm in diameter, and occur as single cells or in pairs. Occurrence of at least two types of cell appendages. Strictly anaerobic. Temperature range for growth 25 °C–40 °C (optimum, 37 °C). Sodium chloride range for growth 0–2.5% (w/v) (optimum, 0.2–1.5%). pH range for growth 6.0–7.7 (optimum, pH 6.5–7.0). Doubling time is 16 h. H 2 /CO 2 , formate, 1-propanol and 2-propanol used for methanogenesis, addition of 0.1% acetate is crucial for growth on substrates different from 1-propanol or 2-propanol. G + C content of DNA is 60.1 mol%. Closely related to Methanofollis ethanolicus JCM15103 T (98.8% 16S rRNA gene sequence similarity). The type strain is CaP3V-MF-L2A T (= DSM 113321 T = JCM 39176 T ), isolated from an oil well in the Cahuita National Park, Costa Rica. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CaP3V-MF-L2A T is MW490723.",
"introduction": "Introduction The genus Methanofollis (Zellner et al. 1999 ) represents one out of six genera within the family Methanomicrobiaceae (Balch et al. 1979 ). With the publication of this genus, the former Methanogenium species M. tationis and M. liminatans were transferred to the novel genus Methanofollis due to distinct patterns of glycolipids, phosphoglycolipids and amino-phosphoglycolipids among others. Furthermore, Methanofollis tationis possesses a pterin that is different from the known methanopterin or sarcinapterin–tatiopterin has not been found in any other methanogen since then (Raemakers-Franken et al. 1991 ). Today, six validly described species are assigned to the genus Methanofollis . These species were isolated from various aquatic environments with different salinities. Methanofollis liminatans was derived from a wastewater reactor in Germany and Methanofollis tationis from a solfataric field in Chile. Methanofollis formosanus (Wu et al. 2005 ) and Methanofollis aquaemaris (Lai and Chen 2001 ) were both isolated from a fish pond in Taiwan. Methanofollis ethanolicus (Imachi et al. 2009 ) was discovered in a lotus field, while the recently described Methanofollis fontis was isolated from a marine sediment near a cold seep (Chen et al. 2020 ). With the increasing number of described species, it became clear that the genus’ capability to use primary and secondary alcohols with two or more C-atoms for methane production is a characteristic feature. CaP3V-MF-L2A T shows a similarity of 98.8% in 16S rRNA gene sequence towards Methanofollis ethanolicus ; however, their physiological characteristics differ significantly. Strain CaP3V-MF-L2A T cannot grow on ethanol or 1-butanol but utilizes 1-propanol and 2-propanol for methanogenesis. Furthermore, strain CaP3V-MF-L2A T is characterized by a smaller cell size, its motility and a significantly shorter generation time. Therefore, we propose the here described strain CaP3V-MF-L2A T as a novel species, Methanofollis propanolicus sp. nov.",
"discussion": "Results and discussion Phylogenetic analysis Bidirectional sequencing (LGC Genomics) resulted in a 16S rRNA gene sequence fragment of 1364 bp. Phylogenetic analysis showed that strain CaP3V-MF-L2A T is affiliated to the genus Methanofollis (Fig. 1 ). Its closest relative is Methanofollis ethanolicus HASU T with a phylogenetic distance of 1.2%. The G + C content was 60.1 mol%. Fig. 1 Phylogenetic position of strain CaP3V-MF-L2A T based on 16S rRNA gene sequence of all Methanofollis species and the type species of remaining genera within the Methanomicrobiaceae . Bootstrap values greater than 90% are displayed. Bar, 2 substitutions per 100 nucleotide positions Morphological and physiological characterization Cells of strain CaP3V-MF-L2A T showed a greenish autofluorescence of factor F 420 typically found in methanogens, stained Gram-negative and were motile. In continuous culture, irregularly shaped cocci with a diameter of 0.8–1.8 μm occurred singly or in pairs. Electron microscopy not only revealed an uneven cell surface but also highly variable shape ranging from conical to circularly dent cells (Fig. 2 ). Cells showed two different types of cell appendages: archaella with a diameter of 12 nm and pili with a diameter of 8 nm. Cells exhibit the typical S-Layer structure of Methanofollis species (Fig. 3 ). Fig. 2 Transmission electron micrograph of Pt/C shadowed cells of strain CaP3V-MF-L2A T show the great variability in cell-shape as well as archaella. Bar, 400 nm Fig. 3 a Transmission electron micrograph of freeze-etched cells of strain CaP3V-MF-L2A T indicating b proteinaceous S-Layer (SL) and an archaellum (AR). Bars, 200 nm Growth of strain CaP3V-MF-L2A T was detected from 20 to 40 °C, with an optimal growth temperature of 37 °C. A pH of 6.0–7.5 supported cell growth, while levels below or above did not. The optimal pH was determined at pH 6.5–7.0. Strain CaP3V-MF-L2A T tolerated sodium chloride concentrations from 0 to 2.5% (w/v), while the optimal range was 0.2–1.5%. Strain CaP3V-MF-L2A T used H 2 /CO 2 , formate, 1-propanol, and 2-propanol as energy source, while acetate was required as a carbon source. This substrate spectrum differed from all other described species of the genus Methanofollis (Table 1 ). Yeast extract had a stimulating effect on cell growth as described for M. ethanolicus and M. liminatans . Acetate as well as the primary and secondary alcohols methanol, ethanol, 1-butanol, 2-butanol, and cyclopentanol did not support cell growth when used alone or in combination with acetate or yeast extract. This was even true for the substrate combination of 5 mM ethanol and 0.01% yeast extract with an incubation time of up to 3 months (which works for Methanofollis ethanolicus) . The doubling time of strain CaP3V-MF-L2A T under optimal physiological conditions was 16 h. Table 1 Characteristics of strain CaP3V-MF-L2A T compared to all other validly described species of the genus Methanofollis 1 2 3 4 5 6 7 Cell morphology Irregular cocci Irregular cocci Irregular cocci Irregular cocci Irregular cocci Irregular cocci/ring shaped Irregular cocci Cell diameter (μm) 0.8–1.8 1.5–3 1.2–2 2–3 1.5–2 0.4–0.5 0.8–1.2 G + C content (mol%) 60.1 54 59.1 60.9 58.4 60 59.5 Temperature (°C) optimum (range) 37 (25–40) 37–40 (25–45) 37 (20 –43) 37 (15–40) 37 (20–42) 40 (25–44) 37 (20–40) pH optimum (range) 6.5–7.0 (6.0–7.5) 7 (6.3–8.8) 6.5 (6.3–8) 7 (6.5–7.5) 6.6 (5.6–7.3) 7 5.9–8.2 (6.7–7.0) NaCl (%) optimum (range) 0.2–1.5 (0–2.5) 0.8–1.2 (0–7) 0–6 (0.5) (0–2.5) 3 (0–4) 0 (0–3.5) 0.17 (0–0.85) Generation time (h) 16 12 13 72 20 7.5 21 Substrates for methanogenesis H 2 /CO 2 + + + + + + + Formate + + + + + + + Ethanol – – – + – – – 1-Propanol + – ND + ND – – 1-Butanol – – ND + ND – – 2-Propanol + – – – – + – 2-Butanol – – – – – + – Cyclopentanol – – – – – + – Growth requirement Acetate + r a + r + s + s + s + s + s Yeast extract + s + r + s + r + s + r ND Motility + – – – – + – Strains: 1, CaP3V-MF-L2A T (data from the present study); 2, Methanofollis tationis OCM 159 T (Zabel et al. 1984 ); 3 , Methanofollis aquaemaris N2F9704 T (Lai and Chen 2001 ); 4 , Methanofollis ethanolicus HASU T (Imachi et al. 2009 ); 5, Methanofollis formosanus ML15 T (Wu et al. 2005 ); 6, Methanofollis liminatans BM1 T (Zellner et al. 1990 , 1999 ; Zellner and Boone 2001 ); 7, Methanofollis fontis FWC-SCC2 T (Chen et al. 2020 ) + positive, – negative; ND not determined, s stimulates growth, r required a Acetate is required as carbon source on substrates other than 1-propanol and 2-propanol The ancient oil well, where strain CaP3V-MF-L2A T was isolated from, represents an open pond with plenty of organic import (e.g., leaves from surrounding fauna). This explains the availability of substrates, such as acetate or propanol. In the natural environment, propanol derives from anaerobic microbial degradation processes. 2-propanol is known to be produced by some saccharolytic Clostridia by the reduction of acetone (Langlykke et al. 1937 ; Kutzenok and Aschner 1952 ; George et al. 1983 )."
} | 2,445 |
22403574 | PMC3289268 | pmc | 5,801 | {
"abstract": "Iron (Fe) is an essential micronutrient for marine organisms, and it is now well established that low Fe availability controls phytoplankton productivity, community structure, and ecosystem functioning in vast regions of the global ocean. The biogeochemical cycle of Fe involves complex interactions between lithogenic inputs (atmospheric, continental, or hydrothermal), dissolution, precipitation, scavenging, biological uptake, remineralization, and sedimentation processes. Each of these aspects of Fe biogeochemical cycling is likely influenced by organic Fe-binding ligands, which complex more than 99% of dissolved Fe. In this review we consider recent advances in our knowledge of Fe complexation in the marine environment and their implications for the biogeochemistry of Fe in the ocean. We also highlight the importance of constraining the dissolved Fe concentration value used in interpreting voltammetric titration data for the determination of Fe speciation. Within the published Fe speciation data, there appear to be important temporal and spatial variations in Fe-binding ligand concentrations and their conditional stability constants in the marine environment. Excess ligand concentrations, particularly in the truly soluble size fraction, seem to be consistently higher in the upper water column, and especially in Fe-limited, but productive, waters. Evidence is accumulating for an association of Fe with both small, well-defined ligands, such as siderophores, as well as with larger, macromolecular complexes like humic substances, exopolymeric substances, and transparent exopolymers. The diverse size spectrum and chemical nature of Fe ligand complexes corresponds to a change in kinetic inertness which will have a consequent impact on biological availability. However, much work is still to be done in coupling voltammetry, mass spectrometry techniques, and process studies to better characterize the nature and cycling of Fe-binding ligands in the marine environment.",
"introduction": "Introduction – Iron Biogeochemistry in the Ocean and the Importance of Iron Speciation Approximately 30% of surface waters in the open ocean are known as high nutrient low chlorophyll (HNLC) regions (Boyd et al., 2007 ). These areas are replete in the macronutrients nitrate and phosphate, but present lower phytoplankton biomass, in terms of chlorophyll concentrations, than expected from residual macronutrient concentrations (Figure 1 ). The restriction of phytoplankton growth in these regions is now acknowledged to be the result of iron (Fe) limitation (Martin and Fitzwater, 1988 ; Boyd et al., 2007 ). Fe is a micronutrient required for proteins involved in fundamental cellular processes, including both photosynthesis and respiration (Raven et al., 1999 ). Despite being the fourth most abundant element in the Earth’s crust (Liu and Millero, 2002 ), dissolved Fe (dFe: <0.2 or 0.45 μm) concentrations in open ocean surface waters typically fall below 0.2 nM, (De Baar and De Jong, 2001 ; Boyd and Ellwood, 2010 ) and dFe generally exhibits a nutrient-type depth profile in the ocean, depicting removal in surface waters by biological uptake, and increased concentrations at depth from remineralization processes occurring through the water column (Boyd and Ellwood, 2010 ). Although low, dFe concentrations in the ocean can be much higher than might be predicted given that the solubility of ferric hydroxide in seawater at pH 8.1 and 25°C has been determined to be as low as 0.01 nM (Liu and Millero, 2002 ). The presence of dFe at concentrations beyond the inorganic solubility of Fe is thought to be facilitated by organic complexation of Fe with stabilizing ligands, which buffer dFe in seawater against hydrolysis and ensuing precipitation (Hunter and Boyd, 2007 ; Boyd and Ellwood, 2010 ). However, the overall physico-chemical speciation of dFe, which encompasses all of its possible physical and chemical forms in seawater, is more complex than implied by the consideration of organic complexation alone. The different physico-chemical fractions of dFe include Fe(II), colloidal, truly soluble, and inorganic iron in addition to organically complexed iron. These different fractions have different environmental and biological mobility (Kuma et al., 1996 ; Maldonado et al., 2005 ; Hunter and Boyd, 2007 ; Kitayama et al., 2009 ; Boyd and Ellwood, 2010 ; Hassler et al., 2011b ). The motivation for understanding the physico-chemical speciation of Fe, therefore, results from a desire to understand how these different fractions influence the overall biogeochemistry of Fe in the oceans. Figure 1 (A) Annualized average nitrate (μM) and (B) composite chlorophyll a (mg L −1 ) distributions observed in surface waters in the global ocean. The nitrate distribution was obtained using data from the World Ocean Atlas 2009 ( http://www.nodc.noaa.gov/OC5/WOA09/pr_woa09.html ), while the chlorophyll a distribution represents the 2009 Aqua MODIS chlorophyll composite ( http://oceancolor.gsfc.nasa.gov/cgi/l3 ). It is important to note that dissolved iron (dFe) is operationally defined by filtration, with early studies employing 0.45 μm or, more recently, 0.2 μm membrane filters (De Baar and De Jong, 2001 ; Cutter et al., 2010 ). However, it has been shown that a significant proportion of dFe is colloidal (Fe colloidal ; Wu and Luther, 1994 ; Cullen et al., 2006 ; Bergquist et al., 2007 ; Kondo et al., 2008 ; Schlosser and Croot, 2008 ; Boye et al., 2010 ). Colloidal Fe is characterized as the difference between the Fe concentration determined in the <0.2 μm fraction (dFe) and the >1 kDa or >0.02 μm fraction, depending on whether cross flow filtration or membrane filtration techniques are used for the determination (Schlosser and Croot, 2008 ). The colloidal fraction is not measured directly, but inferred from the difference between dissolved (<0.2 μm) and soluble (<1 kDa or <0.02 μm) fractions. The mass balances for Fe when considering its physical distribution can be described as F e total = F e particulate + F e colloidal + F e soluble while the mass balance from a chemical perspective might be described as F e total = Fe ′ + FeL + F e inert , where Fe′ represents labile inorganic Fe complexes, FeL represents Fe organic ligand complexes exchangeable within a time scale of <1 day, and Fe inert represents the Fe fraction bound up in matrices that are essentially non-labile. As our analytical methods for the determination of the physico-chemical speciation of Fe tend to focus on either the physical (e.g., Schlosser and Croot, 2008 ; Baalousha et al., 2011 ) or the chemical (e.g., Gledhill and van den Berg, 1994 ; Rue and Bruland, 1995 ; van den Berg, 1995 ; Wu and Luther, 1995 ; Laglera et al., 2007 ; Mawji et al., 2008a ; Velasquez et al., 2011 ) perspective, reconciling these two approaches remains a major challenge to Fe biogeochemists. In recent years there has been a concerted effort to understand more about both the physical partitioning of Fe in the marine environment, and the chemical nature of the Fe ligand pool. The application of filtration with trace metal clean 0.02 μm pore size membrane filtration, ultrafiltration (10 kDa cut offs), and flow field flow fractionation (FFFF) coupled to ultra-violet (UV) and inductively coupled plasma-mass spectrometry (ICP-MS) detection techniques have considerably improved our knowledge of the physical partitioning of Fe in marine waters (Schlosser and Croot, 2008 ; Baalousha et al., 2011 ). Characterization of the FeL pool has been tackled through the utilization of high performance liquid chromatography–electrospray ionization-mass spectrometry (HPLC–ESI-MS) and development of novel electroanalytical techniques (McCormack et al., 2003 ; Laglera et al., 2007 ; Velasquez et al., 2011 ). In parallel to these advances a concerted effort is being made to improve our understanding of the robustness of competitive ligand exchange–adsorptive cathodic stripping voltammetry (CLE–ACSV), the technique most commonly used to determine Fe complexation in seawater (Buck et al., under review;Laglera et al., 2011 ). These advances have indicated that although the absolute physical partitioning determined varies from study to study as a result of the different techniques and filter cut offs, the colloidal Fe pool makes up between 30 and 91% of the dFe pool (Wu and Luther, 1994 ; Nishioka et al., 2001 ; Cullen et al., 2006 ; Bergquist et al., 2007 ; Hurst and Bruland, 2008 ; Kondo et al., 2008 ; Schlosser and Croot, 2008 ; Boye et al., 2010 ). The presence of Fe-binding ligands has been inferred in colloidal and measured in soluble fractions (Wu et al., 2001 ; Boye et al., 2010 ; Thuroczy et al., 2010 ), but both FFFF and ultrafiltration studies indicate that not all of the colloidal Fe (organic or inorganic) is exchangeable or under saturated with respect to Fe (Wu et al., 2001 ; Boye et al., 2010 ; Stolpe and Hassellov, 2010 ; Stolpe et al., 2010 ; Thuroczy et al., 2010 ). The existence of an inert colloidal fraction has broad implications for our understanding of Fe biogeochemistry and its significance in the ocean has yet to be properly assessed. Thus, while Fe soluble might reasonably be expected to include Fe′, FeL, or Fe inert are unlikely to be discreet to Fe soluble , Fe colloidal , or Fe particulate . Even though we have not fully characterized either colloidal or organic Fe associations, efforts to understand the overall effects of Fe speciation on Fe biogeochemistry have been made (Archer and Johnson, 2000 ; Moore et al., 2004 ; Parekh et al., 2005 ; Weber et al., 2005 ; Fan, 2008 ; Moore and Braucher, 2008 ; Tagliabue et al., 2009 ; Fan and Dunne, 2011 ; Tagliabue and Voelker, 2011 ). Modelers have made considerable progress toward capturing the complexity of iron speciation, incorporating inorganic iron scavenging and up to two ligand classes, in order to investigate the large scale implications of iron speciation on ocean productivity and the potential effects of a changing climate (Tagliabue et al., 2009 ; Ye et al., 2009 ; Tagliabue and Voelker, 2011 ). Fe biogeochemical models incorporating Fe speciation tend to highlight the importance of photoreduction processes in determining the dissolved concentrations of Fe in surface waters, while indicating that organic ligands are likely to strongly influence the thermodynamic solubility of Fe (Weber et al., 2005 ; Fan, 2008 ; Tagliabue et al., 2009 ; Tagliabue and Voelker, 2011 ). Furthermore, variations in organic ligand concentration and conditional stability constants have also been shown to influence the residence time and potential bioavailability of Fe in models (Tagliabue et al., 2009 ). Other studies have shown that different organic ligands have different chemical labilities and different susceptibilities to photoreduction (Hutchins et al., 1999 ; Barbeau et al., 2001 , 2003 ; Maldonado et al., 2005 ; Hassler et al., 2011b ). Overall, such studies highlight the need to characterize more fully the physico-chemical speciation of dFe. In this paper, we will review current knowledge of the organic complexation of Fe, the characterization and distributions of specific FeL complexes, and the characterization of colloidal Fe fractions as a first attempt at reconciling the different approaches to understanding the physico-chemical speciation of dFe in seawater. We will not consider the reduced Fe pool, Fe(II), in this review as our understanding of the interactions between Fe(II), Fe(III), and organic matter (e.g., Toner et al., 2009 ; Bligh and Waite, 2010 ) require further investigation. However, it should be born in mind that Fe(II) is likely important to Fe biogeochemistry as it is thought to be highly biologically available and can make up 50–60% of the dFe pool in illuminated surface waters or near sediments, hydrothermal vent systems, or oxygen minimum zones (Hong and Kester, 1986 ; Kuma et al., 1992 ; Johnson et al., 1994 ; Gledhill and van den Berg, 1995 ; Croot et al., 2005 ; Ussher et al., 2007 ; Hansard et al., 2009 ; Sarthou et al., 2011 )."
} | 3,046 |
28380078 | PMC5381785 | pmc | 5,802 | {
"abstract": "Evolution of cooperation and competition can appear when multiple adaptive agents share a biological, social, or technological niche. In the present work we study how cooperation and competition emerge between autonomous agents that learn by reinforcement while using only their raw visual input as the state representation. In particular, we extend the Deep Q-Learning framework to multiagent environments to investigate the interaction between two learning agents in the well-known video game Pong. By manipulating the classical rewarding scheme of Pong we show how competitive and collaborative behaviors emerge. We also describe the progression from competitive to collaborative behavior when the incentive to cooperate is increased. Finally we show how learning by playing against another adaptive agent, instead of against a hard-wired algorithm, results in more robust strategies. The present work shows that Deep Q-Networks can become a useful tool for studying decentralized learning of multiagent systems coping with high-dimensional environments.",
"introduction": "Introduction In the ever-changing world biological and engineered agents need to cope with unpredictability. By learning from trial-and-error an animal, or a robot, can adapt its behavior in a novel or changing environment. This is the main intuition behind reinforcement learning [ 1 , 2 ]. A reinforcement learning agent modifies its behavior based on the rewards it collects while interacting with the environment. By trying to maximize these rewards during the interaction an agent can learn to implement complex long-term strategies. When two or more agents share an environment the problem of reinforcement learning is notoriously more complex. Indeed, most of game theory problems deal with multiple agents taking decisions to maximize their individual returns in a static environment [ 3 ]. Collective animal behavior [ 4 ] and distributed control systems are important examples of multiple autonomous actors in dynamic environments. Phenomena such as cooperation, communication, and competition may emerge in reinforced multiagent systems. While the distributed nature of learning in multiagent systems offers benefits (e.g., inherent parallelism, scalability, or robustness versus failure of some of the agents), new challenges such as how to define good learning goals arise. Also there are few guarantees about the convergence and consistency of learning algorithms [ 3 , 5 , 6 ]. This is so because in the multiagent case the environment state transitions and rewards are affected by the joint action of all the agents. Thus, the value of an agent’s action depends also on the actions of the others and hence each agent must keep track of each of the other learning agents, possibly resulting in an ever-moving target [ 3 , 5 , 6 ]. In general, learning in the presence of other agents requires a delicate trade-off between the stability and adaptive behavior of each agent [ 3 , 5 , 6 ]. Due to the astronomic number of possible states in any realistic environment until recently algorithms implementing reinforcement learning were either limited to simple settings or needed to be assisted by additional information about the dynamics of the environment [ 7 ]. Recently, however, the Swiss AI Lab IDSIA [ 8 ] and Google DeepMind [ 7 , 9 ] have produced spectacular results in applying reinforcement learning to very high-dimensional and complex environments such as video games. In particular, [ 7 , 9 ] demonstrated that AI agents can achieve superhuman performance in a diverse range of Atari video games. Remarkably, the learning agent only uses raw sensory input (screen images) and the reward signal (increase in game score). The proposed methodology, the so called Deep Q-Network (DQN), combines a convolutional neural network for learning feature representations with the Q-learning algorithm [ 10 ]. The fact that the same algorithm was used for learning very different games might suggest it has potential for more general purpose applications [ 7 , 11 ]. The present article builds on the work of [ 7 ]. Instead of a single agent playing against a hardcoded algorithm, we explore how multiple agents controlled by autonomous DQNs learn to cooperate and compete while sharing a high-dimensional environment and being fed only raw visual input. This is an extension to the existing multiagent reinforcement learning studies done in simple grid worlds or with agents already equipped with abstract high-level sensory perception [ 3 , 12 , 13 ]. In particular, using the video game Pong and manipulating the rewarding schemes we describe the agents’ emergent behavior with a set of behavioral metrics. We show that the agents develop successful strategies for both competition and cooperation, depending on the incentives provided by rewarding schemes. We also tune the rewarding schemes in order to study the intermediate states in the progression from competitive to collaborative behavior. Finally, we illustrate how learning by playing against another learning agent results in more robust strategies than those achieved by a single agent trained against a stationary hard-wired algorithm. Agents trained in multiplayer mode perform very well against novel opponents, whereas agents trained against a stationary algorithm fail to generalize their strategies to novel adversaries.",
"discussion": "Discussion Multiagent reinforcement learning has an extensive literature in the emergence of conflict and cooperation between agents sharing an environment [ 3 , 12 , 13 ]. However, most of the reinforcement learning studies have been conducted in either simple grid worlds or with agents already equipped with abstract and high-level sensory perception. In the present work we demonstrated that agents controlled by autonomous Deep Q-Networks (DQNs) are able to learn a two player video game such as Pong from raw sensory data. This result indicates that DQNs might become a useful tool for the decentralized learning of multiagent systems living a high-dimensional environments without the need of manual feature engineering. In particular, we described how the agents learned and developed different strategies under different rewarding schemes, including full cooperative and full competitive tasks. The emergent behavior of the agents during such schemes was robust and consistent with their tasks. For example, under a cooperative rewarding scheme the two Pong agents (paddles) discovered the coordinated strategy of hitting the ball parallel to the x -axis, which allowed them to keep the ball bouncing between them for a large amount of time. It is also interesting to note that the serving time, i.e. the time taken by the agent to launch the first ball in a game, was one of the behavioral variables modified during the learning. We also notice that single-agent training against a stationary hard-wired algorithm exposes the DQN to a limited set of opponent behaviors. In the multiagent setting the strategies of both agents may change considerably during training. This type of training may expose the agents to a more diverse range of opponent behaviors and game situations, thus making them more capable of playing well against an unseen adversary. Discussion of optimal strategies for intermediate rewarding schemes Here, we give a brief discussion on what the optimal strategies would be under different rewarding schemes used in this work. In this theoretical discussion, we consider that both agents are equally skilled and therefore equally likely to win any exchange. In practice we cannot guarantee such equality in all situations, but we do observe that across games the rewards are equally distributed. In the following, we divide this discussion of optimal behaviour to two different phases of the game 1) when the ball is out of play and needs to be served and 2) when the ball is in play. In the first case an agent needs to decide if it is beneficial for it to relaunch the game. With ρ ≤ 0 it is clear that serving is never the optimal choice as any exchange can only lead to negative rewards. In fact, the average expected reward from an exchange is negative for all ρ < 1, because the agents are equal and punishments are bigger than rewards. Nevertheless, in case of ρ > 0, in specific game situations serving might still be the good choice (e.g. when opponent has placed itself unfavourably are the agent is very likely to score). In general, we would expect the average serving time to increase with decreasing ρ . For all non-positive ρ we expect the agents to avoid serving in all situations. This is indeed what we observe on Fig 5c . Let us now consider the case where the ball has already been put into play. Clearly, in the case of ρ ≥ 0 an agent should always try to score. Scoring leads to a positive or zero reward and helps avoid a possible negative reward (conceding) in the future. At the other end of the spectrum, with ρ = −1, scoring is punished as strictly as conceding and the only strategy for minimizing losses is to keep the ball alive. As described above, this leads to cooperative behaviour. With −1 < ρ < 0, the best possible strategy is still to keep the ball alive forever, but the incentive to discover this strategy is reduced. Remember that both agents are independently trying to maximize their own reward. If the agents are not skillful enough or if the ball is flying too fast, keeping the ball in play for a long time is not probable. In such case, the optimal strategy for an agent might be to compete for the lesser penalty ( ρ instead of −1), instead of trying to collaborate. By decreasing the reward difference between scoring and conceding we decrease this incentive to compete. We therefore expected the agent to play on average more cooperatively when ρ is decreased from -0.25 to -1. In our work we suggest that aggressive play can be estimated by the number of wall-bounces per paddle bounce. This metric ( Fig 5b ) does indeed stay equally high for ρ ≥ 0 and has a decreasing trend when ρ is decreased from 0 to -1. Limitations We observe that in the fully competitive (zero-sum) rewarding scheme, the agents overestimate their future discounted rewards. The realistic reward expectation of two equally skillful agents should be around zero, but in most game situations both of our DQNs predict rewards near 0.5 ( Fig 2 , videos in supporting information). Overestimation of Q-values is a known bias of the Q-learning algorithm and could potentially be remedied by using the novel Double Q-learning algorithm proposed by [ 20 ]. Nevertheless notice that biased Q-values do not necessarily mean that the policy would be biased or wrong. In this work we have used the simplest adaptation of deep Q-learning to the multiagent case, i.e., we let an autonomous DQN to control each agent. In general, we expect that adapting a range of multiagent reinforcement algorithms to make use of DQNs will improve our results and pave the way to new applications of distributed learning in high-dimensional environments. A larger variety of metrics might have helped us to better describe the behavior of different agents. More descriptive statistics such as average speed of ball and how often the ball is hit with the side of the paddle would have required analyzing the screen images frame by frame. While probably useful quantitative descriptors of behavior, we were limited to the statistics extractable from the game memory. Some of the above-mentioned descriptors were nevertheless used in qualitative descriptions of behaviour. Future work In the present work we have considered two agents interacting in an environment such as Pong with different rewarding schemes leading them towards competition or collaboration. Ongoing work is devoted to study the feature representation learning achieved by the different types of agents. In particular, one can make use of guided back-propagation [ 21 ] to compare the visual features that activate the hidden nodes of the DQN controllers of competitive and cooperative agents. Using other games such as Warlords we plan to study how a larger number of agents (up to four) organize themselves to compete or cooperate and form alliances to maximize their rewards while using only raw sensory information. It would certainly be interesting to analyse systems with tens or hundreds of agents in such complex environments. This is currently not feasible with the system and algorithms used here. Convolutional neural networks have become the much needed high-level computational framework against which to contrast data-driven hypotheses of visual processing in the brain [ 22 , 23 ]. Similarly, we believe that the success (and limitations!) of network architectures endowed with different capabilities [ 24 – 26 ] provide important insights and constraints for how other cognitive processes occur in the brain. A future direction of the present approach is to study of how communication codes [ 27 , 28 ] and consensus [ 28 – 30 ] can emerge between interacting agents in complex environments without any a priori agreements, rules, or even high-level concepts of themselves and their environment.\n\nDiscussion of optimal strategies for intermediate rewarding schemes Here, we give a brief discussion on what the optimal strategies would be under different rewarding schemes used in this work. In this theoretical discussion, we consider that both agents are equally skilled and therefore equally likely to win any exchange. In practice we cannot guarantee such equality in all situations, but we do observe that across games the rewards are equally distributed. In the following, we divide this discussion of optimal behaviour to two different phases of the game 1) when the ball is out of play and needs to be served and 2) when the ball is in play. In the first case an agent needs to decide if it is beneficial for it to relaunch the game. With ρ ≤ 0 it is clear that serving is never the optimal choice as any exchange can only lead to negative rewards. In fact, the average expected reward from an exchange is negative for all ρ < 1, because the agents are equal and punishments are bigger than rewards. Nevertheless, in case of ρ > 0, in specific game situations serving might still be the good choice (e.g. when opponent has placed itself unfavourably are the agent is very likely to score). In general, we would expect the average serving time to increase with decreasing ρ . For all non-positive ρ we expect the agents to avoid serving in all situations. This is indeed what we observe on Fig 5c . Let us now consider the case where the ball has already been put into play. Clearly, in the case of ρ ≥ 0 an agent should always try to score. Scoring leads to a positive or zero reward and helps avoid a possible negative reward (conceding) in the future. At the other end of the spectrum, with ρ = −1, scoring is punished as strictly as conceding and the only strategy for minimizing losses is to keep the ball alive. As described above, this leads to cooperative behaviour. With −1 < ρ < 0, the best possible strategy is still to keep the ball alive forever, but the incentive to discover this strategy is reduced. Remember that both agents are independently trying to maximize their own reward. If the agents are not skillful enough or if the ball is flying too fast, keeping the ball in play for a long time is not probable. In such case, the optimal strategy for an agent might be to compete for the lesser penalty ( ρ instead of −1), instead of trying to collaborate. By decreasing the reward difference between scoring and conceding we decrease this incentive to compete. We therefore expected the agent to play on average more cooperatively when ρ is decreased from -0.25 to -1. In our work we suggest that aggressive play can be estimated by the number of wall-bounces per paddle bounce. This metric ( Fig 5b ) does indeed stay equally high for ρ ≥ 0 and has a decreasing trend when ρ is decreased from 0 to -1."
} | 4,016 |
36048764 | PMC9436070 | pmc | 5,803 | {
"abstract": "The Hawaiian Archipelago experienced a moderate bleaching event in 2019—the third major bleaching event over a 6-year period to impact the islands. In response, the Hawai‘i Coral Bleaching Collaborative (HCBC) conducted 2,177 coral bleaching surveys across the Hawaiian Archipelago. The HCBC was established to coordinate bleaching monitoring efforts across the state between academic institutions, non-governmental organizations, and governmental agencies to facilitate data sharing and provide management recommendations. In 2019, the goals of this unique partnership were to: 1) assess the spatial and temporal patterns of thermal stress; 2) examine taxa-level patterns in bleaching susceptibility; 3) quantify spatial variation in bleaching extent; 4) compare 2019 patterns to those of prior bleaching events; 5) identify predictors of bleaching in 2019; and 6) explore site-specific management strategies to mitigate future bleaching events. Both acute thermal stress and bleaching in 2019 were less severe overall compared to the last major marine heatwave events in 2014 and 2015. Bleaching observed was highly site- and taxon-specific, driven by the susceptibility of remaining coral assemblages whose structure was likely shaped by previous bleaching and subsequent mortality. A suite of environmental and anthropogenic predictors was significantly correlated with observed bleaching in 2019. Acute environmental stressors, such as temperature and surface light, were equally important as previous conditions (e.g. historical thermal stress and historical bleaching) in accounting for variation in bleaching during the 2019 event. We found little evidence for acclimation by reefs to thermal stress in the main Hawaiian Islands. Moreover, our findings illustrate how detrimental effects of local anthropogenic stressors, such as tourism and urban run-off, may be exacerbated under high thermal stress. In light of the forecasted increase in severity and frequency of bleaching events, future mitigation of both local and global stressors is a high priority for the future of corals in Hawai‘i.",
"conclusion": "Conclusions The 2019 bleaching event had widespread effects on coral reefs across the Hawaiian Archipelago and underscored the rising frequency of thermal stress events not only in the central Pacific, but also around the world. Following the third mass global bleaching event (2014–2017), which resulted in severe mortality, bleaching events have continued to affect reefs globally over the past 5 years including regions such as French Polynesia [ 90 ], Bonaire [ 91 ], and the Great Barrier Reef [ 92 ]. While the 2019 bleaching event in the Hawaiian Islands was not as severe as initially forecasted, future marine heatwaves still harbor the potential for catastrophic impacts. This study highlights the value of large, multi-institutional partnerships to study patterns and processes at spatial scales beyond the scope of any one agency. While the bleaching response was less severe overall across the archipelago in 2019 than in 2014/2015, it was highly variable among sites and taxa—driven largely by the taxonomic susceptibility of the coral assemblages present. Whether the coral communities archipelago-wide exhibited signs of acclimatization to thermal stress is challenging to elucidate, given that potential resilience observed at certain reefs may be directly caused by the massive mortality that followed the 2014/2015 bleaching event and left only the least susceptible taxa present. Further studies should examine changes in coral cover and community composition over time, with an emphasis on collecting taxa-specific size-structure, bleaching and mortality data across a full depth gradient. In light of the forecasted increase in severity and frequency of bleaching events, this work lays important groundwork for predicting the effects of bleaching across space and taxa, and suggests viable management strategies in Hawai‘i for further consideration.",
"introduction": "Introduction Coral bleaching driven by climate-induced marine heatwaves stands as one of the single greatest threats to coral reefs [ 1 ]. As the pace of warming ocean temperatures have risen, so too have the frequency and severity of mass coral bleaching events around the world [ 2 , 3 ]. Over a prolonged period ranging from 4 to 6 weeks, exposure to elevated temperatures 1°C above the local average temperature of the warmest month of the year can often trigger bleaching due to thermal stress [ 4 ], thereby breaking down the symbiotic relationship between corals and the dinoflagellate algae living within their tissues. After disruption of this symbiosis, corals typically have been exposed to significant oxidative stress [ 5 – 7 ], lack a crucial energy source [ 8 ], and become increasingly vulnerable to disease [ 9 , 10 ]. Severe bleaching can result in widespread and immediate partial or full mortality of coral colonies [ 11 , 12 ]. For those corals that survive, the sub-lethal effects of bleaching may interrupt processes of growth and reproduction [ 4 , 13 , 14 ]. As coral reefs worldwide face a barrage of threats imposed by a rapidly warming climate, further understanding of what drives bleaching and how local environmental factors interplay with heat stress to differentially affect coral communities is critical for predicting the state of future reefs. Identifying bleaching resistant coral taxa and reef assemblages can inform local management targets with the goal of supporting coral resilience following repeated bleaching events. The extent of bleaching across reefs in response to thermal stress is often variable and can be a function of local conditions [ 15 ]. Site- and region-specific factors including water flow, weather patterns, irradiance, and community structure [ 16 – 20 ], as well as anthropogenic disturbances due to sedimentation and nutrient enrichment associated with land-based sources [ 21 , 22 ], can impact bleaching extent and severity. Coral taxa exhibit marked variability in bleaching susceptibility. Certain species are able to withstand repeated thermal stress while others are unable to recover after a single bleaching episode [ 23 – 26 ]. Fast growing, branching taxa typically bleach rapidly and undergo whole colony mortality, while slower growing, massive taxa may take longer to bleach and display increased survivorship rates despite remaining bleached for longer time periods [ 24 ]. With continued warming, these hardier and more slowly growing taxa are hypothesized to replace weedy, fast-growing taxa on future reefs [ 24 , 27 ]. While bleaching susceptibility is largely dependent on colony morphology, growth rate, reproduction, and overall life history strategies [ 28 ], bleaching response also varies between coral taxa due to differences in prior thermal stress exposure and acclimatization to particular thermal regimes [ 29 – 31 ]. Moreover, variation in bleaching susceptibility can also be influenced by the composition and characteristics of symbiotic algae residing within the host [ 32 – 34 ]. Growing evidence suggests that corals and their algal symbionts are capable of acclimatization and selective adaptation to thermal stress, resulting in greater bleaching resistance within populations [ 23 , 29 , 30 , 35 , 36 ]. Coral bleaching events in the Hawaiian Archipelago have increased in frequency and severity since 1996 [ 19 , 37 – 39 ]. In 2014, the Northwestern Hawaiian Islands (NWHI) experienced severe thermal stress and high levels of bleaching resulting in loss of coral cover reaching 68% at some sites [ 39 ]. During the 2015 thermal stress event in the main Hawaiian Islands (MHI), catastrophic bleaching was observed, with up to a 71% loss in coral cover on the west coast of Hawaiʻi Island [ 38 ], relative to pre-bleaching values, and close to 50% in both Kāne‘ohe and Hanauma Bay on the island of O‘ahu [ 17 , 40 , 41 ]. In the fall of 2019, the MHI experienced another marine heatwave with some portions of the MHI and NWHI experiencing more than 20 weeks of accumulated thermal stress, resulting in the third major bleaching event recorded in the Hawaiian Islands within a 6-year period. The Hawai‘i Coral Bleaching Collaborative (HCBC) was established following the 2014 bleaching event and includes academic, non-governmental and governmental partners active in research, restoration, conservation, and management of coral reef resources in Hawai‘i. The primary mission of the HCBC is to coordinate coral bleaching surveys across the state to monitor the extent and severity of mass bleaching events, collate and share data about these events to understand their impact, and develop management recommendations for reducing impacts of future events. To document the 2019 bleaching event, HCBC launched a large multi-institutional response consisting of diver visual assessments and image-based surveys across the Hawaiian Archipelago. The goals of this study were to: 1) assess the spatial and temporal patterns of thermal stress; 2) examine taxa-level patterns in bleaching susceptibility; 3) quantify spatial variation in bleaching extent; 4) compare 2019 patterns to those recorded during the 2014 and 2015 bleaching events; 5) determine key natural and anthropogenic predictors (hereafter referred to as drivers) of bleaching in 2019; and 6) explore site-specific management strategies to mitigate future bleaching events using a scenario-based sensitivity analysis.",
"discussion": "Discussion Coral bleaching in 2019 was driven by community composition & environmental factors A gradient of bleaching responses was observed across the MHI during the 2019 event, which contrasted patterns in 2015 when thermal stress was more extreme and more uniform mass bleaching was observed. This variability in bleaching was found to be largely driven by taxonomic susceptibility, consistent with prior studies illustrating the significant impact that the distribution of vulnerable taxa has on the outcome of a bleaching event [ 39 , 55 , 56 ]. The 2019 surveys showed that the coral community assemblage on O‘ahu was relatively more susceptible to bleaching than other islands. On O‘ahu, the most vulnerable taxa may have survived the 2015 event due to the lower thermal stress (0.5°C-weeks) relative to other islands. While lower bleaching and thermal stress was observed on Hawai‘i Island in 2019 compared to 2015, it is important to note that Hawai‘i Island experienced a catastrophic loss of coral cover from the 2015 bleaching event, with mortality recorded in more than half of all stony corals present on the island’s west coast [ 38 ]. The loss of corals likely included taxa more susceptible to bleaching, such as P . meandrina − colonies that averaged 77.6% total post-bleaching mortality following the 2015 event. The resulting communities, therefore, were likely less susceptible overall and when they were exposed to lower acute thermal stress in 2019, they bleached less. While massive Porites colonies are considered stress tolerant in other contexts [ 28 ], certain communities on Hawai‘i Island’s west coast suffered significant post-bleaching mortality of P . lobata and P . evermanni in 2015 (losses of 55.7% and 92.5%, respectively [ 38 ]). In 2019, the bleaching responses of P . lobata and P . lutea were amongst the mildest at each island surveyed. This shift in bleaching response is likely more related to the lower DHW experienced in 2019 vs. 2015, rather than changes in susceptibility operating at the colony-level. While it is hypothesized that coral populations may adapt and/or acclimate to thermal stress [ 23 , 29 , 36 ], we lack the region-wide historical bleaching data that would provide robust support for taxa-specific changes in thermal tolerance. Regional and site-level bleaching may also be strongly driven by physiological variation of the host and symbiont. Fluorescent tissue pigment granules (FPG) are known to protect corals from broad-spectrum solar radiation [ 57 ], and fast-growing pocilloporids have been reported to have lower densities compared to slow-growing massive poritids. In addition, thinner tissues in pocilloporids compared to poritids could make them more vulnerable to thermal stress [ 24 ]. More so, differences in the dominant symbiont genera appear to drive intraspecific variability in colony-level thermal tolerance [ 58 – 60 ]. For example, the bleaching response of Montipora capitata in Kāne‘ohe Bay during the 2015 bleaching event was significantly driven by dominant symbiont genus, with some colonies severely bleaching while others remained unaffected [ 60 ]. Beyond local abiotic conditions, differences in symbionts, together with host genotypic factors, may explain the observed species-level bleaching variability across islands; however, further research is warranted. Although taxonomic susceptibility was the strongest predictor of the 2019 bleaching, thermal stress was a major driver and often played a strong interactive role with other natural and anthropogenic factors. These results are consistent with the bleaching response to thermal stress documented in 2014/2015 [ 39 , 40 ]. The 2019 response was mediated by both historical and acute thermal stress. Importantly, reefs were predicted to bleach more if they experienced both high acute thermal stress and high historical thermal stress, but predicted to bleach less if they experienced high acute thermal stress and low historical thermal stress. These results suggest that previous exposure to bleaching did not result in significant overall acclimation or adaptation to thermal stress events at the reef community level. Moreover, the significant interaction between historical bleaching and susceptibility demonstrated that corals with high susceptibility to bleaching will bleach regardless of their previous bleaching response. Surprisingly, SST variability did not emerge as a significant driver of bleaching in 2019. This is also contradictory to findings in other regions, where coral bleaching is significantly less common in localities with high SST variability [ 15 , 23 , 61 , 62 ]. Other environmental drivers of the 2019 bleaching event included surface light (PAR). This was a positive driver of bleaching during 2019 according to this study and has frequently been implicated in increasing bleaching during periods of elevated thermal stress by amplifying photo inhibition [ 19 , 63 , 64 ]. Light attenuation, or turbidity, can also regulate bleaching by reducing severe irradiance; corals at locations with high turbidity or cloud cover have been found to bleach less [ 37 , 64 , 65 ]. However, turbidity modeled using satellite-derived kdPAR measurements in this study did not emerge as a significant mediator of the 2019 bleaching. The role of depth in bleaching patterns varies considerably across studies. Numerous studies have found decreased bleaching and bleaching-induced mortality with depth, but this effect often varies due to complex and interacting local factors such as coral community composition, temperature, and light attenuation [ 66 , 67 ]. While Couch et al. [ 39 ] found a slight negative relationship between depth and bleaching during the 2014 bleaching event in the NWHI, the 2019 bleaching response in the MHI tended to increase with depth. The modeled significant interaction between susceptibility and depth predicted that the increase in bleaching at depth was more pronounced for corals with higher susceptibility. Our findings are in line with Venegas et al. [ 68 ], who found no meaningful depth refuge from thermal stress down to 38 m in the west and central Pacific Ocean. Also, bleaching thresholds can vary with depth, with higher sensitivities in deeper waters [ 62 , 69 ]. Therefore, in the MHI, taxa more susceptible to bleaching may be found on deeper reefs. Given that the depth gradient in this study did not surpass 17 m and the relatively high water clarity at depth in the MHI, it is unsurprising that we found no evidence of depth as a refuge from bleaching. Anthropogenic stressors exacerbated the 2019 bleaching response Several anthropogenic stressors were correlated with the 2019 bleaching response. Urban run-off, which in this study consisted of a proxy for pollutants such as trash, household chemicals, pharmaceuticals, and oil [ 70 ], was a positive driver of the 2019 bleaching; this relationship was especially detrimental for more susceptible coral taxa. High incidences of coral disease and mortality have been observed on reefs located near areas of run-off [ 71 ]. Many chemicals are detrimental to coral health, with oil being particularly lethal [ 72 ]. The effects of toxic substances may be enhanced at higher temperatures [ 73 ]; during a bleaching event, the rapid degradation of pollutants likely amplifies the negative effects of thermal stress on corals. Tourism and recreation alone did not significantly predict bleaching, but the significant interaction between tourism/recreation and acute thermal stress suggests that the effects of increased tourism/recreation are exacerbated under periods of high thermal stress. In other words, reefs that are exposed to tourism and recreation may bleach more than undisturbed reefs during a bleaching event. The negative impacts of heavy tourism on coral reefs have resulted in higher incidences of damage and disease coupled with lower coral cover observed globally [ 74 , 75 ]. Sewage effluent was negatively correlated with bleaching. However, the input of sewage into coral reef systems has been historically implicated in elevated levels of disease and macroalgae cover, as well as reduced coral cover, growth, and recruitment [ 76 – 78 ]. While the sewage effluent data used in this study is the best available statewide description of nearshore effluent calculated from estimated total nitrogen and phosphorus flux coming from onsite sewage disposal systems, this data fails to include effluent coming from injection wells. An additional caveat to using this effluent data is that the temporal resolution of the data layer notably predates the 2019 bleaching surveys and thus does not include any additional development nor updates to household cesspools following the generation of this data. Yet the negative relationship between bleaching and effluent as suggested by our best-fit model may support an alternative view of ecosystem resilience described by Côté and Darling [ 79 ] in which a degraded ecosystem state increases the abundance of disturbance-tolerant species within a community and boosts the ability of the ecosystem to resist impacts of that disturbance. In this case, we hypothesize that coral communities that persisted in 2019 were able to tolerate high levels of sewage effluent coupled with high thermal stress during the 2015 bleaching event—a sign of positive co-tolerance [ 80 ]. Potential management strategies to mitigate impacts of future thermal stress events In light of the forecasted increase in bleaching events [ 81 ], generating adaptive strategies to mitigate the impacts of these future stress events is essential for coral reef conservation and management. When we simulated a high thermal stress event and perturbed a subset of drivers (that could potentially be manipulated by managers), the impact on predicted bleaching varied substantially across space. This suggests that the effectiveness of these actions may be highly site-specific, which should be interpreted in the context of the operability of these management practices being highly variable across spatial scales [ 82 ]. In Maui and Lānaʻi, reducing urban run-off (modelled by calculating area of impervious surface per watershed as a proxy for trash, household chemicals, oil, etc.) appears to have the greatest positive impact on reefs, particularly along the west Maui coastline. The west Maui shoreline is densely populated with resorts, commercial development, and golf courses, with known land-based sources of pollution (LBSP). Long-term monitoring of coral reefs along this coastline has revealed a decline up to 50% in certain impacted areas [ 83 ]. The reef off Kahekili Beach Park has been particularly affected by LBSP, with multiple studies linking the loss of coral cover and high proliferation of macro and turf algae to a prevailing nutrient imbalance caused by an influx of nutrient-rich wastewater and chemical toxins from the nearby Lāhainā Wastewater Reclamation Facility [ 84 , 85 ]. On O‘ahu, our scenarios suggest that a shift to communities composed of less susceptible coral taxa would play a role in the outcomes of future thermal stress events. Managers should be cognizant of how the dominance of stress-tolerant taxa will effect future restoration efforts, and select coral taxa accordingly. However, a narrow focus on bleaching resistance as a primary target for restoration may lead to less diverse reefs if only a subset of resilient taxa are selected. Data from 2019 surveys point to those taxa that appear less susceptible to bleaching; however, it is important to note that the susceptibility of taxa can change over time. Previous studies have demonstrated that under annual bleaching, the susceptibility of certain taxa can reverse by “turning previous ‘winners’ into ‘losers’” [ 31 ]. As bleaching events in the Hawaiian Archipelago increase in frequency, we may witness further shifts in taxonomic susceptibility and community assemblages thereafter. Along west Hawai’i Island, decreasing surface light (PAR) was the most beneficial management action for reducing predicted bleaching at the majority of sites. Shading has been shown to be a direct and effective means of protection against harmful solar radiation for corals [ 86 , 87 ]. West and Salm [ 88 ] recommended that managers consider this proactive measure in response to future forecasted thermal stress events to mitigate bleaching; however, effectively scaling this effort up to the reef-scale is not yet possible, so these results may have limited applicability for meaningful management of future reef-scale resilience. Rising frequency of bleaching events and changing trends through time While ocean temperature trends have indicated an increase in warming over time [ 3 , 89 ], the 2019 heating event resulted in lower accumulated thermal stress than initially anticipated in the MHI. Thermal stress experienced across the main Hawaiian Islands during the 2019 bleaching event was more moderate than that endured by reefs during the catastrophic 2014/2015 event, and bleaching was either similar or significantly reduced in 2019 compared to 2015. Regardless, marine heatwaves are becoming more frequent and severe across the Hawaiian Archipelago since the first reported bleaching in 1996 ( Fig 1 , [ 39 ]). In the NWHI, the level of thermal stress sustained in 2019 was higher or similar to conditions during previous years, consistent with the warming trend found by Couch et al. [ 39 ] and indicating that repeated thermal stress may start extending across more than just the northern atolls. While NWHI corals continue to bleach during these heatwaves, the lower observed bleaching despite higher thermal stress experienced in the northern atolls (PHR and Midway) may be a sign of acclimation in resilient taxa or due to the elimination of vulnerable individuals that did not survive bleaching events prior to 2019 (this study, [ 39 ])."
} | 5,861 |
39875378 | PMC11775286 | pmc | 5,804 | {
"abstract": "Condensation is a vital process integral to numerous industrial applications. Enhancing condensation efficiency through dropwise condensation on hydrophobic surfaces is well-documented. However, no surfaces have been able to repel liquids with extremely low surface tension, such as fluorinated solvents, during condensation, as they nucleate and completely wet even the most hydrophobic interfaces. Here, we introduce a surface functionalization methodology that enables dropwise condensation of fluorinated refrigerants. This approach, compatible with various substrates, combines low contact angle hysteresis Parylene-C with low surface energy silane (P-HFDS) using a highly scalable atmospheric vapor phase deposition technique. Our experimental results demonstrate that the omniphobic P-HFDS coating facilitates dropwise condensation of both natural refrigerants (water, ethanol, hexane, pentane) and synthetic low-global-warming-potential refrigerants (HCFO R1233zd(E) and HFO R1336mzz(Z)) with surface tension as low as 14.6 mN m −1 at 25°C. The P-HFDS coating improves condensation heat transfer coefficients by 274%, 347%, 636%, and 688% for ethanol, hexane, pentane, and R1233zd(E), respectively, compared to filmwise condensation on uncoated metal surfaces. Additionally, the coating demonstrates long-term durability, sustaining steady dropwise condensation for 170 days without apparent degradation. This work pioneers stable dropwise condensation of multiple refrigerants on a structure-less surface, offering a durable, substrate-independent, and scalable solution for low surface energy coatings.",
"introduction": "Introduction Condensation is a ubiquitous phenomenon used in industrial processes such as steam cycle power generation 1 , electronics thermal management 2 , distillation 3 , natural gas processing 4 , refrigeration, and air conditioning 5 , 6 . The recent increase in fossil fuel consumption due to global development and population growth has heightened the need for more energy-efficient industrial processes to reduce greenhouse gas emissions and mitigate global climate change 7 . Due to its critical role in industrial process efficiency, scientists have explored numerous methods for enhancing condensation in the past century 8 – 12 . Despite significant advancements in enhancing steam condensation using various coatings and surface modifications 13 – 19 , solutions remain elusive for completely wetting low surface tension liquids 20 , especially fluorinated solvents. The majority of industrial processes rely on low-surface tension liquids, natural refrigerants, or synthetic fluorinated refrigerants 11 , 21 , 22 . For instance, alternative energy sources such as biofuels, organic solvents used in chemical synthesis, organic Rankine cycles, and air conditioning technologies depend heavily on the condensation of fully wetting refrigerants 11 , 21 , 23 , 24 . The recent emphasis on reducing the impact of global warming has spurred interest in developing energy-efficient refrigeration systems using low global warming potential (low-GWP) and low ozone-depleting potential (low-ODP) refrigerants 25 , 26 . Development of means to enable the enhanced condensation of these low-GWP refrigerants has the potential to increase system and process energy efficiency and reduce the adverse effects of global warming. Dropwise condensation, where the condensate morphology transitions from a thin liquid film to discrete droplets, enhances condensation heat transfer by lowering the time-averaged droplet size and increasing the exposure of condensing surface for further re-nucleation through increased droplet shedding 8 , 27 , 28 . Nominally, heat exchanger surfaces are easily wetted even by high surface tension liquids such as water (~72 mN m −1 at 25 °C) resulting in filmwise condensation 29 . This occurs because the heat exchanger surface is made of conventional metals (Al, CuNi, and Cu) and their oxides which have high surface energy (>50 mJ m −2 ) 20 , 30 . Various surface modification methodologies have been developed to achieve dropwise condensation. The surface can be chemically modified to lower its surface energy for the formation of discrete condensate droplets. Chemical modification of the free interface to achieve dropwise condensation includes ion implantation 31 , graphene deposition 32 , organic coatings 33 – 38 , and liquid/oil films 39 , 40 . In addition, dropwise condensation can be achieved on a chemically modified surface having low contact angle hysteresis ( θ CAH ), which represents the difference between the droplet advancing contact angle ( θ a ) and receding contact angle ( θ r ) 41 . Lower θ CAH enables dropwise condensation by increasing condensate droplet mobility and reducing droplet departure size 41 . Condensation can be further enhanced by implementing superhydrophobicity and superomniphobicity through the rational micro and nanostructuring of surfaces, which further increase the θ a and reduce the θ CAH 10 , 42 , 43 . Compared to water, low surface tension liquid droplets are either completely wetting or exhibit lower θ a and higher θ CAH on hydrophobic surfaces 44 . As a result, low surface tension liquids exhibit filmwise condensation on conventional hydrophobic surfaces 11 . In the last decade, several super-repellent and superomniphobic surfaces have been developed (micro/nanostructured, reentrant structured, doubly reentrant structured) that can repel some of these low surface tension liquid droplets 45 – 47 . However, even a doubly reentrant structured super-repellant surface that is capable of repelling droplets of very low surface tension fluorinated solvents (FC-72, ∼10 mN m −1 at 25 °C) is defenseless against physical intrusion mechanisms such as condensate nucleation inside cavities 47 . During condensation, these superomniphobic surfaces with reentrant structures suffer from flooding of low surface tension condensates due to spatially random nucleation of nanometric condensate droplets within the structures 48 – 50 , rendering the surfaces nonfunctional for dropwise condensation. Recent studies have demonstrated the possibility to achieve dropwise condensation of low surface tension liquids on lubricant-infused micro/nano-structured surfaces (LIS or SLIPS), surfaces with grafted polymers using initiated chemical vapor deposition (iCVD), patterned quasi-liquid surfaces, polydimethysiloxane-silane (PDMS-silane) coated surfaces, and oleophobic surfaces 11 , 40 , 44 , 48 , 51 – 55 . Although the promotion of dropwise condensation of low surface tension fluids such as ethanol, hexane, and pentane has been achieved on several of the aforementioned coatings, the dropwise condensation of fluorinated solvents such as refrigerants remains elusive and somewhat of a holy grail in condensation physics 20 , 53 . Warming of the earth due to greenhouse gas emissions has become a major environmental concern. Widely used hydrofluorocarbon refrigerants (HFCs) were designated as greenhouse gases under the Kyoto Protocol in 1997 56 . To reduce greenhouse gas emissions, countries have enacted laws to control emissions of HFCs that have high GWP. Recently, hydrofluoroolefin (HFO) and hydrochlorofluoroolefin (HCFO) refrigerants have been developed to have extremely short atmospheric lifetime due to their reactivity with atmospheric hydroxyl radicals 57 , 58 . These refrigerants are environmentally friendly, non-toxic, non-flammable, low GWP, and have relatively high liquid-vapor surface tension. For example, HCFO R1233zd(E) (∼14.58 mN m −1 at 25 °C) and HFO 1336mzz(Z) (∼15.1 mN m −1 at 25 °C), have higher surface tensions than their high-GWP HFC predecessors R134a (∼8 mN m −1 at 25 °C), and R245fa (∼13.6 mN m −1 at 25 °C) 59 – 62 . The phase-out of low surface tension HFC refrigerants and their replacement with higher surface tension HFOs and HCFOs have opened up the possibility of finally developing a refrigerant-phobic surface coating that can achieve the dropwise condensation of a synthetic fluorinated refrigerant 57 , 60 . To overcome previous limitations preventing the dropwise condensation of fluorinated refrigerants, we developed a polymer chemistry that facilitates the manufacturing of robust, conformal, and substrate-independent coatings with low surface energy. By grafting fluoroalkyl silane on Parylene-C, we achieve a very low surface energy, low θ CAH coating which not only exhibits long-term condensation durability but also enables the dropwise condensation of natural refrigerants such as hydrocarbons as well as synthetic low-GWP HCFO refrigerant 1233zd(E), and HFO refrigerant 1336mzz(Z). Our research pioneers the demonstration of dropwise condensation of fluorinated refrigerants, which fundamentally opens up a paradigm shift in the rational design methodology of omniphobic and refrigerant-phobic surfaces.",
"discussion": "Discussion The P-HFDS omniphobic coating developed in this study can sustain stable dropwise condensation of low surface tension liquids on smooth surfaces. The P-HFDS formulation, engineered through a rational design approach, provides a durable, scalable, and versatile method for achieving dropwise condensation of liquids having extremely low surface tension, such as fluorinated refrigerants. By employing sequential surface treatments to synergistically lower surface energy and contact angle hysteresis ( θ CAH ), P-HFDS reliably supports condensation of challenging low surface tension fluids. Unlike traditional methods that depend on geometric surface modifications to increase filmwise condensation area such as adding fins, porous structures, or microscale textures, the P-HFDS coating offers a practical alternative to enhance condensation performance without the need to physically modify or geometrically texture the surface 74 , 75 . Experimental results reveal that the P-HFDS coating enhances the condensation heat transfer coefficient by ~260% for ethanol and up to 688% for R1233zd(E) refrigerant compared to filmwise condensation. Furthermore, the coating demonstrated robust durability, maintaining stable dropwise condensation for 110 days with ethanol, 60 days with steam, and 90 days with R1233zd(E), showing no signs of degradation. The rational design of the P-HFDS coating opens the door for the development of more efficient and compact condensers, benefiting industries like chemical processing, natural gas production, biomass combustion, food processing, and energy systems such as heat pumps and commercial chillers 76 – 81 . The scalable fabrication methods employed to apply the P-HFDS coating, including conformal dip-coating and ACVD, ensure the ability to uniformly deposit the coating on complex geometries, including finned heat exchangers as well as tortuous internal channels. Unlike line-of-sight techniques such as physical vapor deposition, the P-HFDS coating process is ideal for curved and intricate surfaces. Moreover, the P-HFDS coating method is efficient, reducing material waste and chemical use when compared to conventional approaches 11 , 49 , making it a practical option for improving the performance of existing heat exchanger systems on an industrial scale. The omniphobicity of P-HFDS ensures the potential for anti-fouling and anti-scaling applications. Fouling and scaling degrade the efficiency of many components and systems operating in a broad set of industries, including but not limited to: maritime transportation (shipping), water desalination and management, chemical processes and distillation, and building energy systems 82 – 84 . The P-HFDS coating displays properties that are present in effective scale and foul-reducing coatings, including low surface energy, low contact angle hysteresis, and low surface roughness. Synergistically, these properties act to limit foulant and scale nucleation 82 , 83 , 85 . Unlike many of the previously reported anti-fouling and anti-scale coating formulations, P-HFDS can readily be applied to metallic substrates 63 , 82 , 83 , 86 , which make up the majority of scale-forming surfaces in real systems. In the future, it would be interesting to continue the demonstration of dropwise condensation for other synthetic refrigerants, with a focus on reducing the surface tension to even lower values. The two refrigerants demonstrated here (HCFO refrigerant R1233zd(E) and HFO refrigerant R1336mzz(Z)), although representing a breakthrough on their own, were rationally chosen due their potential as drop-in replacements for high-GWP R134a, R245fa, R123 refrigerants in systems such as chillers, organic Rankine cycles, and high-temperature industrial heat pumps 80 , 81 , 87 . Additionally, it would be worthwhile to investigate widely used, more established HFC working fluids (i.e., R134a refrigerant), which also have lower surface tension (i.e., ~8 mN m −1 at 25 °C) and represent a more challenging target for dropwise condensation 59 , 60 . With the recent proposal to limit production and use of polyfluoroalkyl substances (PFASs), it is important to develop environmentally friendly low-surface-energy coating chemistries as replacements that can be applied at scale 88 – 91 . The Parylene-C coating formulation utilized in this study is fluorine-free, non-harmful, and widely used as a biocompatible material. However, the low-energy fluoroalkyl silane (HFDS) does contain fluorinated methyl (CF 3 ) and methylene (CF 2 ) groups. There have been previous initiatives to replace long-chain PFAS with lower molecular weight short-chain PFAS and replacement compounds such as GenX, ADONA, and F53B, which are less persistent, bioaccumulative, and exert a decreased toxic effect. However, the recent emergence of evidence has raised concerns, and further studies need to be conducted to confirm whether these replacements are environmentally or toxicologically acceptable alternatives 92 . The need for environmentally friendly alternatives to PFAS has opened the avenue for additional work to develop hydrogenated low-energy chemistries that can act as alternatives to HFDS in our multilayer formulation for the P-HFDS omniphobic coating. It should be noted that, although we only demonstrate the use of HFDS in this study, other low-energy silanes are also potential alternatives to HFDS in our P-HFDS coating. Basically, MPTS acts as an adhesion promoter due to its intermediate character and thus can serve as an electrostatic glue between the Parylene-C coating and silane interfaces. One end of the MPTS molecule forms an alkene bond with the Parylene-C surface, while the other end forms a siloxane bond with the silane. As a result, due to the MPTS-enabled flexibility in our proposed coating methodology, HFDS can be replaced with a non-PFAS, non-fluorinated silane with comparable surface energy in the future once it has been developed and made available. Until recently, the majority of condensation research was limited to steam condensation enhancement due to the absence of surfaces capable of enabling long-term, durable dropwise condensation of low-surface-tension liquids 20 , 44 . In the case of steam dropwise condensation, a 10X enhancement in h leads to a 2% overall increase in system efficiency for a thermoelectric power plant 93 , 94 . However, for commercial fluorinated refrigerants, trade studies have not been conducted, as there has never been a demonstration of dropwise condensation of a refrigerant. As a result, demonstrating dropwise condensation of refrigerants opens up the potential for future research into more holistic, system-level studies of their impact on economics, component performance, system performance, and power density for existing and potentially new applications. In summary, this study demonstrates the dropwise condensation of ethanol, hexane, pentane, and synthetic refrigerants on an ultra-scalable and facile surface coating developed through the rational combination of Parylene-C and HFDS silane. We show that our developed coating can be applied to conventionally used surfaces such as aluminum, copper, steel, and additively manufactured metals. Condensate droplet analysis of our surface coating demonstrates a 263%, 340%, and 627% enhancement in h compared to filmwise condensation of ethanol, hexane, and pentane on a flat vertical surface, respectively. Ethanol and synthetic refrigerant R1233zd(E) condensation experiments at high saturation pressures on coated metallic tubes showed 260% and 688% enhanced h , respectively, compared to filmwise condensation on bare tubes. The P-HFDS coating is sufficiently robust compared to previously reported low surface energy condenser coatings, maintaining continuous dropwise condensation of ethanol, steam, and synthetic refrigerant for up to 110, 60, and 90 days, respectively. Our surface modification method is substrate-independent, highly scalable, repeatable, and requires small amounts of chemicals compared to previously reported surface modification techniques, such as spray coating, oil infusion, or sol–gel coating. This method can be implemented for components with complex shapes and sizes, presenting retrofit opportunities for existing thermal components. Our work lays the foundation for the development of facile coatings through the careful selection of chemically inert, low θ CAH polymers, further combined with low surface energy polymers to achieve omniphobicity. Our results not only provide guidelines for the development of a previously unexplored class of surface coatings for enhancing condensation heat transfer for low surface tension liquids but also demonstrate methods capable of achieving dropwise condensation of fluorinated refrigerants and solvents."
} | 4,427 |
28101080 | PMC5209367 | pmc | 5,807 | {
"abstract": "Organic farming system and sustainable management of soil pathogens aim at reducing the use of agricultural chemicals in order to improve ecosystem health. Despite the essential role of microbial communities in agro-ecosystems, we still have limited understanding of the complex response of microbial diversity and composition to organic and conventional farming systems and to alternative methods for controlling plant pathogens. In this study we assessed the microbial community structure, diversity and richness using 16S rRNA gene next generation sequences and report that conventional and organic farming systems had major influence on soil microbial diversity and community composition while the effects of the soil health treatments (sustainable alternatives for chemical control) in both farming systems were of smaller magnitude. Organically managed system increased taxonomic and phylogenetic richness, diversity and heterogeneity of the soil microbiota when compared with conventional farming system. The composition of microbial communities, but not the diversity nor heterogeneity, were altered by soil health treatments. Soil health treatments exhibited an overrepresentation of specific microbial taxa which are known to be involved in soil suppressiveness to pathogens (plant-parasitic nematodes and soil-borne fungi). Our results provide a comprehensive survey on the response of microbial communities to different agricultural systems and to soil treatments for controlling plant pathogens and give novel insights to improve the sustainability of agro-ecosystems by means of beneficial microorganisms.",
"introduction": "Introduction Over the past decades, anthropogenic alteration of soils by the increased use of synthetic fertilizers, pesticides and land conversion in order to increase food production is causing unprecedented changes in biodiversity, and thus, rising concern on the sustainability of intensive farming systems. The agriculture intensification has a substantial impact on plant and animal diversity ( Gabriel et al., 2006 ; Jonason et al., 2011 ). However, the effects of agricultural management on below-ground diversity are not well understood ( Li et al., 2012 ). This lack of knowledge is a significant concern because soil-borne microbes, especially bacteria, represent the majority of biodiversity in soil ecosystems and are involved in multiple ecosystem functions, including nutrient cycling ( Pan et al., 2014 ; Navarrete et al., 2015 ) and plant health ( Mazzola, 2004 ; Wakelin et al., 2013 ). The environmental problems associated with the intensification of agriculture have initiated research efforts for conservation strategies. Converting conventional farms to organic farming systems seems to be a potential solution to diminish the loss of biodiversity and increase sustainable food production ( Gonthier et al., 2014 ). Organic farming system consists of low-input agro-ecosystem farms in which plant productivity and ecosystem functionality are based on the natural availability of plant nutrients, use of green manure and biological pathogen control ( Lammerts van Bueren et al., 2002 ). In contrast, conventional farming system relies on intensive use of agrochemicals, such as synthetic fertilizers to increase crop productivity and use of fungicides and pesticides to promote plant protection against pathogens ( Kremen and Miles, 2012 ). Effects of farming systems on microbial communities are complex and time-dependent ( Jonason et al., 2011 ). In general, it has been reported that management practices in organic farming systems change the microbial composition toward a more fast growing community (copiotrophic community) due to nutrients ( Chaudhry et al., 2012 ), promote habitat diversification, increase the diversity and sustainability, and benefit microbial taxa involved in plant health when compared to conventional farming systems ( Esperschutz et al., 2007 ; Sugiyama et al., 2010 ; Reilly et al., 2013 ; Gonthier et al., 2014 ). However, up to date, there are no studies about microbial community heterogeneity, which we refer as microbial community variability, in different farming systems. Although positive effects of organic management have been widely reported ( Liu et al., 2007 ; Ge et al., 2008 ; Jonason et al., 2011 ; Hartmann et al., 2015 ), the effects of farming systems on diversity of microbial communities are complex and commonly controversial ( Kleijn et al., 2001 ). Ge et al. (2008) found an increase in diversity after manure amendment, and other studies reported no differences or decrease in bacterial diversity and richness when organic systems were compared to conventional management ( Liu et al., 2007 ; Reilly et al., 2013 ). Bengtsson et al. (2005) argue that in most cases, organic farming can be expected to benefit the biodiversity, but the effects will differ between organism groups and landscapes. Agro-ecosystems often face problems with plant-pathogens, such as parasitic nematodes (e.g., Pratylenchidae and Meloidogynidae), and soil-borne fungi (e.g., Rhizoctonia solani and Verticillium dahliae ) that affect a large number of important crops ( Back et al., 2002 ). A common method to control these pathogens is the use of chemical pesticides, which are under critical review due potential toxic effect on non-target organisms and environmental pollution ( Oka, 2010 ). Therefore, the development of methods for suppression of pathogens as an alternative to chemical control is an urgent need. These methods can be applied in organic farming systems, but also enable conventional farmers to reduce the use of pesticides. Alternative approaches are organic amendments (compost) ( Mehta et al., 2014 ), cover crops (Asteraceae plants) ( Pudasaini et al., 2006 ), green manure crops (grass-clover) ( Widmer and Abawi, 2002 ), composts or non-composted waste products (chitin) or those based on physical methods (soil disinfestations) ( Mowlick et al., 2012 ). Although these management practices are environmentally friendly, they are expected to induce shifts on microbial diversity and composition ( Mehta et al., 2014 ). At the treatment level, the microbes play an important role in above- and below- ground processes, including their potential contribution to soil suppressiveness ( Cretoiu et al., 2013 ). In this light, the ability to understand and manage microbial community through alternative practices for pathogen control, offer a promising approach to improve sustainable crop production. The broad spectrum of agricultural managements and practices used for plant pathogen control in farming systems limits comparability among different studies ( Liu et al., 2007 ; Xue et al., 2013 ). Up to date, there are few long-term agro-ecosystems experiments comparing organic and conventional farming systems ( Esperschutz et al., 2007 ), and even more seldom are studies that make this comparison on plant pathogen control. This would be ultimately required for evaluating the sustainability of agricultural practice. One exception is the experimental field with Soil Health Treatments (SHTs) in organic and conventional farming initiated in 2006. The SHT experimental site in Vredepeel is a unique experimental field reported in contemporary literature with full-factorial experimental design and replicated experimental plots, where the same soil treatments, crop varieties, crop rotations and fertilization intensities are simultaneously applied in both conventional and organic farming systems under the same sandy soil type. Korthals et al. (2014) have evaluated the potential effects of the different SHTs on plant-parasitic nematode Pratylenchus penetrans , and on soil-borne pathogenic fungus V. dahliae . However, the long lasting responses of the soil microbial community to those different managements and the potential role of microbial community in soil suppressiveness were not studied. In this context, we assessed the bacterial and archaeal communities based on 16S rRNA gene marker by next generation sequencing to examine the response of microbial communities to conventional and organic farming systems and SHTs. The objectives of this study were to address the effect of farming systems and SHTs on (i) soil microbial diversity and presence of pathogen suppressors, and (ii) microbial community heterogeneity. Based on microbial community assessment, we aimed to detect specific structural shifts and identify microbial taxa associated with specific farming system or SHT, which might be useful as a bioindicator of sustainable management of agro-ecosystems and might bring novel insights on soil beneficial agriculture practices for soil health and plant productivity.",
"discussion": "Discussion The SHE represents a unique experiment to assess the response of microbial communities to farming systems (conventional and organic) and Soil Health Treatments (SHTs). This study was limited to temporal sampling (single time point), spatial extent (local scale), and therefore should not be generalized for the farming systems performed in all ecosystems. Although the consistent results in this study provide novel ecological insights into microbial ecology in agro-ecosystems, concrete conclusions are still difficult and need to be confirmed by long-term experiments over distinct environmental conditions, management practices and larger geographic scales. Besides this, the complexity of microbial communities and the technical constraints so far, limited our understanding of the relationship between soil microbiota and agricultural managements. However, using the approach based on high-throughput sequencing of amplified taxonomic markers, we have described the microbial community structure and found that the soil microbiome is more heterogeneous in organic than conventional farming system, and additionally identified potential microbial pathogen suppressors and individual microbial taxon associated with specific management practices. It is difficult to draw robust and generalized conclusions on the effect of systems management on microbial diversity, but an increase in microbial diversity has been repeatedly observed in organic in comparison with conventional system ( Mader et al., 2002 ; Hartmann et al., 2015 ). The increase of microbial diversity in organic systems is strongly associated with the management applied, including the organic amendments and practices related with reduction or absence of chemical inputs and biological plant protection ( Sun et al., 2004 ; Chaudhry et al., 2012 ). The enhancement of microbial diversity also benefits the functional activities and a more heterogeneous distribution of species within the microbial assembly, which implies in a stable and functional redundant community, leading to an ecosystem functionality built on healthier interactions between the different trophic ecosystem levels ( Brussaard et al., 2007 ; Postma et al., 2008 ; Crowder et al., 2010 ; Wagg et al., 2014 ). The decrease of microbial diversity in the conventional system may be explained by the direct or indirect long-term stresses caused by the use of pesticides, fungicides and herbicides used for plant protection. These agrochemicals reduce the total microbial diversity because of the potential to inhibit or eliminate certain groups of microbes and select members adapted or able to growth under conventional farming practices ( El Fantroussi et al., 1999 ; Liu et al., 2007 ; Stagnari et al., 2014 ). Our study revealed consistent farming system effects on microbial community variability, suggesting, for the first time, more heterogeneous community in organic than in conventional system. We suggest that the availability of rich substrate in soil through the introduction of cattle farm yard manure, the biological practices without the interference of synthetic compounds and the presence of weed species provide heterogeneous habitat niches, which can be occupied by a highly variable microbial community resulting in an increase of the beta-diversity. The lower heterogeneity (that is, the lower beta diversity) in microbial community in conventional system is an indication of biotic homogenization, the process of increasing similarity in the composition of communities across an array of taxonomic or functional groups ( Olden et al., 2004 ). Biotic homogenization is a common pattern of the above-ground community in conventional systems ( Gabriel et al., 2006 ), and recently was reported for microbial communities as a response to long-term cultivation ( Montecchia et al., 2015 ). When poor agricultural practices are applied, such as uniformly crop monocultures, fertilization and intensive use of agrochemicals, the chain-reaction of (bio)diversity loss reduce the ecological niches leading to a homogenization of the microbial community and their functional gene pool, altering the ecosystem functioning and reducing the ecosystem resilience ( Olden et al., 2004 ; Constancias et al., 2014 ; Figuerola et al., 2015 ). We acknowledge that the plant species planted in conventional and organic systems between 2006 and 2013 were not the same. This might have some impact on rhizosphere microbial community due to the different exudates released by different plant species. However, in this study we have focused on bulk soils and not on rhizosphere microbiome. Besides the effects of farming systems on microbial community, we hypothesized that there is a legacy effects of the SHTs on diversity. It is expected that the differences between SHTs (e.g., organic matter composition, C/N, physical disturbances) may alter the physical, chemical and biological properties of the soil with consequent shifts in microbial diversity ( Jacquiod et al., 2013 ). However, this study does not support evidence for the occurrence of long-term effects of SHTs on microbial diversity and richness. The first possible explanation is that different SHTs affects microbial diversity only in short-term and this effect may not be observed 3 years after the last application of the different treatments in this study. Some studies suggest a strong and fast resilience of the microbial diversity after a pronounced disturbance on soil community caused by management practices ( Ding et al., 2014 ; Suleiman et al., 2016 ). Second, the continuous long-term farming system can counteract the effects of the soil health treatments, which were applied only twice. It has been suggested that long-term management practices are more likely to greatly influence the microbial community than temporal disturbances in soil ( Buckley and Schmidt, 2003 ). Finally, we believe that the legacy effect of the SHTs occurs in specific microbial groups and cannot be resolved by determining the diversity and heterogeneity of entire microbial community, because shifts in some groups might be compensated by shifts in others. It has been proposed that due to larger availability of organic carbon and nitrogen, organic system should favor copiotrophic bacteria, while oligotrophic should predominate in conventional systems, where the organic carbon quality is low ( Ding et al., 2014 ; Hartmann et al., 2015 ). In this study, we observed that the differences in the structure of microbial communities between conventional and organic farming systems were mainly related to a large fraction of habitat specialist OTUs broadly dispersed across the phylogenetic groups belonging to almost all phyla found in soil. Only few taxonomic groups revealed to respond more uniformly to farming system. For example, most of habitat specialists assigned to Proteobacteria and Euryarchaeota were associated with conventional system and an increase of members belonging to Acidobacteria and Planctomycetes was detected in organic system. These findings are not necessarily surprising, but are an opposite trend toward the copiotrophic-oligotrophic categories expected. However, the rather dispersed OTU association between conventional and organic systems within these taxonomic groups are in agreement with the contrasting behavior of individual members within phyla reported previously ( Rousk et al., 2010 ). Not all members in a taxonomic clade demonstrate the same ecological characteristics, implying that the general lifestyle classification might not be applied for all members in a phylum ( Navarrete et al., 2013 ), and responses to management will occur at lower taxonomic levels rather than at major groups. Proteobacteria have been suggested to be a primarily copiotrophic phylum in soil ( Li et al., 2012 ), while the lifestyle of microbial groups belonging to Euryarchaeota , which are predominately methanogens, are largely unknown ( Angel et al., 2012 ). However, the increased abundance of taxa belonging to these two Phyla in conventional farming system may be promoted by the input of fertilizers, which create copiotrophic environment in nutrient-rich microhabitats and stimulate plant growth, enhancing carbon availability and favoring the growth rate of members of these phyla. Members of Acidobacteria and Planctomyces have been suggested to be adapted to nutrient-poor soils, and the input of organic amendments is expected to inhibit their activity ( Buckley et al., 2006 ; Chaudhry et al., 2012 ). However, Acidobacteria and Planctomyces might be involved in the turnover of soil organic carbon and nutrient availability, pointing out that the manure addition in soil might promote the proliferation of these groups. Microbial communities proved to be sensitive to SHTs. This is an important finding because microbial taxa strongly associated with management practices may help to elucidate the process behind soil suppressiveness. In previous study in the same SHE ( Korthals et al., 2014 ), the SHTs were evaluated within conventional system on the potential effects on plant-parasitic nematode P. penetrans and soil-borne fungi V. dahliae . The combination, chitin, anaerobic soil disinfestation and marigold treatments were more effective in controlling P. penetrans and V. dahliae when compared with chemical control. In contrast, grass-clover, biofumigation, cultivit and compost were not effective alternatives. However, in that study, the bacterial community was not assessed. In this study, we revealed several taxa associated with SHTs distributed among major taxonomic groups, for which we have little or no information about their ecological roles. Therefore, we can only speculate the ecological importance of the detected taxa based on what has been described in previous studies and compare with findings on pathogen control ( Korthals et al., 2014 ). A complete description of the results is beyond the scope of this study and we only focus on some consistent findings and their potential as soil microbe indicators for sustainable practices. In anaerobic soil disinfestation treatment most of habitat specific OTUs were represented by taxa belonging to Bacillales and Clostridialles ( Firmicutes ), whose dominance is linked to their spore-forming capability, a competitive advantage under anaerobic conditions. Members belonging to family Bacillales have been described to be responsible for suppression of soil-borne disease-causing fungi ( Verticillium, Rhizoctonia and Fusarium ) and plant-parasitic nematodes ( Meloidogyne and Pratylenchus ) through production of antimicrobial compound and pore-forming toxins (crystal proteins) ( Wei et al., 2003 ). Thus, this treatment selected Firmicutes taxa that might be involved in suppression of fungi and nematodes. In addition, habitat specific OTUs belonging to phylum Nitrospira , nitrite-oxidizing bacteria, were also associated with this treatment. This may be an indication of previous accumulation of ammonia (NH 3 ) and production of nitrite (NO 2 ), both nitrogenous compounds released due to decomposing of organic material known to play an important role in the suppression of fungi and nematodes ( Tenuta and Lazarovits, 2002 ; Oka, 2010 ). The genus Lysobacter , chitinolytic bacteria, was found to be associated with chitin treatment and have been described to have an important role in soil suppressiveness, with a potential antagonistic property against Rhizoctonia and nematodes plant pathogens ( Tian et al., 2007 ; Postma et al., 2008 ). The genus Virgibacillus , another chitinolytic bacteria ( Cretoiu et al., 2014 ), was also found to be associated with chitin treatment, but its role in soil suppressiveness is not described yet. Chitin is a major component of nematode egg shells and cell wall of most plant-pathogenic fungi, and it is assumed that chitin amendments increase the number of chitinolytic microorganisms and chitinase activity, which in turn suppress nematodes and fungi. Members of Flavobacteriales and Chitinophagaceae associated with marigold may also suppress soil nematodes by their chitinase activity ( Glavina et al., 2010 ; Kharade and McBride, 2014 ), suggesting that besides its ability to produce nematicidal compounds, marigold can also recruit nematode-antagonistic microorganisms ( Hooks et al., 2010 ). The potential plant pathogens antagonists Pasteuria, Pseudomonas and Burkholderiales were associated with cultivit and grass-clover treatments. Bacterial taxa belonging to these groups have been described to be potential against plant-parasitic nematodes and fungi ( Tian et al., 2007 ). However, our results suggest that multiple mechanisms may accounted for an effective soil suppressiveness and the simple presence of taxa with antagonistic behavior against plant pathogens is not a sufficient proof for successful suppression of a pathogen in soil ( Weller et al., 2002 ). Thereafter, the alternative methods to control plant pathogens require more detailed studies in combination with molecular and traditional approaches used in plant pathology and microbiology. Altogether our results indicate that conventional and organic farming systems had a major influence on soil diversity and composition of microbial communities while the effects of the SHTs were of smaller magnitude. Organic farming system promoted beneficial effects on biotic aspects regarding to microbial diversities, richness and community heterogeneity. However, the response of microbial community to farming systems is diverse and complex, and simple conclusions like “organic systems increased the soil biodiversity” may not be directly synonymous with concomitant increase in soil health and plant productivity. Furthermore, impact of the diversity losses in conventional system is not yet known; it is not clear how microbial diversity is related to ecosystem function and whether the changes in diversity we observed are reversible and the long-term consequences remain to be unexplored. Moreover, we detected that there is a legacy of the SHT which selects for treatment-specific microbial members that are consistent with the existing knowledge, but the limited phylogenetic and functional information precludes more definite conclusions about the beneficial impact of individual taxonomic groups with soil suppressiveness. However, the observed shifts in microbial diversity, community structure and individual taxon bring novel insights into the potential of managing the microbial community for sustainable agricultural productivity."
} | 5,842 |
25890272 | PMC4358701 | pmc | 5,808 | {
"abstract": "Background Arginine is a high-value product, especially for the pharmaceutical industry. Growing demand for environmental-friendly and traceable products have stressed the need for microbial production of this amino acid. Therefore, the aim of this study was to improve arginine production in Escherichia coli by metabolic engineering and to establish a fermentation process in 1-L bioreactor scale to evaluate the different mutants. Results Firstly, argR (encoding an arginine responsive repressor protein), speC , speF (encoding ornithine decarboxylases) and adiA (encoding an arginine decarboxylase) were knocked out and the feedback-resistant argA214 or argA215 were introduced into the strain. Three glutamate independent mutants were assessed in bioreactors. Unlike the parent strain, which did not excrete any arginine during glucose fermentation, the constructs produced between 1.94 and 3.03 g/L arginine. Next, wild type argA was deleted and the gene copy number of argA214 was raised, resulting in a slight increase in arginine production (4.11 g/L) but causing most of the carbon flow to be redirected toward acetate. The V216A mutation in argP (transcriptional regulator of argO , which encodes for an arginine exporter) was identified as a potential candidate for improved arginine production. The combination of multicopy of argP216 or argO and argA214 led to nearly 2-fold and 3-fold increase in arginine production, respectively, and a reduction of acetate formation. Conclusions In this study, E. coli was successfully engineered for enhanced arginine production. The ∆adiA , ∆speC , ∆speF , ∆argR , ∆argA mutant with high gene copy number of argA214 and argO produced 11.64 g/L of arginine in batch fermentation, thereby demonstrating the potential of E. coli as an industrial producer of arginine.",
"conclusion": "Conclusion We reported the development E. coli strains overproducing arginine, by targeting genes regulating repression of arginine biosynthesis and competing degradation pathways in addition to amplification of genes for N-acetylglutamate formation and arginine export. The two final strains obtained (SJB009 and SJB010) had the highest arginine yield (1.18 and 0.44 g arg/g glc, respectively) and productivity (0.24 and 0.29 g arg/L/h, respectively) and will be used for further genetic improvement and/or process optimization. The fermentation process developed for the comparison of the different constructs needs to be further optimized regarding fermentation medium, process conditions and process control.",
"introduction": "Effect of introduction of feedback resistant variants of argA and selection of glutamate producing strains In the first rate-limiting step of the arginine synthesis, NAGS, encoded by argA , catalyzes the acetylation of glutamate. To block the feedback inhibition of NAGS the plasmids pKH15 and pKH19, derived from the ASKA- plasmid pCA24N, were transferred into C600 + Δ4 (see Table 1 ) to over-express the feedback resistant variants of argA (H15Y for argA214 and Y19C for argA215 ) under the control of an IPTG-inducible promoter [ 25 ] (Table 1 ). The strain C600 + Δ4 carrying either plasmid pKH15 or pKH19 could not be grown on M9 minimal media containing IPTG without exogenous glutamate supplementation and only weak growth was observed on the same medium when both glutamate and IPTG were absent. This suggested that over-expression of the feedback resistant argA in this strain resulted in glutamate starvation. To overcome this limitation, spontaneous mutants able to grow in the absence of glutamate were selected by plating washed and diluted cell cultures on M9 medium supplemented with IPTG without glutamate. Twelve colonies were picked at random and screened for arginine production based on the bioassay method. The three clones with the highest arginine production were chosen for subsequent work (SJB001, 003 and 004). Table 1 \n Plasmids and strains used in this study \n \n Plasmid/Strain \n \n Relevant characteristics/genotype \n \n Source/Reference \n \n Plasmids \n pKH15 pCA24N (clone JW2786), argA214 \n ASKA- collection [ 26 ], this work pKH19 pCA24N (clone JW2786), argA215 \n ASKA- collection [ 26 ], this work pTrc99a Amp-R, lacIq \n Lab stock pTrcArgP216 pTrc99a with a mutant argP216 allele This work pJB044 pBR322 derived, infA, rop- \n Lab stock [ 27 ] pJB044argA15 pJB044 with argA214 downstream of infA \n This work pJB044p1argA15 Same as pJB044argA15 but with rrsBp1 \n This work pArgObla Arginine bio-sensor plasmid with bla (Amp-R) under transcriptional control of argOp \n This work pArgObla10C Arginine bio-sensor plasmid with bla (Amp-R) under transcriptional control of argOp with mutation in RBS This work pTrcArgP216 pTrc99a with argP216 cloned under transcriptional control of trc promoter This work pJB044argAO pJB044argA15 with argO cloned downstream of argA214 \n This work pJB044argAP pJB044argA15 with argp216 cloned downstream of argA214 \n This work \n Strains \n \n E. coli K-12 C600 \n thr-,1 leuB6, thi-1, lacY1, glnV44, supE44, rfbD1, mcrA1 \n Lab stock [ 28 ] MG1655 \n ilvG -, rfb -50, rph -1 Lab stock C600 + \n Same as C600 but thr \n + \n , leu \n + \n This work C600 + ∆4 Same as C600 + but ∆adiA, ∆speC, ∆speF, ∆argR \n This work pTrcArgP216/C600 + ∆4 C600 + ∆4 with a mutant argP216 allele This work JW3932 Auxotrophic for arginine ∆argH \n [ 29 ] SJB001 Glutamate independent mutant of C600 + ∆4 with pKH15 (clone 2) This work SJB003 Glutamate independent mutant of C600 + ∆4 with pKH19 (clone 2) This work SJB004 Glutamate independent mutant of C600 + ∆4 with pKH19 (clone 4) This work SJB003A SJB003 but no plasmid This work SJB005 SJB003A but ∆argA \n This work SJB015 SJB005 with pJB044argA15 This work SJB025 SJB005 with pJB044p1argA15 This work SJB006 Arginine producing mutant of C600 + ∆4 from biosensor selection, argP216 \n This work SJB007 Derivative of SJB006 from second round biosensor selection This work SJB009 SJB005 with pJB044argAO This work SJB010 SJB005 with pJB044argAP This work Although these three strains were constructed in the same way, fermentations revealed very different growth behavior and arginine production abilities (Table 2 ). Indeed, SJB003 produced more arginine, with a productivity of 0.14 g/L/h and a final arginine concentration (3.03 g/L) significantly higher than that of the other similar mutants. The higher arginine producing capability of SJB003 compared to that of SJB001and 004 indicates that this strain had acquired beneficial mutations during growth under glutamate limitation. SJB003 was therefore chosen as a chassis for further genetic manipulation, although its beneficial mutations were not characterized. Table 2 \n Comparison of the performances of the different \n E. coli \n strains for arginine production by fermentation \n \n E. coli \n strain \n \n Yields \n \n μ (1/h) \n \n Y \n X/S \n (g dcw/g glc) \n \n Y \n P/S \n (g arg/g glc) \n \n Y \n P/X \n (g arg/g dcw) \n \n Q \n P \n (g/L/h) \n \n Arginine (g/L) \n \n Acetic acid (g/L) \n \n Ac/Arg (mol ac/mol arg) \n SJB001 0.14 ± 0.02 0.26 ± 0.02 0.03 ± 0.00 0.10 ± 0.00 0.08 ± 0.00 1.94 ± 0.12 5.57 ± 0.11 8.5 ± 0.36 SJB003 0.14 ± 0.00 0.27 ± 0.01 0.04 ± 0.01 0.15 ± 0.03 0.14 ± 0.02 3.03 ± 0.59 6.12 ± 1.14 6.43 ± 2.36 SJB004 0.13 ± 0.02 0.27 ± 0.01 0.03 ± 0.00 0.11 ± 0.00 0.09 ± 0.00 2.04 ± 0.00 6.15 ± 0.24 8.90 ± 0.36 SJB015 0.04 ± 0.00 0.11 ± 0.00 0.07 ± 0.00 0.50 ± 0.02 0.08 ± 0.00 4.11 ± 0.49 15.85 ± 1.60 11.37 ± 0.22 SJB006 0.17 ± 0.01 0.35 ± 0.01 0.03 ± 0.00 0.09 ± 0.00 0.11 ± 0.00 2.03 ± 0.05 6.24 ± 0.45 9.07 ± 0.42 SJB007 0.16 ± 0.02 0.36 ± 0.01 0.04 ± 0.00 0.11 ± 0.00 0.14 ± 0.00 2.74 ± 0.21 5.31 ± 1.73 5.90 ± 2.31 SJB009 0.04 ± 0.00 0.12 ± 0.01 0.17 ± 0.01 1.18 ± 0.01 0.24 ± 0.01 11.64 ± 0.75 14.56 ± 0.93 3.72 ± 0.48 SJB010 0.09 ± 0.00 0.25 ± 0.01 0.11 ± 0.00 0.44 ± 0.02 0.29 ± 0.01 7.95 ± 0.04 3.14 ± 0.87 1.17 ± 0.33 Aerobic batch fermentations were performed in 1 L bioreactors at 32°C and pH 7; the initial glucose concentration was 70 g/L. μ, growth rate; Y X/S , biomass yield vs. glucose; Y P/S , product yield vs. glucose; Y P/X , product yield vs. cell mass; Q P , volumetric productivity. Results are given as means ± standard deviations. Control fermentations with the parent strain C600 + were also performed. This strain did not yield any arginine (data not shown), which confirmed that the arginine productions displayed by the other strains are the result of their genetic modifications.",
"discussion": "Results & discussion Effect of introduction of feedback resistant variants of argA and selection of glutamate producing strains In the first rate-limiting step of the arginine synthesis, NAGS, encoded by argA , catalyzes the acetylation of glutamate. To block the feedback inhibition of NAGS the plasmids pKH15 and pKH19, derived from the ASKA- plasmid pCA24N, were transferred into C600 + Δ4 (see Table 1 ) to over-express the feedback resistant variants of argA (H15Y for argA214 and Y19C for argA215 ) under the control of an IPTG-inducible promoter [ 25 ] (Table 1 ). The strain C600 + Δ4 carrying either plasmid pKH15 or pKH19 could not be grown on M9 minimal media containing IPTG without exogenous glutamate supplementation and only weak growth was observed on the same medium when both glutamate and IPTG were absent. This suggested that over-expression of the feedback resistant argA in this strain resulted in glutamate starvation. To overcome this limitation, spontaneous mutants able to grow in the absence of glutamate were selected by plating washed and diluted cell cultures on M9 medium supplemented with IPTG without glutamate. Twelve colonies were picked at random and screened for arginine production based on the bioassay method. The three clones with the highest arginine production were chosen for subsequent work (SJB001, 003 and 004). Table 1 \n Plasmids and strains used in this study \n \n Plasmid/Strain \n \n Relevant characteristics/genotype \n \n Source/Reference \n \n Plasmids \n pKH15 pCA24N (clone JW2786), argA214 \n ASKA- collection [ 26 ], this work pKH19 pCA24N (clone JW2786), argA215 \n ASKA- collection [ 26 ], this work pTrc99a Amp-R, lacIq \n Lab stock pTrcArgP216 pTrc99a with a mutant argP216 allele This work pJB044 pBR322 derived, infA, rop- \n Lab stock [ 27 ] pJB044argA15 pJB044 with argA214 downstream of infA \n This work pJB044p1argA15 Same as pJB044argA15 but with rrsBp1 \n This work pArgObla Arginine bio-sensor plasmid with bla (Amp-R) under transcriptional control of argOp \n This work pArgObla10C Arginine bio-sensor plasmid with bla (Amp-R) under transcriptional control of argOp with mutation in RBS This work pTrcArgP216 pTrc99a with argP216 cloned under transcriptional control of trc promoter This work pJB044argAO pJB044argA15 with argO cloned downstream of argA214 \n This work pJB044argAP pJB044argA15 with argp216 cloned downstream of argA214 \n This work \n Strains \n \n E. coli K-12 C600 \n thr-,1 leuB6, thi-1, lacY1, glnV44, supE44, rfbD1, mcrA1 \n Lab stock [ 28 ] MG1655 \n ilvG -, rfb -50, rph -1 Lab stock C600 + \n Same as C600 but thr \n + \n , leu \n + \n This work C600 + ∆4 Same as C600 + but ∆adiA, ∆speC, ∆speF, ∆argR \n This work pTrcArgP216/C600 + ∆4 C600 + ∆4 with a mutant argP216 allele This work JW3932 Auxotrophic for arginine ∆argH \n [ 29 ] SJB001 Glutamate independent mutant of C600 + ∆4 with pKH15 (clone 2) This work SJB003 Glutamate independent mutant of C600 + ∆4 with pKH19 (clone 2) This work SJB004 Glutamate independent mutant of C600 + ∆4 with pKH19 (clone 4) This work SJB003A SJB003 but no plasmid This work SJB005 SJB003A but ∆argA \n This work SJB015 SJB005 with pJB044argA15 This work SJB025 SJB005 with pJB044p1argA15 This work SJB006 Arginine producing mutant of C600 + ∆4 from biosensor selection, argP216 \n This work SJB007 Derivative of SJB006 from second round biosensor selection This work SJB009 SJB005 with pJB044argAO This work SJB010 SJB005 with pJB044argAP This work Although these three strains were constructed in the same way, fermentations revealed very different growth behavior and arginine production abilities (Table 2 ). Indeed, SJB003 produced more arginine, with a productivity of 0.14 g/L/h and a final arginine concentration (3.03 g/L) significantly higher than that of the other similar mutants. The higher arginine producing capability of SJB003 compared to that of SJB001and 004 indicates that this strain had acquired beneficial mutations during growth under glutamate limitation. SJB003 was therefore chosen as a chassis for further genetic manipulation, although its beneficial mutations were not characterized. Table 2 \n Comparison of the performances of the different \n E. coli \n strains for arginine production by fermentation \n \n E. coli \n strain \n \n Yields \n \n μ (1/h) \n \n Y \n X/S \n (g dcw/g glc) \n \n Y \n P/S \n (g arg/g glc) \n \n Y \n P/X \n (g arg/g dcw) \n \n Q \n P \n (g/L/h) \n \n Arginine (g/L) \n \n Acetic acid (g/L) \n \n Ac/Arg (mol ac/mol arg) \n SJB001 0.14 ± 0.02 0.26 ± 0.02 0.03 ± 0.00 0.10 ± 0.00 0.08 ± 0.00 1.94 ± 0.12 5.57 ± 0.11 8.5 ± 0.36 SJB003 0.14 ± 0.00 0.27 ± 0.01 0.04 ± 0.01 0.15 ± 0.03 0.14 ± 0.02 3.03 ± 0.59 6.12 ± 1.14 6.43 ± 2.36 SJB004 0.13 ± 0.02 0.27 ± 0.01 0.03 ± 0.00 0.11 ± 0.00 0.09 ± 0.00 2.04 ± 0.00 6.15 ± 0.24 8.90 ± 0.36 SJB015 0.04 ± 0.00 0.11 ± 0.00 0.07 ± 0.00 0.50 ± 0.02 0.08 ± 0.00 4.11 ± 0.49 15.85 ± 1.60 11.37 ± 0.22 SJB006 0.17 ± 0.01 0.35 ± 0.01 0.03 ± 0.00 0.09 ± 0.00 0.11 ± 0.00 2.03 ± 0.05 6.24 ± 0.45 9.07 ± 0.42 SJB007 0.16 ± 0.02 0.36 ± 0.01 0.04 ± 0.00 0.11 ± 0.00 0.14 ± 0.00 2.74 ± 0.21 5.31 ± 1.73 5.90 ± 2.31 SJB009 0.04 ± 0.00 0.12 ± 0.01 0.17 ± 0.01 1.18 ± 0.01 0.24 ± 0.01 11.64 ± 0.75 14.56 ± 0.93 3.72 ± 0.48 SJB010 0.09 ± 0.00 0.25 ± 0.01 0.11 ± 0.00 0.44 ± 0.02 0.29 ± 0.01 7.95 ± 0.04 3.14 ± 0.87 1.17 ± 0.33 Aerobic batch fermentations were performed in 1 L bioreactors at 32°C and pH 7; the initial glucose concentration was 70 g/L. μ, growth rate; Y X/S , biomass yield vs. glucose; Y P/S , product yield vs. glucose; Y P/X , product yield vs. cell mass; Q P , volumetric productivity. Results are given as means ± standard deviations. Control fermentations with the parent strain C600 + were also performed. This strain did not yield any arginine (data not shown), which confirmed that the arginine productions displayed by the other strains are the result of their genetic modifications. Effect of overexpression of a feedback resistant argA on arginine production To avoid the use of IPTG in an industrial process, it is of interest to place the feedback resistant argA gene under a constitutive promoter. First the SJB003 was cured of the pKH19 plasmid, harboring argA215 , by repeated streaking on Luria Agar (LA) medium without antibiotic, giving rise to SJB003A. The argA214 allele was chosen as the argA variant to be introduced in the strain since we found this allele to be slightly better for arginine productivity in preliminary shake flask experiments (data not shown). To avoid potential recombination with the new argA214 plasmid, the chromosomal copy of the wild type argA gene was deleted in the SJB003A strain, resulting in SJB005. The feedback resistant argA214 was cloned into a high copy number plasmid pJB044 downstream of the infA gene encoding the translation initiation factor IF1. pJB044 carries a tetracycline resistance gene that can be removed by homologous recombination due to the presence of direct repeats flanking the gene, as previously described [ 27 ]. The argA214 gene was placed downstream of the ribosome binding site (RBS) (AGGAGG) either with or without a strong constitutive rRNA promoter (rrsBp1) upstream (Table 1 ). The argA start codon GUG was changed by site-directed mutagenesis to the more efficient AUG codon in both constructs, termed pJB044argA15 and pJB044p1argA15. Strain SJB005 was the host of the pJB044 derived plasmids, resulting in the two IPTG-independent strains SJB0015 and SJB025 that differ only by the absence or presence of a strong rRNA promoter upstream of the argA214 gene respectively. When cultivated in bioreactors, SJB015 displayed a slightly improved arginine production compared to the previous constructs (Table 2 ). In particular, Y P/x was relatively high (0.50 g arg/g dcw). However, cell growth was seriously hampered for this strain and SJB015 had the lowest μ and Y X/S and the final cell density was lower than that of the other strains (data not shown). Consequently the volumetric productivity of SJB015 was relatively low (0.08 g/L/h). In addition, SJB015 produced high levels of acetate. When a DCW of about 7 g/L was reached (Figure 2 d), cell growth stopped, arginine production drastically decreased and the remaining sugar (approximately 30% of the initial glucose) was mainly used for acetate formation (up to 28 g/L). Figure 2 \n Fermentation profile of (a) SJB001, (b) SJB003, (c) SJB004, (d) SJB015, (e) SJB006, (f) SJB007, (g) SJB009 and (h) SJB010. ● glucose, ▲ acetic acid, ■ arginine and ◆ DCW. SJB025 exhibited slow growth on both rich (LA) and minimal medium (M9) with the appearance of some large colonies (data not shown). This suggested that the strong promoter driven argA214 was toxic for the strain, with large colonies representing revertants. 50 of these colonies were screened for fast growing mutants with enhanced ability to produce arginine. However, none had retained this capacity (ascertained by the bioassay method) and consequently, neither these clones nor the parental SJB025 were used for further work. For this strain it is likely that the rate of arginine formation exceeds the capacity of the arginine export system due to the overexpression of argA214 in combination with the presence of a strong promoter upstream of argA214 . The resulting accumulation of arginine inside the cell might have a variety of negative effects on cellular processes, which could explain why cell growth was seriously hampered in SJB025 and the ability for enhanced arginine production easily lost. This is consistent with previous reports, where mutants of C. glutamicum with deletion of lysE , encoding an exporter similar to ArgO [ 30 , 31 ] , were growth inhibited in the presence of intracellular arginine [ 30 - 32 ]. Growth arrest due to intracellular arginine in ∆argO and ∆argP mutants of E. coli has also been reported [ 14 ]. The increased nitrogen flow towards arginine production might also hinder the biosynthesis of other metabolites required for cell growth. Identification of novel mutations for enhanced arginine production using a biosensor To complement the above described rational strain improvement strategies, a selection procedure was employed to select novel or previously unidentified mutants with increased arginine production. C600 + Δ4 carrying the biosensor plasmid pArgObla10C was used for direct selection and screening of arginine accumulating mutants on M9 plates supplemented with 2, 3 and 4 mg/mL Amp. Only mutants with increased expression of the bla gene, most likely through increased transcription of the argOp promoter, can grow on media with Amp concentration higher than 0.6 mg/mL. High Amp resistant mutants were randomly chosen from each Amp concentration and assayed for arginine production using the bioassay method. The isolated mutant showing the highest production was cured of the plasmid by repeated streaking on M9 plates without antibiotic (resulting in SJB006). After the biosensor plasmid removal an improved arginine production was retained, indicating that the acquired increased arginine accumulation was due to chromosomal mutations. Chromosomal sequencing of argA and argP genes in this clone revealed wild type argA and a T647C mutation in argP resulting in a valine to alanine mutation in position 216 (V216A). To assess the effect of the V216A mutation on arginine production the mutant allele argP216 was cloned into a high copy number plasmid downstream of an IPTG inducible promoter (pTrc99a) to give pTrcArgP216. Even without IPTG induction, the arginine accumulation of the strain pTrcArgP216 / C600 + Δ4 was equivalent to SJB006, as based on the bioassay method (data not shown). We thus concluded that the increase in arginine accumulation observed in SJB006 was at least partly due to the presence of the argP216 allele. Selection of mutants with increased arginine accumulation was extended by transforming SJB006 with pArgObla10C anew, and screening on LA plates supplemented with 6, 8 and 10 mg/mL of Amp. Several colonies were assayed for arginine production; the best clone was cured of the biosensor plasmid and used for further work (SJB007). Sequencing of argA and argP showed that SJB007 also carried wild-type argA and no other mutation on the argP gene, other than the V216A mutation present in the parent strain SJB006. Even with only the wild type argA , SJB006 produced similar amounts of arginine as SJB001 and SJB004 during fermentation (Table 2 ). Further, the productivity of SJB006 (0.11 g/L/h) was even slightly higher due to a faster growth. SJB007, which results from a second level biosensor selection, displayed increased arginine production compare to its parent SJB006. This demonstrates the potential effects of the mutation V216A carried by these two strains on the argP gene, but also that there might be some additional unknown mutation in SJB007 promoting arginine production. Effect of co-overexpression of a feedback resistant argA and argP or argO on arginine production The mutant allele argP216 resulted in increased accumulation of arginine. Amongst other physiological functions in the cells, ArgP also controls the transcription of argO. It was therefore of interest to combine overexpression of each of these two genes with the known feedback resistant argA214 allele. The plasmid pJB044argAP was constructed such that the argP216 allele was placed downstream of argA214 , under the control of the RBS sequence AGGAGG. The plasmid pJB044argAO was constructed by placing an argO ORF with the RBS sequence AGGAGG, downstream of argA214 . In addition the inefficient start codon GUG was changed for the canonical AUG. The plasmids pJB044argAO and pJB044argAP were transferred to SJB005, to yield SBJ009 and SJB010 respectively. Slow growth was also observed in the strains having a gene involved in arginine transport overexpressed in combination with the argA214 allele (Figure 2 d, g and h). In particular, SJB009 had almost the same μ and Y X/S as SJB015 and also produced significant amounts of acetate. Nevertheless, cells grew to a somewhat higher density and arginine was steadily formed throughout the whole fermentation. Furthermore, SJB009 had the highest arginine production per amount of cells (1.18 g arg/g DCW), 2 to 13-fold that of the other strains. Consequently this strain yielded the highest final arginine titer (11.64 g/L) at a fair production rate (0.24 g/L/h). Also, a low growth associated with a high Y P/X means that a large part of the glucose is used for arginine formation. SJB009 therefore showed the highest Y P/S (0.17 g arg/g glc) of all strains evaluated. ArgO is directly responsible for the transport of arginine outside the cytoplasm and the high Y P/S might be the result of an immediate excretion, enhanced by ArgO, of the large amount of arginine produced, due to argA214 . Interestingly, despite overexpression of the argP gene, responsible for argO transcription, SJB010 had significantly lower product yields than SJB009, yet higher than the other mutants. However, the cells of this strain grew twice as fast as cells of SJB009 and therefore SJB010 had the highest productivity of all strains (0.29 g/L/h). Unlike SJB015 and SJB009, SJB010 did not form high levels of acetate but produced both acetate and succinate (4–5 g/L). SJB009 and SJB010 are similar to SJB015 except that one of their genes responsible of arginine export has been altered ( argO and argP , respectively). This resulted in an important increase in the final arginine concentration (+183% and +93%), productivity (+200% and +262%) and product yield Y P/S (+143% and +57%) for SJB009 and SJB010, respectively, compared to SJB015. This positive effect of argO and argP overexpression has previously been observed, showing that E. coli strains carrying multicopy yggA + ( argO ) and argP d (S94L mutation) had a greatly increased arginine production as determined from cross-feeding ability on agar plate [ 14 ]. Export has been identified as the rate-limiting step for the production of different amino acids when using C. glutamicum [ 33 - 35 ]. Similarly, it seems that the arginine export system plays a major role for the arginine production by E. coli . Formation of acetate during arginine fermentation All mutants produced acetate as the main by-product. Acetate is formed during the 5 th step of L-arginine biosynthesis from L-glutamate (Figure 1 ). However, for most strains the ratio of ac:arg produced was higher than 1:1 (Table 2 ), which means that acetate was also formed via another pathway. The accumulation of acetate by E. coli , even in aerobic environment, when growing under conditions of high glucose consumption is known as overflow metabolism. It occurs when the rate of glucose consumption is greater than the capacity of the cell to reoxidize the reduced equivalents, i.e. NAD(P)H, generated by glycolysis. Instead of entering the tricarboxylic acid (TCA) cycle, the carbon flux from acetyl-CoA is diverted to acetate, likely to prevent any further NAD(P)H accumulation as only ATP is formed during acetate formation while the TCA cycle generates several reducing equivalents [ 36 , 37 ]. As fermentations were run in batch mode with high initial glucose concentration (70 g/L) overflow metabolism is to be expected. The acetate production depends on the specific glucose uptake rate, with acetate formation occurring only after a certain threshold [ 36 ]. SJB001;3;4;6 and 7 indeed produced large amounts of acetate compared to arginine, which allowed them to have a high glucose uptake (0.44 to 0.54 mol glc/mol dcw/h) and a fast growth (μ > 0.13 h −1 ). SJB009 and SJB010 however had a considerably lower ac/arg ratio, i.e. 3.72 and 1.17 mol/mol, respectively, compared to at least 5.9 mol/mol for the other strains. The glucose uptake was also reduced (0.33 and 0.36 mol glc/mol dcw/h) as well as the growth (μ < 0.09 h −1 ). It is possible that the redirection of carbon and nitrogen toward arginine results in a shortage of other essential amino acids, thereby limiting the growth and the need for fast glucose assimilation. This could also be because the carbon flow from acetyl-CoA is forced toward arginine biosynthesis by the overexpressed argA214 , thereby limiting the formation of acetate from acetyl-CoA. However SJB015 had the highest ac/arg ratio (11.37 mol/mol) despite having a low specific glucose uptake (0.36 mol glc/mol dcw/h). This strain produced 15.85 g/L of acetate, which is comparable to the 14.56 g/L produced by SJB009. It is therefore likely that a large part of the acetate produced by SJB015 comes from the increased carbon flux through the arginine pathway, but that mainly acetate was excreted while arginine accumulated inside the cell due to the export limitation."
} | 6,842 |
35501839 | PMC9063184 | pmc | 5,809 | {
"abstract": "Background Electric energy is not collected and utilized in biobutanol fermentation. The reason is that the yields of electron shuttles and nanowires are not enough to gather and transfer all electrons to the electrode in liquid fermentation. However, the solid matrix of the adsorption carrier may be conducive to the collection and transfer of electrons because of its good adsorption and conductivity. Therefore, this first-attempt study coupled microbial fuel cell (MFC) with adsorption carrier solid-state fermentation (ACSF). In addition, the effect and mechanism of adsorption carrier solid-state fermentation on power generation were explored. Results The power generation performance and fermentation performance were improved by ACSF. The power density by polyurethane and carbon felt carrier solid-state fermentation (PC) was 12 times that by no carrier fermentation (NC). The biobutanol yield of absorbent cotton and carbon felt carrier solid-state fermentation (ACC) was increased by 36.86%. Moreover, the mechanism was explored via metabolic flux analysis, cyclic voltammetry and scanning electron microscopy. The results of metabolic flux analysis showed that more electrons were produced and more carbon flowed to biobutanol production. The cyclic voltammetry results revealed that more riboflavin was produced to enhance extracellular electron transport (EET) by ACSF. The scanning electron microscopy image showed that the adsorption capacity and aggregation degree of bacteria were increased on the electrode and nanowires were observed by ACSF. Conclusions A new fermentation mode was established by coupling MFC with ACSF to improve substrate utilization, which will provide crucial insights into the fermentation industry. In addition, the ACSF is an effective method to enhance power generation performance and fermentation performance. Graphical Abstract",
"conclusion": "Conclusions A coupling system of biobutanol production and MFC has been built in this study. Then, the MFC power generation performance and fermentation performance were evaluated by ACSF. The power density by ACSF was 12 times higher than that by NC. In addition, the biobutanol concentration was increased from 4.47 to 7.08 g/L by ACSF. Furthermore, the mechanism of ACSF intensification method was explored. The metabolic flux analysis revealed that more electrons were generated and more carbon sources flowed to biobutanol production by ACSF. The response current of ACSF was significantly enhanced at − 0.5 V due to the high riboflavin concentration. The SEM revealed that nanowires existed and that the bacteria density and aggregation degree were improved by ACSF.",
"discussion": "Results and discussion Electrical performance of MFCs in adsorption carrier solid-state fermentation Figure 1 a shows the output voltage of MFCs by NC, PC, ACC and cotton fiber and carbon felt (CFC) carrier solid-state fermentation. The output voltage of different fermentation processes had the same trend: updown–updown. Clostridium acetobutylicum was in the adjustment period during the first 6 h after inoculation. It synthesized a large number of substances to adapt to the new environment. Therefore, the output voltage raised rapidly. For example, the output voltage in CFC increased from 623 to 726 mV within 6 h. Then the output voltage reached a maximum at approximately 30 h. At this point, the output voltage in PC was 1.67 times that by NC. Finally, C. acetobutylicum entered the stability period and decline period. The output voltage decreased. Unlike normal bacteria, C. acetobutylicum had two peaks. This is because C. acetobutylicum has two physiological metabolic characteristics [ 5 , 15 ]. The first peak responds to the acid-producing period and the second to the solvent producing period. However, the average output voltages of MFCs by ACSF were all higher than those by NC. The average output voltages by PC, ACC and CFC were 825 mV, 793 mV and 773 mV, respectively. The NC average output voltage was only 452 mV. It is proved that ACSF can improve MFC output voltage. Fig. 1 MFC electrical performance in no carrier fermentation (NC), polyurethane and carbon felt (PC), absorbent cotton and carbon felt (ACC) and cotton fiber and carbon felt (CFC) carrier solid-state fermentation. a output voltage curves, b power density curves, and c polarization curves Figure 1 b shows the power density of MFCs by NC, PC, ACC and CFC. MFCs by PC, ACC and CFC reached higher values than MFC by NC. The highest maximum power density was obtained from the MFC by PC, showing a maximum power density of 231 mW/m 2 . This power density was 91.77% higher than that of MFC by NC. The polarization curve was a common method to measure the internal resistance of MFCs. As shown in Fig. 1 c, the internal resistance of MFCs by NC, PC, ACC and CFC were 326 Ω, 126 Ω, 134 Ω, 160 Ω, respectively. The internal resistance of MFC was closely related to the design of the battery. The ohmic resistance accounted for 83% of the total resistance in the two-chamber MFC. The ohmic resistance mainly comes from the barrier effect of the electrode, electrolyte and membrane on the electron conduction. The adsorption carrier in MFCs can reduce the conduction effect of electrolytes on electrons, thus reducing the internal resistance. Table 1 compares the performance of the dual chamber MFCs reported in the literature. These results indicated that the electrical performance of the MFCs by ACSF was appreciably improved by NC. Table 1 Comparison of the performance of dual chamber MFC reported in the literature with the present study Inoculum source Substrate Volume (L) Time (d) Anode Power density (mW/m 2 ) References Microalga Effluent water from chocolate factory 1 18 Graphite 105.84 [ 33 ] Shewanella haliotis and Aeromonas hydrophilia Luria–Bertani culture 0.2 0.5 Graphite 68.51 [ 34 ] S. oneidensis MR-1 and Rhodococcus sp o-xylene 0.06 8 Carbon brush 92.5 [ 35 ] Bacterial community within ceramic-based MFC fed with human urine Human urine 0.06 20 Carbon veil 36.66 [ 36 ] Anaerobic sludge Synthetic wastewater 0.03 1.6 Graphite felt 264.5 [ 37 ] C. Acetobutylicum Synthetic medium 0.02 2 Graphite 0.33 [ 9 ] C. Acetobutylicum Artificial wastewater 0.5 9 Carbon paper 3.36 [ 10 ] C. acetobutylicum Synthetic medium 0.2 2 Carbon felt 231 This study Fermentation performance of MFCs in adsorption carrier solid-state fermentation The effect of the open circuit, closed circuit and adsorption carrier on fermentation performance was studied. The product concentration is shown in Fig. 2 . The biobutanol yield in the closed circuit was 46.60% higher than that in the open circuit. The electric current can promote bacterial growth, ATP synthesis and protein expression [ 16 ]. Furthermore, the fermentation performance by NC and ACSF was compared. The solvent and biobutanol yield by ACSF was significantly higher than that by NC, and the acid yield was lower than that by NC. The solvent and biobutanol yield of ACC were 11.61 g/L and 7.08 g/L, respectively, which increased by 36.77% and 36.86% compared with NC. The carriers increase the specific surface area, provide more attachment points, and then improve the yield of butanol, which is consistent with the literature report [ 17 ]. In addition, the reason may be that the reabsorption capacity of organic acids was enhanced and the harmful substances around C. acetobutylicum were reduced with adsorption carriers. In addition, C. acetobutylicum can interact with the adsorption carrier to form a cross-linked structure to promote the formation of the cell membrane, improving the tolerance of the strain [ 18 ]. These results showed that the closed circuit and adsorption carrier had a positive effect on fermentation performance. Fig. 2 Solvents and acids concentration of liquid fermentation (LF), no carrier fermentation (NC), polyurethane and carbon felt (PC), absorbent cotton and carbon felt (ACC) and cotton fiber and carbon felt (CFC) carrier solid-state fermentation in MFC Metabolic flux analysis of C. acetobutylicum in adsorption carrier solid-state fermentation The metabolic activity of microorganisms determined the flow of electrons and protons, which affected the performance of electrical MFCs [ 19 ]. Figure 3 shows the metabolic flux distribution of C. acetobutylicum in different fermentation methods. As can be seen, the intracellular absorption and utilization rate of glucose was faster by ACSF. This indicated that the metabolic activity of C. acetobutylicum was faster by ACSF than that by NC. Then, glucose generates pyruvate through glycolysis, which is further degraded to acetyl coenzyme A and hydrogen. The hydrogen produced by ACSF is more than that by NC. The more release of hydrogen also indicates that more electrons are generated in the cell [ 19 ]. The part of the electricity in MFC was stored in acids and alcohols and other parts were used to form electric current. Electrons were mainly generated by the oxidation of glucose to carbon dioxide in anaerobic fermentation. Specifically, 1 mol CO 2 was accompanied by 4 mol electrons. The electron yield was calculated by metabolic flux distribution. The electron production was mainly concentrated in the reaction of acetyl coenzyme A, acetone and acetoin. The electron yields by PC, ACC and CFC were 218.07, 217.83 and 210.44 mmol, respectively. The electron yield by PC was 15.54% higher than that by NC. More electrons released improved NADH yield, which further improved biobutanol yield [ 20 ]. The ACSF flux maps were obviously different from NC. Most notably, electrons were generated by the absorption of organic acids. The rate of organic acids is faster by ACSF. For example, the rate of acetic acid absorption in ACA was 1.82 times that by NC. In addition, 100% pyruvate was used to generate electrons by ACSF, while 0.52% pyruvate was used to produce lactate without electrons. These results indicated that more electrons were generated and more carbon was used for biobutanol production by ACSF. Fig. 3 Distribution of metabolism flux by no carrier fermentation (NC), polyurethane and carbon felt (PC), absorbent cotton and carbon felt (ACC) and cotton fiber and carbon felt (CFC) carrier solid-state fermentation. Green: NC, red: ACSF Cyclic voltammetry analysis of MFCs in adsorption carrier solid-state fermentation The CV was performed to investigate the EET between bacteria and anode in the MFC, as shown in Fig. 4 . Three oxidation peaks of − 0.501 V, 0.922 V, − 0.006 V and two reduction peaks of 0.492 V, − 0.321 V were observed on the CV of PC. However, there was one oxidation peak and two reduction peaks. Since the redox peak number was proportional to the electron shuttles type [ 21 ], the CV results suggested that PC had more kinds of electron shuttles compared to the NC. It is greatly favorable to improve EET performance. The most obvious difference between ACSF and NC was that ACSF had a pair of obvious redox peaks at approximately − 0.5 V, but NC did not. The larger the peak current density of the positive response is, the better the electrical performance of the MCF [ 22 ]. The peak current densities of PC, ACC and CFC were 125.08 mA, 48.86 mA and 14.03 mA, respectively, the trend of which was consistent with the output voltage. This indicated that the EET mediated by the electron shuttle played an important role at approximately − 0.5 V. It has reported that − 0.5 V is a typical redox peak of riboflavin [ 23 ]. In addition, it has been reported that C. acetobutyricum can enhance the EET rate by increasing the secretion of riboflavin [ 24 ]. This indicated that the presence of free riboflavin by ACSF and ACSF can promote C. acetobutylicum to secrete riboflavin [ 23 ]. Riboflavin can promote EET in different ways according to different electron acceptors [ 25 ]. On the one hand, riboflavin can act as an electron shuttle to directly mediate electron transfer. On the other hand, riboflavin can act as a cofactor to improve the EET rate of other electron shuttles [ 26 ]. Therefore, Riboflavin concentration was detected. The riboflavin concentration of ACSF was almost three times that of NC. Surprisingly, the riboflavin concentrations of PC, ACC and CFC were almost the same and inversely proportional to the peak current density. This is likely because the concentration of riboflavin detected included not only extracellular free riboflavin, but also intracellular riboflavin. In addition, riboflavin can regulate the thickness of the cell membrane and exit in the cell membrane. [ 27 ]. For gram-positive bacteria, the thickening of cell membrane will hinder electron transfer [ 28 ]. In addition, a special riboflavin electron transport mechanism may be in C. acetobutyricum [ 24 ]. This needs further study in the future. Fig. 4 Cyclic voltammetry curves of MFCs and riboflavin concentration by no carrier fermentation (NC), polyurethane and carbon felt (PC), absorbent cotton and carbon felt (ACC) and cotton fiber and carbon felt (CFC) carrier solid-state fermentation Anode morphology of MFCs in adsorption carrier solid-state fermentation The microstructure of the anode carbon felt is shown in Fig. 5 . The C. acetobutylicum and metabolites were distributed in fiber surface and space between fibers after fermentation. The number of bacteria by ACSF was more than that by NC. Especially on the anode of PC, the number of bacteria was not only the largest, but also the aggregation degree was highest. Thus, the distance of electron transfer was shortened and EET efficiency was improved. The single fiber was enlarged to study the adsorption of bacteria on fiber. It can be seen from Fig. 6 e that the bacteria were closely linked with carbon fiber, and the electrons were produced and transferred directly to the anode. Some bacteria gathered together to form colony and adsorbed on the electrode of PC. The distance between cells was shortened and increased signal transfer. Furthermore, the colony on the fiber was enlarged to study the relationship between the bacteria. It can be observed that the cells were fusiform. In addition, there are filamentous substances between them, which were nanowires. It twined around the outer surface of the cell and connected with other cells. Therefore, it can transfer the electron between the cells and between cell and medium [ 29 ]. It also indirectly proved that C. acetobutylicum can generate nanowires and transfer the electrons by it. Fig. 5 SEM image of carbon felt anode by no carrier fermentation (NC), polyurethane and carbon felt (PC), absorbent cotton and carbon felt (ACC) and cotton fiber and carbon felt (CFC) carrier solid-state fermentation. a , e , f NC, b CFC, c ACC, d PC Fig. 6 Device photograph of coupling MFC with adsorption carrier solid-state fermentation"
} | 3,713 |
36841903 | PMC10119272 | pmc | 5,810 | {
"abstract": "Characterizing ancient clades of fungal symbionts is necessary for understanding the evolutionary process underlying symbiosis development. In this study, we investigated a distinct subgeneric taxon of Xylaria ( Xylariaceae ), named Pseudoxylaria , whose members have solely been isolated from the fungus garden of farming termites. Pseudoxylaria are inconspicuously present in active fungus gardens of termite colonies and only emerge in the form of vegetative stromata, when the fungus comb is no longer attended (“sit and wait” strategy). Insights into the genomic and metabolic consequences of their association, however, have remained sparse. Capitalizing on viable Pseudoxylaria cultures from different termite colonies, we obtained genomes of seven and transcriptomes of two Pseudoxylaria isolates. Using a whole-genome-based comparison with free-living members of the genus Xylaria, we document that the association has been accompanied by significant reductions in genome size, protein-coding gene content, and reduced functional capacities related to oxidative lignin degradation, oxidative stress responses and secondary metabolite production. Functional studies based on growth assays and fungus-fungus co-cultivations, coupled with isotope fractionation analysis, showed that Pseudoxylaria only moderately antagonizes growth of the termite food fungus Termitomyces , and instead extracts nutrients from the food fungus biomass for its own growth. We also uncovered that Pseudoxylaria is still capable of producing structurally unique metabolites, which was exemplified by the isolation of two novel metabolites, and that the natural product repertoire correlated with antimicrobial and insect antifeedant activity.",
"conclusion": "Conclusion Symbioses of fungi and social insects have independently evolved multiple times in ants, termites, beetles [ 3 ], and bees [ 46 ]. While genome reduction, and concomitant gene loss are commonly observed alongside with increased specialization and interdependencies in intracellular symbiotic bacteria during their transition to obligate symbiosis, examples of features that define fungal symbiotic interdependencies are sparse [ 47 ]. Characterizing features accompanying the evolution of symbiotic fungi is critical to understand symbiotic adaptations and the diversity of life across kingdoms. Capitalizing on the availability of viable cultures from South African termite colonies, we tested if Pseudoxylaria shows features of a termite and comb-associated lifestyle on genomic, transcriptomic and metabolomic levels. In this study, we uncovered genomic evidence for a certain degree of substrate specialization in Pseudoxylaria isolates compared to free-living isolates. Similar to termite-associated clades of the fungal genus Podaxis [ 48 ] and fungal symbiont of attine ants [ 49 ], comparative genome analysis revealed reduced sizes and coding capacities, with a reduced enzymatic capacity to oxidatively degrade recalcitrant plant polymers in all Pseudoxylaria genomes. Although stochastical losses of biosynthetic traits during evolution cannot be excluded, the depletion in specific traits related to saprophytic life styles has likely been driven by a relaxed selection due to the more benign and constant growth conditions (fungus comb) and the availability of fungus-derived carbohydrate and protein-rich biomass. Based on these findings, and analogous to reports from other obligate fungal symbionts of insects [ 3 , 48 ], we conclude that Pseudoxylaria is likely an obligate symbiont adapted to the fungus comb environment of farming termites. While the association of Pseudoxylaria with termites might have provided several fitness benefits to the fungus (presence of a carbon/nitrogen-rich comb substrate, protection from UV radiation by the termite mound, presence of ambient temperatures and humidity), termite-associated strains also face biotic stressors within the comb environment, such as termite weeding, co-occurring bacterial communities, and competition from and natural products produced by the fungal cultivar Termitomyces . We hypothesized that Pseudoxylaria adapted to such comb-specific stressors by having a reduced but specialized secondary metabolome to reduce triggers that could stimulate alarm responses of the fungal mutualist and termites [ 50 ], and the need for specific defense and communication mechanisms to survive. Comparative genome analysis supported the former of these hypotheses as a unique but reduced repertoire of BGCs was present in Pseudoxylaria genomes with a notable reduction in TPSs. In contrast, the co-occurring fungal mutualist Termitomyces has been found to encode above average numbers of TPSs in previous studies, which correlated with the emission of a bouquet of volatile terpenoid products proposed to play roles in the fungal life cycle by exhibiting insect attractant as well as repellant features [ 51 ]. These findings aligned with previous reports on behavioral studies showing that worker castes of O. obesus were able to differentiate between their mutualistic crop fungus Termitomyces and vegetative mycelium of Pseudoxylaria by their volatilome [ 50 ]. Our metabolic analysis demonstrated that Pseudoxylaria also secretes diffusible bioactive and structurally unique natural products as exemplified by the isolation of two novel metabolites [ 43 ]. While neither these or previously identified metabolites had strong antifungal activity, the production of antimicrobial mixtures could still represent a potential benefit in the competition against the co-occurring microbiota and the fungal mutualists to obtain nutrients. In nature, Pseudoxylaria only emerges from weakened or abandoned comb, where the fungus overgrowths the deteriorating fungus garden. We investigated this phenotypic appearance and documented that Pseudoxylaria exhibited not only moderate antagonistic behavior against the termite mutualist Termitoymces without instantaneously killing the fungus (reduced antagonism), but showed signs of fungal biomass conversion. The hypothesis that Pseudoxylaria might harvest nutrients from vegetative Termitomyces mycelium was supported by comparative genome and RNAseq analyses as well as isotope fractionation results. Overall, this study also provides a good starting point to address several unanswered questions as it still remains puzzling how and when Pseudoxylaria enters and remains within the fungus comb, why stromata emerge in the absence of termites (“sit and wait strategy”), and what triggers are required to stimulate germination and growth. This study should also encourage scientists to intensify sampling and sequencing studies on these and other fungal genera to enable broader phylogenomic studies that can address factors driving the evolution of insect-associated fungi in general and termite-associated strains specifically.",
"introduction": "Introduction The Macrotermitinae are the only termite lineage to have acquired fungal symbionts from the genus Termitomyces (family Lyophyllaceae) as their food source [ 1 – 4 ]. Termitomyces is cultivated by workers in cork-like structures termed “fungus combs”, which are maintained in chambers located within the subterranean colony and are also known as “fungal gardens” [ 3 ]. To propagate the food fungus, younger workers ingest plant material alongside with Termitomyces biomass and use excreted lignocellulose and spore-enriched feces to craft new fungus comb on which Termitomyces is able to thrive (Fig. 1A ). Termites have several levels of defense measures to protect this obligate nutritional symbiosis, starting with lower individual levels of hygiene measures to a higher collective level, also called social immunity [ 5 – 8 ]. Despite these preventive measures, fungal gardens inconspicuously host members of a distinct fungal subgenus of Xylaria (Ascomycota: Xylariaceae), commonly referred to as termite-associated Pseudoxylaria [ 9 – 15 ], which only emerge as vegetative stromata from comb material of deteriorating or inactive termite nests (Fig. 1B ) [ 16 ]. While a number of studies have provided insights into their co-evolutionary relation with the fungus-farming termite symbiosis, the ecological role of Pseudoxylaria remains debated [ 1 , 7 ]. Although few reports suggested a commensal role supporting biomass degradation within the comb environment [ 10 , 17 ], other studies analyzing Termitomyces - Xylaria co-cultures hinted towards an antagonistic relation. As free-living Xylaria strains inhibited growth of Termitomyces more intensly than their termite-associated relatives [ 7 , 18 , 19 ], it was postulated that reduced antagonistic behavior might enable Pseudo x ylaria to evade the defense mechanisms of a healthy termite colony, and once conditions are favourable to outcompete the fungal mutualist [ 10 – 16 ]. Fig. 1 Natural growth and occurrence of fungal mutualist Termitomyces and termite-associated Pseudoxylaria strains. A Mature fungus comb from a Macrotermes natalensis colony with spore-containing fungal nodules of a Termitomyces strain, B \n Pseudoxylaria stromata emerging from fungus comb after incubation for eight days in the absence of termites, and C - E axenic fungal cultures of Pseudoxylaria strains isolated from different termite mounds grown on agar plates with, C \n Pseudoxylaria sp. Mn132, D \n Pseudoxylaria sp. X3-2, and E \n Pseudoxylaria sp. Mn153. Driven by the rather anecdotal evidence for the “reduced antagonism hypothesis” and the additional postulation that co-evolved Pseudoxylaria strains might have become a fungus garden substrate specialist over evolutionary time [ 11 – 13 ], we sequenced the genomes of seven and transcriptomes of two Pseudoxylaria isolates to investigate the genomic, transcriptomic and metabolomic basis for symbiotic associations. Whole genome-based comparison with free-living members of this genus uncovered a substantial reduction in genome size and numbers of protein-coding genes, as well as reduced functional capacities, all of which indicated that Pseudoxylaria might have become a dependent symbiont and comb-substrate specialist. By analyzing the secondary metabolite repertoire as well as co-cultivation studies along with isotope experiments, we were further able to solidify the “reduced antagonism hypothesis”.",
"discussion": "Results and discussion Genome reduction is associated with a termite comb-associated lifestyle For our studies, we collected fungus comb samples originating from mounds of Macrotermes natalensis , Odontotermes spp., and Microtermes spp. termites and were able to obtain seven viable Pseudoxylaria cultures (X802 [ Microtermes sp.], Mn132, Mn153, X187, X3-2 [ Macrotermes natalensis ], and X167, X170LB [ Odontotermes spp.], Table S1 - S3 ). To test if a fungus comb-associated lifestyle of Pseudoxylaria was reflected in differences at the genome level, we sequenced the genomes of all seven isolates using a combination of paired-end shotgun sequencing (BGISEQ-500, BGI) and long-read sequencing (PacBio sequel, BGI or Oxford Nanopore Technologies, Oxford, UK). In addition, we sequenced the transcriptomes (BGISEQ, BGI) of two isolates (X802, X170LB). Eleven publicly available genomes of free-living Xylaria (Fig. 2A, B ) were used as reference genomes (Table S4 ). Hybrid draft genomes were comprised on average of 33–742 scaffolds with total haploid assembly lengths of 33.2–40.4 Mb, and a high BUSCO completeness of genomes (> 95 %) with a total number of predicted proteins ranging from 8.8 to 12.1 × 10 3 . The GC content was comparable to reference genomes with 49.7–51.6%. To verify the phylogenetic placement of the isolates, different genetic loci encoding conserved protein sequences (α-actin (ACT), second largest subunit of RNA polymerase (RPB2), β-tubulin (TUB) and the internal transcribed spacer (ITS) were used as genetic markers [ 7 , 13 ]. Fig. 2 Geographic and comparative phylogenomic analysis of termite-associated Pseudoxylaria isolates (strains 1-7) and free-living Xylaria (strains 8–18). A Geographic origins of genome-sequenced free-living Xylaria and termite-associated Pseudoxylaria isolates, B phylogenomic placement based on single-copy ortholog protein sequences, and C comparison of genome assembly length, and numbers of predicted proteins per genome. Phylogenies were reconstructed from ITS sequences and three aligned sequence datasets (RPB2, TUB, ACT) using reference sequences of twelve different taxa (Table S4 – S7 ). Consistent with previous findings, all isolates grouped within the monophyletic termite-associated Pseudoxylaria group [ 9 – 13 ], which diverged from the free-living members of the genus Xylaria (Fig. 2B , Figure S1 – S4 ). As our seven isolates covered a larger portion of the previously reported phylogenetic diversity of the termite-associated subgenus, we elaborated on genomic characteristics of our isolates to uncover features of the termite-associated ecology of Pseudoxylaria . Indeed, comparative genome analysis of the South African Pseudoxylaria isolates with publicly available genomes of free-living Xylaria species of similar genome quality revealed significantly reduced genome assembly lengths in Pseudoxylaria with reduced numbers of predicted genes per genome (Table S4 ). Comparison of the annotated mitochondrial (mt) genomes (Figure S5 , Table S8 ) also indicated that all seven mt genomes were shorter in length (assembly lengths: 18.5–63.8 kbp) compared to the, albeit few, publicly available mitochondrial genomes of free-living species (48.9–258.9 kbp). The reduction in mitochondrial genome size also corresponded to a significantly reduced mean number of annotated genes (7.6) and tRNAs (14.3) in Pseudoxylaria spp. compared to on average 30.0 (annotated genes) and 25.8 (tRNAs) found in free-living species. Analysis of the abundance and composition of transposable elements (TEs), which account for up to 30–35% of the genomes of (endo)parasitic fungi due to the expansion of certain gene families [ 20 , 21 ], showed that the mean total numbers of TEs across Pseudoxylaria spp. genomes were comparable (1530), but the numbers were reduced compared to free-living Xylaria species (3690) (Table S9 ). We also identified high variation in the TE composition across genomes (1.5–9.9 %), comparable to what was observed in free-living Xylaria spp. (1.3–8.1 %), with reductions in long terminal repeat retrotransposons (LTRs: Copia and unknown LTRs) in two inverted tandem repeat DNA transposons (TIRs; CACTA, Mutator and hAT). As Pseudoxylaria spp. contained increased numbers of non-ITR transposons of the helitron class and LTRs of the Gypsy class compared to Xylaria strains, we concluded that Pseudoxylaria exhibits no typical features of an (endo)parasitic lifestyle, but that the overall composition and the reduced numbers of TEs could serve as a fingerprint to distinguish the genetically divergent Pseudoxylaria taxa. Repertoire of carbohydrate-active enzymes indicates specialized substrate use As the fungus comb is mostly composed of partially-digested plant material interspersed with fungal mycelium of the termite mutualist [ 3 ], we anticipated that Pseudoxylaria should exhibit features of a substrate specialist similar to the fungal mutualist Termitomyces , which should be reflected in a Carbohydrate-Active enzyme (CAZyme) repertoire distinguishable from free-living saprophytic Xylaria species [ 22 – 24 ]. In particular, numbers and composition of redox-active enzymes (e.g., benzoquinone reductase (EC 1.6.5.6/EC 1.6.5.7), catalase (EC 1.11.1.6), glutathione reductase (EC 1.11.1.9), hydroxy acid oxidase (EC 1.1.3.15), laccase (EC 1.10.3.2), manganese peroxidase (EC 1.11.1.13), peroxiredoxin (EC 1.11.1.15), superoxide dismutase (EC 1.15.1.1), dye-decolorization or unspecific peroxygenase (EC 1.11.2.1), Table S10 ), which catalyze the degradation of lignin-rich biomass, were expected to differ between free-living strains and substrate specialists [ 22 ]. Identification of CAZymes using Peptide Pattern Recognition (PPR) revealed that Pseudoxylaria genomes encoded on average a reduced number of CAZymes (mean 264) compared to the free-living taxa in the family Xylaria (mean 367 CAZymes, pANOVA; F = 41.4, p = 3.5 × 10 –8 , pairwise p = 1.69 × 10 –7 ) (Fig. 3A, B , Figure S6 ), but similar numbers to those identified in Termitomyces (mean 265, pairwise p = 0.949). Fig. 3 Comparison of carbohydrate-active enzymes (CAZymes) in Xylaria , Pseudoxylaria and the fungal mutualist Termitomyces . A Predicted CAZymes, B Principal Coordinates Analysis (PCoA) of predicted CAZyme families, and C heatmap of representatives CAZyme families in the predicted proteomes of free-living Xylaria , Termitomyces and Pseudoxylaria species. Overall, significant differences in the composition of CAZymes were observed [ 8 ], most notably in the reduction of auxiliary activity enzymes (AA), carbohydrate esterases (CE), glycosyl hydrolases (GH), and polysaccharide lyases (PL). The most significant reduction was observed in the AA3 family (Fig. 3C ), which typically displays a high multigenicity in wood-degrading fungi as many enzymes of this family catalyze the oxidation of alcohols or carbohydrates with the concomitant formation of hydrogen peroxide or hydroquinones thereby supporting lignocellulose degradation by other AA-enzymes, such as peroxidases (AA2). Similarly, although to a lesser extent, reduced numbers within the related AA1 family were detected, which included oxidizing enzymes like laccases, ferroxidases, and laccase-like multicopper oxidases. Along these lines, glycosyl hydrolases of the GH3 and GH5 family, including enzymes responsible for degradation of cellulose-containing biomass and xylose, were less abundant. We also noted that all Pseudoxylaria lacked homologs of the unspecific peroxygenases (UPO; EC 1.11.2.1), while almost all free-living Xylaria spp. and the fungal symbiont Termitomyces harbored at least one or two copies of similar gene sequences. Pseudoxylaria shows reduced biosynthetic capacity for secondary metabolite production A healthy termite colony is engulfed in several layers of social immunity [ 5 , 6 ], which pose a constant selection pressure on associated and potentially antagonistic microbes. As Pseudoxylaria evolved measures to remain inconspicuously present within the comb environment, we hypothesized that one of the possible adaptations to evade hygiene measures of termites could be reflected in a reduced biosynthetic capability to produce antibiotic or volatile natural products, which often serve as infochemicals triggering defense mechanisms [ 25 – 27 ], or as alarm pheromones [ 4 , 28 ]. The biosynthesis of secondary metabolites is encoded in so called Biosynthetic Gene Cluster (BGC) regions. We explored the abundance and diversity of encoded BGCs using FungiSMASH 6.0.0 and manually cross-checked the obtained data set by BLAST to account for possible biases due to varying genome qualities across strains of both groups [ 29 ]. Overall, the herein investigated Xylaria genomes harbored on average 90 BGCs per genome, while Pseudoxylaria encoded on average 45 BGCs (Fig. 4 , Figure S7 ). Fig. 4 Similarity network analysis of biosynthetic gene clusters. Comparative analysis of termite associated-associated Pseudoxylaria isolates (strains 1–7, red circles) and free-living Xylaria (strains 8–18, green circles) with BiG-SCAPE 1.0 annotations (blue hexagon) ACR ACR toxin, Alt alternariol, Bio biotin, Chr chromene, Cyt cytochalasins, Cur curvupalide, Dep depiudecin, Fus fusarin, Gri griseofulvin, Mon monascorubin, MSA 6-methylsalicylic acid, Pho phomasetin, Sol solanapyrone, Swa swasionine, Xen xenolozoyenone, Xsp xylasporins, Xyl xylacremolide. Singletons are not shown. The nature and relatedness of the BGCs were analyzed by creating a curated similarity network analysis using BiG-SCAPE 1.0 [ 30 ]. Overall, 28 orthologous BGCs were shared across all genomes, including the biosynthesis of polyketides like 6-methylsalicylic acid (MSA), chromenes (Chr) and polyketide-non-ribosomal peptide (PKS-NRPS) hybrids like the cytochalasins (Cyt) [ 31 ]. Furthermore, five BGC networks, which were shared by Pseudoxylaria and Xylaria , contained genes encoding natural product modifying dimethylallyltryptophan synthases (DMATS). In contrast, and despite the significant reduction in the biosynthetic capacity within Pseudoxylaria genomes [ 29 ], about 29 BGC networks were unique to Pseudoxylaria and thus could possibly relate to the comb-associated lifestyle (Figure S8 and S9 ). Notably, Pseudoxylaria genomes lacked genes encoding ribosomally synthesized and posttranslationally modified peptides (RiPPs) or halogenases. In comparision, free-living Xylaria spp. harbored at least one sequence encoding a RiPP, and up to two orthologous sequences encoding putative halogenases. In contrast, a reduced average number of terpene synthases (TPS) in Pseudoxylaria (9 TPS) compared to free-living Xylaria (18 TPS) was detected, which included three BGCs encoding TPSs that were unique to Pseudoxylaria . In comparison, genomes of the fungal mutualist Termitomyces were reported to encode for about 20-25 terpene cyclases, but haboured only about two loci containing genes for a PKS and NRPS each [ 24 ]. Manual BLAST searches were conducted to identify BGCs that could be putatively assigned to previously isolated metabolites from Pseudoxylaria ( vide infra Fig. 7 , Figure S8 ) [ 32 , 33 ]. Using e.g., the known NRPS-PKS-hybrid cluster sequence ccs ( Aspergillus clavatus ) of cytochalasins as query, an orthologous BGC, here named cytA , was identified in the cytochalasin-producing strain X802 [ 34 ]. Although the putative PKS-NRPS hybrid and CcsA shared 60 % identical amino acids (aa), the sequences of the accessory enzymes were less related to CcsC-G (45–47% identical aa) and the BGC in X802 lacked a gene of a homologue to ccsB . Similarly, five free-living Xylaria species carried orthologous gene loci ( Xylaria sp. BCC 1067, Xylaria sp. MSU_SB201401, X. flabelliformis G536, X. grammica EL000614, and X. multiplex DSM 110363) supporting previous isolation reports of cytochalasins with varying structural features. Furthermore, three Pseudoxylaria strains (X187, and closely related Mn153, and Mn132) were found to share a highly similar PKS-NRPS hybrid BGC (99–100 % identical aa, named xya ), which likely encodes for the enzymatic production of previously identified xylacremolides [ 32 ]. Four Pseudoxylaria strains (X802, Mn132, Mn153, and X187) also shared a BGC (50–98 % amino acid identity) resembling the fog BGC ( Aspergillus ruber ) [ 35 , 36 ], which putatively encodes the biosynthetic machinery to produce xylasporin/cytosporin-like metabolites. In this homology search, we also uncovered that fog -like BGC arrangements are likely more common than previously anticipated, as clusters with similar arrangements and identity were also found in genomes of Rosellinia necatrix , Pseudomasariella vexata , Stachybotrys chartarum , and Hyaloscypha bicolor (Fig. 4 , Figure S8 ). A detailed analysis of the fog -like cluster arrangements within Pseudoxylaria genomes revealed - similar to homologs of the ccs cluster – variation in the abundance and arrangement of several accessory genes coding for a cupin protein ( pxF ), a short chain oxidoreductase ( pxB ; SDR), and an additional SnoaL-like polyketide cyclase ( pxP ), which could account for the production of strain-specific structural congeners ( vide infra , Fig. 7 ). Change of nutrient sources causes dedicated transcriptomic changes in Pseudoxylaria To further solidify our in silico indications of substrate specialization with comb material as preferred substrate and fungus garden as environment, we analyzed Pseudoxylaria growth on different media (PDA, and reduced medium 1/3-PDA) including comb-like agar matrices (wood-rice medium (WRM), agar-agar or 1/3-PDA medium containing lyophilized (dead) Termitomyces sp. T112 biomass (T112, respectively T112-PDA), PDB covering glass-based surface-structuring elements (GB), Table S11 – S14 ). Cultivation of Pseudoxylaria on agar-agar containing lyophilized biomass of Termitomyces (T112) as the sole nutrient source allowed Pseudoxylaria to sustain growth, although to a reduced extent compared to growth on nutrient-rich PDA medium (Table S3 ). Wood-rice medium (WRM) induced comparable growth rates as observed on PDA and also the appearance of phenotypic stromata. To investigate the influence of these growth conditions on the transcriptomic level, we harvested RNA from vegetative mycelium after growth on comb-like media (WRM, T112, T112-PDA, and GB), PDA, and reduced medium 1/3-PDA (Fig. 5A ). The most significant transcript changes (normalized to data obtained from growth on PDA) were observed for genes coding for specific CAZymes including several redox active enzymes (Fig. 5B ). The 30 most variable transcripts coded for specific glycoside hydrolases (GH), lytic polysaccharide monooxygenases (AA), ligninolytic enzymes, and a glycoside transferase (GT). Similarly, chitinases (CHT2; CHT4; CHI2; CHI4) were upregulated (up to 243-fold on T112) under almost all conditions compared to PDA, but some of these specific transcript changes were exclusive to growth on Termitomyces biomass or artificial comb material (WRM) suggesting the ability to regulate and increase chitin metabolism if necessary [ 37 ]. Fig. 5 Transcriptomic analysis of Pseudoxylaria sp. X802 in dependence of growth conditions. A Representative pictures of Pseudoxylaria sp. X802 growing on PDA, PDB on glass beads (GB), wood-rice medium (WRM), and agar-agar medium containing lyophilized Termitomyces sp. T112 biomass (T112). B Heatmap of the most variable transcripts coding for CAZymes (red), redox enzymes (orange), secondary metabolite-related core genes (green), and more specifically on key genes within the boundaries of cytochalasin (turquoise) and xylasporin/cytosporin BGCs (blue). RNA was obtained from vegetative mycelium after growth on PDA, reduced medium (1/3-PDA), PDB on glass beads (GB), wood-rice medium (WRM), 1/3-PDA-medium enriched with Termitomyces sp. T112 biomass (T112-PDA) and agar-agar medium containing lyophilized Termitomyces biomass (T112). Transcript counts are shown as log 10 transformed transcripts per million (top; TPM). Significance of the changes in transcript counts are compared to control (X802 grown on PDA) and depicted in log -10 transformed p values. When X802 was grown on T112 (agar matrix containing lyophilized Termitomyces sp. T112 biomass), we observed a >400-fold increase in the expression of transcripts encoding glycoside hydrolases in the GH43 family, GH7 (~140-fold), GH3, and GH64 (5–12-fold). Similarly, transcripts for a putative mannosyl-oligosaccharide-α-1,2-mannosidase (MNS1B; 8.2-fold), chitinase CHT4 (2.9-fold), β-glucosidase BGL4 (5.7-fold), and copper-dependent lytic polysaccharide monooxygenase AA11 (1.6-fold) were significantly upregulated. Growth on WRM (wood-rice medium) or T112 ( Termitomyces sp. T112 biomass) also caused a significant upregulation of genes coding for glycoside transferase GT2, glycoside hydrolases GH15, GH3, and aldehyde oxidase AOX1, which indicated the ability to expand the degradation portfolio if necessary. Along these lines, specific transcript levels were reduced when X802 was grown on T112, in particular class II lignin-modifying peroxidases (AA2), carbohydrate-binding module family 21 (CBM21), multicopper oxidases (AA1), secreted β-glucosidases (SUN4), and glycoside hydrolases GH16, and GH128. When the fungus was challenged with lignocellulose-rich WRM medium, higher transcript levels putatively assigned to glutathione peroxidase (GXP2), superoxide dismutase (SOD2), and laccases (LCC5) were observed, which indicated that despite the reduced wood-degrading capacity, Pseudoxylaria activates available enzymatic mechanisms to degrade the provided material and respond to the resulting oxidative stress. Cultivation on GB (glass-based surfaces covered in liquid PD broth) influenced the expression of certain genes coding for glycoside hydrolases (GH64, GH76, GH72, GH128, BGL4) and lytic polysaccharide monooxygenases (AA1, AA2, AA11), presumably enabling the fungus to utilize soluble carbohydrates. To test the hypothesis that the presence of Termitomyces biomass stimulates secondary metabolite production in Pseudoxylaria to eventually displace the mutualist, we also analyzed changes in the transcript levels of core BGC genes that encode the production of bioactive secondary metabolites. Overall, only slight transcript variations were detectable within the most variable expressed genes. (Fig. 5B ). Cultivation on GB, WRM, and T112 media caused lower transcript levels of genes coding for terpene synthase TC1, polyketide synthases (PKS7, PKS8), and the NRPS-like1, while an upregulation of NRPS-like2 on WRM (2.5-fold), and of PKS7 (1.7-fold) on reduced 1/3-PDA medium was observed. Transcript levels of core genes within BGCs assigned to cytochalasines ( cyt ) or xylasporins/cytosporins ( px ), e.g., remained nearly constant, while minor transcript level variations of neighboring genes and reduced transcript levels for pxI (flavin-dependent monooxygenase), pxH (ABBA-type prenyltransferase), pxF (cupin fold oxidoreductase), and pxJ (short-chain dehydrogenase) were detectable. Hence, it was concluded that the presence of Termitomyces biomass only weakly triggers secondary metabolite production in general, but varying transcript levels coding for decorating enzymes could cause substantial structural alterations within the produced natural product composition. It was also notable that transcript levels of the terpene synthase TC1 were downregulated, which could cause a reduced production level of specific volatiles. Pseudoxylaria antagonizes Termitomyces growth and metabolizes fungal biomass The growth behavior of Pseudoxylaria isolates was also analyzed in co-culture assays with Termitomyces . As expected from prior studies, both fungi showed reduced growth when co-cultured on agar plates, often causing the formation of zones of inhibition (ZOI) between the fungal colonies (Fig. 6A–D , Table S11 – S14 ) [ 7 ]. When fungus-fungus co-cultures were maintained for longer than two weeks on agar plates, Pseudoxylaria started to overcome the ZOI and overgrew Termitomyces via the extension of aerial mycelium. The observation was even more pronounced when co-cultures were performed on wood-rice medium (WRM), where Pseudoxylaria remained the only visible fungus after two weeks. Fig. 6 Co-cultivation of Pseudoxylaria sp. X170LB and Termitomyces sp. T112 and results of isotope fractionation experiments. Representative pictures of fungal growth and co-cultivation of A \n Termitomyces sp. T112, B \n Pseudoxylaria sp. X170LB, C co-culture of Pseudoxylaria sp. X802 and Termitomyces sp. T153 exhibiting a ZOI, in which X802 overgrowths T153 in proximity to the interaction zone (red arrow), and D \n Pseudoxylaria sp. X802 growing on the surface of a living Termitomyces sp. T153 culture. E , F Shown is the relative change in the carbon isotope pattern (δ 13 C values, ± standard deviation, with n = 3) of lipid and carbohydrate fractions isolated from fungal biomass of Termitomyces sp. T112, Pseudoxylaria sp. X170LB, and Pseudoxylaria sp. X170LB cultivated on vegetative Termitomyces sp. T112 biomass (T112 ǂ ), or on lyophilized Termitomyces sp. T112 biomass (T112). Fungal strains were grown on E medium with natural 13 C abundance and F medium artificially enriched in 13 C content. To verify whether Pseudoxylaria consumes Termitomyces or even partially degrades specific metabolites present within the fungal biomass, we pursued stable isotope fingerprinting commonly used to analyse trophic relations [ 38 , 39 ]. This diagnostic method relies on measurable changes in the bulk stable isotope composition, because biosynthetic enzymes preferentially convert lighter metabolites enriched in 12 C compared to their heavier 13 C-enriched congeners. This intrinsic kinetic isotope effect results in an overall change in the 13 C/ 12 C ratio of the respective educts and products, in particular in biomarkers such as phospholipid fatty acids, carbohydrates, and amino acids. Using this isotope enrichment effect, we determined the natural trophic isotope fractionation of 13 C in lipids and carbohydrates produced by Termitomyces sp. T112 and Pseudoxylaria sp. X170LB. For clearer differentiation, both fungi were cultivated on PDA medium containing naturally abundant 13 C/ 12 C, Fig. 6E ) and on PDA medium enriched with 13 C-glucose (Fig. 6 F). Lipids and carbohydrates were isolated from mycelium harvested after 21 days (Fig. 6E , Table S15 ). Analysis of fungal carbohydrate and lipid-rich metabolite fractions by Elemental Analysis-Isotope Ratio Mass Spectrometry (EA-IRMS) [ 40 , 41 ] uncovered that under normal growth conditions (full medium), Termitomyces sp. T112 and Pseudoxylaria sp. X170LB showed only a slight negative trophic fractionation of stable carbon isotopes (δ 13 C/ 12 C ratio (expressed as δ 13 C values [‰]), Fig. 6F ) within the carbohydrate fractions (T112: −1.2 ‰; for X170LB: −1.3 ‰), and expectedly a stronger depletion in the lipid fraction (T112: −6.7 ‰, and less pronounced for X170LB: −3.1 ‰). To determine if Pseudoxylaria metabolizes Termitomyces biomass, the isotope pattern of metabolites derived from Pseudoxylaria thriving on living biomass of Termitomyces (T112 ǂ ) was analysed next. Here, an overall positive carbon isotope ( 13 C/ 12 C) fractionation by approximately +0.6 ‰ relative to the control medium was detectable, while the δ 13 C values of lipids remained largely unchanged (Fig. 6F , Table S15 ). These results suggested that Pseudoxylaria might pursue a preferential uptake of Termitomyces -derived carbohydrates. In a last experiment, Pseudoxylaria was grown on lyophilized (dead) Termitomyces biomass (T112) as sole food source. In this experiment, the isotope fingerprint showed converging δ 13 C values of −1.9 ‰ (relative to the media) for both carbohydrate and lipid fractions, which indicated that Pseudoxylaria is able to simultaneously metabolize and cycle carbohydrates as well as lipids resulting in the equilibration of isotopic levels between carbohydrates and lipids. Thus, it was concluded that in nature, Pseudoxylaria likely harvests nutrients firstly from vegetative Termitomyces , and then—if possible—subsequently degrades dying or dead mycelium. Pseudoxylaria produces antimicrobial secondary metabolites Based on the observation that Pseudoxylaria antagonizes growth of Termitomyces , we questioned if the formation of a ZOI might be caused by the secretion of Pseudoxylaria -derived antimicrobial metabolites [ 26 , 42 ]. Thus, we performed an ESI(+)-HRMS/MS based metabolic survey using the web-based platform “Global Natural Product Social Molecular Networking” (GNPS) [ 43 ] to correlate the encoded biosynthetic repertoire of Pseudoxylaria with secreted metabolites. A partial similar metabolic repertoire across the six analyzed strains was detectable and allowed us to match some of the detectable chemical features and previously isolated metabolites to the predicted shared BGCs, such as antifungal and histone deacetylase inhibitory xylacremolides (Xyl; X187/Mn132) [ 32 , 33 ], pseudoxylaramides (Psa; X187/Mn132) [ 32 ], antibacterial pseudoxylallemycins (Psm; X802/OD126) [ 18 ], xylasporin/cytosporins (Xsp; X802/OD126/X187/Mn132) [ 36 ], and cytotoxic cytochalasins (X802/OD126) (Fig. 7A and B ) [ 18 ]. Fig. 7 Comparative metabolomic analysis of six Pseudoxylaria strains (OD126 (red), Mn132 (orange), X170 (black), X187 (green), X3.2 (yellow) and X802 (blue)). A Overview of the GNPS network. Identified metabolite clusters xylacremolides (Xyl; X187/Mn132) [ 32 , 33 ], pseudoxylaramides [ 32 ] (Psa; X187/Mn132), pseudoxylallemycins (Psm; X802/OD126) [ 18 ], xylasporin/cytosporins (Xsp; X802/OD126/X187/Mn132) and cytochalasins (X802/OD126) [ 18 ]. B xylasporin/cytosporin-related cluster formed by nodes from X802 (blue), OD126 (red), X187 (green) and Mn132 (orange). C Chemical structures of natural products isolated from Pseudoxylaria species and related compounds. Red box highlights proposed structures of isolated xylasporin G and I in this study. A cluster that contained MS 2 signals of molecular ions assigned to the cytosporin/xylasporin family, which was shared by at least four strains, caught our attention as a certain degree of structural diversity of xylasporin/cytosporin family was predicted from the comparison of their respective BGCs. The assigned nodes of this GNPS cluster split into two subclusters with only very little overlap between both regions. Analysis of the mass fragment shifts suggested that both subclusters belong to two different families of xylasporin/cytosporin congeners (Figure S9 ). To verify these deductions, we pursued an MS-guided purification of xylasporin/cytosporins from chemical extracts of Pseudoxylaria sp. X187, which yielded xylasporin G (3.23 mg, pale-yellow solid) and xylasporin I (1.75 mg, pale-yellow solid). The sum formulas of xylasporin G and xylasporin I were determined to be C 17 H 22 O 5 (calcd. for [M + H] + C 17 H 23 O 5 + = 307.1540, found 307.15347, −1.726 ppm) and C 17 H 24 O 5 (calcd. for [M + H] + C 17 H 25 O 5 + = 309.1697, found 309.1691, −1.68 ppm) by ESI-(+)-HRMS and were predicted to have six degrees of unsaturation (Fig. 7B , Figure S10 , Table S16 - S17 ). Planar structures were deduced by comparative 1D and 2D NMR analyses, which revealed the presence of an unsaturated polyketide chain that matched the unsaturation degree and the anticipated structural variation from cytosporins (Fig. 7C , Figure S11 - S25 ). To evaluate if Pseudoxylaria -derived culture extracts and produced natural products (e.g., cytochalasins) are responsible for the observed antimicrobial activity, standardized antimicrobial activity assays were performed (Table S17 , S18 and Figure S26 ). As neither culture extracts nor single compounds exhibited significant antimicrobial activity, they could not be held fully accountable for the antagonistic behavior in co-cultures. Thus, we hypothesized that the observed ZOI might be caused by yet unknown effects like nutrient depletion or bioactive enzymes. Pseudoxylaria has a negative impact on the fitness of insect larvae Due to the production of structurally diverse and weakly antimicrobial secondary metabolites, we questioned if mycelium of Pseudoxylaria exhibits intrinsic insecticidal or other insect-detrimental activities, which could discourage or ward off grooming behavior of termite workers. Due to the technical challenges associated with behavioral studies of termites, we evaluated instead the effect of Pseudoxylaria biomass on Spodoptera littoralis , a well-established insect model system and a destructive agricultural lepidopterous pest [ 44 , 45 ]. When S. littoralis larvae were fed with mycelium-covered agar plugs of Pseudoxylaria sp. X802, a clear decrease of the relative growth rate (RGR) and decline in survival was observed (Fig. 8 : treatment D (green), Table S19 , S20 ) compared to feeding with untreated agar plugs (treatment A (black)). In comparison, when larvae were fed with agar plugs covered with the fungal mutualist Termitomyces sp. T153 (treatment B (blue)) an increased growth rate of larvae was observed. Fig. 8 Effect of Termitomyces sp. T153 and Pseudoxylaria sp. X802 mycelia on the relative growth rate and survival of S. littoralis larvae. Insects were fed with either A PDA, B PDA agar plug covered with vegetative Termitomyces sp. T153, C PDA agar plug from which vegetative Termitomyces sp. T153 was removed prior to feeding, D PDA agar plug covered with vegetative Pseudoxylaria sp. X802 mycelium, and E PDA agar plug from which vegetative Pseudoxylaria sp. X802 mycelium was removed prior to feeding. All experiments were performed with 25 replicates per treatment, a duration of 10 days, and larval weights and survival rates were recorded every day. Statistical significances were determined using ANOVA on ranks ( p < 0.001, n = 25) followed by Dunn’s post-hoc test (indicated by different letters at the alpha = 0.05 level). Additionally, S. littoralis larvae were also fed agar plugs that had been cleaned from fungal mycelium prior to feeding to test if secreted metabolites and/or depletion of nutrients within the agar matrix might have an impact on RGR and survival. Here, it was surprising to note that agar-plugs derived from Pseudoxylaria sp. X802 cultures resulted in the death of all treated caterpillars within six days (treatment E (yellow)). In contrast, feeding with agar plugs previously covered with Termitomyces mycelium (treatment C (red)) caused the survival of almost all caterpillars until the end of the experiment, although a slight decline on RGR was observed compared to treatment B (Fig. 8 ). Thus, an overall beneficial nutritional effect of Termitomyces was clearly visible, although a minor negative effect of nutrient depletion within the agar environment during fungal growth could not be fully excluded. Overall, we corroborated from these results that Pseudoxylaria exhibits a pronounced negative effect on insect growth and survival, likely due to the combined effect of harmful metabolite secretion, indigestible fungal mycelium and/or nutrient depletion of the growth environment."
} | 10,526 |
35512710 | null | s2 | 5,811 | {
"abstract": "Biological systems ranging from bacteria to mammals utilize electrochemical signaling. Although artificial electrochemical signals have been utilized to characterize neural tissue responses, the effects of such stimuli on non-neural systems remain unclear. To pursue this question, we developed an experimental platform that combines a microfluidic chip with a multielectrode array (MiCMA) to enable localized electrochemical stimulation of bacterial biofilms. The device also allows for the simultaneous measurement of the physiological response within the biofilm with single-cell resolution. We find that the stimulation of an electrode locally changes the ratio of the two major cell types comprising Bacillus subtilis biofilms, namely motile and extracellular-matrix-producing cells. Specifically, stimulation promotes the proliferation of motile cells but not matrix cells, even though these two cell types are genetically identical and reside in the same microenvironment. Our work thus reveals that an electronic interface can selectively target bacterial cell types, enabling the control of biofilm composition and development."
} | 284 |
19060298 | PMC2651464 | pmc | 5,812 | {
"abstract": "Lotus japonicus , a model legume, develops an efficient, nitrogen-fixing symbiosis with Mesorhizobium loti that promotes plant growth. Lotus japonicus also forms functional nodules with Rhizobium sp. NGR234 and R. etli . Yet, in a plant defence-like reaction, nodules induced by R. etli quickly degenerate, thus limiting plant growth. In contrast, nodules containing NGR234 are long-lasting. It was found that NGR234 initiates nodule formation in a similar way to M. loti MAFF303099, but that the nodules which develop on eleven L. japonicus ecotypes are less efficient in fixing nitrogen. Detailed examination of nodulation of L. japonicus cultivar MG-20 revealed that symbiosomes formed four weeks after inoculation by NGR234 are enlarged in comparison with MAFF303099 and contain multiple bacteroids. Nevertheless, nodules formed by NGR234 fix sufficient nitrogen to avoid rejection by the plant. With time, these nodules develop into fully efficient organs containing bacteroids tightly enclosed in symbiosome membranes, just like those formed by M. loti MAFF303099. This work demonstrates the usefulness of using the well-characterized micro-symbiont NGR234 to study symbiotic signal exchange in the later stages of rhizobia–legume symbioses, especially given the large range of bacterial (NGR234) and plant ( L. japonicus ) mutants that are available.",
"introduction": "Introduction An intense exchange of molecular signals between compatible legumes and rhizobia leads to the establishment of symbioses, in which atmospheric nitrogen is reduced to ammonia that is incorporated into the plants. In return, fixed carbon is supplied to the rhizobia. Nod-factors are the key bacterial signals that allow rhizobia to enter root hairs ( Relić et al. , 1993 , 1994 a , b ; D'Haeze et al. , 1998 ) and re-programme root cell development towards new organs called nodules to accommodate the rhizobia ( Broughton et al. , 2000 ). As they penetrate the root, rhizobia are internalized into cortical cells where they become enclosed in a plant-derived membrane, giving rise to symbiosomes ( Verma and Hong, 1996 ; Jones et al. , 2007 ). Subsequent differentiation of the rhizobia into nitrogen-fixing bacteroids concludes nodule development. Thereafter, a second phase of symbiotic interaction begins that is characterized by a long-lasting intracellular existence of the rhizobia, which requires a continuous metabolite exchange between the two partners ( Lodwig and Poole, 2003 ). Failure to establish successful symbioses, or the breakdown of symbioses, occurs between incompatible legumes and rhizobia. Many examples of rhizobia that attempt to invade legumes exist, but nodule development is often arrested at various levels because one or more of the appropriate symbiotic signals is missing. Bacterial persistence within the host, the second phase of symbiotic interactions, has, however, not been as well studied. Indeed, rhizobial mutants unable to fix nitrogen develop normal nodules. Some days later, however, the symbiosomes show signs of senescence and the nodules degenerate ( Hahn and Studer, 1986 ; Hirsch and Smith, 1987 ). Additionally, Kiers et al. (2003) have shown that inefficiency on the part of the micro-symbiont leads the plant host to restrict the symbiotic interaction. Taken together, these data illustrate that nitrogen fixation by rhizobia is necessary for intracellular persistence within plant tissues. Lotus japonicus is an important model legume that is widely used to study symbiotic interactions with micro-organisms ( Udvardi et al. , 2005 ), and significant advances have been made in the understanding of Nod-factor perception by L. japonicus ( Kistner et al. , 2005 ; Radutoiu et al. , 2007 , and references therein). In the wild, L. japonicus is nodulated by strains of Mesorhizobium loti . Interestingly, M. loti and Rhizobium etli strain CE3 secrete identical Nod-factors ( Cardenas et al. , 1995 ; Lopez-Lara et al. , 1995 ), and R. etli CE3 induces effective nodules on L. japonicus ( Banba et al. , 2001 ). About 3 weeks post-inoculation (wpi) nitrogen fixation suddenly stops and the nodules begin to senesce prematurely. It was suggested that delayed recognition of R. etli CE3 as an ‘irregular’ micro-symbiont of Lotus occurs, and that this triggers some form of plant defence ( Banba et al. , 2001 ). Such transient symbiotic interactions mean that nitrogen fixation is not the only requirement for rhizobial persistence in nodules. Furthermore, they point to the presence of additional plant check-points at later stages of the symbiosis and concomitant signals dedicated to persistence within the plant cells. Perhaps these signals employ mechanisms similar to those used by some pathogenic bacteria to maintain long-lasting (chronic) intracellular infections in their eukaryotic hosts ( LeVier et al. , 2000 ; Rhen et al. , 2003 ; Monack et al. , 2004 ). Rhizobium species NGR234 (hereafter NGR234) is an exceptionally broad host-range micro-symbiont ( Pueppke and Broughton, 1999 ) that utilizes a large diversity of well-characterized symbiotic signals ( Perret et al. , 2000 ). Amongst these are a complex mixture of Nod-factors, some of which are identical to those synthesized by M. loti and R. etli CE3 ( Price et al. , 1992 ). As a probable consequence, NGR234 induces (4–6 wpi) effective nodules on L. japonicus ( Hussain et al. , 1999 ; Pueppke and Broughton, 1999 ). Signs of premature senescence have not been reported. A subsequent study showed that nodules induced by NGR234 were still effective 12 wpi ( Müller et al. , 2001 ) even though NGR234 is not a ‘regular’ micro-symbiont of L. japonicus . This apparent discrepancy was investigated by comparing nodulation of L. japonicus by NGR234 and M. loti MAFF303099 (hereafter MAFF303099) at different stages of the symbiotic interaction.",
"discussion": "Discussion The term Nod + /Fix + is used to describe legume–rhizobia alliances that are fully active symbiotically. If nodules fail to develop with a particular micro-symbiont, a Nod – phenotype results, and if nodules develop but do not fix nitrogen the interaction is called Nod + /Fix – ( Pueppke and Broughton, 1999 ). Neither of these latter two phenotypes is able to sustain plant growth under nitrogen-deficient conditions. In this sense, the L. japonicus –NGR234 interaction falls in the Nod + /Fix + category along with the symbiosis between L. japonicus and its natural micro-symbiont M. loti . Nodules induced by NGR234 develop in a classical manner, infection threads that penetrate root hairs were formed, and levels of nodulin transcripts in L. japonicus nodules were comparable with those found using M. loti as the inoculum. Inoculation by NGR234 or MAFF303099 resulted in similar nodule numbers 4 wpi and transverse sections showed normal structure. Furthermore, nodules containing NGR234 fixed nitrogen, were long-lasting ( Fig. 6 ), and, after several months, could be as large as mature nodules containing MAFF303099 ( Fig. 4 ). Thus despite not being the regular micro-symbiont of L. japonicus , the interaction with NGR234 is stable and clearly different to that reported with R. etli CE3 ( Banba et al. , 2001 ). NGR234 contributes to plant growth under nitrate-limiting conditions and normal nodule functions do not collapse. In comparison with nodules induced on L. japonicus by MAFF303099, those containing NGR234 are only moderately efficient, however, and present clear differences in their ultrastructural organization. Four wpi, almost all symbiosomes were enlarged, and similar in appearance to those induced by nitrogenase-deficient strains ( Hahn and Studer, 1986 ; Regensburger et al. , 1986 ) or to those formed by plant mutants of the Nod + /Fix – category ( Kumagai et al. , 2007 ). Enlarged, starch-containing symbiosomes are frequent in inefficient nodules ( Hirsch et al. , 1983 ; Niehaus et al. , 1993 ; Barsch et al. , 2006 ) and are indicative of poor nitrogen fixation. A thorough examination of nodule sections (4 wpi) revealed rare sectors containing bacteroids tightly enclosed in peribacteroid membranes. The appearance of such sectors was not observed in interactions between L. japonicus and the other (alternative) micro-symbionts R. etli or R. tropici . In these interactions, occasionally ‘tight’ individual symbiosomes were seen, but the vast majority were enlarged (data not shown). As the NGR234-containing nodules matured, enlarged symbiosomes became less predominant, however: tightly enclosed bacteroids dominated, with some infected cells containing only tight symbiosomes. In the development of determinate nodules, such as those found on L. japonicus , there are two stages; first when the infected cells continue to divide and then later when they enlarge. After rhizobia enter the host cells, the infected cells stop dividing and undergo several cycles of endoreduplication, which result in increased cell growth ( Truchet et al. , 1980 ; Bergersen, 1982 ; Brewin, 1991 ). Simultaneously, plants direct the differentiation of rhizobia into bacteroids ( Mergaert et al. , 2006 ). An investigation was carried out to determine whether there could be a correlation with this switch from host cell division to enlargement and the appearance or increase in the numbers of tight symbiosomes. The morphology of symbiosomes from large, extremely mature (7-month-old) nodules where infection events should have stopped argues against this, however. Although some of the infected cells contained only tight symbiosomes, infected cells still containing enlarged symbiosomes interlaced with tight ones were found within the same nodule, in approximately equal numbers ( Fig. 6 ). Thus the correlation between cell enlargement and the formation of tight symbiosomes does not completely parallel maturation of the infected cell. Tight symbiosomes and the nodule sectors containing them are considered to be more active in nitrogen fixation, and the increase in their relative proportion with age correlates with the increased contribution of NGR234 to the growth of L. japonicus . In other words, the initial very loose association between bacteroid and symbiosome membranes, frequently observed in Nod + /Fix – interactions ( Hahn and Studer, 1986 ; Regensburger et al. , 1986 ; Kumagai et al. , 2007 ), does not compromise per se the potential of MG-20–NGR234 to fix nitrogen as the nodules age. It also suggests that the minimal fixed nitrogen supply from these small sectors of active symbiosomes is sufficient to prevent nodule senescence and sustain the development of the whole organ. Thus a critical level of symbiotic activity apparently avoids plant-triggered nodule degradation and subsequently leads to the development of more efficient nodules. For as yet unknown reasons, there is a delay in the ability of NGR234-containing nodules to reach this level of nitrogen fixation. Thus, plant growth is also delayed. Legume growth in nitrate-limiting conditions directly reflects the ability of rhizobia to reduce atmospheric nitrogen to ammonia and thus requires an effective symbiotic association. In the field, symbiotic efficiency is not dependent on interstrain competitiveness, i.e. the ability to develop new fixing organs ( Amarger, 1981 ; Hahn and Studer, 1986 ), and competitive strains are often poorly efficient ( Thies et al. , 1991 ; Sadowsky and Graham, 1999 ). Nitrogen conversion efficiency also varies with legume–rhizobia combinations and environmental conditions ( Vlassak and Vanderleyden, 1997 ; Zahran, 1999 ). These factors mostly alter nodule functioning as opposed to nodule development. Nevertheless, most published data related to the legume–rhizobial symbiosis are focused on nodule initiation/development rather than nodule functionality. Mutations involved in yield modification are often overlooked during genetic screens in comparison with phenotypes showing more dramatic symbiotic phenotypes, e.g. Nod – or Fix – mutants. As a consequence, the mechanisms by which rhizobia establish long-term (nitrogen-fixing) interactions and persist within cells of the legume host remain largely unknown. We suspect that the ineffectiveness of the L. japonicus –NGR234 symbiosis is due to a delay in the establishment of the persistent stage of cell infection. For intracellular pathogens, persistent (or chronic) infection is a phase distinct from that leading to cell invasion and requires specific gene sets ( Rhen et al. , 2003 ). It is possible that rhizobia utilize similar molecular mechanisms, and that after nodule initiation there is a continuation of the molecular dialogue between the symbionts after rhizobial internalization. For L. japonicus it seems that NGR234 has sufficient signals to permit tolerance, whereas R. etli CE3 lacks or has different persistence mechanisms. In conclusion, the interaction between L. japonicus and NGR234 presents all the hallmarks of a typical stable, legume–rhizobia symbiosis and, for this reason, it is possible to study nodule initiation taking advantage of the well-characterized micro-symbiont NGR234 ( Perret et al. , 2000 ). Furthermore the nature of this specific interaction will permit the investigation of rhizobial persistence and plant tolerance strategies within L. japonicus nodules. These are essential aspects of legume–rhizobia interactions often overlooked in classical forward genetic screens."
} | 3,377 |
32442279 | PMC7319574 | pmc | 5,813 | {
"abstract": "Abstract Next-generation sequencing has paved the way for the reconstruction of genome-scale metabolic networks as a powerful tool for understanding metabolic circuits in any organism. However, the visualization and extraction of knowledge from these large networks comprising thousands of reactions and metabolites is a current challenge in need of user-friendly tools. Here we present Fluxer ( https://fluxer.umbc.edu ), a free and open-access novel web application for the computation and visualization of genome-scale metabolic flux networks. Any genome-scale model based on the Systems Biology Markup Language can be uploaded to the tool, which automatically performs Flux Balance Analysis and computes different flux graphs for visualization and analysis. The major metabolic pathways for biomass growth or for biosynthesis of any metabolite can be interactively knocked-out, analyzed and visualized as a spanning tree, dendrogram or complete graph using different layouts. In addition, Fluxer can compute and visualize the k -shortest metabolic paths between any two metabolites or reactions to identify the main metabolic routes between two compounds of interest. The web application includes >80 whole-genome metabolic reconstructions of diverse organisms from bacteria to human, readily available for exploration. Fluxer enables the efficient analysis and visualization of genome-scale metabolic models toward the discovery of key metabolic pathways.",
"introduction": "INTRODUCTION Metabolic reconstructions from whole-genome sequencing and biochemical data aim to determine all metabolic processes occurring within a cell or whole organism, which can then be integrated into genome-scale metabolic models (GEMs) able to predict cellular phenotypes ( 1 , 2 ). Constraint-based analytical methods such as Flux Balance Analysis (FBA) can be applied to such models to predict the steady-state metabolic fluxes during cellular growth ( 3 ) and trace the metabolic flux from input nutrients to biomass growth and output metabolites ( 4 ). Several software tools have been developed for performing FBA ( 5 , 6 ). However, the analysis and visualization of the resultant mathematical solutions is a current challenge due to the large number of reactions that a typical GEM contains. This hinders our ability to identify the global metabolic flux through all reactions in the network and understand how the different metabolites participate in different growth phenotypes. The identification of key chemical routes between metabolites of interest, biomass growth and output bioproducts toward metabolic engineering applications requires novel user-friendly pathway analysis tools able to process and efficiently visualize genome-scale models with thousands of reactions and metabolites. Several tools currently exist for the analysis and visualization of GEMs. The Systems Biology Markup Language (SBML) ( 7 ) is the standard format to specify and store GEMs. Cytoscape is a popular network visualization and analysis desktop tool ( 8 , 9 ), for which several plugins are available for processing SBML metabolic models ( 10 , 11 ). However, the resultant networks including all the reactions and metabolites in a GEM are typically a ‘hairball’ of reactions, making it difficult to visualize and understand individual pathways. The visualization tools ModelExplorer ( 12 ) and Grohar ( 13 ) for metabolic networks have a similar limitation when visualizing complete GEMs. Escher ( 14 ) is one of the most widely used tools for GEM interactive visualization, with an easily accessible web-based interface. However, Escher only displays a sub-set of reactions in the model, using predefined maps to position pre-selected metabolites and reactions. A current extension ( 15 ) is also able to perform FBA through the Escher web application, but is still limited to the subset of metabolites and reactions predefined in the layout map. Similarly, Pathview ( 16 ) and its web implementation ( 17 ) can visualize metabolic pathways for GEMs using predefined layout maps for a subset of reactions, which are obtained from KEGG ( 18 ). MetExplore ( 19 ) is a web application able to import and explore GEMs and visualize them with the MetExploreViz component ( 20 ). However, the application is optimized for visualizing small subsets of reactions and is not suitable for visualizing whole GEMs with an automated clean layout that is easy to explore. There is thus a need for user-friendly tools based on robust graph-theory methods ( 21–24 ) that can automatically visualize with efficient and clean layouts a complete GEM and its FBA solution fluxes directly from an SBML model. Here we present Fluxer, a web-application for the automated computation of GEM graphs based on flux and their interactive visualization with a user-friendly interface. The application is able to take as input an arbitrary GEM specified in SBML format and automatically compute and efficiently visualize the complete network and their metabolic fluxes. The tool performs FBA of the model and includes several algorithms to compute optimized visualizations of complete GEMs, including spanning trees, dendrograms and physics-based force layouts. The application can also compute and display the k -shortest metabolic paths between two reactions or metabolites, using a user-customizable metric for the link weights based on the computed metabolic fluxes. The graph visualizations are interactive, and the user can select any metabolite or reaction as the root for the flux tree, obtain information about the reactions and metabolites such as their chemical structures, and easily adjust the node labels, weight calculations, and inclusion or not of cofactors, zero-flux reactions or cellular localizations in the visualized graph. Fluxer allows any user with no programming experience to exploit the potential of genome-scale metabolic models in their ability to assist in the understanding of the whole metabolic network of an organism and predict specific phenotypes. Fluxer’s combined use of FBA and graph theory for metabolic network visualization and analysis in a user-friendly web-application can pave the way for advancements in applications for metabolic engineering.",
"discussion": "DISCUSSION We presented here Fluxer, a user-friendly web application for the computation, analysis and visualization of flux graphs for GEMs. The tool can perform FBA including specific reaction knockouts and compute metabolic network representations based on spanning trees, k -shortest paths and complete graphs. The different networks can be visualized with tree, dendrogram, radial, dagre and force-based layouts. The web application allows any user to load models in SBML format and interact and customize the different metabolic networks generated. Users require no special training or software installations to access and use the web application. The ability of Fluxer to perform visual comparisons of reaction fluxes makes it a powerful tool for understanding metabolic phenotypes and discovering pathways for metabolic engineering applications. While Fluxer computes graph visualizations centered on a particular root metabolite or reaction of interest, other approaches have been proposed for the global analysis of GEMs. Extreme pathways ( 51 ) represent the set of steady state fluxes that lay at the border of the solution space given by the model constraints. As such, they are useful to analyze the full capability of a metabolic model. Elementary flux modes ( 52 ) are the smallest subset of reactions that allow the system to operate in steady state, giving important insights into the most essential pathways within a metabolic model. The MinSpan algorithm ( 53 ) can decompose complete genome-scale models into their most independent pathways. In this way, it serves as an efficient method to understand the pathway structures that a whole model contains. In contrast, the spanning tree computed in Fluxer is focused on finding the most important pathways that contribute to a single root metabolite or reaction by considering the fluxes obtained from a particular FBA solution. Indeed, the spanning tree computed by Fluxer could be combined in future work with these global model analyses to efficiently visualize their resultant pathway structures. Further future work will extend the current functionality of Fluxer. The capabilities to perform FBA customizations beyond the knockout of reactions will be improved to include different optimization objectives and the ability to change the flux boundaries of any reaction—particularly to specify different uptake rates for different growth media. New graph layouts will be implemented, such as maps to highlight the metabolic composition of a particular phenotype state. Finally, the web server will include the possibility for the user to make public any model uploaded to the application, and then list them in a dynamically updated repository of genome-wide metabolic models. Fluxer provides not only a topological solution for visualizing genome-scale metabolic models, but also specific computational methods to analyze reaction networks and fluxes. The comparison of metabolic network connectivity and motifs across organisms can provide evolutionary insights ( 54 ), an approach that could be enhanced by examining metabolic flux networks such as those computed by the presented application. User-friendly tools, such as Fluxer, are able to analyze complex datasets, networks and mechanistic models ( 55 , 56 ), together with novel visualization and encoding software applications ( 57–59 ), and will be essential for the understanding of complex biological mechanisms ( 60–62 ) and the discovery of novel phenotypes ( 63 , 64 ). In conclusion, Fluxer represents a user-friendly resource to visualize and analyze genome-scale flux networks toward the global understanding of whole-organism metabolism and the advancement of the much sought-after applications in metabolic engineering."
} | 2,498 |
20534342 | null | s2 | 5,814 | {
"abstract": "Modern civilization is dependent upon fossil fuels, a nonrenewable energy source originally provided by the storage of solar energy. Fossil-fuel dependence has severe consequences, including energy security issues and greenhouse gas emissions. The consequences of fossil-fuel dependence could be avoided by fuel-producing artificial systems that mimic natural photosynthesis, directly converting solar energy to fuel. This review describes the three key components of solar energy conversion in photosynthesis: light harvesting, charge separation, and catalysis. These processes are compared in natural and in artificial systems. Such a comparison can assist in understanding the general principles of photosynthesis and in developing working devices, including photoelectrochemical cells, for solar energy conversion."
} | 204 |
25562137 | null | s2 | 5,816 | {
"abstract": "Reconstructing metabolic pathways has long been a focus of active research. Now, draft models can be generated from genomic annotation and used to simulate metabolic fluxes of mass and energy at the whole-cell scale. This approach has led to an explosion in the number of functional metabolic network models. However, more models have not led to expanded coverage of metabolic reactions known to occur in the biosphere. Thus, there exists opportunity to reconsider the process of reconstruction and model derivation to better support the less-scalable investigative processes of biocuration and experimentation. Realizing this opportunity to improve our knowledge of metabolism requires developing new tools that make reconstructions more useful by highlighting metabolic network knowledge limitations to guide future research."
} | 206 |
38391423 | PMC10887993 | pmc | 5,817 | {
"abstract": "Lost circulation control remains a challenge in drilling operations. Self-healing gels, capable of self-healing in fractures and forming entire gel block, exhibit excellent resilience and erosion resistance, thus finding extensive studies in lost circulation control. In this study, layered double hydroxide, Acrylic acid, 2-Acrylamido-2-methylpropane sulfonic acid, and CaCl 2 were employed to synthesize organic-inorganic nanocomposite gel with self-healing properties. The chemical properties of nanocomposite gels were characterized using X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscope, X-ray photoelectron spectroscopy and thermogravimetric analysis. layered double hydroxide could be dispersed and exfoliated in the mixed solution of Acrylic acid and 2-Acrylamido-2-methylpropane sulfonic acid, and the swelling behavior, self-healing time, rheological properties, and mechanical performance of the nanocomposite gels were influenced by the addition of layered double hydroxide and Ca 2+ . Optimized nanocomposite gel AC 6 L 3 , at 90 °C, exhibits only a self-healing time of 3.5 h in bentonite mud, with a storage modulus of 4176 Pa, tensile strength of 6.02 kPa, and adhesive strength of 1.94 kPa. In comparison to conventional gel, the nanocomposite gel with self-healing capabilities demonstrated superior pressure-bearing capacity. Based on these characteristics, the nanocomposite gel proposed in this work hold promise as a candidate lost circulation material.",
"conclusion": "3. Conclusions In summary, this study successfully synthesized self-healing LDH/P(AA-AMPS) NC gels by introducing LDH and Ca 2+ into P(AA-AMPS). LDH and Ca 2+ enhanced the thermal stability, rheological properties, and mechanical properties of self-healed gels. In addition, LDH and Ca 2+ also prolonged the self-healing time of LDH/P(AA-AMPS) NC gels, slowed down the swelling rate, and reduced the adhesion strength. A comprehensive comparison of the performance of LDH/P(AA-AMPS) NC gels determined that the optimal sample had the self-healing time of 3.5 h, the G′ value of 4176 Pa, the swelling rate of 86.3 g/g, and the tensile strength of 6.02 kPa. As the temperature increased, the self-healing time of LDH/P(AA-AMPS) NC gels shortened, and the rheological strength of self-healed gels was slightly reduced. In brines, the self-healing time of LDH/P(AA-AMPS) NC gels was prolonged, with a slight decrease in the rheological strength after self-healing. As the concentration of gel particles increased, the self-healing time of self-healed gel shortened, and the rheological strength strengthened. The pressure-bearing capacity of LDH/P(AA-AMPS) NC gel was 3.5 MPa, surpassing the conventional gel’s pressure-bearing capacity of 1.5 MPa. In conclusion, the synthesized LDH/P(AA-AMPS) NC gels in this study provided a novel LCM with self-healing properties, offering a reference for Lost circulation control.",
"introduction": "1. Introduction Lost circulation refers to the phenomenon in drilling operations where drilling fluid within the well-bore leaks into the formation under the influence of pressure differentials [ 1 , 2 , 3 , 4 ]. Lost circulation not only causes delays in drilling operations and extends drilling cycles but also leads to issues such as well collapse, differential sticking, and even serious drilling accidents such as blowouts [ 5 , 6 , 7 ]. Traditional bridging materials widely used to address lost circulation problems have certain limitations, including poor compatibility with fractures and difficulty in forming high-strength plugging layers. Therefore, there is a need to develop new lost circulation material (LCM) with strong adaptability and excellent resistance to scouring [ 8 , 9 ]. Self-healing gel is a type of polymer material that can self-heal after being damaged, partially or completely restoring its original properties [ 10 ]. Self-healing gels can continuously expand, accumulate, and ultimately self-heal to form an entire gel block, achieving the goal of plugging fractures [ 11 ]. Compared to traditional bridging materials, self-healing gels have advantages such as good matching and stability in forming plugging layers [ 9 ]. Research on self-healing gels has been conducted in enhancing oil recovery and lost circulation control [ 12 , 13 , 14 , 15 , 16 ]. However, challenges such as the strength of self-healing gels in fractures and self- healing time for self-healing still need to be addressed [ 13 , 14 , 17 , 18 ]. Shortening the self-healing time of the self-healing gel can reduce non-productive time (NPT) and reduce the cost of construction. Layered double hydroxide (LDH) is an inorganic compound composed of negatively charged anions and positively charged metal hydroxides [ 19 , 20 , 21 ]. Due to their tunable chemical composition and excellent physical properties, LDH have been used as cross-linking agents in the preparation of nanocomposite (NC) gels [ 22 , 23 , 24 ]. To obtain uniform hydrogels, LDH used as cross-linking agents need to be pre-exfoliated or modified to form dispersed nanosheets [ 25 , 26 ]. Chen et al. prepared LDH/polyacrylamide NC hydrogels by stripping LDH monolayer nanosheets and using them as cross-linking agents [ 27 ]. Zhang et al. directly synthesized monolayer LDH nanosheets and prepared NC hydrogels in situ together with N -isopropylacrylamide monomer [ 28 ]. Currently, research on LDH/polymer NC hydrogels had mostly focused on introducing LDH nanosheets into hydrogels to enhance mechanical performance and stability [ 25 , 28 ]. However, there are few studies on utilizing the reversible dynamic cross-linking between LDH and polymers to form self-healing gels. The positively charged layer of LDH and the abundant hydroxyl groups on the surface can form reversible physical cross-linking such as electrostatic interaction and hydrogen bonding with polymers containing anionic groups. Therefore, the introduction of nanomaterial LDH could not only improve the mechanical properties of the gel but also form a gel with rapid self-healing through reversible dynamic bonds. To avoid the disadvantages of low strength of traditional preformed particle gel and uncontrollable gelation time of in situ formed gels, in this study, LDH was dispersed in the mixed solution of Acrylic acid (AA) and 2-Acrylamido-2-methylpropane sulfonic acid (AMPS), utilizing the dynamic cross-linking properties between LDH and copolymer of AA-AMPS (P(AA-AMPS)) to synthesize NC gels with self-healing properties. The chelation interaction between Ca 2+ and the anion functional group was introduced to further enhance the mechanical properties. The LDH/P(AA-AMPS) NC gels exhibited excellent mechanical and self-healing properties, demonstrating superior pressure resistance compared to conventional gels. Different from the traditional LDH NC gel, the introduction of LDH in LDH/P(AA-AMPS) NC gels not only improves the performance of the gel but also endows the gel with self-healing properties as a dynamic cross-linking agent. Thus, LDH/P(AA-AMPS) NC gels provide a novel LCM for lost circulation control.",
"discussion": "2. Results and Discussion 2.1. Synthesis of LDH/P(AA-AMPS) NC Gels The synthesis of LDH/P(AA-AMPS) NC gels involved two main steps: the dispersion of LDH and the monomer polymerization, as depicted in Figure 1 a. LDH appeared transparent in the AA-AMPS solution with a distinct Tyndall effect ( Figure 1 b), indicating that the solution hadcolloidal properties [ 26 , 29 ]. As shown in Figure 1 c, the transmission electron microscope (TEM) test of LDH in AA-AMPS solution showed a low contrast, indicating that LDH was partially exfoliated in AA-AMPS solution [ 28 ]. This was attributed to the ion exchange between COO − , SO 3 − , and CO 3 2− of LDH. Subsequently, in situ formation of P(AA-AMPS) was initiated by APS adsorbed on LDH [ 26 ]. The LDH/P(AA-AMPS) NC gels were cross-linked through electrostatic interactions, coordination interactions, and hydrogen bonding. Additionally, chelation structures formed by the metal ion with carboxyl groups on the P(AA-AMPS) molecular chains further enhanced the intermolecular cross-linking of the gel. 2.2. Characterization of LDH/P(AA-AMPS) NC Gels X-ray diffraction (XRD) is a typical method for characterizing crystal structures. The XRD patterns of samples with LDH are shown in Figure 2 a. The peaks around 21.1° of AC 6 L 3 and AC 0 L 3 were attributed to the amorphous P(AA-AMPS) [ 30 ]. Furthermore, AC 6 L 3 and AC 0 L 3 exhibited no characteristic diffraction peaks of LDH, indicating the absence of a large number LDH with ordered structure in the gel [ 28 ]. Fourier transform infrared spectroscopy (FT-IR) characterization of functional groups in LDH/P(AA-AMPS) NC gels is presented in Figure 2 b. The LDH showed characteristic bands at 3451 and 1361 cm −1 , which corresponded to the bending vibration of –OH and the CO 3 2− in the LDH interlayer [ 31 ]. For AC 0 L 3 and AC 6 L 3 , the absorption peak at 2931 cm −1 corresponded to the stretching vibration peak of C–H. The absorption peaks at 1699 cm −1 and 1548 cm −1 were attributed to the stretching vibration of C=O and the bending vibration peak of N–H, respectively. The absorption peaks at 1154 cm −1 and 1039 cm −1 represented the infrared characteristic absorption peak of –SO 3 H and the vibration absorption peak of C–H, respectively [ 32 ]. Compared with LDH, the –OH and CO 3 2− peaks of AC 0 L 3 and AC 6 L 3 at 3451and 1361 cm −1 disappeared, indicating that LDH dispersed in the AA-AMPS solution participated in the cross-linking of the gel network [ 30 , 31 , 33 ]. X-ray photoelectron spectroscopy (XPS) characterization of AC 6 L 3 samples was conducted to determine the elemental composition and chemical states. The full spectrum revealed the presence of C, O, Ca Mg, and Al elements in AC 6 L 3 , as seen in Figure 2 c. Figure S1 shows the high-resolution spectra of C 1s, O 1s, and Ca 2p. The high-resolution C 1s spectrum at 284.8 eV, 286.3 eV and 288.7 eV corresponded to C–C, C–O, and O=C–O. Peaks at 530.7 eV, 532.1 eV, 533.5 eV, and 534.3 eV in the O 1s spectrum were attributed to metal-O, C–O, C=O, and O–H [ 34 ]. For Ca 2p, the peaks at 346.9 eV and 350.5 eV corresponded to the characteristic peaks of COO–Ca [ 30 ]. These results indicate the successful incorporation of inorganic nanomaterial LDH and Ca 2+ into the gel network. To investigate the thermal stability of LDH/P(AA-AMPS) NC gels, thermogravimetric analysis (TGA) testing was conducted on AC 0 L 0 and AC 6 L 3 under a nitrogen atmosphere ( Figure 2 d). The TG curve showed that the weight loss of AC 0 L 0 and AC 6 L 3 was divided into three stages of mass loss. For AC 6 L 3 , the weight loss from room temperature to168 °C was 4.09%, attributed to the removal of absorbed and bonded water. From 168 °C to 305 °C, the 25.71% weight loss involved dehydroxylation of LDH, decomposition of carboxyl groups on the molecular chain. Beyond 305 °C, the main chain of sulfonic groups, the destruction of cross-linked network structure, and the layered structure of LDH were disrupted [ 35 , 36 ]. The weight losses of AC 0 L 0 in the three weight loss stages were 5.76%, 38.18%, and 52.06%, respectively. In addition, the residual amount of AC 6 L 3 was higher than that of AC 0 L 0 . This indicates that AC 6 L 3 exhibited superior stability with the introduction of LDH and Ca 2+ . The microstructures of LDH/P(AA-AMPS) NC gels were investigated and scanning electron microscope (SEM) images of the samples after freeze–drying are presented in Figure 3 . AC 0 L 3 sample exhibited a porous and interconnected three-dimensional network structure, while the AC 3 L 3 sample had denser pore sizes. With increasing Ca 2+ content, pore sizes gradually decreased, indicating increased cross-linking. This was attributed to the chelation structure formed by COOH on P(AA-AMPS) polymer chains coordinating with Ca 2+ , enhancing the cross-linking of the samples [ 37 ]. Compared to AC 6 L 0 , AC 6 L 1.5 showed thicker pore walls and fewer pores. Moreover, with an increase in LDH content, pore sizes decreased, and pore walls thickened. This was due to the electrostatic, coordination, and hydrogen bonding interactions between LDH and P(AA-AMPS), enhancing the cross-linking of the samples [ 28 , 29 ]. 2.3. Self-Healing Properties of LDH/P(AA-AMPS) NC Gels The self-healing performance of LDH/P(AA-AMPS) NC gels was the key factor affecting the pressure bearing capacity. Thus, the self-healing performance of LDH/P(AA-AMPS) NC gels was tested by adding a 10% concentration of LDH/P(AA-AMPS) NC gels particles into bentonite mud. As shown in Figure 4 a, at 90 °C, MAA remained as independent gels after expansion, unable to self-heal. AC 0 L 0 has obvious fluidity and cannot fix the shape, while AC 6 L 0 , AC 0 L 3 and AC 6 L 3 were able to self-heal into an entire gel after water absorption and expansion in bentonite mud. Taking AC 6 L 3 as an example, the expanded AC 6 L 3 gel in bentonite mud and bentonite slurry + rhodamine obviously became an entire gel composed of different colors after self-healing, which further proved the self-healing properties ( Figure 4 b). However, as shown in Figure 5 a, the self-healing time of AC 6 L 1.5 at 90 °C was 2 h, longer than the 1 h for AC 6 L 0 . As the amount of LDH added was 3 g, the self-healing time was extended to 3.5 h. Moreover, as the LDH content increased, the self-healing time of AC 6 L 9 was further extended to 7 h. At the same time, the self-healing time of AC 0 L 3 and AC 3 L 3 was 2 h and 3 h, respectively. Further increasing the CaCl 2 addition to 9 g extended the self-healing time to 4.5 h ( Figure 5 b). The introduction of LDH and Ca 2+ increased the cross-linking density of the gel, restricting the movement of molecular chains [ 38 ]. Furthermore, excessive LDH led to shorter polymer chain lengths in the cross-linked polymer, resulting in reduced healing efficiency [ 39 , 40 ]. Rheological and mechanical strengths are crucial indicators for evaluating the stability of gels in fractures. Therefore, the rheological and mechanical properties of the gel after self-healing at 90 °C were tested. Figure 5 c,d shows the strain sweep curves of the self-healed gel in the range of 0% to 1000%. In the early stage of strain increase, the storage modulus (G′) was higher than the loss modulus (G″), indicating predominantly elastic solid characteristics and a stable gel network [ 41 , 42 ]. However, as the strain continued to increase, the G′ gradually decreased, indicating the gradual breakdown of the gel structure [ 43 ]. In the linear viscoelastic region, the G′ value for AC 6 L 0 was 1488 Pa, while the G′ values for AC 6 L 1.5 , AC 6 L 3 , and AC 6 L 4.5 were 3305 Pa, 4176 Pa, and 3848 Pa, respectively ( Figure S2 ). This suggests that with increasing LDH, the G′ value of self-healed gels initially increased and then decreased. This is attributed to the formation of an organic–inorganic NC gel and increased cross-linking degree through introducing LDH, enhancing the rheological strength, while excessive cross-linking negatively affects the rheological performance [ 27 , 28 ]. Additionally, increasing Ca 2+ also improved the rheological performance of the self-healed gel; as the Ca 2+ content increased from 3 g to 9 g, the G′ value of the self-healed gel increased from 3706 Pa to 4416 Pa ( Figure S3 ). This is attributed to the chelation structure formed by Ca 2+ with anionic groups on the P(AA-AMPS) chain [ 44 ]. Figure 5 e,f presents the frequency sweep test results for the self-healed gels in the range of 0.1 to 100 rad/s. With increasing frequency, the G′ value of the self-healed gels showed a noticeable upward trend. This indicates that the difference between moduli changes with frequency, further demonstrating the dynamic cross-linking structure of LDH/P(AA-AMPS) NC gels [ 45 , 46 ]. The tensile performance of the self-healed gels is shown in Figure 6 a,b. As the LDH content increased from 0 to 3 g, the fracture strain of the self-healed gel gradually increased from 183% to 448%, and the fracture stress increased from 1.03 kPa to 6.02 kPa. However, further increasing the LDH amount to 4.5 g resulted in a decrease in fracture strain to 363%, with a decrease in fracture stress to 5.73 kPa. The reason for this change was that excessive LDH lead to over-cross-linking, thereby affecting mechanical properties [ 47 , 48 ]. Meanwhile, increasing Ca 2+ from 0 to 9 also increased the fracture strain of the self-healed gel from 3.41 kPa to 6.56 kPa. This result was consistent with rheological testing and SEM characterization, further indicating that the introduction of LDH and Ca 2+ increases the cross-linking density of LDH/P(AA-AMPS) NC gels, enhancing rheological and mechanical properties. As shown in Figure 6 c, AC 6 L 3 self-healed gel had adhesion properties to the shell and artificial core, which may be attributed to the hydrogen bonding between COOH and the surface of the material [ 49 ]. From the adhesion strength of different self-healed gels to artificial core, shown in Figure 6 d,e, it can be found that the adhesion strength of AC 6 L 3 self-healed gel was 1.94 kPa, and the adhesion strength of self-healed gel decreased with the increase in LDH and Ca 2+ . This was attributed to the introduction of LDH and Ca 2+ , resulting in a decrease in free carboxyl groups, thereby reducing the adhesion strength [ 50 ]. It also reflected the interaction between LDH, Ca 2+ and P(AA-AMPS). Through a comprehensive comparison of self-healing time, rheological, mechanical and adhesion strength of different samples, AC 6 L 3 was identified as the optimal sample and further evaluated. Temperature has a crucial effect on the properties of self-healing gels. The self-healing performance of AC 6 L 3 was investigated at 70 °C, 90 °C, and 110 °C. As shown in Figure 7 a, the self-healing time of AC 6 L 3 at 70 °C was 6.5 h, and as the reaction temperature increased to 90 °C and 110 °C, the self-healing time of AC 6 L 3 shortened to 3.5 h and 2 h, respectively. This was because, as the temperature rose, the migration rate of P(AA-AMPS) molecular chains, Ca 2+ , LDH between contacting gels increased, making P(AA-AMPS) molecular chains more prone to recombine with Ca 2+ and LDH [ 51 ]. The G′ value of AC 6 L 3 self-healed gel at 70 °C and 90 °C was 4251 Pa and 4176 Pa, respectively ( Figure S4 ). However, with the reaction temperature increasing to 110 °C, the G′ value decreased to 3807 Pa ( Figure 7 b). This was attributed to the partial breakdown of the AC 6 L 3 molecular chains’ structure at high temperatures, leading to a decrease in rheological performance. As drilling often encounters saline formations, it was essential to evaluate the self-healing performance of LDH/P(AA-AMPS) NC gels in brines. As shown in Figure 7 c,d, the self-healing time of AC 6 L 3 in 5% NaCl, 10% NaCl, and 2% CaCl 2 was 5.5 h, 9 h, and 5 h, respectively, and the G′ values of self-healed gels were 3719 Pa, 3502 Pa, and 3917 Pa, respectively ( Figure S5 ). Compared with the self-healing performance in salt-free bentonite mud, the self-healing time was prolonged, and the G′ value of self-healed gels was slightly decreased. This was attributed to the fact that the gel’s expansion rate in brines slowed down, and brines affected the coordination between the P(AA-AMPS) and LDH, Ca 2+ . The gel network was disrupted in brines, accelerating dissociation [ 52 , 53 ]. The self-healing performance of AC 6 L 3 at different concentrations in bentonite mud was investigated and the result is shown in Figure 7 e,f. As the AC 6 L 3 gel particle concentration increased from 5% to 20%, the self-healing time decreased from 7.5 h to 2 h, and the G′ value of the self-healed gel increased from 1671 Pa to 5109 Pa ( Figure S6 ). 2.4. Swelling Behavior of LDH/P(AA-AMPS) NC Gels The swelling behavior of different LDH/P(AA-AMPS) NC gels particles in bentonite mud was tested. As shown in Figure 8 a,b, the initial swelling degree of the gel rapidly increased, then gradually decreased, eventually reaching equilibrium. This was primarily due to the reorganization of dynamic bonds within the gel during the swelling process, leading to an increased cross-linking density and a more stable network structure [ 38 ]. Moreover, the equilibrium swelling ratio of AC 6 L 0 , AC 6 L 1.5 , AC 6 L 3 , and AC 6 L 4.5 were 113.6 g/g, 101.5 g/g, 86.3 g/g, and 73.1 g/g, respectively. The equilibrium swelling ratio of AC 0 L 3 , AC 3 L 3 , AC 6 L 3 , and AC 9 L 3 was 106.2 g/g, 97.4 g/g, 86.3 g/g, and 76.5 g/g, respectively. The equilibrium swelling ratio of LDH/P(AA-AMPS) NC gels decreased with increasing LDH and Ca 2+ . This phenomenon was attributed to the interactions between P(AA-AMPS) and LDH, Ca 2+ , resulting in a denser internal network structure of the gel that impedes water infiltration [ 22 , 27 , 54 ]. The swelling behavior of AC 6 L 3 was investigated at different temperatures, and the results are presented in Figure 8 c. The equilibrium swelling ratios of AC 6 L 3 gel particles at 70 °C, 90 °C, and 110 °C were 106.5 g/g, 128.3 g/g, and 136.5 g/g, respectively. As the temperature increased, the equilibrium swelling ratio of AC 6 L 3 gel particles gradually increased, accompanied by a reduction in the time to reach equilibrium swelling. 2.5. Plugging Performance of LDH/P(AA-AMPS) NC Gels The plugging performance is a critical parameter for evaluating LCM. Therefore, the metal slit plate was employed to assess the pressure-bearing capabilities of the LCM. Figure 9 a,b showed the plugging properties of self-healing gel AC 6 L 3 and conventional gel MAA. At 90 °C, the pressure-bearing capacity of AC 6 L 3 reached 3 MPa, surpassing conventional gel MAA with a capacity of only 1.5 MPa. As illustrated in Figure 9 c,d, AC 6 L 3 exhibited the ability to adhere and self-heal, and form an entire gel block in the metal slit plate, while MAA was still an independent gel. Consequently, the self-healed entire gel block can more effectively fill and plug fractures, thereby enhancing the pressure-bearing performance of the LCM [ 11 ]. The plugging mechanism of the self-healing gel AC 6 L 3 in formation fractures was deduced as follows: Initially, the deformable AC 6 L 3 , subjected to pressure differentials, enters the fractures and gradually expands and accumulates. Stimulated by the temperature of leakage layer, molecular chain movements intensify among the inter-contacted yet independent gel segments. P(AA-AMPS), LDH, and Ca 2+ migrated between gel interfaces and progressively reassembled to form dynamic bonds under the cross-linking of electrostatic interactions, coordination interactions, and hydrogen bonding ( Figure 9 e), thereby self-healing into an entire gel block and achieving lost circulation control."
} | 5,742 |
22727142 | PMC3408363 | pmc | 5,819 | {
"abstract": "Background Microbial anaerobic digestion (AD) is used as a waste treatment process to degrade complex organic compounds into methane. The archaeal and bacterial taxa involved in AD are well known, whereas composition of the fungal community in the process has been less studied. The present study aimed to reveal the composition of archaeal, bacterial and fungal communities in response to increasing organic loading in mesophilic and thermophilic AD processes by applying 454 amplicon sequencing technology. Furthermore, a DNA microarray method was evaluated in order to develop a tool for monitoring the microbiological status of AD. Results The 454 sequencing showed that the diversity and number of bacterial taxa decreased with increasing organic load, while archaeal i.e. methanogenic taxa remained more constant. The number and diversity of fungal taxa increased during the process and varied less in composition with process temperature than bacterial and archaeal taxa, even though the fungal diversity increased with temperature as well. Evaluation of the microarray using AD sample DNA showed correlation of signal intensities with sequence read numbers of corresponding target groups. The sensitivity of the test was found to be about 1%. Conclusions The fungal community survives in anoxic conditions and grows with increasing organic loading, suggesting that Fungi may contribute to the digestion by metabolising organic nutrients for bacterial and methanogenic groups. The microarray proof of principle tests suggest that the method has the potential for semiquantitative detection of target microbial groups given that comprehensive sequence data is available for probe design.",
"conclusion": "Conclusions Our results show that both the mesophilic and thermophilic AD process contain a prominent fungal community that survives and grows in anoxic conditions. This suggests that Fungi may metabolise organic nutrients for subsequent use by archaeal and bacterial methanogenic groups, thus contributing to the digesting process and biogas production. The microarray proof of principle testing showed the capability of the technique to profile the microbial composition of AD samples. According to our results, the microarray method is capable of semiquantitative analysis of AD process when comprehensive sequence information is available to support probe design. We expect future metagenomic sequencing of the total genomic content in these environments to enable more accurate probe design and, together with RNA sequencing, to help determining the ecology and metabolic functions of various fungal and other microbial groups present in the AD community.",
"discussion": "Results and discussion Biogas production Anaerobic codigestion of biowaste and sewage sludge was performed with organic loading rates from 1 to 10 kg of VS m -3 d -1 in in mesophilic (M1 and M2) and thermophilic (M3 and M4) conditions. In the steady state conditions, i.e. the biogas production is not changed over time due to the load increase but has reached a constant level, the biogas production at the load of 3 kg VS m -3 d -1 was 680 and 760 liters kg -1 VS -1 in the mesophilic and thermophilic runs, respectively (Table 2 ). In both temperatures the specific biogas production was lower at the loads of 5–8 kgVS m -3 d -1 than that with 3 kg VS m -3 d -1 load. The CH 4 concentration varied between 61.7 -68% in the both runs. The amounts of trace gases, especially ethanol and ammonia, increased in the thermophilic conditions. Overview of microbial diversity in AD Selected samples from the outfeed of meso- (M1 and M2) and thermophilic (M3 and M4) pilot AD reactors at the loading rates of 3 and 5–8 kg VS m -3 d -1 were subjected to microbial diversity analysis using 454 rRNA gene amplicon deep sequencing. A total of 77 189 sequences out of 83 975 sequence reads were classified based on BLASTN results. The total number of sequence reads that passed quality check ranged from 2 000 in Bacteria to almost 17 000 in Fungi per sample (Table 3 ). Figure 2 summarises the most abundant archaeal, bacterial and fungal groups present in the samples. Rarefaction analysis (Additional file 1 ) revealed that the fungal diversity increased together with increasing loading rate and decreasing retention time during the experiment, and Chao1 and Ace [ 27 , 28 ] richness estimates supported this observation (Table 3 ). In Bacteria, the trend in rarefaction analysis was the opposite, thus declining during the digestion process. Richness estimates in the mesophilic process backed up this result whereas in the thermophilic conditions the numbers were contradictory (Table 3 ). In Archaea, the diversity decreased during the experiment in the mesophilic and increased in the thermophilic reactor (Table 3 ). Several studies have shown that mesophilic AD process carries more microbial diversity than thermophilic process and that temperature affects the community composition of microbial communities [ 6 , 44 - 49 ]. In this study, rarefaction analysis (Additional Figure 1 ), richness estimates and diversity indices (Table 3 ) indicated approximately equal diversity in both temperatures. However, at class and genus level more bacterial classes and genera and archaeal genera were found in the mesophilic reactor than in the thermophilic reactor. Based on UPGMA (Unweighted Pair Group Method with Arithmetic mean) clustering [ 50 ] (data not shown), the bacterial and archaeal communities were more similar between the mesophilic samples (M1 and M2) than the thermophilc samples (M3 and M4), suggesting that bacterial and archaeal communities in the study reactors were strongly driven by temperature. In contrast, the fungal communities became more pronounced during the digestion process: the M1 and M3 samples taken in the beginning of the experiment from different reactors were more similar to each other than to M2 and M4 samples, suggesting that organic loading rate is a more important factor in determining the fungal community structure than the process temperature. As the digester was a completely stirred tank reactor, the new feed material is constantly mixed with old material while the mixture is being washed out. The operating time span before sampling was over one HRT in samples M1 and M3 and slightly less one HRT in samples M2 and M4 (Table 1 , Figure 1 ). Due to constant stirring, this difference is not likely to have a major effect on the reactor microbiota. The minimum HRT used in this study was 9–10 days which is approximately the same as the generation time of methanogens and other microbial groups and as such is sufficient for proper decomposition of organic material. The efficiency of the degradation was also illustrated by the fact that no accumulation of degradation intermediates, i.e. VFA, occurred. Bacterial diversity The mesophilc (M1 and M2) and thermophilic (M3 and M4) samples contained in total 15 bacterial phyla. Most commonly found bacterial phyla included Bacteroidetes Firmicutes and Thermotogae , constituting 47%, 24% and 9% of all bacterial sequence reads, respectively. The phylum Bacteroidetes was more abundant in the mesophilic reactor, and the bacterial classes of Flavobacteria Sphingobacteria and Bacteroidia were found solely from the mesophilic reactor. Clostridia and Bacilli , the two classes of Firmicutes , were detected in both reactors but were more prevalent in thermophilic conditions, and Thermotogae was detected exclusively in the thermophilic reactor. Different classes of Proteobacteria and Actinobacteria were found in thermophilic conditions in quite small numbers, but these groups were substantially more abundant in the mesophilic reactor. Spirochaetes Synergistes and Verrucomicrobia were present only in the mesophilic reactor. We also detected several bacterial phyla comprised merely of environmental clones including OP8, OP11, SR1 and TM7. Somewhat concordant results regarding the heterotrophic bacteria in anaerobic digestors have been published before [ 51 - 54 ] . Bacterial phyla Bacteroidetes Firmicutes and Thermotogae are often found in both mesophilic and thermophilic AD processes which reflects their importance in degradation of complex organic compounds [ 6 ]. Bacterial genera frequently encountered in AD include Spirochaeta sp., Clostridium sp., Propionibacterium sp., Thermotoga sp., Arthrobacter sp. and Bacillus sp. [ 8 ]. In the present study, 7% of all bacterial sequence reads were classified to genus level. All in all, we identified a total of 19 bacterial genera. The most common bacterial genus was Clostridium , present in all samples but more abundant in the thermophilic reactor. Genus Clostridium contains species that are capable of anoxic digestion of cellulose and fermenting amino acids, and these bacteria are commonly found in different types of anaerobic digesters [ 55 ]. In several earlier studies members of order Clostridiales have been detected to represent a dominant fraction of bacterial communities in AD and these bacteria are recognised important in biogas production [ 56 - 58 ]. Coprothermobacter sp. and Syntrophomonas sp. were also relatively common, with Coprothermobacter found solely in thermophilic and Syntrophomonas in both reactors. Archaeal diversity We were able to identify 89% of all archaeal reads at phylum level and 34% at genus level. All the Archaea classified at phylum level belonged to phylum Euryarchaeota . This is in agreement with other descriptions of archaeal composition of anaerobic sludge where Euryarchaeota clearly dominate over Crenarchaeota , and orders Methanosarcinales and Methanomicrobiales are known to represent an eminent proportion of the Archaea present [ 59 ]. The two identified methanogenic classes were Methanobacteria and Methanomicrobia . These methanogens were found at both temperatures, although Methanobacteria were more prevalent in the thermophilic conditions (M3 and M4) than in the mesophilic conditions (M1 and M2). These classes represent typical archaeal constituents in methanogenic AD systems [ 54 ]. We identified also six different archaeal genera in our dataset based on BLAST against nr / nt database. Methanosarcina was very abundant, and slightly more common in the mesophilic process. Methanobrevibacter Methanosphaera Methanospirillum and Methanosphaerula were abundant in mesophilic digestor (M1 and M2), while Methanobacterium was detected merely in thermohilic digestor (M3 and M4). In agreement with our study, Goberna and co-workers also found an increase of Methanobacteria in thermophilic AD [ 60 ]. Several studies have shown that Methanosarcina sp., Methanococcus sp. Methanoculleus sp., Methanomethylovorans sp. and Methanobacterium are typically found in anaerobic digesters [ 4 , 6 , 8 - 11 ]. Fungal diversity We identified 85% of the fungal sequences at phylum level and 44% at genus level. The Fungi detected in our study belonged to two phyla, Ascomycota and Basidiomycota . The sequence reads assigned to Ascomycota represented almost 99% of the fungal sequences and consequently, Basidiomycota constituted about 1% of the fungal reads. Saccharomycetes and Eurotiomycetes were the most abundant fungal classes in the whole dataset, constituting 58% and 12% of the fungal sequence reads, respectively. These classes were found in both temperatures, with Saccharomycetes being more abundant in the thermophilic digestor (M3 and M4) and Eurotiomycetes in the mesophilic digestor (M1 and M2) (Figure 2 ). A total of 33 fungal genera were detected. By far the most abundant was Candida, found in both processes at both samplings, but especially prevalently in the thermophilic reactor. The second most common fungal genus, Penicillium , was present in all samples but notably more thriving in the mesophilic reactor where it constituted the majority of all fungal sequence reads. The third most common fungus Mucor was found in all samples as well, but it seemed to prefer elevated thermophilic temperatures. In fact, several fungal groups, like Zygorhynchus Cladosporium and Pseudeurotium were found solely in the thermophilic conditions, whereas for example Rhizomucor Geotrichum and Trichosporon were found exclusively in the mesophilic reactor. The relative abundance of fungal groups like Pichia Saccharomyces Aspergillus Mucor and Candida increased during the digestion process, indicating that these fungal groups not only tolerate the conditions in the reactors but may actually benefit from them. Pichia and Candida are also associated in aerobic digestion [ 61 ]. Schnürer and Schnürer [ 12 ] recently studied fungal survival in anaerobic digestion of household waste and found out that mesophilic temperature did not reduce the amount of culturable fungal colony forming units in the waste, and that thermophilic conditions caused only a slight decrease in the number of fungal viable cells. This phenomenon was not detected in our study, but actually the thermophilic digestor (M3 and M4) contained more fungal diversity in both samplings compared to the mesophilic digestor (M1 and M2, Figure. 2 ). The majority of Fungi are aerobic, but a wide range of them are able to grow in low oxygen conditions. There are also fungi that can survive and grow in anaerobic conditions if an appropriate nutrient source is available. The fungal genera Candida Mucor Penicillium Saccharomyces and Trichoderma , detected in our study, are facultative anaerobes and as such capable of degrading organic material in anoxic environment [ 62 - 64 ]. Thus, these groups can potentially not only survive the anaerobic conditions but also actively contribute to the process by decomposing more complex organic compounds such as lignin and cellulose in the beginning of the degradation. Functional validation of the microarray probes Microarray as a high-throughput platform has the potential for routine microbial analysis of environmental samples [ 65 - 67 ], although detection accuracy of oligomeric probes targeting phylogenetic marker gene may present a challenge in analysing complex communities consisting of a large number of closely related genomes [ 16 ]. Assaying the microbial composition in the AD process would be valuable for in-process monitoring of the microbial content and confirming hygienisation of the end product. To that end, we applied ligation probes that circularize upon target recognition (“padlock probes”) and are subsequently amplified and hybridised on microarray by unique tag sequences (Figure. 3 ). In principle, the method enables detection from unamplified source material and has been previously successfully used for plant pathogen detection on qPCR [ 68 ] and microarray platforms [ 69 ] as well as for gene variant analysis [ 70 , 71 ]. However, to our knowledge, this type of technique has not been applied to profiling complex microbial communities to date. Here, we tested a set of padlock probes to evaluate the potential of the method for AD process monitoring and more generally for microbial community analysis (Figure 4 ). In order to establish the functionality and target sequence specificity of the probes, we used 10 fmol of probe-specific synthetic dsDNA oligos as templates for the probe pool in ligation reactions. Signals from the subset of probes corresponding to the templates present in each pool could be clearly distinguished from signals from the rest of the probes (Additional file 4 ), suggesting a good target sequence specificity. However, the signal intensities of different probes varied considerably at the constant 10 fmol template concentration, probably because of random variability of PCR [ 72 ] and sequence bias of ligation [ 73 , 74 ]. Approximately 10% of the probes were not functional despite their perfect alignment to template. Six probes were non-specific giving false positive signals, despite that they did not have good alignment to any of the templates. To estimate the amount of detectable template, we tested template pools each containing 24 templates, at four different concentrations each. The probe signal intensities correlated with concentration (Additional file 5 ) with the highest concentration (1 fmol/μl/template) giving the highest signals while at the lowest concentration (0.001 fmol/μl/template) practically none of the probes produced detectable signals. Almost all of the probes had consistently lower signals with lower concentrations and the majority of probes were still detectable at 0.01 fmol/μl/template concentration, suggesting that the method may be used for semiquantitative assaying over at least three orders of magnitude. Figure 4 Comparison of sequencing, microarray and qPCR. Performance of probe A123 on samples M1, M2, M3 and M4. ( a ) Relative abundance of sequencing reads corresponding to microarray probe A123 bacterial target groups, ( b ) microarray signal intensities and ( c ) TaqMan assay using the same probe sequence. Microarray analysis of the AD samples To evaluate the microarray’s capability in analysing the AD samples, we performed ligation reactions using about 200 ng of non-amplified sample DNA as template for the probe pool. The microarray signals from the mesophilic samples M1 and M2 and the thermophilic samples M3 and M4 grouped separately and along the gradients of physical and chemical parameters in a similar way as with sequencing data (Figure 5 ) in redundancy analysis [ 16 ]. This suggests that our microarray had the ability to monitor changes in the microbial community structure in response to conditions of the digestor, an important aspect of in-process monitoring of AD status. However, while the grouping with M1 and M2 was comparable to sequencing data, M3 and M4 clustered less clearly separately showing that the microarray was not as accurate in classifying samples as deep sequencing with regard to process loading rate. The reason for this could be that most of the microarray probes did not show detectable signals. The probes were initially designed to match certain phylotypes or phylotype-level OTUs (97% read sequence similarity), but as these typically corresponded to relatively few sequences in the sample material, the target sequence abundances were likely to be below detection limit of the method. Also, specific microarray probes could not always be designed merely on the basis of trimmed 454 sequence reads due to their limited length of 150 nt, which necessitated us to retrieve full-length rRNA genes matching to OTUs from the NCBI nucleotide database. The closest matching gene to an OTU was typically only 94% similar, leaving considerable uncertainty regarding the estimated target specificity of the probes in the context of the AD sample DNA. Probe sequence alignments against the most abundant full-length database rRNA genes identified in the samples showed that many of the probes indeed did not have good matches. As expected under the probe-target sequence mismatch hypothesis, the probes that could be aligned with mismatches to the database rRNA genes were less accurate (Additional file 6 ) than 100% matching probes. Since the probes in the initial specificity tests responded highly accurately to their cognate target oligo pools, it is reasonable to assume that at least some missing signals are explained by unknown sequence differences in the rRNA genes. Secondary structures inherent to rRNA sequences are one possible contributor to probe target recognition [ 75 ] as well. However, we found complementarity within the probe pool only between two sequences (data not shown), but this does not completely rule out the possibility of dimerisation between other probes too, as alignment cannot fully explain oligo hybridisation behaviour. However, with 100% match to target sequences the signals were more consistent. Figure 4 shows microarray signals of a probe matching to several full length rRNA genes of uncultured bacterial groups, and corresponding relative number of 454 reads of these targets. The signals correlated with read number and TaqMan RT-qPCR signals obtained using the same probe sequence, thus verifying the microarray results. This proof of principle data suggests that the microarray method is capable of semiquantitative assaying of target microbial groups, provided the target sequences constitute at least 1% of total DNA in the sample as measured by amplicon sequence reads. Furthermore, the results show that sensitivity of the padlock method is clearly better compared to the traditional ligation detection reaction (LDR), which requires PCR amplification of the target sequences first, and is not able to detect targets directly from source DNA [ 66 ]. Figure 5 Ordination of microbial composition together with physical and chemical parameters of AD samples. Redundancy analysis (RDA) was used to explore the main trends in the data. The canonical axes represent principal components. Sample (M1-M4) locations relative to each other indicate their similarity in the ordination space. Red squares indicate microbial groups in sequence data ( a and b ) and probes in microarray data ( c and d ), with the numbers indicating the microarray probes listed in the Additional file 2 . Only the most abundant groups or strongest probe signals were included in the analysis. Blue arrows indicate the physical and chemical parameters used as constraining variables in the analysis (from Tables 1 and 2 ). The length and position of an arrow illustrates its significance on the canonical axes."
} | 5,414 |
22043295 | PMC3197185 | pmc | 5,820 | {
"abstract": "Biofuels derived from algal lipids represent an opportunity to dramatically impact the global energy demand for transportation fuels. Systems biology analyses of oleaginous algae could greatly accelerate the commercialization of algal-derived biofuels by elucidating the key components involved in lipid productivity and leading to the initiation of hypothesis-driven strain-improvement strategies. However, higher-level systems biology analyses, such as transcriptomics and proteomics, are highly dependent upon available genomic sequence data, and the lack of these data has hindered the pursuit of such analyses for many oleaginous microalgae. In order to examine the triacylglycerol biosynthetic pathway in the unsequenced oleaginous microalga, Chlorella vulgaris , we have established a strategy with which to bypass the necessity for genomic sequence information by using the transcriptome as a guide. Our results indicate an upregulation of both fatty acid and triacylglycerol biosynthetic machinery under oil-accumulating conditions, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of an unsequenced microalga.",
"conclusion": "Conclusions The prevalence of microalgal translational gene regulation necessitates higher-level omic analyses at the protein level in order to fully elucidate changes in gene expression under varying conditions. However, proteomic analysis of unsequenced microalgae is clearly limited by the lack of flexibility in fragment matching. Our results underscore how much more powerful proteomic analysis can be when accurate sequence information is available, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of unsequenced microalgae. Strain improvement strategies targeting increased lipid accumulation and productivity as well as improved understanding of the relevant basic biology will be critically enhanced by utilization of our transcriptomic sequence data combined with proteomic abundance data. We have focused our initial investigation of differential protein expression upon dramatically different lipid accumulation states (10% vs. 60% fatty acid) in N-replete and deplete C. vulgaris . These analyses indicate that the fatty acid and TAG biosynthetic pathways are dramatically upregulated (TAG>fatty acid) under nitrogen limitation. Data from intermediate accumulation states will likely provide a wealth of additional information with regards to the stages at which gene and protein-expression are initiated. Carbon flux analyses, glycerolipid speciation, and metabolomic analysis will ultimately need to be initiated to complement comparative transcriptomic and proteomic analyses, in order to fully assess flux through lipid-relevant pathways of interest on a comprehensive systems biology level.",
"introduction": "Introduction Oleaginous microalgae produce substantial amounts of neutral lipids, primarily comprised of triacylglycerides (TAGs) of favorable fatty acid chain length, making them an ideal feedstock for conversion to biodiesel or renewable diesel and jet fuel [1] , [2] , [3] . Many of these species can also grow rapidly under a large range of environmental conditions, such as varied light intensity, temperature, and nutrient availability [1] , [2] . Microalgae are also capable of growth on non-arable land using a variety of water sources, including fresh, brackish, saline, and waste water [1] , [4] , [5] . At current production levels, oleaginous microalgae also have the potential to produce 1–2 orders of magnitude more oil per acre than soybeans, the most common US oilseed crop (∼50 gal acre −1 y −1 ) [6] . The Department of Energy funded an almost 20-year effort, known as the Aquatic Species Program (ASP), aimed at developing algal biofuels. The ASP was terminated, however, in 1996 because the projected cost of algal biofuels could not compete at that time with the low price of petroleum. Now, after more than a decade, microalgae have again risen to international prominence for their potential to contribute to the global liquid fuel demand. However, despite the great promise of algae-based fuels, our understanding of algal lipid metabolism, particularly the regulation of biosynthetic pathways of fatty acids and TAGs, as well as the metabolism affecting carbon partitioning, is still largely inadequate. Furthermore, the lack of available genome sequence information limits the development of basic biological understanding required for strain improvement of unsequenced microalgae. Rapidly developing post-genomic, systems biology approaches such as transcriptomics, proteomics, and metabolomics have become essential for understanding how microorganisms respond and adapt to changes in their physical environment. The application of such high-throughput approaches could greatly accelerate the commercialization of algal-derived biofuels by providing the framework for hypothesis-based strain improvement programs, built on an improved fundamental understanding of the specific pathways and regulation of networks involved in algal oil production. However, a systems biology approach to investigating oleaginous microalgal metabolism remains relatively unexplored especially in unsequenced organisms. To date, the nuclear genomes of only about ten eukaryotic microalgae have been sequenced ( http://genome.jgi-psf.org ), and though a few of these strains may have some properties of interest for algal biofuels, most, if not all of them, were chosen to be sequenced for reasons other than biofuel production. “Higher level -omic” analyses – transcriptomics, proteomics, and metabolomics – of microalgae have largely focused upon those species with sequenced genomes (for an extensive review, please refer to Jamers, 2009). For example, the transcriptome of the model green alga, Chlamydomonas reinhardtii , was recently characterized under nutrient-replete, anaerobic H 2 -producing, and sulfur-depleted growth conditions [7] , [8] , [9] . Benning and coworkers have also extensively examined the nitrogen-deprivation stress response and its effects upon lipid accumulation in C. reinhardtii , through comparative transcriptomics and lipid droplet proteomics [10] , [11] . However, the best cellular lipid accumulation noted for this organism is only ∼20% in wild type cultivars, though ∼40% total lipids have been reported for starchless mutants [12] , [13] . Many unsequenced wild-type strains have been reported to make more than 50% total lipids under these same growth conditions [1] . It is likely that the dearth of microalgal genomics data has dissuaded biologists from pursuing higher-level systems biology analyses of unsequenced yet potential commercially relevant oleaginous microalgae. Transcriptomic analyses focused upon elucidating specific metabolic pathways have, up until recently, employed microarrays generated from cDNA libraries, or sandwich hybridization assays, as opposed to utilization of the high-throughput, massively parallel, cDNA-sequencing techniques currently available for global transcriptome analysis [14] , [15] , [16] . On the other hand, Rismani-Yazdi et al. [17] just reported 454 pyrosequencing to carry out the first de novo transcriptomic sequencing and annotation of the unsequenced microalgae Dunaliella tertiolecta . This work marks a meaningful advance in the pursuit of higher-level systems biology approaches with unsequenced, oleaginous microalgae, and demonstrates the capability of using transcriptomic data to identify pathways and targets of interest for metabolic engineering and functional genomic analyses in non-model microalgae. Despite its power, transcriptomic analysis does not adequately define the control points for metabolic regulation. This is especially true in algae where post-transcriptional regulation is not well understood. For example, translational regulation of chloroplast gene expression occurs in a number of microalgae and higher plants [18] , [19] , [20] , [21] , [22] , [23] . Thus, a more complete systems biology analysis is required to provide useful hypotheses for strain improvement strategies. Proteomics can bring us closer to this goal, but the availability of proteome data from unsequenced microalgae is also sparse. Such proteomic analyses have typically targeted specific cellular components (sub-proteomes such as chloroplasts) and yielded relatively low orthologous identification rates [16] , [24] , [25] . For example, Wang et al. [25] employed a cross-species protein identification strategy in order to examine the proteome of the Haematococcus pluvialis cell wall. For the identification of proteins with low sequence identity, a conserved motif and domain strategy was also implemented. They observed that approximately one half of the proteins examined failed to be recognized in protein databases. This was attributed to amino acid substitutions and/or post-translational modifications, both of which dramatically reduce the probability of cross-species proteomic identifications. Such analyses underscore the need for increased sequence information on diverse microalgae. As we present in this communication, utilization of a de novo sequenced transcriptome can provide the necessary sequence information to pursue such proteomic analyses. After considering the fact that of the over 40,000 species of microalgae identified to date, fewer than a dozen microalgal genome sequences are available, it is not surprising that elucidation of the key pathways and networks regulating lipid accumulation remains limited [1] . An integrated systems biology examination of these organisms will be critical in order to understand the unique, strain-specific mechanisms of lipid accumulation, and to develop strategies required to engineer improved strains with enhanced lipid production. Furthermore, genetic engineering of unsequenced strains will require identification of unique promoter and untranslated region (UTR) sequences for targeted overexpression or silencing of target genes [22] . Transcriptomics and proteomics cannot provide complete sequence data for promoters and UTRs, but can identify genes with desirable expression patterns, thereby directing strain-engineering strategies to a small region rather than the entire genome. And so, with the full potential of transcriptomics and proteomics largely dependent upon genome sequence availability, many promising algal strains have been left unexplored. The oleaginous green alga, Chlorella vulgaris , has been extensively studied due to its relatively fast growth rate and its value as both a food supplement and potential biofuel feedstock. Additionally, C. vulgaris has also recently been examined in light of the genus' biomedical relevance, demonstrating anti-oxidant and anti-tumorigenic properties, as well as having value in increasing vascular and immune function [26] , [27] , [28] , [29] . C. vulgaris also accumulates >50% lipid under nutrient-deplete conditions, with a favorable fatty acid profile for biodiesel production. Finally, many reports have been published describing successful genetic transformation of Chlorella cultivars [30] , [31] . Taken together, we have concluded that this is an ideal platform to explore algal lipid metabolism and the biosynthetic pathways involved in fatty acid and TAG biosynthesis in oleaginous algae. To date, however, there is no genome sequence available for C. vulgaris (although a genome sequence has been published for the presumably related strain, C. variablis NC64A [32] ), hindering the further development of this organism as a food, fuel and biomedical resource. We have taken a different approach and set out to demonstrate the utility of bypassing the genome sequencing step by taking advantage of current high-throughput technologies in order to pursue direct, higher-level systems biology analyses. Herein, we have built upon the de novo transcriptome sequencing approach, and set out to conduct a comparative global transcriptomic and proteomic study of the microalga, C. vulgaris UTEX 395, chosen after screening all ten C. vulgaris cultivars in the UTEX Algae Culture Collection for growth rate and lipid accumulation capability, under conditions that induce high oil production. These conditions have been optimized to yield greater than 60% fatty acid accumulation based on dry cell weight when the algae were grown under nutrient-deplete conditions. cDNA from C. vulgaris was sequenced using Illumina technology and de novo transcriptome assembly was performed using a combination of readily available software and newly generated bioinformatic tools. The proteomic analysis was subsequently undertaken utilizing the assembled C. vulgaris transcriptome as a search model. This work marks the first comprehensive proteomic investigation of lipid accumulation in an unsequenced microalga, as well as the first utilization of a de novo assembled transcriptome as a search model for proteomic analysis in an unsequenced microalga. Our results indicate that this approach can provide a powerful and effective search model for proteomic analysis. Our efforts demonstrate the feasibility of bypassing the bottleneck of genomic sequencing, opening the door for a comprehensive systems biology examination of other unsequenced oleaginous microalgae.",
"discussion": "Discussion Chlorella vulgaris UTEX395 as a commercially relevant algal strain Selection of suitable algal strains will be a critical step in realizing the full potential of commercial-scale photosynthetic algal cultivation for biofuel and bioproduct production. An ideal production strain will have the attributes of fast growth at acceptable cell density, cultivation robustness, and high lipid-accumulation capacity and productivity [12] . Genetic engineering, though perhaps problematic from a regulatory or community acceptance standpoint, can, at a minimum, help establish upper limits to lipid productivity and guide classical genetics or breeding programs. In addition, the glycerolipid profile of a strain will have a large impact upon the lipid's potential as a biofuel feedstock, as chain length and degree of unsaturation are critical for conversion to both biodiesel and renewable diesel and jet fuel. For example, a C18:3 fatty acid requires 7 moles of hydrogen per mole of fatty acid ester for full saturation, while C18:0 requires only 4 moles of hydrogen. As such, the glycerolipid profile of a microalgal species can ultimately have a significant impact on production costs (Robert McCormick, NREL, personal communication). The green alga, C. reinhardtii , has been extensively examined as a model organism due to its ease of cultivation in a laboratory setting and its ability to be genetically manipulated. As such, investigation of C. reinhardtii has perhaps contributed more to the elucidation of the fundamental underpinnings of microalgal biology than any other microalga to date. However, this species lacks the intrinsic high-lipid productivity of many oleaginous microalgae. For example, under nitrogen-deplete conditions, wild-type C. reinhardtii starchless mutant only produces ∼20% lipid on a dry cell weight basis [12] . Conversely, the fatty acid profile, production, and accumulation capacity of C. vulgaris under nitrogen-deplete conditions suggests it presents an ideal feedstock for biodiesel production. Nitrogen limitation dramatically increases the desirable C18:1 fatty acid content at the expense of less desirable C18:3, C18:2, C16:1 and C16:0 content ( Figure 1c ). As such, nutrient-limiting conditions result in the accumulation of an oil having improved properties as a feedstock for biodiesel or renewable diesel and jet fuel. Our experimental conditions also yield ∼60% fatty acids on a dry cell-weight basis, nearly 3-fold higher than wild type C. reinhardtii , suggesting it is an excellent model system to examine changes in gene and protein expression under conditions that induce high-oil accumulation. We and our co-workers have had limited success reproducing the transformation techniques with C. vulgaris reported by Jarvis and Brown [31] and Chow and Tung [30] , but we have recently improved on these methods and have developed a simple and reproducible transformation protocol (J.J. Lee and Y.-C. Chou, unpublished results). The concurrent development of systems biology and genetic tools will help establish C. vulgaris as a viable model organism for algal biofuels development. The utility of a de novo assembled transcriptome as a proteomic search model Proteomic analysis using orthologous sequence databases presents a unique challenge in that it requires nearly identical m/z values (±1–2 Da) between the search model and peptides of interest in order to positively match an equivalent m/z ratio of statistical significance. As such, a single amino acid differential between a search model sequence and a peptide fragment sequence of interest can often result in a failure to produce a statistically significant match, leaving significant gaps in protein identification. One example of an absence caused by a single amino acid differential was observed for ACAT. Using Chlorophyta sequence databases, ACAT was not identified via proteomic analysis, yet was successfully identified using the C. vulgaris transcriptome as a search database. ACAT peptides identified via mass spectrometry using the C. vulgaris transcriptome were aligned against all available Chlorophyta ACAT sequences. Peptide sequence alignment shows just a single amino acid differential between C. vulgaris and Chlorophyta peptides in two instances, both corresponding to a species of same genus, Chlorella variabilis , demonstrating both the limitation of using orthologous databases for peptide identification and the advantage of using a de novo assembled transcriptome as a search database ( Figure 5 ). Improved identification capability using the transcriptome as a search database was further underscored on a more global pathway mapping scale, examining the fatty acid and TAG biosynthetic pathways. Orthologous searching identified only three enzyme components of the fatty acid biosynthetic pathway, and none of the TAG enzymatic components. Conversely, utilization of the C. vulgaris transcriptome as a search database allowed us to identify all enzymatic components of the fatty acid and TAG biosynthetic pathways. It is clear from these data that using the de novo assembled transcriptome dramatically improves proteomic identification capabilities. These results might not have been expected if the assembly or annotation of the C. vulgaris transcriptome was weak, or if the Chlorophyta database provided higher sequence identity. This is an important observation because it most notably confirms our hypothesis, and at the same time provides a measure of quality of our de novo assembly and annotation. Finally, utilization of the de novo assembled C.vulgaris transcriptome allowed for identification and differentiation of critically important protein isoforms. Though no protein isoforms were identified for the TAG biosynthetic pathway, homomeric and heteromeric ACCase isoforms, as well as multiple KAS isoforms, were identified during the annotation stage. Isoform differentiation can have a dramatic impact upon strain engineering strategies. For example, it has been suggested that overexpression of cytosolic homomeric ACCase, coupled with plastidial sub-cellular localization, as opposed to overexpression of the more complex, multi-subunit heteromeric plastidial isoform, may be a simpler and more efficient means to increase fatty acid content in oleaginous organisms [50] . Targeted strain improvement efforts and complete pathway analyses will thus be greatly facilitated by the isoform identification and maximal identification coverage a de novo assembled transcriptome search database affords. The gene ontology analysis was encouraging because it indicated that all classes of proteins were equally represented in the proteome in comparison to the transcriptome, including proteins that would be expected to reside mainly in the insoluble fraction. But it is also a warning that the majority of transcribed genes were not found in the proteome. It is likely that this failure to identify most transcribed gene products is due to some combination of quantitative limits to the GeLC-MS methodology and to post-transcriptional regulation. The distinction between these two possibilities can have a major impact on the quantitative proteomic analysis, especially as it would be applied to hypothesis-driven strain improvement programs and will need to be evaluated on a gene-by-gene basis for pathways of interest. Future work involving quantitative transcriptomic analysis, insoluble proteomic analysis, and more focused searches for specific missing proteins will help shed light on this missing piece of the puzzle. Changes in Fatty Acid and TAG Biosynthetic Pathways in a High-Lipid State As observed previously for C. cryptica \n [51] , our analysis also found upregulation of ACCase under lipid-accumulating conditions. More importantly, though, our current work also established that the majority of the other fatty acid biosynthetic pathway components are upregulated under nitrogen-depletion and concurrent lipid accumulation. Interestingly, AMPK was down-regulated under high-lipid producing conditions. AMPK is proposed to serve as a fatty acid beta-oxidation “metabolic master switch,” acting as a direct ACCase inhibitor (and indirect carnitine palmitoyltransferase (CPT-1) activator in higher eukaryotes) [52] . This lends potential insight into the regulation of fatty acid synthesis through rate-limiting ACCase activity and concurrent increase in beta-oxidation. Overexpression of the ACCase gene in both C. cryptica and N. saprofila failed to significantly increase lipid accumulation [53] . A number of mechanisms have been proposed to explain this observation, including post-transcriptional regulation and feedback inhibition. It is possible AMPK also played a critical role in driving the equilibrium between acetyl-CoA and malonyl-CoA in the reverse direction, ultimately slowing the rate of fatty acid biosynthesis and increasing the rates of fatty acid beta-oxidation. The activity of AMPK under nitrogen-replete and nitrogen-deplete conditions warrants further investigation. The possibility must be considered that our decision to harvest cultures at the onset of stationary phase yielded cells that were past their peak in abundance of the enzymatic components of the fatty acid biosynthetic pathway (even though many significant increases were observed). Resultant late stage down-regulation of synthesis may explain the decrease observed for KAR under nitrogen-limited conditions. As synthesis of fatty acid biosynthetic machinery is shut down, protein turnover is likely to occur, as cells may be under a state of high catabolic activity in order to recycle internal nitrogen stores. However, protein abundance is not directly reflective of protein expression rates or activity. Thus, it is possible that KAR enzymatic rates remain unchanged despite decreased abundance. It is also unclear whether the increased abundance of fatty acid biosynthetic components (and decrease in KAR abundance) is due to altered rates of mRNA expression and translation, or mRNA and protein turnover. A more complete integrated systems biology analysis, incorporating transcriptomic, proteomic, and metabolomic data will be necessary to fully elucidate potential flux bottlenecks in the fatty acid pathway. Our results demonstrate that TAG biosynthetic machinery abundance is upregulated significantly higher than the fatty acid synthesis machinery in a high-lipid accumulation state. This massive upregulation can again likely be attributed to the late-stage harvest of nitrogen-deplete cells. At this stage, cells have neared stationary phase, having exhausted internal nitrogen stores. Photosynthetic energy and carbon fixation can continue (albeit with presumably altered efficiency indicated by the reduction in pigmentation in the starved, chlorotic cells, Figure 1a ). The most effective diversion of the fixed carbon and reducing equivalents generated by photosynthesis is their conversion to TAGs (as reflected in fluorescence imaging of neutral lipids, Figure 1d ). As observed in C. reinhardtii lipidomics analysis, acyltransferases were amongst the most abundant lipid droplet-associated proteins observed [11] . As such, with the intracellular space largely encompassed by neutral lipids, it is to be expected that we would observe significant abundance of TAG-related acyltransferases. The dramatic differential between fatty acid biosynthetic and TAG biosynthetic components may imply TAG biosynthesis may also play a significant role in the rate-limiting production of neutral lipids, suggesting future studies aimed at strain improvement might be focused upon overexpression of TAG biosynthetic components in addition to fatty acid biosynthetic components. Notably, none of the TAG biosynthetic machinery was identified under nitrogen-replete conditions, suggesting TAG biosynthesis is either largely in an “off state” or at very low levels in early growth phase, and the majority of the 10% fatty acids observed under nitrogen-replete conditions is derived from structural membrane phospholipids. Interestingly, we observed steady-state abundance of DAGK between nitrogen-replete and nitrogen-deplete conditions. DAGK catalyzes the conversion of DAG to PA via phosphorylation of DAG, and its activity has been shown to increase upon activation of the phosphoinositide (PI) pathway. It is, therefore, proposed to function as a termination cue in the formation of DAG [48] . Steady state abundance may be indicative of steady-state activation of the PI pathway, potentially pointing to a minimal baseline phospholipid production level, required for cell viability. We hypothesize that future analyses using intermediate harvest points will lead to a less pronounced differential between fatty acid and TAG biosynthetic components, with an increased abundance of fatty acid components and a decrease in abundance of TAG components prior to nitrogen exhaustion. Future analyses will therefore be focused upon intermediate accumulation, which will allow for abundance mapping throughout the lipid accumulation cycle and help clarify the rates of TAG component expression. Concurrently, quantitative analyses of PA, DAG, and TAG will lend further insight into the flux through the TAG pathway, as well as temporal regulation throughout the lipid accumulation cycle. Conclusions The prevalence of microalgal translational gene regulation necessitates higher-level omic analyses at the protein level in order to fully elucidate changes in gene expression under varying conditions. However, proteomic analysis of unsequenced microalgae is clearly limited by the lack of flexibility in fragment matching. Our results underscore how much more powerful proteomic analysis can be when accurate sequence information is available, and demonstrate the utility of a de novo assembled transcriptome as a search model for proteomic analysis of unsequenced microalgae. Strain improvement strategies targeting increased lipid accumulation and productivity as well as improved understanding of the relevant basic biology will be critically enhanced by utilization of our transcriptomic sequence data combined with proteomic abundance data. We have focused our initial investigation of differential protein expression upon dramatically different lipid accumulation states (10% vs. 60% fatty acid) in N-replete and deplete C. vulgaris . These analyses indicate that the fatty acid and TAG biosynthetic pathways are dramatically upregulated (TAG>fatty acid) under nitrogen limitation. Data from intermediate accumulation states will likely provide a wealth of additional information with regards to the stages at which gene and protein-expression are initiated. Carbon flux analyses, glycerolipid speciation, and metabolomic analysis will ultimately need to be initiated to complement comparative transcriptomic and proteomic analyses, in order to fully assess flux through lipid-relevant pathways of interest on a comprehensive systems biology level."
} | 7,126 |
22676290 | null | s2 | 5,821 | {
"abstract": "The layer-by-layer (LbL) assembly of polyelectrolyte pairs on temperature and pH-sensitive cross-linked poly(N-isopropylacrylamide)-co-(methacrylic acid), poly(NIPAAm-co-MAA), microgels enabled a fine-tuning of the gel swelling and responsive behavior according to the mobility of the assembled polyelectrolyte (PE) pair and the composition of the outermost layer. Microbeads with well-defined morphology were initially prepared by synthesis in supercritical carbon dioxide. Upon LbL assembly of polyelectrolytes, interactions between the multilayers and the soft porous microgel led to differences in swelling and thermoresponsive behavior. For the weak PE pairs, namely poly(L-lysine)/poly(L-glutamic acid) and poly(allylamine hydrochloride)/poly(acrylic acid), polycation-terminated microgels were less swollen and more thermoresponsive than native microgel, whereas polyanion-terminated microgels were more swollen and not significantly responsive to temperature, in a quasi-reversible process with consecutive PE assembly. For the strong PE pair, poly(diallyldimethylammonium chloride)/poly(sodium styrene sulfonate), the differences among polycation and polyanion-terminated microgels are not sustained after the first PE bilayer due to extensive ionic cross-linking between the polyelectrolytes. The tendencies across the explored systems became less noteworthy in solutions with larger ionic strength due to overall charge shielding of the polyelectrolytes and microgel. ATR FT-IR studies correlated the swelling and responsive behavior after LbL assembly on the microgels with the extent of H-bonding and alternating charge distribution within the gel. Thus, the proposed LbL strategy may be a simple and flexible way to engineer smart microgels in terms of size, surface chemistry, overall charge and permeability."
} | 456 |
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