pmid stringlengths 8 8 | pmcid stringlengths 8 11 ⌀ | source stringclasses 2
values | rank int64 1 9.78k | sections unknown | tokens int64 3 46.7k |
|---|---|---|---|---|---|
33149289 | PMC7116757 | pmc | 2,550 | {
"abstract": "The growing importance of applications based on machine learning is driving the need to develop dedicated, energy-efficient electronic hardware. Compared with von-Neumann architectures, brain-inspired in-memory computing uses the same basic device structure for logic operations and data storage 1 – 3 , thus promising to reduce the energy cost of data-centric computing significantly 4 . While there is ample research focused on exploring new device architectures, the engineering of material platforms suitable for such device designs remains a challenge. Two-dimensional materials 5 , 6 such as semiconducting MoS2 could stand out as a promising candidate to face this obstacle thanks to their exceptional electrical and mechanical properties 7 – 9 . Here, we explore large-area grown MoS2 as an active channel material for developing logic-in-memory devices and circuits based on floating-gate field-effect transistors (FGFET). The conductance of our FGFETs can be precisely and continuously tuned, allowing us to use them as building blocks for reconfigurable logic circuits where logic operations can be directly performed using the memory elements. After demonstrating a programmable NOR gate, we show that this design can be simply extended to implement more complex programmable logic and functionally complete sets of functions. Our findings highlight the potential of atomically thin semiconductors for the development of next-generation low-power electronics."
} | 368 |
33149289 | PMC7116757 | pmc | 2,550 | {
"abstract": "The growing importance of applications based on machine learning is driving the need to develop dedicated, energy-efficient electronic hardware. Compared with von-Neumann architectures, brain-inspired in-memory computing uses the same basic device structure for logic operations and data storage 1 – 3 , thus promising to reduce the energy cost of data-centric computing significantly 4 . While there is ample research focused on exploring new device architectures, the engineering of material platforms suitable for such device designs remains a challenge. Two-dimensional materials 5 , 6 such as semiconducting MoS2 could stand out as a promising candidate to face this obstacle thanks to their exceptional electrical and mechanical properties 7 – 9 . Here, we explore large-area grown MoS2 as an active channel material for developing logic-in-memory devices and circuits based on floating-gate field-effect transistors (FGFET). The conductance of our FGFETs can be precisely and continuously tuned, allowing us to use them as building blocks for reconfigurable logic circuits where logic operations can be directly performed using the memory elements. After demonstrating a programmable NOR gate, we show that this design can be simply extended to implement more complex programmable logic and functionally complete sets of functions. Our findings highlight the potential of atomically thin semiconductors for the development of next-generation low-power electronics."
} | 368 |
33149289 | PMC7116757 | pmc | 2,551 | {
"abstract": "The growing importance of applications based on machine learning is driving the need to develop dedicated, energy-efficient electronic hardware. Compared with von-Neumann architectures, brain-inspired in-memory computing uses the same basic device structure for logic operations and data storage 1 – 3 , thus promising to reduce the energy cost of data-centric computing significantly 4 . While there is ample research focused on exploring new device architectures, the engineering of material platforms suitable for such device designs remains a challenge. Two-dimensional materials 5 , 6 such as semiconducting MoS2 could stand out as a promising candidate to face this obstacle thanks to their exceptional electrical and mechanical properties 7 – 9 . Here, we explore large-area grown MoS2 as an active channel material for developing logic-in-memory devices and circuits based on floating-gate field-effect transistors (FGFET). The conductance of our FGFETs can be precisely and continuously tuned, allowing us to use them as building blocks for reconfigurable logic circuits where logic operations can be directly performed using the memory elements. After demonstrating a programmable NOR gate, we show that this design can be simply extended to implement more complex programmable logic and functionally complete sets of functions. Our findings highlight the potential of atomically thin semiconductors for the development of next-generation low-power electronics."
} | 368 |
33149289 | PMC7116757 | pmc | 2,551 | {
"abstract": "The growing importance of applications based on machine learning is driving the need to develop dedicated, energy-efficient electronic hardware. Compared with von-Neumann architectures, brain-inspired in-memory computing uses the same basic device structure for logic operations and data storage 1 – 3 , thus promising to reduce the energy cost of data-centric computing significantly 4 . While there is ample research focused on exploring new device architectures, the engineering of material platforms suitable for such device designs remains a challenge. Two-dimensional materials 5 , 6 such as semiconducting MoS2 could stand out as a promising candidate to face this obstacle thanks to their exceptional electrical and mechanical properties 7 – 9 . Here, we explore large-area grown MoS2 as an active channel material for developing logic-in-memory devices and circuits based on floating-gate field-effect transistors (FGFET). The conductance of our FGFETs can be precisely and continuously tuned, allowing us to use them as building blocks for reconfigurable logic circuits where logic operations can be directly performed using the memory elements. After demonstrating a programmable NOR gate, we show that this design can be simply extended to implement more complex programmable logic and functionally complete sets of functions. Our findings highlight the potential of atomically thin semiconductors for the development of next-generation low-power electronics."
} | 368 |
34289233 | PMC8913906 | pmc | 2,552 | {
"abstract": "Summary Biorefineries have a pivotal role in the bioeconomy scenario for the transition from fossil‐based processes towards more sustainable ones relying on renewable resources. Lignocellulose is a prominent feedstock since its abundance and relatively low cost. Microorganisms are often protagonists of biorefineries, as they contribute both to the enzymatic degradation of lignocellulose complex polymers and to the fermentative conversion of the hydrolyzed biomasses into fine and bulk chemicals. Enzymes have therefore become crucial for the development of sustainable biorefineries, being able to provide nutrients to cells from lignocellulose. Enzymatic hydrolysis can be performed by a portfolio of natural enzymes that degrade lignocellulose, often combined into cocktails. As enzymes can be deployed in different operative settings, such as separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF), their characteristics need to be combined with microbial ones to maximize the process. We therefore reviewed how the optimization of lignocellulose enzymatic hydrolysis can ameliorate bioethanol production when Saccharomyces cerevisiae is used as cell factory. Expanding beyond biofuels, enzymatic cocktail optimization can also be pivotal to unlock the potential of non‐ Saccharomyces yeasts, which, thanks to broader substrate utilization, inhibitor resistance and peculiar metabolism, can widen the array of feedstocks and products of biorefineries.",
"conclusion": "Conclusions Biorefineries will play a pivotal role in the development of a sustainable global bioeconomy. Efficient biorefineries will integrate biomass, bespoke enzyme cocktails and specific cell factories. Although the traditional focus has been on cell factory design, the critical need to exploit second‐generation biomasses to achieve sustainability is now a major driver of research. Indeed, this leads to somewhat of a paradigm shift since, in these scenarios, the substrate achieves equal importance to the product: a process that does not use residual biomasses will struggle to deliver sustainability. This adds a substantial variable that was little considered in traditional fermentations and first‐generation bioprocesses. It also creates new opportunities since there is increased scope to exploit microbial diversity, and in the case of yeasts, non‐ Saccharomyces yeasts because of their properties related to substrate specificity, inhibitor tolerance and growth parameters. The metabolism of these yeasts can then be exploited to produce new products, often with better efficiency that S . cerevisiae . The third pillar in second‐generation biorefineries is the enzyme that links the biomass to the cell factory. This is a crucial area of ongoing investigation that considers the enzymes and how they are applied in either SHF or SSF processes. The use of enzyme cocktails to treat LCBs is now in routine, but considerable work is still required to decide on the best enzyme formulation, and the range of options is still too limited. It is important to recognize that the best enzyme cocktail is the one that can deliver the optimum sugar mix to a specific cell factory microbe when starting from a particular LCB source. This is also possible because of the improved knowledge on yeast biodiversity and the constant development on enzymes potential. It is implicit in this that considering any of the components in isolation cannot achieve the best outcome. At the moment, each bioprocess needs to be designed from first principles, but it is hoped that, as experience grows, it will become possible to develop framework principles that facilitate rational selection of the components of a biorefinery. In this case, it will be possible to reduce the cost and time needed to develop new second‐generation biorefineries, including those that operate on a modest scale.",
"introduction": "Introduction Biorefineries can be described as ‘the sustainable processing of biomass into a spectrum of marketable products (food, feed, materials, chemicals) and energy (fuels, power, heat)’ (IEA Bioenergy Task42, 2014 ). Biorefineries indeed aim to provide a broad portfolio of products alongside classical bio‐based molecules such as biofuels or biogas (European Commission, 2018 ; Rosales‐Calderon and Arantes, 2019 ; Stegmann et al ., 2020 ). Consistently, they are considered as one of the key technologies in the circular bioeconomy scenario, presenting different opportunities and challenges across countries, as they need to be organically integrated in the territories’ landscape and infrastructure. In order to widen possible outcomes of biorefineries and, in some cases, minimize environmental impacts, it is possible to exploit microorganisms, the so‐called microbial cell factories, whose role is to convert the provided biomass(es) into the desired product(s) (Dahiya et al ., 2018 ). As a consequence, it is crucial that nutrients released from biomasses can match microbial requirements. In the case of lignocellulosic biomasses (LCBs), constituted by cellulose, hemicellulose and lignin in different ratios, a pre‐treatment step to open‐up the recalcitrant macromolecular structure is followed by enzymatic hydrolysis. This step is preferred to chemical treatment (Galbe and Wallberg, 2019 ) (e.g. acid) as enzymes operate under conditions that are more compatible with microbial growth. Different hydrolyses can generate different mixtures of sugars and other nutrients, both in terms of composition and relative quantities. Notably, enzymes can be applied in two quite distinct processes, namely, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), in order to match microbial cell factories’ characteristics (Kawaguchi et al ., 2016 ). Here, we recapitulate recent literature on this subject, and our aim is to underline the tight correlation between biomass, enzymes and yeast cell factories: optimization of the enzymatic cocktail and its operative conditions can unlock the potential of a lignocellulosic biomass and/or of a yeast cell factory (Fig. 1 ). This synergy is crucial for assessing or improving the viability of the overall process and therefore its economic feasibility (Pellis et al ., 2018 ). This review is a qualitative description on the importance of a link between LCBs composition, choice of enzyme cocktail and selection of yeast species and strains that need to be considered in an integrated fashion to enable the development of an efficient process. Fig. 1 Overview of the processes/factors involved in the conversion of lignocellulosic biomass into final products in second‐generation biorefineries. For each step of the overall process (coloured boxes), the main parameters to be considered when establishing a (yeast‐based) biorefinery are indicated under the dotted line. Pre‐treatment is needed to weaken the intertwined structure of LCBs prior to enzymatic hydrolysis. In SHF, hydrolysis and fermentation are performed as sequential steps, whereas in SSF, they are combined into a single one."
} | 1,775 |
30870453 | PMC6417678 | pmc | 2,553 | {
"abstract": "Non-methanotrophic bacteria such as methylotrophs often coexist with methane-oxidizing bacteria (methanotrophs) by cross-feeding on methane-derived carbon. Methanol has long been considered a major compound that mediates cross-feeding of methane-derived carbon. Despite the potential importance of cross-feeding in the global carbon cycle, only a few studies have actually explored metabolic responses of a bacteria when cross-feeding on a methanotroph. Recently, we isolated a novel facultative methylotroph, Methyloceanibacter caenitepidi Gela4, which grows syntrophically with the methanotroph, Methylocaldum marinum S8. To assess the potential metabolic pathways in M . caenitepidi Gela4 co-cultured with M . marinum S8, we conducted genomic analyses of the two strains, as well as RNA-Seq and chemical analyses of M . caenitepidi Gela4, both in pure culture with methanol and in co-culture with methanotrophs. Genes involved in the serine pathway were downregulated in M . caenitepidi Gela4 under co-culture conditions, and methanol was below the detection limit (< 310 nM) in both pure culture of M . marinum S8 and co-culture. In contrast, genes involved in the tricarboxylic acid cycle, as well as acetyl-CoA synthetase, were upregulated in M . caenitepidi Gela4 under co-culture conditions. Notably, a pure culture of M . marinum S8 produced acetate (< 16 μM) during growth. These results suggested that an organic compound other than methanol, possibly acetate, might be the major carbon source for M . caenitepidi Gela4 cross-fed by M . marinum S8. Co-culture of M . caenitepidi Gela4 and M . marinum S8 may represent a model system to further study methanol-independent cross-feeding from methanotrophs to non-methanotrophic bacteria.",
"introduction": "Introduction Microbial methane oxidation plays an important role in the global methane cycle. Aerobic methane-oxidizing bacteria (methanotrophs) are key players in aerobic and micro-aerobic environments. The development of stable isotope probing has highlighted that methane-derived carbon is incorporated not only by methanotrophs but also by non-methanotrophic bacteria (methylotrophs or others) in diverse environments, and suggests the global importance of cross-feeding interactions in methane oxidation [ 1 , 2 , 3 , 4 ]. Methanol is synthesized in the periplasm of methanotrophs, and therefore, may diffuse out of the cell [ 5 ]. Hence, it is generally speculated that the co-existence of methanotrophs and non-methanotrophic bacteria mainly involves the cross-feeding of methanol excreted by methanotrophs to non-methanotrophic bacteria [ 2 ]. Methanol-dependent cross-feeding between methanotrophs and non-methanotrophic bacteria has been studied since the 1970s [ 6 , 7 ]. Using isolates from lake sediments, Krause et al. [ 8 ] demonstrated that methanol excreted by a methanotroph is indeed the sole carbon and energy source for the co-existing obligate methylotroph that can utilize methanol or methylamine exclusively. On the other hand, some methanotrophs are also known to excrete some organic acids [ 9 ]. It is speculated that these organic acids are derived from dead cells. Recently, Kalyuzhnaya et al. [ 10 ] demonstrated that Methylomicrobium alcaliphilum of Gammaproteobacteria release organic compounds, such as formate and acetate, via fermentation-based methanotrophy. This suggested that cross-feeding of methane-derived carbon from a methanotroph to non-methanotrophic bacteria may not be mediated solely by methanol and may involve more diverse processes. However, this has not been extensively explored yet. Recently, we obtained a methane-oxidizing enrichment culture from marine sediments of Kagoshima Bay, Japan and isolated a novel facultative methylotroph, Methyloceanibacter caenitepidi Gela4 [ 11 ] and a novel gammaproteobacterial methanotroph, Methylocaldum marinum S8 [ 12 ]. M . caenitepidi Gela4 grows on a wide range of substrates and can grow syntrophically with M . marinum S8 using methane added as the sole carbon source. The co-culture of M . caenitepidi Gela4 and M . marinum S8 would be a suitable model system for addressing the nature of a facultative methylotroph cross-feeding on a methanotroph. In this study, we aimed to reveal the metabolic pathways employed by a marine facultative methylotroph cross-feeding on a methanotroph using genome analysis, RNA sequencing (RNA-Seq), and chemical analysis.",
"discussion": "Discussion In the current study, we compared gene expression of the facultative methylotroph M . caenitepidi Gela4 grown in pure culture with methanol as the sole carbon source or co-cultured with the methanotroph M . marinum S8 with methane as the sole carbon source. The cross-feeding compound from methanotrophs to non-methanotrophic bacteria has been often considered as methanol excreted by methanotrophs. Krause et al. (2017) [ 8 ] has examined this interrelationship in detail. The authors conducted transcriptome analysis of two obligate methylotroph strains in co-culture with the methanotroph Methylobacter tundripaludum and demonstrated that the obligate methylotroph relies solely on methanol in co-culture with M . tundripaludum . In the current study, we examined for the first time the response of a facultative methylotroph, M . caenitepidi Gela4, which can assimilate a wide range of substrates in addition to methanol, such as acetate, ethanol, formate and succinate [ 11 ], grown in co-culture with a methanotroph by transcriptome analysis. We discovered that the expression levels of nearly half of the genes of M . caenitepidi Gela4 were altered during co-culture ( Fig 4 , Table 1 ). Of note, genes for acetyl-CoA synthesis, the EMC pathway and TCA cycle were upregulated during co-culture cultures ( Fig 3 , Table 1 ). Considering that acetyl-CoA is a starting compound of the EMC pathway and TCA cycle, compounds that can be used for the acetyl-CoA synthesis are cross-fed from M . marinum S8 to M . caenitepidi Gela4. One possible source of acetyl-CoA in co-culture is acetate. Indeed, expression of acetyl-coenzyme A synthetase increased with a log2FC value of 3.57. Some methanotrophs are known to produce organic acids, e.g. formate and acetate [ 10 , 32 ]. For example, gammaproteobacterial methanotroph, Methylomicrobium alcaliphilum , has been shown to produce organic acids under oxygen-limited conditions [ 10 ]. The pure culture of M . marinum S8 produced up to 16 μmol/L of acetate during growth ( Fig 4B ). Pyruvate dehydrogenase (sS8_5306) and acetate kinase (sS8_2955) were found in the M . marinum S8 genome, suggesting that M . marinum S8 is also capable of ‘fermentation-based methanotrophy’ as M . alcaliphilum . These results suggested that acetate is a carbon source for M . caenitepidi Gela4 in co-culture. Increased relative expression level of acsA to hpr in M . caenitepidi Gela4 grown with acetate as well as in co-culture was confirmed by real-time PCR analysis ( Table 2 ), supporting that M . caenitepidi Gela4 utilizes acetate in co-culture. Two genes (GL4_0878 and 2347) were found to encode proteins homologous to acetate transporter of M . extorquens (ActP) with 26–27% amino acid identities. However, there was no significant difference in the expression levels of these two genes between pure culture and co-culture conditions. Functional acetate transporter in M . caenitepidi Gela4 should be identified in future studies. Another possible source of acetyl-CoA is acetaldehyde. Indeed, the most highly upregulated gene of M . caenitepidi Gela4 co-cultured with M . marinum S8 was that encoding the aldehyde dehydrogenase gene. However, relatively high expression of aldA was observed not only in co-culture but also in M . caenitepidi Gela4 grown with acetate ( Table 2 ), suggesting that aldA is involved in acetate metabolism. Substrate specificity of aldA in M . caenitepidi Gela4 and its role in acetate metabolism should be investigated in future studies. While formate was also produced in M . marinum S8 at 8 μmol/L, it was not considered as a cross-feeding compound as M . caenitepidi Gela4 produced a higher concentration of formate (23 μmol/L). Our results demonstrated that methanol assimilation pathways were downregulated in M . caenitepidi Gela4 in co-culture conditions ( Fig 3 ). It is possible that low concentrations with a high turnover would be sufficient to maintain cross-feeding. However, the concentration of methanol, both in pure culture of M . marinum S8 and co-culture, was below the detection limit (< 310 nmol/L), which is more than 1,000 times lower than the previous report on M . tundripaludum cross-feeding methanol (1.24 mmol/L) [ 8 ]. Surprisingly, the methanol concentration in the culture supernatant of M . marinum S8 was also much lower than the values reported previously for other methanotrophic species (0.01–1.00 mmol/L) [ 33 ]. Considering that a higher concentration of acetate (16 μmol/L) was produced by M . marinum S8, methanol may not be a primary carbon source for M . caenitepidi Gela4 in co-culture with M . marinum S8, if any, in contrast to the previous study on an obligate methylotroph [ 8 ]. M . alcaliphilum produces acetate under oxygen-limited conditions [ 10 ]. In this study, relative expression of acsA of M . caenitepidi Gela4 in co-culture was also high in the early exponential phase ( Table 2 ), which is not considered to be an oxygen-limited condition, although the difference was not significant by t-test. Methanol was not detected, but 8.5 μmol/L of acetate was detected in early exponential phase of M . marinum S8, suggesting that M . caenitepidi Gela4 may utilize acetate not only in the late exponential phase. Further study is required to elucidate the mechanism of acetate production and effects of environmental conditions such as oxygen concentration on acetate excretion by M . marinum S8. XoxF is a rare-earth element-dependent methanol dehydrogenase [ 34 ]. Although we did not supplement the growth medium with rare-earth elements (Inductively coupled plasma-mass spectrometry analysis confirmed that the concentration of La, Ce, Nd, and Pr in the growth medium was < 1 μg/kg), M . caenitepidi Gela4 expressed three xoxF genes at relatively high RPKMs, and their expression was altered in co-culture with the methanotroph ( S5 Fig ). This was in contrast to mxaF genes that remained highly expressed. The expression of xoxF genes of Methylotenera mobilis 13 was also altered in co-culture [ 8 ]. Although further studies are required to elucidate the functions of these genes, the results suggested that xoxF genes may be involved in the response to a co-culture conditions, such as, low methanol concentration. It has long been considered that the cross-feeding compound between methanotrophic and non-methanotrophic bacteria is primarily methanol. Our results presented here suggest that this is not always the case. Organic acids such as acetate can also be an important cross-feeding compound depending on the species and perhaps on environmental conditions. M . marinum S8 and M . caenitepidi Gela4 is a good co-culture model to further study methanol-independent cross-feeding from methanotrophs to non-methanotrophs."
} | 2,839 |
34160888 | PMC8541784 | pmc | 2,554 | {
"abstract": "Summary Lignins are cell wall‐located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar ( Populus tremula × P. alba ). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR‐Cas9‐generated cse1 and cse2 single mutants had a wild‐type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced‐lignin content translated into a fourfold increase in cellulose‐to‐glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p ‐coumaroyl, 5‐hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl‐shikimates, and show that CSE is a promising target to improve plants for the biorefinery.",
"introduction": "Introduction Lignocellulosic biomass is a promising renewable feedstock for the production of bio‐based chemicals and fermentable sugars (Marriott et al ., 2016 ; Van de Wouwer et al ., 2018 ). The cell wall polysaccharides can be hydrolysed to monosaccharides by saccharification, after which the monosaccharides can be fermented to ethanol or other products by specific microorganisms (Vanholme et al ., 2013b ). However, efficient saccharification is hindered by the presence of lignin in the secondary‐thickened cell walls, as lignin physically prevents the hydrolytic enzymes from accessing the cellulose surface and it also adsorbs the saccharification enzymes (Jørgensen et al ., 2007 ; Mansfield et al ., 1999 ). Biomass pretreatments are used to reduce the recalcitrance and therefore improve the saccharification efficiency. Nevertheless, these pretreatments are a costly step in the production of bio‐based products (Aden et al ., 2002 ; Vanholme et al ., 2013b ). Alternatively, plants can be engineered to contain less lignin and/or lignin with a different composition to reduce biomass recalcitrance (Chanoca et al ., 2019 ; Chen and Dixon, 2007 ; De Meester et al ., 2018 ; de Vries et al ., 2018 ; Eudes et al ., 2014 ; Halpin, 2019 ; Mansfield et al ., 2012 ; Van Acker et al ., 2014 ; Van Acker et al ., 2013 ; Wilkerson et al ., 2014 ; Zhang et al ., 2012 ). In angiosperms, lignin is mostly derived from the monolignols coniferyl alcohol and sinapyl alcohol, with traces of p ‐coumaryl alcohol. These monolignols are synthesized via the general phenylpropanoid and monolignol‐specific pathways (Boerjan et al ., 2003 ; Bonawitz and Chapple, 2010 ; Freudenberg, 1965 ; Vanholme et al ., 2019 ; Vanholme et al ., 2010 ). After their biosynthesis, the monolignols are deposited in the cell wall, where they are oxidized to radicals by laccases and peroxidases, and subsequently polymerized through combinatorial radical–radical coupling to create the lignin polymer (Berthet et al ., 2011 ; Ralph et al ., 2019 ; Ralph et al ., 2004 ; Zhao et al ., 2013 ). Upon incorporation into the lignin polymer, p ‐coumaryl alcohol, coniferyl alcohol and sinapyl alcohol give rise to p ‐hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units, respectively. In addition to these three main monolignols, an increasing number of other aromatic compounds has been shown to couple into the lignin polymer (Mottiar et al ., 2016 ; Ralph et al ., 2019 ; Vanholme et al ., 2019 ). The biosynthesis of the two main monolignols coniferyl alcohol and sinapyl alcohol passes through caffeoyl‐CoA. Caffeoyl‐CoA can be produced from caffeoyl shikimate by p ‐HYDROXYCINNAMOYL‐CoA:QUINATE/SHIKIMATE p ‐HYDROXYCINNAMOYLTRANSFERASE (HCT) (Franke et al ., 2002 ; Hoffmann et al ., 2004 ; Hoffmann et al ., 2003 ; Schoch et al ., 2001 ; Shadle et al ., 2007 ). Alternatively, it can be produced from p‐ coumaric acid via either 4‐COUMARATE 3‐HYDROXYLASE (C3H (Barros et al ., 2019 )) or an enzyme complex involving p ‐COUMAROYL SHIKIMATE 3΄‐HYDROXYLASE/CINNAMATE 4‐HYDROXYLASE (C3΄H/C4H), followed by the action of 4‐COUMARATE:CoA LIGASE (4CL) (Chen et al ., 2011 ). In addition to these routes, it was shown that the HCT‐catalysed conversion of caffeoyl shikimate into caffeoyl‐CoA could be bypassed by the combined activities of CAFFEOYL SHIKIMATE ESTERASE (CSE) and 4CL in Arabidopsis (Vanholme et al ., 2013c ). A loss‐of‐function mutant of CSE in Arabidopsis showed up to 36% reduced‐lignin content and a 30‐fold increase in the relative amount of H units in the lignin polymer (Vanholme et al ., 2013c ). Stacking a C3H silencing construct with the cse loss‐of‐function mutation in Arabidopsis resulted in a further reduction in lignin content and an increase in the H unit frequency, as compared to the cse mutant (Barros et al ., 2019 ). The essential role of CSE in monolignol biosynthesis has also been proven in M. truncatula and P. tremula × P. alba (Ha et al ., 2016 ; Saleme et al ., 2017 ). The loss‐of‐function mutation of CSE in M. truncatula caused more severe phenotypes than those caused by the loss‐of‐function mutation in Arabidopsis, such as severe dwarfism, reduction of lignin levels by 80% and levels of up to 84% H units in the lignin, as opposed to approximately 6% H units in the wild type (WT) (Ha et al ., 2016 ). Poplar has two CSE homologs, CSE1 (corresponding to Potri.001G17500) and CSE2 (corresponding to Potri.003G059200), that share 92% identity in amino acid sequence based on the genome sequence of P. tremula × P. alba 717‐1B4 from AspenDB (Xue et al ., 2015 ; Zhou et al ., 2015 ). Simultaneous down‐regulation of CSE1 and CSE2 by a hairpin approach in P. tremula × P. alba resulted in residual expression levels as low as 35% and 15%, respectively, a 25% reduction in lignin amount, and a twofold increase in H unit content (Saleme et al ., 2017 ). Although the saccharification efficiency of the hpCSE was 30% higher than that of the WT, this increase was much smaller than the fourfold improvement observed with the Arabidopsis cse2 loss‐of‐function mutant (Vanholme et al ., 2013c ). We hypothesized that the modest increase in saccharification efficiency and the small elevation in H unit content in comparison with the Arabidopsis cse2 were caused by the residual CSE expression in the hairpin lines. Additionally, the hairpin strategy down‐regulated both CSE genes simultaneously, hindering the understanding of potentially differential roles for each of the two genes individually. Here, we investigated the role of CSE1 and CSE2 in P. tremula × P. alba , both independently and simultaneously, via the generation of knockout mutants through CRISPR‐Cas9. Growth, lignin analysis and metabolic profiling of the mutants provided evidence that CSE1 and CSE2 are partially redundant in poplar. By comparative metabolic profiling of WT and double mutants and in vitro enzyme kinetics, we obtained evidence for another metabolic layer in the grid‐like phenylpropanoid pathway. Furthermore, the large increases in saccharification efficiency in cse1 cse2 further supported the contention that CSE is a promising target for engineering trees for the biorefinery.",
"discussion": "Discussion Biosynthesis of phenylpropanoids and lignin in cse1 cse2 mutant poplar We have previously shown that down‐regulation of CSE in poplar using a hairpin approach resulted in up to 25% reduced‐lignin content, a 100%‐110% increase in H units and a 62%‐91% increase in saccharification efficiency on a plant basis, depending on the pretreatment (Saleme et al ., 2017 ). However, the relatively modest increases in H units and saccharification efficiency compared with the Arabidopsis cse2 mutant suggested that a full knockout of CSE generated by CRISPR‐Cas9 could result in stronger reductions in lignin content and associated improvements in saccharification efficiency (Vanholme et al ., 2013c ). In addition, CRISPR‐Cas9 generated cse knockout mutants could reveal the role of the individual CSE genes. Here, we show that lignin content is not affected in poplar cse1 and cse2 mutants, and is reduced in cse1 cse2 mutants by about 35% in comparison with WT. The remaining 65% lignin in cse1 cse2 mutants imply other routes towards the biosynthesis of lignin in the absence of functional CSE in poplar. One possible route towards coniferyl and sinapyl alcohol could be through HCT. The activity of recombinant poplar HCTs involved in lignification (HCT1 and HCT6) is very low towards caffeoyl shikimate (Wang et al ., 2014 ), but because caffeoyl shikimate increased to very high levels in the cse1 cse2 mutants, HCT enzymes might work near their V \n max in these mutants. Despite that, accumulation of caffeoyl shikimate ( 16 ) and caffeoyl shikimate hexoside ( 17 ) in cse1 cse2 mutants showed that HCT was unable to convert all caffeoyl shikimate to caffeoyl‐CoA and that part of it was detoxified via glycosylation or converted to other hydroxycinnamoyl‐shikimate derivatives. A second possible route towards coniferyl and sinapyl alcohol is the bypass via either the recently characterized ascorbate peroxidase C3H enzyme or the C3′H/C4H heteromeric complex, whereby p ‐coumaric acid is converted to caffeic acid (Barros et al ., 2019 ; Chen et al ., 2011 ). A third possibility is that a CSE‐like protein has partially taken over the role of CSE1 and CSE2, as discussed previously (Ha et al ., 2016 ; Saleme et al ., 2017 ). Although the frequency of H units had increased by sevenfold in cse1 cse2 mutants (based on NMR data), the total incorporation of H units remained relatively low (approximately 4% of the total H+G+S units), whereas in M . truncatula , a knockout of CSE dramatically increased the frequency of H units in lignin from 6% up to 84% and in Arabidopsis from almost 0% up to 44% (Ha et al ., 2016 ; Vanholme et al ., 2013c ). A possible reason for the relatively low frequency of H units in the lignin in cse1 cse2 mutant poplars in comparison with the M. truncatula mutant could be that CCR2 in poplar is not efficient in catalysing the reaction from p ‐coumaroyl‐CoA to p ‐coumaraldehyde [ k \n cat / K \n m of 2.51 µ m /min for feruloyl‐CoA vs 0.15 for p ‐coumaroyl‐CoA (Wang et al ., 2014 )], whereas for M. truncatula CCR2, p ‐coumaroyl‐CoA is actually the preferred substrate with the highest k \n cat / K \n m [0.90 versus 0.40, 0.37 and 0.49 µ m /min for feruloyl‐CoA, sinapoyl‐CoA and caffeoyl‐CoA respectively (Zhou et al ., 2010 )]. Nevertheless, for Arabidopsis the preferred substrate is feruloyl‐CoA [ k \n cat / K \n m of 4.52 µ m /min vs 3.03 and 0.72 for sinapoyl‐CoA and p ‐coumaroyl‐CoA, respectively (Baltas et al ., 2005 )]. However, the k \n cat / K \n m is (relatively) still much higher for Arabidopsis CCR1 compared with poplar CCR2. These observations could suggest that CCR (and/or CAD) cannot efficiently catalyse the biosynthesis of p ‐coumaryl alcohol in poplar, resulting in a relatively low accumulation of H units in the cse1 cse2 knockouts. The cse1 cse2 lines accumulate several hydroxycinnamoyl‐shikimates in xylem and bark tissues, such as p ‐coumaroyl shikimate ( 15 ), caffeoyl shikimate ( 16 ), feruloyl shikimate ( 18 ), 5‐hydroxyferuloyl shikimate ( 66 ), sinapoyl shikimate ( 22 ) and several derivatives thereof ( 17, 20, 21 , 23 , 63 ‐ 65, 67 ). The accumulation of these hydroxycinnamoyl‐shikimates suggests that they are also substrates of CSE. Indeed, our enzyme kinetic studies of PtrCSE1 and PtrCSE2 showed that both CSE1 and CSE2 are able to convert shikimates other than caffeoyl shikimate, such as p ‐coumaroyl, feruloyl and to a lesser extent also sinapoyl shikimate in vitro (Table 3 , Figure S9 ). Together with the observed accumulation of the hydroxycinnamoyl‐shikimates and their derivatives in cse1 cse2 mutant lines, the enzyme kinetics hint at the existence of another metabolic layer in the phenylpropanoid pathway, in which hydroxylation and methylation occur at the shikimate ester level, besides the free‐acid level, the CoA‐thioester level and the aldehyde level, as recently suggested (Saleme et al ., 2017 ). Moreover, cell lysates of yeast lines co‐expressing Populus nigra HCT1 and Arabidopsis thaliana 4CL4 were able to convert caffeic and ferulic acid to the corresponding shikimate esters, illustrating that Populus nigra HCT1 is able to use caffeoyl‐CoA and feruloyl‐CoA (formed by 4CL4 from the acids) as substrates, albeit to a lesser extent than p‐ coumaroyl‐CoA (Vanholme et al ., 2013a ). These results suggest a route in the phenylpropanoid pathway from the acids, through the CoA‐thioester intermediates, towards the shikimate esters that, in their turn, could be converted back to their acid forms by the CSE enzymes and in this way potentially form a substrate cycle. Such substrate cycles play an important role in cellular homeostasis, allowing for fluctuations in the cycle without directly affecting other fluxes in the metabolic network (Sridharan et al ., 2015 ), and in this particular case could be important in maintaining the levels of free CoA by regulating the accumulation of CoA‐ester intermediates such as caffeoyl‐CoA. \n CSE1 and CSE2 are partially redundant in poplar Both CSE promoters confer similar expression patterns (Figure S3 a–l), and both CSE proteins were shown to have similar relative activities towards caffeoyl shikimate (Ha et al ., 2016 ; Figure S9 ), suggesting that CSE1 and CSE2 are redundant in poplar. Despite the normal development and lignin accumulation of the cse1 and cse2 mutants, they show metabolic shifts in xylem and bark, including the accumulation of the CSE substrate, caffeoyl shikimate. Furthermore, cse2 mutants showed more differential compounds in both bark and xylem tissues in comparison with cse1 mutants, demonstrating that the redundancy of CSE1 and CSE2 is incomplete at the metabolic level. These results, in combination with the difference in catalytic efficiency of PtrCSE1 and PtrCSE2 (Table 3 ), suggest that CSE1 and CSE2 might be undergoing subfunctionalization. Moreover, our data indicate that the conversion of caffeoyl shikimate to caffeic acid (and potentially the conversion of p ‐coumaroyl shikimate, feruloyl shikimate and sinapoyl shikimate to p ‐coumaric acid, ferulic acid and sinapic acid, respectively) is not the rate‐limiting step in the pathway flux to lignin in WT plants, as its decrease in efficiency in the cse1 and cse2 mutants, substantiated by the product accumulation, does not result in notable changes in lignin amount. \n CSE as a target for engineering low‐lignin trees for the biorefinery A ~fourfold improvement in cellulose‐to‐glucose conversion was observed in cse1 cse2 poplars, independent of pretreatment. However, due to the significant biomass penalty this improvement does not persist when the glucose yield is expressed on a plant basis. Nevertheless, if the yield penalty of the plants could be overcome, CSE ‐edited trees would represent a significantly improved feedstock for the biorefinery. The vessel cell morphology is compromised in cse1 cse2 lines, as observed in several other plants with perturbations in the lignin biosynthetic pathway (Coleman et al ., 2008 ; Jones et al ., 2001 ; Leplé et al ., 2007 ; Schilmiller et al ., 2009 ; Vanholme et al ., 2013c ; Voelker et al ., 2010 ). This is a possible reason for the observed yield penalty, either because the function of the vessel cell wall is impaired or because its physico‐chemical defects signal a stress response (Gallego‐Giraldo et al ., 2020 ; Gallego‐Giraldo et al ., 2018 ; Ha et al ., 2021 ; Muro‐Villanueva et al ., 2019 ); restoring lignification in the vessel cells of Arabidopsis lignin mutants has been successfully used to recover the vessel cell morphology and overall biomass, while still maintaining the enhanced saccharification efficiency of the mutant biomass (De Meester et al ., 2018 ; Vargas et al ., 2016 ; Yang et al ., 2013 ). However, implementing this strategy in trees might not be straightforward to rescue the biomass yield penalty. It has been shown that in poplar, the fibres adjacent to vessels have a similar lignin composition as the vessels, suggesting that monolignols diffuse from vessels to fibres (Gorzsás et al ., 2011 ). Although it is not clear to what extent this diffusion could re‐lignify poorly lignified fibres, such as in the Arabidopsis cse mutants, it should be plausible to screen for transgenic lines that have normal‐shaped and lignified vessels but hypolignified fibres. Such biomass would still present advantages over wild‐type biomass. Down‐regulation of CSE1 and CSE2 expression in poplar through RNA interference ( hpCSE ) did not result in a yield penalty, even though the lignin amount was reduced by 25% (Saleme et al ., 2017 ). This suggests that there are no phenotypic effects as long as the lignin amount stays above a certain threshold in CSE ‐down‐regulated lines. The saccharification efficiency of the hpCSE ‐down‐regulated poplars was increased by 31% when no pretreatment was used (Saleme et al ., 2017 ), whereas that of the cse1 cse2‐ mutated poplars had an increase of 320% when no pretreatment was applied. To achieve a high saccharification efficiency while at the same time avoiding a yield penalty, instead of knocking out CSE1 and CSE2 , weak alleles may be generated via CRISPR‐Cas9, as recently illustrated for the CINNAMOYL‐CoA REDUCTASE 2 gene by De Meester et al . ( 2020 )."
} | 4,551 |
38682447 | PMC11200011 | pmc | 2,555 | {
"abstract": "Abstract The development of soft electronics and soft fiber devices has significantly advanced flexible and wearable technology. However, they still face the risk of damage when exposed to sharp objects in real‐life applications. Taking inspiration from nature, self‐healable materials that can restore their physical properties after external damage offer a solution to this problem. Nevertheless, large‐scale production of self‐healable fibers is currently constrained. To address this limitation, this study leverages the thermal drawing technique to create elastic and stretchable self‐healable thermoplastic polyurethane (STPU) fibers, enabling cost‐effective mass production of such functional fibers. Furthermore, despite substantial research into the mechanisms of self‐healable materials, quantifying their healing speed and time poses a persistent challenge. Thus, transmission spectra are employed as a monitoring tool to observe the real‐time self‐healing process, facilitating an in‐depth investigation into the healing kinetics and efficiency. The versatility of the fabricated self‐healable fiber extends to its ability to be doped with a wide range of functional materials, including dye molecules and magnetic microparticles, which enables modular assembly to develop distributed strain sensors and soft actuators. These achievements highlight the potential applications of self‐healable fibers that seamlessly integrate with daily lives and open up new possibilities in various industries.",
"conclusion": "3 Conclusion A stretchable thermoplastic elastomer TPU was synthesized via a step‐growth polymerization reaction, and the introduction of disulfide bonds endowed it with superior self‐healing properties. The synthesized TPU was employed in the thermal drawing process to fabricate a STPU fiber, thereby enabling the mass production of multifunctional self‐healing fibers. Furthermore, real‐time monitoring of the transmission spectrum variation of the STPU material during the self‐healing process enabled investigation into its healing speed, thereby establishing a standardized approach for monitoring and characterizing self‐healable materials. Additionally, STPU fibers with diverse peak absorption were fabricated by doping dye molecules, offering promising applications in distributed strain sensors. Finally, leveraging the inherent self‐healing capabilities of TPU allowed for the construction of soft magnetic actuators with various geometrical configurations to facilitate versatile motions. This work is expected to advance efforts in the processing, characterizing, and functionalizing of self‐healable materials. Also, by incorporating self‐healing attributes, this study is anticipated to prolong the operational lifespan of soft fiber devices and broaden their applications in diverse fields such as sensing and soft robotics.",
"introduction": "1 Introduction Elastic and stretchable fiber devices have garnered much attention due to their high flexibility, elasticity, mechanical toughness, and ease of fabrication. [ \n \n 1 \n , \n 2 \n \n ] The vigorous development of elastic and stretchable fibers makes up for the shortcomings of traditional silica fibers that are fragile and limited in strain, especially when applied in wearable textiles and optomechanical sensing applications. [ \n \n 3 \n , \n 4 \n , \n 5 \n , \n 6 \n , \n 7 \n , \n 8 \n \n ] So far, these fibers have shown promising performance in strain sensors, [ \n \n 9 \n , \n 10 \n , \n 11 \n \n ] temperature sensors, [ \n \n 12 \n \n ] robotics and automation, [ \n \n 13 \n , \n 14 \n \n ] in vivo optogenetic modulations, [ \n \n 15 \n , \n 16 \n \n ] optoelectronic probes, [ \n \n 17 \n , \n 18 \n , \n 19 \n \n ] and energy storage. [ \n \n 20 \n , \n 21 \n , \n 22 \n , \n 23 \n \n ] A variety of materials have been used for fabricating elastic and stretchable fibers, such as polydimethylsiloxane, [ \n \n 24 \n \n ] hydrogels, [ \n \n 25 \n , \n 26 \n \n ] and biomaterials. [ \n \n 27 \n , \n 28 \n \n ] However, these thermoset elastomers typically require shaping into fibers before curing, as they become difficult to soften and reshape once crosslinked. [ \n \n 29 \n , \n 30 \n \n ] Furthermore, due to thermoset elastomers' inherent low mechanical strength, handling ultra‐thin fibers still poses challenges. Thermoplastic elastomers (TPEs) with melt‐processability offer a potential solution to overcome these limitations. TPEs are polymers that become soft and moldable above their glass transition temperature ( T g), making them suitable for various polymer processing techniques. [ \n \n 31 \n , \n 32 \n , \n 33 \n \n ] They are compatible with various processing techniques to produce long fibers. For example, the thermal drawing technique commonly used for glass fiber production involves heating a macroscopic preform, which is then drawn into a kilometer‐long microstructure fiber while preserving its geometry but reducing the cross‐sectional dimension. [ \n \n 34 \n , \n 35 \n \n ] To date, the thermal drawing technique has been exploited to produce TPE fibers and shown unique endowments, enabling the mass fabrication of multimaterial fibers with fine diameters and delicate structures. [ \n \n 36 \n , \n 37 \n , \n 38 \n \n ] \n Another primary concern is the weakened properties of elastic and stretchable fibers when damaged. Inspired by nature, self‐healable materials can recover their physical properties after damage. [ \n \n 39 \n \n ] Two main self‐healing mechanisms exist: extrinsic approaches to encapsulate healing agents and chemical approaches to incorporate supramolecular chemistry and dynamic bonds. [ \n \n 40 \n , \n 41 \n \n ] Encouragingly, the self‐healing property has been successfully introduced into TPEs through the dynamic exchange of disulfide bonds, metal coordination bonds, and hydrogen bonds, exploiting application scenarios for elastic and stretchable fibers. [ \n \n 42 \n , \n 43 \n \n ] Tan et al. fabricated covalently cross‐linked self‐healable ionogel fibers by melt‐spinning approach and innovatively demonstrated their application in flexible electronic devices. [ \n \n 44 \n \n ] However, most self‐healable fibers are primarily fabricated by molding or spin‐coating, limiting their large‐scale manufacturing and application opportunities. [ \n \n 45 \n , \n 46 \n , \n 47 \n , \n 48 \n \n ] In addition, current characterization methods for self‐healable materials mainly fall into two categories: direct visual observation of the material morphology or indirect measurement of the functional integrity. [ \n \n 49 \n , \n 50 \n , \n 51 \n \n ] There are significant differences between various test methods, so it is difficult to compare the healing effects of different self‐healable materials horizontally. Moreover, most current characterization methods are performed at certain points in the healing process and lack real‐time monitoring. Hence, there is an urgent need to establish a unified standard for quantifying the speed and time of self‐healing, especially for real‐time monitoring systems. [ \n \n 52 \n \n ] \n This work employs the thermal drawing technique with mass production capability to manufacture self‐healable multifunctional fibers. Specifically, a self‐healable thermoplastic polyurethane (STPU) material is synthesized, containing dynamic reversible aromatic disulfide bonds, hard asymmetric alicyclic segments, and soft polytetramethylene ether glycol (PTMEG) segments. The self‐healing property of the synthesized material relies on the disulfide bond exchange reaction, while the hard segment domains enhance the mechanical performance. Subsequently, the synthesized material is molded into a cuboid‐shaped preform and then drawn into a microscopic functional fiber through the thermal drawing technique. Next, taking inspiration from commonly used optical fiber testing, the self‐healing process is quantitively studied by real‐time monitoring of the fiber transmission spectrum throughout the segment cutting, resplicing, and healing phases. Multifunctional capabilities are demonstrated: 1) distributed strain sensors are achieved by incorporating methylene blue and rhodamine 590 dye molecules, and 2) magnetic self‐healable soft actuators with four distinct modes of deformation and locomotion. These results highlight the potential applications of self‐healable fibers and unlock further opportunities across various fields in material characterization, fiber device fabrication, sensing, and soft robotics.",
"discussion": "2 Results and Discussion 2.1 Fabrication of Self‐Healable Multifunctional Fiber via Thermal Drawing The STPU material is synthesized through a two‐step reaction. To start, the prepolymer was obtained by step‐growth polymerization of thoroughly dried PTMEG and isophorone diisocyanate in DMAc at 70 °C. Subsequently, the mixture was cooled to 40 °C and further polymerized with bis(4‐hydroxyphenyl) disulfide. The detailed information can be found in Figures S1–S5 (Supporting Information). Due to dynamic disulfide bonds, the synthesized polymer exhibits disulfide metathesis and automatic repair capabilities when subjected to damage. In addition, incorporating loosely packed asymmetric alicyclic segments into soft PTMEG segments can effectively enhance mechanical performance while maintaining self‐healing properties. [ \n \n 53 \n \n ] \n Then, in order to construct a preform applicable to the fiber thermal drawing process, a DMAc solution with synthetic STPU was poured into a designed cuboid‐shaped PTFE mold after the synthetic procedure ( Figure \n \n 1 a ). Then, the preform material was subjected to continuous heating, gradually increasing the temperature from 60 to 130 °C throughout 48 h, to facilitate evaporation of DMAc. A well‐shaped preform was obtained after being baked in a vacuum oven at 130 °C for 24 h. Figure 1 Fabrication of self‐healable fibers via preform‐to‐fiber thermal drawing technique. a) The as‐synthesized STPU material forms into a preform through a molding method. Then, the preform is fed into the tube furnace of the fiber drawing tower, where the bottom of the preform undergoes controlled softening and necking down under an externally applied force. Finally, the fiber is collected onto a cylindrical bobbin. b) Schematic diagram illustrating the self‐healing process after damage, wherein reversible metathesis of broken disulfide bonds facilitates the repair of cut materials. c) The surface of the self‐healable fiber healed in 2 h at room temperature against scratching. d) The self‐healable fiber exhibited outstanding strength against external forces after the instant healing. Next, the prepared preform was loaded into a two‐zone vertical tube furnace on a fiber drawing tower (Figure 1a ). During the thermal drawing process, the bottom portion of the preform was softened by the prescribed heating temperature and underwent necking by the applied external force. Subsequently, the preform was severed from the necking region, and its end was elongated to affix onto a pair of rotating rollers below for drawing. The material's viscosity ensures that the fiber's cross‐sectional shape aligns consistently with the preform. The viscosity property of the STPU was determined using a rheometer (TA HR10) under steady shear and dynamic oscillatory conditions, with in situ heating from 90 to 150 °C. Figure S6 (Supporting Information) shows the complex shear viscosity (η*), the storage (G′), and the loss (G′′) modulus. Both G′ and G″, as well as |η*|, decrease as temperature rises. A transition point is observed where G′ and G″ curves intersect. Below this point, elastic behavior dominates; above it, flow is favored by G″ prevailing. This crossover point satisfies the rheological requirements for thermal drawing, making the synthesized STPU suitable for processing through this method. The preform was fed down into the furnace at a speed of 1 mm min −1 , while the fiber was drawn at a speed of 0.4 m min −1 by the rotating rollers. Finally, the self‐healable fiber with a cross‐sectional dimension of 1 mm × 0.5 mm was collected by a cylindrical bobbin. The fabricated fiber exhibits superior self‐healing properties at room temperature, owing to the disulfide metathesis, as shown in Figure 1b . To study the self‐healing performance of the fabricated TPU fibers, the surface of the self‐healable fiber was scratched and healed in 2 h at room temperature. Optical microscope images of the STPU surface scratched by the tip of a tweezer also show that the X‐shaped scratch gradually shallows with time and almost disappears after 2 h (Figure 1c ). Moreover, a routine cut and healed method was conducted to confirm the self‐healing capability of the fabricated fiber. As illustrated in Figure 1d , self‐healable fiber was divided into three sections, respliced, and immediately stretched from both ends. Although complete healing of the fiber surface requires more time, the fiber was partially healed and was strong enough to resist external forces. Additionally, a dolphin stamp was printed on the STPU film. The disappearing process of the pattern was vividly displayed under microscopic observation, further revealing the self‐healing ability of the synthetic material ( Figure \n \n 2 a ). Figure 2 Self‐healing performances of the fabricated STPU fibers and their surface. a) Optical microscopy images of dolphin‐patterned STPU surface during the healing process. b) Stress–strain curves of the original STPU fiber and respliced fibers after different healing times. Inset: Photograph of STPU fiber attached to cardboard during stretching test. c) Schematic diagram of the formation process of a self‐healable net. The cut STPU fibers are divided into two groups and vertically stacked in a parallel arrangement, resulting in immediate net formation through disulfide bond metathesis. d) Photograph of STPU fibers self‐healed to form a net. The net can lift a 50 g apple steadily. The mechanical strength of the fabricated STPU fiber was measured by a uniaxial tensile test. Specifically, an STPU fiber with a cross‐sectional dimension of 0.5 mm × 0.3 mm was affixed to cardboard using glue at both ends, leaving a hanging portion of 5 mm in length. Subsequently, the sample was subjected to axial tension using a testing machine equipped with a 10 N load cell, applying a constant speed of 5 mm min −1 (the inset of Figure 2b ). As depicted in Figure 2b , the mechanical behavior of the pristine STPU fiber aligns more closely with the stress‐strain model observed in softened glassy polymers, [ \n \n 54 \n \n ] albeit exhibiting greater strain capacity. Following the yield point, necking occurs, and stress rises significantly during the latter half of the curve until the ultimate failure is reached. The fracture strength of the STPU fiber is measured to be 1.05 MPa, slightly lower than the traditional thermally drawn fibers previously reported. [ \n \n 17 \n , \n 36 \n , \n 37 \n \n ] One possible explanation is the low molecular weight of TPU. For example, the molecular weights of commercial TPEs are generally hundreds of thousands or even higher, while the molecular weight of our synthesized TPU is only ≈40 000. Notably, this STPU fiber demonstrates exceptional stretchability, with an elongation rate as high as 1600%. To investigate its self‐healing performance, multiple samples were prepared by attaching STPU fibers onto cardboard, cutting them in half, and realigning them. Tensile tests were carried out under the same conditions after different healing periods at room temperature. The fracture stress and strain of STPU fibers increase with the extension of healing time, as illustrated in Figure 2b . After a healing period of 3 h at room temperature, the STPU fiber can withstand a fracture stress of 0.89 MPa and reach a fracture strain of 1550%. This indicates that the mechanical property of the STPU fiber is almost restored through self‐healing within a relatively short duration. The fiber was cut and spliced into a net configuration to further validate its mechanical strength and self‐healing capability for practical applications (Figure 2c ). Due to the dynamic reaction of the disulfide bonds, immediate self‐healing occurred at the junctions upon connection. Subsequently, a 50 g apple was placed on the net, demonstrating its stable supporting capability for real‐life scenarios (Figure 2d ). 2.2 Real‐Time Monitoring Transmission Spectra of the Self‐Healing Process In addition to the conventional indirect characterization of the self‐healing performance, an optical approach is proposed for quantitatively characterizing the time and speed of self‐healing. Specifically, an STPU sample was cut into two pieces and placed in the optical path between a white light source (Fiber‐Lite DC950 Illuminator) and a miniature spectrometer (Ocean Optics USB2000+) ( Figure \n \n 3 a ). Real‐time monitoring of the variation in transmitted light during the self‐healing process was performed. As depicted in Figure 3b , the black curve represents the transmission spectrum after dividing the sample into two sections. Compared to the pre‐cut spectrum (the gray curve), the normalized transmission intensity experiences a decrease due to the diffuse reflection caused by the roughness at the cut cross‐section. Since no absorption peak exists for STPU within the 550–750 nm range (Figure S5 , Supporting Information), variations in spectral decline at different wavelengths correspond to changes in relative spectral radiance from the light source, [ \n \n 55 \n \n ] with maximum change observed at 699 nm where the intensity is the highest, decreasing from 0.72 to 0.56. With the gradual self‐healing of the cut cross‐section, the transmission spectrum gradually recovers. After 6 min, half of the reduced transmission intensity is restored (the blue curve in Figure 3b ). The recovery speed gradually slows, and after 3 h, the transmission spectrum almost entirely coincides with the pre‐cut curve, indicating that the STPU sample is completely self‐healed. The real‐time variations in transmission intensity at different wavelengths were recorded to further investigate the self‐healing process. As presented in Figure 3c , four representative wavelengths are selected. Taking 550 nm as an example, the transmission intensity increases rapidly in the first few minutes and then gradually slows down. The noise in the spectrum decreases with longer wavelengths due to the increased relative spectral radiance of the incident light at the corresponding wavelength. Obviously, the transmission spectra at different wavelengths exhibit identical varying tendencies. Self‐healing occurs instantly after re‐splicing the STPU and slows down until complete healing is achieved. This self‐healing process elucidates why the fiber can immediately withstand external tension upon reconnection. As shown in Figure 1c , partial healing occurs internally despite surface cracks still being present. Next, derivative analysis is performed on the transmission spectra to determine recovery speeds during the self‐healing process. As shown in Figure 3d , all spectra exhibit maximum recovery speeds at the beginning, dramatically decreasing over time until stabilizing at relatively low values after 30 min. The observed trend of self‐healing speed obtained from transmission spectra aligns with previously reported disulfide displacement reaction rates found in literature since self‐healing relies on disulfide metathesis. [ \n \n 56 \n \n ] \n Figure 3 Direct monitoring of the self‐healing process in real‐time. a) Schematic diagram of the spectrometer setup for monitoring the self‐healing process. b) Transmission spectra recovery along the STPU block self‐healing process. c) The transmission spectra at 550, 600, 650, and 690 nm over time in the self‐healing process. d) The differentiation of the transmission spectra reveals the recovery speed during the self‐healing process: it heals the fastest at the beginning, and the healing speed decreases with time. Inset: The smoothed recovery speed within 30 min. The method of recording the transmission spectrum variation to characterize the self‐healing process of materials exhibits notable merits and promising prospects. First, this method provides a standardized measurement to assess the light‐permeable material's self‐healing speed and recovery extent as the spectral change is visualized and quantifiable. Secondly, unlike other methods that select several points during the self‐healing process for testing, this approach enables real‐time and coherent observation. Thirdly, the approach is simple, only requiring self‐healable materials to be placed along the optical path. Finally, this method can theoretically be applied to all light‐permeable materials. Encouragingly, most self‐healable materials are transparent, including hydrogels, biomaterials, and self‐healable TPEs. In addition, light sources and spectrometers can be replaced with other wavelength ranges, such as ultraviolet, infrared, etc., further expanding the range of characterizable materials. Therefore, this method has the potential to be applied to a broader range of self‐healable materials. 2.3 Distributed Strain Sensor Based on Self‐Healable Fibers As the spectral changes in these fibers can accurately reflect the strains induced by human motion, elastic and stretchable optical fibers have gained significant attention. As discussed, STPU fibers exhibit a superior stretchability of 1600% strain, making them a promising candidate for strain‐sensing applications. In addition, the self‐healing property of STPU enables the modular assembly of different STPU fibers with distinct absorption peaks into a distributed strain sensor configuration. To demonstrate the feasibility of this approach, two dye molecules with varying absorption spectra were separately incorporated into synthetic STPU solutions before molding. These solutions were then poured into rectangular‐shaped molds and subjected to vacuum heating for DMAc solvent evaporation. Figure S7 (Supporting Information) illustrates three types of resulting STPU films: original film without doping, rhodamine 590‐doped film, and methylene blue‐doped film, each having a thickness of 120 µm. The black line represents the absorption spectrum of the undoped STPU film, exhibiting no prominent absorption peak. In contrast, rhodamine 590‐doped and methylene blue‐doped STPU films exhibit absorption peaks at wavelengths of 533 and 631 nm, respectively. Next, rhodamine 590 and methylene blue‐doped STPU fibers were fabricated. The transmission spectrum of the original STPU fiber is described by the green line in Figure S9 (Supporting Information). Notably, compared to the original fiber's transmission spectrum, the transmission spectrum of rhodamine 590‐doped fiber shows a significant drop within the wavelength range from 500 to 600 nm (orange line), indicating light absorption within this range. Similarly observed for methylene blue‐doped fiber (blue line), its transmission spectrum decreases within a wavelength range from 550 to 660 nm. The broad wavelength drop range is reasonable because the light propagates throughout the entire length of the fiber. Figure S10 (Supporting Information) displays the normalized absorption spectra extracted from the transmission spectra, corresponding to the absorption spectra obtained from UV‐vis tests. Initially, the transmission spectrum alterations in the original STPU fiber during stretching were documented. Subsequently, a comparative analysis was conducted to examine the spectral changes in the dye molecular‐doped fibers under similar stretching conditions ( Figure \n \n 4 a ). Figure S8 (Supporting Information) shows the experimental setup where the STPU fiber is placed between a light source and a spectrometer while being fixed on two moveable stages for left and right stretching. The transmission spectra variation during stretching is shown in Figure 4b , indicating that the absorption of the fiber increases with the change in fiber length. The normalized loss of the STPU fiber exhibits a linear relationship with the propagation length (Figure 4c ), indicating its suitability as a strain sensor. Subsequently, an original STPU fiber was spliced together with a methylene blue doped STPU fiber, followed by healing at room temperature for 3 h to fabricate a distributed strain sensor featuring a 4 mm dye‐doped region (Figure S11a , Supporting Information). This strain sensor is realized by recording the changes in the transmission spectra during the application of strain. The strain sensor was affixed onto a translation platform to perform stretching experiments, and the left displacement table was adjusted to stretch the undoped region. Each increment of stretching is set at 0.8 mm until reaching 8 mm after five repetitions. Subsequently, the right displacement table was adjusted to stretch the methylene blue‐doped area to 8 mm (Figure S11a , Supporting Information). As shown in Figure 4d , stretching the methylene blue‐doped region leads to a more pronounced increase in the normalized loss than stretching the undoped area (Figure S12 , Supporting Information). Taking the absorption at 631 nm as the sensing indicator, the sensor provides a nearly linear response within a strain range of 100%. The difference in the normalized loss for the undoped region is 0.11/ε, whereas, for the methylene blue‐doped region, it reaches 0.31/ε (Figure 4e ). This preliminary demonstration showcases a trustworthy distributed strain sensor that utilizes the self‐healing characteristic of STPU fiber. Similarly, an original STPU fiber and a rhodamine 590 doped STPU fiber were spliced together for strain sensing application (Figure S11b , Supporting Information). When the doped region is stretched to 1× strain, it results in twice the normalized loss compared to the undoped region (Figure 4f ; Figure S13 , Supporting Information). Taking the absorption at 533 nm as the sensing indicator, the difference normalized loss is 0.13/ε and 0.02/ε, respectively (Figure 4g ). Figure 4 Methylene blue and rhodamine 590 doped STPU fiber for distributed strain sensing. a) Schematic of the testing approach. b) Transmission spectra variation of STPU fiber during stretching. c) The nearly linear increase of the normalized loss against propagation length. d) Normalized absorption spectra variation of the fabricated distributed strain sensor when the strain is applied to the undoped and methylene blue‐doped sensor regions. e) The nearly linear increase of difference loss against the applied strain. f) Normalized absorption spectra variation of the fabricated distributed strain sensor when the strain is applied to the undoped region and rhodamine 590 doped sensor region. g) A nearly linear increase in difference loss with respect to the applied strain. 2.4 Magnetic STPU Soft Actuators In addition to dye molecules, magnetic particles can be incorporated into STPU to achieve multifunctional expansion. Compared with stiff materials, STPU, as an exemplary elastomer, possesses a low elastic modulus and is accessible to dope, making it a compelling candidate for soft actuators. For magnetic‐driven soft actuators, magnetic particles are embedded into the polymer matrix to fabricate magnetically responsive soft materials. [ \n \n 57 \n \n ] Moreover, the magnetic particles‐embedded soft actuator exhibits unique and desirable features, including rapid response and remote actuation under a magnetic field. [ \n \n 58 \n , \n 59 \n \n ] In this work, to achieve various shape deformations and enable locomotion of the STPU elastomer‐based magnetic soft actuators, oriented magnetic microparticles are incorporated into the STPU polymer matrix. Initially, the magnetic microparticles are distributed randomly in the polymer matrix after mixing. Subsequently, they tend to change from a disordered arrangement to align along the direction of the applied magnetic field, as illustrated in Figure \n \n 5 a . Upon solidification, the oriented magnetized STPU elastomer is procured, wherein the soft polymer matrix has successfully immobilized the linearly aligned iron microparticles. Throughout our experiment, the magnetization direction of the magnetic microparticles is intentionally chosen to coincide with the horizontal plane of the STPU elastomer specimen. Figure 5 Magnetic STPU soft actuators. a) Schematic of the magnetic chain alignment in the TPU elastomer under the magnetic field. b) Opposite magnetization direction (180°) spliced strips‐based soft actuator with bending deformation under the x–y plane magnetic field. c) Vertically magnetization direction (90°) spliced fiber‐based soft actuator with the rolling motion under the x–z plane rotating magnetic field. d) Möbius ring spliced strip‐based soft actuator with distorting deformation under the x–y plane magnetic field. e) Butterfly‐shaped inspired spliced soft actuator under an x–z plane oscillating magnetic field. Leveraging the inherent self‐healing properties of TPU, soft magnetic actuators with various geometrical configurations were constructed by splicing different modules together via the disulfide bonds. Ordinarily, the self‐healable magnetic soft actuator will endure magnetic torque until the oriented magnetic microparticles in the polymer matrix align with the direction of the applied external magnetic field (Figure S14 , Supporting Information). Ordinarily, the self‐healable magnetic soft actuator will endure magnetic torque until the oriented magnetic microparticles in the polymer matrix align with the direction of the applied external magnetic field. Here, four types of magnetically responsive soft actuators are presented: bending, rolling, distorting, and beating. For the first type, Figure 5b illustrates a magnetic soft actuator exhibiting bending deformation. By bisecting STPU strips laden with the oriented magnetic microparticles inverting the right segment and then adhesively splice joining the left and right sections through self‐healing, opposite magnetization directions (180°) spliced STPU strips based soft actuator is successfully obtained. When subjected to an external magnetic field, this horizontally resting soft actuator first erects itself, then bends and recovers when the magnetic field rotates at an angle in the x–y plane, as demonstrated in Movie S1 (Supporting Information). Such bending deformation is caused by magnetic torque under the magnetic field, owing to the opposite magnetization directions within the soft actuator strip structure. For the second type, when an oriented magnetic STPU fiber with a circular cross‐section is bisected and the right half rotated by 90° before reassembling via the self‐healing process, a vertically spliced fiber‐based soft actuator is obtained. This type of self‐healable soft actuator achieves a rolling motion under the 360° rotating magnetic field in the x–z plane and moves forward along x direction as depicted in Figure 5c . The soft robot rolls forward owing to the pulling force and the torque under the rotating and straightforward magnetic field (Movie S2 , Supporting Information). For the third type, the Möbius ring, a captivating structure, is constructed by twisting the oriented magnetic STPU strip and then splicing the two ends together. The magnetic Möbius ring stands upright when the magnetic field is off but transitions to rest flat, beginning its distorting deformation when the magnetic field rotates at an angle in the x–y plane, as shown in Figure 5d . The orientation distribution of magnetic microparticles in the strip alters due to the twist of the 2D circular structure, enabling the Möbius ring spliced strip soft actuator to exhibit distortion and folding deformations (Movie S3 , Supporting Information). For the fourth type, a butterfly‐shaped inspired structure is constructed. Two rectangular magnetic STPU blocks are spliced into assembly with the pristine STPU block in the middle to form a butterfly‐shaped soft actuator by the self‐healing process. This soft actuator achieves the beating motion under an oscillating magnetic field in the x–z plane, reminiscent of a butterfly flapping its wings, as represented in Figure 5e and Movie S4 (Supporting Information). The controllable deformation and locomotion of these magnetic STPU‐based soft actuators demonstrate the immense potential to serve as integral components in soft robotics applications, including artificial muscles, autonomous soft robots, and soft grippers."
} | 8,135 |
30307662 | PMC6282539 | pmc | 2,557 | {
"abstract": "Abstract Methanogenic communities play a crucial role in carbon cycling and biotechnology (anaerobic digestion), but our understanding of how their diversity, or composition in general, determines the rate of methane production is very limited. Studies to date have been correlational because of the difficulty in cultivating their constituent species in pure culture. Here, we investigate the causal link between methanogenesis and diversity in laboratory anaerobic digesters by experimentally manipulating the diversity of cultures by dilution and subsequent equilibration of biomass. This process necessarily leads to the loss of the rarer species from communities. We find a positive relationship between methane production and the number of taxa, with little evidence of functional saturation, suggesting that rare species play an important role in methane‐producing communities. No correlations were found between the initial composition and methane production across natural communities, but a positive relationship between species richness and methane production emerged following ecological selection imposed by the laboratory conditions. Our data suggest methanogenic communities show little functional redundancy, and hence, any loss of diversity—both natural and resulting from changes in propagation conditions during anaerobic digestion—is likely to reduce methane production.",
"introduction": "1 INTRODUCTION Consistent with the large body of work on plant communities (Grime, 1997 ; Hector et al., 1999 ; Hooper, Adair, & Cardinale, 2012 ; Nielsen, Ayres, Wall, & Bardgett, 2011 ; Tilman, 1997 ), microbial diversity can have a positive role in a range of community functions, including aerobic respiration, litter decomposition and plant growth (Bell, Newman, Silverman, Turner, & Lilley, 2005 ; Delgado‐Baquerizo et al., 2016 ; Handa et al., 2014 ; Philippot et al., 2013 ; Wagg, Bender, Widmer, & van der Heijden, 2014 ). Strongly positive diversity–function relationships imply little functional redundancy of community members, and hence, loss of diversity resulting from environmental change may have considerable impact on community function (Jax, 2005 ; Loreau, 1998 ). One of the key microbial ecosystem functions where the role of diversity has not been experimentally investigated is methanogenesis: methane production resulting from the anaerobic conversion of H 2 , CO 2 and short chain fatty acids by archaeal methanogens (Ferry, 2012 ). Methane is both a potent greenhouse gas and a renewable resource from organic waste; therefore, determining causal links between microbial community diversity, composition and methanogenesis is important. Research investigating the links between methanogenesis and microbial diversity has been correlational. Studies of methanogenesis in natural soil communities have reported positive correlations between methane production (from an incubated soil sample) and the diversity of both methanogens and the total bacterial/archaeal communities (Wagner, Zona, Oechel, & Lipson, 2017 ; Yavitt, Yashiro, Cadillo‐Quiroz, & Zinder, 2012 ). However, any conclusions are potentially confounded by other environmental variables, such as pH, that can have a major role on community structure (Fierer et al., 2012 ; Hesse et al., 2018 ) and methanogenesis (Wagner et al., 2017 ). Other studies have focussed on correlations between community structure and methanogenesis under “common garden” laboratory conditions, where environmental factors are better controlled. The largest of these, involving 150 samples (Venkiteshwaran et al., 2017 ), showed no relationship between diversity and function, but in this case, the composition of communities differed in many ways in addition to diversity, and biomass was not controlled for. As a consequence, there is a clear need to conduct manipulative experiments where causal links between diversity and methanogenesis can be determined. Manipulating diversity of methanogenic communities is nontrivial: They are typically very complex, consisting of varied taxa, most of which cannot be easily grown in pure culture or even cultivated at all. This makes the factorial manipulation of diversity at ecologically relevant levels almost impossible. Diversity can, however, be manipulated by dilution (Hernandez‐Raquet, Durand, Braun, Cravo‐Laureau, & Godon, 2013 ; Philippot et al., 2013 ; Salonius, 1981 ), which necessarily results in the loss of rare species relative to common species. Here, we conduct such a dilution manipulation across six orders of magnitude on a methanogenic ancestral community obtained by mixing twelve separate communities. We have previously shown that mixing multiple communities maximizes the function and diversity in the mix (Sierocinski et al., 2017 ), thus using the mix maximized our chance of generating a highly functional community in the process. We allowed the biomass of the diluted cultures to become re‐established over months in laboratory reactors and then densities equalized between treatments. Methane production was subsequently measured over six weeks in laboratory anaerobic digesters. In an attempt to assess the importance of diversity of rare species relative to other differences in community composition, we also investigated correlations between diversity and methanogenesis in natural communities isolated from a range of industrial anaerobic digesters and associated feedstock environments (sewage, silage, slurry, etc) over eight weeks. A number of studies suggest that novel propagation conditions impose selection pressures can result in large changes in the composition of methanogenic communities (De Vrieze et al., 2015 ; Mladenovska, Dabrowski, & Ahring, 2003 ; Regueiro et al., 2012 ; Vanwonterghem et al., 2014 ), and hence, we determined community composition at the start and end of the experiment.",
"discussion": "4 DISCUSSION We investigated the link between microbial diversity and the rate of methane production in natural and manipulated communities. We found no correlations between any aspect of community composition at the start of the experiment and methane production across the 12 natural communities. However, after eight weeks of propagation in laboratory anaerobic digesters, there was a loss of diversity within communities and communities had converged. At this point, we found a positive relationship between methane production, species (OTU) richness, bacteria and methanogen density. We obtained the same qualitative results in communities where diversity was manipulated by dilution over six orders of magnitude. This suggests that decreasing species richness in methanogenic communities will reduce methane production and that this effect is robust to variation in species composition present in natural communities. Manipulating diversity by dilution has limitations. Most obviously, it confounds diversity with species identity to some extent, in that dilution of communities results in the loss of predominantly rare taxa. As a consequence, the results suggest that methane production decreases with the increasing loss of rare species, rather than the loss of random taxa. To put this into context, the loss of half of the community made up by the rarest species results in approximately 50% reduction in gas production. Dilution also had the effect of increasing within‐treatment beta diversity, which could limit the interpretation of analyses. This increase in beta diversity is presumably the result of increased stochasticity in community assembly when taxa are at lower frequencies as a result of dilution. The relationship between gas production and species richness in the dilution experiment showed little functional saturation (an exponent of 0.43 for the relationship) compared to most diversity–function studies (O'Connor et al., 2017 ). By contrast, the exponent of the gas production‐species richness relationship in the correlational study was extremely high (~4), suggesting an accelerating relationship. However, this very high value likely reflects an overestimation of species richness of the poorer‐performing communities. Specifically, poor‐performing communities had the greatest net loss of OTUs through time, and this loss may be underestimated because of residual DNA of dead cells and the presence of OTUs that were not yet driven to extinction. Our study supports the growing body of evidence that rare species play an important role in the community function (Lynch & Neufeld, 2015 ; Mouillot et al., 2013 ). Both our studies that suggest large numbers of rarer species support higher densities of acetoclastic methanogens: methane‐producing Archaea locked into mutualisms with acetate‐producing bacteria (Ferry, 2012 )), which are locked into syntrophic cross‐feeding interactions with acetate‐producing bacteria. Precisely why this might be is unclear, but recent theory suggests that growth under low energy conditions (as is the case under anaerobic conditions when oxygen is not used as the final electron receptor) is typically thermodynamically constrained, and results in a high diversity of metabolic niche specialists. This is because there a selective advantage to use a substrate in different way to competitors (negative frequency‐dependent selection (Clarke, 1979 ), to avoid thermodynamic inhibition of metabolism resulting from the build up of waste products (Großkopf & Soyer, 2016 ). More generally, thermodynamic constraints may help to explain why diversity seems less important for aerobic (Nielsen et al., 2011 ) than anaerobic (e.g., methanogenesis and denitrification; Philippot et al., 2013 ) functions in communities approaching natural levels of diversity. Finally, it is also possible that genetic variation within species, which would have been reduced by dilution and perhaps during propagation of the natural communities, could have contributed to the results. For example, recent work suggests that within‐species diversity associated with rapid adaptation can play a major role in the structure of natural soil microbial communities (Gómez et al., 2016 ). The composition of the communities we investigated was broadly typical of methanogenic communities (Nelson, Morrison, & Yu, 2011 ; Yang et al., 2014 ; Yu, Lee, & Hwang, 2005 ), with Firmicutes , Bacteroides and Proteobacteria being the main phyla. However, consistent with other studies (De Vrieze et al., 2015 ; Demirel & Yenigün, 2006 ; Elbeshbishy, Nakhla, & Hafez, 2012 ; Mladenovska et al., 2003 ; Regueiro et al., 2012 ; Town, Links, Fonstad, & Dumonceaux, 2014 ; Vanwonterghem et al., 2014 ), we observed a convergence of communities through time. This was associated with an increase in Firmicutes and a decline in Bacteriodetes reads through time in the 12 natural communities. The most predominant group in the Firmicutes , Clostridia, is known for their cellulolytic and amylolytic activity (Nelson et al., 2011 ). Our medium was based on starch and cellulose, making Clostridia perfect candidates for the hydrolysis steps of fermentation within the system. Another reason for the increase in Firmicutes could simply be selection against them during sampling: Firmicutes have low oxygen tolerance (Kampmann et al., 2012 ), and while every care was taken during sampling , initial communities were inevitably exposed to air in the field. It is less clear why Bacteriodetes were selected against in the laboratory‐scale anaerobic digesters, but their reduction in frequency is consistent with increased biogas production: Bacteroidetes are associated with the production of propionate and other short fatty acids, which can lead to disturbances in anaerobic digester system (Gallert & Winter, 2008 ). It was difficult to draw any firm conclusions about the role of specific taxa in gas production, beyond the positive effect of acetoclastic methanogens. However, in the natural converged communities, poor gas production was associated with the presence of Pseudoramibacter , Oscillospira, Bacteroides uniformis and Enterobacteriaceae . These species are typically associated with animal gut microbiomes, where they putatively are responsible for fermentation of glycans to butyrate (Benítez‐Páez, Gómez del Pulgar, & Sanz, 2017 ). It is possible that our medium, rich in meat extract, contributed to the enrichment of these species. The lack of animal host able to metabolize butyrate may have to its accumulation, detrimental to the functioning of the communities not capable of coping with it. OTUs that were overrepresented in the more diverse communities in the dilution experiment could plausibly have important roles: Coriobacteriacea have been suggested before to play a role in breaking down aromatic compounds in (Noguchi, Kurisu, Kasuga, & Furumai, 2014 ); Ruminococcus are involved in cellulolytic and xylolytic activity (Jia, Wilkins, Lu, Cai, & Lee, 2016 ); and Peptococcus are speculated to be acetate‐producing syntrophic partners of acetoclastic methanogens (Tang, Shigematsu, Morimura, & Kida, 2005 ). The importance of rare species in determining the productivity of methanogenic communities has potentially important implications. First, communities may take a relatively long time to achieve maximal levels of methane production following environmental changes, given that key beneficial rare species may not be present. This is in contrast to aerobic communities where function is typically restored to high levels following environmental change because of functional redundancy within communities (Martiny et al., 2006 ; Strickland, Lauber, Fierer, & Bradford, 2009 ). Second, from a biotechnological perspective, we demonstrate, like research before us, that the starting inoculum plays a crucial role (De Vrieze et al., 2015 ; Elbeshbishy et al., 2012 ). Unfortunately, our results show that knowledge of the starting inoculum a priori may prove uninformative as the importance of community composition only becomes apparent after ecological selection imposed by the specific anaerobic digester conditions. This problem can be circumvented by inoculating multiple communities in the starting culture (Sierocinski et al., 2017 ). In summary, our results suggest that there is little functional redundancy in methanogenic communities, and hence, any loss of diversity will likely reduce methane production. Moreover, given that microbes appear to be dispersal‐limited to some extent (Bell, 2010 ), the potential for methanogenic communities to adapt to changing conditions is likely to be constrained by their starting composition."
} | 3,657 |
35694474 | PMC9178951 | pmc | 2,558 | {
"abstract": "Superhydrophobic\nsurfaces have great potential for various applications\nowing to their superior dewetting and mobility of water droplets.\nHowever, the physical robustness of nano/microscale rough surface\nstructures supporting superhydrophobicity is critical in real applications.\nIn this study, to create a superhydrophobic surface on copper, we\nemployed copper electrodeposition to create a nano/microscale rough\nsurface structure as an alternative to the nanoneedle CuO structure.\nThe rough electrodeposited copper surface with a thin Teflon coating\nshows superhydrophobicity. The enhancement of dewetting and mobility\nof water droplets on copper surfaces by electrodeposition and hydrophobization\nsignificantly improved the condensation heat transfer by up to approximately\n78% compared to that of copper substrates. Moreover, the nano/microscale\nrough surface structure of the electrodeposited copper surface exhibits\nbetter tolerance to physical rubbing, which destroys the nanoneedle-structured\nCuO surface. Therefore, the condensation heat transfer of the superhydrophobic\nelectrodeposited copper surface decreased by only less than 10%, while\nthat of the nanoneedle-structured CuO surface decreased by approximately\n40%. This suggests that an electrodeposited copper surface can lead\nto the stable performance of superhydrophobicity for real applications.",
"conclusion": "4 Conclusions A multifunctional superhydrophobic\nsurface can be fabricated using\na thin Teflon coating on an electrodeposited copper surface. The microscale\nroughness of the copper deposit increases with an increase in the\nelectrodeposition duration, thereby enhancing the dewetting and mobility\nof the water droplet. This enhancement contributes to the easy roll-off\nof condensed water droplets on the cold surface; thus, the surface\nof an electrodeposited copper layer with a Teflon coating shows a\nsignificant improvement in the condensation heat transfer. Moreover,\nthe rough microscale structure fabricated by copper electrodeposition\nhas a better tolerance against physical contacts that destroy the\nrough surface structure, such as rubbing, compared to that of the\nnanoneedle CuO structure, which is generally used to fabricate superhydrophobic\nsurfaces on copper. Therefore, the superhydrophobic electrodeposited\ncopper surface shows stable condensation heat transfer. In contrast,\nthe superhydrophobic nanoneedle CuO surface is significantly damaged\nby surface rubbing, which causes significant debilitation in condensation\nheat transfer.",
"introduction": "1 Introduction Condensation is a ubiquitous\nphenomenon in the natural environment\nand is applied to various engineering systems, such as water harvesting,\nenergy conversion, and heat management systems. 1 − 3 In particular,\nthe condensation heat transfer caused by the release of latent heat\nto the surface of the condenser at a lower temperature than that of\nvapor, where the phase change from vapor to liquid occurs, is significant\nin heat-exchange systems. The condensed liquid water forms a film\nor droplet depending on the wettability of the cold surface. Condensed\nwater forms a film on a highly wettable (hydrophilic) surface, while\nwater droplets are formed on a dewettable (hydrophobic) surface. 4 − 7 Condensed water easily spreads to form a liquid film on the hydrophilic\ncold surface, which can act as a thermal barrier to inhibit the heat\ntransfer between the ambient and cold solid surfaces. 8 , 9 Moreover, the unremovable water film causes local accumulation of\ncontaminants and corrosion, which degrades heat transfer. In contrast,\ncondensed water forms droplets on the hydrophobic surface so that\nthe dewetted cold solid surface remains heat-transferred from the\nambient. 10 , 11 In addition, because the contact area of\na water droplet on a hydrophobic surface depends on the surface physical\nmorphology, the mobility of water droplets on the surface can be enhanced. 12 , 13 Therefore, it is possible to remove condensed water droplets by\ngravital sliding or rolling along the surface, thereby exposing the\ndewetted cold solid surface to ambient conditions for continuous condensation. 14 , 15 Such effects enable hydrophobic surfaces to show a more enhanced\ncondensation heat transfer than that of hydrophilic water-wettable\nsurfaces. Therefore, the hydrophobization treatments of metallic materials\ncan enhance the efficiency of heat exchangers, water harvesting and\ndesalination, environmental control, and power generation. 16 Copper and its alloys are among the most\npromising metallic materials\nin applications related to heat transfer because of their high thermal\nconductivity, ductility, and weldability. 17 , 18 Therefore, surface treatment and hydrophobization techniques that\nenhance condensation heat transfer have significant potential for\nvarious applications of copper. In addition, various strategies realizing\nhydrophobicity on copper surfaces have been widely explored because\nthe dewetting surface provides anticontamination and anticorrosion. 19 , 20 A coating with low-surface energy materials and control of the surface\nmorphology of copper are required to create a superhydrophobic surface\non copper. Thin layers of fluorocarbon materials (for example, Teflon,\nFDTS, and calcium stearate), which have a negligible effect on the\nsurface morphology, are used to reduce the surface energy of copper. 21 − 23 According to the Cassie–Baxter rendering, sharp micro/nanoscale\nsurface morphologies were created on copper for extremely high contact\nangles and mobility of water droplets. 24 , 25 Various techniques,\nincluding anodizing, chemical etching, thermal oxidation, and photolithography,\nhave been applied to build nano- and microscale porous surface structures\nsupporting superhydrophobicity. 26 − 28 In particular, a sharp nanoneedle\nCuO formed by simple chemical and thermal treatments has been widely\napplied to realize superhydrophobicity and enhance condensation heat\ntransfer of copper and its alloys. 29 , 30 However, the\nnanoneedle CuO structure weakly adheres to copper and is brittle;\nthus, the surface structure is easily destroyed by slight physical\ndamage, such as smooth rubbing with a finger. Therefore, the nanoneedle-structured\nCuO surface is unsuitable for real applications, such as condensation\nheat transfer, where long-term operation and maintenance are critical.\nFor better durability, multistep electrodeposition of copper or electrodeposition\nin ionic liquids is introduced to create the superhydrophobic surface,\nbut the fabrication processes are not suitable for practical applications\nwith scalability. 31 − 33 In this study, we employed a single-step electrodeposition\nof copper\nto create a nano/microsharp morphology on a copper substrate (CS)\nfor the fabrication of durable superhydrophobic surfaces with enhanced\ncondensation heat transfer. Copper is one of the most widely used\nmetals for electrodeposition in practical fields. The physical morphology\nof the copper substrate can be controlled by a simple modification\nof processing parameters, such as the chemical composition of the\nelectrolyte, current density, temperature, agitation, and additive.\nWe adopted an electrodeposition condition to create a rough surface\nmorphology of the copper layer, which was subsequently hydrophobized\nwith a thin layer of poly(tetrafluoroethylene). Such a rough copper\nsurface structure is expected to show better tolerance against physical\ndamage than that of the nanoneedle CuO surface. In addition, the hydrophobized\ncopper layer formed by electrodeposition had a lower thermal resistance\nthan that of the nanoneedle CuO surface. The condensation heat transfer\nof the fabricated hydrophobic rough copper surface was evaluated and\ncompared with that of the nanoneedle CuO surface. Moreover, we tested\nits tolerance against physical damage to maintain stable condensation\nheat transfer.",
"discussion": "3 Results and Discussion 3.1 Fabrication of the Superhydrophobic\nCopper-Electrodeposited\nSurface The wettability of a hydrophobic surface depends\non the roughness with respect to Cassie–Baxter rendering. 36 , 37 The morphology and roughness of the sample surfaces with copper\nelectrodeposition and the Teflon coating were characterized using\nSEM and AFM ( Figure 2 ). The copper substrate (CS) had linear grooves formed by mechanical\npolishing. In addition, the Teflon coating used in this study only\nhas a thickness of a few nanometers, so the hydrophobizing coating\ndoes not significantly affect the surface morphology. 38 , 39 In this study, we used a low-concentration electrolyte for copper\nelectrodeposition and PEG as an additive, causing film growth in the\npreferred orientation of copper crystals. 27 , 40 , 41 Thus, randomly rough copper surface structures\n( Figure 2 a–d)\nare created by electrodeposition, and the rough structure grows with\nan increase in electrodeposition time. The formation and growth of\nrandomly rough surface structures contributed to the increase in surface\nroughness. The average roughness ( R a )\nincreased from 90 ± 13 nm (for the copper substrate, CS) to 271\n± 25 nm after 10 min of electrodeposition (ED10), which further\nincreased to 387 ± 31 and 845 ± 94 nm by copper electrodeposition\nfor 20 min (ED20) and 30 min (ED30), respectively. Figure 2 Surface morphology of\n(a) copper substrate and electrodeposited\ncopper surface for (b) 10, (c) 20, and (d) 30 min. (i) SEM and AFM\nimages from (ii) perspective view and (iii) top view. (e) Averaged\nroughness from AFM images. According to the Cassie–Baxter state, roughness is critical\nfor improving the dewetting of hydrophobic surfaces formed by the\nTeflon coating with stability under hot and humid conditions. 42 , 43 Therefore, the Teflon coating on electrodeposited copper surfaces\nwith different average roughness values results in varying wettability\nand mobility of water droplets. The apparent contact angle and contact\nangle hysteresis (advancing contact angle–receding contact\nangle) were measured to estimate the wettability and mobility of water\ndroplets, respectively ( Figure 3 ). In addition, water droplets mixed with a blue dye on the\nsample are shown in Figure 3 . The apparent contact angle and contact angle hysteresis\nof the water droplet on the copper substrate were 87.4 ± 3.5\nand 73.3 ± 12.9°, respectively. The copper electrodeposition\ncreating a randomly rough surface structure reduces the contact area\nof the water droplet on the solid surface, which significantly increases\nthe contact angle with an increase in the surface roughness, such\nas 142.4 ± 2.3, 154.2 ± 4.2, and 168.4 ± 3.7°\nfor 10, 20, and 30 min of copper electrodeposition, respectively.\nThe contact line of the three-phase interface (solid/air/liquid) also\naffects the adhesion between the two phases (water and solid surface);\nthus, the mobility of water droplets is enhanced on a rough hydrophobic\nsurface. 44 , 45 Therefore, the contact angle hysteresis\ndecreases with an increase in the average roughness, such as 18.9\n± 1.4, 6.3 ± 3.1, and 2.3 ± 0.8° for 10, 20, and\n30 min of copper electrodeposition, respectively. In particular, the\nsurface fabricated by 30 min of copper electrodeposition and its Teflon\ncoating exhibited superhydrophobicity with extremely low wettability\nand high mobility of water droplets. These results suggest that superhydrophobicity\ncan be realized on copper using electrodeposition without surface\noxidation of copper-forming nanoneedle-structured CuO, which is generally\nused to fabricate superhydrophobic surfaces on copper. 29 , 46 Figure 3 Image\nof water droplets on (a) copper substrate and hydrophobic\ncopper surface electrodeposited for (b) 10 (ED10), (c) 20 (ED20),\nand (d) 30 (ED30) min. CA, CA_adv, and CA_rec indicate the static\ncontact angle, advancing contact angle, and receding contact angle,\nrespectively. (e) Averaged static contact angle and contact angle\nhysteresis. 3.2 Condensation\nHeat Transfer The condensation\nheat transfer of four types of samples (CS, ED10, ED20, and ED30)\nwas tested using the setup shown in Figure 1 with changing coolant temperature. The coolant\ntemperature controlled the temperature of the surface, and the condition\nof the chamber was maintained at 90 °C with hot water at the\nbottom. We measured the stabilized temperature of each location in\nthe meter bar ( Figure 1 ), and Figure 4 shows\nthe temperature profile in the meter bar. The temperature gradient\n(slope of temperature vs. distance in Figure 4 ) was estimated by linear fitting and is\nsummarized in Table 1 . 47 The sample surface temperature was\ncalculated considering the linear relationship between the temperature\nand location in the meter bar ( Table 1 ). Even though the thermal conductivity of Teflon (poly(tetrafluoroethylene))\nis 0.25 W/m·K, which is relatively very lower than that of copper,\nthe Teflon layer shows negligible thermal resistance on the copper\nsurface, due to its extremely low thickness (∼2 nm). 48 The sample surface had a lower temperature with\na decrease in the coolant temperature; thus, the temperature difference\nbetween the vapor and sample surface increases, thereby enhancing\nwater condensation. In addition, the decrease in the coolant temperature\ncontributed to an increase in the temperature gradient in the meter\nbar. When the coolant temperature was 70 °C, the temperature\ngradients were −0.23, −0.30, −0.30, and −0.35\nfor CS, ED10, ED20, and ED30, respectively, showing no significant\ndifference. However, the hydrophobic surface with improved mobility\n(low contact angle hysteresis) of the water droplet shows a significantly\nenhanced temperature gradient with a decrease in the coolant temperature.\nIn particular, the temperature gradient of ED30 was higher than that\nof CS by approximately 1.78-fold at a coolant temperature of 10 °C,\nwhile ED30 shows approximately 1.55-fold higher temperature gradient\nthan that of CS at a coolant temperature of 50 °C. Hydrophobized\ncopper electrodeposition with high mobility of water droplets also\nincreases the surface temperature ( Table 1 ) for coolant temperatures below 50 °C.\nIn contrast, no significant difference was observed in the surface\ntemperature at a coolant temperature of 70 °C. Figure 4 Temperature profile in\nthe meter bar during the condensation test\nfor (a) copper substrate and hydrophobic copper surface electrodeposited\nfor (b) 10 (ED10), (c) 20 (ED20), and (d) 30 (ED30) min with a coolant\ntemperature of 10 (CT10), 30 (CT30), 50 (CT50), and 70 (CT70) °C. Table 1 Measured and Estimated Data from the\nCondensation Heat Transfer Test for the Copper Substrate (CP) and\nHydrophobic Copper Surface Electrodeposited for 10 (ED10), 20 (ED20),\nand 30 (ED30) min coolant\ntemp 10 °C 30 °C 50 °C 70 °C sample CP ED10 ED20 ED30 CP ED10 ED20 ED30 CP ED10 ED20 ED30 CP ED10 ED20 ED30 temp gradient (°C/cm) –0.97 –1.30 –1.47 –1.73 –0.67 –0.93 –1.03 –1.31 –0.47 –0.53 –0.68 –0.73 –0.23 –0.30 –0.30 –0.35 surface temp (°C) 15.9 ± 1.0 16.9 ± 0.9 17.3 ± 1.0 18.0\n± 0.9 34.6 ± 0.8 35.8 ±\n1.1 35.9 ± 1.1 36.2 ± 0.8 52.5 ± 1.0 52.8 ± 1.1 53.2 ± 0.9 53.3 ± 0.7 71.1\n± 1.0 70.9 ± 1.0 71.2 ±\n0.8 70.8 ± 1.2 The increased temperature gradient in the meter bar\nand surface\ntemperature of the sample due to the enhanced dewetting and mobility\nof water droplets on the hydrophobized copper electrodeposition indicates\na change in the heat transfer in the copper meter bar. The transferred\nheat ( Q ) from the sample surface to the coolant through\nthe meter bar can be calculated using the following equation 49 , 50 1 where λ, Δ T ,\nand d are the thermal conductivity of the copper\nmeter bar (391.1 W/(m·K)), temperature difference, and distance\nbetween thermocouples, respectively. 17 The\ntemperature gradient summarized in Table 1 corresponds to Δ T / d ; thus, the heat flux through the copper meter\nbar can be calculated. 51 , 52 Figure 5 shows the calculated heat flux (condensation\nheat transfer) as a function of the temperature difference between\nthe water vapor and the sample surface. The surface appearances of\nthe sample with condensed water droplets are shown in Figure 5 . Figure 5 Condensation heat transfer;\n(a) estimated heat flux and appearance\nof condensed water on (b) copper substrate and hydrophobic copper\nsurface electrodeposited for (c) 10 (ED10), (d) 20 (ED20), and (e)\n30 (ED30) min. A lower temperature of the sample\nsurface than that of water vapor\ncauses the condensation of water on the surface. Thus, the latent\nheat of condensation is released on the surface, which is transferred\nto the meter bar and then to the coolant to condense more water on\nthe sample surface, producing a greater heat flux with a higher temperature\ngradient in the meter bar. In addition, the higher the latent heat\nby condensation, the higher the surface temperature of the sample.\nFor the coolant temperature of 70 °C, because the temperature\ngradients of the samples did not show any significant difference,\nthe heat flux by condensation heat transfer showed a similar value\nfor each sample. However, with a decrease in the coolant temperature\nfrom 70 to 10 °C, the heat flux on the CS increases by more than\n4.2-fold. Nevertheless, the hydrophobized copper-electrodeposited\nsurfaces (ED10, ED20, and ED30) showed a more significant increase\nin heat flux than for the CS. The decrease in the coolant temperature\nfrom 70 to 10 °C increases the condensation heat flux by more\nthan 4.3-, 4.9-, and 5.5-fold for ED10, ED20, and ED30, respectively.\nIn particular, ED30 with the lowest contact angle hysteresis showed\nthe most significant increase in the heat flux with the highest surface\ntemperature. Therefore, the temperature difference between the water\nvapor and sample surface ( T vap – T surf ) slightly decreases with the condensation\nheat transfer. The shape and mobility of water droplets on a\nsuperhydrophobic\ncopper surface enhance the condensation heat transfer. The CS surface,\neasily wettable by water, shows widespread water droplets ( Figure 5 b) as a filmwise\ncondensation. Moreover, the water droplet is almost immobile on the\nCS surface, and the water film wetting the copper surface is not easily\nremoved by gravity. However, the condensed water shows a spherical\nshape on the hydrophobic copper surfaces ( Figure 5 c–e), indicating dropwise condensation\ndue to their dewetting property, which shows a high apparent contact\nangle of the water droplet. Despite the spherical shape of the condensed\nwater droplets, the hydrophobic surfaces demonstrate different sizes\nof water droplets. ED10 showed the largest number of water droplets\npinned on the surface. The size of the water droplets was smaller\non the ED20 surface than in the case of ED10. The gravity of a water\ndroplet on a vertically inclined hydrophobic surface provides a force\nto roll off the droplet along the surface so that a small droplet\nis easily mobile on the surface with high mobility of water droplets\n(low contact angle hysteresis). 53 , 54 Therefore, the condensed\nwater droplet on the ED20 surface with a lower contact angle hysteresis\nthan that of ED10 cannot be grown up to the droplet size on the ED10\nsurface. In the case of ED30 with the lowest contact angle hysteresis,\na significant area of noncondensed copper surface is exposed to ambient\nand smaller condensed water droplets than in the case of ED20. These\nresults are attributed to the frequent roll-off of the condensed water\ndroplet with a small size. A rolling condensed water droplet combines\nwith other droplets along the rolling path; thus, a larger area of\nthe cold copper surface can be exposed to humid conditions to initiate\nthe nucleation of water condensation. These results indicate that\ncoating with a thin hydrophobic material and controlling the surface\nmorphology enhance water mobility, significantly improving the condensation\nheat transfer. To examine the stability of the Teflon layer,\nthe contact angle\nof water droplets on ED30 is measured after testing condensation heat\ntransfer up to 7 days ( Figure 6 a) and exposing to air up to 24 days ( Figure 6 b). During the condensation heat transfer\ntesting, the contact angle of the water droplet on ED30 is consistently\nmaintained at 170–175°, showing the superhydrophobicity.\nMoreover, the exposure of ED30 to air for a month does not affect\nthe contact angle of 170–175°. These results indicate\nthat the Teflon coating on roughly electrodeposited copper stably\nshows superhydrophobicity both under humid and air conditions over\ntime. Figure 6 Contact angle of water droplets on the ED30 surface after (a) condensation\nheat transfer test up to 7 days and (b) exposure to air up to 24 days. 3.3 Tolerance to Physical Damage Nanoneedle\nCuO (NNC) structures are generally used to fabricate superhydrophobic\nsurfaces on copper substrates. Moreover, the enhancement of condensation\nheat transfer is achieved owing to its exceptional water droplet mobility.\nWe compared the condensation heat transfer of the superhydrophobic\ncopper-deposited surface with that of the NNC surface. In addition,\nthe tolerance of the superhydrophobic copper-electrodeposited and\nNNC surfaces to mechanical damage was evaluated using a rubbing test\n(DIN EN ISO 11640) with an elastomer. The changes in the surface morphology,\ndistribution of fluorine, and wettability by the rubbing test are\nshown in Figure 7 .\nIn the case of the superhydrophobic electrodeposited copper surface,\nthe rough-structured surface is stronger than the elastomer; thus,\nno significant damage is found on the surface, and only worn elastomer\nparticles adhere to the rough structure. Moreover, no significant\nchange in fluorine distribution indicating the coated Teflon on the\nsurface is observed. Such elastomer particles adhered to the rough\nstructure inhibit the dewetting of the hydrophobic surface, so the\napparent contact angle of ED30 decreases from 168.4 ± 3.7 to\n158.6 ± 3.9°, indicating that the surface is still superhydrophobic.\nThe superhydrophobic NNC surface showed a significant change in the\nsurface structure and wettability by the rubbing test. Owing to the\nbrittle nature of CuO, most of the sharp nanoneedle structure, which\neffectively supports the Cassie–Baxter interface for superhydrophobicity,\nis destroyed by rubbing, so the distribution of fluorine is significantly\ndecreased, indicating the removal of the coated Teflon layer. For\nthese reasons, the apparent contact angle of the water droplet on\nthe damaged NNC surface is significantly decreased from 174.2 ±\n1.4 to 132.6 ± 3.3°. These results imply that a simple physical\ncontact can easily degrade the superhydrophobicity of the NNC-structured\nsurfaces. Figure 7 Surface morphology of (a) hydrophobic copper surface electrodeposited\nfor 30 min (ED30) and (b) hydrophobic nanoneedle copper oxide surface\n(i) before and (ii) after rubbing. (c) Averaged static contact angle\nand contact angle hysteresis. The degradation of hydrophobicity by damage to the micro/nanostructure\ncan also deteriorate the condensation heat transfer; thus, we measured\nthe condensation heat transfer of the superhydrophobic ED30 and NNC\nsurfaces with and without the rubbing test ( Figure 9 ). The temperature gradients and surface\ntemperatures are summarized in Table 2 . The intact NNC demonstrated higher temperature gradients\n(slope of temperature vs. distance in Figure 8 ) and surface temperature than those of ED30\nfor each coolant temperature, indicating better condensation heat\ntransfer of the NNC surface than that of the ED30 surface. These results\nare in good agreement with the NNC surface, showing better mobility\nand dewetting of water droplets than those of the ED30 surface ( Figure 7 ). Despite such superior\nhydrophobicity and condensation heat transfer, the damaged NNC showed\nsignificantly reduced temperature gradients by more than 35% than\nthe entire surface because the physical damage to NNC by rubbing with\nan elastomer significantly deteriorates the dewetting and mobility\nof water droplets. However, regardless of the physical damage, the\nED30 surface showed a less-significant reduction in the temperature\ngradient by less than 10% compared to that of the entire surface.\nMoreover, the temperature gradients of the damaged ED30 were higher\nthan those of the NNC with damages for each coolant temperature. Figure 8 Temperature\nprofile in the meter bar during the condensation test\nfor (a) hydrophobic copper surface electrodeposited for 30 min and\n(b) hydrophobic nanoneedle copper oxide surface (i) before and (ii)\nafter rubbing at a coolant temperature of 10 (CT10), 30 (CT30), 50\n(CT50), and 70 (CT70) °C. Table 2 Measured and Estimated Data from the\nCondensation Heat Transfer Test for the Hydrophobic Copper Surface\nElectrodeposited for 30 min (ED30) and Hydrophobic Nanoneedle Copper\nOxide (NNC) with and without Surface Rubbing coolant temp 10 °C 30 °C 50 °C 70 °C ED30 NNC ED30 NNC ED30 NNC ED30 NNC sample intact damage intact damage intact damage intact damage intact damage intact damage intact damage intact damage temp gradient (°C/cm) –1.73 –1.57 –1.87 –1.03 –1.31 –1.2 –1.49 –0.93 –0.67 –0.66 –0.8 –0.5 –0.35 –0.32 –0.4 –0.26 surface temp (°C) 18.0 ± 0.9 16.9 ± 1.0 18.3 ± 0.7 15.7\n± 1.0 36.2 ± 0.8 35.5 ±\n1.3 37.3 ± 0.3 35.3 ± 0.9 53.1 ± 0.7 52.8 ± 1.1 53.7 ± 0.5 52.1 ± 1.0 70.8\n± 1.2 70.9 ± 1.1 71.7 ±\n0.6 70.6 ± 1.3 Figure 9 illustrates the condensation heat transfer\ncalculated\nfrom the temperature gradients in Table 2 and the surface appearance during condensation.\nIn the case of the NNC surface, the condensation heat transfer values\nare 4.8 ± 0.6, 9.7 ± 1.2, 18.1 ± 0.8, and 2.3 ±\n1.6 (kW/m 2 ) for the coolant temperatures of 10, 30, 50,\nand 70 °C, respectively, which are higher than those of the ED30\nsurface by less than 15%. In addition, the temperature difference\nbetween the humid atmosphere and the sample surface of NNC is lower\nthan that of the ED30 surface. These results indicate that the superhydrophobic\nNNC has superior condensation heat transfer compared to that of the\nsuperhydrophobic electrodeposited copper surface. Nevertheless, a\nphysical rubbing test destroying the brittle nanoneedle CuO structure\ndiminishes the dewetting and mobility of water droplets; thus, the\nsize of the condensed water droplet pinned on the surface significantly\nincreases ( Figure 9 b). Such changes in the wetting of the condensed water decrease the\ncondensation heat transfer by more than 35%. Although the condensation\nheat transfer of the entire ED30 surface is slightly lower than that\nof the entire NNC surface, the dewetting and mobility of water droplets\non the damaged ED30 surface are better than those on the NNC surface.\nTherefore, smaller condensed water droplets were pinned on the damaged\nED30 surface ( Figure 9 c) than on the damaged NNC surface ( Figure 9 b). These results indicate that the superhydrophobic\nsurface fabricated on an electrodeposited copper surface has superior\nphysical contact tolerance compared to that of the superhydrophobic\nsurface with a nanoneedle CuO structure. Owing to the robustness of\nthe rough structure by copper electrodeposition, a stable surface\nwith dewetting and mobility of water droplets can be enabled against\nphysical contacts, which may destroy the rough surface structure of\nNNC, supporting the superhydrophobicity. Therefore, the condensation\nheat transfer values of the damaged ED30 surface are higher than those\nof the damaged NNC surface by more than 22%, such as 3.9 ± 1.0,\n8.0 ± 1.2, 14.5 ± 0.6, and 19.0 ± 0.4 (kW/m 2 ) for the coolant temperatures of 10, 30, 50, and 70 °C, respectively.\nStable performance against unwanted physical contacts and physical\nrobustness of the surface structure are two of the most important\ncharacteristics for the practical application of hydrophobic surfaces.\nTherefore, although the hydrophobic performance was slightly less\nthan that of the previous nanoneedle CuO structure, the physically\nrobust rough structure by copper electrodeposition can be a potential\ncandidate for practical applications of hydrophobic surfaces, including\ncondensation heat transfer. Moreover, since hydrophilic or hydrophilic/hydrophobic\nhybrid surfaces provide benefits in boiling heat transfer, the application\nof a durable rough electrodeposited copper surface with scalability\nand practical feasibility can be extended to heat exchange using boiling\nheat transfer. 55 − 60 Figure 9 Condensation\nheat transfer of the damaged surface; (a) estimated\nheat flux and appearance of condensed water on (b) hydrophobic copper\nsurface electrodeposited for 30 min and (c) hydrophobic nanoneedle\ncopper oxide surface (i) before and (ii) after rubbing."
} | 7,163 |
25757097 | PMC4408185 | pmc | 2,559 | {
"abstract": "B acillus subtilis is a widespread and diverse bacterium t exhibits a remarkable intraspecific diversity of the ComQXPA quorum-sensing (QS) system. This manifests in the existence of distinct communication groups (pherotypes) that can efficiently communicate within a group, but not between groups. Similar QS diversity was also found in other bacterial species, and its ecological and evolutionary meaning is still being explored. Here we further address the ComQXPA QS diversity among isolates from the tomato rhizoplane, a natural habitat of B . subtilis , where these bacteria likely exist in their vegetative form. Because this QS system regulates production of anti-pathogenic and biofilm-inducing substances such as surfactins, knowledge on cell–cell communication of this bacterium within rhizoplane is also important from the biocontrol perspective. We confirm the presence of pherotype diversity within B . subtilis strains isolated from a rhizoplane of a single plant. We also show that B . subtilis rhizoplane isolates show a remarkable diversity of surfactin production and potential plant growth promoting traits. Finally, we discover that effects of surfactin deletion on biofilm formation can be strain specific and unexpected in the light of current knowledge on its role it this process.",
"introduction": "Introduction It was already suggested by Darwin ( 1859 ) that intraspecific diversity increases the species adaptive potential to changing conditions, simply by making the population better prepared for the unexpected. Recently, this assumption was confirmed experimentally for various species, such as eelgrass where diversity within species contributes to its survival in fluctuating environments (Hughes and Stachowicz, 2004 ) and for Pseudomonas aeruginosa where strain diversity was shown to increase its stress resistance in biofilms (Boles et al ., 2005 ). Moreover, experiments reveal that genetically uniform populations of bacteria can readily diverge, especially in structured environments that offer various niche opportunities (Rainey and Travisano, 1998 ; Poltak and Cooper, 2011 ). Bacillus subtilis , ubiquitous and highly diverse Gram-positive bacterium, has been most often isolated in the form of heat resistant spores from soil and plant rhizosphere, as well as from other habitats such as aquatic systems, animal guts and various foods (Earl et al ., 2008 ; Mandic-Mulec and Prosser, 2011 ). Its intraspecies diversity is reflected by high number of ecotypes (Koeppel et al ., 2008 ; Stefanic et al ., 2012 ; Kopac et al ., 2014 ) and mirrored in diversification of distinct ‘communication’ groups or pherotypes (Tran et al ., 2000 ; Tortosa et al ., 2001 ; Ansaldi et al ., 2002 ; Stefanic and Mandic-Mulec, 2009 ), which are defined as groups of bacteria that are able to communicate through signalling molecules (peptides) that elicit a response in strains sharing the same pherotype but not (or to significantly lesser extent) in those of a different pherotype (Ansaldi et al ., 2002 ). Stefanic and colleagues ( 2012 ) proposed that pherotype diversity could be an adaptation to ecological diversity and showed that one pherotype dominates an ecotype with other pherotypes being present with lower frequency within an ecotype. Therefore, additional studies are needed to better understand the pherotype puzzle and its ecological meaning. Diversification into pherotypes is coupled to striking polymorphisms of the ComQXPA quorum-sensing (QS) system (Tran et al ., 2000 ; Ansaldi et al ., 2002 ; Stefanic and Mandic-Mulec, 2009 ). It was shown that pherotypes can coexist in soil at a millimetre scale (Stefanic and Mandic-Mulec, 2009 ) and that the communication diversification is under Darwinian selection (Ansaldi and Dubnau, 2004 ) and is present also in other Bacillus species that encode the comQXPA homologues loci (Dogsa et al ., 2014 ). Still, it is not clear how this diversification is manifested in other parts of the genome and how it is adaptive for the species. The ComQXPA lingual system operates the QS (Fuqua et al ., 1994 ), a process where secreted signalling molecules (ComX) accumulate to critical concentration and by activation of cognate receptors (ComP), trigger the expression of target genes. More precisely, ComX signal is initially synthesized as 55 amino acid-long prepeptide that is processed by ComQ and secreted from the cells (Magnuson et al ., 1994 ; Ansaldi et al ., 2002 ; Schneider et al ., 2002 ). Signal production by B. subtilis serves as a negative feedback mechanism which modulates QS response of producing cells (Oslizlo et al ., 2014 ). When ComX accumulates, it activates ComP receptor, which then phosphorylates response regulator ComA, and this one in turn modulates the transcription of many genes (Weinrauch et al ., 1996 ; Comella and Grossman, 2005 ). Microarray studies revealed that srfAA-D operon encoding the surfactin synthetase (Nakano et al ., 1988 ) accounts for the most affected target by the ComQXPA regulon (Comella and Grossman, 2005 ). Surfactin is a powerful lipopeptide biosurfactant and an antibiotic that acts against many bacteria and fungi, including plant pathogens like Pseudomonas syringae (Bais et al ., 2004 ). In addition, surfactin by inducing a potassium leakage in the cells (Lopez et al ., 2009 ) indirectly serves as a signal that triggers biofilm formation, which is essential for colonization of roots (Beauregard et al ., 2013 ; Zeriouh et al ., 2013 ) and protection of plants against pathogens (Bais et al ., 2004 ; Chen et al ., 2013 ; Zeriouh et al ., 2013 ). In fact, B. subtilis is regarded to be a PGPR (plant growth-promoting rhizobacterium) and has been well known for its biocontrol potential (Barea et al ., 2005 ; Berg, 2009 ; van Elsas and Mandic-Mulec, 2013 ). It was even proposed that the vegetative form of this species is normally associated with plant roots and that soil is predominantly inhabited by its dormant spores (Norris and Wolf, 1961 ). If this holds true, it is of major importance to complement our current knowledge on the communication diversity of B. subtilis isolates that live on plant root surfaces (rhizoplane), especially because the diverse ComQXPA system controls the expression of biocontrol agents, like surfactin (Nakano et al ., 1988 ; Zeriouh et al ., 2013 ). PGPR are widely accepted as ecofriendly alternatives to chemical pesticides, and they have been in commercial use for several years (Nakkeeran et al ., 2005 ). Knowledge on QS diversity in rhizoplane and on how this diversity manifests in QS-regulated traits could then contribute to optimal design of PGPR-based formulations. Most work on B. subtilis ecology has been performed by studying spores isolated from soil (Mandic-Mulec and Prosser, 2011 ). Here, we use a set of B. subtilis isolates and close relatives isolated from tomato rhizoplane to address the genetic and functional diversity of spore formers. Our aim was to examine whether different QS pherotypes of B. subtilis vegetative cells can coexist within the rhizoplane of a single plant. We further addressed whether being a member of a certain pherotype in the rhizoplane manifests in similar expression of known ComQXPA-regulated biocontrol properties like production of surfactin or biofilm formation. In addition, we compare direct plant growth promotion and potential PGP traits between isolates derived from a single plant or within a pherotype. We find that B. subtilis strains living on roots of a single plant carry diverse QS pherotypes and are highly diverse with respect to their biocontrol potential. The strains show differences in biofilm formation, surfactin production and other PGPR traits and most interestingly, behave differently after silencing of the srfA operon. We discuss what such intraspecies diversity could mean for the bacteria, for the plant and for biocontrol by rhizoplane communities.",
"discussion": "Discussion Bacillus subtilis strains that persist in soil are classified to three to four distinct QS groups – pherotypes (Ansaldi et al ., 2002 ; Stefanic and Mandic-Mulec, 2009 ). As plant rhizosphere was proposed the main habitat of B. subtilis vegetative existence (Norris and Wolf, 1961 ), we asked here whether the pherotype diversity is also found among rhizoplane isolates. We confirmed that different QS groups (pherotypes) can coexist on roots of a single plant. This was shown by comQ sequence analysis and by specific induction of QS response. Despite low number of B. subtilis isolates per plant (6 isolates from plant 16 and 4 isolates from plant 14), we found a comparable diversity, manifested in three pherotypes, which was previously observed for soil millimetre scale (Stefanic and Mandic-Mulec, 2009 ), confirming that comQXP diversity is widespread and easy to find. This again brings up the fundamental question on adaptive role of this diversity, which is also found in other gram-positive bacteria, including Staphylococcus aureus , Streptococcus pneumoniae (Pozzi et al ., 1996 ; Ji et al ., 1997 ; Whatmore et al ., 1999 ; Carrolo et al ., 2014 ) and B. cereus (Slamti and Lerecius, 2005 ). Stefanic and colleagues ( 2012 ) found that most but not all B. subtilis strains of the same ecotype belong to the same pherotype and proposed that pherotypes may at least in part be under ecological selection. Rhizoplane strains were not analysed for ecotype association, but their high phenotypic diversity (e.g. variability in surfactin production and other biocontrol properties) suggests that many of them may be ecologically distinct. Interestingly, isogenic strains of S. pneumoniae of distinct pherotypes differed in their ability to form biofilms, possibly because of pherotype-dependent strength of QS signalling (Carrolo et al ., 2014 ). Here we found no correlation between QS type (pherotype) and the expression of a QS-regulated trait – surfactin production. This is in contrast to observation by Carrolo and colleagues ( 2014 ), who noticed such interrelationship, but it should be stressed that they used isogenic strains, which only differed in pherotypes. We used wild isolates of different genetic backgrounds that may additionally influence the surfactin synthesis and secretion and dominate over pherotype association. Strong variability in surfactin production among rhizoplane isolates, even among isolates of a single plant, may also influence social life of this species. Because surfactin is secreted and probably shared between neighbouring B. subtilis strains, it may, under certain conditions, serve as a public good (West et al ., 2007 ). Differences in surfactin production could also result in disproportions in metabolic investment and fitness among strains (Oslizlo et al ., 2014 ), allowing weak surfactin producers to benefit from strong producers when plant pathogen invades and needs to be opposed. Coexistence of social and less social phenotypes was previously found in neighbouring strains of Myxococcus xanthus (Kraemer and Velicer, 2014 ) or P. aeruginosa (Wilder et al ., 2011 ). In case of M. xanthus it was shown that ‘social’ strains can promote the persistence of less social isolates without negative effects on the social's fitness, but sometimes when highly abundant, the less social strains can decrease fitness of the whole community (Kraemer and Velicer, 2014 ). It was recently suggested that spatial segregation facilitates the evolution of cooperation in B. subtilis biofilms (van Gestel et al ., 2014 ). Therefore, high spatial segregation of different pherotypes might help to stabilize cooperative traits and public goods production, like surfactin. Consequently, selection for cooperation might lead to assortment and diversity of pherotypes even at small distances. In addition, it was recently discovered that B. subtilis colonizes hyphae of Aspergilus niger where surfactin expression is downregulated by these fungi (Benoit et al ., 2014 ). Could then the decreased level of surfactin secretion in certain B. subtilis strains (like T16-2 orT14-4) be an adaptation to peaceful coexistence with fungi, which are also common rhizoplane inhabitants? It is not known how presence of poor surfactin producers influences the performance of the whole B. subtilis community in fighting plant pathogens, and it is an interesting problem of sociomicrobiology that could be addressed in the future. It is also worth noting that in certain isolates (like T16-10 or T16-2), the presence of surfactin showed no influence on biofilm biomass and in others (like T16-8 and T16-5) even exerted a negative influence on biofilm biomass. It is known that surfactin serves as a paracrine signal for biofilm formation (Lopez et al ., 2009 ). Our results, however, indicate that surfactin role in biofilm formation may be more complex and strain specific. This result also further supports previous assumptions about high genetic and phenotypic diversity of strains sharing a pherotype. Moreover, because surfactin is required for root colonization (Bais et al ., 2004 ; Chen et al ., 2013 ), it remains to be tested how the observed differences in surfactin production translate into root-colonization abilities of the B. subtilis isolates. Also, it will be interesting to address whether the ΔsrfA mutants (T16-2; T16-5: T16-5: T16-8 and T16-10) lose the ability to colonize the plant roots, despite different effects of srfA deletion on pellicle biofilms. We observed only moderate diversity at the level of biofilm formation. Although we did not look into biofilm-related gene expression, our results are in line with previous data showing that genetic diversity within B. subtilis is especially high with respect to antibiotic-related genes (like surfactin) and low with respect to biofilm-related genes (Earl et al ., 2008 ). The reason for the later may be associated with attached growth being essential for rhizocompetence and probably represents a competitive advantage in rhizoplane. While our strain isolation strategy does not allow us to speculate on the original spatial distribution among the Bacillus spp. isolates on roots, the results confirm that different pherotypes reside on roots of a single plant. Stefanic and colleagues ( 2012 ) proposed that ratios of pherotypes continuously cycle in nature because of induction of costly products released by high-frequency pherotype and temporary advantage of the low-frequency pherotype. Therefore, the diversity of pherotypes could be naturally selected by means of social conflict based on release of costly products (Eldar, 2011 ; Stefanic et al ., 2012 ). However, because members of one pherotype dramatically differ in QS-regulated biocontrol traits, direct effects on plant growth and other potential PGP behaviours (IAA secretion, production of siderophores or phosphate solubilization), pherotype diversity would not threaten the biocontrol function of the Bacillus community – eventually each pherotype contains a strong surfactin producers, or/and direct plant growth promoters. Therefore, the diversity of pherotypes on plant roots may promote coexistence of different strains and thus positively influence the biocontol potential of this species. It was previously shown that plant growth promotion by B. subtilis depends on production of volatile 2,3-butanediol (Ryu et al ., 2003 ). Because we found no correlation between PGP effects and analysed secretions (IAA, siderophores and phosphatases), it is possible that volatile molecules could influence the growth of a model plant in our experiment; however, this awaits further studies. The idea to use microbes as biocontrol agents emerged many years ago, and many studies screening for PGP properties of rhizosphere and rhizoplane isolates were performed. Some strains were patented and its commercial use is steadily increasing (Maheshwari, 2011 ). Although it is known that diverse community of microbes determines the plant health, PGPR-based preparations are still based on monocultures (Maheshwari, 2011 ). There were several successful attempts of applying multispecies formulations (Raupach & Kloepper 1998 ; Singh et al ., 1999 ), but sometimes, simply because of interspecies competition, the effects can be just opposite to the expectations (Chiarini et al ., 1998 ; de Boer et al ., 1999 ). In fact, in terms of sharing a niche, one should rather expect a competition instead of a synergy between microbial species (Foster and Bell, 2012 ). It is therefore important to bring more attention to intraspecies genetic and phenotypic diversity in the rhizosphere and rhizoplane, where next to strong indirect competition, more cooperation, especially within a pherotype, would be predicted. Testing biocontrol properties or plant growth-promotion effects of mixed B. subtilis communities should be the next step to answer this question. We show here that B. subtilis residing on roots differ in QS pherotypes, potential PGPR traits and the ability to influence the growth of A. thaliana . We believe this study opens new interesting questions about the role of strain diversity in arms race between B. subtilis and plant pathogens, or in interactions with host plant. We also believe that applying diverse strains of one genus or species carrying diverse biocontrol properties should be considered as an alternative to non-monoculture-based biocontrol agents design."
} | 4,396 |
29430211 | null | s2 | 2,560 | {
"abstract": "The processes used to create synthetic spider silk greatly affect the properties of the produced fibers. This paper investigates the effect of process variations during artificial spinning on the thermal and mechanical properties of the produced silk. Property values are also compared to the ones of the natural dragline silk of the "
} | 83 |
18952513 | null | s2 | 2,561 | {
"abstract": "This review focuses on the self-assembly of macromolecules mediated by the biorecognition of peptide/protein domains. Structures forming alpha-helices and beta-sheets have been used to mediate self-assembly into hydrogels of peptides, reactive copolymers and peptide motifs, block copolymers, and graft copolymers. Structural factors governing the self-assembly of these molecules into precisely defined three-dimensional structures (hydrogels) are reviewed. The incorporation of peptide motifs into hybrid systems, composed of synthetic and natural macromolecules, enhances design opportunities for new biomaterials when compared to individual components."
} | 164 |
39286993 | PMC11840469 | pmc | 2,562 | {
"abstract": "Abstract It is generally assumed that contact angle hysteresis of superhydrophobic surfaces scales with liquid–solid contact fraction, however, its experimental verification has been problematic due to the limited accuracy of contact angle and sliding angle goniometry. Advances in cantilever‐based friction probes enable accurate droplet friction measurements down to the nanonewton regime, thus suiting much better for characterizing the wetting of superhydrophobic surfaces than contact angle hysteresis measurements. This work quantifies the relationship between droplet friction and liquid–solid contact fraction, through theory and experimental validation. Well‐defined micropillar and microcone structures are used as model surfaces to provide a wide range of different liquid–solid contact fractions. Micropillars are known to be able to hold the water on top of them, and a theoretical analysis together with confocal laser scanning microscopy shows that despite the spiky nature of the microcones droplets do not sink into the conical structure either, rendering a diminishingly small liquid–solid contact fraction. Droplet friction characterization with a micropipette force sensor technique reveals a strong dependence of the droplet friction on the contact fraction, and the dependency is described with a simple physical equation, despite the nearly three‐orders‐of‐magnitude difference in liquid–solid contact fraction between the sparsest cone surface and the densest pillar surface.",
"conclusion": "3 Conclusion To conclude, we have explored how droplet sliding friction relates to liquid–solid contact fraction on regular micropillar and ‐cone surfaces. A theoretical consideration was presented to show that the cone surfaces used in this work have dense enough arrays of microcones so that they can fully support droplets and only the cone tip vertices become wetted. Confocal laser scanning microscopy imaging also supports this finding by showing that there is no observable difference in the plastron height in relation to the cone height. Therefore, the liquid–solid contact fraction of the microcone surfaces used in this work can be calculated based on the tip geometry, similar to the micropillar surfaces. Droplet contact line friction measurements with the micropipette force sensor technique revealed extremely small friction values down to 0.2 µN mm −1 for the sparsest microcone surface C5 and showed that the contact line friction and liquid–solid contact fraction relates to each other via a model developed earlier by Reyssat and Quéré. [ \n \n 11 \n \n ] The model was also found to simultaneously fit the contact line friction of microcone as well as micropillar surfaces that have liquid–solid contact fractions extending almost over three orders of magnitude from 0.06% to 36.8%. The good fit of the model enables us to use it for estimating even smaller liquid–solid contact fraction such as for bSi surfaces with random conical microstructures. However, we note that irregular, anisotropic, or re‐entrant surface geometries may alter scaling between droplet friction and liquid–solid contact fraction, calling for additional research on this topic. Nevertheless, our work deepens the knowledge of how droplets behave on conical microstructures, which may help in designing surfaces for applications such as anti‐icing where minimal contact between water and the surface is beneficial.",
"introduction": "1 Introduction The ability to control how water wets a solid surface enables applications like self‐cleaning, [ \n \n 1 \n \n ] anti‐icing, [ \n \n 2 \n , \n 3 \n \n ] anti‐fogging, [ \n \n 4 \n , \n 5 \n \n ] and drag reduction. [ \n \n 6 \n , \n 7 \n , \n 8 \n \n ] Common for these examples is that they all require minimal adhesion or friction between water and the surface, which usually is achieved by making the surface hydrophobic. Barthlott et al. found in the 1970′s that extreme hydrophobicity is achieved when the surface has microscopic roughness combined with hydrophobic surface chemistry, e.g., lotus leaves have ca. 10 µm sized papillae covered with hydrophobic wax. [ \n \n 9 \n \n ] On such surfaces, water wets only the vertices of the microstructures (i.e., remains in Cassie wetting state), which leads to minimal contact and adhesion between water and the surface (i.e., minimal liquid–solid contact fraction, LS–CF), enabling water to roll off from the surface even at minor tilt angles. [ \n \n 10 \n \n ] This phenomenon is termed superhydrophobicity and is usually defined as a water contact angle exceeding 150° and a droplet roll‐off angle less than 5° or 10°. Over the years, numerous artificial superhydrophobic surfaces have been developed based on various nano‐ and microstructure designs. [ \n \n 11 \n , \n 12 \n , \n 13 \n , \n 14 \n , \n 15 \n , \n 16 \n , \n 17 \n , \n 18 \n , \n 19 \n \n ] One notable design type is a periodic array of micropillars (see Figure \n 1 a ), as it has provided a simple model design to study how various geometrical parameters affect different wetting properties of droplets in the Cassie state. Earlier works for example by Reyssat & Quéré and Qiao et al. show that water droplet roll‐off angle and sliding friction become smaller when the LS–CF is reduced by tuning the micropillar size and spacing, yet the lowest LS–CF they considered was still fairly high (≈5%) having substantial contact angle hysteresis beyond 10°. [ \n \n 11 \n , \n 12 \n \n ] Another notable design type is nano‐ and micro‐sized cones, spikes, or needles (see Figure 1b ), in particular, due to the extremely low droplet sliding friction shown for this surface design. [ \n \n 20 \n \n ] These surfaces have much smaller tip vertices than micropillars, suggesting that the extremely small sliding friction of the droplet is due to a small LS–CF. However, an open question remains on how droplets actually “sit” on the sharp tips; whether they can remain on the tip vertices or whether they sink partly inside the structure. Thus, their LS–CF has either remained unknown or has been estimated via contact angles from the Cassie‐Baxter equation, [ \n \n 21 \n , \n 22 \n \n ] which may be inaccurate due to theoretical assumptions behind that equation [ \n \n 23 \n \n ] and difficulties of accurate contact angle measurements of superhydrophobic surfaces, [ \n \n 24 \n , \n 25 \n \n ] and no work for determining the relationship between LS–CF and droplet sliding friction has been performed earlier on conical surface structures. Figure 1 a) On micropillar surfaces, droplets can remain atop the pillars without observable sinking ( z = 0), and therefore the LS–CF (φ) can be calculated based on pillar width and spacing. The resulting friction is relatively high. [ \n \n 11 \n , \n 12 \n \n ] b) The liquid–solid contact fraction on microcone surfaces is more difficult to determine since droplets may sink below the cone tip level ( z ≥ 0). Nevertheless, the resulting friction can be very low at least for certain surfaces with micrometer sized cones. [ \n \n 20 \n \n ] \n In this work, we quantify in detail the relationship of LS–CF and droplet sliding friction (specifically contact line friction, CLF) by investigating the friction on both regular micropillar and microcone surfaces that are superhydrophobic. We first theoretically investigate how water droplets sit on the microcone surfaces and note that droplets prefer to remain atop the cone tips as long as the cones are densely enough spaced. In addition, we use confocal laser scanning microscopy (CLSM) imaging to determine the height of the air layer (called plastron) trapped between the solid surface and the droplet and observe that plastron height approximately equals cone height determined with electron microscopy, which supports the theoretical prediction that droplets remain atop the cone tips. This allows calculation of the LS–CF of droplets on cone surfaces based on the regular surface geometry, revealing that LS–CF as low as 0.06% is enough to support droplets atop the cone tips. Water on such surfaces is extremely mobile and it is practically impossible to determine droplet sliding friction via sliding angle or contact angle hysteresis measurements due to their limited accuracies, [ \n \n 24 \n , \n 25 \n \n ] and more advanced cantilever‐based wetting characterization techniques are needed. [ \n \n 20 \n , \n 26 \n , \n 27 \n , \n 28 \n , \n 29 \n , \n 30 \n , \n 31 \n , \n 32 \n \n ] For that reason, we characterize the sliding friction with the micropipette force sensor (MFS) technique [ \n \n 20 \n , \n 28 \n , \n 30 \n , \n 33 \n \n ] and show that the droplet CLF on the microcone surfaces is one to two orders of magnitude lower than on micropillar surfaces. Yet, the droplet CLF on both surface textures scales similarly with the LS–CF regardless of the large, nearly three orders of magnitude difference in the LS–CF. This relation between droplet friction and liquid–solid contact fraction can be described via a simple physical equation. We find that our results help understanding droplet mobility on conical microstructures and thus how microcone surfaces would perform under various applications that benefit from minimal contact between water and the surface.",
"discussion": "2 Results and Discussion We prepared five microcones (labeled as C1 to C5) and four micropillar (labeled as P1 to P4) sample surfaces with periodic arrays of the microstructures, details in Experimental Section. Figure \n 2 \n illustrates the geometry design of the surfaces. Cone surfaces have square lattice and pillar surfaces have hexagonal lattice, and the varying parameter in both structure types is the spacing between the cones or pillars. The structure dimensions of the cone and pillar surfaces were determined with scanning electron microscopy (SEM), see Table \n 1 \n and Figures S1,S2 (Supporting Information). Tips of the cones have the aluminum mask remaining from the cone fabrication, see Figure 2d , and thus all the cone samples from C1 to C5 have rather consistent tip size and shape despite the very small length scales of the tips. The prepared cone surfaces were made superhydrophobic by coating them with an octyltrichlorosilane (OTS) self‐assembled monolayer coating and the pillar surfaces with a fluoropolymer coating, details in the Experimental Section. The prepared samples are superhydrophobic, and droplets roll off easily. More detailed wetting characterization will be presented later in this paper, as we will first focus on how stationary droplets sit on the surfaces. Figure 2 Schematics of a) microcone and b) micropillar array geometries. d \n tip is tip diameter, h is height, and s is spacing between the pillars/cones, measured from cone center to center, and β \n cone is the cone tip half angle. c) SEM image of the cone surface C2. d) High magnification of a typical cone tip, image from surface C2. e) SEM image of the P3 pillar surface. SEM images of the other cone and pillar surfaces are in Figure S1,S2 (Supporting Information). Table 1 Dimensions of the cone and pillar surfaces were determined with SEM. Values are averages obtained from multiple cones/pillars at different locations on the surface ( n = 8–12) and the error margins represent standard deviation. Sample Spacing \n s \n [µm] Height \n h \n [µm] Tip diameter d \n tip [µm] Tip half angle β \n cone [°] C1 0.96 ± 0.01 3.68 ± 0.02 0.096 ± 0.006 4.3 ± 0.4 C2 1.44 ± 0.01 3.72 ± 0.01 0.106 ± 0.007 4.7 ± 0.3 C3 1.92 ± 0.02 4.02 ± 0.01 0.111 ± 0.004 6.2 ± 0.3 C4 2.87 ± 0.02 4.16 ± 0.01 0.128 ± 0.017 3.0 ± 0.3 C5 3.84 ± 0.06 3.96 ± 0.06 0.108 ± 0.008 5.9 ± 0.6 P1 19.5 ± 0.1 35.9 ± 0.1 12.41 ± 0.03 – P2 24.5 ± 0.1 37.7 ± 0.2 12.03 ± 0.03 – P3 34.4 ± 0.1 38.9 ± 0.1 12.52 ± 0.05 – P4 58.6 ± 0.1 40.4 ± 0.3 12.42 ± 0.08 – John Wiley & Sons, Ltd. 2.1 How do Droplets Sit on Micropillar and ‐Cone Surfaces? It is well known for the micropillar surfaces that water sits on top of the pillars (i.e., remains in the Cassie state), as long as droplets are placed gently on them. [ \n \n 34 \n , \n 35 \n \n ] On the contrary, droplets may sink into the cone surfaces and the amount depends on the dimensions of the cones as has been shown by Lecointre et al. [ \n \n 5 \n \n ] Here, we can use a similar theoretical approach. For static droplets sitting on the periodic square array of micrometric‐sized cones, the contact line tension force opposes droplets sinking into the structure. The force scales with the length of the contact line, which grows the deeper the droplet sinks into the cone structure, as the cones become wider. The “critical width” of the cones ( d \n crit , see Figure \n 3 a ) enough to prevent droplets from further sinking in this case\n \n (1) \n d crit = R cos θ adv − β 1 + 4 s 2 π R 2 cos 2 θ adv − β − 1 \n where R is the droplet radius, s is the cone spacing, β is the cone sidewall angle, and θ \n adv is the advancing contact angle related to the surface chemistry of the cones, and Equation 1 is valid when θ \n adv − β > 90° (see Note S1 Supporting Information for derivation of Equation 1 ). Equation 1 predicts that for all surfaces C1–C5 ( θ \n adv = 113°, corresponding OTS advancing contact angle, see Experimental Section), the value d \n crit remains mostly below 100 nm for droplets with radius between 0.3 mm and 1.5 mm (Figure S3 , Supporting Information). Figure 3 a) On ideally sharp cones, Equation 1 predicts droplet meniscus to sink to a level where the cone becomes wide enough (horizontal diameter d \n crit ) to prevent the meniscus from further sinking. β \n cone represents cone sidewall angle. b) On cones with blunt spherical tips, droplet meniscus can remain atop the tips with minimal vertical sinking. Equations (1) , (2) predict the critical width d \n crit and critical latitude angle β \n crit needed to support droplet meniscus from further sinking. c) Difference of cone tip critical width d \n crit calculated from Equations (1) , (2) in relation to tip width d \n tip obtained from SEM as function of droplet radius R . d) Critical latitude angle β calculated from Equation 1 and Equation 2 as function of droplet radius R . Data points in c) and d) represent average values, and error bars represent standard deviation of Equations (1) , (2) solved via Monte Carlo simulations ( n = 3 × 10 6 ), see Note S2 (Supporting Information). Dashed lines in c) and d) represent linear interpolation between data points and serve as guide for eye. The value of d \n crit in this droplet size range is smaller than the tip diameter of the cones on surfaces C1–C5, meaning that the droplet meniscus would wet only the spherical cone tips. Equation 1 can also be applied to investigate how much the droplet meniscus would wet the spherical cone tips when taking into account that also β depends on the sinking depth. If assuming perfectly spherical tips (Figure 3b ) β represents latitude angle of the spherical tip, and a simple relation between horizontal width d of the cone and its latitude angle β can be made,\n \n (2) \n d z = d tip cos β z \n where z represents the sinking depth and d \n tip the diameter of the spherical cone tip. Combining Equation 1 and Equation 2 yields that for all surfaces C1–C5, the critical diameter enough to prevent the sinking of the droplet meniscus is close to the tip diameter while the “critical slope” β \n crit is ≈ 20°, see Figures 3c,d . Thus, the meniscus would wet the cones similarly as sketched in Figure 3b . In reality, the cone tips are not fully spherical but are somewhat flattened instead, which causes d \n crit to be slightly wider than predicted by Equation 1 and Equation 2 . Nearly the whole cone tip is wetted, so an approximation can be made that the droplet meniscus roughly wets a circular area with a diameter equivalent to the cone tip diameter. The theoretical consideration thus predicts that the droplets would not sink into the cone surfaces C1–C5. We decided to use confocal laser scanning microscopy (CLSM, details in Experimental Section) for an experimental check on whether droplets remain atop the cone tips or not. Figure \n 4 a shows the schematics of the experimental setup. By moving the microscope stage in the vertical direction, the CLSM can be focused accurately to different heights at the sample surface. There are two highly reflective planes in the system: the substrate bottom surface and the air‐water interface (called meniscus), see Figures 4b,c (images of all surfaces are shown in Figures S4,S5 , Supporting Information). The distance between these two planes (i.e., the plastron height) can be read from the CLSM stage position. As light refracts at the air‐water interface, a correction\n \n (3) \n h plastron ≈ Δ z stage n 2 n 1 \n based on Snell's law of refraction must be applied to convert the stage travel distance between the cone bottom surface and the meniscus levels (Δ z \n stage ) to plastron height ( h \n plastron ). n \n 1 and n \n 2 are the refractive indices of water and air, respectively. Derivation and error estimation of Equation 3 are presented in Note S3 (Supporting Information). Figure 4 a) Schematics of the confocal microscopy setup. A droplet is attached to the lens and imaging occurs through the droplet that is in contact with the sample surface. Images are obtained with 0.2 µm spacing of focal planes starting from below the substrate surface ( z \n stage = 0) up to bulk water ( z \n stage = 15.6 µm). The pinhole limits the light collection only to the plane of focus (i.e., no light is collected from elsewhere). b) Example images i–v shown at different focal planes: ii is from the substrate surface bottom, iv is from the air–water interface and i, iii, and v are below the substrate surface, from the air layer, and from the bulk droplet, respectively, where no reflection of light occurs, thus forming dark images. The scale bar length is 5 µm. The images are from surface C2, other surfaces are presented in Figure S4 (Supporting Information). c) Mean brightness of the z ‐stack frames at different focal planes. i–v mark the planes of images in b. Mean reflection intensities as a function of z \n stage of other cone surfaces are shown in Figure S5 (Supporting Information). d) Comparison of cone height read from SEM images (left bars, data from Table 1 ) and measured plastron height (right bars). Values of plastron height represent average ( n = 3) and error bars of the plastron height are ± 10%, see Notes S2,S3 (Supporting Information). For all surfaces C1–C5, the calculated plastron height is ≈10% higher than the cone height determined with SEM, see Figure 4d . Naturally, the plastron height cannot be larger than the cone height, but we ascribe this difference to the errors related to the CLSM measurements (see Notes S2,S3 , Supporting Information). Nevertheless, these measurements show that water droplets either do not sink into the cone structures or sink only marginally (in the order of 100 nm) at maximum in any of the surfaces C1–C5. Therefore, the earlier theoretical estimation that the droplet meniscus remains at the tip vertices appears to be valid (additional discussion in Note S4 , Supporting Information). The CLSM imaging also shows that the meniscus is rather flat (see Figure S5 , Supporting Information) meaning that the meniscus does not sag significantly between the cones. This is in line with the theoretical prediction of the difference in critical latitude angle β \n crit and advancing contact angle θ \n adv : their difference is nearly 90°, meaning that the meniscus is close to horizontal level when touching the cone tips. 2.2 Droplet Friction Characterization with Micropipette Force Sensor As mentioned earlier, the prepared cone surfaces C1–C5 and pillar surfaces P1–P4 are superhydrophobic, and the droplets on them are very mobile. Such surfaces are practically impossible to characterize accurately with the standard wetting characterization tool, contact angle goniometry, due to inaccuracy in determining the droplet baseline. [ \n \n 24 \n , \n 25 \n \n ] Backholm et al. developed a force‐based method that utilizes a micropipette force sensor (for measuring the droplet sliding friction on highly superhydrophobic surfaces, [ \n \n 20 \n , \n 28 \n \n ] and we use the same method in this work. The schematics of the measurement setup are illustrated in Figure \n 5 a , and a detailed description of the procedure can be found in Experimental Section. Figure 5 a) Schematics of the micropipette force sensor measurement setup. b) An example side view image of the droplet during a force scan. The dashed blue line represents the pipette rest position. The deflection is exaggerated in the image for visualization purposes, the real deflection is only tens of micrometers. c) A force versus time graph of a single scan. The force zero level is set to the average of forward and reverse scan forces determined in the regions marked by the red dashed lines. d) Measured CLF of pillar and cone surfaces as a function of droplet contact region diameter. The solid lines represent linear fits (through the origin) to the data. The dark shaded regions represent the 95% confidence intervals of the fit and the light shaded region the 95% prediction intervals of individual measurements. e) CLF force normalized by contact region diameter as a function of LS–CF. The solid black line represents a fit of Equation 6 to the cone and pillar data where \n ξ \n = 1.63. The dashed black lines represent fits to the cone data only ( \n ξ \n = 1.44, lower dashed line) and pillar data only ( \n ξ \n = 1.90, upper dashed line). The grey area represents an extrapolation of the fit ( \n ξ \n = 1.63) to the lower LS–CF regime. The LS–CF of the bSi surface is predicted with the fit (crossing of the fit and the reported average F \n µ / D value). [ \n \n 41 \n \n ] In d and e, each data point represents a single MFS scan, and information on the error bars is in Note S2 (Supporting Information). In short, the droplet is dragged along the sample surface with the micropipette cantilever that has a known stiffness k \n p . The friction force causes the bending of the micropipette (see Figure 5b ), and the amount of deflection Δ x relates to pulling force F via Hooke's law for spring force ( F = − k \n p Δ x ). Dragging the droplet on the sample surface from one point to another and back to the original location yields a force curve as shown in Figure 5c . As no other net forces are acting on the droplet in the horizontal direction, the pipette pulling force must relate directly to the sliding friction of the droplet during the constant motion phase. The sliding velocity is kept small to minimize internal viscous losses, [ \n \n 36 \n , \n 37 \n \n ] so the sliding friction practically equals to CLF force F \n µ , which relates to contact angle hysteresis via equation\n \n (4) \n F μ = 24 π 3 γ D cos θ rec − cos θ adv , \n where γ is the liquid surface tension, D is the droplet contact region diameter and θ \n adv and θ \n rec are the advancing and receding contact angles, respectively. [ \n \n 38 \n , \n 39 \n , \n 40 \n \n ] The factor 24π −3 holds when the droplet contact region remains circular. [ \n \n 39 \n , \n 40 \n \n ] \n The MFS experiments were performed for all surfaces C1–C5 and P1–P4, and Figure 5d shows the determined CLF force of each measurement. It is evident from the results that the friction force scales linearly with the droplet contact region diameter, as predicted by Equation 4 . Droplet size independent contact line friction ( F \n µ / D ) of each sample surface is obtained via linear regression analysis. For the cones, the values range between 0.18–0.99 µN mm −1 , corresponding to contact angle hysteresis of only ca. 5°–10° when assuming an advancing contact angle of 180°. [ \n \n 34 \n \n ] The pillar surfaces have F \n µ / D values ranging between 5.8–19.9 µN mm −1 , so significantly higher than those of the cone surfaces. On the other hand, in our earlier work, we found black silicon (bSi) surfaces (irregular conical microstructure, see Figure S6 , Supporting Information) with the same OTS coating as used for C1–C5 to have the F \n µ / D value of 0.025 ± 0.005 µN mm −1 , [ \n \n 41 \n \n ] which is significantly lower than for those of the regular cone surfaces C1–C5. 2.3 Effect of Liquid–Solid Contact Fraction on Contact Line Friction The main difference between these three types of surfaces is their topography which causes water to have different liquid–solid contact fractions on them. As discussed above, the water sits solely on the cone tips for all the surfaces C1–C5. Based on SEM images, the cone tip is a flattened sphere, and the LS–CF can be approximated as φ ≈ π d \n tip \n 2 /4 s \n 2 (see Note S4 (Supporting Information) for a discussion on this approximation) and the values range between 0.06%–0.79%. The pillar surfaces have much higher LS–CF as the pillar tops are much wider, and the values range between 4.1%–36.8%. Reyssat & Quéré derived a relation between contact angle hysteresis and liquid–solid contact fraction when the liquid–solid contact points are circular and periodic:\n \n (5) \n cos θ rec − cos θ adv = ξ 4 φ ln π φ \n where ξ is a parameter related to how strongly the liquid adheres to the solid (e.g., due to surface chemistry). [ \n \n 11 \n \n ] Combining Equation 4 and Equation 5 gives a relation:\n \n (6) \n F μ D = 6 π 3 γ ξ φ ln π φ \n and Figure 5e shows that cone and pillar surfaces indeed do follow the relation between F \n µ / D and φ when ξ = 1.63 despite the nearly three orders of magnitude difference between the LS–CF of the surfaces. In addition, parameter ξ seems not to be significantly sensitive for the microstructure vertex size as pillar diameter is ca. 500‐fold compared to cone tip diameter, nor it is significantly sensitive for the surface chemistry, as θ \n adv / θ \n rec on smooth surfaces of OTS and fluoropolymer are 113°/106° [ \n \n 41 \n \n ] and 111°/85°, [ \n \n 42 \n \n ] respectively. Fitting Equation 6 separately for cones yields ξ = 1.44 and correspondingly for pillars ξ = 1.90, leading only to a minor difference of the fit as visualized with the dashed lines in Figure 5e . The bSi surface has a random surface geometry in the sub‐micrometer length scale that prevents direct calculation or measurement of the LS–CF. Despite Equation 6 being derived for periodic contact points, it should also apply for randomly spaced contact points as long as the spatial variations in the LS–CF remain in the micrometer length scales. Therefore, Equation 6 also enables predicting the LS–CF of the bSi surface, and with ξ = 1.63, a droplet on the bSi surface would have an average LS–CF of only 0.007%–0.020%. This is almost an order of magnitude less than for the sparsest cone surface C5 which has the lowest LS–CF of the cone surfaces, and such an LS–CF fraction has not been reported for any surfaces earlier."
} | 6,739 |
36580382 | PMC9809966 | pmc | 2,563 | {
"abstract": "ABSTRACT Synthetic communities grown in well-controlled conditions are an important tool to decipher the mechanisms driving community dynamics. However, replicate time series of synthetic human gut communities in chemostats are rare, and it is thus still an open question to what extent stochasticity impacts gut community dynamics. Here, we address this question with a synthetic human gut bacterial community using an automated fermentation system that allows for a larger number of biological replicates. We collected six biological replicates for a community initially consisting of five common gut bacterial species that fill different metabolic niches. After an initial 12 hours in batch mode, we switched to chemostat mode and observed the community to stabilize after 2–3 days. Community profiling with 16S rRNA resulted in high variability across replicate vessels and high technical variability, while the variability across replicates was significantly lower for flow cytometric data. Both techniques agree on the decrease in the abundance of Bacteroides thetaiotaomicron , accompanied by an initial increase in Blautia hydrogenotrophica . These changes occurred together with reproducible metabolic shifts, namely a fast depletion of glucose and trehalose concentration in batch followed by a decrease in formic acid and pyruvic acid concentrations within the first 12 hours after the switch to chemostat mode. In conclusion, the observed variability in the synthetic bacterial human gut community, as assessed with 16S rRNA gene sequencing, is largely due to technical variability. The low variability seen in HPLC and flow cytometry data suggests a highly deterministic system.",
"introduction": "Introduction The human gut microbiome is a complex and dynamic ecosystem, containing numerous microbes that perform different functions. They produce essential nutrients such as vitamins and short-chain fatty acids (SCFA). 1 , 2 In addition, the human gut microbiome plays an important role in the regulation of the host immune system, which is essential for health. 3 Longitudinal cohort studies show that the composition of the human gut microbiome as assessed with fecal samples does not change substantially when unperturbed, 4 , 5 but that day-to-day variation is nevertheless visible. This poses the question whether short-term variation is due to small changes in the environment (e.g. diet or circadian rhythms of the host), 6 , 7 the intrinsic stochasticity of the system (e.g. variable growth and death rates), or technical variability. Here, our goal is to investigate the reproducibility of the dynamics of a defined human gut bacterial community to quantify the variability in gut microbial abundances in well-controlled conditions. To do this, we need to work with a single source of inoculum and control all external sources that influence microbial dynamics. The latter is only possible in a bioreactor where atmosphere, temperature, and pH are kept constant, and the process can be operated in chemostat conditions in which a steady state can be reached. This requires a continuous culture to regularly add fresh nutrients and remove waste products. In such well-controlled conditions, the remaining variation is either due to community dynamics (i.e. stochasticity, but also nearby alternative stable states or chaos 8 ), short-term evolution (not explored here), or technical variability of measurements. Although variability of microbial gut community dynamics can also be assessed with fecal slurries, 9 , 10 working with a defined community makes it easier to understand the mechanisms driving the dynamics as it is less complex than fecal slurries, lacking archaea, fungi, and viruses. Bioreactor systems range in complexity from coupled vessels mimicking the human intestinal system to well plates, 11 , 12 , 33 representing a trade-off in realism and control versus the number of replicates that can be achieved. Although well plates combined with serial transfer are claimed to emulate chemostat conditions, they instead represent a sequential batch process (i.e., a series of (short) batch experiments). Traditional chemostats in bench-top bioreactors adopt a continuous inflow of fresh medium and outflow of broth keeping the volume constant. A series of such chemostats (with different specific process parameters) have been coupled to mimic the intestinal tract. 11 Since such bench-top chemostat bioreactors are cost-, time- and labor-intensive, replicate time series of defined human gut microbial communities in such chemostats are rare. The few that have been collected show that gut communities reach a stable state within a few days and that the dynamics are reproducible, but that there is some variability across biological replicates. 14–16 However, the number of replicates in these experiments is small and cell densities are usually not measured, such that conclusions rely on relative instead of absolute abundances. Here, we monitored microbial community composition in six parallel bioreactors in chemostat mode to investigate the variability of the dynamics of a defined human gut community and obtained total cell counts via flow cytometry. The community was composed of gut bacterial species with different metabolic roles, including the butyrate producer Roseburia intestinalis DSM 14610 (RI), 17 the acetogen Blautia hydrogenotrophica DSM 10507 (BH), 18 , 19 the lactate producer Collinsella aerofaciens RCC 1377 (CA), 20 and the succinate producers Bacteroides thetaiotaomicron DSM 2079 (BT) 21 and Prevotella copri DSM18205 (PC). 22 The two latter species are also representatives of two different enterotypes. 23 , 24 We calculated the total number of cells in each sample using flow cytometry and systematically assessed the technical variability of 16S rRNA gene sequencing. As an alternative to the latter, we also applied supervised classification to flow cytometry data to classify events by species. This method has already been validated in the context of gut bacteria, where a synthetic community comprised four bacteria (RI, BH, BT, and FP) was evaluated. 25 In both cases, we observed a reproducible change in composition before the community stabilized.",
"discussion": "Discussion Here, we investigated the dynamics of a defined community of human gut bacteria in continuous culture in a comparatively large number of replicates, while maintaining a high degree of environmental control. To our knowledge, this is the first time that the dynamics of a synthetic human gut bacterial community is investigated in chemostat mode using an automated multi-bioreactor system. A limitation of the current study is the frequent contamination in monocultures despite their location inside a HEPA-filtered anaerobic chamber. We adopted a stricter cleaning routine for the validation experiment, which reduced contamination in the community below the detection level. Another limitation is that the growth stage of the inoculated strains (18 h pre-cultures) may affect the dynamics of the community, which was not further explored here. We found that species abundances and metabolite concentrations in the community were reproducible across replicates, in agreement with previous results. 14 , 16 However, we also observed that the technical variability of 16S rRNA gene sequencing exceeded the variability across vessels. Since we repeated the entire sequencing protocol three times, we do not know whether variation comes from DNA extraction, PCR, or the sequencing runs. We positioned blanks randomly and differently across replicate well plates, to avoid bias due to well-to-well contamination. Previous studies using mock communities have shown that most of the technical variation is due to extraction and amplification and not from the sequencing step itself. 26 , 27 The high technical variability of 16S rRNA gene sequencing limits how much biological variability we can see. Therefore, we also applied an alternative method for the analysis of community composition based on flow cytometry data, implemented in CellScanner. Here, we found that trajectories across biological replicates were more similar to each other than they were for 16S rRNA sequencing data. Together with the low variation of metabolite data, this suggests that the technical variability of 16S rRNA sequencing inflates the variability observed with sequencing data and that the true biological variability is lower. However, CellScanner results are also potentially biased, since only the first monoculture time point was usable for BT and CA because of subsequent contamination. Changes in cell structure due to physiological changes from batch to continuous mode or species interactions could not be considered when training the classifiers. Thus, neither 16S rRNA gene sequencing nor CellScanner may have captured the exact composition. Alternative techniques such as whole-genome shotgun and full-length 16S rRNA gene sequencing are worth mentioning in this context, but they are work-intensive and it is questionable whether they reduce the technical variability compared to 16S rRNA gene sequencing. Quantitative PCR with species-specific primers is another alternative that delivers absolute abundances, which were observed previously to agree in sum with flow cytometry counts. 33 The advantage of supervised classification of flow cytometry data is that it gives absolute abundances without DNA extraction. However, like species-resolved quantitative PCR, it does not scale to complex communities. 25 Obtaining reliable species-specific cell counts in a high-throughput manner remains a major challenge when exploring community dynamics. Both CellScanner and 16S rRNA gene sequencing agree on the main trend, namely a decrease in BT and an increase in other species, notably BH. BT grows faster than BH in batch. In chemostat, BT cannot sustain its high growth rate given the glucose feed and its abundance therefore declines. Other species are less dependent on glucose and exploit alternative carbon sources such as pyruvate. This is especially true for BH, which did not consume glucose in the first 12 h but lowered the pyruvate and trehalose concentrations instead. Interestingly, in an unintended invasion experiment of BH versus BT (initially, a BT monoculture), BH dominated at 67 h (Supplementary Figure 7A). However, BT won over BH in a two-species co-culture where a chemostat was emulated through serial transfer. 13 This may be due to the fact that the medium in the serial-transfer experiment contained starch in addition to sugar, which may have given BT, a known carbohydrate specialist, 28 , 29 an edge. In our experiments, BT displays the behavior of a pioneer species that quickly exploits an easily accessible carbon source but is then replaced, to an extent, by slower growing but metabolically more versatile species. This also fits observations in cohort studies, which show that the Bacteroides 2 enterotype dominates infant microbiota after weaning 30 but is less frequent in adults. PC disappeared quickly from the community; after 25 hours the abundance was already bordering the detection limit of 16S rRNA gene sequencing. We do not know whether it was surviving in the community with a cell density below the detection limit, whether it was washed out, or whether it was outcompeted by other species in the community. A serial transfer experiment in a similar medium previously showed that BT outcompetes PC. 13 Transient dynamics in which a species loses its dominant position was also observed in two other continuous cultures with defined human gut communities. In both cases, the community was first dominated by Escherichia coli , which then declined in favor of Bacteroides thetaiotaomicron . 14 , 15 The observations described here are to our knowledge the first instance of a defined human gut community where a Bacteroides species decreases while other gut species increase and as such may contribute to understanding the enterotype shifts observed in fecal time-series data. The metabolite consumption and production patterns agree with previous observations and with predictions based on metabolic reconstructions from the AGORA database (Supplementary Table S2). 31 For instance, we saw a decrease in formate in the community. It has been shown before that formate produced in co-culture can be converted to acetate by BH using the Wood-Ljungdahl pathway. 32–34 It is a challenge to disentangle the contributions of different species to metabolite concentrations in a community. Although meta-omics can provide clues of pathway activation in a community context in different conditions, 35 it does not measure metabolite uptake and production rates. We saw that some species-metabolite correlations computed from co-culture data agree with monoculture data (e.g. BH consumes pyruvate, trehalose, and formate and produces acetate in monoculture and is negatively correlated with the three former and positively with the latter for CellScanner data) but there are also several correlations that do not reflect consumption/production (such as the negative correlation of BH with succinate, which is an indirect relationship due to the decrease of BT, the only succinate producer). Species may alter their metabolism when encountering low levels of their preferred nutrient and/or in the presence of interaction partners, 32 and thus determining who produces and consumes what in this community is a task for the future. Our data suggest that the community reached a steady state. We also note that all replicates followed similar trajectories i.e. only one state was reached. It is possible that this community does not have alternative stable states (i.e. it is not multi-stable). Alternatively, the different stable states for the species combination could be too far apart for small variations to trigger a change in community state. A different community state might also be too rare to see using six replicates. Further research is necessary to test whether initial differences in species abundances or perturbations would alter the final state reached. In conclusion, we have shown that the defined human gut community investigated here is a highly deterministic system where transient dynamics (succession) is reproducible, and the observed variation is due in large part to the technical variability of 16S rRNA gene sequencing data. This further encourages the development of mathematical models for gut microbial communities."
} | 3,633 |
26161139 | PMC4496950 | pmc | 2,566 | {
"abstract": "Background Lignocellulosic biomass has the potential to be a major source of renewable sugar for biofuel production. Before enzymatic hydrolysis, biomass must first undergo a pretreatment step in order to be more susceptible to saccharification and generate high yields of fermentable sugars. Lignin, a complex, interlinked, phenolic polymer, associates with secondary cell wall polysaccharides, rendering them less accessible to enzymatic hydrolysis. Herein, we describe the analysis of engineered Arabidopsis lines where lignin biosynthesis was repressed in fiber tissues but retained in the vessels, and polysaccharide deposition was enhanced in fiber cells with little to no apparent negative impact on growth phenotype. Results Engineered Arabidopsis plants were treated with the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate 1-ethyl-3-methylimidazolium acetate ([C 2 C 1 im][OAc]) at 10 % wt biomass loading at either 70 °C for 5 h or 140 °C for 3 h. After pretreatment at 140 °C and subsequent saccharification, the relative peak sugar recovery of ~26.7 g sugar per 100 g biomass was not statistically different for the wild type than the peak recovery of ~25.8 g sugar per 100 g biomass for the engineered plants (84 versus 86 % glucose from the starting biomass). Reducing the pretreatment temperature to 70 °C for 5 h resulted in a significant reduction in the peak sugar recovery obtained from the wild type to 16.2 g sugar per 100 g biomass, whereas the engineered lines with reduced lignin content exhibit a higher peak sugar recovery of 27.3 g sugar per 100 g biomass and 79 % glucose recoveries. Conclusions The engineered Arabidopsis lines generate high sugar yields after pretreatment at 70 °C for 5 h and subsequent saccharification, while the wild type exhibits a reduced sugar yield relative to those obtained after pretreatment at 140 °C. Our results demonstrate that employing cell wall engineering efforts to decrease the recalcitrance of lignocellulosic biomass has the potential to drastically reduce the energy required for effective pretreatment. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0275-2) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusion The impact of engineering secondary cell wall structure in Arabidopsis with a selective reduction of lignin and an enhancement of cellulose accumulation was evaluated in terms of pretreatment efficacy, sugar yields, and energy requirements. The reduced lignin Arabidopsis engineered lines resulted in high levels of monomeric sugar release at lower pretreatment temperatures as compared to the wild type. Ionic liquid pretreatment of the engineered Arabidopsis using [C 2 C 1 im][OAc] at 70 °C for 5 h resulted in improved saccharification efficiency and increased hemicellulose recovery for the pretreated biomass and produced similar total sugar yields as compared to those obtained after pretreatment at 140 °C for 3 h. The similar sugar recovery obtained for the engineered lines at the lower temperature pretreatment supports the hypothesis that reducing lignin can reduce the necessary severity of pretreatment needed and increased polysaccharide deposition can increase glucose recovery on a mass basis. Secondary cell wall regulatory networks are only partially understood and seem to be conserved across many species from dicot to monocot plants [ 30 – 32 ]. For example, an Arabidopsis nst1/nst3 double T-DNA insertional mutant lacking expression of both NST1 and NST3 transcription factors that control secondary cell wall deposition in fiber cells could be complemented by the expression of NST1 transcription factor orthologs derived from poplar or rice under the control of the Arabidopsis NST1 promoter [ 33 , 34 ]. This had an effect on the ASR amounts between the engineered lines, which could be important for pretreatment and sugar recovery. This suggests that a similar approach for cell wall engineering could be implemented into other vascular plant species to enhance polysaccharide deposition in secondary cell walls. The different levels of sugar recovery between LLHPL1 and LLHPL2 demand further investigations into the optimal expression levels and patterns of C4H and NST1. Using this selective strategy to reduce lignin deposition and enhance carbohydrate composition of specific cellular structures in a more diverse group of vascular plants could create higher yielding feedstocks that require less energy to process, thereby, improving the overall economics of biofuel production.",
"discussion": "Results and discussion Mature, senesced stems (corresponding to the main stems and side branches depleted of seeds and cauline leaves) from multiple plants of the WT , LLL , LLHPL1 , and LLHPL2 Arabidopsis lines grown under the same conditions were collected and milled, and the chemical composition was quantified. As previously reported, all the lines ( LLL , LLHPL1 , and LLHPL2 ) harboring the pVND6::C4H construct, exhibit a significantly lower lignin content (12.9 to 14 %) compared to that of WT (19.1 %) and had no visible phenotypic differences (Table 1 , Fig. 1 ) [ 21 ]. As expected, LLHPL1 shows an increase in the amount of both glucose 30.4 % and xylose 16.1 % present versus WT (26.1 and 11.4 % respectively). The LLHPL2 showed only a minor increase in xylose, 11.7 %, for the bulk composition and a significant decrease in the amount of glucose present, 22.1 %, where previously it was found to have a significant increase on a per plant scale [ 21 ]. Both the LLL and LLHPL2 engineered Arabidopsis lines exhibit a significant increase in acid soluble residue (ASR), while LLHPL1 had an increase in glucose with little change in ASR compared to WT (Table 1 ). Table 1 Initial compositional analysis for each Arabidopsis engineered line studied Untreated composition % Glucose % Xylose % Lignin % ASR \n WT \n 26.1 ± 0.1 11.4 ± 0.1 19.1 ± 0.3 43.4 ± 0.5 \n LLL \n 23.0 ± 0.7 ** \n 10.8 ± 0.2 12.9 ± 0.8 ** \n 53 ± 2 ** \n \n LLHPL1 \n 30.4 ± 0.4 ** \n 16.1 ± 0.5 ** \n 13.7 ± 0.6 ** \n 40 ± 2 \n LLHPL2 \n 22.1 ± 0.5 ** \n 11.7 ± 0.1 14 ± 2 ** \n 53 ± 2 ** \n There was an overall significant difference in the concentration of glucose, xylose, lignin, and ASR (acid soluble residue, ash, protein) F (3,12) = 150.87, P < 0.0001, F (3,12) = 340.36, P < 0.0001, F (3,12) = 28.65, P < 0.0002, F (3,12) = 100.54, P < 0.0001. ANOVA with a Tukey’s HSD post-hoc test was used to determine overall statistics, and results of the comparison to WT from the Tukey’s HSD post-hoc test are shown in the table. Values expressed ± SD \n ** \n P < 0.01 Fig. 1 Compositional profile of the four Arabidopsis engineered lines ( WT , LLL , LLHPL1 , LLHPL2 ) We pretreated the WT and the engineered strains with [C 2 C 1 im][OAc] at 10 % (w/w) biomass loading at 140 °C for 3 h (Fig. 2 ) [ 8 , 10 , 27 , 28 ]. The pretreated slurry was washed with water as an anti-solvent, precipitating a solid. The lignin concentrations of the pretreated solids from the reduced lignin lines were confirmed to be significantly lower than WT (~20 % lignin in the engineered lines and ~30 % lignin in the WT , Table 2 ) with insignificant differences in the amount of glucose and xylose removed for the engineered lines (Table 2 ). The WT had a significantly higher glucan recovery in the after IL pretreatment, as compared to the engineered lines where glucan recoveries of 86, 70, and 74 % were quantified for LLL , LLHPL1 , and LLHPL2 , respectively. Less than 50 % of xylan was recovered in the solids after pretreatment for all of the Arabidopsis lines tested (Table 2 ), and all three of the reduced lignin lines had a significant increase in ASR in the recovered biomass after IL pretreatment as compared to the WT (Table 2 ). Fig. 2 Mass balance of [C 2 C 1 im][OAc] pretreatment of the four Arabidopsis lines ( WT , LLL , LLHPL1 , and LLHPL2) at 140 °C for 3 h. Mass balance adjusted to 100 g starting biomass. Values presented as ±SD Table 2 Percent recovered solid composition after pretreatment at 140 °C for 3 h with [C 2 C 1 im][OAc] at 10 % (w/w) biomass loading as a percent of starting biomass Solids recovery 140 °C 3 h % Glucose % Xylose % Lignin % ASR \n WT \n 101 ± 6 47 ± 3 82 ± 5 17 ± 2 \n LLL \n 86 ± 7 39 ± 3 68 ± 6 23 ± 1 * \n \n LLHPL1 \n 70 ± 3 ** \n 34 ± 1 * \n 81 ± 14 38 ± 5 ** \n \n LLHPL2 \n 74 ± 10 ** \n 44 ± 6 70 ± 13 28 ± 3 ** \n Pretreated solids composition 140 °C for 3 h % Total solids % Glucose % Xylose % Lignin \n WT \n 52 ± 3 51 ± 1 10 ± 2 30 ± 2 \n LLL \n 43 ± 4 46 ± 2 10 ± 2 20 ± 2 * \n \n LLHPL1 \n 52 ± 2 41 ± 6 11 ± 1 21 ± 4 ** \n \n LLHPL2 \n 44 ± 6 37 ± 3 12 ± 3 21 ± 3 ** \n All values presented as ±SD. There was an overall significant difference in % recovery of glucose, xylose, and ASR (acid soluble residue, ash, and protein) in the recovered solids, F (3,12) = 12.86, P < 0.002, F (3,12) = 7.37, P < 0.01 and F (3,12) = 32.87, P < 0.0001. There was a non-significant difference in the lignin recovery in the solids, F (3,12) = 1.03, P = 0.43. Composition of recovered solids after pretreatment with [C2C1im][OAc] for 140 °C 3 h at 10 % (w/w) biomass loading. Glucose, xylose, and lignin reported as a percent of recovered biomass ±SD. There was an overall significant difference in % total solids and lignin, F (3,12) = 5.08, P < 0.05, F (3,12) = 9.74, P < 0.005. There was no overall significance for the % composition glucose or xylose F (3,12) = 6.22, P = 0.05, F (3,12) = 0.35, P = 0.79. ANOVA with a Tukey’s HSD post-hoc test was used to determine overall statistics, and results of the comparison to WT from the post-hoc test are shown in the table \n * \n P < 0.05; ** \n P < 0.01 The recovered solids from the Arabidopsis lines after [C 2 C 1 im][OAc] pretreatment were then saccharified using a commercial cellulase (CTec2) and hemicellulase (HTec2) enzyme mixture [ 10 ]. The yields of glucan after saccharification for LLL , LLHPL1 , and LLHPL2 were >95 % and significantly higher than those obtained from samples with no pretreatment (Table 3 ). There was no difference in the saccharification efficiency for xylan yields between the three modified plant lines. This resulted in final glucose yields of 69 to 87 % recovery in terms of the initial amount present in the samples before pretreatment. These glucose yields were not significantly different between the WT , LLL , and LLHPL2 samples but were significantly lower for glucose and xylose released from LLHPL1 compared to the WT, as well as xylose released from the LLL sample (Table 3 , Fig. 2 ). Table 3 Enzymatic saccharification efficiency of Arabidopsis engineered line versus pretreatment condition Enzymatic saccharification 10 % loading for 72 h % Glucose % Xylose % Glucose recovery % Xylose recovery \n WT \n Untreated 31 ± 3 17 ± 3 31 ± 3 17 ± 3 10 %, 70 °C, 5 h 67 ± 20 1.0 ± 0.3 62 ± 11 1.0 ± 0.2 10 %, 140 °C, 3 h 84 ± 6 87 ± 2 84 ± 1 41 ± 2 \n LLL \n Untreated 46 ± 4 ** \n 33 ± 1 ** \n 46 ± 4 ** \n 33 ± 1 ** \n 10 %, 70 °C, 5 h 76 ± 7 46 ± 5 ** \n 76 ± 5 46 ± 4 ** \n 10 %, 140 °C, 3 h 95 ± 4 92 ± 7 82 ± 4 35 ± 4 ** \n \n LLHPL1 \n Untreated 48.4 ± 0.7 ** \n 30.2 ± 0.2 ** \n 48 ± 0.4 ** \n 30 ± 0.3 ** \n 10 %, 70 °C, 5 h 79 ± 4 ** \n 58 ± 3 ** \n 63 ± 4 48 ± 2 ** \n 10 %, 140 °C, 3 h 99 ± 7 * \n 83 ± 9 69 ± 3 * \n 30 ± 3 \n LLHPL2 \n Untreated 53 ± 3 ** \n 31 ± 1 ** \n 53 ± 3 ** \n 31 ± 1 ** \n 10 %, 70 °C, 5 h 81 ± 4 ** \n 55 ± 5 ** \n 79 ± 1 * \n 58 ± 1 ** \n 10 %, 140 °C, 3 h 117 ± 6 ** \n 89 ± 6 87 ± 8 39 ± 2 Enzymatic saccharification efficiency reported as percent of theoretical in the saccharification (released as percent from pretreated biomass) and final recovery % from concentration in initial solids (sugar recovery * enzymatic efficiency), from the cellulose and hemicellulose mixtures CTec2 and HTec2 (20 mg/g and 2 mg/g loading for 72 h). All values presented as ±SD. There was an overall significant difference of the % glucose and xylose released from untreated biomass during enzymatic saccharification between the WT and the three engineered lines, F (3,12) = 30.59, P < 0.0001, F (3,12) = 66.83, P < 0.0001, xylose for the 70 °C pretreated biomass, F (3,12) = 139.36, P < 0.0001, and glucose for the 140 °C pretreated biomass, F (3,12) = 18.57, P < 0.001. There was a non-significant differences for the % saccharification efficiency for glucose for the 70 °C pretreatment F (3,12) = 0.95, P = 0.46 and for xylose for the 140 °C pretreatment F (3,12) = 0.85, P = 0.50. There were significant differences both the glucose and xylose recoveries at each pretreatment condition, untreated (F (3,12) = 30.6, P < 0.0001, F (3,12) = 66.8, P < 0.0001), 70 °C (F (3,12) = 4.35, P < 0.05 F (3,12) = 355.29, P < 0.0001) and 140 °C (F (3,12) = 7.93, P < 0.01, F (3,12) = 9.38, P < 0.01). ANOVA with a Tukey’s HSD post-hoc test was used to determine overall statistics, and results of the comparison to WT from the post-hoc test are shown in the table \n * \n P < 0.05; ** \n P < 0.01 All of the Arabidopsis samples were observed to swell during IL pretreatment at 140 °C for 3 h (see Additional files 1 , 2 , 3 , and 4 : Movies 1–4). The observed rate of dissolution due to [C 2 C 1 im][OAc] pretreatment, however, was slower for the WT than the engineered lines (Fig. 3 , Additional file 5 : Figure S1). Due to the relatively minor differences observed in the rate and extent of dissolution at 140 °C, the temperature was reduced to 70 °C to determine if there were any significant differences observed in swelling and dissolution between the WT and LLHPL2 . At this set of pretreatment conditions, there was an initial swelling step observed after 1 h of pretreatment, followed by the onset of extensive swelling after 3–4.5 h (Additional file 6 : Figure S2, Additional files 7 and 8 : Movie 5 and 6). Based on these results, a pretreatment incubation of 5 h at 70 °C was selected as the new pretreatment condition (Fig. 4 ). Fig. 3 Confocal fluorescence imaging of Arabidopsis during [C 2 C 1 im][OAc] pretreatment at 140 °C. Autofluorescence of 100 μm slices of the stems from four Arabidopsis lines during [C 2 C 1 im][OAc] pretreatment at 140 °C over 4.3 h. Horizontal panels show the different Arabidopsis lines. Vertical panels show the progression of the time course of [C 2 C 1 im][OAc] pretreatment on Arabidopsis with a temperature ramp from ambient conditions to 140 ± 5 °C occurring during time 0 to 46 min, scale bar 500 μm Fig. 4 Confocal fluorescence imaging of Arabidopsis during [C 2 C 1 im][OAc] pretreatment at 70 °C for 11 h. Heating occurred during ramp from room temperature to 70 °C during the first 30 min of imaging. Horizontal panels show comparison of WT versus the engineered line LLHPL2 while the vertical panels show selected images of the time course ( a , b ) 0, ( c , d ) 5 h, ( e , f ) 10 h, scale bar 50 μm The Arabidopsis lines WT , LLL , LLHPL1 , and LLHPL2 were pretreated in [C 2 C 1 im][OAc] at 70 °C for 5 h. The pretreated plant biomass was then precipitated and analyzed for composition (Fig. 5 , Table 4 ). All of the lines had significantly lower solid recoveries (70.7 to 80.6 %) than those of the WT (96 %, Table 4 ), yet the three engineered lines had similar glucose and xylose recoveries in the pretreated solids as the WT ( WT >94 % glucose, >106 % xylose, relative to initial biomass, Table 4 ). Furthermore, all of the Arabidopsis lines had minimal lignin removal (between 3 to 11 %) after pretreatment (Table 4 ). Fig. 5 Mass balance of [C 2 C 1 im][OAc] pretreatment of the four Arabidopsis lines ( WT , LLL , LLHPL1 , and LLHPL2) at 70 °C for 5 h. Mass balanced adjusted to 100 g starting biomass. Values presented ±SD Table 4 Percent recovered solid composition after pretreatment at 70 °C for 5 h with [C 2 C 1 im][OAc] at 10 % (w/w) biomass loading as a percent of starting biomass Pretreated biomass solids recovery 70 °C 5 h % Glucose % Xylose % Lignin % ASR \n WT \n 94 ± 10 106 ± 14 97 ± 15 94 ± 20 \n LLL \n 101 ± 2 102 ± 5 94 ± 8 66 ± 10 \n LLHPL1 \n 80 ± 10 84 ± 8 97 ± 5 59 ± 20 \n LLHPL2 \n 98 ± 4 106 ± 8 89 ± 7 50 ± 3 * \n Pretreated biomass composition 70 °C 5 h % Total solids % Glucose % Xylose % Lignin \n WT \n 96 ± 10 26 ± 4 13 ± 2 19 ± 3 \n LLL \n 80.6 ± 0.9 ** \n 31 ± 1 13.7 ± 0.7 15 ± 2 \n LLHPL1 \n 74 ± 1 * \n 33 ± 4 19 ± 2 * \n 18.1 ± 0.9 \n LLHPL2 \n 70.7 ± 0.7 * \n 29 ± 2 17 ± 1 * \n 17 ± 1 Values presented as ±SD. There was an overall significant difference in % recovery of glucose and ASR (acid soluble residue, ash, and protein) in the recovered solids, F (3,12) = 5.01, P < 0.03 and F (3,12) = 4.07, P < 0.05. There was a non-significant difference in the % xylose and lignin recovery in the solids, F (3,12) = 3.89, P = 0.06 and F (3,12) = 0.44, P = 0.73. Composition of recovered solids after 70 °C 5 h [C2C1im][OAc] pretreatment. Glucose, xylose, and lignin were reported as the relative composition of recovered biomass. Pretreatment was done at 10 % (w/w) biomass. There was an overall significant difference in % total solids and xylose, F (3,12) = 11.52, P < 0.005, F (3,12) = 8.48, P < 0.01. There was no overall significance for the % composition glucose or lignin F (3,12) =2.57, P = 0.13 and F (3,12) = 3.1, P = 0.09. ANOVA with a Tukey’s HSD post-hoc test was used to determine overall statistics, and results of the comparison to WT from the post-hoc test are shown in the table \n * \n P < 0.05; ** \n P < 0.01 The recovered solids from the different Arabidopsis lines after [C 2 C 1 im][OAc] pretreatment at 70 °C for 5 h were then saccharified. While there was less than 11 % removal of lignin, glucose yields of 76, 79, and 81 % were obtained for LLL , LLHPL1 , and LLHPL2 , respectively, and the saccharification efficiency was significantly greater for LLHPL1 and LLHPL2 than that of WT (67 %, Table 3 ). The resulting release of glucose relative to initial levels in the biomass was 62 % of the initial glucose for the WT , 76 % for the LLL , 63 % for the LLHPL1 , and 79 % for the LLHPL2 (Table 3 , Fig. 5 ). There was minimal detectable xylose released (1 %) during saccharification for the WT ; however, the three engineered lines had a significantly higher xylose yields of 46 to 58 %. In addition to the high recovery of glucose (63–79 %) and xylose (46–58 %) at the lower pretreatment temperature, the enhanced concentration of cellulose and hemicellulose per gram of starting biomass resulted in higher monomeric sugar release in all of the engineered lines (Figs. 2 , 5 , and 6 ). Both LLHPL1 and LLHPL2 have significantly increased total sugar recovery (27.3 and 24.2 g total sugar per 100 g starting biomass) as compared to the 16.2 g total sugar per 100 g starting biomass of the WT (Fig. 6 , Additional file 9 : Tables S1 and S2). Fig. 6 Comparison of glucose and xylose recovery after enzymatic saccharification as a percent of original biomass for [C 2 C 1 im][OAc] pretreatment. Glucose and xylose recovery after 70 °C for 5 h and 140 °C for 3 h compared to the untreated (ut) for all of the Arabidopsis lines. There was an significant difference in total sugar released per starting biomass between the Arabidopsis lines at each pretreatment temperature, untreated (F (3,12) = 72.44, P < 0.0001), 70 °C (F (3,12) = 19.45, P < 0.0005), and 140 °C (F (3,12) = 5.86, P < 0.05). This was in part due to significant differences between groups in glucose recovery per starting biomass for all three pretreatment conditions untreated (F (3,12) = 47.2, P < 0.0001), 70 °C (F (3,12) = 7.86, P < 0.01), and 140 °C (F (3,12) = 6.62, P < 0.01). There was also significant difference in xylose recovery per starting biomass between the lines for two of the three pretreatment conditions untreated (F (3,12) = 134.12, P < 0.0001) and 70 °C (F (3,12) = 404.71, P < 0.0001). There was not a significant difference in xylose release per starting biomass at 140 °C (F (3,12) = 3.43, P = 0.07). ANOVA with a Tukey’s HSD post-hoc test and the Tukey’s HSD post-hoc test are shown in the figure for the comparison to WT (total sugar, P < 0.05, *; P < 0.01, **; glucose, P < 0.05, +; P < 0.01, ++; xylose, P < 0.05, −; P < 0.01, --), additional post-hoc test comparisons reported in Additional file 9 : Table S1 and S2 While there are similar recoveries and enhanced total sugar release, the saccharification kinetics are slower for the biomass pretreated at 70 °C than those pretreated at 140 °C (Table 5 ). After pretreatment at 70 °C for 5 h, the initial rate of glucose release for the WT was 86 mg/L/min, and the rates for the three engineered lines were between 40 to 52 mg/L/min. The rate of xylose release was below the detectable limit for WT , while the initial rate of release for xylose was significantly higher, between 46 to 68 mg/L/min, for the engineered lines. As the composition of both LLHPL1 and LLHPL2 are different, so were the rates of sugar released during saccharification. [C 2 C 1 im][OAc] pretreatment at 70 °C for 3 days has been shown previously to release less sugar than pretreatment at 140 °C for 3 h [ 29 ]. The reduced lignin Arabidopsis lines, however, all show increased sugar release after pretreatment at 70 °C, highlighting the impact of plant cell wall modifications on pretreatment severity and related energy requirements. Table 5 Rate of enzymatic saccharification as calculated by release during the first 30 min of enzymatic hydrolysis with both the cellulase and hemicellulase mixtures CTec2 and HTec2 Rate of enzymatic saccharification 10 % loading at 72 h Rate glucose Rate xylose (mg/L/min) (mg/L/min) \n WT \n Untreated 43 ± 2 51 ± 10 10 % 70 °C, 5 h 86 ± 16 n.d. 10 % 140 °C, 3 h 196 ± 7 96 ± 42 \n LLL \n Untreated 30 ± 0.5 41 ± 7 10 % 70 °C, 5 h 41 ± 7 * \n 46 ± 1 ** \n 10 % 140 °C, 3 h 255 ± 10 * \n 154 ± 33 \n LLHPL1 \n Untreated 54 ± 7 58 ± 20 10 % 70 °C, 5 h 52 ± 8 * \n 68 ± 9 ** \n 10 % 140 °C, 3 h 271 ± 13 * \n 221 ± 16 * \n \n LLHPL2 \n Untreated 19 ± 10 41 ± 20 10 % 70 °C, 5 h 40 ± 16 ** \n 62 ± 13 ** \n 10 % 140 °C, 3 h 221 ± 16 146 ± 50 Values presented ±SD. There were significant differences between Arabidopsis lines for both the initial glucose and xylose rates for solids pretreated at 70 °C (glucose, F (3,12) = 8.8, P < 0.01 and xylose, F (3,12) = 43.6, P < 0.0001), and solids pretreated at 140 °C (glucose, F (3,12) = 7.35, P < 0.05 and xylose, F (3,12) = 5.66, P < 0.05). There was no significant difference of initial rate of xylose release between the groups in untreated (xylose, F (3,12) = 0.62, P = 0.62), but there was a significant difference between groups for initial rate of glucose release (glucose, F (3,12) = 11.22, P < 0.05). ANOVA with a Tukey’s HSD post-hoc test was used to determine overall statistics, and results of the comparison to WT from the post-hoc test are shown in the table \n n.d. not detectable \n * \n P < 0.05; ** \n P < 0.01"
} | 5,778 |
27697682 | null | s2 | 2,567 | {
"abstract": "Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can substitute for petroleum-based plastics in a variety of applications. One avenue to commercial PHA production involves coupling waste-based synthesis with the use of mixed microbial consortia (MMC). In this regard, production requires maximizing the enrichment of a MMC capable of feast-famine PHA synthesis, with the metabolic response induced through imposition of aerobic-dynamic feeding (ADF) conditions. However, the concept of PHA production in complex matrices remains unrefined; process operational improvements are needed, along with an enhanced understanding of the MMC. Research presented herein investigated the effect of aeration on feast-famine PHA synthesis, with four independent aeration state systems studied; MMC were fed volatile fatty acid (VFA)-rich fermented dairy manure. Regardless of the aeration state, all MMC exhibited a feast-famine response based on observed carbon cycling. Moreover, there was no statistical difference in PHA synthesis rates, with q"
} | 261 |
17239231 | PMC1794243 | pmc | 2,570 | {
"abstract": "Background The origin of eukaryotic cells was one of the most dramatic evolutionary transitions in the history of life. It is generally assumed that eukaryotes evolved later then prokaryotes by the transformation or fusion of prokaryotic lineages. However, as yet there is no consensus regarding the nature of the prokaryotic group(s) ancestral to eukaryotes. Regardless of this, a hardly debatable fundamental novel characteristic of the last eukaryotic common ancestor was the ability to exploit prokaryotic biomass by the ingestion of entire cells, i.e. phagocytosis. The recent advances in our understanding of the social life of prokaryotes may help to explain the origin of this form of total exploitation. Presentation of the hypothesis Here I propose that eukaryotic cells originated in a social environment, a differentiated microbial mat or biofilm that was maintained by the cooperative action of its members. Cooperation was costly (e.g. the production of developmental signals or an extracellular matrix) but yielded benefits that increased the overall fitness of the social group. I propose that eukaryotes originated as selfish cheaters that enjoyed the benefits of social aggregation but did not contribute to it themselves. The cheaters later evolved into predators that lysed other cells and eventually became professional phagotrophs. During several cycles of social aggregation and dispersal the number of cheaters was contained by a chicken game situation, i.e. reproductive success of cheaters was high when they were in low abundance but was reduced when they were over-represented. Radical changes in cell structure, including the loss of the rigid prokaryotic cell wall and the development of endomembranes, allowed the protoeukaryotes to avoid cheater control and to exploit nutrients more efficiently. Cellular changes were buffered by both the social benefits and the protective physico-chemical milieu of the interior of biofilms. Symbiosis with the mitochondial ancestor evolved after phagotrophy as alphaproteobacterial prey developed post-ingestion defence mechanisms to circumvent digestion in the food vacuole. Mitochondrial symbiosis triggered the origin of the nucleus. Cilia evolved last and allowed eukaryotes to predate also on planktonic prey. I will discuss how this scenario may possibly fit into the contrasting phylogenetic frameworks that have been proposed. Testing the hypothesis Some aspects of the hypothesis can be tested experimentally by studying the level of exploitation cheaters can reach in social microbes. It would be interesting to test whether absorption of nutrients from lysed fellow colony members can happen and if cheaters can evolve into predators that actively digest neighbouring cells. Implications of the hypothesis The hypothesis highlights the importance of social exploitation in cell evolution and how a social environment can buffer drastic cellular transformations that would be lethal for planktonic forms. Reviewers This article was reviewed by Eugene V Koonin, Purificación López-García, and Igor Zhulin."
} | 770 |
30287755 | PMC6313629 | pmc | 2,571 | {
"abstract": "Generating chemical energy carriers and bulk chemicals from solar energy by microbial metabolic capacities is a promising technology. In this long-term study of over 500 days, methane was produced by a microbial community that was fed by the mono-substrate glycolate, which was derived from engineered algae. The microbial community structure was measured on the single cell level using flow cytometry. Abiotic and operational reactor parameters were analyzed in parallel. The R-based tool flowCyBar facilitated visualization of community dynamics and indicated sub-communities involved in glycolate fermentation and methanogenesis. Cell sorting and amplicon sequencing of 16S rRNA and mcrA genes were used to identify the key organisms involved in the anaerobic conversion process. The microbial community allowed a constant fermentation, although it was sensitive to high glycolate concentrations in the feed. A linear correlation between glycolate loading rate and biogas amount was observed (R 2 = 0.99) for glycolate loading rates up to 1.81 g L −1 day −1 with a maximum in biogas amount of 3635 mL day −1 encompassing 45% methane. The cytometric diversity remained high during the whole cultivation period. The dominating bacterial genera were Syntrophobotulus , Clostridia genus B55_F, Aminobacterium, and Petrimonas . Methanogenesis was almost exclusively performed by the hydrogenotrophic genus Methanobacterium .",
"conclusion": "5. Conclusions Glycolate is clearly a suitable substrate for several anaerobic microorganisms eventually leading to methane production. The experimental set up and the use of flow cytometry and the connected bioinformatic evaluations disclosed an ingenious and cytometrically diverse ecosystem where mainly the dominant sub-communities jointly contributed to a successful carbon conversion to methane. The high cytometric community structure diversity seems to be an important factor for the continuation of the process. Flow cytometry also reveals vulnerable community states [ 66 ], as found in this study for biogas productivity and acid accumulation, which emerged when the glycolate loading rate surpassed 1.8 g L −1 day −1 , a condition that can be easily controlled in a mono-feed operating system. Cell sorting and amplicon sequencing uncovered that the conversion of glycolate to methane was performed mainly by glycolate-fermenting clostridia of the genus Syntrophobotulus in syntrophy with hydrogenotrophic methanogens of the genus Methanobacterium.",
"introduction": "1. Introduction The replacement of fossil fuels by renewable energy sources is essential to mitigate global warming. The conversion of biomass into gaseous or liquid biofuels is generally considered sustainable [ 1 ], but the energy conversion efficiency is low [ 2 ]. However, most biofuels are currently produced from energy crops grown on valuable agricultural land, thus competing with food and feed production [ 3 ]. While microalgae are an alternative feedstock for biofuel production [ 4 ] their utilization includes high costs due to the investment for the photobioreactors and operation for stirring, harvesting or transport to biorefinery plants [ 5 ]. A new approach [ 6 , 7 ] proposed a concept in which photosynthetic energy is mainly conserved in the form of glycolate by a controlled balance between carboxylation and oxygenation by the ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) in Chlamydomonas reinhardtii. The produced glycolate is efficiently excreted by the microalgae because its metabolization via the C2 cycle is blocked [ 7 ]. The concept minimizes both metabolic and economic costs of glycolate production. In the present study, we investigated whether the excreted glycolate can be efficiently converted to methane by a subsequent anaerobic digestion process. Consortia of syntrophic bacteria and methanogenic archaea that can convert glycolate to methane have been already described [ 8 , 9 ]. While several aerobic degradation pathways of glycolate are well known such as the dicarboxylic pathway in Escherichia coli [ 10 , 11 ], the glycerate pathway in E. coli [ 12 ], Pseudomonas sp. [ 13 ] and Azotobacter chroococcum [ 14 ], and the β-hydroxyaspartate pathway in Micrococcus denitrificans [ 15 ], the metabolization of glycolate under anaerobic conditions is less well explored. Only a few isolates have been described for anaerobic glycolate conversion such as Desulfofustis glycolicus and Syntrophobotulus glycolicus [ 16 ], Moorella sp. strain HUC22-1 [ 17 ], Moorella thermoacetica [ 18 ], and Lachnospiraceae strain 19gly4 [ 19 ], which use the malyl-CoA-pathway [ 20 ]. Some of the fermentation products, i.e., hydrogen and carbon dioxide, formate or actetate, can be directly converted to methane. The set-up proposed in this study relies on glycolate as mono-substrate for methane production [ 6 , 7 , 8 , 9 ]. Other mono-substrates such as acetate [ 21 ], butyrate [ 22 ], propionate [ 23 ], and glucose [ 24 ] were already shown to be suitable substrates for continuous methane production. However, glycolate could be problematic for the process due to the potentially small group of anaerobic glycolate utilizers. Moreover, in contrast to the anaerobic oxidation of propionate or butyrate, which is only possible by syntrophic interaction of proton-reducing bacteria and hydrogen-scavengers such as hydrogenotrophic methanogens, glycolate can be expected to be fermented directly to acetate by homoacetogens such as Moorella via the Wood-Ljungdahl pathway [ 17 ] or to other fermentation products such as succinate and acetate by single strains such as Lachnospiraceae strain 19gly4 [ 19 ] and thus its degradation to acetate does not necessarily require the involvement of methanogens. In that case, conversion to methane would rely on the presence of acetoclastic methanogens. However, glycolate can also be exploited by hydrogenotrophic methanogens together with syntrophic proton-reducing bacteria that are needed to perform the oxidation of glycolate to glyoxylate and further to carbon dioxide and hydrogen [ 9 ]. Usually, biogas is produced by natural microbial communities from complex substrates. These microbial systems behave dynamically and convey short reaction times to external changes [ 25 ]. Molecular tools are typically used to analyze the microbial community composition [ 26 , 27 ]. However, these methods have limitations for routine applications especially when fast dynamics, which require dense sampling procedures over longer time scales, are expected. Missing sampling points can aggravate for instance association analyses by using Cytoscape [ 28 ] and CoNet [ 29 ], which help find functional key organisms in microbial communities. Flow cytometry is an alternative approach, especially since bioinformatic tools are now available that enable the interpretation of fast shifts of microbial community structures using flowCHIC [ 30 ] and flowCyBar [ 31 ]. These tools grant the accurate quantitative analysis of cell abundance variation and allow via correlation analyses with abiotic data to attribute metabolic functions to specific sub-communities [ 32 , 33 ]. The aim of this study was to challenge an anaerobic digester community to continuously convert the mono-substrate glycolate to methane at high turnover rates and over long time periods. Possible positive or negative influences of reactor parameters on biogas production and the function of microbial key players that contribute to either glycolate fermentation or methanogenesis were of interest to obtain means for process control.",
"discussion": "4. Discussion Biogas reactors are usually fed with plant biomass such as energy crops, grass silage or landscape conservation material, or organic waste, such as manure, biowaste, agro-industrial residues or wastewater sludge [ 50 ]. The process comprises four metabolic steps executed by different microbial consortia, i.e., hydrolysis, acidogenesis, acetogenesis, and methanogenesis [ 50 ]. However, anaerobic glycolate metabolization for methane production is a new concept based on basically infinite resources when connected to an algae reactor producing glycolate from CO 2 and sun light [ 7 ]. Such a biogas production process does not need the hydrolysis step and immediately starts with acidogenesis producing several intermediates such as glyoxylate, propionate, and acetate. The products of glycolate fermentation can be either used directly by methanogens (in case of acetate, H 2 , and CO 2 ) or further metabolized by syntrophic bacteria (e.g., in case of propionate). The microbial degradation of glycolate usually starts with the oxidation to glyoxylate, which can be further metabolized by three different pathways: (i) The conversion to glycerate, which can be used to form pyruvate and eventually various fermentation products such as acetate, formate, H 2 and CO 2 [ 12 ]; (ii) the formation of malate by malate synthase and further conversion to pyruvate and subsequent fermentation products as mentioned in (i) [ 8 ]; and (iii) the formation of oxaloacetate via the β-hydroxyaspartate pathway [ 15 ]. An alternative pathway in Firmicutes is the direct fermentation of glycolate to acetate and succinate without an external electron acceptor as described for a Lachnospiraceae bacterium [ 19 ]. Besides glyoxylate and acetate, we detected propionate as intermediate, which is not an intermediate of the aforementioned pathways but can be formed from succinate [ 51 ]. Propionate is a critical intermediate in anaerobic digestion as its degradation depends on the syntrophic interaction of propionate-oxidizing bacteria and hydrogenotrophic methanogens that keep the hydrogen partial pressure sufficiently low to enable propionate oxidation to acetate [ 52 ]. In the relatively stable phases 4 and 6 of our system, we measured methane contents of 39–45% in the biogas produced from glycolate. Biogas from conventional anaerobic digesters usually has higher methane contents. Substrates such as cattle manure and maize silage produce methane contents up to 55–70% [ 50 ]. The differences are caused by the more reduced state (higher C:O ratio) of complex organic substrates. However, complex substrates are prone to cause fluctuations in methane production due to altering composition or the presence of inhibiting components, while the mono-substrate glycolate might have the advantage to always produce stable CH 4 amounts, which may facilitate process control in future. For complete glycolate conversion to biogas and without considering microbial biomass turnover and acid by-production, the theoretical stoichiometric ratio of CH 4 to CO 2 is 3:5 assuming that all electrons of the glycolate would be used for the formation of methane. However, we clearly found biomass turnover as verified by the cytometric pattern analysis. Thus, glycolate was also used for biomass synthesis and subsequently microbial biomass was also converted to methane. The relatively high amounts of organic acids were mainly produced in instable phases of the process and thus represent intermediates that accumulated transiently during instable process periods. We need to state that the 526-day study was performed as a single approach but the 178 biological samples taken in addition with the even higher numbers of abiotic data allowed the verification of trends in community behavior by avoiding indiscriminate sample handling and interpretation. It was surprising to discover the high diversity of our community on the mono-substrate glycolate, which revealed as much as 29 sub-communities present during the 526 days of cultivation. The structure of the community was evaluated using only the 18 most dominant sub-communities, which were by no means constant in cell abundance and changed in high proportions. Natural communities can be expected to behave very differently when grown on mono-substrates. However, so far there is no experience around on how communities develop under such conditions. Cytometric cluster 1 cells were highly abundant by quota and can be assumed, due to the performed correlation analyses ( Figure 5 ), to be major functional contributors to biogas production but also to acetate and propionate production. In contrast, correlation of cluster 2 cells with abiotic data revealed a tight connection to glyoxylate formation. Cluster 3 cells did not show high correlation values and are therefore assumed to be of lower importance in glycolate fermentation. Therefore, only gates from cell clusters 1 and 2 were used for amplicon sequencing of 16S rRNA and mcrA genes revealing a snapshot of the phylogenetic composition of the sub-communities in G1-G3, G6, G8, G25, and G26 ( Figure 4 ). Sub-communities G2, G3, G6, and G26 contained substantial shares of a bacterial OTU assigned to the genus Syntrophobotulus , which comprises the syntrophic glycolate-oxidizing species S. glycolicus that converts glycolate to H 2 and CO 2 in a methanogenic co-culture [ 8 , 9 , 16 ]. Together with the observed dominance of hydrogenotrophic methanogens, this result suggests that a substantial portion of glycolate was directly converted to methane by a syntrophic consortium of Syntrophobotulus and Methanobacterium . However, the sub-communities G1–G3 were dominated by the clostridial genus B55_F, the physiological function of which is yet unknown. This genus was detected in substantial proportions in all sub-communities except G25. Based on the known metabolic diversity of the class Clostridia, it can be speculated that it might be a homoacetogen that utilized glycolate directly to form acetate, as described for Morella [ 17 , 18 ], a succinate and acetate-producing fermenter as described for a Lachnospiraceae species [ 19 ], or a syntrophic acetate-oxidizing bacterium (SAOB) that degrades acetate formed from glycolate by other fermenting bacteria. As almost no acetoclastic methanogens were detected, syntrophic acetate oxidation must be the major acetate sink in the system. The mcrA primers we used to detect methanogens were already described in 2002 [ 37 ] and thus might have failed to detect more recently described taxa such as Methanomassiliicoccales, Candidatus Methanofastidiosales and Candidatus Verstraetearchaeota. However, these newly described methanogens are probably obligate methylotrophs [ 53 ] and thus we did not expect that they play a major role in our system. An abundant genus in sub-communities G1–G3 was Petrimonas , members of which have been described as carbohydrate-fermenting bacteria that produce acetate and propionate [ 54 , 55 ]. Being involved in acidogenesis in biogas reactors [ 55 ], this genus might be attributed to microbial biomass decay in our system. Another genus that was predominant in G8 but also quite abundant in G6 and G26 was Aminobacterium . Species of this genus have been isolated from anaerobic digesters and ferment amino acids to acetate and propionate or oxidize amino acids in syntrophy with Methanobacterium that was the prevailing methanogen in our reactor [ 56 , 57 , 58 ]. A similar physiology has been described for the genus Anaerobaculum , which was also present in most sub-communities with abundances of up to 9%. Species of this genus are strictly anaerobic, chemo-organotrophic bacteria fermenting several sugars, peptides, and organics acids to acetate and hydrogen [ 59 , 60 , 61 ]. Neither glycolate nor one of the metabolites detected in our reactor (glyoxylate, acetate, or propionate) was described to be a substrate of Aminobacterium or Anaerobaculum species. Thus, it is more likely that these members of the phylum Synergistes were related to microbial biomass decay together with fermenting species of the phylum Bacteroidetes (i.e., mainly Petrimonas ), and that acetate and propionate accumulated during instable process periods due to biomass turn-over rather than being fermentation products of glycolate. However, propionate could be also formed as fermentation product from succinate provided that our reactor harbored a bacterium fermenting glycolate to succinate and acetate as described for the Lachnospiraceae sp. strain 19gly4 [ 19 ]. Despite propionate concentrations transiently increased during instable process periods, we did not find any of the classical syntrophic propionate oxidizers that are usually involved in propionate turn-over in biogas reactors digesting complex biomass (e.g., Syntrophobacter or Pelotomaculum ). However, an OTU affiliated to the Cloacimonetes (previously known as candidate division WWE1) was detected in most sub-communities with abundances between 1% and 3%. While the only described species of this phylum (Candidatus Cloacamonas acidaminovorans ) has been described as syntrophic amino acid degrader based on its genome, it was also discussed as facultative syntrophic propionate oxidizer [ 62 ]. However, more detailed analyses such as metagenomics or proteomics would be required to unravel the ecophysiological functions of the several phylotypes in the sub-communities. Nevertheless, the limited taxonomic information we retrieved from MiSeq amplicon sequencing implies that glycolate is most likely converted to methane by syntrophic consortia of Syntrophobotulus and Methanobacterium , as previously described [ 8 , 9 , 16 ]. High biogas production is of huge commercial interest; therefore, we started two attempts to increase the biogas productivity. The increase of the glycolate loading rate is one way, and we found indeed higher biogas production with increasing GLR. However, this strategy often fails [ 63 ] for high organic loading rates as was also found for our system in phase 2. Acid concentrations increase in a biogas reactor due to the low metabolic capacity of the methanogenic archaea, which are usually present at low cell concentrations and cannot adapt fast enough to new conditions because of their long generation times. In turn, the increasing acid concentrations further inhibit the methane production by archaea [ 64 , 65 ]. Therefore, as the metabolism of methanogens is favored by neutral to slightly alkaline pH values a second strategy was to increase the biogas production by adjusting the pH of the glycolate feed suspension to seven. By this way, forestalling a pH drop in the reactor at prospective higher glycolate loading rates was anticipated. However, although the reactor pH reached values well above 8, biogas productivity decreased (phase 5) and a dramatic increase in acetate and propionate concentrations was observed. Additionally, the CH 4 content of the biogas increased. The neutralization of the glycolate feed suspension may have led to a shift in the carbonate buffer system due to the higher pH and consequently to lower CO 2 contents in the biogas. It is also conceivable that the change in the pH glycolate feed led to an increased decay of biomass changing the gas composition in favor of CH 4 . Nevertheless, the overall biogas productivity decreased. While a higher CH 4 to CO 2 ratio would be of interest for the industry, a reduction of the biogas productivity by half is obstructive."
} | 4,793 |
30804382 | PMC6389951 | pmc | 2,572 | {
"abstract": "To prevent the settlement and/or the growth of fouling organisms (i.e. bacteria, fungi or microalgae), benthic sessile species have developed various defense mechanisms among which the production of chemical molecules. While studies have mostly focused on the release of chemical compounds by single species, there exist limited data on multi-species assemblages. We used an integrative approach to explore the potential interactive effects of distinct assemblages of two corals species and one giant clam species on biofouling appearance and composition. Remarkably, we found distinct biofouling communities suggesting the importance of benthic sessile assemblages in biofouling control. Moreover, the assemblage of 3 species led to an inhibition of biofouling, likely through a complex of secondary metabolites. Our results highlight that through their different effect on their near environment, species assemblages might be of upmost importance for their survival and therefore, should now be taken into account for sustainable management of coral reefs.",
"introduction": "Introduction Benthic sessile communities are under direct and constant pressure of their surrounding environment. Therefore, many species have developed various mechanisms such as direct and indirect biotic interactions to maximize their survival 1 . Each of these interactions can be either positive or negative and are responsible to a large extent for structuring communities 2 . Most studies on bioactive products have focused on the competition and defense processes that may lead to the discovery and commercialization of new bioactive metabolites 3 . In sessile coral reef organisms, numerous studies have identified a wide variety of secondary metabolites such as terpenoids (e.g. siphonodictine 4 ), saponins (e.g. muricins 5 ), macrolides (e.g. latrunculins 6 ), and steroids (e.g. verumbsteroids 7 ) that can reduce the growth or even lead to the death of neighboring species. The production of such secondary metabolites, mainly non-polar, synthesized constitutively or inducible 8 , strongly influences sessile organisms’ behaviors in response to local competition or predation pressures. The latter are particularly pervasive in marine biofouling environments where strong developmental and organism-environment interactions take place 9 . Biofouling is an ubiquitous phenomenon in the marine environment and a common feature of a wide variety of natural and artificial structures 10 . Biofouling formation typically starts with the adhesion of dissolved organic matter onto a surface which leads to physiochemical changes 11 and to the development of a bacterial mat or early biofilm layer 12 . Successfully settled bacterial communities may influence the successive settlement of micro-algae (e.g. diatoms, cyanobacteria), fungi and protists, which are all precursors for colonization by larger fouling organisms or macrofouling 9 . Biofouling formation onto a substrate, its growth rate and the type of species able to colonize this habitat are modulated by various interactions and involve complex biochemical, behavioral or physical mechanisms 9 , 13 . The external environment plays an important role in the biofouling formation, in particular through physical and chemical conditions 14 , 15 . For example, the availability of nutrients will play a critical role in the selection of early colonizers and the successive community composition of the biofouling 16 . Light and temperature increase also play important roles in enhancing the propagation of biofilm and biofouling 17 , 18 . Besides these environmental factors, many antifouling agents have been characterized from sessile marine organisms, in particular antifouling molecules produced by sponges, soft corals, and seaweeds 12 , 19 , 20 . Microorganisms associated with marine algae and invertebrates, such as epibionts, also possess antifouling potential 12 . Despite the growing knowledge that biofouling-associated antifouling compounds are damaging to both larval and adult stages of hard corals and giant clams 21 – 23 , there exist very limited data on the actual processes and the specific molecules involved in antifouling activities of multiple interactive coral reef species. There is now growing evidence that hard corals release antifouling active substances 3 , which can confer different competitive abilities to coral species against algae 24 . Studies on Antarctic marine benthos have shown that by bringing together different defense strategies, mainly regulated by chemical interactions such as deterrent or repellent molecules, species assemblages create a complex model of interaction which may help protect Antarctic organisms from competition, for space and fouling pressure notably 25 . In coral reef ecosystems, the variety of antifouling compounds produced by sessile organisms, even among coral genera, prompted us to study the importance of species biodiversity and interaction on coral reefs’ fitness. Our work investigated their potential cumulative effect on biofouling formation and their ability to minimize the dramatic impact that uncontrolled biofouling expansions (i.e shifts from coral to algal dominated reefs) may have on coral reef ecosystems. Thus, in this study we combined cytological, metagenomic and metabolomic approaches to explore the interactive effects of multiple benthic species assemblages on the biofouling appearance and composition under normal and thermal stress conditions. Two common Indo-Pacific scleractinian coral species, Pocillopora damicornis and Acropora cytherea , and the giant clam Tridacna maxima were artificially grouped in distinct assemblages, exposed to varying water temperatures, and their influence on algal biofouling formation was investigated.",
"discussion": "Discussion This is the first study demonstrating that biofouling formation is strongly influenced by surrounding benthic sessile species. In particular, we show that in its most severe form, the resulting effect of a species-specific assemblage is biofouling inhibition. Biofouling is a complex sequential process, which can be modulated by many abiotic and biotic factors, ranging from external factors to intrinsic influence of the marine taxa participating in biofouling formation itself. Among these factors, nutrients are known to influence growth and productivity of various organisms such as bacteria and algae. In our study, nutrient contents in the seawater of each aquarium were compatible with those observed in previous nutrient analyses from the Moorea lagoon 26 , 40 . An increase of silicates was notably observed during the wet season as well as after strong rains 41 , as was the case for all aquariums of the A experiment. This link with external parameters can be explained by the design of our experiment, which consisted of an open-circuit with a water flow of 20 L/h per aquarium of 40 L. Therefore, the lagoon seawater composition mainly accounted for the significant variation of nutrient composition between the 3 experiments. As the effect of species assemblage on biofouling development was reproducible, this nutrient discrepancy between experiments could not account for the observed differences in biofouling development. Studies have shown that bacteria, plankton and phytoplankton as well as dissolved organic and inorganic molecules are filtered from seawater and ingested by corals and bivalves 42 , 43 . This feeding can lead to a depletion of 30 to 45% of total chlorophyll a and picoplankton above a scleractinian coral dominated reef when compared with adjacent waters 42 . Tridacna species are able to filter up to 58% of algal cells from their surrounding water 44 . Moreover, a recent study has shown that corals are able to feed on diatoms such as Thalassiosira sp. 43 . In light of these results, and taking into account that giant clams filter large amounts of seawater (e.g. water filtration rate of one Tridacna crocea is 2-3 L/h) 44 , filtering activities could reduce the number of species participating in biofouling development and so could disrupt some steps of biofouling formation. Therefore, whenever another heterotrophic species is added to an assemblage, the community of microorganisms within the aquariums’ seawater could be modified (e.g. additive depletion of microorganisms). Such modifications could account for the differences in biofouling composition and development observed between assemblages, the stronger impact of these additive depletion effects leading to the inhibition of biofouling (e.g. lack of key species required for biofouling progression). Biofouling development was strongly inhibited in the three species assemblages, PAT, of which aquarium water was depleted of 47 bacteria, 2 diatoms and 3 algae genera when compared to the other assemblages. Among these lacking organisms, some bacterial taxa e.g. Rhodobacteraceae that are important biofilm precursors 45 , could be required for effective biofouling formation. Further studies of giant clam and coral filter-feeding species interactions will be helpful to better understanding their respective putative roles in depleting the microorganisms involved in biofouling progression. However, our experimental conditions with high seawater renewal and successive samplings led to four organisms per species at the end of the experiments. Therefore, corals and giant clams’ nutrition alone cannot be responsible for the disappearance of biofouling key species. Even though relevant, this hypothesis is not sufficient to account for the observed difference in biofouling development. In contrast to ingested filtered organisms, those associated with invertebrate species, bacteria and microalgae, have been well characterized. Bacteria have been found living on the surface, in the tissues and in the mucus of coral species 46 – 48 . Because mucus contains a lot of inorganic phosphate and dissolved organic carbon, it is suspected as an effective inducer of bacterial growth 49 . Moreover, the diversity and quantity of the hosted bacteria differs not only between species but also according to their environment 50 . Dobrestov and collaborators 12 have shown that numerous bacteria associated with corals possess an anti-bacterial activity, and antibacterial compounds in coral mucus have notably been found to select specific bacterial population leading to an antifouling effect 51 . Interestingly, 7 bacterial species were specifically found in PAT assemblages. Among them, bacteria belonging to the genus Lysobacter which are able to degrade biofilm and could contribute to the observed antifouling effect 52 . Corals and giant clams are known to produce bioactive compounds such as sterols which are described as chemical defensive substances 53 , 54 . Indeed, living corals are free of fouling and this particularity come from the production of secondary metabolites, especially sterols playing key roles in allelopathy with the ability to inhibit the growth of organisms around them 54 . Thereby, a fouling-resistant composition, non-toxic for the environment and applicable on immersed equipment, was made with sterols 55 . Even though no specific metabolite to the PAT assemblage was detected, suggesting that no new compound is produced in this context, 5 VIPs characterizing the PAT assemblage were highlighted. Among them, one was assigned to carotenoids and 4 to lipids. The carotenoid, identified as peridinin, is a specific pigment found in Symbiodinium dinoflagellates from corals and giant clams 35 . Interestingly, like most of the carotenoids, peridinin exhibits an inhibitory activity even at low concentration. As peridinin can inhibit cell proliferation and can exhibit a cytotoxic activity 56 , 57 , it might contribute to the biofouling inhibition observed in PAT assemblage. Moreover, among the lipids identified, one was assigned to brassicasterol and two to DGDG. Brassicasterol is found in various marine organisms such as cnidarians, sponges or diatoms 58 – 60 . In addition to having significant anti-inflammatory properties 60 , this sterol exhibits activity against organisms such as protozoans 61 . Furthermore, DGDG are also found in marine organisms, especially in Symbiodinium from both corals and giant clams 62 , 63 . Digalactosyldiacylglycerols possess strong nitric oxide inhibitory properties leading to antiviral activity 64 , 65 . Moreover, DGDG have been identified as antifouling agent demonstrating repellent activity notably of blue mussels 66 . These data suggest that a synergism of secondary metabolites might lead to antifouling activity as it has already been assumed in a previous study 67 . Therefore, in the present study, either due to their abundance as VIPs or to their synergism, carotenoid and lipids might be responsible for the biofouling inhibition specifically observed in PAT assemblage. In this regard, as mucus is continuously released by both corals and giant clams 68 , we cannot exclude that mucus flocs and/or secreted molecules such as antibiotics could adhere to the aquarium’s walls, playing a role in biofouling production. This effect will be, as discussed for nutrition, dependent on the different species present in the aquarium, with possible positive or negative interactions. Regardless of the mechanisms involved in this inhibition, our experimental results suggest that biofouling develops differently, depending on the type and complexity of the sessile species assemblages and this may have important implications in coral reef ecology. Moreover, these phenotypes were maintained throughout experimental trials, even during the five days short thermal stress, suggesting that occasional increases in seawater temperature are not sufficient to induce differences of biofouling appearances. Global warming, ocean acidification, and local human impacts are considered as the main causes of coral reefs deterioration globally 69 – 71 . The combination of these factors usually prevent resilience of coral reefs, and favour the shift from coral-dominated to algal-dominated reef ecosystems 72 – 74 . Our discovery of an assemblage dependent antifouling activity is all the more important that biofouling inhibition occurs even in brief increased temperature conditions. Another major point of our results is that the loss of one species in a three species assemblage leads to a change in the algal inhibition spectrum, the two species assemblage being less active against or more prone to algal development. Despite the timeliness of understanding ecological shifts towards algal-dominated tropical habitats, a gap of knowledge still exists about the mechanisms governing these shifts 75 . It is well known that coral reefs taxa, e.g. scleractinian corals species, are differentially sensitive to environmental stressors 76 – 78 and that, among sessile species, giant clams are notably more resistant than corals to sea water warming 79 , 80 . Therefore, environmental stress raises sequential and differential loss of species. This selective loss of species may impact the structuration of coral reefs benthic communities, leading to a change in the species interaction network and therefore, as exemplified by our work, might strongly impact their defence mechanisms against prerequisite steps of algal development. On the contrary, maintaining the diversity may temper this coral-algal shift. Hence, our study pinpoints that sessile coral reef species diversity, distribution and fitness might strongly influence this coral-algal shift, underlining the putative dramatic consequences of the decrease in biodiversity and the health of coral reefs. Beyond the biofouling phenotypes, with its extreme form translating into biofouling inhibition, the present study also highlighted the functional interactions resulting from sessile species assemblages. Our data showed that specific communities of bacteria, diatoms and algae characterized the biofouling composition of each assemblage. The bacterial functional profiling revealed an increase of “stores polyhydroxybutyrat” in PA assemblage that could be linked to a need of energy storage molecule or to a stress 81 . In line with this interpretation, microbiomes in PA assemblages included species of Pseudoalteromonaceae that are able to degrade a form of polyhydroxybutyrat referred to as poly-3-hydroxybutyrate 82 . Other functions such as sulfur reducer, iron oxidizer, streptomycin producer and cellobiose degrading were, respectively, strictly found in T, PAT, P and PA. Additionally, some specific functions such as nitrite reducer and atrazine metabolism for T and PAT or degrade aromatic hydrocarbon for PAT, P and PA, are shared by some but not all of the assemblages. Therefore, the presence of nitrite reducer activities in both PAT and T assemblages could account for the systematically higher level of NH 4 + in these assemblages. Such common features between some specific assemblages highlight the potential cumulative functional effect of the addition of one species in an assemblage, such as degrading aromatic hydrocarbons produced by P in PAT and PA as well as some emergent functions such as iron oxidizer in PAT. Therefore, all these combinations of functions provide a specific capacity to an assemblage for responding to changing environmental conditions. Such a trait can be related to the beneficial effect of plant assemblages for the growth, productivity and protection against pathogens and others, such as in permaculture where combinations of plants are used to enhance resistance and productivity 83 . Considering the resulting functional aspects of various species assemblages, with some positive and/or negative interactions, together with their effect on biofouling development, our data support the notion that marine species assemblages should be integrally part of future coral reefs restoration plans as is already the case for terrestrial restoration."
} | 4,485 |
24586260 | PMC3929350 | pmc | 2,574 | {
"abstract": "A distinct succession from a hydrolytic to a hydrogeno- and acetotrophic community was well documented by DGGE (denaturing gradient gel electrophoresis) and dHPLC (denaturing high performance liquid chromatography), and confirmed by qPCR (quantitative PCR) measurements and DNA sequence analyses. We could prove that Methanosarcina thermophila has been the most important key player during the investigated anaerobic digestion process. This organism was able to terminate a stagnation phase, most probable caused by a decreased pH and accumulated acetic acid following an initial hydrolytic stage. The lack in Methanosarcina sp. could not be compensated by high numbers of Methanothermobacter sp. or Methanoculleus sp., which were predominant during the initial or during the stagnation phase of the fermentation, respectively.",
"introduction": "Introduction Irrespective of the disputed contribution of man to the global warming, the dramatic effects per se and the involvement of gases like CO 2 and CH 4 are unquestionable. Therefore it has (or should have) become an important global goal to reduce uncontrolled greenhouse gas emissions. One possibility to do so (and to fulfill the Kyoto Protocol) is to increase the portion of renewable energy sources like biogas. Therefore and before the background of a decreasing availability and increasing costs of fossil energy sources, the European Union has decided that by the year 2020 about 5% of the total energy budget should be derived from biogas production (De Vrieze et al, 2012; EC, 2011). No wonder that both, the number and capacity of biogas plants have steadily increased during the last decades [1] . Unfortunately, most of these plants are designed on the basis of empirical data and quite often unexpected and unexplainable fluctuations in fermenter performance occur [2] , [3] and even recent publications come to the conclusion that the engaged microorganisms still work within a ‘black box’ [4] . However, there has been significant progress in identifying and investigating microbial key players of anaerobic fermentations especially since culture independent techniques have become increasingly available in microbiology [1] , [5] , [5] – [9] . In a former investigation we could prove Methanosarcina sp. to be a key player during thermophilic biogas production – especially during the recovery after disturbed fermentations [7] , a finding which corresponds with similar investigations [10] , [11] . It was possible to prove that inoculation with Methanosarcina sp. could successfully restart or at least accelerate the restoration process after a disturbance of the fermentation [12] . Despite this progress a couple of questions remain unsolved, especially those connected with the microbial succession during optimal and malfunctioning. Thus, within the present investigation we used different methods to characterize the microbial succession during a batch fermentation. These methods comprised both, fingerprint and analytical approaches and especially focused on the abundance of Methanosarcina sp., and its correspondence with the biogas production, as we assumed that a lack of Methanosarcina sp. might cause severe disturbance during thermophilic digestion.",
"discussion": "Results and Discussion \n Figure 1 shows the course of the batch fermentation with respect to the most important fermenter properties including quality and quantity of biogas. At each of the sampling days 66 mL of the culture broth was withdrawn and kept frozen at −20°C till the whole process was finished. Afterwards, concentrations of VFAs were determined within all samples. On the basis of the process parameters, we decided to investigate samples from t = 0, 4, 18, 26 and 41, with molecular approaches and PLFA analysis in detail. At these days – indicated by dashed lines in Figure 1 – distinct changes in fermentation performance occurred, and thus, differences in microbiology should become obvious. 10.1371/journal.pone.0086967.g001 Figure 1 Fermenter performance. pH-values, qualitative and quantitative properties of biogas (A) and concentrations of VFAs (B) during the fermentation. Gas production rate (grey background) is given in A and B to ease the comparison. Dashed lines at t = 0, 4, 18, 26, 41 outline the samples which were additionally investigated by molecular approaches. Start up At the very beginning of the fermentation there was a distinct decrease in pH from about 7.5 to 6.6 connected with a sharp increase in the concentrations of H 2 and CO 2 in the headspace and the concentrations of acetic and butyric acid in the sludge. Altogether, this obviously reflects the high metabolic activity of hydrolytic and acetogenic microorganisms, resulting in about 40% CO 2 , 17% H 2 , 15 mM acetic acid and 4 mM butyric acid within only one single day. The production of appreciable amounts of iso-butyric acid and propionic acid took more time and concentrations reached approximately 1.5 mM and 3 mM at t = 1 and t = 4, respectively. At these levels the two acids remained remarkably constant till the second phase of high biogas production occurred. PLFA analyses showed high initial concentrations of long polyunsaturated fatty acids, pointing to a high abundance of eukaryotic cells, possibly deriving from plant material introduced to the fermenter sludge. This explanation seems quite probable as these fatty acids completely disappeared within four days of fermentation. \n Figure 2 shows the DGGE patterns of nucleic acids amplified with archaea-specific primers. Results prove a distinct dominance of Methanothermobacter thermoautotrophicus and Methanothermobacter wolfei at t = 0. These two members of Methanothermobacter as well as all other organisms, which will be discussed within the present paper, exactly matched the reference lines of the respective pure culture (as far as available) and/or were identified by sequencing. The dominance of Methanothermobacter sp. confirms previous investigations of the thermophilic fermenter where the inoculum was taken from for the present investigation, and where M. wolfei has been proven to be the dominant methanogenic organism [9] . Both species of Methanothermobacter are efficient hydrogenotrophic organism (following reaction 1, shown in Table 1 ) and so this efficient pathway of methanogenesis started after a very short lag phase of not more than two days. This was also proven by a very sharp decrease in the concentration of H 2 , which fell beneath the detection limit (0.005%) again within one week. However, conditions for the initial dominant species seemed to become unfavorable as these organisms completely disappeared till t = 4 and another hydrogenotrophic organism, Methanoculleus thermophilus , became increasingly abundant. A very sharp increase in the abundance of M. thermophilus was also proven by dHPLC analyses, which again proved to be an efficient fingerprint method for investigating post-PCR mixtures of nucleic acids [22] . 10.1371/journal.pone.0086967.g002 Figure 2 Archaeal DGGE. DGGE of archaeal PCR-products out of samples taken at day = 0, 4, 18, 26 and 41. Assignment of different bands: 1 Methanothermobacter thermoautotrophicus , 2 Methanothermobacter wolfei , 3 Methanoculleus thermophilus , 4 Methanosarcina sp., 5 Thermoplasma sp., 6 Methanosarcina thermophila , 7 Methanosarcina thermophila . 10.1371/journal.pone.0086967.t001 Table 1 Thermodynamic properties of selected reactions at standard and at in situ conditions *) . Reaction Standard conditions (kJ reaction −1 ) \n In situ conditions *) (kJ reaction −1 ) (1) 4H 2 +HCO 3 \n − +H + →CH 4 +3H 2 O −135.5 −43.4 (2) Butyric acid − +2H 2 O→2 acetic acid − +H + +2H 2 \n 48.2 −8.3 (3) Acetic acid − +H 2 O→CH 4 +HCO 3 \n − \n −31.0 −20.7 Reaction (1) plus two times reaction (2) resulting in (4) Butyric acid − +4H 2 O→2CH 4 +2HCO 3 \n − +H + +2H 2 \n −13.8 −49.7 *) 52°C and real concentrations of gases and VFAs according to [26] . \n Figure 3 shows the course of the abundance of M. thermophilus during the whole fermentation. The DNA from the peaks was gathered after HPLC analysis, sequenced and proven to derive from M. thermophilus (100% identity). Obviously, this methanogen could rapidly respond to the harsh initial conditions and to a certain extent better handle the steadily increasing concentrations of VFAs accompanied with the decreasing pH. Besides that, the hydrogenotrophic methanogenesis has once again turned out to be more efficient, not only under standard but also under in situ conditions (Reaction 1 vs. 3, Table 1 ) as proven in an earlier investigation [26] . 10.1371/journal.pone.0086967.g003 Figure 3 dHPLC of Methanoculleus sp. dHPLC signals [mV] of Methanoculleus sp. within PCR-products of different samples taken at day = 0, 4, 18, 26 and 41. Quantitative analyses proved the total DNA content to be maximal at t = 0, probably because of the above mentioned input of eukaryotic cell material and thus nucleic acids. Contrary, the numbers of total archaea, which we could prove to be equivalent to methanogens in this environment, were minimal at t = 0 and distinctly increased till t = 4 ( Figure 4 ). 10.1371/journal.pone.0086967.g004 Figure 4 qPCR and archaeal diversity. Content of DNA, copy numbers of Archaea and Methanosarcinales determined via qPCR and Shannon index (bars) on the basis of archaeal DGGE bands before the background of gas production (see Figure 1 ). Stagnation After t = 4 the number of methanogens remained constant till t = 18 ( Figure 4 ) and thus mark a stagnation phase of approximately 14 days, during which nearly no further methane was produced. Concentrations of CH 4 , CO 2 and H 2 remained constant at 30%, 50% and <0.005%, respectively, and also the pH remained unchanged at a low level of about 6.7 ( Figure 1A ). The only parameters which changed within this phase were the concentrations of acetic and butyric acid ( Figure 1B ). Whereas the first one steadily increased during this whole phase the latter one was characterized by a distinct degradation starting at t = 14. This degradation of butyric acid was nearly the only sign of microbial activity within this phase of fermentation. As the degradation of butyric acid usually follows the reaction 2, shown in Table 1 , this degradation should additionally account for the increasing concentration of acetic acid ( Figure 1B ). It is important to notice that this reaction is endergonic under standard conditions but becomes slightly exergonic under in situ conditions (52°C and real pH and concentrations of gases and VFAs) [26] . Furthermore, there is a syntrophic connection with acetate-degrading organisms (e.g. reaction 3, Table 1 ) so that the sum of the reactions (Reaction 4, Table 1 ) becomes exergonic, both under standard and even more under realistic conditions. And indeed, the acetic acid oxidation resulted in a distinct production of methane at the end of the stationary phase ( Figure 1 ). When concentration of acetic acids exceeds about 32 mM the oxidation of propionic acid is hampered leading to a second appearance of H 2 which might favor hydrogenotrophic methanogens. Microbial analyses proved that M. wolfei and M. thermoautotrophicus have completely disappeared, that the dominance of Methanoculleus sp. steadily decreased and that Methanosarcina thermophila very slightly appeared in several strains during this stagnation phase ( Figure 2 ). Obviously, there is a connection between the bad fermenter performance and low gas production during the stagnation phase on the one hand, and the lack of an acetoclastic methanogenic organism, being able to efficiently use the high amounts of acetic acid on the other hand. However, the growth rate of M. thermophila was obviously quite low or somehow suppressed, which resulted in a long lag phase of acetoclastic methane production. Although Methanosarcina sp. was first detectable at t = 4, it took this strain a long time, till t = 26, until it became dominant. The DGGE band quantification did not only point to the dominance of M. thermophila but also to a greater archaeal diversity at t = 26 as several weak and unidentified bands appeared. Obviously, M. thermophila is a very robust acetotrophic methanogen and it was the only one in our investigation which was able to handle high concentrations of acetic acid (35 mM) and the corresponding low pH values, confirming results from the literature [27] , [28] . The distinct drop in pH seems to be the reason for the break in CH 4 production, which only Methanosarcina sp. was able to resolve. In former investigations always using the very same inoculum from the 750 000 L large-scale fermenter, sometimes a break with a hampered gas production occurred and sometimes the second phase of gas production was directly connected to the first hydrolytic phase [7] , [12] . The reason for this different and hardly predictable behavior is not clear yet but we assume that it corresponds with the presence or lack of Methanosarcina sp. Slight differences in buffer capacities of the media resulting in different extents of the pH decreases and thus different growth rates of Methanosarcina sp. might be a possible explanation. Another possibility, however not likely, might be that Methanosarcina sp. did not grow on acetic acid in the original fermenter sludge and thus had to adapt its metabolism towards the acetoclastic instead of methylo- or hydrogenotrophic pathway leading to a lag-phase. [29] observed that the pregrowth conditions for Methanosarcina spp., which define the pathway for methanogenesis, had a significant impact on the occurrence and duration of the lag-phases. Also a very recent investigation proved that bioaugmentation of enriched inocula with Methanosarcina sp. led to an improved start-up of digestions suffering from high acetic acid loads [30] . Results from PLFA analyses point to an increase of fatty acids typical for gram positive bacteria and confirm the absence of eukaryotic cells during the stagnation phase (data not shown). These latter results clearly proved that anaerobic fungi like Neocallimastix sp., which are sometimes discussed to be engaged in anaerobic digestion [31] , [32] should not play any role during the investigated fermentation. Second phase of efficient methane production As mentioned above, at t = 14 the degradation of butyric acid started but it was not before the rapid degradation of acetic acid, started at t = 19, that the second increase in gas production occurred ( Figure 1B ). Within a few days the concentration of CH 4 increased to the final concentration of about 80%, whereas the content of CO 2 decreased to about 15%. Finally the cumulative gas production reached about 14 L standing for about 850 mL gas per gram of carbon, which is a remarkable result compared with gas yields, known from literature [33] , [34] . Interestingly, H 2 became detectable again at the end of the stagnation phase. It should be derived from the degradation of VFAs (see Table 1 ) and might have promoted the growth of hydrogenotrophic methanogens. As several bands appeared in the DGGE, in all three independent DNA-extractions, and because the number of operons per organisms (probably not more than three) should be constant for a single organism, we think that the bands represent different species of Methanosarcina sp. or at least different strains of M. thermophila . It is important to keep in mind that Methanosarcina sp. is a very versatile methanogen with respect to its substrates because it is able to use all four known methanogenic pathways, which are the hydrogenotrophic, acetoclastic, methylotrophic and the methyl reduction way [11] , [35] , [36] . Altogether, our results as well as the referred literature, emphasize the potential of Methanosarcina sp. as the central key player under high organic loads or deteriorated conditions, as it was the case during this second phase of gas production. Although within complete different habitats – an abandoned coal mine and in a rice field – [37] and [35] could also prove Methanosarcinales to govern CH 4 formation by utilizing acetic acid rather than H 2 . Accompanying the distinct degradation of acetic acid, the pH rose again and reached a level of about 7.4. Obviously this was favorable for a greater variety of methanogens, apart from M. thermophila . The Shannon index calculated on the basis of DGGE-data proved the highest archaeal diversity at t = 26. Besides M. thermophila and M. thermophilus a further organism could be identified by all the methods applied, namely Thermoplasma sp. or at least some closely-related archaeon representing a non methanogenic organism, which is usually known for its extremophilic way of living [38] . However, despite the occurrence of Thermoplasma sp. and despite the high archaeal diversity, M. thermophila remained the dominant organism, and also the gas production rate reached its optimum at this time ( Figure 1 ). At t = 26 the concentration of DNA increased again and the number of archaea (determined via qPCR) reached its maximum ( Figure 4 ). Final phase At the end of the fermentation the gas production ceased and the concentrations of VFAs, CH 4 and CO 2 were at a constant level. All other parameters describing abundance and activities of the engaged microorganisms distinctly decreased and reached final minima. The distinct succession from a hydrolytic to a hydrogeno- and acetotrophic community was well documented by DGGE and dHPLC and confirmed by qPCR measurements as well as sequencing data. PLFA analyses in contrast seemed to be of limited evidence in anaerobic systems due to the uncertainties in assignment of specific fatty acids to microbial groups. However, within the present investigation we could prove that there were only very few key players engaged in the investigated digestion, i.e. Methanothermobacter thermoautotrophicus and M. wolfei at the beginning, Methanoculleus thermophilus during the intermediate and to minor quantities in the second phase of high gas production, and Methanosarcina thermophila most dominant during the second phase of high gas production. Members of the genera Methanosarcina and Methanosaeta are the only methanogens able to degrade acetic acid. While Methanosarcina sp. usually dominates at high acetic acid concentrations because of its high conversion rates and low affinity, Methanosaeta sp. dominate at opposite conditions due to its high affinity but low conversion rates [10] , [11] , [30] . In our investigation threshold values for acetoclastic methanogenesis were found to be around 0.6 mM, which corresponds to findings of [39] who determined for M. barkeri and M. mazei 1.2 and 0.4 mM, respectively, whereas distinct lower values (0.07 mM) were calculated for Methanosaeta sp.. Additionally, in an early, anyway excellent work, kinetics of Methanosarcina sp. MSTA-1 was investigated [38] . Under optimum temperature and pH conditions Km for acetate kinase and threshold values for acetate were 10.7 and 0.7 mM respectively, thus again confirming our data. Besides, Methanosarcina sp. was shown to have a wide pH-range optimum for growth, and slightly acidic conditions even seem to induce increased growth rates [40] . Overall, Methanosarcina thermophila seems to be the most important methanogen in the investigated environment, as it was able to terminate a stagnation phase, most probably caused by a decreased pH and accumulated acetic acid. Thus, besides the inoculation with Methanosarcina sp., an adaptation of fermenter conditions towards properties favorable for this organism might be a promising possibility to skip phases of low gas production and to optimize CH 4 yields during anaerobic fermentation. Nevertheless, further research is required, also with respect to potential for up-scaling and applicability."
} | 5,003 |
38549615 | PMC10972764 | pmc | 2,575 | {
"abstract": "Due to the complicated metabolic and regulatory networks of l -serine biosynthesis and degradation, microbial cell factories for l -serine production using non-model microorganisms have not been reported. In this study, a combination of synthetic biology and process optimization were applied in an ethanologenic bacterium Zymomonas mobilis for l -serine production. By blocking the degradation pathway while introducing an exporter EceamA from E. coli , l -serine titer in recombinant Z. mobilis was increased from 15.30 mg/L to 62.67 mg/L. It was further increased to 260.33 mg/L after enhancing the l -serine biosynthesis pathway. Then, 536.70 mg/L l -serine was achieved by removing feedback inhibition with a SerA mutant, and an elevated titer of 687.67 mg/L was further obtained through increasing s erB copies while enhancing the precursors. Finally, 855.66 mg/L l -serine can be accumulated with the supplementation of the glutamate precursor. This work thus not only constructed an l -serine producer to help understand the bottlenecks limiting l -serine production in Z. mobilis for further improvement, but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways such as amino acids or terpenoids.",
"conclusion": "4 Conclusion In this work, the l -serine tolerance of Z. mobilis was investigated by constructing various recombinant strains for l -serine production. Z. mobilis can generally tolerate up to 40 g/L of l -serine. Blockage of l -serine degradation pathways and the introduction of exporter EceamA were effective in improving l -serine production. Additionally, metabolic engineering strategies to enhance the biosynthesis pathway, remove feedback inhibition, and increase serB copies and l -serine precursors were combined to promote l -serine production in Z. mobilis . Lastly, glutamate supplementation was found to further elevate l -serine production of S02A9B5 to 855.66 mg/L; this indicated a 55.6-fold increase compared to the parental strain. This work thus not only lays a solid foundation for constructing an l -serine producer of Z. mobilis in the future, but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways.",
"introduction": "1 Introduction Serine has broad applications in food, cosmetic, as a nutritional additive, and in pharmaceutical industries with fast-growing market demands [ 1 , 2 ]. It plays a crucial role in various biological processes, including one-carbon unit (C-1) metabolism, protein synthesis, purine and pyrimidine synthesis, as well as cellular membrane production and processing [ 3 ]. Direct fermentative production of l -serine, rather than extraction from protein hydrolysates, chemical synthesis, and enzyme or expensive precursor glycine has indeed garnered significant attentions in recent years [ 2 , [4] , [5] , [6] , [7] ]. It offers a promising alternative to traditional methods as a more cost-effective, sustainable, and scalable approach for meeting the increasing demand for this valuable amino acid in various industries. However, l -serine production with high titers still faces several challenges even in well-studied model species such as Escherichia coli and Corynebacterium glutamicum , since l -serine plays critical roles in many biochemical reactions as an important intermediate in the central metabolic pathway [ 2 ]. Furthermore, the serine cycle with its unique characteristic of naturally evolved oxygen-insensitive pathway can synthesize acetyl-CoA (the C2 building block) from multiple groups of C1 compound without carbon loss [ [8] , [9] , [10] ]. Therefore, the production and application of l -serine still face several challenges, especially in non-model microorganisms that are not naturally amino acid producers, due to its complicated regulatory network and significance in numerous cellular reactions. There are two major l -serine degradation pathways in both E. coli and C. glutamicum. Serine hydroxymethyl transferase (SHMT, encoded by glyA ) catalyzes the conversion of serine to glycine while transferring one carbon unit to tetrahydrofolate (THF), which is an important cofactor required for C1-metabolism. After blocking its degradation pathway by deleting sdaA , sdaB, tdcG and glyA , and overexpressing l -serine biosynthesis pathway genes along with the cysteine/homoserine transporter EamA, a recombinant strain of E. coli MG1655 produced 11.7 g/L l -serine [ 11 ], and l -serine production was further promoted to 37 g/L after 52 h of fermentation with the application of metabolic engineering and adaptive laboratory evolution strategies [ 12 ]. Then, through translation initiation optimization, an industrialized E. coli ALE-5 (DE3) reached 50 g/L serine in fed batch fermentations, which was the highest titer reported thus far [ 13 ]. The gene of glyA for glycine biosynthesis can be deleted in E. coli with two alternative threonine degradation pathways, while it was essential in C. glutamicum [ 2 , 7 ]. Although wild-type C. glutamicum ATCC13032 cannot accumulate l -serine, C. glutamicum SYPS-062 screened naturally can generate up to 6.65 g/L l -serine from sugar [ 14 ]. Subsequently, various genetic manipulations and fermentation strategies were applied alone or together to improve l -serine production in C. glutamicum [ [15] , [16] , [17] , [18] ] . The highest l -serine titer of 43.9 g/L with a yield of 0.44 g/g sucrose was achieved in C. glutamicum A36 by overexpressing serE gene encoding a novel exporter and l -serine synthetic pathway key genes of serA Δ197 , serC , and serB [ 19 ]. Despite of significant progress achieved on l -serine production in model microorganisms of E. coli and C. glutamicum , there are still many challenges that must be addressed in other microbial cell factories, such as aeration requirements, dissolved oxygen level, fermentation efficiency and cost of production. To date, the fermentation processes for the l -serine production in model microorganisms require continuous aeration to maintain the metabolic activity, which are energy-intensive and will increase operational costs. Zymomonas mobilis is a non-model generally regarded as safe (GRAS) strain, which is the only known microorganisms possessing an anaerobic Enter-Doudoroff (ED) pathway with many excellent characteristics, such as high sugar utilization efficiency at broad pH ranges (3.5–7.5) [ 20 ]. With the rapid technology advancement in systems and synthetic biology, native and exogenous CRISPR-Cas genome editing toolkits [ 21 , 22 ] as well as systems for biological parts identification and characterization [ 23 ] have been established in Z. mobilis . Although various recombinant strains have been constructed to produce platform biochemicals such as cellulosic ethanol, lactate, acetoin, isobutanol, 2,3-butanediol (2,3-BDO ) , and poly-3-hydroxybutyrate (PHB ) [ 21 , [24] , [25] , [26] , [27] , [28] ], there are limited reports on engineering Z. mobilis for the production of biochemicals involved in sophisticated regulations such as amino acids. Z. mobilis possesses the unique anaerobic ED pathway, which can only gain one net ATP molecule per consumed glucose. In addition, the formation of l -serine by consuming 3-phosphoglycerate leads to decreased ATP formation, thereby limiting the maximal l -serine productivity. In this study, Z. mobilis was utilized in this study to explore the bottleneck for producing high titer of l -serine in a non-model microorganism that is not naturally suitable for amino acid production due to its low ATP generation, which can help explore the pathway compatibilities among different microbial hosts for future rational design of synthetic microbial cell factories. l -serine tolerance of Z. mobilis was evaluated. Subsequently, various metabolic engineering and synthetic biology strategies were applied for enhanced l -serine production to address the aforementioned challenges ( Fig. 1 ). These efforts aim not only to help generate recombinant strains for anaerobic l -serine production, but also to establish a foundation for further engineering of Z. mobilis for higher l -serine production and other biochemicals with sophisticated metabolic and regulatory pathways. Fig. 1 Schematic diagram of l -serine biosynthesis from glucose in Z. mobilis , and metabolic engineering strategies for efficient production of l -serine employed in this study. Relevant reactions are represented by the proteins and genes. Red forks mean gene deletion, and red triangle denote repression of genes with CRISPRi. The red star represents enzyme modification to remove feedback inhibition. G3P : Glycerate-1,3P; KDPG : 2-Keto-3-deoxy-6-phosphogluconate; EceamA : l -serine transporter; GpmA : glycerate 3-phosphate mutase; GlyA : serine hydroxymethyl transferase; PGA : 3-P- d -Glycerate; 2-PGA : 2-phosphoglycerate; PEP : Phosphoenolpyruvate; P-Serine : 3-Phosphoserine; Pdc : pyruvate decarboxylase; Pgk : phosphoglycerate kinase; SdaA : l -serine deaminase; SerA1 : 3-phosphoglycerate dehydrogenase; SerB : phosphoserine phosphatase; SerC : phosphoserine aminotransferase. Fig. 1"
} | 2,317 |
38286984 | PMC10825196 | pmc | 2,576 | {
"abstract": "Efforts to produce aromatic monomers through catalytic lignin depolymerization have historically focused on aryl–ether bond cleavage. A large fraction of aromatic monomers in lignin, however, are linked by various carbon–carbon (C–C) bonds that are more challenging to cleave and limit the yields of aromatic monomers from lignin depolymerization. Here, we report a catalytic autoxidation method to cleave C–C bonds in lignin-derived dimers and oligomers from pine and poplar. The method uses manganese and zirconium salts as catalysts in acetic acid and produces aromatic carboxylic acids as primary products. The mixtures of the oxygenated monomers are efficiently converted to cis,cis -muconic acid in an engineered strain of Pseudomonas putida KT2440 that conducts aromatic O -demethylation reactions at the 4-position. This work demonstrates that autoxidation of lignin with Mn and Zr offers a catalytic strategy to increase the yield of valuable aromatic monomers from lignin.",
"introduction": "Introduction Lignin is the most abundant natural source of aromatic units, comprising 15–30 wt% of lignocellulosic biomass 1 , 2 , and many contemporary efforts are pursuing lignin valorization via depolymerization to aromatic monomers 1 , 3 – 7 . Multiple depolymerization strategies have been developed that cleave aryl–ether C–O bonds very effectively 3 – 6 , 8 ; however, the various C–C linkages in lignin, formed during its biosynthesis 9 or from condensation reactions during lignocellulose processing 6 , 10 , are much more difficult to cleave 11 . To date, very few catalytic methods have been reported to cleave C–C inter-unit linkages in lignin. A notable example uses a Ru/NbOPO 4 catalyst that cleaves both C–O and C–C bonds and yields deoxygenated lignin oils with up to 32 wt% monomers. This system generates monocyclic products in significantly higher yields (up to 153 mol%) than those accessible from nitrobenzene oxidation, a method that primarily cleaves C–O linkages 12 , 13 . Wang et al. reported that up to 13 wt% of phenol can be obtained through tandem C–O and C(aryl)–C bond cleavage using a multifunctional catalyst comprising CuCl 2 /AlCl 3 and Ru/CeO 2 14 . Similarly, Han and colleagues reported an 8 wt% yield of phenol with a zeolite catalyst 15 . Stahl and colleagues reported oxidative catalytic fractionation of lignin affording C–C cleavage products 16 , and Waldvogel and colleagues showed vanillin could be obtained by oxidation of kraft lignin with peroxodicarbonate 17 . Other methods may generate products of C–C cleavage, but mechanistic information is lacking, the monomers are formed yields below values that can be obtained through C–O cleavage alone, or the processes require multiple steps and proceed in lower yields 18 – 21 . Lignin oil from reductive catalytic fractionation (RCF) is an ideal substrate to explore C–C bond cleavage in lignin due to the near-theoretical C–O bond cleavage that occurs during this process, resulting in an oil wherein the dimers and oligomers are only linked by C–C bonds (Fig. 1 ) 10 , 22 – 24 . Recently, Samec and coworkers demonstrated that a TEMPO + -derived oxidant [TEMPO= (2,2,6,6-tetramethylpiperidin-yl)−1-oxyl] is able to produce 1.9 mmol of 2,6-dimethoxybenzoquinone/g of oligomers derived from RCF of birch wood 25 . The reaction uses 400 wt% of TEMPO + oxidant with respect to the lignin-derived substrate, although the oxidant could be regenerated electrochemically and re-used to afford similar product yields in subsequent cycles. Fig. 1 Representative lignin C–C bonds. β-1, β-5, β-β, and 5-5 carbon–carbon bond linkages found in oligomers of RCF oil. Despite these promising reports, there remains a need for catalytic systems that can cleave C–C bonds in lignin to utilize lignin more effectively and offer product flexibility for lignin valorization to useful products. One option is autoxidation, a radical-chain process that is widely used in the commodity chemical industry (e.g., in the production of phenol from cumene 26 , cyclohexanol/cyclohexanone 27 , terephthalic acid and other aromatic carboxylic acids 28 – 32 , and alkyd paints 33 , 34 ). Co, Mn, and other metal ions are often used as catalysts in these processes, leveraging their ability to break down hydroperoxides into reactive alkoxyl radicals via the Haber-Weiss reaction 28 , 31 , 35 . The resulting alkoxyl radicals can undergo C–C bond cleavage via β-scission. Seminal reports from Partenheimer demonstrated production of aromatic carboxylic acids from 3,4-dimethoxytoluene, as a simple lignin model, and from various lignin sources using Co/Mn/Br and Co/Mn/Zr/Br catalyst systems, respectively 36 , 37 . In two recent studies, we demonstrated C–C bond cleavage in mixed plastics and an acetyl-protected poplar RCF oligomer substrate using a Co/Mn autoxidation catalyst system paired with a N- hydroxylphthalimide or bromide co-catalyst, respectively 38 – 40 . In an attempt to develop catalyst systems that use more abundant and sustainable metals, here we present an autoxidation catalyst for C–C bond cleavage that avoids the need for cobalt 41 . Further, we sought to eliminate bromide as it is corrosive and necessitates the use of expensive titanium-clad reactors due to its corrosivity 29 . Studies comparing oxidations of an acetyl-protected lignin model demonstrated Mn is not active enough for appreciable C–C bond cleavage. However, we observed C–C bond cleavage with Mn when studying the methyl-protected analog, 4-propylveratrole, in the presence of a Zr co-catalyst. Thus, in this work we use a homogeneous Mn and Zr aut-oxidation catalyst system to cleave the C–C bonds in oligomers of methylated pine (primarily G-type lignin) and poplar (G- and S-type lignin) RCF oils, along with the model compounds 1 – 6 shown in Fig. 2 . We demonstrate that this Mn/Zr catalyst system produces a mixture of bio-available monomers that can be converted to cis,cis -muconic acid using biological funneling 38 , 42 – 45 , a strategy that utilizes genetically engineered microbes to convert mixtures of compounds into a single target product, with a strain engineered to demethylate the methyl-stabilized phenol 42 , 46 . An overview of the workup procedure from biomass to cis,cis -muconic acid is shown in Supplementary Fig. 1 . Fig. 2 Lignin models. Structures of model monomers and dimers 1 – 6 were used in this study.",
"discussion": "Discussion These results demonstrate the catalytic production of bio-available monomers through C–C bond cleavage, as shown unambiguously using oligomers prepared from pine and poplar RCF oils, and their biological conversion to a single product, cis,cis -muconic acid. This Mn/Zr autoxidation catalyst system allows cleavage of the aryl propane units in 3 of the 4 types of carbon–carbon linkages present in lignin, shown in Fig. 1 (β-1, β−5, β-β). While oxidations of lignin models and lignin have been studied with Co/Mn/Br and Co/Br catalysts 36 , 37 , 55 , the target of those studies was not C–C bond cleavage. Rather, the goal was to increase monomer yields from the oxidation of β-O-4-containing substrates by acetylation of the generated phenolic products in situ prior to their degradation under the oxidation conditions. Conversely, our study presents a method to overcome the inherent limit of monomer yields from C–O cleavage alone by catalytically cleaving C–C bonds in lignin oligomers. This is accomplished by effecting β-scission of the high-energy radical intermediates generated via autoxidation. We further describe methods that enable the identification and quantification of a mixture of oxygenated products along with a gene editing protocol that enables its biological conversion using engineered strains of Pseudomonas putida . While promising, the overall process performed in batch reactors is limited by the need for stabilization chemistry and limited product stability, as observed in our model compound oxidations in Figs. 3 – 5 . Although autoxidation catalysis for lignin warrants phenol stabilization, there are several options that could facilitate progressing this chemistry further. One approach to improve the viability of lignin methylation could be to use a flow system with dimethyl carbonate as a methylating reagent 56 – 59 . Further, the regeneration of dimethyl carbonate from carbon dioxide is an active area of study and would enable a circular methylation process 60 – 62 . Flow chemistry could also be used to overcome issues with aromatic decomposition. The products could be continuously removed from the reaction in a flow reactor, which could enable lignin oxidation without the need to separate out the initial monomers generated from RCF. Alternatively, if the RCF monomers with propyl chains are sufficiently valuable, other separation methods could be employed including advanced chromatographic techniques and membrane separations 63 – 66 that allow monomer separation and preparation of RCF dimers and oligomers. Mechanistic details of Co/Mn/Br/Zr-mediated autoxidations in acetic acid have been studied for decades, but to date, there is not a universally accepted model. This is likely because kinetic parameters vary substantially with the reaction conditions and substrate 67 – 70 . Dimeric and trimeric acetate complexes of Co(III) and Mn(III) have been proposed as intermediates 71 – 74 , and differences in the activity between various Co(III) species have been observed spectroscopically along with the decay of active Co(III) to more stable forms 28 , 74 – 77 . Less reactive forms have been proposed to exist as bridged hydroxo Co(III) dimers 75 – 77 , as well as hydroxo-substituted oxo-centered trimers 74 . But their interconversion with less hydroxylated structures at elevated temperatures has been described by varying water concentrations 71 . Regarding Zr, its role in the oxidation of RCF oligomers is not obvious based on product yields and distributions. In the industrial Co/Mn/Br mediated autoxidation of alkylbenzenes, Zr is used as a co-catalyst, especially for the oxidation of poly(alkyl)benzenes 28 . Several hypotheses based on kinetics data involving Co have been proposed to explain the increase in activity when Zr is added 28 , 30 , 47 , 68 , 78 – 81 . Chester et al. proposed from kinetics models that Zr forms heterobimetallic complexes with Co(III), thereby preventing decay to more stable Co(III) complexes 78 . Partenheimer speculated that Zr affects the selectivity of toluene oxidation by promoting hydroperoxide coordination and dehydration to benzaldehyde and preventing the precipitation of MnO 2 47 , 48 . While it is unclear whether these models can be extrapolated to our Mn/Zr catalyst and lignin substrate, we did observe an increase in selectivity toward reactions that produce the desired acid and aldehyde products as opposed to deleterious side reactions. It should be noted that Partenheimer also observed a selectivity difference in oxidations of hydroxymethylfurfural. With Zr, double the yield of 2,5-diformylfuran was observed 82 . The Br co-catalyst in Co/Mn/Br also differs significantly from our Mn/Zr catalyst. In the former system, Br initiates the radical-chain process by H-atom transfer. In the absence of Br, it has been proposed that Mn(III) generates radicals by single-electron transfer from the electron-rich lignin aromatics via the loss of a proton from a radical cation intermediate 83 . It has been suggested that Co(III) is generated from Co(II) in Co/O 2 /AcOH systems by adventitious peroxide 84 . Oxidation specifically allows the production of bio-available compounds that can be funnelled into a single product, in this case, cis,cis -muconic acid, a platform chemical used in the production of biopolymers including nylon, nylon derivatives, and polyethylene terephthalate (PET), as well as performance-advantaged composites 85 – 92 . Translation of the CYP199A4 system from R. palustris HaA2 provides a catabolic pathway for p -methoxylated aromatics in engineered P. putida , and the use of oxidized RCF oligomers offers an example of biological funnelling of a heterogenous, lignin-derived stream toward a single product. The oxidized RCF oligomer streams were provided to P. putida at dilute concentrations, but the strains rapidly utilized the major monomer constituents (veratrate and veratraldehyde), indicating that tolerance to and conversion of higher substrate loadings is within the performance capability of these strains, as we have demonstrated for similar feedstocks and engineered pathways 93 , 94 . Although our system does not produce appreciable monomers from 5-5 dimers, a metabolic pathway for cleavage of the 5-5 C–C bond has been reported 95 – 97 . The resulting monomers—equivalents of vanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate (CHPD), in the case of 5,5′-dehydrodivanillate cleavage—could then be directed toward central metabolism or engineered pathways for muconate production in P. putida 97 , 98 . Going forward, bioprocess development could be combined with metabolic engineering or evolution strategies to improve the performance of these biocatalysts, increasing the yield of muconate or similar products 93 , 94 , 99 – 102 from lignin. We have demonstrated C–C bond cleavage in lignin using a Mn and Zr catalyst system in acetic acid, as shown with oligomers derived from pine and poplar lignin RCF oils. The method uses molecular oxygen as the oxidant and phenol-stabilization chemistry and yields of 1.01(4) and 0.96(2) mmol of monomers/g of oligomer substrate were obtained for pine and poplar, respectively. The oxygenated and bio-available product stream produced from pine oligomer oxidation was converted in biological systems using P. putida KT2440 to cis,cis -muconate."
} | 3,444 |
36085516 | PMC9666503 | pmc | 2,578 | {
"abstract": "Drought disrupts soil microbial activity and many biogeochemical processes. Although plant-associated fungi can support plant performance and nutrient cycling during drought, their effects on nearby drought-exposed soil microbial communities are not well resolved. We used H 2 18 O quantitative stable isotope probing (qSIP) and 16S rRNA gene profiling to investigate bacterial community dynamics following water limitation in the hyphospheres of two distinct fungal lineages ( Rhizophagus irregularis and Serendipita bescii ) grown with the bioenergy model grass Panicum hallii . In uninoculated soil, a history of water limitation resulted in significantly lower bacterial growth potential and growth efficiency, as well as lower diversity in the actively growing bacterial community. In contrast, both fungal lineages had a protective effect on hyphosphere bacterial communities exposed to water limitation: bacterial growth potential, growth efficiency, and the diversity of the actively growing bacterial community were not suppressed by a history of water limitation in soils inoculated with either fungus. Despite their similar effects at the community level, the two fungal lineages did elicit different taxon-specific responses, and bacterial growth potential was greater in R. irregularis compared to S. bescii -inoculated soils. Several of the bacterial taxa that responded positively to fungal inocula belong to lineages that are considered drought susceptible. Overall, H 2 18 O qSIP highlighted treatment effects on bacterial community structure that were less pronounced using traditional 16S rRNA gene profiling. Together, these results indicate that fungal–bacterial synergies may support bacterial resilience to moisture limitation.",
"conclusion": "Conclusion As global precipitation patterns change, it is important to understand how drought influences ecological functions and microbial interactions, both during and after water limitation. Plant-associated fungi are known to support plant growth and nutrition in droughted soils, but their simultaneous effects on the soil bacterial communities that mediate nutrient cycling and other critical terrestrial processes remain poorly explored. With H 2 18 O qSIP, we show that plant-associated fungi have a protective effect on bacterial communities exposed to water limitation. Both the AM fungus R. irregularis and the Sebacinales fungus S. bescii facilitated greater growth potential, growth efficiency, and diversity of actively growing hyphosphere bacteria in drought-affected soil. While these divergent fungal lineages stimulated responses of differing magnitude, the broad patterns were similar, suggesting that the dominant underlying mechanisms may be conserved across a substantial portion of the bacterial community rather than limited to interactions with a small number of bacterial taxa. Remarkably, both R. irregularis and S. bescii had a protective effect on hyphosphere bacteria exposed to water limitation in a “live” soil, which may have included functionally redundant fungal lineages. This finding is relevant for practical evaluation of fungal inoculants, whose ability to persist and elicit a positive effect in natural settings is not well-established. Additionally, our work indicates that H 2 18 O qSIP is a useful approach in challenging systems such as the terrestrial hyphosphere, where microbial dynamics may be difficult to detect with traditional 16S rRNA gene profiling. Together, our findings demonstrate that context-dependent multipartite relationships support bacterial resilience to water limitation and may promote post-drought recovery.",
"introduction": "Introduction Drought alters plant productivity [ 1 ], soil microbial biomass [ 2 , 3 ] and community composition [ 4 , 5 ], greenhouse gas emissions [ 6 ], and many critical biogeochemical processes [ 7 ]. Plant–microbial mutualisms mitigate plant drought response and may aid in post-drought recovery through a variety of mechanisms [ 8 , 9 ]. In particular, mutualistic root-associated fungi—such as mycorrhizal fungi, which form a symbiosis with most terrestrial plant families [ 10 ]—can support plants during drought by facilitating water transport [ 11 ], soil aggregation [ 12 ], root growth [ 13 ], plant nutrient uptake [ 14 ], photosynthesis [ 15 , 16 ], and stomatal conductance [ 15 – 17 ]. While mycorrhizal fungi may also influence the microbial communities that mediate nutrient cycling and other processes in drought-affected soil, multipartite plant–fungal–bacterial feedbacks remain poorly quantified [ 9 ]. The soil hyphosphere—the region that surrounds fungal hyphae—is a hotspot for fungal–bacterial interactions that influence microbial community composition [ 18 – 22 ], nutrient cycling [ 20 – 27 ], and plant growth [ 27 , 28 ]. Interactions between soil bacteria and plant-associated hyphae are likely shaped by resource dynamics. Plants share up to 20% of photosynthates with their mycorrhizal symbionts [ 29 , 30 ], which can rapidly transport these resources to surrounding bacteria [ 24 , 31 ]. Because fungi may explore a volume of soil that is two orders of magnitude greater than the area explored by plant roots [ 32 ], they may exert a substantial effect on soil microbiome structure and function. Distinct bacterial communities form in proximity to different fungal lineages [ 18 , 19 , 31 ]. However, we have a limited understanding of how different plant-associated fungi may shape the soil microbiome’s response to environmental stress. Investigation of fungal–bacterial interactions in hyphosphere soil is methodologically challenging because the hyphosphere is small, dynamic, and may not exert a detectable effect on soil microbial community composition or activity when assessed at a “bulk” scale [ 18 ]. Furthermore, a substantial proportion of soil DNA may represent inactive organisms or extracellular “relic” DNA [ 33 , 34 ]. Stable isotope probing (SIP) coupled with 16S rRNA gene profiling can distinguish active organisms from those that are inactive by tracing isotope incorporation into newly synthesized microbial DNA [ 35 , 36 ]. With quantitative SIP (qSIP), we can estimate taxon-specific growth based on shifts in DNA buoyant density caused by heavy isotope incorporation [ 37 , 38 ]. This enables sensitive detection of actively growing taxa, even in samples where a substantial quantity of DNA belongs to dead or dormant organisms. In experimentally challenging environments like the hyphosphere, qSIP has the potential to provide novel insight into taxon-specific activity and microbial growth potential following experimental treatments. In this study, we investigated how two plant-associated fungal lineages—the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis and the Sebacinales fungus Serendipita bescii —mediate bacterial growth potential following water limitation in a marginal soil planted with Panicum hallii (Hall’s panicgrass), a model species closely related to the bioenergy crop switchgrass. Both AM and Sebacinales fungi associate with a wide range of plant species, including switchgrass [ 39 – 41 ], and support plant growth and nutrition during drought [ 42 – 44 ]. However, R. irregularis has a reduced enzymatic repertoire [ 45 , 46 ] and depends largely upon host-provided C and microbial transformation of nutrient sources into bioavailable forms [ 27 , 47 , 48 ]. In contrast, S. bescii and other Serendipita lineages are facultative symbionts with a broader enzymatic repertoire that enables direct resource acquisition from both living plants and detritus [ 49 – 51 ]. We hypothesized that both fungi would mitigate the effects of moisture limitation, but that bacterial community composition and growth potential would be distinct in the soils colonized by each fungus.",
"discussion": "Discussion We found that plant-associated fungi have a protective effect on bacterial communities exposed to water limitation, and that bacterial responses to different fungal lineages are distinct. Because plant biomass was similar across all conditions investigated, we attribute differences in bacterial community composition and growth potential to direct effects of moisture history and fungal inocula, rather than to indirect effects mediated by plants. We found that H 2 18 O qSIP highlighted treatment differences that were not apparent through traditional 16S rRNA gene profiling. This demonstrates the utility of DNA qSIP for investigation of the soil hyphosphere and other systems in which it is difficult to discern a microbial signal above a complex background community. Soil water limitation suppresses bacterial growth potential and growth efficiency We observed a significant decrease in bacterial growth potential following 3 months of moisture limitation, but little effect of moisture history on respiration potential. Together, these responses resulted in a substantial reduction in bacterial growth efficiency. This may reflect a trade-off between microbial stress tolerance and growth in drought-affected soil [ 76 ]. Reduced microbial growth efficiency can occur when biota prioritize essential metabolic activities over cellular growth and replication [ 7 , 56 , 76 – 78 ]. Slowed growth also helps bacteria persist in the presence of antibiotics [ 79 , 80 ], which can accumulate in dry soils as microorganisms compete for limited resources [ 81 ]. We measured lower growth potential across a broad range of bacterial lineages present in our water-limited soils. Interestingly, this included several monoderm taxa (often referred to as Gram-positive organisms) belonging to the phyla Actinobacteria , Firmicutes , and Chloroflexi . Monoderm taxa have thick cell walls and lack an outer membrane, which can protect them against oxidative damage under dry conditions [ 7 , 82 – 85 ]. Several studies report that monoderms maintain greater abundance and activity in dry soils compared to diderms (also known as Gram-negative organisms, such as most of the taxa belonging to the phyla Acidobacteria, Bacteroidetes, Proteobacteria , and Verrucomicrobia ) [ 7 , 84 , 85 ]. In addition to the protection conferred by the structure of their cellular envelope, monoderms may also be poised to outcompete other organisms in drought-affected soils through their capacity to produce antibiotics [ 81 , 84 , 86 ] or utilize complex C substrates that remain available following water limitation [ 2 , 81 ]. Our results suggest that even monoderm taxa that are considered relatively drought tolerant can be negatively affected by water limitation. Plant-associated fungi support bacterial resilience in drought-affected soil, but fungal–bacterial relationships are context dependent While water limitation had a broad suppressive effect in uninoculated soils, many of the bacteria in soils inoculated with either R. irregularis or S. bescii maintained similar growth potential following cultivation under either water-replete or water-limited conditions. This protective fungal effect extended throughout the bacterial community, and affected many taxa that are often considered drought susceptible. Relative abundances of Bacteroidetes, Planctomycetes, Verrucomicrobia , and many Proteobacteria and Acidobacteria have been shown to decrease following drought [ 4 , 5 , 87 ]. However, we found that many ASVs belonging to these phyla sustained similar growth potential in fungal-inoculated soils, regardless of moisture treatment. This suggests that R. irregularis and S. bescii modified edaphic conditions in some way that broadly supported bacterial resilience to water limitation. Plant-associated fungi can exude plant-derived C [ 24 , 29 – 32 ], promote biofilm formation [ 88 ], enhance soil aggregation through their interactions with other soil biota [ 14 , 89 ], and facilitate bacterial transport through soil [ 90 ]. Together, these fungal-mediated processes could help maintain soil connectivity, microbial activity, and nutrient cycling under water-limited conditions, thereby preventing bacterial dormancy and death despite a substantial decline in soil moisture. By supporting bacterial function in drought-affected soil, plant-associated fungi may counteract the destabilizing effect of moisture stress [ 91 ] and improve capacity for recovery once moisture is restored. In contrast to their synergistic effects in water-limited soils, R. irregularis and S. bescii appeared to suppress bacterial growth potential following water-replete conditions. This demonstrates that the relationship between plant-associated fungi and hyphosphere bacteria is context-dependent, and not entirely mutualistic. Plant-associated fungi are known to compete with other biota for N [ 92 ] and P [ 20 , 21 ], and can suppress microbial decomposers [ 23 , 93 ]. Similarly, bacteria can inhibit mycorrhizal proliferation [ 20 , 21 , 92 ]. Putative bacterial predators have also been found in greater abundance on extraradical mycorrhizal hyphae than in surrounding soil [ 18 ]. We did not investigate the potential mechanisms of suppressive interactions between bacteria and plant-associated fungi. However, our observation that bacterial growth potential depends on moisture history in fungal-inoculated soil indicates that there are trade-offs between fungal and bacterial growth. In this trade-off, fungi may limit bacterial growth under resource-replete conditions, but promote bacterial growth under resource-limited conditions. Similar context-dependency is well-documented in other mutualisms [ 94 ]. In our system, context-dependency may indicate a stabilizing ecological effect exerted by multipartite hyphosphere interactions. Since R. irregularis and S. bescii were not actively associating with their plant hosts in our H 2 18 O qSIP assay, we attribute these results to fungal effects that had occurred during the preceding 3-month greenhouse experiment. Although the qSIP assay might have caused a nutrient flush from the perturbed soil and biota, CO 2 efflux from the fungal-inoculated soils was not significantly greater than from the uninoculated soils. Therefore, we conclude that differences in bacterial growth potential, growth efficiency, and diversity of the actively growing community present in fungal-inoculated soil were related to preceding fungal effects on the soil environment rather than to bacterial decomposition of fungal necromass. Magnitude of bacterial response is fungal lineage-dependent While both fungal lineages supported bacterial resilience, R. irregularis elicited a stronger positive response than S. bescii —both with respect to the number of ASVs and the magnitude of individual responses. Distinct microbial consortia associate with different mycorrhizal lineages [ 18 , 19 , 31 , 89 ]. Although empirical evidence remains sparse, different mycorrhizal exudate profiles, growth habits, and other functional traits may shape the composition and activity of the surrounding microbial community [ 48 , 89 , 95 , 96 ], a phenomenon that has been documented more extensively for root-microbe interactions [ 97 – 99 ]. Lower bacterial growth potential and growth efficiency in S. bescii- compared to R. irregularis -inoculated soils may be due to S. bescii ’s wider enzymatic repertoire, which could accelerate decomposition (as indicated by higher CO 2 efflux) or heighten competitive interactions with other soil biota. Additionally, higher gene copy numbers of R. irregularis compared to S. bescii detected in hyphosphere soil suggest that R. irregularis colonization levels were more robust. Greater fungal proliferation could be correlated with greater resource distribution, enhanced soil structure, or other conditions that support bacterial growth. Together, these findings suggest that diverse fungal lineages promote bacterial resilience to water limitation, but that the individual taxa and magnitude of taxon-specific response to each fungus is distinct."
} | 4,005 |
30691012 | PMC6462935 | pmc | 2,579 | {
"abstract": "Heterocysts are specialized cells that differentiate in the filaments of heterocystous cyanobacteria. Their role is to maintain a microoxic environment for the nitrogenase enzyme during diazotrophic growth. The lack of photosynthetic water oxidation in the heterocyst puts special constraints on the energetics for nitrogen fixation, and the electron transport pathways of heterocyst thylakoids are slightly different from those in vegetative cells. During recent years, there has been a growing interest in utilizing heterocysts as cell factories for the production of fuels and other chemical commodities. Optimization of these production systems requires some consideration of the bioenergetics behind nitrogen fixation. In this overview, we emphasize the role of photosynthetic electron transport in providing ATP and reductants to the nitrogenase enzyme, and provide some examples where heterocysts have been used as production facilities.",
"conclusion": "5. Conclusions The bioenergetics of the thylakoid membranes in heterocysts is still far from being as well known or investigated as that of the “normal” photosynthesizing cyanobacterial cells. The growing interest for heterocysts as cell factories evokes questions about energy utilization by heterocystous cyanobacteria, and about how it can be made more efficient for biotechnological applications. The aim of this review was to cast light over different aspects of heterocyst bioenergetics and to provide some starting points for further engineering endeavors.",
"introduction": "1. Introduction Oxygenic photosynthesis revolutionized life on earth by providing an endless source of energy and electrons for carbon dioxide fixation and by changing the composition of the atmosphere that enabled the development of multicellular life forms. Thylakoid membranes, the membrane system of oxygenic photoautotrophs, convert solar energy to biochemical energy via an electrochemical potential that drives ATP synthesis. The enzyme composition of thylakoid membranes is for the most part very similar in cyanobacteria, algae, and higher plants. Algae and higher plants have separate cellular compartments for photosynthesis and respiratory energy conversion. In cyanobacteria, the thylakoid membrane, where oxygen is produced, and the cell membrane, where most of the respiratory enzymes are located, are discrete zones of the same continuous membrane system [ 1 ]. Some of the respiratory enzymes are equally distributed between the two membrane domains in cyanobacteria, making the bioenergetics of these organisms complex and fascinating. Several cyanobacterial strains are able to fix atmospheric nitrogen into ammonia. This is made by the enzyme nitrogenase, in an extremely ATP-demanding reaction: N 2 + 8 H + + 8 e − + 16 MgATP → 2 NH 3 + H 2 + 16 MgADP + 16 P i . (1) To protect the nitrogenase from being inactivated by oxygen, nitrogen fixation has to be kept separated from photosynthetic oxygen formation either spatially or temporally. Some filamentous strains keep the nitrogenase away from the oxygen rich surroundings by differentiating a fraction of the cells into so-called heterocysts, where nitrogen fixation can take place in a safely microoxic environment [ 2 , 3 ]. In the heterocysts, Photosystem II (PSII) is necessarily inactivated, so the electron source for nitrogen fixation is provided by vegetative cells in the filament. Carbohydrates originating from photosynthetic carbon fixation are transported from the vegetative cells into the heterocysts where they are metabolized by the enzymes in the oxidative pentose phosphate cycle, generating NADPH [ 4 , 5 , 6 ]. Exactly which pathway the reducing equivalents take from there before they are fed to the nitrogenase by ferredoxin is still a matter of debate [ 7 ]. This question has gained in importance during the past decade, as heterocysts have become attractive candidates for being used as host compartments for oxygen-sensitive biosynthetic production [ 8 , 9 ]. To engineer non-native energy-consuming processes so that they are energy-efficient and non-harmful to the host, it is helpful to understand the bioenergetics of heterocysts and how reductants are utilized. The aim of this review is to shed some light on energy flows in the heterocyst and to indicate new research directions."
} | 1,074 |
39824798 | PMC11742011 | pmc | 2,580 | {
"abstract": "Effective heat dissipation remains a grand challenge for energy-dense devices and systems. As heterogeneous integration becomes increasingly inevitable in electronics, thermal resistance at interfaces has emerged as a critical bottleneck for thermal management. However, existing thermal interface solutions are constrained by either high thermal resistance or poor reliability. We report a strategy to create printable, high-performance liquid-infused nanostructured composites, comprising a mechanically soft and thermally conductive double-sided Cu nanowire array scaffold infused with a customized thermal-bridge liquid that suppresses contact thermal resistance. The liquid infusion concept is versatile for a broad range of thermal interface applications. Remarkably, the liquid metal infused nanostructured composite exhibits an ultra-low thermal resistance <1 mm² K W -1 at interface, outperforming state-of-the-art thermal interface materials on chip-cooling. The high reliability of the nanostructured composites enables undegraded performance through extreme temperature cycling. We envision liquid-infused nanostructured composites as a universal thermal interface solution for cooling applications in data centers, GPU/CPU systems, solid-state lasers, and LEDs.",
"introduction": "Introduction Efficient heat dissipation is essential for saving energy and preventing malfunction in all kinds of energy-dense devices and systems 1 – 4 . Globally, data centers consume about 240–340 terawatt hours of electricity annually (1–1.3% of total electricity used on earth each year), which will continue to grow with the development of cloud computing and artificial intelligence 5 , 6 . Cooling demands account for as much as 33–40 % of data center energy usage and consume billions of tons of water per year 7 . Meanwhile, heat fluxes of high-power electronics, such as solid-state lasers, wide bandgap transistors, and phased array radars, have reached unprecedented ~ 1 kW cm −2 \n 8 , which, without effective thermal solutions, will lead to rapid device performance and lifetime degradation 9 . As heterogeneous integration becomes increasingly inevitable in future electrical devices and systems 10 , thermal resistance at interfaces has emerged as a critical bottleneck for efficient thermal management 11 , 12 . While thermal interface materials (TIMs), such as solders, greases, compounds, and pads, have been widely applied to alleviate thermal interface resistance 13 , they must exhibit both a low bulk thermal resistance and a low contact thermal resistance to ensure optimal thermal performance. In addition, TIM should possess mechanical softness to effectively absorb the thermal stress resulting from the mismatch in coefficient of thermal expansion (CTE) at the interface 14 , 15 . Meeting these stringent requirements poses formidable challenges in the development of a high-performance multifunctional TIM. Traditional solders and solderable nanostructured TIMs have high thermal conductivity and low contact resistance but rely on high-temperature soldering processes limited to specific material surfaces, which hinders their applications as universal joints 15 – 18 . Although polymer composite TIMs such as thermal greases and compounds have high compliance, they struggle with “pump-out” issues, which cause poor reliability, and low thermal performance due to the scattered heat conduction among dispersed conductive fillers 13 , 14 , 19 . Compared with liquid-form thermal greases/compounds, solid-state thermal pads are more programmable on the material alignment and thereby can potentially achieve a higher thermal conductivity 20 , 21 . However, the substantial thickness (typically hundreds-of-μm) caused by the fabrication limitations and direct (“dry”) thermal contact with substrates result in high bulk and contact thermal resistances, compromising their overall thermal performance. To date, there is still a lack of a universal high-performance TIM that can meet the crucial demands of thermal management. Here, we demonstrate a printable liquid-infused nanostructured composite (LINC) as a universal high-performance TIM. As shown in Fig. 1a , a LINC is comprised of a unique mechanically compliable and thermally conductive double-sided Cu nanowire (CuNW) array scaffold infused with a customized thermal-bridge liquid. Vertically aligned CuNWs with a typical diameter of 200 nm and height of ~ 25 µm are grown on both sides of a thin Cu foil (~ 10 µm thick) by scalable templated electrochemical deposition (Fig. 1b–d and Supplementary Fig. S1 ). Also, the CuNWs have high uniformity with a height difference <3 µm, as shown in Fig. 1b and Supplementary Fig. S2 . The double-sided CuNW arrays, alongside the center Cu foil, function as a conductive and compliant scaffold due to the high thermal conductivity of Cu and the high aspect ratio (>100) of CuNWs. The high compliance of CuNW arrays ensures that they conform to the morphology of an object surface like “nano-springs”, thereby serving as efficient heat flow channels across the interface. By transforming the nanowire-surface point-to-point “dry” contact into nanowire/liquid-surface composite contact, our novel liquid infusion method can effectively bridge the CuNW tips thermally to a target surface and therefore largely suppress the contact resistance of LINCs, considering that the thermal conductivity of liquids is usually more than one order of magnitude larger than that of air. When employing a high thermal conductivity infused liquid, such as liquid metal, it also contributes to the substantial reduction of the bulk thermal resistance of LINCs. Hence, in contrast with existing TIM studies that primarily focus on reducing the bulk resistance, LINCs simultaneously achieve a low bulk resistance and a low contact resistance, both of which are equally crucial aspects in practical applications. Moreover, the infused liquid is multifunctional and highly customizable for a broad range of applications. As shown in Fig. 1e, f , when using non-adhesive liquids (e.g., non-volatile solvents, liquid metals, etc.), LINCs allow for pre-packaging and provide high reworkability, facilitating convenient and repeated uses (Supplementary Movie S1 ). Unlike the high-temperature soldering process with restricted material compatibility, the utilization of a low-viscosity liquid adhesive (e.g., resin, super glue, etc.) in LINCs enables thermal bonding of two substrates at room-temperature (Supplementary Fig. S3a, b ), which not only maintains strong structural integrity at the interfaces akin to soldering but also expands its applicability to non-solderable materials such as polymers and ceramics. Fig. 1 Liquid-infused nanostructured composites (LINCs). a Schematic illustration of LINCs, where an ultra-thin, mechanically compliant, and thermally conductive double-sided copper nanowire (CuNW) array scaffold is infused with a customized thermal-bridge liquid. CuNW arrays comply with the target surface like “nano-springs” and serve as efficient heat flow channels across the interface. Infused liquid effectively bridges the CuNW tips thermally to the target surface and, therefore, largely suppresses the contact resistance of LINCs. The multifunctional thermal-bridge liquid allows for pre-packaging provides reworkability when using non-adhesives, and enables thermal bonding of two substrates at room temperature akin to soldering but with extended applicability to non-solderable materials when using adhesives, therefore is highly customizable for a broad range of applications as a universal high-performance thermal interface material (TIM). b Top-view atomic force microscopic image of the CuNW scaffold. c Cross-sectional scanning electron microscopic (SEM) image of the CuNW scaffold, scale bar, 20 μm. Pictures of ( d ) CuNW scaffold, ( e ) Glycerol-LINC, ( f ) Liquid-Metal-LINC, at the size of ~ 30 × 30 mm.",
"discussion": "Results and discussion Thermal and mechanical characterization of LINCs We implement the well-established frequency-domain thermoreflectance (FDTR) method 22 – 24 to measure the bulk thermal properties of the CuNW scaffold in LINCs (Supplementary Text S1 ). The FDTR method is a pump-probe optical technique in which the modulated pump beam excites the sample while the probe beam measures the changes in the temperature-dependent reflectance. The phase lag between the pump and probe lasers is measured as a function of the modulation frequency, where the thermal properties including in-plane and cross-plane thermal conductivities and thermal interface resistance of the sample can be extracted by fitting the measured data to a 2D heat conduction model for multilayer thin films (Supplementary Fig. S4 ) 22 , 24 . A typical data plot is presented in Fig. 2a with a photo of the tested sample included as inset. Figure 2b summarizes thermal conductivity measurements of the CuNW layer fitted from the phase-frequency plots obtained from 9 different locations on the sample. The average thermal conductivity of the CuNW layer is determined to be 70.4 ± 13.9 W m −1 K − 1 , corresponding to a thermal resistance of 0.36 ± 0.07 mm 2 K W − 1 (Supplementary Text S2 and Table S1 ). The measured thermal conductivity of the 10 μm Cu foil in the middle of LINCs is 376.6 ± 37.7 W m − 1 K − 1 with an effective bulk resistance of 0.027 ± 0.003 mm 2 K W − 1 . The thermal contact resistances at CuNW/Cu-base interfaces are determined to be 0.027 ± 0.005 mm 2 K W − 1 . The overall effective thermal conductivity of the CuNW scaffold is 76.0 ± 13.6 W m −1 K − 1 , corresponding to the thermal resistance of 0.79 ± 0.14 mm 2 K W − 1 . Therefore, CuNW layers give rise to the majority (91.1%) of the total thermal resistance of the CuNW scaffold. In addition to vertical heat conduction, the CuNW scaffold also has a large lateral thermal conductivity of 69.5 ± 16.1 W m −1 K − 1 because of the highly conductive Cu-base layer in the middle and the laterally cross-linked CuNWs on both sides (Supplementary Text S3 ). The Cu-base layer further performs as a lateral heat spreader to increase heat flux uniformity and mitigate the local hot spot issue. Therefore, LINCs can synergistically regulate heat flow along an interface in both vertical and lateral directions, offering an advantage over vertically aligned nanowires 16 – 18 , graphene 20 , 21 , 25 , CNTs 26 , 27 , polymer fibers 28 , and semiconductor pillars 29 , 30 that mainly regulate heat conduction in the vertical direction. Fig. 2 Thermal and mechanical characterization. a Typical frequency-domain thermal reflectance (FDTR) data plot and the best least-square fitting to the heat transfer model. Inset: picture of tested sample and 9 test spots marked in blue, scale bar, 5 mm. b Histograms summarizing thermal conductivity of the copper nanowire (CuNW) array layer extracted from the FDTR measurements at different test spots, error bar data listed in Supplementary Information Table S1 . c Schematic showing the principle of ASTM D5470 standard thermal measurements. d Pressure-dependent thermal resistance of liquid-infused nanostructured composites (LINCs) without and with different thermal-bridge liquids, tested under the ASTM D5470 standard, in which the Liquid-Metal-LINC shows an extremely low thermal resistance (<1 mm 2 K W −1 ) above 50 Psi. Two commercially available thermal pastes with reported thermal conductivities of 8.5 W m K − 1 and 14.2 W m K − 1 are measured in the same setup as references, error bar data accessible in Source Data. e Stiffness characterization of an isolated CuNW bundle using in situ cylindrical flat punch indentation. Inset: Scanning electron microscopic image of the nanoindentation process on an isolated nanowire bundle, scale bar, 5 μm. f Displacement vs Stiffness curve in the continuous stiffness measurement showing the stiffness results for single-sided and double-sided films each from three different indentation locations, where the double-side film shows about half the stiffness measured from the single-sided film, indicating the structural benefit of the double-sided design. Source data are provided as a Source Data file. Following the ASTM D5470 standard, the overall thermal resistance of LINCs, including both bulk and contact resistances, is characterized through the 1D thermal measurement, as depicted in Fig. 2c . A pressure-dependent thermal performance of LINCs with different infused liquids is obtained through progressively increasing the mechanical load from approximately 20 to 100 Psi. As illustrated in Fig. 2d , the overall thermal resistances of the bare CuNW scaffold, Glycerol-LINC, and Liquid-Metal-LINC consistently decrease with increased pressure. Under 100 Psi, the bare CuNW scaffold shows a large overall thermal resistance of 28.72 ± 1.14 mm² K W − 1 , which is notably suppressed to 10.53 ± 0.36 mm² K W − 1 with glycerol infusion, and remarkably, <1.0 mm² K W − 1 with liquid metal infusion. Two commercially available thermal pastes with reported thermal conductivity of 8.5 W m −1 K − 1 and 14.2 W m −1 K − 1 are measured in the same setup as references, showing thermal resistance of 3 ~ 6 mm 2 K W − 1 . Our Liquid-Metal-LINC achieves an ultra-low thermal resistance <1.0 mm² K W − 1 under the ASTM D5470 standard at the pressure of 50 Psi, surpassing the previously reported TIMs. We also measure the mechanical characteristics of CuNW arrays via in situ cylindrical flat punch (diameter ~ 10 μm) indentation monitored under SEM (Fig. 2e , Supplementary Text S4 , and Movie S2 ). Since the CuNW array in this work is partially cross-linked (Fig. 1c ), we use focus ion beam milling to obtain an isolated CuNW bundle with a diameter of ~ 10 µm so that the influence from the cross-linked CuNW structure on the indentation experiment can be eliminated. With in situ monitoring under SEM, the cylindrical flat punch is located right on top of the isolated CuNW bundle. As a result, the array of CuNWs with an aspect ratio of ~ 125 exhibits a low Young’s modulus of ~ 2.5 GPa, which is about two orders of magnitude lower than that of bulk Cu. Furthermore, the stiffness of the double-sided CuNW array film is found to be only half that of the monolayer CuNW array structure (Fig. 2f ), which can thereby better accommodate the object morphology. In situ power cycling and extreme temperature cycling of LINCs Both power and temperature cycling experiments are conducted to evaluate the long-term reliability of LINCs. We establish an in situ power cycling setup (Fig. 3a, b ) equipped with a ceramic heater and an active air-cooler. In our power cycling tests, the CTE mismatch between the metal heat sink and the ceramic heater is ~ 4 times. With a constant cooling power, the heater is periodically turned on and off to cycle the TIMs from room temperature to an equilibrium temperature of >100 o C (Fig. 3c ). A high heating power density of ~ 46 W/cm 2 is applied to magnify the temperature difference between the ambient and equilibrium (maximum) heater temperature, \\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}$$\\Delta T={T}_{{heater}}-{T}_{{ambient}}$$\\end{document} Δ T = T h e a t e r − T a m b i e n t , which directly indicates the thermal performance of the TIMs. The degradation of the TIM performance can be observed in real-time through monitoring \\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}$$\\Delta T$$\\end{document} Δ T . As shown in Fig. 3d for >2600 cycles, our Liquid-Metal-LINC shows no observable degradation during the entire test period. The high reliability of our Liquid-Metal-LINC may be attributed to its composition as a metal composite, which eliminates “dry-out” issues. In addition, the hydrophilicity and friction with CuNWs can effectively trap the infused liquid, preventing it from being pumped out. As shown in Supplementary Fig. S5 , pure liquid metal tends to form droplets and leak from the interface, while our Liquid-Metal-LINC retains the minor squeezed liquid metal and mitigates its movement. Fig. 3 In situ power cycling and temperature cycling tests of liquid-infused nanostructured composites (LINCs). a Real image, and ( b ) a schematic of the in situ power cycling test setup for testing the cycling stability of thermal interface materials (TIMs). c Typical cycling temperature profile of Liquid-Metal-LINC. d Measured temperature difference between the ambient and equilibrium heater temperature for >2600 power cycles. The Liquid-Metal-LINC shows high reliability with no obvious performance degradation. e Picture of the assembly mounted on a Linkam stage equipped with a liquid nitrogen pump to control the temperature precisely and rapidly. f 1000-cycle accelerated aging test under an extreme temperature range from − 55 °C to 125 °C using the Linkam stage. g Schematic showing the temperature history measurement before and after temperature cycles, where a thermal infrared microscope is used to monitor the top surface temperature of the carbon-steel substrate when the built-in 38 W ceramic heater is turned on. h Real-time carbon-steel substrate surface temperature data. The Liquid-Metal-LINC assembly shows stable thermal performance after 1000 cycles. Source data are provided as a Source Data file. We carry out a 1000-cycle temperature cycling test on our Liquid-Metal-LINC under an extreme temperature range from − 55 °C to 125 °C, which is an industrial standard test condition. As shown in Fig. 3e and Supplementary Fig. S6 , we first apply the Liquid-Metal-LINC between a carbon-steel substrate and a ceramic heater, which gives rise to a ~ 4-time CTE mismatch. The whole assembly is then integrated onto a Linkam stage equipped with a liquid nitrogen pump for precise and rapid temperature control, with the carbon-steel surface in thermal contact with the stage. For each cycle, it takes 2 min to rise from − 55 o C to 125 o C and 30 s for dwelling, then 2 min for ramping down to − 55 o C and maintaining the temperature for another 30 s (Fig. 3f ). To characterize the thermal performances of the Liquid-Metal-LINC before and after temperature cycling, we employ a thermal infrared microscope to monitor the temperature history of the carbon-steel surface when turning on the built-in ceramic heater to provide ~ 38 W heating power to the assembly, shown in Fig. 3g . Under the same heating condition, Fig. 3h shows the real-time temperature data of the hot spot before and after the temperature cycling test. The temperature rises of the Liquid-Metal-LINC sample before (0 cycle) and after 1000 cycles almost overlap with each other, which indicates consistent thermal transport behavior and, thus, high reliability. As a reference experiment, we also measure the temperature rise of a commercial high-performance graphite thermal pad based on the same assembly, which exhibits a more sluggish temperature rise due to the larger thermal resistance. Considering that the viscosity of the infused liquid may change with temperature and impact the LINC performance, we further conducted a high-power constant heating experiment on the Epoxy-Resin-LINC (Supplementary Figs. S7a, b ). We specifically choose an epoxy resin with low initial viscosity and negligible shrinkage after setting. A hydraulic cooler is used to maintain a low working temperature, thus enabling slow curing of the epoxy resin. Throughout the curing process, its viscosity changes from 300 cP to infinity (fully cured solid). In Supplementary Fig. S7c , there is no observable temperature change during the curing process, indicating a negligible impact of viscosity change on the thermal performance. Mechanical reliability of LINCs under repeated thermal stress is further studied via a repeated compression test on a Glycerol-LINC with results presented in Supplementary Figs. S8 , S9 , and Text S5. A rebound effect on thermal performance in the LINC sample is observed when pressure is released, clearly demonstrating the “nano-spring” effect. However, gradual degradation of this “nano-spring” behavior is observed with repeated compression, suggesting that some nanowires may undergo plastic deformation, permanently conforming to the surface roughness of the object under high pressure (e.g., 80 psi). Demonstration of CPU cooling and liquid thermal bridge We directly apply the LINC on a CPU (~ 29 × 29 mm 2 ) for heat dissipation tests (Fig. 4 ). A home-built open desktop equipped with a 65 W standard office-level CPU and an air cooler is assembled as the test platform with consistent heat flow and mounting pressure (Fig. 4a ). We evaluate the cooling performance of LINCs by monitoring the temperature of the CPU at full load (monitored to be consistently ~ 69 W) using its built-in temperature sensors under similar ambient temperature (<1 o C fluctuation) and cooling conditions. Here, LINCs are tested under the CPU platform with different infused liquids to elucidate the thermal transport mechanism and the function of thermal-bridge liquids. When using the CuNW scaffold solely as a TIM, the contacts between the nanowire tips and the target surface are limited to point-to-point “dry” thermal contacts (Fig. 4b ), which results in a high CPU temperature of ~ 70 o C due to the large thermal contact resistance (Fig. 4c ). When glycerol is applied as the thermal-bridge liquid on the same CuNW scaffold (Fig. 4c ), the CPU temperature is significantly decreased to ~ 60 o C. Since the thermal conductivity of glycerol (~ 0.3 W m −1 K − 1 ) is two orders of magnitude lower than the CuNW scaffold and barely contributes to the bulk thermal conductivity of the composite layer, the substantial improvement in thermal performance is mainly attributed to the infused liquid that thermally bridges the nanowire tips and the target surface with a low contact resistance (Fig. 4b ). When using adhesives such as epoxy resin and polydimethylsiloxane (PDMS) for permanent bonding, the resulting full-load CPU temperature is also ~ 60 o C (Supplementary Fig. S3c ). To verify the contribution of the thermal-bridge liquids, evaporative isopropanol alcohol (IPA) is applied with the same CuNW scaffold. We observe that the full-load CPU temperature first stabilizes at ~ 62 o C, for ~ 4 min but then rises to over 67 o C indicating the loss of bridging effects after the evaporation of IPA. In Fig. 4c , it is evident that a liquid with higher thermal conductivity, such as water (~ 0.6 W m −1 K − 1 ), enhances the overall performance of LINCs (~ 58 o C) as compared to a liquid with lower thermal conductivity, like glycerol or IPA, by further reducing the contact resistance. To maximize the thermal performance of LINCs, the LINC infused with a liquid metal further decreases the CPU temperature to ~ 56 o C in our test platform. We benchmark the Liquid-Metal-LINC with commercially available state-of-the-art TIMs in Fig. 4d . Compared with Reference TIM 2 (high-performance phase-change material, reported thermal conductivity: ~ 8.5 W m −1 K − 1 ) and Reference TIM 3 (high-performance thermal grease, reported thermal conductivity: ~ 14.2 W m −1 K − 1 ), our Liquid-Metal-LINC demonstrates superior thermal performance by further reducing the CPU temperature for >2 o C, which corresponds to a chip-to-coolant efficiency improvement for >5%. With the non-volatile solvents (e.g., glycerol, oil), LINCs can be pre-packaged for easy applications and achieve high reworkability. As shown in Fig. 4e and Supplementary Movie S1 , the CPU test platform using the Glycerol-LINC is disassembled and assembled more than 20 times to simulate the reconfiguring process. Consistently, steady-state CPU temperatures of ~ 60 o C are observed before and after the process, indicating the stable performance of LINCs with robust reworkability. In Fig. 4f , we estimate the contribution of the TIM performance evolution using a thermal model (Supplementary Text S6 ). For modern high-end chips with high thermal design power density (e.g., NVIDIA AD102, ~ 1.3 W mm − 2 ), the thermal interface resistance reduction from 4 mm 2 K W − 1 to 1 mm 2 K W − 1 can contribute to up to 4 K reduction on the interfacial temperature gap and benefit the chip cooling, especially for data centers where the chip-to-coolant temperature difference is typically smaller than 30 K. Fig. 4 Demonstration of CPU cooling tests. a Home-built open desktop test station equipped with a 65 W standard office level CPU and an air cooler, right-side pictures showing the thermal grease and LINCs applied on the tested CPU. b Schematics showing the mechanism of the thermal bridging effect from the infused liquid, where the nanowire-surface point-to-point “dry” contact is transformed into the nanowire/liquid-surface composite contact with largely suppressed contact resistance. c Temperature data of the tested CPU at full load when using liquid-infused nanostructured composites (LINCs) without and with different thermal-bridge liquids. Thermal conductivity ( k ) of air, IPA, glycerol, water, and liquid metal is around 0.024, 0.140, 0.287, 0.600, and 73 W m K − 1 , respectively. The improved heat dissipation with increased k and the observed two-stage temperature behavior of the IPA bridged interface clearly verify the thermal transport mechanism by thermal-bridge liquids. d Temperature data of the tested CPU at full load when benchmarking state-of-the-art thermal interface materials (TIMs) with the Liquid-Metal-LINC. Reference TIMs 1, 2, and 3 are widely used thermal greases and phase change material pads with claimed thermal conductivities of 3.8, 8.5, and 14.2 W m K − 1 , respectively. e Temperature data of the tested CPU at full load using the Glycerol-LINC before and after 20-time assembling and disassembling. The stable thermal performance indicates the robust reworkability of LINCs. f Thermal model simulating the interfacial temperature gap reduction at different power densities, where a ~ 4 K temperature reduction would occur when the TIM thermal resistance is decreased from 4 mm 2 K W − 1 to 1 mm 2 K W − 1 . Source data are provided as a Source Data file. In summary, we report a printable LINC as a versatile and high-performance TIM. In the LINC scheme, the high thermal conductivity (>76 W m −1 K − 1 ) of the CuNW scaffold achieves a low bulk thermal resistance, whereas the infusion of thermal-bridge liquids effectively mitigates the contact resistance and results in a significant reduction of overall resistance from 28.72 ± 1.14 mm² K W − 1 for the bare CuNW scaffold to an ultra-low value of <1.0 mm 2 K W − 1 for the Liquid-Metal-LINC under ASTM D5470 standard tests. With a compliant and hydrophilic CuNW array on both sides serving as a “nano-spring” and retaining the infused liquid, LINCs demonstrate stable performance in both >2600 power cycles and 1000 temperature cycles over wide temperature ranges and large CTE mismatch. In the CPU cooling test, our Liquid-Metal-LINC demonstrates overperformed thermal performance as compared to state-of-the-art TIMs. The highly customizable nature of thermal-bridge liquids allows LINCs to be either mechanically bonded like soldering or highly reworkable as a thermal pad. Overall, LINCs facilitate a substantial reduction in interfacial thermal resistance while maintaining robust reliability. LINCs thus show high potential for enhancing the thermal management of energy-dense devices and systems, contributing to future energy efficiency and sustainability."
} | 7,012 |
40001153 | PMC11863533 | pmc | 2,581 | {
"abstract": "Background A transgenic strain of Escherichia coli has been engineered to directly assimilate gaseous CO 2 into its biomass through hydrogen-powered anaerobic respiration. This was achieved by expressing key components of the reverse tricarboxylic acid (rTCA) cycle, including genes encoding α-ketoglutarate: ferredoxin oxidoreductase (KOR) and ATP-dependent citrate lyase (ACL) from Chlorobium tepidum . These enzymes were selected for their essential roles in enabling CO 2 fixation and integration into central metabolism. Results This study found that KOR alone can support cellular maintenance under chemolithotrophic conditions, while additional expression of ACL enhances CO 2 assimilation. Using isotopic 13 CO 2 tracing, it was demonstrated that KOR alone facilitates CO 2 assimilation into TCA metabolites. However, co-expression of ACL with KOR redirected carbon fluxes from TCA cycle toward essential metabolic pathways, particularly those involved in protein and nucleotide biosynthesis. Compared to KOR alone, ACL co-expression significantly increased isotopic enrichments in amino acids (e.g., methionine, threonine, glycine) and nucleotides (e.g., deoxythymidine, deoxycytidine). These results suggest that ACL supports the synthesis of nitrogen-containing metabolites when inorganic nitrogen is sufficient, while KOR alone sustains core metabolic functions under chemolithotrophic conditions. Conclusions This study demonstrates a novel strategy to engineer E. coli for CO 2 fixation using only one or two heterologous enzymes under chemolithotrophic conditions. These findings reveal the minimal genetic and nutritional requirements for CO 2 assimilation and provide insights into metabolic flux partitioning in engineered strains. This research paves the way for sustainable applications in carbon fixation and biotechnological innovation. Supplementary Information The online version contains supplementary material available at 10.1186/s13036-025-00489-w.",
"conclusion": "Conclusion This study is the first to successfully engineer a carbon-fixing E. coli strain capable of cellular maintenance under chemolithotrophic conditions with hydrogen-powered anaerobic respiration by adding only one enzyme. The expression of KOR facilitates CO 2 assimilation and carbon fluxes retention within the TCA cycle (Fig. 5 a). Moreover, the additional expression of ACL further directs carbon fluxes into essential cellular processes such as protein and nucleotide biosynthesis (Fig. 5 b). The metabolic pathways assembled in the transgenic E. coli strains are deemed essential and represent a minimal configuration for cell living, closely reflecting core metabolism functions. \n Fig. 5 An overview of the potential metabolic fate of 13 CO 2 during the TCA cycle, cellular protein biosynthesis, nucleotide biosynthesis, and the impact on fatty acid production in transgenic E. coli K12 strains expressed ( a ) KOR or ( b ) KOR with ACL compared to vector control strain, respectively. A two-tailed t-test was used for statistical analysis, **** indicates p < 0.005 (highly statistically significantly difference);\n*** indicates p < 0.01; ** indicates p < 0.05 (statistically significantly difference); * indicates p < 0.1 (borderline statistically significantly difference). Two heterologous enzymes, KOR and ACL, play distinct roles in this carbon fixing system. KOR primarily fixes CO 2 through the rTCA cycle, maintaining carbon flow within the TCA cycle. ACL further redirects and shifts the carbon flux of CO 2 out of TCA cycle towards cellular protein and nucleotide biosynthesis. Metabolites from carbohydrate and TCA cycle are denoted in black, amino acids in blue, fatty acids in orange, and labeled 13 CO 2 in red. Hetero-expressed enzymes, KOR and ACL, are represented in purple and yellow, respectively. (SAFA: saturated fatty acids; MUFA: mono-unsaturated fatty acids; TFA: total fatty acid; PPRP: phosphoribosyl pyrophosphate; dC: deoxycytidine; dT: deoxythymidine; dG: deoxyguanosine; dA: deoxyadenosine)",
"discussion": "Results and discussion The present study provides new insights into the role of KOR in supporting transgenic E. coli under chemolithotrophic mode through hydrogen-powered anaerobic respiration. KOR expression facilitated CO₂ assimilation and helped retain carbon fluxes within the TCA cycle. Furthermore, the additional expression of ACL preferentially redirected carbon fluxes from the TCA cycle and further channeled them into essential metabolic pathways, including those for protein and nucleotide biosynthesis. These pathways likely play critical roles in sustaining essential reactions under nutrient-limited conditions. The implications of these findings are discussed below. α-ketoglutarate: ferredoxin oxidoreductase (KOR) supported cellular maintenance of Escherichia coli under chemolithotrophic mode Transcriptome analysis under chemolithotrophic mode was conducted using CO 2 as the sole carbon source after two transgenic E. coli strains were constructed. The heterologous genes were well-expressed in two transgenic strains K ( korAB genes) and KA ( korAB and aclBA genes) (Table 1 b). In enzymatic activity assays, α-ketoglutarate oxidoreductase activity encoded by KOR was significantly higher in the K (49.6 ± 12.2 nmol/min/mg protein, p = 0.002) and KA (47.0 ± 16.6 nmol/min/mg protein, p = 0.008) strains compared to that of the vector control strain (strain VC, < 0.01), confirming the functionality of the heterologous enzymes in these transgenic strains (Additional file 1: Table S1). The activities of isocitrate dehydrogenase and isocitrate lyase showed no significant difference between strains VC and K due to their natural presence in E. coli [ 45 , 59 ]. However, four enzymes including α-Ketoglutarate oxidoreductase, isocitrate dehydrogenase, isocitrate lyase, and succinyl-CoA synthetase exhibited higher activity in strain KA compared to strain VC. Notably, succinyl-CoA synthetase activity was significantly higher in the strain KA (9072 ± 522) compared to strain K (5457 ± 674, p = 0.008). This increase in activity may be attributed to enhanced acetyl-CoA production driven by ACL expression. Acetyl-CoA, a substrate of malate synthase A (encoded by the aceB gene), promotes glyoxylate consumption, which is also a product of isocitrate lyase. As a result, isocitrate lyase activity was marginally higher in strain KA (17.3 ± 3.5, p = 0.073) than in strain VC, though not significantly different in strain K (23.2 ± 12.6, p = 0.280). The promotion of isocitrate lyase likely increased succinate levels, which serve as the substrate for succinyl-CoA synthetase. This cascade of metabolic changes may explain the significantly enhanced succinyl-CoA synthetase activity observed in strain KA. To evaluate the role of rTCA machinery under chemolithotrophic conditions, two transgenic E. coli strains (K and KA) and a strain with an empty vector (VC) were cultured in an organic-free medium (devoid of organic compounds) under conditions with and without CO 2 supply. The medium was supplemented with hydrogen as the electron donor and nitrate as the electron acceptor for anaerobic respiration. None of the transgenic strains could survive without CO 2 (Fig. 1 c, hollow symbols). However, KOR-expressing strains (K and KA) showed an increase in microbial counts under chemolithotrophic conditions with CO 2 supply, in contrast to the VC strain (Fig. 1 b, solid symbols). These findings indicate that KOR expression can support cellular maintenance under chemolithotrophic conditions through hydrogen-powered anaerobic respiration. In previous studies, we introduced four rTCA enzymes into E. coli JM109, which successfully assimilated CO 2 with glucose supplementation [ 40 , 60 ]. Yu et al. introduced five heterologous enzymes (fructose-1,6-bisphosphatase, phosphoribulokinase, fructose-bisphosphate aldolase, ribulose bisphosphate carboxylase, transketolase, and glyceraldehyde-3-phosphate dehydrogenase) from the CBB cycle that enabled CO 2 fixation in the presence of a glucose supply [ 61 ]. Gleizer et al. engineered an evolved E. coli strain with three additional enzymes including: ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), phosphoribulokinase through the RuBisCO system, and formate dehydrogenase, enabling autotrophic growth using formate as an energy source [ 62 ]. These approaches demonstrated that engineered carbon-fixing E. coli strains require multiple heterologous enzymes and supplementation with glucose or formate to accomplish the carbon fixation recycling system and maintain cell viability. This study is the first to successfully engineer a carbon-fixing E. coli capable of sustaining cellular maintenance under chemolithotrophic conditions by introducing a single enzyme (KOR) from rTCA cycle, with CO₂ as the sole carbon source and hydrogen as the energy source. By limiting the heterologous enzyme expression to KOR only, this work achieved carbon fixation, cellular maintenance, and the production of essential metabolites under chemolithotrophic conditions. Constraint-based flux balance analysis of the reverse tricarboxylic acid cycle machinery in Escherichia coli In-silico modeling was employed to explore potential pathways for CO 2 assimilation in E. coli via the rTCA machinery. The predicted pathways for CO 2 assimilation in the E. coli strains are illustrated in Additional file 1: Fig. S1. Additional file 1: Tables S2-S4 present the stoichiometric matrix results used in the flux balance analysis aimed at maximizing the biomass reaction rate. The model predicted that CO 2 assimilated by KOR predominantly follows the pathway from glyoxylate to tartronate semialdehyde to glycerate pathway, regulated by glyoxylate carboligase (encoded by the gcl gene). To validate the importance of this pathway for transgenic cell survival, this study assessed the cell growth of the gcl gene knock-out strains under organic-free conditions in the presence of CO 2 . None of the gcl gene knock-out strains could survive under these conditions (Additional file 1: Fig. S1b), highlighting the essential role of this pathway. However, further isotopic labeling experiments are needed to quantify the actual metabolic fluxes in transgenic strains. Expression of α-ketoglutarate: ferredoxin oxidoreductase (KOR) significantly enhanced the incorporation of CO 2 in tricarboxylic acid cycle metabolites Strains K and KA were cultured under organic-free conditions with 13 CO 2 supplementation. The potential and actual incorporations of 13 CO 2 into TCA cycle metabolites are depicted in Fig. 2 . Compared to strain KA, strain K exhibited significantly higher enrichments of 13 CO 2 in citrate ( p = 0.004), succinate ( p = 0.008), pyruvate ( p = 0.016), α-ketoglutarate ( p = 0.005), and malate ( p = 0.007). Increased TCA metabolite concentrations were also observed compared to strain VC, especially in fumarate ( p = 0.089), malate ( p = 0.013), and citrate ( p = 0.025). These results indicate that KOR expression alone effectively enhances the retention of carbon fluxes within the TCA cycle, whereas the addition of ACL might divert the carbon flow away from the TCA (Fig. 2 ; Table 2 ). Thus, KOR alone and not KOR + ACL is more effective in maintaining CO 2 fluxes within the TCA cycle compared to KOR + ACL. Further investigation was conducted to explore the role of ACL. \n Fig. 2 An overview of the potential metabolic fate of carbon during CO 2 carboxylation in TCA cycle. Isotopic labeling experiments from 13 CO 2 demonstrate the preferential TCA cycle metabolites synthesis and metabolites concentration in transgenic E. coli K12. Data are expressed as means standard ± deviation, n = 3-4/group. A two-tailed t-test was used for statistical analysis, **** indicates p < 0.005 (highly statistically significantly difference); *** indicates p \n< 0.01; ** indicates p < 0.05 (statistically significantly difference); * indicates p < 0.1 (borderline statistically significantly difference). Strains were supplemented with 10% 13 CO 2 and 90% H 2 , re-filled 10% of 13 CO 2 per 12 hr for 48 hr incubation. Isotopic enrichments derived from 13 CO 2 are detected in the M + 1 specie of various organic acid. The labeled 13 CO 2 are marked in red asterisk * marks. Hetero-expressed enzymes: KOR & ACL are marked in purple and yellow, respectively \n \n Table 2 Isotopic labeling experiments from 13 CO 2 demonstrate the preferential TCA cycle metabolites synthesis and metabolites concentration in transgenic E. Coli K12 Pyr 1 Pyr + 1 2 Lac Lac + 1 Strain VC 3 42.89 ± 12.27 0.048 ± 0.024 637.39 ± 121.82 0.0020 ± 0.0018 Strain K 4 36.89 ± 14.14 0.079 ± 0.001 499.13 ± 179.39 0.0014 ± 0.0017 P-value 6 0.584 0.074 0.306 0.696 Strain KA 5 30.15 ± 4.36 0.032 ± 0.023 404.89 ± 123.97 0.0019 ± 0.0018 P-value 6 0.165 0.417 0.081 0.981 KA vs. K 7 0.470 0.016 0.474 0.715 α-KG α-KG + 1 Suc Suc + 1 Strain VC 0.35 ± 0.07 0.010 ± 0.009 2.67 ± 0.75 0.0001 ± 0.0002 Strain K 0.27 ± 0.12 0.135 ± 0.023 2.83 ± 0.41 0.1387 ± 0.0487 P-value 0.346 < 0.001 0.719 0.002 Strain KA 0.24 ± 0.06 0.010 ± 0.009 1.70 ± 0.11 0.0020 ± 0.0034 P-value 0.097 0.604 0.075 0.303 KA vs. K 0.715 0.005 0.006 0.008 Fum Fum + 1 Mal Mal + 1 Strain VC 0.48 ± 0.21 0.008 ± 0.0001 1.16 ± 0.50 0.002 ± 0.002 Strain K 0.78 ± 0.11 0.022 ± 0.002 2.43 ± 0.39 0.023 ± 0.001 P-value 0.089 < 0.001 0.013 < 0.001 Strain KA 0.75 ± 0.08 0.019 ± 0.008 2.82 ± 0.46 0.006 ± 0.008 P-value 0.105 0.056 0.013 0.431 KA vs. K 0.703 0.499 0.281 0.007 Cit Cit + 1 Cit + 2 Strain VC 1.57 ± 0.77 0.0003 ± 0.0005 0.003 ± 0.002 Strain K 6.11 ± 2.33 0.1338 ± 0.0386 0.012 ± 0.006 P-value 0.025 0.004 0.052 Strain KA 5.49 ± 0.70 0.0006 ± 0.0011 0.014 ± 0.008 P-value 0.001 0.659 0.086 KA vs. K 0.631 0.004 0.790 Data are expressed as mean ± SD ( n = 3–4/group) and compared by Student’s t-test 1 The quantitation (pmol/log cfu) in Pyr (pyruvate), Lac (lactate), α-KG (α-ketoglutarate), Suc (succinate), Fum (fumarate), Mal (malate), Cit (citrate) produced by E. coli are shown in grey background 2 The enrichments in Pyr (pyruvate), α-KG (α-ketoglutarate), Suc (succinate), Fum (fumarate), Mal (malate), Cit (citrate) derived from 13 CO 2 are shown 3 Strain VC: E. coli K12 vector control 4 Strain K: E. coli K12 korAB (+) strain 5 Strain KA: E. coli K12 korAB (+) & aclBA (+) strain 6 Statistically significantly compared to the strain VC at p < 0.05; borderline statistical significance compared to strain VC at 0.05 < p ≦ 0.1 7 Statistically significantly compared to the strain K at p < 0.05; borderline statistical significance compared to strain K at 0.05 < p ≦ 0.1 The TCA cycle is a crucial metabolic pathway that integrates carbohydrate, fat, and protein metabolism [ 63 ]. Compared to strain VC, the transgenic strain K expressing KOR demonstrated the supporting cellular maintenance under chemolithotrophic conditions. This finding underscores the importance of KOR within the TCA cycle for CO 2 assimilation. The fact that a single enzyme (encoded by two genes) can drive the core reactions in the TCA cycle is noteworthy. α-ketoglutarate, the product of the KOR reaction, is one of the five universal metabolic precursors, as shown through chemical reactions [ 38 ], suggesting that the reactions encoded by the korAB genes closely resemble metabolic processes. The in - silico modeling of strain K proposed an alternative metabolic pathway where glyoxylate is converted to glycerate 2-phosphate. The glyoxylate shunt is an anabolic pathway within the TCA cycle in E. coli , which synthesizes components of proteins and the pyrimidines under limited carbon source incubation [ 64 , 65 ]. This pathway may support cell survival under nutrient-scarce conditions. Metabolic studies further confirmed that KOR expression enhanced the metabolic flux toward glyoxylate, accumulating key metabolites such as citrate, malate, and fumarate. With only one exogenous enzyme for CO 2 fixation, E. coli can activate the glyoxylate shunt and replenish critical TCA metabolites. The activation of the glyoxylate shunt by KOR expression may mimic the metabolic pathways of ancient microbes [ 38 ]. Additional ATP-dependent citrate lyase (ACL) expression rewired carbon flux of CO2 Although the in-silico model predicted that CO 2 flux rates in the TCA cycle would be higher in the strain expressing both KOR and ACL compared to the strain expressing KOR alone (Additional file 1: Table S4), the experimental results were different. In the experiments using a stable isotope tracer, strain K retained a significantly higher amount of labeled CO 2 in α-ketoglutarate + 1, succinate + 1, malate + 1, and citrate + 1, resulting in increased succinate production compared to strain KA (Fig. 2 ; Table 2 ). This discrepancy could be due to differences between the modeling and actual situations. The flux balance analysis and models used [ 66 ] did not account for cofactors (nicotinamide adenine dinucleotide (NAD + ), NADP + , ATP, and others), water, or H + , assuming these were sufficiently supplied and did not limit biomass synthesis. Additionally, the anaplerotic reactions from the TCA cycle were not included in the current model, and the actual experiments provided complementary insights to the in - silico model. Many biosynthetic reactions utilize the TCA cycle molecules as substrates; however, the model did not incorporate the metabolic pathways for amino acid and protein, fatty acid, or nucleotide biosynthesis. The experiments showed that while the transgenic E. coli expressing ACL accumulated fewer CO 2 fluxes within the TCA cycle compared to the KOR-only strain, wild-type E. coli did not express ACL, and this cannot cleave citrate into acetyl-CoA and oxaloacetate. The additional expression of ACL in strain K appeared to redirect CO 2 flux toward oxaloacetate and aspartate. Given that aspartate is a proteinogenic amino acid [ 67 ] vital for protein synthesis in E. coli [ 68 ], this study further examined the incorporation of CO 2 into cellular proteins. “Co-expression of ATP-Dependent Citrate Lyase (ACL) and α-Ketoglutarate: Ferredoxin Oxidoreductase (KOR) exhibited limited effect on fatty acid production ACL catalyzes the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate to oxaloacetate and acetyl-CoA, which are key precursors for the biosynthesis [ 69 ] of fatty acids, cholesterol, and acetylcholine [ 70 , 71 ]. The possible role of ACL expression in promoting fatty acid synthesis in the transgenic strains was investigated under anaerobic conditions. However, the experiments showed that the concentrations of fatty acid (Additional file 1: Fig. S2a) and SCFAs (Additional file 1: Fig. S2b) did not differ between strains VC and KA. Additionally, the concentration of butyrate (ng/log cfu) in strain KA (63.37 ± 9.42) was lower than strain VC (79.94 ± 3.02, p = 0.044), suggesting that ACL expression did not promote fatty acid synthesis under such a scarce environment. As for the short-chain fatty acids, strain K exhibited a trend of lower levels of formate, acetate, and propionate compared to strain VC and KA ( p < 0.1), while butyrate levels were significantly lower ( p < 0.05). This may indicate that the expression of KOR helps keep CO₂-derived fluxes within the TCA cycle at the expanse of reduced synthesis of SCFAs. A previous study showed that E. coli can resorb acetate for converting it to acetyl-CoA via acetyl-CoA synthetase (Acs) and utilized it to energy and biosynthetic components through the TCA cycle and the glyoxylate shunt [ 72 ]. This aligns with the observed TCA cycle metabolite profile in strain K. To sustain KOR activity, strain K appears to use SCFAs as substrates to replenish its carbon source, reflecting a metabolic adaptation to maintain carbon flux under chemolithotrophic conditions. Co-expression of KOR and ACL restored SCFA levels to values comparable to those of strain VC. Although additional ACL expression in the presence of KOR did modestly enhance SCFA, it did not enhance long chain fatty acid synthesis. Rather, additional ACL expression help re-direct CO 2 metabolic fluxes into nucleotide and protein synthesis. ATP-dependent citrate lyase (ACL) and α-ketoglutarate: ferredoxin oxidoreductase (KOR) expression assisted 13 CO 2 carbon fixation in protein biosynthesis ACL cleaves citrate into oxaloacetate and acetyl-CoA, both of which are directly linked to fatty acid turnover in E. coli [ 73 ]. The similar contents of fatty acid among strains VC, K, and KA suggested that the CO 2 assimilated by KOR was not used for fatty acid with the addition of ACL and that ACL may divert the flow of carbon into other critical metabolic pathways. Consequently, carbon fluxes in cellular protein biosynthesis were experimentally tracked (Fig. 3 ). As data shown in Fig. 3 ; Table 3 , strain KA exhibited enhanced amino acid enrichments of cellular protein hydrolysates compared to strain K, including threonine + 1, phenylalanine + 1, glycine + 1, isoleucine + 1, methionine + 1, serine + 1, glutamate + 1, cysteine + 1, and alanine + 1. These results suggested that the expression of heterologous KOR with additional ACL, facilitated an increase in CO 2 incorporation in protein biosynthesis. \n Fig. 3 Incorporation of CO 2 into cellular proteins. The intermediates of the TCA cycle, oxaloacetate and α-ketoglutarate, are metabolized to produce various amino acids, which are subsequently incorporated into cellular proteins. Isotopic labeling experiments using 13 CO 2 demonstrate the preferential synthesis of proteinogenic amino acids in transgenic E. coli K12. Data are presented as means ± standard deviation, with n = 3 independent biological samples. A two-tailed t-test was used for statistical analysis, **** indicates p < 0.005 (highly statistically significantly difference); *** indicates p < 0.01; ** indicates p < 0.05 (statistically significantly difference); * indicates p < 0.1 (borderline statistically significantly difference). Strains were supplemented with 10% 13 CO 2 and 90% H 2 , with 10% of 13 CO 2 replenished every 12 h during the 48 h incubation. Isotopic enrichments derived from 13 CO 2 are detected in the M + 1 specie of various amino acids. The labeled 13 CO 2 are marked in red asterisk * marks. Hetero-expressed enzymes KOR and ACL are indicated in purple and yellow, respectively \n \n Table 3 Isotopic labeling experiments from 13 CO 2 demonstrate the preferential proteinic amino acid synthesis in transgenic E. Coli K12 Asp + 1 1 Glu + 1 Lys + 1 Thr + 1 Strain VC 2 0.017 ± 0.001 0.000 ± 0.001 0.011 ± 0.001 0.022 ± 0.002 Strain K 3 0.037 ± 0.002 0.006 ± 0.001 0.019 ± 0.003 0.030 ± 0.000 P-value 5 < 0.001 0.001 0.006 0.001 Strain KA 4 0.052 ± 0.004 0.012 ± 0.002 0.019 ± 0.002 0.037 ± 0.003 P-value 5 < 0.001 0.001 0.002 0.003 KA vs. K 6 0.003 0.013 0.943 0.034 Ile + 1 Met + 1 Gly + 1 Ser + 1 Strain VC 0.0005 ± 0.0005 0.004 ± 0.001 0.020 ± 0.001 0.024 ± 0.004 Strain K 0.0038 ± 0.0004 0.011 ± 0.000 0.028 ± 0.001 0.027 ± 0.002 P-value 0.001 0.001 0.001 0.316 Strain KA 0.0044 ± 0.0.0019 0.015 ± 0.001 0.025 ± 0.002 0.030 ± 0.002 P-value 0.026 < 0.001 0.0440 0.048 KA vs. K 6 0.632 0.001 0.078 0.064 Cys + 1 Ala + 1 Tyr + 1 Phe + 1 Strain VC 0.009 ± 0.001 0.0061 ± 0.0008 0.010 ± 0.000 0.0018 ± 0.0004 Strain K 0.016 ± 0.001 0.0056 ± 0.0004 0.027 ± 0.002 0.0044 ± 0.0002 P-value 0.001 0.418 < 0.001 0.001 Strain KA 0.049 ± 0.012 0.0080 ± 0.0015 0.015 ± 0.003 0.0082 ± 0.0026 P-value 0.004 0.134 0.001 0.014 KA vs. K 6 0.008 0.063 0.003 0.067 Data are expressed as mean ± SD ( n = 3/group) and compared by Student’s t-test 1 The enrichments in Asp (aspartate), Glu (glutamate), Lys (lysine), Thr (threonine), Ile (isoleucine), Met (methionine), Gly (glycine), Ser (serine), Cys (cysteine), Ala (alanine), Tyr (tyrosine), Phe (phenylalanine) derived from 13 CO 2 are shown 2 Strain VC: E. coli K12 vector control 3 Strain K: E. coli K12 korAB (+) strain 4 Strain KA: E. coli K12 korAB (+) & aclBA (+) strain 5 Statistically significantly compared to the strain VC at p < 0.05; borderline statistical significance compared to strain VC at 0.05 < p ≦ 0.1 6 Statistically significantly compared to the strain K at p < 0.05; borderline statistical significance compared to strain K at 0.05 < p ≦ 0.1 Expressing ACL in strain K may have been expected to increase fatty acid synthesis due to the greater availability of precursors for fatty acid elongation. However, in this study, the addition of ACL in strain K did not enhance fatty acid production (Additional file 1: Fig. S2). This outcome may be influenced by the presence of inorganic nitrogen (NaNO₃) in the culture medium. Nitrogen limitation has been shown to promote fatty acid accumulation in various organisms [ 74 – 76 ]. It is plausible that when nitrogen is abundant, carbon fluxes are more likely to support the synthesis of nitrogen-containing compounds rather than lipid accumulation. These results suggest that carbon flow in strain KA may be redirected toward other metabolic pathways, such as the production of oxaloacetate, a precursor of aspartate. Aspartate is a key compound involved in the synthesis of various metabolites, including amino acids and nucleotides [ 77 , 78 ]. This redirection aligns with the observed role of ACL in promoting protein biosynthesis over fatty acid production under chemolithotrophic conditions in the presence of NaNO₃. Consequently, the effects of KOR and ACL expression on CO₂ incorporation into nucleotides were further investigated. ATP-dependent citrate lyase (ACL) and α-ketoglutarate: ferredoxin oxidoreductase (KOR) expression assisted 13 CO 2 carbon fixation in nucleotides biosynthesis Nucleotides, essential for RNA and DNA synthesis and as the primary energy donors for cellular processes [ 79 ], were investigated for their biosynthesis in the presence of ACL and KOR. L-aspartate, a substrate of pyrimidine biosynthesis catalyzed by the allosteric enzyme, aspartate transcarbamoylase, is regulated both homotrophically by L-aspartate and heterotrophically by nucleotide effectors such as ATP, CTP (cytidine triphosphate) [ 80 ], and UTP (uridine triphosphate) in the presence of CTP [ 81 – 83 ]. It was speculated that ACL also promote nucleotides biosynthesis by increasing the supply of aspartate, a substrate for nucleotides biosynthesis. Although E. coli naturally incorporates CO 2 slightly in nucleotide biosynthesis (Fig. 4 ) [ 78 , 84 ], it cannot use CO 2 as the sole carbon source for cell growth. When the selected carbon-fixing genes were introduced to the rTCA cycle, the transgenic E. coli strains could incorporate more CO 2 into nucleotide structures under nutrient-scarce environments. \n Fig. 4 Incorporation of CO 2 into nucleotides. Effect of hetero-expressed enzymes promote CO 2 incorporation into nucleotides in E. coli K12. Data are expressed as means standard ± deviation, n = 3 independent biological samples. A two-tailed t-test was used for statistical analysis, **** indicates p \n< 0.005 (highly statistically significantly difference); *** indicates p \n< 0.01; ** indicates p < 0.05 (statistically significantly difference); * indicates p < 0.1 (borderline statistically significantly difference). Strains were supplemented with 10% 13 CO 2 and 90% H 2 , re-filled 10% of 13 CO 2 per 12 hr for 48 hr incubation. Isotopic enrichments derived from 13 CO 2 are detected in the M + 1 or M + 2 specie of various nucleotides. The labeled 13 CO 2 are marked in red asterisk * marks. Hetero-expressed enzymes: KOR & ACL are marked in purple and yellow, respectively. In deoxythymidine and inosine-5'-monophosphate biosynthesis pathway, 13 CO 2 was not incorporated into nucleotide through reverse TCA cycle which are marked in pink asterisk * marks. (Asp: aspartate; Gly: glycine; Gln: glutamine; For: formate; PPRP: phosphoribosyl pyrophosphate; UTP: Uridine triphosphate; IMP: inosine-5'-monophosphate; dC: deoxycytidine; dT: deoxythymidine; dG: deoxyguanosine; dA: deoxyadenosine) \n A comparison of nucleotide biosynthesis between the two transgenic strains revealed differences. Compared to strains VC and K, the isotopic enrichments in deoxythymidine + 1 (dT + 1) increased by 29% and 48%, respectively, in strain KA. Similarly, the isotopic enrichments in deoxycytidine + 1 (dC + 1) increased by 26% and 38%, respectively, in strain KA compared to strains VC and K. Additionally, in the purine biosynthesis pathway, the isotopic enrichments in deoxyadenosine + 1 (dA + 1) increased by 134% and 29%, respectively, in strain KA compared to strains VC and K, while enrichment in dA + 2 was increased by 401% in strain KA compared to strain VC. The enrichments of deoxyguanosine + 1 (dG + 1) increased by 115% and 26% in strain KA compared to strains VC and K, respectively, with dG + 2 being significantly enriched in strain KA compared to that in strain VC (Fig. 4 ; Table 4 ). These results suggested that the co-expressions of KOR, and KOR with ACL promoted the incorporation of CO 2 for the biosynthesis of pyrimidines and purine in E. coli , with the highest CO 2 incorporation was detected in strain KA. \n Table 4 Effect of hetero-expressed enzymes promote CO 2 incorporation into nucleotides in E. Coli K12 dC + 1 1 dC + 2 dT + 1 dT + 2 Strain VC 2 0.035 ± 0.003 0.041 ± 0.019 0.042 ± 0.002 0.019 ± 0.001 Strain K 3 0.032 ± 0.003 0.035 ± 0.009 0.036 ± 0.001 0.016 ± 0.003 P-value 5 0.231 0.647 0.027 0.142 Strain KA 4 0.044 ± 0.004 0.045 ± 0.007 0.054 ± 0.005 0.018 ± 0.002 P-value 5 0.024 0.783 0.016 0.386 KA vs. K 6 0.009 0.226 0.004 0.430 dG + 1 dG + 2 dA + 1 dA + 2 Strain VC 0.047 ± 0.003 0.000 ± 0.000 0.027 ± 0.001 0.004 ± 0.002 Strain K 0.080 ± 0.002 0.007 ± 0.001 0.049 ± 0.003 0.007 ± 0.001 P-value < 0.001 < 0.001 < 0.001 0.131 Strain KA 0.101 ± 0.005 0.010 ± 0.003 0.063 ± 0.003 0.011 ± 0.002 P-value < 0.001 0.005 < 0.001 0.009 KA vs. K 6 0.002 0.166 0.006 0.021 Data are expressed as mean ± SD ( n = 3/group) and compared by Student’s t-test 1 The enrichments in dC (deoxycytidine), dT (deoxythymidine), dG (deoxyguanosine), dA (deoxyadenosine) derived from 13 CO 2 are shown 2 Strain VC: E. coli K12 vector control 3 Strain K: E. coli K12 korAB (+) strain 4 Strain KA: E. coli K12 korAB (+) & aclBA (+) strain 5 Statistically significantly compa-red to the strain VC at p < 0.05; borderline statistical significance compared to strain VC at 0.05 < p ≦ 0.1 6 Statistically significantly compared to the strain K at p < 0.05; borderline statistical significance compared to strain K at 0.05 < p ≦ 0.1 In E. coli and other organisms, CO 2 , formate, and glycine are involved in synthesizing the purine ring [ 85 ] (Fig. 4 ). The model indicated an advantage of CO 2 incorporation into purines upon KOR expression. Furthermore, the additional expression of ACL significantly impacted purine synthesis from CO 2 . These findings align with the increased enrichment of glycine in cellular proteins and cytoplasmic amino acids. Glycine is a crucial source for purine formation [ 84 ], suggesting that ACL and KOR facilitate CO 2 incorporation into purines by improving glycine availability. For pyrimidines, CO 2 , aspartate, glutamate, and acetate serve as carbon sources [ 84 ] (Fig. 4 ). Compared to purines, the effects of KOR and ACL on pyrimidines biosynthesis were relatively moderate. These findings are consistent with the similar enrichment of aspartate in cellular protein and cytoplasmic amino acids. Notably, KA exhibited higher enrichments of deoxy-cytosine + 1 and deoxy-thymidine + 1 compared to K and VC, which may partially result from increased CO 2 incorporations into glutamate. In this study, carbon-fixing E. coli transgenic strains capable of assimilating CO₂ via rTCA machinery were successfully constructed. The results demonstrate that expressing either KOR alone or KOR in combination with ACL is sufficient to support cellular maintenance and CO₂ assimilation in E. coli under chemolithotrophic conditions. The engineered metabolic pathways in these transgenic strains represent the minimal and essential requirements for sustaining cellular life, closely reflecting the core metabolic functions necessary for survival. However, achieving indefinite growth of E. coli with CO₂ as the sole carbon source remains a significant challenge. According to a previous study [ 2 ], α-ketoglutarate: ferredoxin oxidoreductase (KOR) catalyzes the carboxylation of succinyl-CoA to α-ketoglutarate and coenzyme A, utilizing CO₂ and two moles of reduced ferredoxin as substrates. We hypothesize that the primary limitation lies in the insufficient availability of reduced ferredoxin to sustain KOR activity under nutrient-scarce conditions. To address this bottleneck and move closer to fully autotrophic growth, future research should focus on increasing the availability of reduced ferredoxin. Potential strategies include engineering pathways to enhance ferredoxin reduction or exploring methods for external supplementation of reducing power. These efforts could pave the way for transgenic strains capable of achieving complete autotrophic growth in fully organic-free environments."
} | 8,367 |
38516083 | PMC10952061 | pmc | 2,582 | {
"abstract": "Oceans and salt lakes contain vast amounts of uranium. Uranium recovery from natural water not only copes with radioactive pollution in water but also can sustain the fuel supply for nuclear power. The adsorption-assisted electrochemical processes offer a promising route for efficient uranium extraction. However, competitive hydrogen evolution greatly reduces the extraction capacity and the stability of electrode materials with electrocatalytic activity. In this study, we got inspiration from the biomineralisation of marine bacteria under high salinity and biomimetically regulated the electrochemical process to avoid the undesired deposition of metal hydroxides. The uranium uptake capacity can be increased by more than 20% without extra energy input. In natural seawater, the designed membrane electrode exhibits an impressive extraction capacity of 48.04 mg-U per g-COF within 21 days (2.29 mg-U per g-COF per day). Furthermore, in salt lake brine with much higher salinity, the membrane can extract as much uranium as 75.72 mg-U per g-COF after 32 days (2.37 mg-U per g-COF per day). This study provides a general basis for the performance optimisation of uranium capture electrodes, which is beneficial for sustainable access to nuclear energy sources from natural water systems.",
"conclusion": "Conclusions In summary, to improve the extraction capacity and the durability of uranium-capture electrodes with electro-catalytic activity, we got inspiration from the biomineralization of marine bacteria in high-salinity environments, and biomimetically regulated the electrochemical process. This regulation could reduce the competitive reaction between uranium reduction and hydrogen evolution, preventing the deposition of earth metal hydroxides. In this way, the uranium uptake capacity of the designed S-COF membrane could be increased by more than 20% without extra energy input. This process also endowed the membrane electrode with stability. In a long-term test, the S-COF membrane showed an excellent extraction capacity of 48.04 mg-U per g-COF in seawater within 21 days and 75.72 mg-U per g-COF in salt lake brine within 32 days, which is impossible to achieve using physicochemical adsorption. This way of optimization provides a new strategy for mining uranium from unconventional resources.",
"introduction": "Introduction The development of nuclear power is indispensable for global clean energy transitions to cope with current carbon emissions and future fossil-fuel exhaustion. 1–3 Annual nuclear capacity additions to 2050 are expected to grow four times in the Net Zero Emissions by 2050 Scenario (NZE) announced by the International Energy Agency (IEA). 4 Uranium is a critical element in the nuclear industry. Owing to the limited reserves and uneven distribution of uranium resources on land, it is important to look for alternative uranium sources to ensure a sufficient supply. 5 Oceans are estimated to contain more than 4.5 billion tons of uranium, which can ensure continued nuclear energy delivery. 6 However, extracting uranium at a concentration of 3.3 μg L −1 from the hypersaline and complex marine environment is an enormous challenge. 7–11 From a cost perspective, chemical adsorption has long been recognised as the preferred method for uranium extraction from seawater (UES). 12–14 This method typically usually relies on the number of accessible binding sites and their affinity for uranium, which is limited by adsorption thermodynamics. 15–17 However, in environments with higher salinity, such as seawater desalination rejects or salt lake brine, adsorbents perform poorly because of increased competition from other ions, even if these brine samples have significantly higher uranyl concentrations. 18–22 Electrochemical methods are a substitute for traditional chemical adsorption for UES. 23–26 Highly efficient electrodes for electrocatalytic uranium deposition, which directly determines external energy consumption, are critical in this process. Hydrogen evolution under high salinity is a major obstacle to continuous uranium extraction. A half-wave pulse method has been reported to prevent water splitting at the electrode. 23 However, when using electrocatalytic materials as the cathode to further improve the efficiency of uranium extraction, the possible enhancement of hydrogen evolution would cause stronger competition between uranium reduction and water splitting, especially when the metal concentration is extremely limited. Accordingly, the precipitation of hydroxides resulting from hydrogen evolution will seriously reduce the uranium uptake capacity and durability of the electrode. 27 Marine bacteria are single-celled organisms that live in the ocean and account for more than twenty percent of ocean biomass. 28 Some of these bacteria could reduce heavy metals to lower valence states, being not affected by the high salinity of marine environments. 29–31 Drawing inspiration from this special biological phenomenon, exquisitely regulating the process of electrochemical uranium capture is expected to avoid hydroxide deposition under a high salinity background when using electrocatalytic materials, further improving the extraction efficiency and operation life of electrode materials ( Fig. 1 ). Fig. 1 Schematic of the biological reduction/mineralization of marine bacteria and the bio-inspired electrochemical regulation for continuous uranium capture from oceans and salt lakes. Here, we report a membrane electrode (denoted as S-COF membrane) composed of single-walled carbon nanotubes (SWCNTs) and sp 2 carbon-conjugated covalent organic framework fibers (AO-g-C 34 N 6 -COF) for efficient uranium extraction from seawater and salt lake brine. The superior chemical stability of the C \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 C bonds enables amidoxime functionalisation in alkaline solutions for introducing specific uranium-binding sites. The high specific surface area, abundant selective uranium-binding sites, and spontaneous electron transfer from SWCNTs to the fully π-conjugated frameworks synergistically endow the membrane with an efficient electrocatalytic reduction of uranium. In addition, for the serious hydroxide deposition caused by the competitive hydrogen evolution, we mimic the biological reduction/mineralization of marine bacteria under high salinity and assess the effect of different electrical signal parameters to realize biomimetic mineralization. By avoiding the deposition of metal hydroxides, we successfully increase the uranium uptake capacity and durability of the electrodes. The membrane electrode exhibited a high capacity of 48.04 mg-U per g-COF (2.29 mg-U per g-COF per day) during a long-term test for up to 21 days in natural seawater. Furthermore, it demonstrated outstanding performance in salt lake brine and exhibited a capacity of 75.72 mg-U per g-COF within 32 days (2.37 mg-U per g-COF per day), and the uranium capture performance remains stable.",
"discussion": "Results and discussion Synthesis and characterization The synthesis of g-C 34 N 6 -COF and its amidoxime functionalization are described in the Methods section. The Fourier transform infrared spectra of the monomers and g-C 34 N 6 -COF in Fig. S1 (ESI) † confirm the generation of the carbon–carbon double bond, whose characteristic peak is at 1627 cm −1 . The stretching vibration of trans –HC CH– in the fingerprint region at 975 cm −1 demonstrates the trans -configurations of olefin linkages. The COF demonstrates a feature of full π-conjugation along with C C bond connection, leading to improved electrochemical activity. Compared to common imine-linked COFs, g-C 34 N 6 -COF linked by olefinic bonds has better chemical stability, making it possible to convert cyano groups into amidoxime groups in alkaline solutions ( Fig. 2a and S2, ESI † ). 32–34 As shown in Fig. 2b , the disappearance of the nitrile peak at 2221 cm −1 and the emergence of both the C N peak at 1643 cm −1 and the N–O peak at 871 cm −1 illustrate the successful functionalization of g-C 34 N 6 -COF. We characterised this process using energy-dispersive spectroscopy (EDS) elemental line scans. There was an apparent oxygen signal on the AO-g-C 34 N 6 -COF fibres, which was related to the introduction of amidoxime groups ( Fig. 2c ). Fig. 2 Amidoxime functionalization of g-C 34 N 6- COF. (a) Structural unit of g-C 34 N 6 -COF and AO-g-C 34 N 6 -COF. (b) Fourier transform infrared spectra of g-C 34 N 6 -COF and AO-g-C 34 N 6 -COF. The disappearance of the nitrile peak and the appearance of new characteristic peaks related to amidoxime confirm the successful functionalization of g-C 34 N 6 -COF. (c) Energy dispersive spectroscopy elemental line scan of AO-g-C 34 N 6 -COF. The signal of oxygen proved the introduction of amidoxime groups. The inset shows the transmission electron microscopy of AO-g-C 34 N 6 -COF and the length of elemental analysis. The scale bar is 500 nm. (d) PXRD patterns for g-C 34 N 6 -COF and AO-g-C 34 N 6 -COF. The peak shifts demonstrate that amidoxime functionalization leads to lattice changes. (e) Side views of final optimized models of g-C 34 N 6 -COF and AO-g-C 34 N 6 -COF. The interlayer shifting and interlayer spacing variation could be observed. Scanning electron microscopy was used to investigate the topography of the synthesised COFs. The fibrous g-C 34 N 6 -COF showed a uniform diameter of ∼100 nm and a length of several microns, which is consistent with previous literature (Fig. S3, ESI † ). After amidoxime functionalization, the fibrous morphology was preserved (Fig. S4, ESI † ), which makes it easy to fabricate membranes via vacuum filtration. Gas adsorption measurements were performed to investigate the porosities of the powdered g-C 34 N 6 -COF and AO-g-C 34 N 6 -COF samples. N 2 adsorption measurements (at 77 K) revealed permanent porosities. The Brunauer–Emmett–Teller surface areas of g-C 34 N 6 -COF and AO-g-C 34 N 6 -COF were 719 and 584 m 2 g −1 , respectively (Fig. S5, ESI † ). The pore size distribution curves were obtained from the adsorption branches using the QSDFT method (Fig. S6, ESI † ). Gas adsorption measurements demonstrated that after functionalization, the specific surface area remained high, and the pore structure was preserved. Given the extremely low concentration of uranium in oceans, a high surface area is necessary to provide abundant adsorption sites during the early stages of electrodeposition, which is an advantage of COFs. Thermogravimetric analysis was performed to study the thermal stabilities of the two COFs (Fig. S7, ESI † ). Compared with g-C 34 N 6 -COF, AO-g-C 34 N 6 -COF had an additional step starting at approximately 200 °C before the degeneration of the backbone, corresponding to the degradation of the amidoxime group. Overall, thermal stabilisation was preserved, enabling it to cope with temperature fluctuations during uranium recovery. Powder X-ray diffraction (PXRD) measurements were used to evaluate the crystallinity of the two COFs and the possible influence of amidoxime functionalization on the crystal structure ( Fig. 2d ). In the experimental PXRD profile of g-C 34 N 6 -COF, a strong peak at 5.69° along with relatively weaker peaks at 9.05°, 10.83° and 26.10° were assigned to (100), (110), (200), and (001) planes, respectively. The experimental PXRD pattern is in good agreement with the Pawley refinement of the AA-eclipsed layer stacking model, with reliability parameters R wp = 0.99% and R p = 0.78%. After converting the cyano groups into amidoxime, the PXRD pattern of AO-g-C 34 N 6 -COF was comparable to that of g-C 34 N 6 -COF. The (110) and (200) diffraction peaks shifted slightly to higher angles, whereas the (001) diffraction peak shifted to a lower angle. This indicates that the steric hindrance arising from the introduction of the functional group resulted in slight interlayer shifting and interlayer spacing variation. Geometric optimisation of the model of AO-g-C 34 N 6 -COF also displayed a degree of lattice change (Fig. S8, S9, Tables S1 and S2, ESI † ). The calculated layer spacing increased from 3.65 Å to 3.73 Å ( Fig. 2e ). Interlayer shifting correlated with the introduction of groups has recently been reported. This lattice deformation can lead to a quasi-AA stacking structure that is similar, but not identical, to AA stacking. 35 Electronic properties and membrane fabrication The electronic properties of the two COFs were investigated using photophysical and electrochemical methods. Their ultraviolet-visible diffuse reflectance spectra displayed broad absorption bands in the UV and visible regions. The spectrum of AO-g-C 34 N 6 -COF showed a slight blueshift of the adsorption edge compared with that of g-C 34 N 6 -COF, indicating that amidoxime functionalization affected the π-conjugated structure to some extent. However, overall, the extended π-conjugated skeleton was well preserved (Fig. S10 † ). Accordingly, the optical band gaps were calculated using the Kubelka–Munk function, and g-C 34 N 6 -COF demonstrated an optical band gap of 1.38 eV, while AO-g-C 34 N 6 -COF had an optical band gap of 1.64 eV ( Fig. 3a ). Mott–Schottky measurements were conducted to further estimate the semiconductor behaviour of AO-g-C 34 N 6 -COF. 36 The positive slopes indicated typical n-type semiconductor characteristics, suggesting that the majority carriers are electrons ( Fig. 3b ). Density functional theory (DFT) calculations using the Perdew–Burke–Ernzerhof functional predicted that AO-g-C 34 N 6 -COF had an indirect bandgap of 1.58 eV, which was close to the measured optical band gap ( Fig. 3c ). The DFT-calculated band structures had large band dispersions along the out-of-plane direction (Γ–X, Γ–L, Γ–N, and Γ–R) in both the valence and conduction bands. 37,38 In contrast, the in-plane direction (Γ–Y, Γ–Z, and Γ–M) exhibited different band dispersions. These results suggest that the charge transport in AO-g-C 34 N 6 -COF might be anisotropic. The corresponding partial density-of-states curves illustrate that the bands mainly originate from the 2p-orbitals. Collectively, these results indicate that AO-g-C 34 N 6 -COF is a narrow band gap organic semiconductor. According to a recent study, n-type semiconductors have a universal self-gating characterization that favours cathodic reactions, 39,40 implying that AO-g-C 34 N 6 -COF has the potential for electrocatalytic uranium extraction from seawater or salt-lake brine. Fig. 3 Electronic properties and characterization. (a) Band gaps of g-C 34 N 6 -COF and AO-g-C 34 N 6 -COF determined from the Kubelka–Munk-transformed reflectance spectra. The amidoxime functionalization process led to the widening of the band gap. (b) Mott–Schottky plots for g-C 34 N 6 -COF and AO-g-C 34 N 6 -COF measured in 0.2 M Na 2 SO 4 (pH 6.8) with an Ag/AgCl reference electrode. The positive slopes suggested that the COF is a typical n-type semiconductor. (c) Electronic band structures and corresponding partial density-of-states of AO-g-C 34 N 6 -COF determined using DFT calculations. The Brillouin zone is shown on the right. (d) Digital photograph of a free-standing S-COF membrane. The membrane presented impressive flexibility and can be highly curved. (e) Scanning electron microscopy image showing that the AO-g-C 34 N 6 -COF fibres are tightly tangled in the nanotubes. The scale bar is 500 nm. (f) Differential charge density distribution of the AO-g-C 34 N 6 -COF/graphene heterointerface calculated using DFTB+. Yellow and blue areas represent charge density increase and decrease, respectively. (g) Plane-average electron difference perpendicular to the AO-g-C 34 N 6 -COF/graphene heterointerface. Given its fibrous morphology, AO-g-C 34 N 6 -COF can be easily mixed with commercially available carboxylated single-walled carbon nanotubes (SWCNTs) and dispersed in organic solvents. A self-supporting S-COF membrane was prepared on a nylon filter using vacuum-assisted filtration. The content (20%) of COF is a balance between its uranium extraction capacity and membrane mechanical properties. More COF would result in the membrane becoming brittle (Fig. S11, ESI † ). As shown in Fig. 3d , the S-COF membrane exhibited excellent flexibility. Hydrogen bonds between the carboxyl and amidoxime groups can improve the mechanical strength of the membrane. The top-view SEM image shows that AO-g-C 34 N 6 -COF was tightly tangled in the carbon nanotubes ( Fig. 3e ). To estimate the electron transport performance of the composite membrane, we used the density-functional tight-binding (DFTB+) method, which is an approximate density functional theory method based on the tight-binding approach, to analyse the electronic interactions. For computational expediency, the SWCNTs were simplified to a periodic graphene substrate, on which the geometry-optimised AO-g-C 34 N 6 -COF was modeled. 41,42 As shown by the differential charge density distribution in Fig. 3f and plane-average electron difference in Fig. 3g , the graphene substrate (electron-deficient, blue colour) injects delocalised electrons into AO-g-C 34 N 6 -COF (electron-rich, yellow colour), resulting in an n-doping effect on the COF. Delocalised electrons serving as excessive carriers can change the electrochemical activity of the COF. 43 This process can be mainly attributed to the support effects in carbon-supported catalysts, which is the reason why we choose carbon nanotubes as the component. 44 Extraction performance in uranium-spiked seawater Cyclic voltammetry (CV) was performed to measure the uranium reduction activities. The CV scan curves at a scan rate of 10 mV s −1 of natural seawater spiked with 50 ppm and 100 ppm uranyl nitrate at pH 8.2 are shown in Fig. 4a . In comparison with seawater without extra uranium( vi ), additional reduction peaks at −0.65 V SCE and anodic peaks at −0.46 V SCE were observed, corresponding to uranium reduction/oxidation reactions. It is worth mentioning that this membrane electrode based on AO-g-C 34 N 6 -COF has a more positive reduction potential than those previously reported for semiconductor/carbon-based electrodes for uranium extraction from seawater measured under similar experimental conditions. 23–25,45 This means that energy consumption can be further reduced when an electric field is applied. Fig. 4 Uranium extraction performance of the S-COF membrane in spiked seawater. (a) Cyclic voltammograms of uranium-spiked natural seawater with concentrations of 50 ppm and 100 ppm compared to unspiked seawater. (b) Schematic of the pulse applied to the working electrode. The amplitude and the duty ratio were adjustable parameters. (c) Effective values of current and the corresponding uranium extraction amount of the electrode when pulses were applied with different amplitudes and duty ratios. (d) Digital photographs of the S-COF membrane before (top) and after (bottom) extracting uranium at 5 V voltage with a 75% duty ratio. The membrane was wrapped tightly by white deposits. (e) Schematic of the current density regulation strategy. The S-COF membrane was bonded to graphite papers of different sizes to control the current density. (f) Uranium extraction from spiked seawater using the S-COF membrane with varying substrate sizes. The error bars represent the standard deviation ( n = 3). The digital photographs correspond to the S-COF membrane with substrate sizes of 2 cm × 2 cm (top panel), 3 cm × 3 cm (middle panel), and 4 cm × 4 cm (bottom panel) after extracting uranium for 24 h. (g) High-resolution X-ray photoelectron spectroscopy profile of the S-COF membrane used for uranium uptake by chemical adsorption and electrodeposition. The 4f 5/2 and U 4f 7/2 peaks shifted to lower binding energy, indicating the reduction reaction of U( vi ). In order to investigate the role of selective adsorption and spontaneous electron transfer in adsorption-assisted electrochemical enrichment, a series of control experiments were carried out. Two pieces of control membranes with g-C 34 N 6 -COF (SWNT-COOH membrane with g-C 34 N 6 -COF) and without COF (SWNT-COOH membrane) were prepared. Three membranes were tested in seawater with various uranium concentrations by applying a square-wave pulse with a frequency of 400 Hz and a duty ratio of 75% for 24 h (Fig. S12, ESI † ). When the uranium concentration is high, three membranes all showed the ability to extract uranium and the membrane having AO-g-C 34 N 6 -COF had the highest capacity. The facilitation effect of COFs gradually increased with the uranium concentration decreased. When the uranium concentration was low at 1 ppm, the SWNT-COOH membrane could hardly extract uranium from seawater. The SWNT-COOH membrane with g-C 34 N 6 -COF retained some extraction capacity and the S-COF membrane still performed robustly. The results indicate that spontaneous electron transfer cooperates with selective adsorption in complex environments to promote effective uranium extraction from seawater through electrochemical methods. To further evaluate the uranium uptake performance, the S-COF membrane was cropped into 1 cm × 1 cm pieces and clamped using a glassy carbon clip as the working electrode. A graphite rod was used as the counter electrode. A series of square-wave pulses at a frequency of 400 Hz were applied to the membrane electrode. In these experiments, the voltage amplitude and duty ratio were set as adjustable parameters ( Fig. 4b ). We recorded the instantaneous current using a multimeter when the electrode worked for 5 min to evaluate the variations in current density caused by parameter adjustment. The uranium extraction system was operated continuously in uranium-spiked natural seawater (∼8 ppm) for 24 h, and the extraction amount was determined based on changes in the concentration of uranium in the solution. The uranium concentrations were measured from ultraviolet-visible absorption spectra (Fig. S13, ESI † ). As shown in Fig. 4c , the measured current was approximately proportional to the duty ratio. When the voltage amplitude was 4 V, the uranium extraction amount increased with increasing duty ratio and reached a maximum value of 675.9 mg-U per g-membrane at a 75% duty cycle. This phenomenon is reasonable because higher duty cycles imply higher input energy. When the voltage amplitude was 5 V, the extracted uranium continued to increase as the duty ratio increased from 50% to 60%. However, the extraction amount decreased anomalously at higher duty ratios. To determine the reason for this decline in electrode performance, we recorded the macroscopic changes at the electrode surface over time. When a voltage of 5 V (75% duty ratio) was applied, the electrode was quickly wrapped by large amounts of white deposits in the first hour ( Fig. 4d ). In contrast, deposition on the electrode at 4 V (75% duty ratio) was significantly slower. Its wrapping state was not as compact as that at 5 V until electrolytic deposition continued for 24 h (Fig. S14, ESI † ). This suggests that although high-frequency pulses could reduce water splitting to some extent and there were no visible bubbles on the electrode, hydrogen evolution was still not completely avoided. Fast electron transfer depletes the uranyl cations near the electrode and causes concentration polarisation. Further polarisation of the electrode promotes a competitive hydrogen evolution reaction and changes the pH near the electrode, leading to the deposition of alkaline earth metal hydroxides. These compact deposits prevent uranyl cations from migrating to the electrode, thereby reducing uranium extraction. To address the negative effects of undesired deposition on the electrochemical process, we tried to further decrease the operating voltage. However, we found it difficult to achieve a balance between keeping high uptake capacity and avoiding hydroxide deposition. Although the surface deposition gradually slowed down, the uranium extraction capacity of the electrode also decreased substantially, diminishing its practicality. Hence, we attempted to regulate the current density of the membrane without changing the working voltage. We used graphite paper as the substrate to support the S-COF membrane ( Fig. 4e and S15, ESI † ). The introduction of the inert area would lower the local current density, decreasing the reaction rate of the electrode. In this way, the concentration polarization of uranium could be decreased, reducing the competition between uranium reduction and hydrogen evolution. The cumulation and expulsion of hydroxide ions could gradually reach an equilibrium, averting the generation of deposition. The current density was controlled by varying the substrate size to 2 cm × 2 cm, 3 cm × 3 cm, and 4 cm × 4 cm, and the size of the S-COF membrane was fixed at 1 cm × 1 cm. The test device is shown in Fig. S16 (ESI). † The possible influence of the substrate on the uranium extraction amount was excluded using graphite paper alone as the working electrode. Fig. S17 (ESI) † shows that the uranium concentration in the spiked seawater remained virtually unchanged under the same conditions. The current changes also demonstrated this phenomenon. The measured instantaneous current was only slightly enhanced with an increase in the substrate (Fig. S18, ESI † ). These slight variations were mainly due to an increase in the non-faradaic current. We studied the dynamics of composite electrodes with different substrate sizes during uranium extraction ( Fig. 4f ). The inset shows the partially enlarged kinetic curves. It can be seen that the kinetics of uranium extraction on the electrode with a size of 2 cm × 2 cm was the most rapid during the first 5 h because it had the maximum current density. However, the electrode was gradually wrapped by undesired deposits, and the amount of extracted uranium slowed. There was almost no increase in this amount for the remaining time. The digital photograph in Fig. 4f (top panel) shows the S-COF membrane to which dense deposits were attached after the electrode was operated for 24 h. When the substrate size was 3 cm × 3 cm (middle panel), the amount of deposition was less than that of the electrode with a 2 cm × 2 cm substrate, indicating that some space remained in the membrane for uranium transport and further extraction. Correspondingly, the electrode had a higher uranium extraction amount of 718.72 mg-U per g-membrane after running for 24 h. The kinetic curve also maintained a gradual increase, indicating that electrodeposition continued. When a 4 cm × 4 cm substrate was used, no deposits were observed on the surface of the membrane (bottom panel). Although this composite electrode had the slowest uranium extraction rate during the first 5 h, it exhibited a maximal extraction amount of 819.35 mg-U per g-membrane at 24 h, and the enrichment process did not stop. The excellent uranium uptake capacity was approximately 17 times that of chemical adsorption (Fig. S19, ESI † ). The scanning electron microscope (SEM) images of the membrane surface reveal that, with regulated current density, the micro-voids on the surface are maintained, while without current density regulation, these voids become clogged with deposits (Fig. S20, ESI † ).The above experiments suggest that an appropriate current density reduction for decreasing the electrode reaction rate could increase the uranium extraction amount by avoiding undesired deposits, which could also increase the life of the electrode. High-resolution X-ray photoelectron spectroscopy (XPS) and X-ray diffraction were used to characterise the uranium species produced during the electrocatalytic process. The S-COF membrane was used to extract uranium from uranium aqueous solutions (50 ppm) via traditional chemical adsorption and electrodeposition for 12 h. The electrical uranium extraction was performed under a nitrogen atmosphere after deoxygenation of the solution to eliminate the impacts of oxygen reduction. As shown in Fig. 4g , the U 4f peaks of uranium on the electrocatalytic membrane were shifted more significantly to lower binding energies than those of the adsorption membrane, indicating an decrease in the valence state of U. 46 Raman spectroscopy was also used to identify the uranium species (Fig. S21, ESI † ). It can be seen that the sample extracting uranium under the N 2 atmosphere showed characteristic peaks from UO 2 at ∼230 cm −1 and 445 cm −1 . 47 The generation of U( iv ) is generally considered to come from the disproportionation of unstable U( v ) intermediates: 48 2U V O 2 + + 4H + → U 4+ + U VI O 2 2+ + H 2 O To further investigate the uranium species generated in natural seawater, we performed the extraction process in real seawater spiked with 1000 ppm uranium. A large amount of yellow powder was produced, which was attached to the membrane (Fig. S22, ESI † ). The collected powder was identified as a Na 2 O(UO 3 ·H 2 O) x species according to the PXRD result (Fig. S23, ESI † ). Similar results have been reported in related work. 24,49 Uranium extraction performance in unspiked natural water The uranium extraction performance of the S-COF membrane was investigated using natural seawater and salt lake brine ( Fig. 5a ). Given the extremely low concentration of uranium in seawater and the possible concentration fluctuation caused by the pre-filtration step, we monitored the concentration variation in seawater and salt lake water during each operational period. For experiments using seawater, 2 mg of the membrane was composited with a 5 × 5 cm substrate as the working electrode. Seawater (2 L) was treated with the electrode for 24 h during each period and then replaced with fresh seawater. This process lasted 21 days. As shown in Fig. 5b , the initial concentration of uranium fluctuated slightly near 3.3 ppb, and apparent decreases in concentration were observed during each extraction period. In the long term, the magnitude of the decline became progressively smaller, perhaps because the performance attenuation of the electrode was more evident in an environment containing a low concentration of uranium. Fig. 5 Uranium extraction performance of the S-COF membrane in natural seawater and salt lake brine. (a) Schematic illustration showing the sampling locations and physicochemical properties of natural seawater and salt lake brine. (b) Initial and final uranium concentrations in seawater during each extraction period. 2 L of seawater was treated by the electrode for 24 h per treatment. The whole process lasted 21 days. The error bars represent the standard deviation ( n = 3). (c) Initial and final uranium concentrations in the salt lake brine during each extraction period. The electrode treated 500 mL of brine for 4 days per treatment. The error bars represent the standard deviation ( n = 3). (d) Cumulative uranium extraction amount of the S-COF electrode from seawater. (e) Cumulative uranium extraction amount of the S-COF electrode from salt lake brine. Encouraged by the outstanding uranium-extraction properties of the S-COF membrane, we further tested its performance in real salt-lake brine from Chaerhan Salt Lake, which has a salinity much higher than seawater. The membrane weighed 2 mg, and the substrate was maintained at 5 × 5 cm. During each period, the electrode was treated with 500 mL salt lake brine for four days, and the entire process lasted 32 days. Although the uranium concentration was approximately 50.0 ppb, the extremely high alkali metal content in the brine occupied the adsorption sites, leading to unsatisfactory uranium extraction performance using the traditional chemical adsorption method (Fig. S24, ESI † ). However, when the S-COF membrane was employed as the working electrode with voltage pulses, the uranium concentration in the brine showed a clear decreased during each extraction period ( Fig. 5c ). The cumulative extraction amount was calculated according to the uranium concentration variations ( Fig. 5d and e ), which was 12.01 mg-U per g-membrane (48.04 mg-U per g-COF, 2.29 mg-U per g-COF per day) for seawater and 18.93 mg-U per g-membrane (75.72 mg-U per g-COF, 2.37 mg-U per g-COF per day). The extraction capability of this S-COF membrane far exceeded that of previously reported chemical adsorbents or photocatalysts based on olefin-linked COFs. At the same time, this is currently the longest running time based on the electrochemical method for continuous uranium extraction from seawater compared to what has been reported so far (Table S3, ESI † ). There were no deposits on the S-COF membrane surface after the uranium extraction process in natural water and salt lake brine (Fig. S25, ESI † ), indicating long-term service performance. The continuous uranium capture performance and the long-term stability of the electrodes prove that this biomimetic electrochemical regulation is an effective strategy to promote the application potential of electroactive materials in harsh environments with high salinity. We also evaluate the economic viability of the electrode along with the electrical extraction process. The cost of preparing the S-COF membrane was estimated to be ≈52 USD per g. The electricity cost was calculated to be ≈3.40 × 10 −3 USD per g-U using seawater and 2.15 × 10 −3 USD per g-U using salt lake brine. Given the continuous electrical power consumption of the electrochemical method, future research should focus on the higher working efficiency of electrodes and cheaper electrical energy."
} | 8,507 |
29565975 | PMC5863959 | pmc | 2,583 | {
"abstract": "The complex morphologies observed in many biofilms play a critical role in the survival of these microbial communities. Recently, the formation of wrinkles has been the focus of many studies aimed at finding fundamental information on morphogenesis during development. While the underlying genetic mechanisms of wrinkling are not well-understood, recent discoveries have led to the counterintuitive idea that wrinkle formation is triggered by localized cell death. This work examines the hypothesis that the material properties of a biofilm both power and control wrinkle formation within biofilms in response to localized cell death. Using an agent-based model and a high-performance platform ( Biocellion ), we built a model that qualitatively reproduced wrinkle formation in biofilms due to cell death. Through the use of computational simulations, we determined important relationships between cellular level mechanical interactions and changes in colony morphology. These simulations were also used to identify significant cellular interactions that are required for wrinkle formation. These results are a first step towards more comprehensive models that, in combination with experimental observations, will improve our understanding of the morphological development of bacterial biofilms.",
"introduction": "Introduction Bacteria live in almost every environment. While they are critical drivers of biogeochemical cycles and ecosystem dynamics, some bacteria are major threats to human health [ 1 – 3 ]. Bacterial cells can attach to a surface and form a multicellular aggregate, referred to as a biofilm, which increases their survival [ 1 ]. Biofilms protect bacteria from attack by the immune system and by antibiotics and are responsible for many infections caused by implanted medical devices [ 4 ]. One of the main reasons survival is improved in biofilms is due to their complex morphologies. How bacterial assemblies develop complex morphologies has been a question pursued by many scientists. Many researchers have recently focused on the formation of wrinkles in bacterial colonies because the analysis of wrinkle formation provides fundamental information on how structural patterns can develop [ 5 – 10 ]. Many bacterial colonies have a complex morphology characterized by an elaborate organization of connecting wrinkles (see Fig 1 ). It has been shown that these wrinkles participate in liquid transport within the colony by forming permeable channels connected in a radial network [ 11 ]. The liquid-filled channels can carry nutrients, waste, and signaling molecules. Importantly, in some bacterial colonies, such as those formed by Vibrio cholerae [ 12 ], the presence or absence of wrinkles distinguishes between virulent and benign states. 10.1371/journal.pone.0191089.g001 Fig 1 Topside of a Bacillus subtilis colony showing a complex interlocking wrinkled pattern. Figure adapted from Jers et al. [ 20 ]. Wrinkles can form in tissues through mechanical instabilities that are generated by constrained growth of tissues with specific elastic properties [ 13 – 16 ]. This physical mechanism has been suggested for the development of many wrinkled or undulated morphologies, such as the wrinkled morphology of the brain, tubular organs, and some biofilms [ 17 – 19 ]. A novel mechanism of wrinkle formation was revealed in a recent study by Asally et al. [ 9 ] which showed that localized cell death initiates wrinkle formation in Bacillus subtilis colonies. The abundant extracellular polymeric substance (EPS) produced by cells plays a critical role in wrinkle formation, underlying the formation of local regions of cell death and providing a mechanical support that resists compressive forces stemming from cell displacement driven by cell growth and cell division. Cell death disrupts the integrated network of cells and EPS within the biofilm, providing an outlet for compressive stress [ 9 ]. Complexly organized biofilms start from a single bacterium adhering to a surface. The bacterium secretes a glue-like protein that attaches it more tightly to the substratum. Upon division, the daughter cells are cemented together and to the substratum [ 8 ]. These cell-cell and cell-surface bonds, coupled with the pressure arising from population growth, push the expanding colony into a quasi-stable state in which unrelaxed forces are dampened by the rigid structure of biofilm. This rigid structure is formed by the EPS that wraps around the cells and provides the biofilm both mechanical support and resilience against environmental stresses [ 21 – 24 ]. Significantly, EPS production is essential for biofilm wrinkling [ 22 , 25 , 26 ]. A quasi-stable state is reached between 24 to 48 hours of biofilm development when the colony appears as a smooth, disk-like structure [ 9 ]. Continued growth leads to the formation of an intricate colony-wide pattern of cell death at the colony-substratum boundary in response to nutrient depletion, high cell density, and waste accumulation (see Fig 1 in Asally et al. [ 9 ]). In regions of cell death, the colony detaches from both, the substratum and surrounding cells, and the biomass converges to the areas opened by the dying cells. This leads to buckling of the colony into a complex pattern of interlocking wrinkles illustrated in Fig 1 . The focus of this study is on the transition from a smooth, stiff colony under compression to a complex wrinkled morphology triggered by localized cell death. This study does not consider the development of the smooth compressed colony or model how cell death patterns emerge, but begins with the initiation of realistic patterns of cell death at the colony-substratum interface. We developed an agent-based model that considers cells and associated EPS as single agents to study the formation of 3D cellular structures that result from the interplay of cell death and biomechanical forces, and implemented this model using the Biocellion simulation framework [ 27 ]. Agent-based modeling is becoming a popular modeling framework to investigate the influence of mechanical properties on biological systems, including biofilms [ 28 – 32 ]. Agent-based approaches allow the integration of inter- and intra-cellular interactions and the exploration of cellular heterogeneity [ 33 ]. Our aims were to: a) test the hypothesis that cellular mechanical approaches allow the integration of intracellular interactions, b) explore how cellular interactions can both power and control wrinkle formation in biofilms in response to localized cell death, c) to learn how changes in mechanical properties of biofilms affect the structure of wrinkles, and d) to identify the intercellular interactions needed to form wrinkles.",
"discussion": "Discussion To form a multicellular aggregate, such as a biofilm, cells interact via a complex interplay between biochemical signaling and biomechanical forces. However, these interactions are still poorly understood. Investigating the morphogenesis of model biological systems, such as biofilms, is important for understanding and formalizing the common patterns seen in more complex systems and organisms. Recent studies showed that cell death triggered by biochemical stress combined with a relaxation phase of biomechanical stress, plays a critical role in the initiation of wrinkles in biofilms. We developed an agent-based model to evaluate the effect of cellular level mechanical interactions on wrinkle formation due to cell death. We modeled mechanical interactions through the implementation of elastic bonds between pairs of agents and also between agents and the agar surface. In this model, an agent is a cell and the surrounding EPS. Cell death was modeled by removing agents from the system. Because instantaneous deletion of cells may be a more drastic perturbation than encountered in biological systems, we have also performed simulations in which cells are gradually removed from the system. We found that abrupt or gradual removal of cells produced the same final results (see S1 Text in Supporting Information). By implementing the cellular processes of mechanical interactions and cell death, we were able to qualitatively recapitulate the process of wrinkle formation that are observed in colonies of Bacillus subtilis [ 9 ]. Although this simple model performed well, a more complete model would include other cellular events, such as cell density change due to cell division, cell motility, the effect of waste molecules and nutrients, and heterogeneity of EPS production. We first aimed to investigate the role of bond stiffness on wrinkle morphology. We found that bond stiffness is the major modulator of colony stiffness. By changing the bond stiffness, we simulated colonies with distinct mechanical behaviors. Colonies that generate wrinkles when perturbed by cell death have a mechanical behavior characterized by a linear elastic regime followed by plastic-like behavior. Colonies without plastic-like behavior did not form wrinkles after cell death. Moreover, colonies simulated with higher stiffness generated wider and higher wrinkles, in good agreement with observations of bacterial colonies [ 9 ]. In the simulations, the relationship between wrinkle height and stiffness is more pronounced for wider cell death regions. Furthermore, decreasing the height of the cell death region resulted in larger wrinkles, even when cell death regions are relatively small. This non-intuitive result suggests a complex interplay between the geometry and volume of the cell death region and wrinkle morphology. In actual biofilms, it is likely that the volume of cell death regions is influenced by colony stiffness, which will require a model that relates cell death to mechanical stress. We performed simulations that correspond to different sets of parameters that characterize our model. This approach helped us identify properties beside mechanical stiffness that determine wrinkle formation. Our results suggest that small cell death regions are less likely to trigger wrinkles in colonies with low cell densities. Moreover, the size of cell death regions determines the morphology of the wrinkles; large cell death regions produce multiple wrinkles with specific wavelengths, whereas smaller cell death regions generate a single wrinkle on top of the region of cell death. Our results also show that cell-cell adhesion is essential for wrinkle formation. However, while cell adhesion to the agar substratum influences colony morphology, its suppression does not completely prevent the formation of wrinkles. The wrinkle formation simulated in this study represents one specific morphological feature of the whole colony. Future work will include expanding the current simulation to a larger spatial scale, including other biological events such as cell division, the effects of nutrients and waste molecules, as well as intracellular gene regulatory networks that modulate the primary determinants of wrinkling, cell adhesion and cell death."
} | 2,748 |
35759507 | PMC9269948 | pmc | 2,584 | {
"abstract": "The topology of metabolic networks is recognisably modular with modules weakly connected apart from sharing a pool of currency metabolites. Here, we defined modules as sets of reversible reactions isolated from the rest of metabolism by irreversible reactions except for the exchange of currency metabolites. Our approach identifies topologically independent modules under specific conditions associated with different metabolic functions. As case studies, the E . coli i JO1366 and Human Recon 2.2 genome-scale metabolic models were split in 103 and 321 modules respectively, displaying significant correlation patterns in expression data. Finally, we addressed a fundamental question about the metabolic flexibility conferred by reversible reactions: “Of all Directed Topologies (DTs) defined by fixing directions to all reversible reactions, how many are capable of carrying flux through all reactions?”. Enumeration of the DTs for i JO1366 model was performed using an efficient depth-first search algorithm, rejecting infeasible DTs based on mass-imbalanced and loopy flux patterns. We found the direction of 79% of reversible reactions must be defined before all directions in the network can be fixed, granting a high degree of flexibility.",
"introduction": "Introduction A genome-scale metabolic model (GeM) is a comprehensive mathematical representation of an organism’s metabolism [ 1 , 2 ]. To date, GeMs for more than 6,000 organisms, including all model organisms, have been reconstructed [ 3 – 8 ]. This network representation is widely employed to study the metabolic phenotype of cells with applications ranging from strain development, modelling interactions among multiple cells or organisms, understanding human diseases to the study of evolutionary processes [ 8 – 13 ]. GeMs describe all metabolic capabilities of an organism, i.e., all biochemical reactions that can carry flux under any condition. These detailed models contain thousands of reactions, which can confound more detailed studies of network properties and functions. A common strategy to overcome this limitation is to focus the analysis on one or a few model subsystems. Subsystems have been defined by conventional biochemical pathways in online databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) [ 14 ] and BioCyc [ 15 ]. Subsystems have been used to map omics data [ 16 ] and for model reduction [ 17 ], yet their definition is arbitrary and identical for all organisms. Recognising the diversity and uniqueness of the metabolism in individual organisms, a more satisfying alternative would be to generate model subsystems in an unsupervised manner relying exclusively on the specific topology of the studied metabolic network. The topology of metabolic networks has been widely studied by graph theory methods. Early work by Barabasi and colleagues concluded that metabolic networks are scale-free, hierarchical networks with highly connected modules overlapping known metabolic functions [ 18 , 19 ]. However, these analyses did not consider the nature of the edges and it soon became apparent that the extremely short average pass length observed was realized through cofactors (e.g., ATP, NADH, NADPH), whereas the flow of carbon from a substrate to a product often is quite long. Following a more biologically meaningful interpretation of the network topology, by excluding currency metabolites (cofactors and moieties) and accounting for directionality of irreversible reactions, Ma [ 20 , 21 ] observed that metabolic networks can be broken into a modest number of strong networks (i.e., networks where each metabolite can be reached from every other metabolite). The network arranged as a directed bow-tie structure with a substrate subset connected to a product subset through a giant strong component corresponding to central carbon metabolism [ 20 , 22 ]. Another approach for inferring and studying metabolic modules/pathways is based on structural (stoichiometric) analysis [ 23 – 26 ]. For this task, classical Elementary Flux Modes (EFMs) has been adapted for enumerating flux patterns in metabolic subnetworks (i.e., modules) under biomass-optimal growth [ 23 , 25 ], incorporating even loopless criteria [ 27 ] avoiding thermodynamically infeasible flux cycles [ 24 ]. While these approches have yielded deep insights about the flexibility and functioning of metabolic newtorks, their applicability still remains limited to small- to medium-sized models. This work presents a novel approach to generate topologically independent metabolic modules exploiting the network topology and directionality constraints. The E . coli iJO1366 [ 4 ] and Recon 2.2 [ 7 ] GeMs were subdivided in topologically independent modules and evaluated for their biological relevance under specific growth conditions. The clustering approach provides fundamental insights into the role and flexibility conferred to metabolic networks by reversible reactions. We quantitatively estimated the network flexibility by counting in each module the number of feasible Directed Topologies (DTs), which represent consistent flux solutions [ 28 ] where all reactions carry flux, and hence, the directions are fixed. Notably, these DTs are maximal pathways known as Flux Topes (FTs) [ 23 ], which have been recently applied for exploring the flexibility of optimal network states, and correcting thermodynamically infeasible cycles [ 29 ]. Under the assumption of ‘thermodynamic’ isolation, the (Cartesian) product of the DTs of the different modules provides an unprecedented upper bound estimate of the ‘topological’ degree of freedom of the network.",
"discussion": "Discussion Metabolic networks are inherently modular [ 19 , 20 , 22 ]. This modular nature provides a means for simplifying structural and functional analysis of large-scale metabolic networks. Early work described the network topology using an undirected graph with no consideration of the nature of the edges, hereby yielding artificial short path lengths and an ambiguous structure. By excluding currency metabolites and accounting for directionality of reactions, metabolic networks have been previously described having a bow-tie structure with a substrate subset connected to a product subset through a giant strong component corresponding to central carbon metabolism [ 20 , 21 ]. The deliberate omission of energy/redox co-factors was critical for the identification of thermodynamically isolated modules. Clearly, these modules are coupled through energy and redox to other reactions, however, the coupling is to the tightly maintained global pool rather than between any two individual reactions. Arguably energy/redox homeostasis–maintaining energy charge and the ratios of various redox partners–is a more global regulatory principle (see for example [ 42 ]) than the modules identified. Conversely, assuming that coupling through energy/redox links individual reactions would speak against modularity, e.g., suggest that all gene regulation is globally coordinated with no modularity, which is clearly not accurate (operons in bacteria is a clear example of modularity). The thermodynamic isolation hereby employed focus on is the isolation achieved by irreversible reactions–commonly subject to allosteric regulation–that ensures the products have no impact on substrate concentrations or the reactions upstream of the substrates. Importantly, once modules have been indentified, currency metabolites (redox and co-factors) may be reincorporated to the respective reactions if desired. For example, a kinetic model of a module would typically include cofactors as fixed concentration external metabolites [ 43 , 44 ]. It is for the sole purpose of identifying modules that currency metabolites are reversibly removed. Altogether, the identified modules unveiled the modular organization of the reversible reactions of the E . coli iJO1366 and the Recon 2.2 GeMs into metabolic modules connected by irreversible reactions. The resulting organization resembles conventional metabolic pathways and subsystems known for these organisms, but in this case, they emerged from the topological features of each network leveraged by a novel clustering approach. The approach unravelled hundred and three nearly isolated modules for the E . coli network growing aerobically in media with glucose as sole carbon source. The majority of the modules contained only one structural reversible reaction, whereas thirty contained more than one. For comparison, Ma and Zeng [ 20 ] found 29 strong components that include no less than three metabolites. When applying the clustering approach to a much larger reconstruction, Recon 2.2, a large number of antiporter transport reactions (e.g., the amino acid “harmonizers” such as LAT1) were removed, which artificially connect different parts of metabolism [ 35 ]. After the removal of antiporters, the model was subdivided into 321 thermodynamically isolated modules. The six largest modules in the human model displayed known metabolic functions similar to those found in E coli , namely: TCA cycle, glycolysis, pentose phosphate pathway, fatty acids, nucleotides, sugars and amino acids metabolism. The biological relevance of the identified modules was demonstrated through gene expression analysis, which showed that the correlation between of reversible reactions of distance 2 was significantly higher between reactions within a cluster and low between reactions in separate clusters. The majority of correlated enzymes catalysing reactions within the same module are highly correlated (more than 0.8 absolute value correlation across 278 transcription datasets in the E . coli model and more than 0.5 absolute value correlation across 17,382 RNA-seq samples in the Human model). In contrast, the majority of absolute correlations between reactions in different modules concentrated around zero supporting the presence of the inferred underlying modular structure. The E . coli iJO1366 metabolic network flexibility was studied using the identified modules. An efficient depth-first search algorithm using simple infeasible mass balance and loopless rules was developed to explore the topological flexibility of the modules by enumerating all feasible DTs. We note that this amounts to enumerating all the flux topes in these (currency-free) subnetworks [ 23 ]. The analysis revealed only a weak coupling between structural reversible reactions in the largest module, which points to an overall high topological flexibility providing a high degree of robustness [ 45 ]. Strong coupling was only found between some boundary (exchange) and internal reactions consuming a common metabolite, which is known to be the case as exchange reactions can exert massive coupling and blocking of reactions at the boundary of metabolic networks [ 31 ].This observation is true across the modules. Assuming that modules operate independent of each other, the topological degree of freedom of the E . coli iJO1366 model was determined to be 200 (79%) out of a theoretical maximum of 248. This number represents an upper bound on the number of directionalities that must be determined to fix the topological state of the metabolic network. A more exact estimate would be obtained by enumerating all the feasible DTs in the entire network as whole, which is unfortunately impractical at this scale [ 23 ]. Still, we can conclude that except for linear pathways, reversible reactions operate practically independent of each other, granting both flexibility and robustness against internal and external perturbations [ 22 , 34 , 46 , 47 ]."
} | 2,907 |
28713623 | null | s2 | 2,585 | {
"abstract": "Synthetic microbial consortia are conglomerations of multiple strains of genetically engineered microbes programmed to cooperatively bring about population-level phenotypes. By coordinating their activity, the constituent strains can display emergent behaviors that are difficult to engineer into isogenic populations. To do so, strains are engineered to communicate with one another through intercellular signaling pathways. As a result, the regulatory networks that control gene transcription throughout the population are sensitive to the extracellular concentration of the signaling molecules, and hence the relative densities of constituent strains. Here, we use computational modeling to examine how the behavior of a synthetic microbial consortium results from the interplay between the population dynamics governed by cell growth and the internal transcriptional dynamics governed by cell-to-cell signaling. Specifically, we examine a synthetic microbial consortium in which two strains each produce signals that down-regulate transcription in the other. Within a single strain this regulatory topology is called a \"co-repressive toggle switch\" and can lead to bistability. We find that in a two-strain synthetic microbial consortium the existence and stability of different states depends on the population-level dynamics of the interacting strains. As the two strains passively compete for space within the colony, their relative fractions can fluctuate and thus alter the strengths of intercellular signals. These fluctuations can drive the consortium to alternative equilibria. Additionally, if the growth rates of the strains depend on their transcriptional states, an additional feedback loop is created that can generate relaxation oscillations. These findings demonstrate that the dynamics of microbial consortia cannot be predicted from their regulatory topologies alone, but also is determined by interactions between the strains."
} | 487 |
29500726 | PMC5834416 | pmc | 2,586 | {
"abstract": "Bacterial strains were isolated from the sediments of the Baltic Sea using ferulic acid, guaiacol or a lignin-rich softwood waste stream as substrate. In total nine isolates were obtained, five on ferulic acid, two on guaiacol and two on a lignin-rich softwood stream as a carbon source. Three of the isolates were found to be Pseudomonas sp. based on 16S rRNA sequencing. Among them, isolate 9.1, which showed the fastest growth in defined M9 medium, was tentatively identified as a Pseudomonas deceptionensis strain based on the gyrB sequencing. The growth of isolate 9.1 was further examined on six selected lignin model compounds (ferulate, p -coumarate, benzoate, syringate, vanillin and guaiacol) from different upper funneling aromatic pathways and was found able to grow on four out of these six compounds. No growth was detected on syringate and guaiacol. The highest specific growth and uptake rates were observed for benzoate (0.3 h −1 and 4.2 mmol g CDW −1 h −1 ) whereas the lowest were for the compounds from the coniferyl branch. Interestingly, several pathway intermediates were excreted during batch growth. Vanillyl alcohol was found to be excreted during growth on vanillin. Several other intermediates like cis,cis -muconate, catechol, vanillate and 4-hydroxybenzoate from the known bacterial catabolic pathways were excreted during growth on the model compounds. Electronic supplementary material The online version of this article (10.1186/s13568-018-0563-x) contains supplementary material, which is available to authorized users.",
"introduction": "Introduction Lignin is one of the most plentiful biopolymers on Earth. It is a complex alkyl–aromatic heteropolymer found in the plant cell wall, which provides strength and protection to terrestrial plants. Currently, technical lignin is produced from either the pulp/paper industries and/or cellulosic ethanol biorefineries, where it is mainly used as an energy source to produce steam and electricity. Lignin is underexploited as a chemical feedstock because of its heterogeneity and intractable structure (Ayyachamy et al. 2013 ). The valorization of lignin to produce renewable fuels and chemicals is important to further develop the biorefineries (Beckham et al. 2016 ; Camarero et al. 2014 ; Rodriguez et al. 2017 ). To allow lignin to be used as a substrate for bioconversion, a depolymerization step is essential to generate a combination of monomeric, dimeric and oligomeric lignin-based compounds (Rinaldi et al. 2016 ; Xu et al. 2014 ). These can be further catabolized into various bio-based compounds of economic value for lignocellulosic biorefineries (Abdelaziz et al. 2016 ; Salvachúa et al. 2015 ). Lignin in nature is degraded mainly by extracellular peroxidases and laccases, secreted by white-rot and brown-rot fungi. The action of these enzymes on lignin results in a diverse range of low molecular weight aromatic fragments (Martínez et al. 2005 ). Due to the widespread availability of these aromatic molecules in the environment, several microbes have developed catabolic pathways for these (Makela et al. 2015 ). There have been many studies on a variety of fungal species, which can utilize lignin-related compounds with the help of redox mediators (Martínez et al. 2005 ). However, their commercial application is limited by slow growth rates and difficulties related to fungal genetic manipulation (Bugg et al. 2011a ). In addition to fungi, there are several bacterial species capable of catabolizing lignin or lignin-related aromatic compounds (Brown and Chang 2014 ; Bugg et al. 2011b ). Bacteria have certain benefits over fungi, e.g. easy genetic engineering, fast growth and ease of cultivation. Reported bacteria with ability to convert aromatics include Pseudomonas putida KT2440 (Jiménez et al. 2002 ; Ravi et al. 2017 ), Cupriavidus necator JMP134 (Pérez-Pantoja et al. 2008 ), Rhodococcus opacus (Zhao et al. 2016 ), Rhodococcus jostii RHA1 (Ahmad et al. 2011 ), Acinetobacter baylyi ADP1 (Barbe et al. 2004 ), Amycolatopsis sp. 75iv2 (Brown et al. 2011 ), Sphingomonas sp. strain SYK-6 (Masai et al. 2007 ) and Streptomyces viridosporus T7A (Ramachandra et al. 1988 ). Most likely, there are other bacteria with aromatic metabolizing capacities, which are yet to be identified. Recently, there has been an extensive search for more bacterial species from several natural or man-made environments, which exhibit certain lignin-degrading abilities thanks to the secretion of oxidoreductases, etherases and other enzymes (Picart et al. 2016 ; Taylor et al. 2012 ). Some of the reported organisms are, e.g. Klebsiella and Pseudomonas spp. from compost samples (Ravi et al. 2017 ), Cupriavidus basilensis B-8 (Shi et al. 2013 ) and Comamonas sp. B-9 from eroded bamboo slips (Chen et al. 2012 ), Bacillus pumilus and Bacillus atrophaeus from biodiversity-rich rainforest soil (Huang et al. 2013 ) and Trabulsiella sp. isolated from termite gut (Suman et al. 2016 ). It is essential not only to isolate these organisms, but also to characterize their inherent aromatic metabolism in order to assess their potential—as hosts or donor of pathways—for lignin valorization. In the present study, our initial aim was to isolate and identify easily culturable bacterial species from sediments in the Baltic Sea, close to the wastewater stream of a sulfite pulp production plant in northern Sweden (Kramfors). In this environment, lignin-rich residuals were deposited and accumulated between 1907 and 1977 (Apler et al. 2014 ). Lignin-enriched wastewater effluents have a damaging effect to the aquatic ecosystems because of their toxic chemical compounds and also due to the recalcitrance of this polymer in natural environments (Berryman et al. 2004 ). Therefore bacterial species present in such polluted sediments are likely to possess aromatic metabolic capacities, and hence are interesting to investigate (Priyadarshinee et al. 2016 ). The second objective was to further examine the metabolism of aromatic compounds by the most interesting isolates. In particular, one of the isolates, identified as Pseudomonas sp. 9.1 was grown on six lignin model compounds (ferulate, p -coumarate, benzoate, syringate, vanillin and guaiacol) representing the main branches of the upper funneling catabolic pathways (Fig. 1 ) and specific growth rates, specific uptake rates and by-product formation from the aromatic compounds were quantified. The selected isolate, under the same initial aromatic compounds concentration, was found to behave quite differently from the previously studied P. putida KT2440 (Ravi et al. 2017 ) since it excreted several pathway intermediates. Further experiments were conducted with the excreted intermediates and the conversion rates of various upper funneling branches leading to β-ketoadipate pathway were assessed. Fig. 1 Major upper funneling pathways for the bacterial metabolism of lignin model compounds. The model compounds used in this study are indicated in bold. Metabolic intermediates that can be subjected to aromatic ring cleavage are indicated in dashed boxes",
"discussion": "Discussion In the present study, a bacterial strain with extensive capacity for catabolism of aromatic compounds was isolated and physiologically characterized. The method of isolation, i.e. culture-dependent screening on either lignin or lignin-derived molecules from an interesting environment, is a widely used method to find lignin-utilizing prokaryotes (Tian et al. 2014 ) and proved successful also here. Most of the bacterial isolates (five out of nine) from the enrichment cultures carried out in this study, belong to the γ - proteobacteria class, particularly to the genus Pseudomonas , the versatility of which to degrade and grow on lignin-related aromatic compounds and even technical lignins is widely acknowledged (Bandounas et al. 2011 ; Bugg and Rahmanpour 2015 ; Narbad and Gasson 1998 ). Three Gram-positive isolates from the Bacillaceae family were also detected by this growth-dependent method, namely Bacillus licheniformis , B. safensis and Lysinibacillus macroides . Many species of the genus Bacillus were previously found in deep-sea sediments in a similar screening carried out by Ohta et al. ( 2012 ), and most of them were also metabolically active toward lignin-related aromatic compounds. Finally, one Rhodococcus erythropolis strain, an actinomycete belonging to the high G+C Gram positive bacteria, was found as well. This is not surprising, since a number of rhodococci including R. erythropolis have been described among the most efficient bacterial lignin degraders (Ahmad et al. 2010 ) and are also able to metabolize the low-molecular weight compounds coming from lignin degradation (Taylor et al. 2012 ). Some of the bacterial isolates found in this work did not show ability to grow in liquid cultures with the model compounds utilized for their isolation (Additional file 1 : Figs. S1 and S2). The initial growth on the screening plates might be due to the presence of intracellular storage compounds accumulated during the revitalization stage in rich medium. Characterizing the aromatic metabolism, both in terms of catabolic pathways and the capacities of these pathways, is important to enable the design of efficient lignin conversion processes. The growth on low-molecular-weight lignin model compounds is relevant since the catabolism of such compounds is essential after, or along with, lignin mineralization (Bandounas et al. 2011 ). In the present study, six aromatic model compounds from the main different upper funneling pathways of lignin conversion were selected as carbon sources: vanillin, guaiacol and ferulate from the coniferyl branch; p -coumarate from the coumaryl branch; benzoate from the benzoyl branch and syringate from the sinapyl branch (Fig. 1 ). The coniferyl and coumaryl branches converge at protocatechuate (except for guaiacol which is converted to catechol), the benzoyl branch continues through catechol, whereas the sinapyl branch proceeds down to 3- O -methylgallic acid or even further to gallic acid. Protocatechuate and catechol enter the β-ketoadipate pathway ending with acetyl-CoA and succinyl-CoA, while 3- O -methylgallic acid/gallic acid undergoes ring cleavage and degrades to oxaloacetic acid and pyruvic acid. All these products either are the TCA (tricarboxylic acid) cycle intermediates or eventually enter the TCA cycle to promote cell growth. The isolate 9.1, tentatively identified as P. deceptionensis was able to take up four out of the six tested lignin model compounds as sole sources of carbon. The highest specific growth and uptake rates were measured for benzoate (Table 4 ), indicating that the benzoyl branch is the fastest of the upper funneling pathways in this isolate. The second fastest uptake occurred in the p -coumaryl branch with uptake rates of p -coumarate and 4-HBA around 2.9 mmol g CDW −1 h −1 . The coniferyl branch operated at a somewhat lower flux rate, with uptake rates of ferulate, vanillate and vanillyl alcohol lower than 2 mmol g CDW −1 h −1 . This might be due to the presence of phenylmethyl ether linkage in the coniferyl branch, which is comparatively difficult to breakdown (Okamura-Abe et al. 2016 ). No growth was obtained for syringate and guaiacol. The difficulty in metabolizing highly methoxylated substrates such as those present in the syringyl branch is most likely due to the complex enzymatic systems required for its demethylation. The O -demethylase systems for dimethoxylated compounds are well described in the case of Sphingobium sp. SYK-6, Acetobacterium woodii , Acetobacterium dehalogenans and Moorella thermoacetica (Berman and Frazer 1992 ; Kaufmann et al. 1998 ; Naidu and Ragsdale 2001 ). They consist of two methyl transferases which convey the methyl groups from the aromatic substrate (e.g. veratrol, syringate) to an accepting cofactor like tetrahydrofolate (H 4 F), often with the involvement of a corrinoid protein. These systems usually show relatively high substrate specificity, and also require continuous supply and enzymatic recycling of the cofactor; moreover, when a corrinoid protein is involved, spontaneous oxidation of its cobalt group can take place, and ATP-consuming enzymatic regeneration of this oxidized catalyst will be necessary (Siebert et al. 2005 ). Additionally, the demethylation reactions can result in the formation of formaldehyde or formic acid (Masai et al. 2007 ; Overhage et al. 1999 ), toxic intermediates that require further processing (Hibi et al. 2005 ), which can contribute to the slower metabolism of these compounds (Additional file 1 : Figs. S3 and S4). There was a short lag phase of approximately 15 h for ferulate, p -coumarate and benzoate (Fig. 2 ), necessary for adaption to the new carbon source and expression of required enzymes after prior growth on glucose (Kurosawa et al. 2015 ). A much longer lag phase, around 100 h, for growth on vanillin (Fig. 2 ) and no growth on syringate for 200 h, was observed. The antimicrobial effect of both compounds is well documented for many organisms. The concentration of vanillin and syringate required to inhibit the growth the Escherichia coli LY01 by 50% is 0.9 and 1.6 g/L respectively (Zaldivar et al. 1999 ). The mechanism of inhibition can be either bacteriostatic or bactericidal. The increased uptake of nucleic acid stain upon exposure of E. coli to vanillin suggests that the integrity and stability of the cell membrane was affected, leading to cell lysis (Fitzgerald et al. 2004 ). In another study, the addition of syringic acid at the concentration of IC 80 (80% inhibitory concentration), proved to destroy the membrane integrity by increasing membrane leakage by three fold (Zaldivar and Ingram 1999 ). However, in our case, no major cell lysis (i.e. no decrease in OD) was observed when exposed to vanillin or syringate, which suggests that the inhibition was bacteriostatic. A quite long lag phase before growth on vanillin, might explain the time required by the organism to repair the cell damage and overcome the inhibition (Rolfe et al. 2012 ). Interestingly, Pseudomonas sp. isolate 9.1 while growing on some model compounds excreted certain pathway intermediates. This contrasts to previous observations with P. putida strains (Ravi et al. 2017 ). It appears that isolate 9.1 is not regulated to maintain the same flux throughout the pathway to the same extent as P. putida . With the exception of cis,cis -muconate, all the excreted intermediates were later consumed, thereby enabling the complete utilization of the carbon source, although in a di- or tri-auxic growth pattern. Cis,cis -muconate and catechol were excreted during growth on benzoate, but while catechol was subsequently taken up, cis,cis -muconate remained unconsumed (Fig. 2 c). Possibly Pseudomonas sp. isolate 9.1 lacks or has a defective version of the gene encoding the transporter involved in the uptake of cis,cis -muconate, which in Acinetobacter and other Pseudomonas strains has been identified as mucK (Williams and Shaw 1997 ), a member of the family 15 of MFS transporters (Pao et al. 1998 ). It is not clear how the accumulated cis , cis -muconate is excreted outside of the cell without a functional transporter, but there may be other transporters able to move this compound across the cell membrane with lower specificity. Cis,cis -muconate can be used as a precursor to adipic acid, one of the monomers in nylon-6,6 (Vardon et al. 2015 ) and has therefore attracted quite some interest. Bioconversion of benzoate to cis,cis -muconate was explored in P. putida KT2440-JD1 (van Duuren et al. 2012 ). Also in this P. putida strain, the maximum uptake rate of benzoate was higher than the production rate of cis,cis -muconate, which is the reason for the excretion of benzoate derivatives in the cells. Excessive uptake of benzoate is also a reason for the excretion of catechol, which reduces the expression of the ben operon leading to the decreased conversion of benzoate to cis,cis -muconate (van Duuren et al. 2012 ). Accumulation of catechol in the culture media might be toxic to the organism since catechol is unstable at pH 7 and produces reactive oxygen species. The oxidized species spontaneously form colored polymers (Subramanian and Worden 1993 ; Sudarsan et al. 2016 ; Yang et al. 2014 ), which give experimental problems to study catechol as a sole source of carbon in growth experiments, also in the present work. Somewhat unexpectedly, vanillyl alcohol was excreted during growth of Pseudomonas sp. isolate 9.1 on vanillin (Fig. 2 vanillin). As vanillin is a potential inhibitor to many microorganisms, the conversion of vanillin to less toxic intermediates is quite common (Gallage and Møller 2015 ). In bacteria, especially pseudomonads, vanillin is normally converted to vanillate (Graf and Altenbuchner 2014 ; Ravi et al. 2017 ). However, under growing conditions, P. fluorescens B56 was able to convert vanillin to vanillate with a small amount of vanillyl alcohol being excreted (Asm et al. 1988 ). In addition, a genetically modified Pseudomonas strain (vanillin dehydrogenase deletion mutant) was able to produce vanillyl alcohol via ferulic acid and vanillin, when eugenol was given as a sole carbon source (Takasago Perfumery Co Ltd 1993 ). Although the intention of genetic modification was to produce vanillin from eugenol, the strain further converted some amount of vanillin to vanillyl alcohol, which proves the existence of such activity under certain conditions. The conversion of vanillin to vanillyl alcohol is more common as a part of the detoxification process in eukaryotes such as Phanerochaete chrysosporium or Saccharomyces cerevisiae , (Nguyen et al. 2015 ; Stentelaire et al. 1997 ). The uptake rate of vanillin by isolate 9.1 was 3.19 mmol g CDW −1 h −1 , whereas the uptake of vanillate was only 1.95 mmol g CDW −1 h −1 . The excess uptake of vanillin was apparently channeled to vanillyl alcohol, which was subsequently giving rise to a diauxic growth pattern. Another observation was that Pseudomonas sp. isolate 9.1 had a relatively stable stationary phase after the carbon source was depleted (Figs. 2 , 3 ). This is in contrast to our previous work with P. putida KT2440, in which case there was a rapid decrease in the biomass concentration directly after the depletion of the carbon source without a stable stationary phase (Ravi et al. 2017 ). In conclusion, the isolate 9.1, tentatively identified as Pseudomonas deceptionensis based on gyrB alignment, appears to possess most of the major upper funneling pathways necessary for the conversion of lignin-related aromatic compounds. Growth on model compounds does not mean that the organism will necessarily convert lignin or depolymerized lignin. However, it is a prerequisite for such conversion to occur. Further work will show if this isolate or other bacterial strains are able to metabolize or partially convert different types of depolymerized lignin, which is necessary for the establishment of efficient processes in the future biorefineries, paving the way for a complete exploitation of the forestry and agricultural resources."
} | 4,849 |
35630551 | PMC9143230 | pmc | 2,588 | {
"abstract": "Zwitterionic polymers as crucial antifouling materials exhibit excellent antifouling performance due to their strong hydration ability. The structure–property relationship at the molecular level still remains to be elucidated. In this work, the surface hydration ability of three antifouling polymer membranes grafting on polysiloxane membranes Poly(sulfobetaine methacrylate) (T4-SB), poly(3-(methacryloyloxy)propane-1-sulfonate) (T4-SP), and poly(2-(dimethylamino)ethyl methacrylate) (T4-DM) was investigated. An orderly packed, and tightly bound surface hydration layer above T4-SP and T4-SB antifouling membranes was found by means of analyzing the dipole orientation distribution, diffusion coefficient, and average residence time. To further understand the surface hydration ability of three antifouling membranes, the surface structure, density profile, roughness, and area percentage of hydrophilic surface combining electrostatic potential, RDFs, SDFs, and noncovalent interactions of three polymers’ monomers were studied. It was concluded that the broadest distribution of electrostatic potential on the surface and the nature of anionic SO3- groups led to the following antifouling order of T4-SB > T4-SP > T4-DM. We hope that this work will gain some insight for the rational design and optimization of ecofriendly antifouling materials.",
"conclusion": "4. Conclusions In this work, we investigated the surface hydration of three antifouling membranes—T4-DM, T4-SP, and T4-SB—by a series of molecular dynamics simulations. Dipole orientation distribution, diffusion coefficient, and average residence time revealed an orderly, packed, and tightly bound surface hydration layer above T4-SP and T4-SB antifouling membranes. The surface structure, density profile, surface roughness, and area percentage of hydrophilic surface provide further details regarding the strong surface hydration of T4-SP and T4-SB from the membranes’ aspect. The side chains of T4-SP and T4-SB were more stretched in hydrated state due to their high hydration ability, which can cause steric repulsion and prevent adsorption. Their surfaces are relatively rough, which can bind much more water or even let water penetrate into the internal voids of the membrane. To further understand the surface hydration ability of three antifouling membranes, solvation free energy, electrostatic potential, RDFs, SDFs, and noncovalent interactions of three monomers were analyzed. T4-SB monomer has the broadest distribution of electrostatic potential on the surface, resulting from the separated negatively and positively charge center and largest water coordination number for its zwitterionic architecture. Its exposed negative charge center SO 3 − group can form hydrogen bonds with surrounding water molecules and the shielded positive charge center N can also bind water molecules through weak vdW interaction. The simulation data suggest the hydration ability of monomers ranks in terms of T4-SB > T4-SP > T4-DM. Since the surface hydration layer serves as a physical and energy barrier during the prevention of protein adsorption, we believe their antifouling ability ranks in terms of T4-SB > T4-SP > T4-DM, which is consistent with experiments.",
"introduction": "1. Introduction The adsorption and accumulation of fouling organisms on surface of materials, i.e., marine biofouling, is a major problem faced by ships and offshore facilities [ 1 , 2 ]. The annual cost of increased fuel consumption, cleaning, maintenance, and repair of ships caused by marine biofouling is as high as billions of dollars [ 3 , 4 ]. Early marine antifouling coatings mainly used biotoxic tributyltin (TBT) antifouling paints, which killed marine organism larvae or spores through the release of antifouling agents to achieve antifouling purposes [ 5 , 6 ]. However, traditional antifouling paints are highly toxic for many aquatic organisms and have caused severe damage to the environment. The development of ecofriendly antifouling coatings is gradually becoming a research hotspot in this field [ 7 , 8 , 9 ]. Among them, protein-resistant antifouling material that inhibits the settlement of proteins is a relatively promising one [ 10 ], such as poly (ethylene glycol) (PEG), zwitterionic polymers [ 11 ] (poly (Sulfobetaine methacrylate), pSBMA, or poly (Carboxybetaine methacrylate), pCBMA). For example, Jiang’s group [ 12 , 13 , 14 ] has been engaged in biofouling research for a long period and synthesized a series of zwitterionic polymers. On the one hand, they used molecular simulation methods to reveal the antifouling mechanism of materials on the microscopic level. On the other hand, they carried out application research on this basis to design and synthesize new antifouling materials. Zheng and coworkers [ 15 , 16 , 17 ] investigated the antifouling properties of zwitterionic polymer brushes, polyacrylamide, and hydroxyalkyl acrylamides using combined molecular dynamics and steered molecular dynamics, believing that the carbon space and anionic groups have distinct effects on their antifouling performance. The state key laboratory of marine corrosion and protection in China has also synthesized a series of antifouling coatings by grafting zwitterionic sulfobetaine methacrylate (T4-SB) or anionic sulfonate methacrylate (T4-SP), which have the property of inhibiting adsorption of proteins on the surface of polysiloxane material (T4). These materials have a good antifouling effect on fouling organisms such as diatoms. We found that the static adsorption number of diatoms in the T4-SP antifouling material is 15/mm 2 (4% of the T4 antifouling material) in the experiment; for T4-SB, the static adsorption number of diatoms is 9/mm 2 (2% of the T4 antifouling material), which significantly improved the antifouling performance of the silicone material. The adsorption of protein on surface is affected by many factors [ 18 , 19 , 20 , 21 ], among which the factors favorable for adsorption mainly include the enthalpy loss from the van der Waals and electrostatic attraction between protein and surface, and the entropic gain from the removal of hydration layer at the surface of material and protein. The disadvantages include the enthalpy gain required for the dehydration of surface and protein, protein’s conformation adjustment, as well as the entropic loss from protein adsorption and exposure of hydrophobic regions. The hydration layer above the surface of the antifouling material plays a crucial role from the antifouling perspective [ 22 ] because it provides the physical and energy barriers that must be overcome during protein adsorption. To confirm the structure of the hydration layer above the surface of antifouling materials, many experimental studies have been carried out. For example, Leng et al. [ 23 , 24 ] confirmed that there is a tightly bounded and regularly ordered hydration layer above zwitterionic antifouling membrane compared with polymer membrane without antifouling ability using sum frequency generation (SFG) vibrational spectroscopy. Paul et al. [ 25 ] directly observed the structure of hydration layer above the surface of epoxy organosilane modified silica nanoparticles and unmodified silica nanoparticles by frequency modulation−atomic force microscopy. Combined with molecular dynamics simulations, a more continuous and thicker hydration layer structure was found on the surface of modified silica particles, which endows the material with a better antifouling ability. In this work, we will compare the antifouling ability of three polymer antifouling membranes (T4-DM, T4-SP, T4-SB) using molecular dynamics simulation at the molecular level through the hydration layer. We hope this work will provide theoretical support for the subsequent design and optimization of related antifouling materials.",
"discussion": "3. Results and Discussion 3.1. Properties of Antifouling Membranes 3.1.1. Density Profiles The simulated configurations of three antifouling membranes at dry and hydrated states are illustrated in Figure S1 . We can clearly see that there are no significant differences between T4-DM membrane under dry and hydrated states, while for T4-SP and T4-SB membranes, many side chains extend to water phase. This indicates that the side chains of T4-SP and T4-SB have a better hydrophilicity. Besides this, the compression of these chains during adsorption of foulant would reduce the conformation possibility, which is entropically unfavorable, subsequently causing steric repulsion and preventing adsorption [ 10 ]. To quantitatively study the structure of three antifouling membranes, the density profile along z-axis was calculated, as shown in Figure 3 . The results were derived from the last 5 ns trajectory. The density profile was symmetrized around the membrane center to obtain a better result. The density profiles of T4-DM in dry and hydrated states almost overlapped. As for T4-SP and T4-SB membranes, the density profile of hydrated state broadened compared with that of dry state (more obvious for T4-SB membrane), which is consistent with the configurations in Figure S1 . The density of water in T4-SB is higher than that of T4-SP, and even higher than that of T4-DM, which indicates that the side chains of T4-SB can attract extra water molecules compared with those of T4-SP and T4-DM. We then deduce that the hydrophilicity of the antifouling membranes follows the order of T4-SB > T4-SP > T4-DM. 3.1.2. Surface Roughness Since the density profile is a statistical average of the entire membrane layer, it cannot reflect the local specific structural information of membranes. To further analyze the detailed surface structure, contour maps of the upper surface of three antifouling membranes in hydrated states were sketched, as shown in Figure 4 . To define the surface of membrane, the simulation box was divided into grids with 0.4 nm × 0.4 nm resolution in xy plane. Atoms with the largest or smallest z-axis were selected as the top atoms to define the membrane surface. It can be seen from Figure 5 that T4-DM membrane’s surface is relatively flat, while T4-SP has more peaks and valleys than T4-DM. As for T4-SB, the contour lines are the densest, indicating that the order of surface roughness is T4-SB > T4-SP > T4-DM. To quantify the surface roughness of three antifouling membranes, the root mean square roughness R was introduced [ 37 ]: R = ∑ i = 1 N ( Z i − Z ¯ ) 2 N \nwhere Z i is the z-coordinate of the atoms exposed in the outermost layer in each grid point and Z ¯ is the average value of the z-coordinates of all the atoms exposed on the outermost surface. Both the up and down surfaces of three antifouling membranes in dry and hydrated states are calculated and listed in Table 1 . The data suggested there is little difference between dry and hydrated states for T4-DM. The roughness in hydrated state follows the order of T4-SB > T4-SP > T4-DM, which is consistent with Figure 3 and Figure 5 . Obviously, the greater the roughness of the surface, the more hydrophilic sites were exposed, and the more water molecules could be bound. 3.1.3. Hydrophilicity In addition to the influence of surface roughness on surface hydration, the hydrophilicity and hydrophobicity of the surface determine the surface hydration ability directly. The hydrophilic and hydrophobic surface area of each antifouling membrane were calculated from the last 5 ns trajectory, as shown in Figure 5 . During calculation, the atomic charge between −0.2 and 0.2 was considered as the hydrophobic surface area, and the other is the hydrophilic surface area. The hydrophilic surface area and its proportion of all three antifouling membranes increased in hydrated state. The total surface area does not change much between dry and hydrated states, which is consistent with the configuration in Figure S1 . The total surface area, especially the hydrophilic surface area, of T4-SP and T4-SB both increased significantly when immersed in water, which suggests that they have a strong hydration ability. 3.2. Properties of Surface Hydration Layer 3.2.1. Structural Properties After the above structural analysis of the antifouling membranes, it was found that the surface hydration ability of the three antifouling membranes was T4-SB > T4-SP > T4-DM. We also noticed that with the increase in surface hydration ability, more water molecules can penetrate into the matrix of membrane from the density profiles in Figure 3 . To examine the structure of water molecules near the interface of antifouling membranes, we calculated the cosine of the angle between dipole of water and z-axis at different distances from the surface, as shown in Figure 6 . Obviously, for a random distribution, the cos θ should be close to 0 [ 38 ]. In the T4-DM membrane system, only water molecules close to membrane have a certain orientation, while water molecules farther away are randomly distributed. In the T4-SP system, the dipole orientation of surface water molecules slightly decreased to 0 after 2 nm, while in the T4-SB system, there is still a long-distance arrangement of water molecules even beyond 2 nm away from the surface. This observation is consistent with Leng’s experiment [ 23 , 24 ], where ordered water molecules were found at zwitterionic pSBMA surfaces. 3.2.2. Dynamic Properties The antifouling membranes can also affect the hydration layer’s dynamic properties beside the structure of surface water molecules. We calculated the distribution of the average residence time of water molecules within 0.5 nm of antifouling membrane surfaces, as shown in Figure 7 b. The average residence time means how long water molecules can stay near the surface of the antifouling membrane on average [ 39 ]. It reflects the stability of the hydration water layer of the antifouling membrane or, in other words, the hydration ability of antifouling membranes [ 40 ]. Figure 7 a shows the trajectory of one hydration layer water molecule above T4-SB membrane. The calculated average residence time is shown in Table 2 . It can be seen that the average residence time increased from T4-DM and T4-SP to T4-SB, indicating that the binding effect of antifouling membranes on their surface hydration layers increased. The diffusion behavior of surface hydration layer water molecules above three antifouling membranes was investigated. The mean square displacement (MSD) of surface hydration layer water molecules is shown in Figure 8 . Their diffusion coefficients were then calculated according to Einstein’s equation and collected in Table 2 . It can be seen that the diffusion coefficients of surface hydration layer water molecules above three antifouling membranes gradually decreased from T4-DM and T4-SP to T4-SB, indicating that the mobility of water molecules decreased or the binding effect from the antifouling membranes increased, which is consistent with the previous analysis. 3.3. Hydration Mechanisms—From the View of Monomers 3.3.1. Solvation Free Energy We have analyzed and compared the structural properties of the antifouling membranes and the structural and dynamic properties of their hydration water layers from the overall antifouling membranes’ view. The order of surface hydration ability or antifouling ability, T4-SB > T4-SP > T4-DM, was obtained. Next, we analyze the mechanisms for the difference in hydration ability from the monomer’s view, which serves as a model for the antifouling polymer membrane [ 38 ]. Solvation free energies were calculated for three monomers at M05-2X/6-31 g* level, as collected in Table 3 . The negative of solvation free energy indicates all three monomers have a high affinity with water. The order of solvation free energy follows the order of T4-SB > T4-SP >> T4-DM, which is consistent with previous analysis. 3.3.2. Electrostatic Potential Electrostatic potentials of the three monomers were calculated and mapped on their van der Waals surfaces [ 41 ], as shown in Figure 9 . The molecular polarity, polar, and nonpolar surface area were also calculated, as shown in Table 3 [ 42 ]. The surface area with |ESP| <= 10 kcal/mol was considered as nonpolar surface area while the others were considered as polar surface area. It can be seen that the negative charge center of T4-DM monomer is located at the N atom. Since T4-SP monomer has a negative charge, the overall electrostatic surface is negative, and mainly concentrated on the sulfonate group. In the zwitterion T4-SB monomer, the negative charge center is located in the sulfonate group and the positive charge center is located at the N atom. Though the MPI of T4-SP was the largest, the T4-SB has the largest polar surface area, which can combine with more water. Combining with the distribution of areas occupied by different electrostatic potential regions in Figure 9 b, it can be seen that the distribution of electrostatic potential on the surface of T4-SB monomer is the broadest, which is conducive to the electrostatic interaction with other polar molecules such as water [ 43 ]. 3.3.3. Radial Distribution Function To further understand the hydration ability of antifouling polymers’ monomers, another molecular dynamics simulation was conducted. Three monomers were solvated in 4 × 4 × 4 nm 3 water box, respectively; then, 50 ns NPT simulations were performed. After that, the radial distribution functions (RDFs) of the water molecules or Na + around the polar groups of three monomers and their coordination number were calculated, respectively, as shown in Figure 10 . The RDFs can reflect the intermolecular structure and interactions between center atoms and surrounding water molecules. Two peaks were found in the RDF curve, indicating that two hydration layers were formed, which corresponded to the first hydration layer that consisted of bound water and the second hydration layer made up of trapped water; this agrees with Paul’s experiment [ 25 ]. According to Figure 10 a,b, SO 3 − groups in T4-SP and T4-SB have similar hydration ability and are stronger than the N group in T4-DM and T4-SB. Meanwhile, the peaks of g(r) N-OW in T4-DM were lower than those in T4-SB and also the coordination number of the first hydration shell from Figure 11 c,d, indicating a better packed hydration shell around N in T4-SB. The number of water molecules tightly bonded to three monomers were also calculated and collected in Table 3 . Consequently, the T4-SB antifouling membrane presents a more hydrophilic behavior than T4-SP and T4-DM. 3.3.4. Spatial Distribution Function Though the RDFs can reflect the hydration effect of hydrophilic groups in three monomers on water molecules, the calculation of RDFs is based on the spherical averaging of the water molecules around the hydrophilic group, which neglects the spatial distribution of the water molecules. Therefore, the spatial distribution function (SDF) of water molecules around hydrophilic groups was calculated, shown in Figure 11 . From this, we can see that there is only a ribbonlike distribution around the carbonyl oxygen in DM monomer, while the distribution of water molecules around the N atom cannot be shown under current isosurface. In the SP monomer, there are three spherical crown water molecule distribution areas in the direction of three S–O bonds, which is obviously caused by the hydrogen bond formed between the O atom in SO 3 − group and the water molecules. Similar structures were also found in SB monomer. Besides this, there is a ribbonlike distribution of water molecules around the N atom. 3.3.5. Noncovalent Interactions To fundamentally understand the different hydration ability of three antifouling monomers, aNCI (averaged noncovalent interaction) analysis [ 44 , 45 ] was conducted, shown in Figure 12 . The green area in the figure indicates that van der Waals interaction is dominant. Blue area indicates that there is a strong hydrogen bond interaction. The red area indicates that there is a strong steric hindrance effect. In DM monomer, as the negative charge center N atom was shielded by surrounding methyl groups, it can only interact with water molecules through weak vdW interactions. In T4-SP and T4-SB monomers, water molecules can directly form hydrogen bonds with the exposed O atoms, which plays a key role in their strong hydration ability. Besides that, the extra positive charge center N atom can also interact with water molecules through weak vdW interactions such as N in the T4-DM monomer. Therefore, the hydration abilities of three antifouling polymers are in the order of T4-SB > T4-SP > T4-DM."
} | 5,149 |
38594291 | PMC11004001 | pmc | 2,589 | {
"abstract": "We demonstrate a highly biomimetic spiking neuron capable of fast and energy-efficient neuronal oscillation dynamics. Our simple neuron circuit is constructed using silicon–germanium heterojunction based bipolar transistors ( HBT s) with nanowire structure. The HBT has a hysteresis window with steep switching characteristics and high current margin in the low voltage range, which enables a high spiking frequency (~ 245 kHz) with low energy consumption (≤ 1.37 pJ/spike). Also, gated structure achieves a stable balance in the activity of the neural system by incorporating both excitatory and inhibitory signal. Furthermore, inhibition of multiple strengths can be realized by adjusting the integration time according to the amplitude of the inhibitory signal. In addition, the spiking frequency can be tuned by mutually controlling the hysteresis window in the HBT s. These results ensure the sparse activity and homeostasis of neural networks.",
"conclusion": "Conclusions We have successfully developed a highly biomimetic spiking neuron composed of four components. The heterogeneous bandgap structure of HBT results in the formation of hysteresis with high current margin in the low voltage region. By taking advantage of these hysteresis characteristics, the periodic IF behavior can be operated reliably at high frequency (~ 245 kHz) with low energy consumption (≤ 1.37 pJ/spike). Through modulation of inhibitory signals, inhibition is implemented in multiple strengths, thereby effectively regulating excessive firing. Additionally, the threshold can be adjusted to modulate the spiking frequency by controlling the gate bias of HBT s. These features play an important role in the sparse activity and homeostasis of neural networks. Consequently, our developed neuron can be a strong candidate for realizing fast and energy-efficient neuromorphic systems.",
"introduction": "Introduction Brain-inspired spiking neural networks (SNNs) have emerged as a promising platform for neuromorphic hardware due to their remarkable energy efficiency 1 – 3 . In SNNs, numerous spiking neurons act as the basic information processing unit of SNNs and transfer signals between synapses. Therefore, spiking neurons with highly compact and energy efficiency are crucial to implement SNNs in hardware. In addition, to enhance the performance of SNNs, several spike-based coding techniques and architectures have implemented biomimetic functions at the neuron level. Inhibition can prevent overfitting of neural networks by suppressing the firing rates of highly activated neurons. This helps the network generalize better to new inputs 4 – 6 . Another essential function, namely the tunable threshold, can induce sparse activity in SNNs by emulating the brain stimulus activation. This enables dynamic modulation of neural coding precision, potentially saving significant energy by selectively increasing firing rates only at specific times and locations as required 7 – 9 . In addition, this function provides robust immunity against artificial neurons with threshold deviations, ensuring the homeostasis 10 – 12 . The complex neuronal behavior has been emulated through CMOS-based circuits, which typically consist of numerous transistors and capacitors, requiring a large footprint area and power consumptions 13 – 16 . To overcome these problems, spiking neurons with simple structures have been reported by applying various silicon 17 – 25 and non-silicon devices 26 – 29 . PD-SOI MOSFET based neurons provided a means of incorporating integration and threshold triggering operation using the floating body effect without a capacitor 19 – 22 . However, these neurons require external circuit for signal conversion and reset process, which results in large power consumption 30 . Recently, the single MOSFET neuron devices have been reported that can realize neuronal behavior without both capacitors and external circuitry. However, these single-device neurons consume large power and have small internal capacitance, limiting their ability to integrate large amounts of synaptic signals 23 – 25 . Non-silicon devices such as memristors and ferroelectric field effect transistor (FeFET) neurons have also been reported due to their steep switching characteristics and scalable structures. However, these neurons have difficulties in controlling their properties consistently in large-area fabrication. Moreover, the resistance change according to the constant voltage pattern is non-linear, which can make practical application difficult 26 – 29 . In conclusion, these reported spiking neurons still operate with large energy consumption for periodic neural oscillations incorporating biomimetic functions. In this work, we proposed a novel spiking neuron using silicon–germanium (SiGe) based heterojunction bipolar transistors ( HBT s) for low energy applications and implementation of biomimetic functions. Our simple spiking neuron consists of four components (2 HBT s, 1 resistor, and 1 capacitor) to realize the periodic integrate-and-fire (IF) behavior without external reset circuit. The latch-up voltage, voltage width and current gain were investigated according to germanium content of p -base region. The hetero-bandgap structure of HBT amplifies the positive feedback gain of hysteresis in the low voltage range through improved hole storage capability and impact-ionization coefficient. This hysteresis characteristic enables integration, threshold triggering, and self-reset processes to run entirely within a low voltage range, resulting in low spiking energy consumption. The hysteresis properties of HBT were utilized to analyze neuronal function for various synaptic inputs. The inhibition of multiple strengths was demonstrated though the control of firing latency achieved by modulation of inhibitory signals. Furthermore, the spiking frequency was tuned by controlling the voltage width of hysteresis of HBT s."
} | 1,477 |
35024581 | PMC8733261 | pmc | 2,590 | {
"abstract": "Summary Polymer memristors are preeminent candidates for low-power edge computing paradigms. Poly[chalcogenoviologen- alt -triphenylamine] (PCVTPA) has been synthesized by direct coupling of chalcogeno-viologen as electron acceptor and 4-(bromomethyl)-N-(4-(bromo-methyl)phenyl)-N-phenylaniline as electron donor. The introduction of chalcogen atoms (S, Se, Te) into viologen scaffolds can greatly improve electrical conductive, electrochemical, and electrochromic properties of the materials when compared with the conventional viologens. Taking PTeVTPA as an example, the as-fabricated electronic device with a configuration of Al/PTeVTPA/ITO exhibits excellent multilevel storage and history-dependent memristive switching performance. Associated with the unique memristive behavior, the PTeVTPA-based device can not only be used to emulate the synaptic potentiation/depression, the human's learning and memorizing functions, and the transition from short-term synaptic plasticity to long-term plasticity but also carry out decimal arithmetic operations as well. This work will be expected to offer a train of new thought for constructing high-performance synaptic biomimicking and neuromorphic computing system in the near future.",
"conclusion": "Conclusions A proof-of-concept PCVTPA-based polymer memristor with a configuration of Al/PCVTPA/ITO exhibited excellent history-dependent memristive switching and multilevel storage performance. Associated with the unique memristive behavior, the as-fabricated device can be used to not only emulate the human's learning and memorizing functions but also execute decimal arithmetic operations of addition, subtraction, multiplication, and division. These results demonstrated that the PCVTPA polymer showed a great potential in constructing high-performance intelligent computing system in the near future.",
"introduction": "Introduction The human memory mainly benefits from the natural evolution of neural networks that exhibit several outstanding properties such as massive parallel processing, in-memory computing architecture, event-driven operation and others. Since the discovery of the first real memristor at HP Labs in 2008 ( Strukov et al., 2008 ), great efforts have been devoted to developing novel memristive functional materials and devices to construct artificial neural networks for neuromorphic computation and emulate the physiological functions (e.g., the learning, memorizing, forgetting, decision-making, and judging actions) of biological synapses ( Chen et al., 2014 ; Zhang et al.,2018 , Zhang et al., 2019 , 2020 , Zhang et al., 2021 ; Van De Burgt et al., 2018 ; Choi et al.,2018 , 2020 ; Liu et al.,2016 , 2018 ; Li et al., 2017 ; Wan et al., 2020 ; Kim et al., 2018 ; Wang et al., 2014 , 2015 ; Ren et al., 2020 ; McFarlane et al., 2020 ). Recently, it was found that the negative photoconductance effect observed impressively in the high resistance state branch of the resistive switching memory enabled the memristor function to be extended to both memory logic display and multistate data storage ( Zhou et al., 2021 ). The combination of multiple physical properties (memristive and capacitive) in a single device could also prefigure potential multifunctional applications ( Sun et al., 2020 ). The memristor-based brain-like intelligent computing system not only can improve significantly the computing capability of modern computer systems via massive parallelism at very low power consumption but also can overcome the von Neumann bottleneck (i.e., the limited throughput between the memory and central processing unit) when dealing with data-intensive tasks. A large number of inorganic materials are used to construct the memristor devices and still play a dominant role in fabricating memristors with superior processing efficiency and enormous storage capacity so far ( Choi et al., 2018 ; Li et al., 2017 ; Wan et al., 2020 ; Kim et al., 2018 ). In comparison with the inorganic and organic small molecule counterparts, polymer materials show higher intrinsic flexibility and ease for solution processing. The solution processing capability allows them to be handled via low-cost techniques of dip coating, blade casting, spray coating, spin coating, roller-coating, and even ink-jet printing, making the fabrication procedure of electronic and optoelectronic devices potentially more economical without the involvement of vacuum deposition procedure ( Chen et al., 2014 ; Liu et al., 2018 ; Ling et al., 2008 ; Lin et al., 2014 ; Liu and Chen, 2011 ). Since 2005, polymer memories have been proposed to revolutionize electrical applications by providing extremely inexpensive, lightweight, and transparent modules that can be fabricated onto plastic, glass, or the top layer of the complementary metal-oxide semiconductor circuits. Similar to these inorganic materials, some polymer functional materials, including metal-containing polymers, polymer-based multi-component redox systems, and pure polymers, have also been found to exhibit excellent memristive performance in recent years ( Chen et al., 2014 ; Zhang et al.,2018 , Zhang et al., 2019 , 2020 , Zhang et al., 2021 ; Wang et al.,2014 , 2015 ; Liu et al., 2016 ; Ren et al., 2020 ; McFarlane et al., 2020 ; Pincella et al., 2011 ; Bandyopadhyay et al., 2011 ). With these polymer memristors, one can observe nonlinear transmission characteristics similar to that of a biological synapse. More importantly, their memristive performance can be easily tuned through innovative molecular design cum synthesis strategy. Polymer memristors are preeminent candidates for low-power edge computing paradigms. In our pioneering works, we reported a proof-of-concept polymer memristive processing-memory unit that demonstrates programmable information storage and processing capabilities ( Zhang et al., 2019 ). By using polyfluorene functionalized with triphenylamine and ferrocene moieties side chains as an active layer, the as-fabricated electronic device, which shows excellent memristive switching and multilevel memory performance, is capable of executing multilevel memory; decimal arithmetic operations of addition, subtraction, multiplication, and division; as well as simple Boolean logic operations. Recently, we designed and synthesized a two-dimensional conjugated polymer PBDTT-BQTPA ( Zhang et al., 2021 ). This material-based electronic device exhibited a rapid transition from the OFF state to the ON state within ∼32 ns. More importantly, we successfully achieved a record high 90% production yield of polymer memristors with low power and miniaturization potentials. The reliability of the as-fabricated memristor was greatly enhanced by the delocalized resistive switching, making downscaling of the device to 100-nm scale possible for low-power edge computing applications. We also observed for the first time the self-rectified memristive effect in a metal-free soluble organic oligomer PFD-8CN ( Wang et al., 2015 ). It would be quite desirable to integrate programmable multilevel storage, synaptic biomimicking, and neuromorphic computing in the same electronic device. As one of the most promising candidates for energy, electrochromism, gas separation and storage, and electronic and optoelectronic applications, viologens (Vs) composed of conjugated bi-/multipyridyl groups show a number of unique properties such as reversible redox behavior, ionic and localized conjugation, bio-sensitive response, and electrochromic and radical-rich features ( Mi et al., 2019 ; Kortz et al., 2019 ; Ding et al., 2019 ; Zhang et al., 2017 ; Yang et al., 2021 ; Sluysmans et al., 2020 ; Lipke et al., 2017 ; Li et al.,2018 , 2019 ). For reduction of Vs, all of their three reductive states (neutral, radical cation, and dication) are thermodynamically stable. Also, Vs can be used as a strong electron-withdrawing unit to construct donor-acceptor-type organic/polymeric functional materials. On the other hand, triphenylamine (TPA) and its derivatives have been widely applied in the fields of optoelectronics and electronics owing to their 3D steric profile, low ionization potentials, inner redox activity, and excellent UV light-harvesting ability ( Fang and Yamamoto, 2004 ; Zhang et al., 2018 ; EI-Khouly et al., 2009 ; Ego et al., 2002 ). Based on these backgrounds, in this work, we designed and synthesized a novel conjugated donor-acceptor polymer, poly[chalcogenoviologen- alt -triphenylamine] (PCVTPA, including PSVTPA, PSeVTPA, and PTeVTPA), by direct coupling of 4-(bromomethyl)-N-(4- (bromomethyl)phenyl)-N-phenylaniline as electron donor and chalcogenoviologen as electron acceptor, as shown in Scheme 1 . The introduction of chalcogen atoms (S, Se, Te) into viologen scaffolds can greatly improve electrochemical, electrical conductive, and electrochromic properties of the materials ( Lipke et al., 2017 ; Li et al., 2019 ). Like Vs, poly (chalcogenoviologen) (PCV) also exhibits good electron-withdrawing characteristics ( Lipke et al., 2017 ; Li et al., 2019 ; Stolar et al., 2016 ). As a result, the as-prepared Al/PCVTPA/ITO sandwich structure device exhibited excellent history-dependent memristive switching performance. With this device one can not only easily realize the biological stimuli of the “learning→forgetting→ re-learning→re-forgetting” processes associated to the human's learning/memory functions but also execute decimal arithmetic operations of addition, subtraction, multiplication, and division. These results demonstrate that this material shows the great potential in constructing artificial neural networks for neuromorphic computation. As comparison, we also synthesized poly[4,4-bipyridine- alt -triphenylamine] (PBPTPA) under the same experimental conditions. Unlike PCVTPA, PBPTPA did not show any apparent memristive effect. Scheme 1 Synthesis route and Schematic illustration (A) Synthesis of PCVTPA (PSVTPA, PSeVTPA, PTeVTPA) and PBPTPA. (B) Schematic illustration of the Al/polymer/ITO memristor and the biological synapse.",
"discussion": "Results and discussion Both the PCVTPA and PBPTPA polymers are highly soluble in acetonitrile and N , N -dimethylformamide (DMF). Their weight-average molecular weights/polydispersity indexes, which were determined with a gel permeation chromatograph PL-GPC50 using polystyrene standards eluting with DMF, are 4.7× 10 3 /1.00 for PSVTPA, 6.4× 10 3 /1.05 for PSeVTPA, 5.9× 10 3 /1.21 for PTeVTPA, and 7.8× 10 3 /1.10 for PBPTPA, respectively. The formation of the C=N + -C bond in the polymer structure has been confirmed by X-ray photoelectron spectroscopy (XPS) ( Figure S2 ). The N1s core-level XPS spectra of these polymers showed two peaks of nitrogen functionalities at 401.69 (the N in C=N + -C bond) and 399.60 eV (the N in C-N bond). Also, the S2p, Se3d, and Te3d core-level XPS spectra of PCVTPA showed S functionalities at 169.23 (2P 1/2 ) and 168.02 (2P 3/2 ) eV, Se functionalities at 57.51 (3d 3/2 ) and 56.57 (3d 5/2 ) eV, and Te functionalities at 584.99 (3d 3/2 ) and 574.69 (3d 5/2 ) eV. These results demonstrated the successful synthesis of both the PCVTPA and PBPTPA polymers. From Figure 1 A, it can be clearly seen that the UV-vis absorption spectrum of the PBPTPA film showed a strong absorption peak at 313 nm, followed by a broad absorption peak centered at 485 nm. The former can be assigned to TPA, whereas the latter corresponded to the typical absorption of bipyridines. Similarly, the PCVTPA film also exhibited the main absorption peaks from the TPA moieties and the chalcogenoviologen moieties. With the changes of chalcogen atoms introduced into viologen scaffolds from S to Se to Te, the absorption band assigned to the viologen moieties were found to be shifted to the longer wavelength (389 nm→415 nm→475 nm) owing to the gradually weakened electron-drawing ability from S to Se to Te. Upon excitation with laser light at 310 nm, all the samples exhibited a strong emission band that is located at 373 nm for PSVTPA, 374 nm for PSeVTPA, 375 nm for PTeVTPA, and 378 nm for PBPTPA ( Figure 1 B), respectively. The thermal properties of the samples were investigated by thermogravimetric analysis (TGA) in nitrogen atmosphere ( Figure 1 C). As a result, the onset decomposition temperature for the thermal bond cleavage of the samples was found to be 178°C for PSVTPA, 322°C for PSeVTPA, 346°C for PTeVTPA, and 353°C for PBPTPA, respectively. The thermal stability of PTeVTPA is higher than those of PSVTPA and PSeVTPA but smaller than that of PBPTPA without chalcogen atoms. Figure 1 Basic characterization of polymers (A) Normalized UV-vis spectra of the thin film samples. (B) Fluorescence spectra of the samples in DMF (λ ex = 310 nm). (C) TGA curves of the samples. Redox potentials (versus Ag/Ag + ) of the thin film samples, which were measured in deaerated dichloromethane containing recrystallized tetrabutylammonium hexafluorophosphate (TBAPF 6, 0.05M) at room temperature, were found to be E red 1,1/3 = −1.08 V, E red 2,1/3 = −0.63 V, and E red 3,1/3 = +0.82 V for PSVTPA; E red 1,1/3 = −1.03 V, E red 2,1/3 = −0.45 V, and E red 3,1/3 = +0.75V for PSeVTPA; E red 1,1/3 = −1.04 V, E red 2,1/3 = −0.54 V, and E red 3,1/3 = +0.79V for PTeVTPA. Similar to these materials mentioned above, redox potentials (versus Ag/Ag + ) of the PBPTPA thin film also showed three redox potentials: E red 1,1/3 = −1.81 V, E red 2,1/3 = −1.21 V, and E red 3,1/3 = +0.50 V. As reported in the literature ( Woodward et al., 2017 ; Vermeulen and Thompson, 1992 ; Oh et al., 2017 ), when a voltage is applied to Vs, these materials can exhibit apparent color changes as a result of the two-step reversible one-electron reduction that occurred in the system (V 2+ + e − →V +· ; V +· + e − →V 0 ). Similarly, PCVTPA also exhibited reversible color changes under an applied voltage. For example, the PTeVTPA film showed blue at +1 V (PTeVTPA 3+· ), red at 0 V (PTeVTPA 2+ ), green at −0.7 V (PTeVTPA 1+· ), and orange at −1.2 V (PTeVTPA 0 ), respectively. The corresponding electronic absorption spectra of the PTeVTPA film at different sweep voltages are shown in Figure S3 A. In the neutral state (PTeVTPA 0 ), its absorption spectrum showed three main absorption peaks at 299, 363, and 556 nm. The absorption peaks of PTeVTPA at different oxidation states are located at 299, 360 (shoulder peak), 500, and 708 nm for PTeVTPA 1+· ; 299, 357, and 510 nm for PTeVTPA 2+ ; and 299, 359 (shoulder peak), 490, and 689 nm for PTeVTPA 3+· , respectively. For better understanding of the absorption spectra measured experimentally, we also used time-dependent density functional theory (TD-DFT) to simulate the above electronic absorption spectra ( Figure S3 b). All the computational work was carried out by using B3LYP functional and def2-SVP basis set including Grimme's D3 dispersion correction (Becke-Johnson damping) ( Frisch et al., 2016 ; Grimme et al., 2011 ). As a result, the predicted absorption spectra were in good coincidence with the spectra observed in the thin film sample. Furthermore, it was found that the dipole moments of PTeVTPA at different oxidation states are 12.40 Debye for PTeVTPA 3+· , 18.17 Debye for PTeVTPA 2+ , and 14.41 Debye for PTeVTPA +· ( Figure S4 ). In the neutral state (PTeVTPA 0 ), the calculated dipole moment is only 0.77 Debye. In general, both the intensive electron delocalization and strong dipole moment of molecule in a D-A polymer system would be able to effectively stabilize the conductive charge-transfer state and consequently produce the non-volatile nature of the high-conductivity state in an electronic memory device. From Figure 2 A, one can see a pinched hysteretic loop confined to the first and third quadrants of the I-V plane in the Al/PTeVTPA/ITO device. This result suggests that the PTeVTPA-based electronic device exhibits a typical memristive performance. When the electronic device was applied seven consecutive positive sweep voltages from 0 to 0.5 V and then back to 0 V, the observed device current was found to increase incrementally from 0.27 to 0.67 mA (read at 0.2 V). After that, the device was applied seven consecutive negative voltage sweeps of 0 V→-0.5 V→ 0 V, and the device current decreases gradually from −0.89 to −0.42 mA (read at −0.2 V). During the whole voltage sweeping processes, a smoother change of the material conductance or the device current was observed. In contrast to the above observation, for the bistable electrical switching devices reported in the literature, their switching effect usually follows steep conductance or current jumps ( Liu et al., 2018 ; Chen et al., 2012 ; Lin et al., 2014 ). Similarly, both the Al/PSVTPA/ITO and Al/PSeVTPA/ITO devices also showed memristive effect ( Figures 2 B and 2C). Among these three devices, however, the changes in resistance states and the boundary between the pinched hysteretic loops observed in the Al/PTeVTPA/ITO device are much clearer than the other two devices. Unlike PCVTPA-based devices, the PBPTPA-based device displayed unstable memristive behavior ( Figure 2 D). These results demonstrated that the introduction of chalcogen atoms (S, Se, Te) into viologen scaffolds could greatly improve memristive performance of the resultant materials. Furthermore, by using consecutive multilevel conductance switchings, one can easily realize nonlinear transmission characteristics exhibited by a biological synapse ( Figure S5 ), and modulation and memory of the synaptic weight, which refers to the conductance of the two-terminal device, or the strength or amplitude of a connection between two nodes in neuroscience. Figure 2 The current-voltage characteristics of the Al/polymer/ITO devices (A) PTeVTPA. (B) PSVTPA. (C) PSeVTPA. (D) PBPTPA. The effect of film thickness on the current-voltage characteristics of the Al/PTeVTPA/ITO devices has been observed. The film thickness was varied from 37 to 85 nm and to 120 nm. As shown in Figure 3 , all the devices showed a pinched hysteretic loop confined to the first and third quadrants of the I-V plane. The main difference between these devices lies in the degree of an increase (or decrease) in the magnitude of the current signal between two sweeps. For example, the device was applied seven consecutive positive voltage sweeps of 0 V→ 0.5 V→ 0 V; the current change observed between the first sweep and the seventh sweep (ΔI, read at 0.5V) was 1.9 mA (2.4 mA→4.4 mA) at 37 nm, 1.0 mA (1.1 mA→2.1 mA) at 85 nm, and 0.4 mA (1.3 mA→1.7 mA) at 120 nm, respectively. A similar change tendency was also observed when the device was applied seven consecutive negative voltage sweeps of 0 V→-0.5 V→ 0 V. These results demonstrate that the film thickness has only a little bit of influence on the change in the device current under the same experimental conditions. In contrast to the as-fabricated memristor mentioned above, for a typical bistable switching and nonvolatile rewritable memory device, the ON/OFF current ratio will increase with increasing film thickness, whereas their turn-on and turn-off voltages almost kept unchanged ( Fan et al., 2017 ). Considering that the oxygen vacancies (oxygen anions) from the ITO electrode will diffuse into the switching layer under external electric field, we replaced the ITO electrode with the Au electrode. As shown in Figure S6 , the as-fabricated Al/PTeVTPA/Au device showed almost the same memristive performance as that exhibited by the Al/PTeVTPA/ITO device. This result suggested that the migration of oxygen vacancies from the ITO electrode to the active layer has no obvious influence on the polymer-based device performance. Figure 3 The effect of active layer thickness on device performance (A–C) The effect of film thickness on the current-voltage characteristics and SEM images of the Al/PTeVTPA/ITO devices with different active layer thicknesses. (A and D) 37 nm. (B and E) 85 nm. (C and F) 120 nm. Resistive switching performance of the polymer memories mainly arise from changes in the intrinsic properties of the switching media. Although several switching mechanisms such as reduction-oxidation interactions, charge transfer, conformation change, and phase change have been proposed ( Liu et al., 2018 ), less direct physical evidence is available to support these proposed mechanisms so far due to the lack of advanced in situ characterization and analysis tools. Therefore, it is still difficult to identify the operation mechanism of the polymer-based memory device and/or memristor at the moment ( Jeong et al., 2012 ). In the present study, a possible operation mechanism might concern the redox process of the as-prepared polymer film under an applied electric field. At the initial stage (no voltage was applied to the device), the polymer backbone is in a positive divalent state (e.g., PTeVTPA 2+ ). When a positive sweep voltage was applied to the device, the polymer was gradually oxidized (or partially oxidized) to PTeVTPA 3+· , and consequently the charge carrier concentration in the system increased, giving rise to the increase of the device current, as observed in the first quadrant of the I-V plane. When the device was applied a negative sweep voltage, with increasing the applied voltage, PTeVTPA 3+· would undergo a one-step or multiple-step electrochemical reduction reaction process (e.g., PTeVTPA 3+· → PTeVTPA 2+ → PTeVTPA 1+· → PTeVTPA 0 ). As a result, the charge carrier concentration in the system gradually reduced, followed by the dropping of the device current with increasing the sweep times, as shown in the third quadrant of the I-V plane. To explore the influence of stimulus frequency and duration on synaptic weights, we successfully emulated the spike-rate dependent plasticity of biological synapses ( Markram et al., 1998 ; Li et al., 2013 ) by increasing the frequencies of the voltage pulses applied to the Al/PTeVTPA/ITO device ( Figures 4 A and 4B). During the experiments, the stimulus frequency was changed from 1 to 20 Hz, whereas the numbers of the pulse stimulations was fixed at a constant of 10. As seen from Figure 4 A, the more frequently the device acting as biological synapse was being stimulated, the higher the device currents observed in this study became. It was also found that, at the frequency of 1 or 2 Hz, the device current changes with 10 pulse stimulations were very small. With further increasing the frequency, the observed current changes started to become more apparent or remarkable. At a frequency of 20 Hz and the stimulation numbers of 10, the device current changes reached up to 51.99 μA when compared with the stimulation number of 1. These results suggest that the as-prepared memristor can serve as a high-pass filter. Figure 4 C shows the device current as a function of the pulse durations at a frequency of 1 Hz and the stimulation numbers of 5, suggesting the good stability of the device. Considering that the sub-threshold potentiation in neurons is associated with both paired pulse facilitation (PPF) and short-term synaptic plasticity (STP), we also established such functionality in our device. From Figure 4 D, it can be seen that the PPF shows two characteristic timescales ( van de Burgt et al., 2017 ), with τ 1 of 71 ms and τ 2 of 280 ms. These two values are very close to those exhibited by biological synapses ( Zucker and Regehr, 2002 ). Figure 4 Frequency-dependent synaptic potentiation of the Al/PTeVTPA/ITO memristor (A) Evolution of the device current. (B) Evolution of the device current change (ΔI = In- I1) at different frequencies. (C) Current with five pulse stimulations at different pulse durations (inset: schematic diagram of the applied pulse). (D) Short-term potentiation and paired pulse facilitation. The amount by which the synaptic weight is temporarily modified depends on the time interval between two short pulses. An exponential fit is applied to obtain two characteristic timescales. The inset is a schematic diagram of how such biasing is typically realized. The synaptic potentiation and depression, which are regarded as the neurobiological basis of the brain memory functions, may be realized through action potential spikes. When the device was applied the consecutive negative voltage pulses or positive stimuli, respectively, synaptic weight can be effectively depressed or potentiated ( Figure 5 A). The synaptic potentiation and relaxation processes are found to compete with each other during the biological stimuli. Figure 5 B showed the retention curves for synaptic weight at different numbers of identical voltage pulse stimulations, from which one can see that the synaptic weight undergoes the fast decay at the beginning and then gradually tends to fatten out. This result implies that the numbers of identical voltage pulse stimulations with same amplitude, period, and duration can greatly influence the memory loss or retention performance of the device. Basically, the human memory mainly comes from STP and long-term potentiation/plasticity (LTP). Long-term memory can be sustained for a long time (e.g., several days, several months, even several years), whereas short-term memory only lasts for a very short time (e.g., several seconds or minutes). Even an accidental electrical shock or decay rapidly with time may easily interrupt or destroy short-term memory. As shown in Figure 5 C, with increasing the pulse stimulation numbers the relaxation time constant (τ) was changed from 4.51 s@10 pulses to 4.98 s@20 pulses to 6.81 s@30 pulses to 7.40 s@40 pulses to 9.35 s@50 pulses and to 10.36 s@60 pulses. This finding makes the transition from short-term memory to long-term memory possible in our device. And more a “learning → forgetting → re-learning” process shown in human daily life has been successfully explored in this study ( Figures 5 D–5G). When a stimulation of 50 consecutive voltage pulses was applied to the device, the device current was found to increase gradually with the increase of the pulse numbers ( Figure 5 D). This is similar to a “learning” process observed during the human learning. Once the power was cut off, the device current dropped gradually to an intermediate state within 200 s ( Figure 5 E). This process can be regarded as a “forgetting” process. And then we applied a stimulation of 35 consecutive pulses to the device again, and the current returned to its end level from the first learning stage ( Figure 5 F). Such a “re-learning” process is similar to the transition process from STP to LTP, by which the human memory capability can be significantly strengthened. Afterward, the as-fabricated device went through a “re-forgetting” process again ( Figure 5 G). By comparison with the device current detected at the end of the first “forgetting” process, which is about 0.36 mA, the device current observed at the end of the “re-forgetting” process reached up to 0.41 mA, much higher than that of the former. This suggests that the “re-forgetting” speed slows down greatly when compared with the first “forgetting” speed. Surprisingly, it only took 15 voltage pulses for the device current to be recovered to the same higher level as that shown at the end level from the first learning stage ( Figure 5 H). These results demonstrated that the voltage pulses applied to the device would become fewer and fewer with an increasing number of “re-learning” processes. Figure 5 Simulation of human brain memory behavior (A) The current in response to a series of positive and negative voltage stimulations showing the respective potentiation and depression of the device synaptic connection. (B) Experimental (symbols) and fitted (solid lines) memory retention performance after being subjected to different numbers of identical voltage pulse stimulations. (C) Evolution of the relaxation time constant (τ) and the stabilized synaptic weight (I0) along with the stimulating numbers. I(t) = I 0 +Aexp(-t/τ). (D–H) demonstration of the “learning-forgetting-relearning” process. The amplitude, duration, and period of the voltage pulses are 1 V, 10 ms, and 1 s, respectively. The current responses are monitored with a small voltage of 0.2 V. From the above discussion, we can see that our device shows a consecutive resistive switching effect at any device current levels. This result is very interesting and can be used to implement arithmetic addition, subtraction, multiplication, and division. When a stimulation of consecutive voltage pulses was applied to the device, as shown in Figure 6 A, whatever positive sweeping or negative sweeping, the relationship between the pulse numbers and the device currents read at ±0.5V is perfectly linear. This relationship guarantees the normal operation of the device as a decimal abacus. It was also found that the pinched hysteretic loops shown in Figures 2 A–2C are highly symmetric. For calibration, an X-X = 0 operation is carried out in pulse mode to guarantee the calculating accuracy ( Figure 6 B). After a stimulation of 10 consecutive positive pulses (0.5 V and 10 ms) was applied to the device, the observed device current reached 0.43 mA. As such, when a current of ∼0.43 mA is read, the number 10 will be counted in future calculation. By setting the device current of ≤0.2 mA as the initial state of the device, the decimal numbers of 0–10 can be indexed proportionally. When a subsequent stimulation of 10 consecutive negative pulses was applied, the device current got back to the initial state again. With such an operation, one can easily realize the subtraction operation of 10–10 = 0 and calibrate the as-fabricated device for accurate decimal arithmetic operations. In this device, the application of positive voltages, which can result in the increase of the device current, was used to make addition operations, whereas the negative voltages applied to the device correspond to the subtraction function of an “abacus.” By monitoring the current changes with respect to the initial state of the device, the quantity of the input pulse stimuli can be counted. Figure 6 Demonstration of arithmetic computing with the Al/PTeVTPA/ITO memristor (A) Linear relationship between the device currents read at ± 0.5 V and voltage sweeping numbers. (B) Calibration of the memristor with the operation of 10–10 = 0 for decimal arithmetic calculations. (C) Commutative addition conducted with the PTeVTPA device. (D) Subtraction conducted with the PTeVTPA device. (E) Multiplication conducted with the PTeVTPA device. (F) Fractional division conducted with the PTeVTPA device. The application of a succession of six consecutive positive pulses (0.5 V and 10 ms) followed by an extra train of four consecutive positive pulses with the same duration and amplitude as those of the former produces a device current of ∼0.43 mA, which exactly confirms 6 + 4 = 10 ( Figure 6 C). When we reverse the sequence of the two sets of input pulse signals, the device current also reached ∼0.43 mA under the same experimental conditions. These results verify such a commutative law that X + Y=Y + X (6 + 4 = 4 + 6 = 10). After the preloading of 10 positive pulses (0.5 V and 10 ms), which gives a device current of ∼0.43 mA, the sequential application of four consecutive negative pulses and a following train of six consecutive negative pulses resulted in that the device current is down back to the initial state again ( Figure 6 D). This confirmed 10−6−4 = 0. Reversing the loading order of the input signals can also reduce the device current to the value at the initial state (10−4−6 = 0), which confirms that X−Y−Z = X−Z−Y (10−6−4 = 10−4−6 = 0). These findings demonstrated the commutative subtraction operation. Similarly, the accumulative addition operation-based multiplication also obeys the commutative law ( Figure 6 E). The result obtained from the application of two sets of five positive pulses is the same as that from the application of five sets of two positive pulses, assuring that X×Y=Y×X (2 × 5 = 5 × 2 = 10). The fractional division is based on the combination of the subtraction and addition operations. For example, we can carry out the division operation of 6÷4 with our device ( Figure 6 F). The device was first reset to the initial state, and then was applied six consecutive positive voltage pulses, followed by another series of four negative pulses, to perform 6−4. As a result, a remainder of 2 and an integer quotient of 1 were achieved. Considering the remainder 2 is smaller than the divisor 4, we added two series of 9 positive voltage pulses to the device to replace 2 with “2 + 2×9” (2 × 10). Subsequently repeated subtraction of −4 for five times (20− 4− 4− 4− 4− 4) made the device current back to the initial state again. The fractional division calculation was then terminated. These results demonstrate that the decimal number of 5 was at the tenths position and the quotient of 6÷4 = 1.5 (1 + 0.5). Conclusions A proof-of-concept PCVTPA-based polymer memristor with a configuration of Al/PCVTPA/ITO exhibited excellent history-dependent memristive switching and multilevel storage performance. Associated with the unique memristive behavior, the as-fabricated device can be used to not only emulate the human's learning and memorizing functions but also execute decimal arithmetic operations of addition, subtraction, multiplication, and division. These results demonstrated that the PCVTPA polymer showed a great potential in constructing high-performance intelligent computing system in the near future. Limitations of the study This work is mainly focused on the design and synthesis of new chalcogenoviologen and triphenylamine-based alternative copolymer for constructing high-performance synaptic biomimicking and neuromorphic computing system. This material shows excellent history-dependent memristive switching and multilevel storage performance. The memristive performance of the polymers mainly arises from changes in the intrinsic properties of the switching media. In contrast to the inorganic materials-based memory and memristor, the proposed main switching mechanisms of the polymer-based devices are reduction-oxidation interactions, charge transfer, conformation change, and phase change. However, less direct physical evidence is available to support these proposed mechanisms so far owing to the lack of advanced in situ characterization and analysis tools. Therefore, it is still difficult to identify the operation mechanism of the polymer-based memristors at the moment. Future work should be focused on the design of new high-performance conjugated polymer functional materials for memristors, exploration of the operation mechanism, and determination of the relationship between the structural parameters and the memristive response while seeking to optimally combine materials and devices."
} | 8,716 |
33380776 | null | s2 | 2,592 | {
"abstract": "Piezoelectric nanomaterial-polymer composites represent a unique paradigm for making flexible energy harvesting and sensing devices with enhanced devices' performance. In this work, we studied various metal doped ZnO nanostructures, fabricated and characterized ZnO nanoparticle-PVDF composite thin film, and demonstrated both enhanced energy generation and motion sensing capabilities. Specifically, a series of flexible piezoelectric nanogenerators (PENGs) were designed based on these piezoelectric composite thin films. The voltage output from cobalt (Co), sodium (Na), silver (Ag), and lithium (Li) doped ZnO-PVDF composite as well as pure ZnO-PVDF samples were individually studied and compared. Under the same experimental conditions, the Li-ZnO based device produces the largest peak-to-peak voltage (3.43 Vpp) which is about 9 times of that of the pure ZnO based device, where Co-ZnO, Na-ZnO and Ag-ZnO are 1.2, 4.9 and 5.4 times, respectively. In addition, the effect of doping ratio of Li-ZnO is studied, and we found that 5% is the best doping ratio in terms of output voltage. Finally, we demonstrated that the energy harvested by the device from finger tapping at ~2 Hz can charge a capacitor with a large output power density of 0.45 W/cm"
} | 313 |
22037345 | null | s2 | 2,593 | {
"abstract": "Synthetic biologists combine modular biological \"parts\" to create higher-order devices. Metabolic engineers construct biological \"pipes\" by optimizing the microbial conversion of basic substrates to desired compounds. Many scientists work at the intersection of these two philosophies, employing synthetic devices to enhance metabolic engineering efforts. These integrated approaches promise to do more than simply improve product yields; they can expand the array of products that are tractable to produce biologically. In this review, we explore the application of synthetic biology techniques to next-generation metabolic engineering challenges, as well as the emerging engineering principles for biological design."
} | 179 |
40235473 | PMC11998799 | pmc | 2,594 | {
"abstract": "Abstract \nClimate change has caused drastic declines in corals. As sessile organisms, corals acclimate to environmental shifts through genome-wide changes in gene expression, epigenetic modifications, and alterations in microbiome composition. However, alternative splicing (AS), a conserved mechanism of stress response in many organisms, has been under-explored in corals. Using short-term acute thermal stress assays, we investigated patterns of AS in the scleractinian coral\n Acropora cervicornis \nduring response to low (33°C), medium (35°C), and high (37°C) heat stress and subsequent overnight recovery. Our findings demonstrate reproducible dynamic shifts in AS of at least 40 percent of all genes during response to heat treatment and the recovery phase. The relative proportion of AS increased in response to heat stress and was primarily dominated by intron retention in specific classes of transcripts, including those related to splicing regulation itself. While AS returned to baseline levels post-exposure to low heat, AS persisted even after reprieve from higher levels of heat stress, which was associated with irreversible loss of photosynthetic efficiency of the symbiont. Our findings demonstrate that, although animals, corals are more plant-like in their likely usage of AS for regulating thermal stress response and recovery."
} | 337 |
20865717 | null | s2 | 2,595 | {
"abstract": "No abstract available"
} | 5 |
39499271 | PMC11599339 | pmc | 2,599 | {
"abstract": "Plant invasions are impacting alpine zones, altering key mutualisms that affect ecosystem functions. Plant–mycorrhizal associations are sensitive to invasion, but previous studies have been limited in the types of mycorrhizas examined. Consequently, little is known about how invaders that host rarer types of mycorrhizas may affect community and ecosystem properties. We studied invasion by an ericoid mycorrhizal host plant ( Calluna vulgaris L., heather) in alpine tussock grasslands in New Zealand. We investigate the effects of increasing C. vulgaris density on the plant and soil microbial community and on mycorrhization in the dominant native species ( Chionochloa rubra Z., red tussock), an arbuscular mycorrhizal host. We show that variation in plant community composition was primarily driven by invader density. High invader densities were associated with reductions in C. rubra diameter and in the cover, richness and diversity of the subordinate plant community. Belowground, we show that higher invader densities were associated with lower rates of mycorrhization in C. rubra and higher proportional abundance of the fungal lipid biomarker 18:2ω6 but had little effect on total microbial biomass, which may suggest increased ericoid mycorrhizal and fine root biomass in high C. vulgaris density stands. Our data suggest that disruption of native plant–arbuscular mycorrhizal networks may contribute to the competitive success of C. vulgaris , and that the dramatic decline of C . rubra with invasion reflects its relatively high mycorrhizal dependence. By exploring invasion of a plant with a less common mycorrhizal type, our study expands knowledge of the ecosystem consequences of biological invasions. Supplementary Information The online version contains supplementary material available at 10.1007/s00442-024-05632-w.",
"introduction": "Introduction Alpine ecosystems form isolated pockets of biodiversity above the treeline covering ~ 5% of the terrestrial surface (Cavieres et al. 2014 ). Warming is leading to changes in plant community composition whereby low elevation species, particularly woody species, are colonizing alpine areas, shrinking an already spatially restricted ecosystem (Halloy and Mark 2003 ; Gottfried et al. 2012 ; Pauli et al. 2012 ; Lamprecht et al. 2018 ; Steinbauer et al. 2018 ). Alpine ecosystems are characterized by cold temperatures and short growing seasons which limit plant growth and nutrient availability by slowing decomposition (Pauchard et al. 2016 ; Frazier and Brewington 2020 ). The stress-tolerant (Grime 1977 ) and often highly endemic (Körner 1995 ) alpine plants adapted to these harsh abiotic conditions (Chapin III and Shaver 1985 ; Brooker 2006 ) exhibit relatively little trait variation over environmental gradients (Rixen et al. 2022 ) and are over-represented among those species experiencing population declines and range contraction (Rumpf et al. 2018 ). By contrast, some woody species are expanding in grasslands and cold biomes in locations worldwide (Myers-Smith et al. 2011 ; Elmendorf et al. 2012 ; Mod and Luoto 2016 ; Stevens et al. 2017 ) and have become a particular threat to alpine grasslands (Komac et al. 2011 ; Brandt et al. 2013 ; Espunyes et al. 2019 ). As climatic warming continues to alleviate the harsh abiotic conditions in mountains, plant–plant interactions are likely to shift from net facilitative to more competitive (Greenlee and Callaway 1996 ; Brooker 2006 ; Maestre et al. 2009 ). Human-mediated plant introductions are a global phenomenon (Hulme 2009 ) and while only a few species become invasive, these now affect even the most remote ecosystems, including alpine zones (Alexander et al. 2016 ; Clavel et al. 2021 ). Currently, nearly 200 non-native plant species occur in alpine ecosystems and their impacts are expected to increase due to multiple drivers, including climate change (Alexander et al. 2016 ; Pyšek et al. 2020 ). The direct negative effects of invaders on native plant species are well-characterized; competition for space and resources (Gioria and Osborne 2014 ) often leads to the reduced local abundance and richness of natives (Vilà et al. 2011 ; Bradley et al. 2019 ); and, in extreme cases, species extinctions (Bellard et al. 2016 ). Invaded plant communities are associated with altered habitat structure, animal behaviors and trophic networks as well as altered ecosystem functions, services, and disturbance regimes (Pyšek et al. 2020 ). Nevertheless, the impacts of plant invasions are complex and varied (Vilà et al. 2011 ). Plant symbiotic fungi are increasingly recognized as critical determinants of the ecological impacts of invasive plant species (Pringle et al. 2009 ; Dickie et al. 2017 ). Mycorrhizal symbioses are ubiquitous often positive plant–fungal interactions that are obligate for many plant species (Smith and Read 2008 ). Root colonization by mycorrhizas directly affects plant nutrition and performance, while the extraradical mycorrhizal mycelium affects soil functioning, modifying the distribution of carbon (C) through the soil profile, affecting soil aggregation and the structure of the soil microbial community (Barceló et al. 2020 ). Invasive plants can disrupt the mycorrhizal symbioses of native plants, contributing to their invasion success (Wolfe et al. 2008 ; Barto et al. 2011 ; Meinhardt and Gehring 2012 ). High invader densities can reduce mycorrhizal colonization in native hosts , negatively affecting their survival and growth, and altering plant and fungal community composition and associated ecosystem properties and functions (Meinhardt and Gehring 2012 ; Grove et al. 2017 ; Sapsford et al. 2022 ). While mycorrhizas can be critical in the establishment and spread of invaders worldwide (Dickie et al. 2010 , 2017 ; Policelli et al. 2019 ), previous studies have favored invaders that host arbuscular, ectomycorrhizas or are non-mycorrhizal (Zubek et al. 2016 ; Grove et al. 2017 ). Consequently, little is known of the ecosystem impacts of invaders that host less common mycorrhizas. Invaders of different mycorrhizal types are likely to have different impacts on ecosystem functions. Mycorrhizas play foundational roles in ecosystem C cycling (Bardgett and van der Putten 2014 ; Tedersoo et al. 2020 ), by influencing plant production and litter quality, being a key C source to other microbes, controlling soil organic matter turnover (Hawkes et al. 2008 ; Bardgett and van der Putten 2014 ) and ultimately the size of the soil C sink (Orwin et al. 2011 ; Clemmensen et al. 2013 ; Averill et al. 2014 ). Mycorrhizal types differ in their effects on ecosystem C cycling, with ectomycorrhiza (EcM) and ericoid mycorrhiza (ErM) fungi obtaining greater C per unit nitrogen than arbuscular mycorrhizal fungi (AMF) (Orwin et al. 2011 ; Phillips et al. 2013 ; Soudzilovskaia et al. 2015 ). Where plant invasions or woody plant expansions cause changes in the dominance of mycorrhizal types, we may expect them to have larger effects on ecosystem properties. Indeed, during plant invasions, mycorrhizal mismatch, whereby native and invasive species host different types of mycorrhizal fungi, is generally associated with significant ecosystem impacts (Grove et al. 2017 ), but this has been explored in only a few systems. Calluna vulgaris (heather, Ericaceae) is a European shrub common to bog, heathland and alpine ecosystems. C. vulgaris is invasive in New Zealand’s Tongariro National Park (TNP), where it has become a common and often dominant member of the plant community over roughly a third (265/798 km 2 ) of the landscape (Rogers and Leathwick 1996 ; Effah et al. 2020 ). Invasion by C. vulgaris is detrimental to the native red tussock, Chionochloa rubra , which forms the tussock grassland ecosystems formerly typical of non-forested areas of the park (Chapman and Bannister 1990 ; Rogers and Leathwick 1996 ). High densities of C. vulgaris are associated with pollination limitation and suppressed seed masses in a native ericaceous shrub (Giejsztowt et al. 2020 ), altered herbivorous insect assemblages (Keesing 1995 ), and successional trajectories (Rogers and Leathwick 1996 ) in former C. rubra tussock grasslands, but we know little about concomitant effects on below-ground components of the ecosystem. C. vulgaris is facultatively ErM species (Fitter and Peat 1994 ) that is considered to be generalist in its ErM interactions, allowing it to thrive in broad edaphic conditions (Perotto et al. 2012 ; van Geel et al. 2020 ). Mycorrhizal facilitation by native ErM species may contribute to the establishment of C. vulgaris in inter-tussock spaces in TNP (Chapman and Bannister 1990 ; Orlovich and Cairney 2004 ). C. vulgaris expansion is viewed as the primary cause of C. rubra decline in TNP (Chapman and Bannister 1990 ) and C. vulgaris is predicted to exhibit range expansion under warming (Giejsztowt 2019 ) , potentially leading to greater competitive effects of C. vulgaris on C. rubra in future (Brooker and Callaghan 1998 ; Choler et al. 2001 ; Callaway et al. 2002 ; Maestre et al. 2009 ). This study focuses on invasion of an alpine tussock grassland by the woody ErM host plant C. vulgaris. We investigate how a gradient of C. vulgaris density affects the surrounding plant community, mycorrhization in the dominant native host ( C. rubra ), and the soil microbial community. Our study took place in a comparably little-studied ecosystem; an alpine zone in the Southern hemisphere. We hypothesized (Hyp 1 ) that increasing C. vulgaris density leads to a shift in the composition of the plant community through the suppression of C. rubra and other native species. As a consequence of mycorrhizal mismatch between native C. rubra and invasive C. vulgaris , we hypothesized (Hyp 2 ) that higher densities of the invader would be associated with a reduction in AMF colonization in the roots of C. rubra. Finally we hypothesized (Hyp 3 ) high densities of invasive ErM C. vulgaris would be associated with reduced AMF abundances in soil.",
"discussion": "Discussion We studied the effects of increasing C. vulgaris density on the plant and soil microbial community and on mycorrhization in the dominant native species, C. rubra , an AM host in alpine tussock grasslands in New Zealand. We found that C. vulgaris density was the major driver of variation in plant community composition, with higher invader densities associated with reduced cover of subordinate plant species, especially shrubs and ferns, as well as altered morphology of C. rubra tussocks. Among subordinate plant species, these changes reflected reduced richness and diversity and an increase in the proportional abundance of AM hosts at the expense of ErM hosts. Belowground, we found high invader densities were associated with lower rates of AMF mycorrhization in C. rubra and a higher proportional abundance of the biomarker for soil fungi, which include ErM, but had little effect on the biomass of AMF or on total soil microbial biomass. In what follows, we discuss these results in detail and consider the potential for mycorrhizal mismatch to be contributing to C. vulgaris invasion, converting these tussock grasslands into shrublands. We tested the hypothesis that increasing C. vulgaris density leads to a shift in the composition of the plant community through the suppression of C. rubra and other native species. Our ordination and PERMANOVA revealed that variation in plant community composition among study plots was primarily driven by C. vulgaris density. Higher invader densities were associated with a three-fold reduction in the total cover of subordinate plant species, primarily caused by significant reductions in the cover of native shrubs and ferns. Native ericaceous shrubs were more severely affected by increasing densities of C. vulgaris than native AM shrubs, suggesting that greater niche overlap with the invader led to their competitive exclusion. These shifts in plant cover and community composition corresponded to 42–50% reductions in indices of plant species richness and diversity between the low and high C. vulgaris density plots. Moreover, they were associated with a loss of plant functional diversity as two of the six functional groups (native ferns, native sedges and rushes) were absent from the high C. vulgaris density plots. Our finding agrees with that of a recent review that found reduced functional diversity of native species at high invader densities (Renault et al. 2022 ). Our data suggest that C. vulgaris invasion into alpine tussock grasslands in New Zealand leads to homogenization of the alpine flora, effects that are analogous to those induced by the upward shift of invasive plants as climate warms in the Swiss Alps (Jurasinski and Kreyling 2007 ). Our study plots were centered on C. rubra individuals to facilitate its root sampling and to ensure that it was present in every plot, because our preliminary field observations suggested that C. rubra became much less abundant in high-density C. vulgaris stands. Our measurements of plant morphology for C. rubra revealed a significant decline in plant diameter across the C. vulgaris density gradient. However, we found no significant effect on plant height or the plot-level cover of C. rubra . This suggests that C. rubra responds to increasing competition from C. vulgaris by reducing its lateral spread and exhibiting a more upright stature, maintaining its maximal height to persist in the densest C. vulgaris thickets. While physical space is not a consumable resource, it is important for gaining access to light, water, and nutrients, all of which can determine the success of the plant (Gioria and Osborne 2014 ). In a classical study of uninvaded grasslands of the closely related Chinochloa rigida , tussock height and diameter vary synchronously across elevational gradients (Mark 1969 ). The decoupling of plant height and diameter in C. rubra tussocks across the C. vulgaris density gradient may suggest that C. rubra is altering its morphology to avoid competitive exclusion by C. vulgaris as affected through light limitation. Together with the dramatic changes in plant community composition over the C. vulgaris density gradient, these morphological changes in C. rubra provide strong support for our first hypothesis and suggests pairwise competition by C. vulgaris often leads to competitive exclusion of C. rubra , and other native species in these alpine tussock grasslands. The ferns and sedges/rushes functional categories consist of species associated with the wettest microhabitats of the red tussock ecosystem (Mark 2021 ). Therefore, the exclusion of these species from the highest density C. vulgaris plots may indicate that high invader densities are associated with drier soils. Since we placed all study plots at flat locations, this may suggest that high-density C. vulgaris thickets alter site moisture locally, potentially drying soils through increased vegetative evapotranspiration. Two mechanisms could potentially contribute to this change; first, C. rubra is more conservative in its water use, especially at lower soil moisture, than C. vulgaris leading to measured transpiration rates in Chinocholoa tussock grasslands that are less than half that reported for C. vulgaris shrublands (Miranda 1982 ; Campbell and Murray 1990 ; Espie 1999 ). Second, higher leaf areas are likely to be achieved in densely invaded areas, creating more surface area for evapotranspiration. In our study, total vegetative cover achieved the highest values in the high C. vulgaris density category, and woody shrub invasion increases evapotranspiration in the Swiss Alps (Bergh et al. 2018 ) and across grassland ecosystems of the American South-West (Shen et al. 2022 ). Unfortunately, we did not measure soil moisture and therefore we cannot exclude the alternative explanation that the highest density C. vulgaris stands simply did not occur in the wettest microsites in our study. However, C. vulgaris has broad edaphic and hydrologic tolerances and it commonly occurs in moist to wet habitats throughout its native range (Rayner et al. 1911 ; Rayner 1913 ) and in TNP (Chapman and Bannister 1990 ), so it is unlikely that excessive soil moisture limited C. vulgaris density in our study. Therefore, our results suggest that one possible mechanism through which increasing C. vulgaris density leads to a shift in the composition of the plant community is by excluding the most drought sensitive native species by promoting drier soils although this conclusion requires further experimental verification. The mycorrhizal type of an invader can be a major determinant of its ecological impacts (Pringle et al. 2009 ; Grove et al. 2017 ). Facultatively mycorrhizal species like C. vulgaris exhibit wider ecological niches in the European flora (Gerz et al. 2018 ), and this is associated with their greater occupied ranges there (Menzel et al. 2017 ). Invaders often reduce mycorrhization of native species (Busby et al. 2013 ; Zubek et al. 2016 ; Řezáčová et al. 2021 ) and these effects tend to be large where mycorrhizal mismatch occurs (Wolfe et al. 2008 ; Meinhardt and Gehring 2012 ). Supporting our second hypothesis, we show that higher densities of C. vulgaris, an ErM invader, reduced AMF colonization in the native grass C. rubra . We also found a negative relationship between C. vulgaris density and C. rubra diameter. A meta-analysis of AMF hosts showed that response ratios of plant biomass and P content respond significantly positively to percent root length colonization (Treseder 2013 ). Therefore, the reduction in AMF colonization in C. rubra that we observed over the invader density gradient is likely to have had negative effects on its mineral nutrition and biomass in these poorly fertile alpine tussock grasslands, contributing to its smaller diameter in the high C. vulgaris density stands. We are aware of no previous studies demonstrating mycorrhizal disruption of an AM host by an ErM invader. However, a review by Grove et al. ( 2017 ) found that non-mycorrhizal invaders reduced root colonization in both AM or EcM hosts while AM invaders reduced or had no effect on root mycorrhizal colonization of EcM plants. Our study extends these results, demonstrating a reduction in root colonization of an AM host due to mycorrhizal disruption by and ErM invader. We observed a negative correlation between C. rubra cover and soil nutrients, indicating that C. rubra was most abundant on the most infertile soils in our study. AMF colonization of native grasses is generally negatively related to soil nutrient availability in grassland and alpine ecosystems (de Mesquita et al. 2018 ; Jones and French 2021 ), and therefore we might expect higher AMF colonization of C. rubra in those lower nutrient soils, although we were unable to test this. On the other hand, while the total cover of all plant functional groups declined over the C. vulgaris density gradient, AM hosts, and especially AM shrubs, increased their proportional abundance in the highest density C. vulgaris stands. Taken together with the strong decline in AMF colonization in C. rubra over the C. vulgaris density gradient, these findings point to a higher mycorrhizal dependence of C. rubra relative to other AM hosts in this ecosystem. Likewise, in alpine meadows of the Russian Caucasus, dominant AM hosts tended to have higher mycorrhizal colonization and lower relative growth rates in the absence of mycorrhizal colonization than subordinate species, implying that they were more dependent on mycorrhizal symbionts (Elumeeva et al. 2018 ). Further, theoretical work exploring the mechanism of mycorrhizal disruption by invaders found strongest declines in mycorrhization in native plants when densities of the invader and mycorrhizal dependence of the native species were both high (McCary et al. 2019 ). The reduction of AM host cover in high-density C. vulgaris stands may have reduced AMF dispersal to C. rubra via root-to-root contact, the primary mechanism of AMF infection (Read et al. 1976 ; Powell 1979 ). Indeed, mycorrhizal host displacement by an invader is associated with reductions in mycorrhizal colonization in remaining hosts in other systems (Tanner and Gange 2013 ; Zubek et al. 2016 ), which would be expected to have had proportionally greater effects on C. rubra than co-occurring AM hosts, if its mycorrhizal dependence is high. Therefore, it seems probable that the highly negative effect of C. vulgaris invasion on C. rubra noted here and in previous studies (Chapman and Bannister 1990 ) reflects mycorrhizal disruption of the plant–AMF network, which is particularly detrimental for C. rubra relative to other AMF hosts in this alpine tussock grassland. We found no support for our third hypothesis that higher densities of C. vulgaris would be associated with reduced soil AMF abundances. This suggests that mycorrhizal disruption by invasive C. vulgaris was insufficient to reduce AMF biomass in soils despite that it drove strong and significant declines in AM host species cover and intraradical AMF structures in C. rubra across the C. vulgaris density gradient. As obligate biotrophs, AMF allocate greater biomass to plant roots than soils, especially when considered by volume or mass of substrate (Hiiesalu et al. 2014 ) and consequently, AMF are more difficult to detect in soil relative to plant roots (Ngosong et al. 2012 ). Moreover, even in highly AMF dominated systems, AMF typically account for only a small proportion of total soil microbial biomass (Hiiesalu et al. 2014 ). In our study system, average microbial biomass across all sites was low and the AMF biomarker was detected in less than a third (22/75) of samples, limiting our ability to detect effects of increasing C. vulgaris density on AMF biomass. However, the non-significant but negative relationship between the likelihood of NLFA 16:1ω5 detection and C. vulgaris density revealed by our glm may indicate a trend toward less AMF biomass in soils of heavily invaded areas. While previous studies do not generally examine AMF biomass in both roots and soils as we have done, more studies appear to report reduced AMF colonization in native species than reduced AMF biomass in soils with invasion (Bunn et al. 2015 ). Regardless, the strong decline in intraradical AMF structures in C. rubra together with no significant concomitant decline in NLFA 16:1ω5 in soils across the C. vulgaris density gradient suggests that the primary mechanism of mycorrhizal disruption of C. vulgaris on C. rubra occurs through reduced root-to-root spread of AMF to C. rubra , rather than by reduced germination of soil AMF spores. Studying invasion by an ErM host, we found no change in total microbial biomass but a significant increase in the fungal biomarker 18:2ω6 over the C. vulgaris density gradient. These results may suggest an increase in the ratio of fungi to bacteria in soils with C. vulgaris invasion, which agrees with the effect of C. vulgaris on soil microbes in a mesocosm study (Witt and Setälä 2010 ). A caveat of our use of the PLFA 18:2ω6 to indicate fungal biomass in soils is that this lipid also occurs in plant roots (Zelles 1997 ; Laczko et al. 2004 ). However, a quantitative assessment of root contributions to the 18:2ω6 contents of soil was found to be negligible (less than 0.61%) in a beech forest (Kaiser et al. 2010 ). In this study, we located the two soil cores directly under the focal C. rubra plants in each plot to maximize the likelihood of sampling C. rubra roots rather than those of co-occurring species. Nevertheless, soil cores contained the roots of other species, including C. vulgaris, which are very fine and prone to breakage during soil processing. Small fragments of fine roots are unlikely to have been entirely removed by sieving. Therefore, we cannot exclude the possibility that the significant increase we observed in 18:2ω6 over the C. vulgaris density gradient reflects an increase root as well as fungal biomass in soils. While 18:2ω6 is a general fungal biomarker (Zelles 1997 ), its concentration in root-free soils is considered a sensitive indicator of changes in ErM and EcM biomass (Nilsson et al. 2005 ). We recorded no EcM plant species in plots, and while the dual AM–EcM shrub Leptospermum scoparium was common, it was unlikely to have hosted EcM fungi at our sites given the absence of other EcM hosts (Cooper 1976 ; Moyersoen and Fitter 1999 ). Therefore, the increase of PLFA 18:2ω6 over the invader gradient is most likely to reflect increasing ErM and root biomass associated with C. vulgaris in our study, although we cannot exclude the possibility that fungi with other trophic modes (e.g., saprobes or DSE) contributed to this change. Similarly, invasion by rose into coastal dunes increased the biomass of its mycorrhizal symbiont in soils (Stefanowicz et al. 2019 ). Mycorrhization in C. vulgaris is negatively related to soil nutrient availability (Caporn et al. 1995 ) and positively related to plant aboveground biomass, but not plant tissue nutrient content when grown in low nutrient soils (Strandberg and Johansson 1999 ). Soils in the study region are nutrient poor. Therefore, our findings may suggest that C. vulgaris uses its ErM to compete successfully with native plants for growth-limiting soil nutrients, which it allocates to the growth of aboveground biomass, effectively competing with native species for space and light as evidenced by the altered morphology of C. rubra over the invasion gradient. Larger C. vulgaris shrubs with higher leaf area are able to shuttle more C belowground to their roots and ErM symbionts, which could then increase in biomass in densely invaded soils. Presumably these larger networks of root and ErM biomass further enhance the mineral nutrition of C. vulgaris , generating a positive feedback on the invasion cycle. In conclusion, we have shown that invasion by ErM C. vulgaris into tussock grasslands formerly dominated by AM C. rubra is associated with reduced mycorrhization in the native host, which appears to have high mycorrhizal dependency. High densities of the invader were associated with the increased abundance of PLFA 18:2ω6 but no significant increase in bacterial biomarkers, suggesting an increased ratio of fungi to bacteria in these soils which may reflect increased biomass of ErM roots and extraradical mycelium with invasion. This shift in the soil community appears to have enhanced competition by C. vulgaris for growth-limiting soil nutrients, enabling it to increase aboveground biomass, effectively competing with C. rubra and other native species for space and light. Dense C. vulgaris thickets were associated with reduced plant species and functional diversities, due in part to the decline or absence of species associated with wet microhabitats, which may reflect reduced soil moisture due to higher vegetative evapotranspiration by C. vulgaris . Reduced cover of native AM hosts appears to be more important than reduced soil AMF inoculum in affecting mycorrhizal disruption of C. rubra and its symbionts by C. vulgaris . Taken together, our data point to a role for mycorrhizal mismatch to be among the mechanisms contributing to invasion by an ErM host into an AM dominant system, effectively converting these C. rubra tussock grasslands into shrublands with reduced diversities of native species."
} | 6,949 |
36864107 | PMC9981606 | pmc | 2,600 | {
"abstract": "The global degradation of coral reefs is steadily increasing with ongoing climate change. Yet coral larvae settlement, a key mechanism of coral population rejuvenation and recovery, is largely understudied. Here, we show how the lipophilic, settlement-inducing bacterial pigment cycloprodigiosin (CYPRO) is actively harvested and subsequently enriched along the ectoderm of larvae of the scleractinian coral Leptastrea purpura . A light-dependent reaction transforms the CYPRO molecules through photolytic decomposition and provides a constant supply of hydrogen peroxide (H 2 O 2 ), leading to attachment on the substrate and metamorphosis into a coral recruit. Micromolar concentrations of H 2 O 2 in seawater also resulted in rapid metamorphosis, but without prior larval attachment. We propose that the morphogen CYPRO is responsible for initiating attachment while simultaneously acting as a molecular generator for the comprehensive metamorphosis of pelagic larvae. Ultimately, our approach opens a novel mechanistic dimension to the study of chemical signaling in coral settlement and provides unprecedented insights into the role of infochemicals in cross-kingdom interactions.",
"introduction": "Introduction Profound changes in coral reef communities are recognized at increasing frequency and severity, often resulting in strong declines in biodiversity and ecosystem functioning. As climate change continues, the destruction of coral reefs is expected to worsen, with serious consequences for the livelihoods of several hundred million people 1 – 7 . Adult corals in particular have shown to be more vulnerable to climate change, while juveniles appear to possess a broader physiological plasticity, in part due to their greater flexibility in exchanging beneficial symbiotic dinoflagellates 8 – 10 . Thus, the solution for fighting coral decline may not be found in the parent population but rather in the recruitment of juvenile coral generations, capable to adapt to constantly fluctuating conditions in a rapidly changing Anthropocene. Sexual reproduction enables scleractinian corals to generate myriads of pelagic larvae that must recruit onto suitable substrates to achieve their permanent sessile life stage. This process – i.e . , coral settlement – is characterized by the attachment to a desired surface followed by a rapid metamorphic event that transforms larvae into sessile, benthic recruits. Current research has intensified its focus on the early life-stages of corals and highlights the idea of inductive cues as a requirement to induce the cascade of attachment and subsequent metamorphosis in coral larvae. These cues can be of varying nature and include, for example, light 11 – 13 , reef sound 14 and surface structure 15 . However, many studies have proven the efficacy of live crustose coralline algae (CCA) as biological, settlement-inducing substrate for larvae of various coral species 16 – 18 . Moreover, microbial mats, covering the surface of marine hard substrates such as CCA, but also monospecific bacterial biofilms have received considerable attention as potent coral settlement inducers 19 – 24 . Particularly the genus Pseudoalteromonas features a variety of species capable of stimulating settlement in coral larvae and other marine invertebrates 19 , 23 , 25 – 29 . These insights greatly improved todays understanding of coral settlement, but we are still missing mechanistic insights into this morphogenic reaction on the molecular level. In rare cases, the inductive activity towards coral recruits was attributed to a mixture of chemical molecules produced by the CCA holobiont 30 – 32 . Isolated coral settlement inducing compounds from CCA or associated microorganisms often remain poorly described 26 , 33 and only a few have been fully characterized 34 – 36 . To date, there are only two settlement inducing compounds derived from bacteria described in the literature: tetrabromopyrrole (TBP) and cycloprodigiosin (CYPRO). TBP is a brominated secondary metabolite isolated from the CCA-associated Pseudoalteromonas sp. PS5 strain, that induced metamorphosis (mostly without prior attachment) of Pacific Acropora millepora larvae 25 , but complete settlement of Caribbean Porites astreoides , Orbicella franksi , A. palmata and Pacific Leptastrea purpurea larvae at varying levels 28 , 37 . CYPRO is a reddish alkaloidal pigment that was isolated along with other prodiginines from the CCA-associated bacterium Pseudoalteromonas rubra #1783. It induced complete settlement of L. purpurea larvae in a light- and concentration dependent manner 29 . Although TBP and CYPRO are chemically fully defined, both compounds lack further functional characterization in the coral settlement process and their mode of action remains unknown. Thus, the present study sought to investigate the interaction between the settlement cue CYPRO and coral larvae and, in particular, to decipher possible molecular traits that explain the efficacy of CYPRO. Building on our previous work 23 , 29 , 37 – 40 , we here provide an unprecedented visualization of the uptake of a chemical settlement inducer by coral larvae and reveal an underlying molecular mechanism that promotes the complex transformation of pelagic larvae to sessile recruits. Ultimately, our study provides unique insights into the role of infochemicals in coral reproductive biology and opens a chemo-mechanistic element in the study of coral settlement.",
"discussion": "Discussion We have demonstrated that the lipophilic pigment CYPRO produced by the CCA-associated bacterium Pseudoalteromonas rubra #1783 triggers settlement in larvae of the stony coral Leptastrea purpurea . It does so by being actively collected and subsequently enriched within the outer larval surface tissue. We further revealed that the light-driven degradation of CYPRO in attached larvae is accompanied by a steady production of µM levels H 2 O 2 , resulting in metamorphosis of the larvae on the substrate. µM levels of H 2 O 2 added to seawater induced only rapid ‘nonsense metamorphosis’ – a phenomenon bearing mild resemblance to natural metamorphosis, but without prior attachment and being eventually fatal to coral larvae. Furthermore, H 2 O 2 rapidly induced ‘nonsense metamorphosis’ within 12 h of complete darkness, excluding light as a secondary cue in this morphogenic reaction. The here presented results suggest that the physico-chemical properties of CYPRO allow its active uptake and translocation along the outer tissue of coral larvae. Other prodiginines, a family of highly bioactive pigmented alkaloids of which CYPRO is a member 43 , 44 , such as prodigiosin and 2-methyl-3-hexyl prodiginine did not induce settlement in L. purpurea and in turn were toxic in a concentration-dependent manner 29 . In comparison, CYPRO lacks the hydrophobic alkyl chain and instead contains a six-membered ring, fused to the alkyl pyrrole (Figure S2 ). We propose that this structural feature could affect the uptake and solubility of CYPRO in the larval bilayer membrane – similarly to highly membranophilic quinones, carotenoids and squalenes 45 . Yet, the dominant presence of CYPRO at the oral section of a settling larva could also be a side effect of active tissue rearrangement during larval metamorphosis 46 . Nevertheless, our findings are intriguing since CYPRO was detected in the same location as GFP, which recently has been identified to fine-tune the light microclimate in corals 47 and reported to scavenge reactive oxygen species (ROS) such as H 2 O 2 48 . Although it is not trivial to investigate, possible interactions between both compounds cannot be excluded. In fact, interactions between photosensitive ROS-producing pigments like CYPRO and ROS-scavenging GFPs are likely to occur under certain light conditions and could correlate with the settlement success of coral larvae. We here suggest that the distinct cascade of events, i.e., active uptake, translocation, molecular utilization and successive H 2 O 2 production through photodegradation is a key factor for the effectiveness of CYPRO as a settlement inducer. In comparison to water-soluble H 2 O 2 , CYPRO is highly hydrophobic and thus actively harvested by the larvae from the well bottom (Fig. 1 ). Upon light exposure, the absorbed pigment is locally converted to H 2 O 2 , and larval attachment and metamorphosis are carried out consecutively 29 . In contrast, directly applied H 2 O 2 can be homogeneously encountered throughout the entire well volume. Thus, µM additions of H 2 O 2 (Fig. 3 ) induced metamorphosis in coral larvae, but only in the water column and without prior attachment (‘nonsense metamorphosis’), likely, since larvae do not encounter high local H 2 O 2 concentrations as in the case of CYPRO. That is, our results contrast recent studies that reported synergistic effects of multiple morphogens, often of lipid and polysaccharide origin, in corals and other marine model organisms 26 , 49 – 52 . However, their mechanisms have not been described and it is debatable if the latter compounds are general metamorphic cues for cnidarian larvae. Conversely, there is supporting evidence for CYPRO-induced settlement via ROS production, since H 2 O 2 has been shown to elicit settlement in various larvae of Caribbean scleractinian corals 53 and here in Pacific corals as well. Since H 2 O 2 is involved in the regulation of many reproductive processes 54 – 56 it is tempting to examine whether the cascading photochemical production of ROS by chromophoric compounds such as CYPRO is a phenomenon with widespread ecological significance in coral settlement. These considerations led us to propose a conceptual model that incorporates insights from the current and past studies 23 , 29 , 39 , 40 (Fig. 4 ). Microbial biofilms composed of settlement-inducing bacteria such as Pseudoalteromonas assemble on marine hard substrates and continue to produce photoactive chemicals such as the pigment CYPRO. During phases of high light intensity, the photosensitive molecules degrade and may establish an H 2 O 2 gradient that attracts coral larvae. Following the gradient, coral larvae encounter, accumulate and utilize intact lipophilic compounds such as CYPRO for the subsequent transformation. Eventually, the light-driven degradation of accumulated pigments releases elevated intracellular levels of H 2 O 2 that likely energize the costly metamorphosis into coral recruits. In the reef environment, additional morphogens and environmental parameters such as light quality and intensity might further diversify recruitment mechanisms and enhance settlement success rates of coral larvae. In summary, our findings let us hypothesize that the metamorphic cue CYPRO may act as a molecular battery which, through successive photodegradation, can facilitate a constant intracellular supply of elevated H 2 O 2 levels over an extended period within the larval tissue, ultimately fueling the metamorphosis of L. purpurea larvae. If this chemical interaction indeed happens in vivo remains yet to be tested, however, our data likely supports such a scenario and provides a conceptual framework for future investigations. Accordingly, we substantiate the potential of bioactive settlement cues and encourage future coral conservation efforts to be mindful of external physical factors. This study has revealed the intricate interplay between organisms, biomolecules and abiotic factors, and has demonstrated that the photodegradation of a reactive compound is indeed the driving force behind the rate-limiting conversion of coral larvae to polyps. More research is needed to assess the ecological significance of CYPRO across scleractinian diversity, but preliminary data with Acropora tenuis larvae indicated settlement with a broadcast spawning species as well. Our approach is opening an unprecedented mechanistic element to the study of chemical signaling in coral larvae settlement and potentially other marine invertebrates. Figure 4 Conceptual model of cycloprodigiosin (CYPRO) and H 2 O 2 induced settlement in coral larvae. Microbial biofilms consisting of settlement-inducing bacteria such as Pseudoalteromonas rubra assemble on marine hard substrates and continue to produce bioactive pigments like CYPRO ( Step 1 ). During phases of high light intensity, the photosensitive pigments bleach and establish an H 2 O 2 gradient that attracts coral larvae ( Step 2 ). Following the gradient, coral larvae encounter, accumulate and utilize intact lipophilic pigments for the following transformation ( Step 3 ). Eventually, the light-driven degradation of accumulated pigments releases elevated intracellular levels of H 2 O 2 that seems to be crucial for the transformation into coral recruits ( Step 4 ) and eventually juvenile polyps."
} | 3,208 |
23541503 | null | s2 | 2,602 | {
"abstract": "Fatty acid metabolism is an attractive route to produce liquid transportation fuels and commodity oleochemicals from renewable feedstocks. Recently, genes and enzymes, which comprise metabolic pathways for producing fatty acid-derived compounds (e.g. esters, alkanes, olefins, ketones, alcohols, polyesters) have been elucidated and used in engineered microbial hosts. The resulting strains often generate products at low percentages of maximum theoretical yields, leaving significant room for metabolic engineering. Economically viable processes will require strains to approach theoretical yields, particularly for replacement of petroleum-derived fuels. This review will describe recent progress toward this goal, highlighting the scientific discoveries of each pathway, ongoing biochemical studies to understand each enzyme, and metabolic engineering strategies that are being used to improve strain performance."
} | 229 |
23852978 | null | s2 | 2,605 | {
"abstract": "Spiking neuron models are used in a multitude of tasks ranging from understanding neural behavior at its most basic level to neuroprosthetics. Parameter estimation of a single neuron model, such that the model's output matches that of a biological neuron is an extremely important task. Hand tuning of parameters to obtain such behaviors is a difficult and time consuming process. This is further complicated when the neuron is instantiated in silicon (an attractive medium in which to implement these models) as fabrication imperfections make the task of parameter configuration more complex. In this paper we show two methods to automate the configuration of a silicon (hardware) neuron's parameters. First, we show how a Maximum Likelihood method can be applied to a leaky integrate and fire silicon neuron with spike induced currents to fit the neuron's output to desired spike times. We then show how a distance based method which approximates the negative log likelihood of the lognormal distribution can also be used to tune the neuron's parameters. We conclude that the distance based method is better suited for parameter configuration of silicon neurons due to its superior optimization speed."
} | 300 |
27492680 | PMC4974511 | pmc | 2,606 | {
"abstract": "The ability for magnetite to act as a recyclable electron donor and acceptor for Fe-metabolizing bacteria has recently been shown. However, it remains poorly understood whether microbe-mineral interfacial electron transfer processes are limited by the redox capacity of the magnetite surface or that of whole particles. Here we examine this issue for the phototrophic Fe(II)-oxidizing bacteria Rhodopseudomonas palustris TIE-1 and the Fe(III)-reducing bacteria Geobacter sulfurreducens , comparing magnetite nanoparticles ( d ≈ 12 nm) against microparticles ( d ≈ 100–200 nm). By integrating surface-sensitive and bulk-sensitive measurement techniques we observed a particle surface that was enriched in Fe(II) with respect to a more oxidized core. This enables microbial Fe(II) oxidation to occur relatively easily at the surface of the mineral suggesting that the electron transfer is dependent upon particle size. However, microbial Fe(III) reduction proceeds via conduction of electrons into the particle interior, i.e. it can be considered as more of a bulk electron transfer process that is independent of particle size. The finding has potential implications on the ability of magnetite to be used for long range electron transport in soils and sediments.",
"conclusion": "Conclusions We have shown that magnetite can undergo microbial redox processes as both nanoparticles and microparticles. Whilst microbial Fe(II) oxidation appears to be highly sensitive to the surface area to volume ratio of the magnetite, this relationship does not appear to be valid for microbial Fe(III) reduction. Instead we suggest that microbial Fe(III)-reducers drive an electron hopping mechanism through the particles, thus rendering it a bulk dependent effect. Such differences between surface/bulk processes could potentially have significant impact on ( i ) the identification of biomarkers and changes to redox conditions in the rock record using magnetic signatures; ( ii ) the suitability of magnetite in mediating long range intercellular electron exchange within soils and sediments; ( iii ) use of magnetite as a remediation agent for treating wastewater or contaminated soils and sediments.",
"discussion": "Discussion Surface vs bulk reactions One of the most striking results of this study is the difference in stoichiometry of both the Nanomag and Micromag starting materials observed by XMCD compared to Mössbauer spectroscopy. The amount of Fe(II)/Fe(III) ratio determined by XMCD ( x ≈ 0.64–0.75) was far in excess of the expected stoichiometric value of 0.5. In comparison, the Mössbauer data indicated that both nanoparticles and microparticles were slightly oxidized ( x ≈ 0.38–0.43). The XMCD signal is surface sensitive with an exponentially decreasing probing depth of 4.5 nm, whilst Mössbauer works via transmission and will probe the entire crystal. This suggests that the surfaces of the particles are highly enriched in Fe(II) compared to their cores. Scanning transmission electron microscopy–high angle annular dark field (STEM-HAADF) mapping was applied on Micro-ox, because their larger crystal sizes were more accessible to this technique, and confirmed such a surface layer to be present ( Fig. 6 ). This is in agreement with previous studies that show a tendency for magnetite nanoparticles in aqueous solution to spontaneously segregate electron density to the particle-solution interface, and that this tendency can persist through redox reactions arising from changes in solution conditions or solutes. For example, oxidation by acidic dissolution through dilution of the initial particle suspensions (~pH 8) in NaHCO 3 buffer (pH 7) produces an oxidized interior (as shown by XRD data) while the surface region remains enriched in Fe(II) relative to Fe(III) (as shown by XMCD data) 9 . With subsequent oxidation by R. palustris TIE-1, both XMCD and Mössbauer support oxidation of the mineral phases, in line with previous studies 8 9 13 , with the changes more pronounced in the XMCD measurements. This enables us to conclude that the microbial Fe(II) oxidation is highly dependent upon the surface properties of the particles, and it is thus likely that surface stoichiometry will play a significant role in the ability for Fe(II)-oxidizing bacteria to oxidize magnetite. This hypothesis is further supported by the fact that the difference is more pronounced in the case of the nanoparticles than in the microparticles, which highlights the importance of the surface area to volume ratio effect. However, in the case of reduction, the results of both Mössbauer and XMCD are less clear despite the fact that it has previously been shown that bacteria are capable of reducing magnetite 10 11 13 37 . The XMCD data suggest that the Fe(II)/Fe(III) ratio at the particle surface is very similar if not slightly oxidized after exposure to the Fe(III)-reducing bacteria. However, using Mössbauer spectroscopy, it could be seen that the Fe(II)/Fe(III) ratio for the whole magnetite particle did in fact increase as a result of microbial Fe(III) reduction. These differences between the XMCD and Mössbauer suggest that the surfaces of the particles remain relatively unmodified by the bacteria, whilst particle cores are reduced, consistent with some type of electron transport through the surface into the mineral lattice 35 . The high abundance of surface Fe(II) in these synthetic magnetites is likely one of the reasons why the Fe(II)-oxidizing and Fe(III)-reducing bacteria behave so differently with respect to the surface and the bulk. As the ratio of Fe(II)/Fe(III) determined using XMCD (i.e., the ratio at the magnetite surface) appears to be high, it is possible that the magnetite surfaces are saturated with a supply of Fe(II), which the Fe(II)-oxidizer R. palustris TIE-1 directly accesses and converts to Fe(III); this simultaneous accumulation of an Fe(III) product at the surface results in a larger net decrease in the surface Fe(II)/Fe(III) ratio, whilst the core remains relatively intact. The surface saturation with Fe(II), however, may also hinder the electron transfer by Fe(III)-reducing bacteria as the surfaces of the magnetite particles are already so highly reduced that any further reduction of the surface is not or hardly possible. Instead, we suggest that an electron transport process must be enlisted to enable reduction to proceed, transferring charge through the surface via an electron hopping mechanisms to the core of the magnetite and leading to the overall reduction of the core of the particles, a process that could manifest the observed changes to the magnetic susceptibility. Magnetic properties The low temperature magnetic properties of the samples reveal broad similarities between the reduced and starting materials as compared to the oxidized samples. The most notable changes occur in the RT-SIRM data for Nano-ox. Whilst the initial change in remanence magnetization is the smallest for Nano-ox samples on cooling, the warming curve shows dramatic differences to the other samples. These data suggest that low-temperature magnetic methods can distinguish magnetite particles which have undergone oxidation (through either microbial or abiotic pathways) from those that have not been oxidized. However, the reduction of both magnetite nanoparticles and microparticles does not yield significant differences in low temperature magnetic properties, which potentially rules it out as a diagnostic tool for such processes. Notwithstanding, it is evident that reduction does lead to significant changes to the room temperature volume dependent magnetic susceptibility ( Fig. 1b ), which could potentially have a profound impact on the use of this technique for recording changes to the magnetic content of soils and sediments. Changes in susceptibility might not simply be confined to the principle of increased or decreased magnetic material, but may also be a reflection of microbial processes that are currently poorly understood (see recent review by Maxbauer et al . (2016) 38 . An increasing number of studies suggest that bacteria have a direct influence on magnetic properties of soils and sediments 39 , and low temperature magnetometry appears to be one of the more useful and sensitive characterization tools in this line of study 40 . Thus, there is a close link between microbiology and geophysics, specifically environmental- and rock magnetism, and even simple metrics such as magnetic susceptibility may provide an important signature that could be used as an indicator for redox conditions in the past and present 41 42 . Environmental implications The results presented here have several implications in terms of the interactions of bacteria with magnetite and other redox active Fe minerals in the environment. Perhaps most important is the relevance to the processes by which long range electron transport is mediated by extracellular electron transfer. For instance, the different methods through which Fe(III)-reducing bacteria are able to use solid electron acceptors, such as ferrihydrite, require either direct contact (operating over distances <1.8 nm), the production of chelators (nm-μm), use of electron shuttles (nm-μm), or the formation of nanowires, which are appendages that appear to facilitate electron transport over several micrometers. However, recently a number of different studies have attempted to learn more about these types of long range electron transport 43 and also the potential role of iron minerals (including magnetite) in facilitating it 44 . A recent study suggested that sulfide minerals can even enable centimetre-long electron transport in marine sediments 45 . Our results support the idea that mixed valent iron minerals, such as magnetite, can potentially play a much more important role in electron transfer than previously considered. In the context of microbial Fe(II) oxidation, it is clear that surface stoichiometry plays a major role in the ability of Fe(II)-oxidizers to use magnetite as an electron donor, and that oxidation is perhaps unlikely to occur in locations where abiotic oxidation is able to induce maghemitization of the mineral surface. By contrast, we observe that microbial Fe(III) reduction can take place regardless of the size of the magnetite (i.e., microparticles and nanoparticles can both be relatively easily reduced), and that any large magnetite crystals could potentially be used as a terminal electron acceptor for Fe(III)-reducing bacteria. Finally, these findings also have implications for the use of magnetite, predominantly in the form of nanoparticles, for environmental remediation strategies of waste water and contaminated soils and sediments 46 . Specifically, nano-magnetite has been previously shown to react with a variety of heavy metals (e.g., Cr and U) as well as other compounds (e.g., ArNO 2 ), making it a suitable candidate for environmental remediation 14 15 47 48 . However, the reactivity of magnetite is highly dependent upon its Fe(II) content, with reduction rates significantly inhibited by decreasing Fe(II)/Fe(III) stoichiometry 16 17 . Thus, the microbial oxidation or reduction of magnetite demonstrated in this study could significantly impact the redox reactivity at the mineral surface and directly influence its remediation capabilities 49 ."
} | 2,827 |
35727971 | PMC9245687 | pmc | 2,608 | {
"abstract": "Significance Combining (photo)electrochemical platforms with CO 2 -fixing bacteria as “living” biocatalysts has realized the highly selective reduction of CO 2 to C 2 + products, such as acetate. This approach also enables the downstream conversion of the initial CO 2 product to a higher-value one. We report an advance on this concept by coculturing primary CO 2 -fixing bacteria producing acetate with secondary N 2 -fixing bacteria that employ the acetate to reduce N 2 to NH 3 and to generate a bioplastic. The symbiotic coculture can be controlled electrochemically and modularly tuned to generate a desired product stream. We foresee that this platform could be expanded to produce several additional products, including bioplastics, biofuels, and sugars, from only CO 2 , N 2 , H 2 O, and electricity.",
"discussion": "Results and Discussion In nature, a symbiosis exists between legume plants and nitrogen-fixing Rhizobia ( Fig. 1 ) ( 47 ). Legumes provide an anerobic microenvironment in their root nodules and supply rhizobia with organics, such as malate ( 48 ). Rhizobia metabolize the organic substrates and in turn, fix sufficient N 2 for themselves and the host plant. Taking inspiration from nature, we identified the bacterium R. palustris that is capable of assimilating acetate to fuel cell functions, including N 2 fixation ( 49 ). As demonstrated in SI Appendix , Fig. S1 , R. palustris initiates acetate metabolism by activating acetate to acetyl coenzyme A (acetyl-CoA), which feeds into the tricarboxylic acid (TCA) cycle with a glyoxylate shunt. Acetyl-CoA is combined with oxaloacetate to form citrate. After recombining citrate to isocitrate, it is divided into succinate and glyoxylate. Succinate is oxidized to malate, yielding ubiquinol, while glyoxylate is joined with acetyl-CoA to form malate. Malate is then oxidized to oxaloacetate-generating nicotinamide adenine dinucleotide and ubiquinol. Oxaloacetate is either incorporated back into the TCA cycle or decarboxylated to phosphoenolpyruvate to be used in biosynthesis. In addition to providing ammonia, metabolic N 2 reduction serves as an electron sink allowing for the oxidization of molecular redox shuttles generated through the TCA cycle ( 50 ). Therefore, acetate is directly responsible for providing reductive energy to nitrogenase. Concurrently, R. palustris could also condense two acetyl-CoA molecules to hydroxybutyryl-CoA, the monomer for PHB. Fig. 1. Bioinspired biohybrid coculture design. Rhizobia inhabit the anoxic root nodules of legumes, where they are provided with organics (e.g., malate) by the legume. The rhizobia in turn fix N 2 to nitrogenous compounds that the legume uses for growth. In our design, an SiNW/ S. ovata ensemble generates acetate from CO 2 , taking the place of the legume. Similar to rhizobia, R. palustris uses the acetate as a feedstock to convert N 2 to NH 3 . Solar energy powering the biohybrid platform is symbolized by the ray of light where ℏν specifically signifies the light energy. ( Inset ) Image depicts vigna unguiculata root nodules. Image credit: Harry Rose (photographer), licensed under CC BY 2.0 . As a result of the adept acetate metabolism in R. palustris , we posit that we could integrate R. palustris into our SiNW– S. ovata biohybrid system, thus mimicking the plant/bacteria symbiosis fixing both CO 2 and N 2 to value-added products ( Fig. 1 ). Overall, (photo-)electrochemical reducing equivalents power S. ovata -mediated CO 2 to acetate conversion; this acetate is then consumed by R. palustris to enable N 2 fixation and PHB production. Characterization of Individual S. ovata and R. palustris \n nifA * Cultures. Before combining S. ovata and R. palustris , we characterized each strain’s growth and product outputs in separate cultures. We inoculated S. ovata in autotrophic medium (pH 6.8, 35 °C) at three sequential starting optical densities (OD): I (starting OD 545 of 0.15), II (starting OD 545 of 0.225), and III (starting OD 545 of 0.6) with an H 2 /CO 2 headspace ( Fig. 2 A ). Over the first 24 h, III reached an acetate concentration of 34 mM, while I and II reached 25 and 15 mM, respectively ( Fig. 2 B ). These results indicated that over the first day, acetate production was proportional to culture density. All cultures achieved a similar acetate concentration on the second day (∼52 mM) and a maximum of ∼60 mM on the third day, regardless of OD. At this point, the pH of the cultures was 5.2, which is lower than the optimal pH range for S. ovata ( SI Appendix , Fig. S2 ) ( 51 ). The production of acetate presented a self-inhibitive problem; the accumulated acetate became toxic to cells as it acidified the pH of the media to an intolerable level. Each culture set grew by OD 545 ∼ 0.2, and the stationary stage coincided with media acidification, indicating that metabolic activity is paused in a low-pH environment. Therefore, a mechanism for acetate sequestration could maintain a neutral pH and thus, enable sustained growth and metabolic activity of S. ovata cultures over a longer period. Fig. 2. Monoculture characterization of S. ovata and R. palustris . ( A ) Biomass and ( B ) acetate tracking of autotrophic S. ovata monocultures I, II, and III inoculated at sequentially increasing starting optical densities of OD 545 = 0.15, OD 545 = 0.225, and OD 545 = 0.6, respectively. Diazotrophic, photoheterotrophic R. palustris nif A * provided with synthetic acetate (2.5 to 15 mM) and photoautotrophic R. palustris TIE-1 WT monocultures supplied with an H 2 /CO 2 /N 2 headspace. Daily measurements of ( C ) biomass, ( D ) total nitrogen, and ( E ) ammonia. Error bars represent one SD of three independent measurements. Next, we characterized the photoheterotrophic growth of the R. palustris nif A * mutant under diazotrophic conditions, providing synthetic acetate as a carbon substrate with a pure N 2 headspace. As previously noted, R. palustris nif A * expresses nitrogenase genes constitutively. This is a result of a mutation in the Q-linker region between the NifA N-terminal GAF and AAA + domains ( 50 ). The R. palustris nif A * mutant was first identified by Rey et al. ( 52 ) following adaptative evolution to isolate R. palustris mutants with enhanced biohydrogen production, as H 2 is produced obligatorily along with NH 3 by nitrogenase. Importantly, the R. palustris nif A * mutant does not metabolize H 2 , which prevents any competition with S. ovata for H 2 ( 53 ). Secretion of NH 3 into the culture medium by R. palustris nif A * under N 2 -fixing conditions has been previously detected ( 54 ). Genetic engineering of the NifA gene has also been undertaken in different N 2 -fixing bacteria strains to obtain extracellular NH 3 ( 55 ). We plotted the growth curves of R. palustris nifA * under diazotrophic conditions provided with increasing acetate concentrations from 2.5 to 15 mM ( Fig. 2 C ). The greatest culture density was achieved in the culture with the highest initial acetate concentration; thus, the biomass production was dictated by acetate. As a comparison, we cultured R. palustris TIE-1 wild type ( TIE-1 WT) under diazotrophic and autotrophic conditions (N 2 /CO 2 /H 2 ) ( 56 ). R. palustris TIE-1 WT failed to reach a comparable density as the photoheterotrophic cultures within the same experimental time frame. Afterward, using a Kjeldahl-type nitrogen digestion assay, we were able to quantify the total fixed nitrogen in each culture set ( Fig. 2 B and D ). The amount of fixed nitrogen directly correlated with the density of each culture; thus, the concentration of acetate prescribed the overall quantity of nitrogen that was fixed. We tracked the acetate concentration in an R. palustris nif A * culture and found that the acetate was categorically consumed by the midexponential phase ( SI Appendix , Fig. S3 ). Furthermore, we determined that even if the growth of an R. palustris nif A * culture plateaus and the initial acetate is completely consumed, we can induce further growth by adding more acetate ( SI Appendix , Fig. S4 ). This establishes that R. palustris nif A * culture activity can be extended by continuous addition of acetate. Finally, we investigated the ability of R. palustris nif A * to generate extracellular NH 3 . We utilized an established fluorescence-based assay to detect NH 3 coupled with proton nuclear magnetic resonance ( 1 H-NMR) confirmation ( 57 ). In order to maximize the amount of NH 3 recovery, we performed a short selection experiment. R. palustris nif A * was divided into cultures grown photoheterotrophically under diazotrophic conditions, and the culture with the highest concentration of extracellular NH 3 was reinoculated successively over at least five generations ( SI Appendix , Fig. S5 ). This boosted the NH 3 recovery by an order of magnitude. We found that the amount of extracellular NH 3 produced by R. palustris nif A * was inversely proportional to the initially available acetate (i.e., the cultures with 2.5 mM acetate had the highest concentration of extracellular NH 3 ) ( Fig. 2 E ). This observation may be explained by the fact that cultures with higher acetate concentration have access to more carbon for protein biosynthesis through which NH 3 is depleted rapidly. As previously noted, the acetate was fully consumed before the culture reached its maximum OD, pointing to a metabolic stage during which all the acetate has been converted to carbon intermediates. The amount of NH 3 consumed by protein biosynthesis depends on the abundance of carbon intermediates. Therefore, the amount of extracellularly secreted NH 3 decreases with a larger pool of carbon intermediates, which stem from acetate. Nonetheless, more focused study is required to fully elucidate this observation. Favorably, we can conclude that the initial acetate input can be used to direct nitrogen products, either nitrogenous biomass or NH 3 . We also conducted an experiment consisting of growing R. palustris nif A * under varying N 2 headspace pressures ( SI Appendix , Fig. S6 ). We confirmed that N 2 pressurization did not have an outsize effect on cell concentration below 175 kPa. The cell concentration at 200 kPa was slightly increased, although experiments at higher pressures could not be safely conducted. The concentration of extracellular NH 3 demonstrated an increasing trend up to 175 kPa. Moreover, we noted H 2 coproduction (247 ± 26 ppm) in N 2 -fixing cultures of R. palustris nif A * as H 2 is an obligatory by-product of nitrogenase-mediated N 2 hydrogenation. We investigated monocultures of S. ovata and R. palustris \n nifA* to characterize the behavior of each strain under different culturing conditions and to understand how to set up a symbiotic coculture. R. palustris \n nifA * could consume acetate generated from CO 2 and H 2 by S. ovata and thus, prevent culture acidification, whereas S. ovata could benefit by obtaining free NH 3 and H 2 from R. palustris \n nifA *-mediated N 2 fixation. Characterization and Manipulation of S. ovata and R. palustris \n nifA * Cocultures. Cocultures involving at least one N 2 -fixing bacterium have been demonstrated. These consortia have shown the ability of the N 2 -fixing strain to provide its partner strain with necessary ammonia. LaSarre et al. ( 54 ) established a cross-feeding mutualism, wherein R. palustris consumed fermentative products from E. coli and subsequently released ammonia promptly taken up by the E. coli . Smith and Francis ( 58 ) conceived of a coculture in which a genetically engineered photosynthetic cyanobacterium fixed CO 2 and provided an N 2 -fixing organism with fixed carbon products. Here, we construct a coculture between S. ovata and R. palustris nif A *. The advantages of our design are the inclusion of S. ovata , capable of fixing CO 2 to acetate innately with reducing equivalents sourced from H 2 or from a (photo-)electrochemical process. This lends the ability to direct the coculture products by managing the available substrate gases and the magnitude and duration of the photoelectrochemical process. We formulated a fixed carbon and nitrogen free minimal medium amenable for both S. ovata and R. palustris nif A * ( SI Appendix , Fig. S7 ). Our first approach involved inoculating S. ovata and R. palustris nif A * concurrently in four sets of culture tubes (at OD 600 of ∼0.4) ( Fig. 3 A and C ). The initial cell ratio of S. ovata to R. palustris nif A * was ∼6:1 (determined by correlating OD to cell counts), and the headspace was pressurized to 175 kPa with an H 2 /CO 2 /N 2 (40:20:40%) gas mixture. We tracked OD, acetate, total fixed nitrogen, and ammonia concentrations during the experimental period. Over the course of the experiment, the original headspace gas mixture was exchanged to pure N 2 at different time points in each of the four coculture sets to observe coculture growth dynamics under different headspace conditions. The four coculture sets exhibited similar OD growth over the first 3 d. At this time point, less than 1.25 mM acetate was measured in all respective cocultures, and no ammonia was detected, which pointed to successful cross-feeding. The headspace gas mixture was exchanged to pure N 2 in the first coculture set after 3 d ( Fig. 3 A , blue curves and C , blue bar). By day 10, the remaining three cocultures with H 2 /CO 2 /N 2 headspace continued to exhibit growth more than doubling the initial OD, while the coculture with a pure N 2 headspace showed markedly lower growth rate. Only trace concentrations of acetate and ammonia were detected in all the cocultures. On day 10, the headspace gas mixture in a second coculture was exchanged to pure N 2 ( Fig. 3 A , red curves and C , red bar). By day 19, the two remaining cocultures with H 2 /CO 2 /N 2 headspace had continued their linear growth trend, while the two coculture sets with N 2 headspaces had nearly plateaued. Following this observation, circa 0.5 mM acetate was measured in the cocultures with H 2 /CO 2 /N 2 headspaces, while no acetate could be detected in the cocultures with pure N 2 headspaces, pointing to S. ovata inactivity due to the lack of H 2 . On day 19, considerable amounts of NH 3 were detected in both cocultures with pure N 2 headspaces, illustrating that NH 3 cross-feeding ceased as S. ovata became inactive. The headspace of one of the two remaining cocultures with H 2 /CO 2 /N 2 headspaces was exchanged to pure N 2 on day 19 ( Fig. 3 A , green curves and C , green bar). Final sampling was conducted on day 33 and demonstrated that the coculture with H 2 /CO 2 /N 2 headspace grew to surpass OD 600 2.0, while cell densities in the cultures with pure N 2 headspaces plateaued ( Fig. 3 A , purple curves and C , purple bar). The coculture with the H 2 /CO 2 /N 2 headspace proliferated past the densities achieved by either strain in a monoculture. The media did not become acidic as acetate was not allowed to accumulate, which sustained growth of the coculture. Critically, the S. ovata in the coculture remained active over the entire 33-d-long experiment if H 2 was available, exhibited by a detectable amount of acetate on the final sampling day and an increase in total fixed nitrogen in the coculture, which was fueled by acetate. Continued coculture growth over >1 mo can be taken as proof of cross-feeding between each species, with R. palustris nif A * taking up acetate and S. ovata consuming ammonia. No significant amount of ammonia could be measured in the coculture with H 2 /CO 2 /N 2 headspace, whereas concentrations in the hundreds of micromolar were measured in the cocultures after they were switched to N 2 headspaces. Fig. 3. S. ovata and R. palustris nifA * coculture characterization. ( A ) S. ovata and R. palustris nifA * inoculated concurrently. ( B ) S. ovata inoculated 24 h before R. palustris nifA * is introduced into the culture. The legend signifies the day on which the headspace was exchanged from H 2 /CO 2 /N 2 to pure N 2 for each condition. Color-matching dotted lines and arrows illustrate the day of headspace exchange on each individual plot. C and D depict the summary of the results on the last sampling day for ( C ) concurrent S. ovata and R. palustris nifA * inoculation and for ( D ) 24-h S. ovata incubation prior to R . palustris nifA * introduction. Error bars represent one SD of three independent measurements. While a successful S. ovata and R. palustris nif A * coculture was realized, we sought to accelerate the culturing time with a modified approach. As evidenced by the lack of a typical exponential growth phase in the first cocultures, acetate could be a limiting factor. Therefore, we inoculated S. ovata first with an H 2 /CO 2 (80:20%) headspace for 24 h to produce an initial acetate reservoir. Since acetate production for S. ovata is a growth-linked process, the pure S. ovata cultures were initially provided with 100 μM NH 3 to ensure high acetate production. This supplement was completely consumed within the first 24 h. R. palustris nif A * is responsible for most of the coculture growth as it grows photoheterotrophically on acetate and S. ovata grows autotrophically with limited ammonia. We determined that S. ovata has very meager growth when only supplemented with limited NH 3 ( SI Appendix , Fig. S7 B ). Corroborating this point, LaSarre et al. ( 54 ) found that the N 2 -fixing organism dominates in an NH 3 -deficient coculture, which in their study, was demonstrated as R. palustris dominated growth when cocultured with E. coli . Therefore, our 24-h preinoculation of S. ovata ensured a high acetate concentration (∼15 mM) and no extra ammonia upon the introduction of R. palustris nif A * to accelerate coculture growth ( Fig. 3 B and D ). The growth in these cocultures over the first 6 d was exponential in contrast to the first cocultures. Similar to the first coculture experiments, the headspace was exchanged in one coculture on day 2 ( Fig. 3 B , blue curves, and D , blue bar), but the growth continued a similar trajectory as in the others. By day 6, the acetate reservoir had been depleted, and the coculture growth rates slowed down significantly. On days 6 and 9, the second ( Fig. 3 B , red curves, and D , red bar) and third ( Fig. 3 B , green curves, and D , green bar) cocultures had their headspaces exchanged for N 2 , respectively. By the final sampling on day 14, the cocultures with an N 2 headspace had plateaued, while the remaining coculture with H 2 /CO 2 /N 2 headspace maintained steady growth, albeit slowed from its initial phase ( Fig. 3 B , purple curves, and D , purple bar). Extracellular ammonia was present in all the cocultures with an N 2 headspace. The 24-h S. ovata incubation strategy allowed the coculture to reach an OD 600 of 2.0 in half the time (14 vs. 28 d). We plotted the densities when the headspaces were exchanged to pure N 2 and performed a regression analysis ( SI Appendix , Fig. S9 ). Notably, the slopes of both curves are similar, although the y intercept is greater for the curve belonging to the 24-h S. ovata preincubation cocultures. This points to comparable steady-state growth dynamics, although prior incubation of S. ovata for 24 h grants the coculture much faster initial growth (two times faster biomass accumulation). Altogether these results indicate that S. ovata and R. palustris nif A * developed a robust consortium with cross-fed acetate and ammonia. Second, coculture dynamics can be directed as evidenced by the changes that occurred after headspace exchange. When reducing equivalents, such as H 2 , exist, S. ovata supported R. palustris nif A * with sufficient acetate, resulting in quick consumption of acetate, biomass accumulation, and no free ammonia in the culture. After losing access to H 2 , S. ovata activity became minimal, and free ammonia by R. palustris nif A * accumulated due to limited carbon sources. Electrochemically and Solar-Powered S. ovata and R. palustris \n nifA *-Based CO 2 and N 2 Fixation. We employed a custom-made H cell–type electrochemical reactor, which we reconditioned with metal clamps and supporting silicone o rings to withstand pressurization. The total volume was 160 mL. A TiO 2 - and Ni-coated degenerate SiNW 2.25-cm 2 cathode was fabricated as previously described ( 17 ). Pt wire and Ag/AgCl were used as counter and reference electrodes, respectively. The electrolyte was composed as previously noted with some modifications ( SI Appendix ). S. ovata was cultured as previously reported, except that the cells were washed three times anaerobically in fresh electrolyte after culturing autotrophically in minimal medium and thereafter, introduced into the cathodic chamber of the electrochemical cell with 100 μM NH 3 mimicking the previous coculture experiment. We undertook the bioelectrochemical experiments in two stages. First, we generated reducing equivalents on the cathode to power S. ovata acetogenesis and produce acetate. Thereafter, we ceased electrochemical inputs and introduced R. palustris nif A *, which consumed acetate for energy to fix N 2 and produce PHB. We undertook this staged approach to determine productivity and efficiency metrics for each coculture product. For example, if we had inoculated S. ovata and R. palustris nif A * at the same time, we would not have been able to detect acetate, as it would have been instantly consumed by R. palustris nif A *. Accurately determining the concentration of acetate is paramount in this initial demonstration, as the coculture behaves according to its culturing conditions with acetate concentration directing the makeup of N 2 products, namely NH 3 or nitrogenous biomass. We allowed for three increasing amounts of charge to pass through the electrodes, as these would translate to increasing acetate outputs ( Fig. 4 A and B ). For acetate, the lowest charge (99 ± 12 coulombs) resulted in an FE of 103% with a current density of 0.29 mA/cm 2 . Similarly, a charge of 166 ± 20 coulombs translated to an FE of 93% and a current density of 0.35 mA/cm 2 , while the experiment with the highest charge of 235 ± 15 coulombs rendered an FE and current density of 91% and 0.35 mA/cm 2 , respectively. After R. palustris nif A * was introduced, the FEs for nitrogen in biomass increased proportionally with charge from 13.7 to 17.4% and to 19.1%. The corresponding current densities demonstrate a similar trend, increasing from 0.049 to 0.058 mA/cm 2 and to 0.067 mA/cm 2 . Whereas nitrogenous biomass scaled with charge, NH 3 production revealed a dissimilar trend with the highest FE and current density peaking at 6.3% and 0.023 mA/cm 2 , respectively, for 166 ± 20 coulombs. The efficiency of ammonia production fell off with increasing charge. This trend mirrored that of the R. palustris nif A * monoculture experiment, which exhibited an inverse proportionality between the starting amount of acetate and the concentration of extracellular NH 3 ( Fig. 2 E ). Therefore, the nitrogen products can be regarded as tunable because the amount of electrochemical charge passed to the culture dictated their composition. McKinlay and Harwood ( 59 ) determined an optimum production yield of 21 ± 3 mol H 2 per 100 mol of acetate fed to R. palustris nif A *. As H 2 is obligately generated alongside N 2 reduction by nitrogenase, we can stoichiometrically deduce that their nitrogen fixation yield was 42 mol fixed nitrogen per 100 mol acetate. Our R. palustris nif A * monoculture yielded 31.4 ± 10.5 mol fixed nitrogen per 100 mol acetate, while electrochemical induction results in 28.6 ± 6.8 mol fixed nitrogen per 100 mol acetate. When benchmarking our production metrics with those obtained by McKinlay and Harwood ( 59 ), it is apparent that there is an opportunity for improvement, which could increase the average electricity to fixed nitrogen efficiency 1.5-fold. Fig. 4. Electrochemically supported S. ovata and R. palustris nifA *. ( A and B ) FEs and current densities for coculture products acetate, nitrogenous biomass, and ammonia. ( C ) Fully solar-powered coculture via an external photovoltaic device with solar to chemical efficiencies. ( D ) Scanning electron microscopy micrograph of the SiNW array with S. ovata and R. palustris nifA *. Individual bacteria on the front-most plane are identified and color-highlighted based on morphology ( S. ovata is yellow, and R. palustris is pink). Error bars represent one SD of three independent measurements. Importantly, R. palustris also synthesized PHB as a form of carbon and energy storage. We measured 1.04 ± 0.04 mg/L PHB (0.8% carbon yield), which was commensurate with previous accounts ( 60 ). PHB production could be maximized to up to 30% of the dry cell weight by limiting access to N 2 ( 49 ). Therefore, switching to a pure CO 2 headspace (instead of N 2 ) during coculturing could direct PHB synthesis from acetate, offering more tunable products for this coculture platform. Although not verified in this study, R. palustris has been reported to produce CH 4 simultaneously with NH 3 , thus possibly positioning the coculture platform to offer another carbon product ( 61 ). Additionally, other forms of exploitable carbon storage products have also been observed for R. palustris , including glycogen and trehalose ( 49 ). Moreover, isotope-labeled 13 C control experiments were employed ( SI Appendix , Fig. S10 ). To further confirm N 2 fixation, we inoculated R. palustris nif A * into liquid cultures with argon (Ar) degassed medium. We then filled the headspaces with 15 N 2 and Ar and provided acetate to each. Notably, the culture with Ar exhibited negligible growth, and the culture with 15 N 2 accumulated biomass through 15 N 2 fixation ( SI Appendix , Fig. S11 A ). 1 H-NMR results confirm no 14 NH 3 in the Ar culture and 15 NH 3 in the 15 N 2 culture ( SI Appendix , Fig. S11 B ). The absence of 14 NH 3 in cultures with Ar and 15 N 2 headspaces and the lack of growth in the Ar culture point to curtailed external contaminations. Following the confirmation of an electrochemically supported platform, we investigated whether our system would allow for solar-driven CO 2 and N 2 fixation. We connected our electrochemical cell housing the bacterial coculture to a photovoltaic device, as shown in diagram in Fig. 4 C , and followed a process analogous to the electrochemical experiments. Similarly to our previous report, we used a commercially available multijunction Si solar cell (V oc = 4.7 V, I sc = 4.4 mA under one sun illumination) to drive the reaction ( 17 ). The light intensity was set at 25 mWcm −2 using an National Renewable Energy Laboratory (NREL)-calibrated Si photodiode. The system produced acetate with a solar to chemical efficiency of 1.78 ± 0.13% over 2 d, and upon introduction of R. palustris nif A *, it was able to fix N 2 into nitrogenous biomass and ammonia with efficiencies of 0.51 ± 0.05% and 0.08 ± 0.007%, correspondingly. To the best of our knowledge, no other platform has been reported for full solar-driven CO 2 and N 2 fixation. Finally, we assessed the SiNW array used to support the bacterial culture by scanning electron microscopy. We noticed the presence of two distinct bacterial morphologies. One was bulbous, and the other was more tubular in appearance. We proceeded to culture R. palustris nif A * and S. ovata individually on SiNWs and examined them by scanning electron microscopy ( SI Appendix , Fig. S12 A and B ). We observed that the bulbous morphology can be attributed to S. ovata , while R. palustris nif A * presents as longer and thinner. These observations of the individual strain morphologies agree with the literature ( 51 , 52 ). Next, we centrifuged down a mature coculture at 12,000 rpm and found layering, with the longer, heavier, and pink R. palustris nif A * underneath the shorter, lighter, and beige S. ovata as evident by the distinct colors of each strain ( SI Appendix , Fig. S12 C ). A qualitative approximation of the final cell ratio is 2:3 S. ovata to R. palustris nif A *, having been seeded at an initial 6:1 S. ovata to R. palustris nif A * cell ratio. This result corroborates that most of the coculture growth stems from the N 2 -fixing heterotrophic R. palustris , as has previously been claimed ( 54 ). Finally, we were able to qualitatively identify each bacterial strain on the front-most plane of a scanning electron microscopy micrograph as highlighted by false coloring in Fig. 4 D ( SI Appendix , Fig. S12 D ). Fluorescence-activated cell sorting or a fluorescence in situ hybridization assay needs to be performed to quantitatively report individual bacterial populations in future work. Overall, we demonstrate a biohybrid coculture capable of fixing CO 2 and N 2 to value-added products. We namely show that bio-produced acetate from CO 2 could be used to power N 2 fixation in a separate whole-cell catalyst. By assigning CO 2 and N 2 fixation to two separate biocatalysts, we are able to maximize efficiency and rate. However, not only is acetate a fuel for N 2 fixation, but it also provides a substrate for bioplastic synthesis and potentially manifold other carbon products owing to the metabolic versatility of R. palustris . Moreover, employing two separate biocatalysts in synergy allowed us to direct product outputs through electrochemistry and headspace inputs. We strongly believe that advances in bioelectrochemistry will arise from studying and engaging bacterial communities. Organisms in nature cooperate to thrive, and we must emulate these associations in biohybrid systems to tackle environmental challenges."
} | 7,499 |
25237788 | null | s2 | 2,609 | {
"abstract": "Compost-assisted phytostabilization has recently emerged as a robust alternative for reclamation of metalliferous mine tailings. Previous studies suggest that root-associated microbes may be important for facilitating plant establishment on the tailings, yet little is known about the long-term dynamics of microbial communities during reclamation. A mechanistic understanding of microbial community dynamics in tailings ecosystems undergoing remediation is critical because these dynamics profoundly influence both the biogeochemical weathering of tailings and the sustainability of a plant cover. Here we monitor the dynamics of soil microbial communities (i.e. bacteria, fungi, archaea) during a 12-month mesocosm study that included 4 treatments: 2 unplanted controls (unamended and compost-amended tailings) and 2 compost-amended seeded tailings treatments. Bacterial, fungal and archaeal communities responded distinctively to the revegetation process and concurrent changes in environmental conditions and pore water chemistry. Compost addition significantly increased microbial diversity and had an immediate and relatively long-lasting buffering-effect on pH, allowing plants to germinate and thrive during the early stages of the experiment. However, the compost buffering capacity diminished after six months and acidification took over as the major factor affecting plant survival and microbial community structure. Immediate changes in bacterial communities were observed following plant establishment, whereas fungal communities showed a delayed response that apparently correlated with the pH decline. Fluctuations in cobalt pore water concentrations, in particular, had a significant effect on the structure of all three microbial groups, which may be linked to the role of cobalt in metal detoxification pathways. The present study represents, to our knowledge, the first documentation of the dynamics of the three major microbial groups during revegetation of compost-amended, metalliferous mine tailings."
} | 505 |
35093526 | null | s2 | 2,610 | {
"abstract": "A variety of chemical and biological processes have been proposed for conversion of sustainable low-cost feedstocks into industrial products. Here, a biorefinery concept is formulated, modeled, and analyzed in which a naturally (hemi)cellulolytic and extremely thermophilic bacterium, Caldicellulosiruptor bescii, is metabolically engineered to convert the carbohydrate content of lignocellulosic biomasses (i.e., soybean hulls, transgenic poplar) into green hydrogen and acetone. Experimental validation of C. bescii fermentative performance demonstrated 82% carbohydrate solubilization of soybean hulls and 55% for transgenic poplar. A detailed technical design, including equipment specifications, provides the basis for an economic analysis that establishes metabolic engineering targets. This robust industrial process leveraging metabolically engineered C. bescii yields 206 kg acetone and 25 kg H"
} | 225 |
23898300 | PMC3722494 | pmc | 2,613 | {
"abstract": "Coral reef communities are undergoing marked declines due to a variety of stressors including disease. The sea fan coral, Gorgonia ventalina , is a tractable study system to investigate mechanisms of immunity to a naturally occurring pathogen. Functional studies in Gorgonia ventalina immunity indicate that several key pathways and cellular components are involved in response to natural microbial invaders, although to date the functional and regulatory pathways remain largely un-described. This study used short-read sequencing (Illumina GAIIx) to identify genes involved in the response of G. ventalina to a naturally occurring Aplanochytrium spp. parasite. De novo assembly of the G. ventalina transcriptome yielded 90,230 contigs of which 40,142 were annotated. RNA-Seq analysis revealed 210 differentially expressed genes in sea fans exposed to the Aplanochytrium parasite. Differentially expressed genes involved in immunity include pattern recognition molecules, anti-microbial peptides, and genes involved in wound repair and reactive oxygen species formation. Gene enrichment analysis indicated eight biological processes were enriched representing 36 genes, largely involved with protein translation and energy production. This is the first report using high-throughput sequencing to characterize the host response of a coral to a natural pathogen. Furthermore, we have generated the first transcriptome for a soft (octocoral or non-scleractinian) coral species. Expression analysis revealed genes important in invertebrate innate immune pathways, as well as those whose role is previously un-described in cnidarians. This resource will be valuable in characterizing G. ventalina immune response to infection and co-infection of pathogens in the context of environmental change.",
"conclusion": "Conclusions Here we report the generation of the first transcriptome for a soft coral species. In addition, we also report the results of the first RNA-seq experiment of a coral exposed to a microbial pathogen. We annotated a large number of immune-related genes, with many of these genes being differentially expressed in response to Aplanochytrium , including those involved in pattern recognition, anti-microbial peptide production, and wound repair. Enrichment analysis also identified processes (protein translation and energy production) involved in the host response to Aplanochytrium species. With this information, we now have a basis for comprehensively studying the immune response of G. ventalina to pathogens. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.",
"introduction": "Introduction Infectious diseases, caused by a variety of pathogens, are contributing to the decline of coral reefs worldwide (reviewed by Sutherland et al., 2004 ; Harvell et al., 2007 ; Bourne et al., 2009 ) by threatening biodiversity, causing marked population declines, and changing community structure (Harvell et al., 2002 ). Corals, like other invertebrates, defend against pathogenic invaders using the innate immune system, an ancient defense system found in both invertebrates and vertebrates. In cnidarians, both genomic and functional studies indicate the existence of key innate immune components including pathogen recognition, signaling cascades, and effector responses [reviewed by (Miller et al., 2007 ; Dunn, 2009 ; Augustin and Bosch, 2010 ; Palmer and Traylor-Knowles, 2012 )]. Targeted approaches suggest a key role of immune receptors (e.g., modified Toll like receptors, (Bosch et al., 2009 ) anti-microbial peptides (e.g., Bosch et al., 2009 ; Vidal-Dupiol et al., 2011 ), and the inflammatory cascade (Mydlarz et al., 2008 ) in the cnidarian response to pathogens. The sea fan coral, Gorgonia ventalina , underwent large-scale declines in the Caribbean in the 1990s (reviewed by Burge et al., 2013 ) as a result of “Aspergillosis” caused by the fungal pathogen Aspergillus sydowii (Smith et al., 1996 ; Geiser et al., 1998 ). A second sea fan pathogen, an Aplanochytrium spp, a marine stramenopile protist (Order Labyrinthulomycetes) was also recently described, isolated, and cultured, (Burge et al., 2012a ) where clear damage to the host has been noted in association with Labyrinthulomycete cells including longitudinal tearing of the host gorgonin (skeleton) and degradation of the host polyps (Burge et al., 2012a , 2013 ). The well-characterized disease ecology and the identification and culture of multiple natural pathogens has made the sea fan one of the best-studied corals the in the context of host immunity (e.g., Kim and Harvell, 2004 ; Mydlarz et al., 2008 ). To date, the response to pathogen exposure or disease in G. ventalina has focused on measurement of effector enzymes (e.g., prophenoloxidase, peroxidase, chitinase, catalase, and antifungal and antibacterial peptides) (Douglas et al., 2007 ; Mydlarz and Harvell, 2007 ; Couch et al., 2008 ; Mydlarz et al., 2008 ) and pathological responses using histology (Petes et al., 2003 ; Mydlarz et al., 2008 ; Burge et al., 2012a ). These studies demonstrate cellular and systemic responses that play a critical role in sea fan immune function. For instance, the inflammatory response of amoebocytes to infections in G. ventalina includes production of prophenoloxidase enzymes that enable the formation of a melanin barrier within the sea fan skeleton (Mydlarz et al., 2008 ), which is the primary observed pathological response of sea fans to both fungal and Labyrinthulomycete infection (Petes et al., 2003 ; Mydlarz et al., 2008 ; Burge et al., 2012a ). While functionally important and supported by studies in other invertebrates, these effector responses are a limited portion of the sea fan immune response and it is unclear what pathways and regulatory networks are ultimately responsible for their initiation. A transcriptomics approach employed to study sea fan immune physiology would provide a comprehensive understanding of how sea fans recognize and respond to pathogens, information that will expand our knowledge of innate immunity in cnidarians. The aims of this study were to generate a transcriptome for the sea fan coral and to characterize the sea fan host immune response to the Aplanochytrium spp. (24 h post exposure) using RNA-Seq analysis. The transcriptome data generated for G. ventalina will enable future studies focusing on key functional aspects of sea fan immunology, including environmental drivers of disease and host immunity, co-infection dynamics mediated by the immune system, and mechanisms of immune priming.",
"discussion": "Discussion Here we report the first transcriptome generated for a gorgonian coral species, G. ventalina . We characterized 40,142 contigs based on functional annotation. This data will provide an important resource for further targeted studies on immune physiology of G. ventalina . Analysis of the G. ventalina transcriptome as compared to other cnidarian transcriptomes ( A. millepora and N. vectensis ) indicated a similar number of shared contigs between each cnidarian transcriptome. Additionally, G. ventalina contigs included a high percentage of significant matches to orthologs shared across metazoans indicating the transcriptome is relatively complete. We conducted the first comprehensive transcriptomic characterization of a cnidarian exposed to a natural pathogen. Validation of differentially expressed genes performed using RT-qPCR (on a subset of genes) indicates strong similarity of individual sea fan gene expression using qPCR to our pooled Illumina sequencing data, similar to validation of pooled Illumina results by Altincicek et al., 2013 and qPCR corroboration of other RNA seq studies (e.g., Meyer et al., 2011 ). We will describe host response based on statistically relevant measures: functionally enriched categories and individual immune genes detected by DESeq analysis. Functional enrichment analysis Based on functional enrichment analysis, the majority of the enriched genes encode ribosomal proteins involved in translation. All of these genes were up-regulated in the Aplanochytrium - exposed sea fans, indicating an up-regulation of the host protein response. This pattern has also been documented in other invertebrates' exposed to pathogens [i.e., clams (Gestal et al., 2007 ); abalone (Travers et al., 2010 ); urchins (Nair et al., 2006 )]. Although the up-regulation of ribosomal gene products is not a specific response to pathogens and is likely a general response to stress, ribosomal proteins may be important regulators of metabolism during pathogen exposure (Travers et al., 2010 ). Four of the five categories with the highest fold enrichment include genes primarily involved in production of energy in the mitochondria, indicating an increased energy demand to mount an effective immune response (Nayak et al., 2011 ) or general stress response. Herein, genes involved in the production of reactive oxygen species (ROS) (NADH-ubiquinone oxidoreductase chain 1 and cytochrome c oxidase subunits) were among enriched processes in sea fans exposed to the Aplanochytrium . Additionally, NADH-ubiquinone oxidoreductase chain 1 expression was validated with RT qPCR. Up-regulation of NADH-ubiquinone oxidoreductase 1 and cytochrome c oxidase subunits suggest energy production in the mitochondria through oxidative phosphorylation. In other invertebrate host-pathogen systems, cytochrome oxidases have been shown to be up-regulated in response to immune stimulation [i.e. shrimp: (James et al., 2010 ); abalone: (van Rensburg and Coyne, 2009 ); clams: (Gestal et al., 2007 )], and in fact, evidence suggests a successful immune response in abalone may depend on electron transport (van Rensburg and Coyne, 2009 ). Herein, the electron transport chain had the highest fold enrichment (23X). Oxidative phosphorylation also leads to the production and release of ROS, which is an important pathogen-killing mechanism in invertebrate immunity and has been shown to be a critical component of the immune response of many invertebrates, including mosquitoes (Molina-Cruz et al., 2008 ) and abalone (van Rensburg and Coyne, 2009 ). Immune genes and role in cnidarian immunity We also identified a number of immune-related genes that were differentially expressed between control and Aplanochytrium -exposed sea fans, including those that code for proteins involved in pattern recognition molecules, antimicrobial peptides, and wound repair (refer back to Figure 3 ). Although these genes were not classified into significantly enriched biological processes, they nonetheless represent genes that play an important role in immune-related processes and should be investigated in more detail. We therefore devote the remaining discussion to these genes and their potential role in cnidarian immunity. Pattern recognition molecules Pattern Recognition Molecules (PRM), also known as Pattern Recognition Receptors (PRRs), recognize and bind to conserved components of microbial cell walls (e.g., lipopolysaccharide of Gram-negative bacteria). This binding results in a signaling cascade that leads to the induction of effector molecules and cellular components of immunity (Ferrandon et al., 2007 ; Dunn, 2009 ). Many of the immune-related genes that were up-regulated (e.g., Tachylectin-5A and Protein G7c) or down-regulated (e.g., Neuronal pentraxin-2) serve as PRMs. Expression of Tachylectin-5A, Protein G7c and Neuronal pentraxin-2 were validated using RT qPCR. Tachylectin-5a belongs to the fibrinogen related domain (FReD) superfamily. Members of the FReD superfamily have been described in many invertebrate taxa including cephalochordates, and are important in defense processes such as agglutination, pathogen recognition, bacterial lysis, histocompatibility, and parasite defense (reviewed by Hanington and Zhang, 2011 ). Tachylectin-5A is a plasma lectin isolated from the horseshoe crab, Tachypleus tridentatus , and agglutinates human erythrocytes as well as Gram-positive and Gram-negative bacteria (Gokudan et al., 1996 ). FReD superfamily members are well-studied in mosquito-parasite interactions where RNAi silencing experiments indicate that they are key in clearing bacterial infections (Dong and Dimopoulos, 2009 ). Furthermore, in snail-trematode interactions, the diversity of FReD superfamily members within individuals has implied somatic diversification (Zhang et al., 2004 ) and may be associated with resistance (Stout et al., 2009 ). Tachylectin homologs have been described from EST libraries of anemones and corals (tachylectin-2; Hayes et al., 2010 ) and Hydractina (tachylectin-1), although neither of these putative tachylectin's contain the FReD domain found in Tachylectin 5A or the putative sea fan Tachylectin. While the Hydractina tachylectin-like gene has been suggested to play a role in developmental metamorphosis and not immunity (Mali et al., 2006 ), work by Hayes and colleagues indicates positive selection of Oculina tachylectin-2, which is disproportionately observed in immune genes, suggesting an immune-related role for this gene (Hayes et al., 2010 ). Given that the putative sea fan Tachylectin was up-regulated upon pathogen exposure and possesses a FReD domain, we hypothesize that the sea fan tachylectin functions in host defense. Antimicrobial peptides Activation of specific PRMs such as the toll like receptors (TLRs) leads to production of antimicrobial peptides in innate immunity. Antimicrobial peptides described in microbial defense in cnidarians include hydramacin-1, arminin 1a, and periculin-1 in Hydra (reviewed by, Augustin and Bosch, 2010 ), damicornin in a hard coral (Vidal-Dupiol et al., 2011 ), and aurelin in jellyfish (Ovchinnikova et al., 2006 ). Transcripts identified in this study are homologous to anti-microbial peptides including arenicin-2 from a polycheate worm which has anti-microbial activity again gram positive, negative, and fungi (up-regulated) (Ovchinnikova et al., 2004 ), and royalisin from a honeybee which processes activity against gram positive bacteria and fungi (and not against gram negative activity to date) (down-regulated) (Fujiwara et al., 1990 ). We can speculate that the arenicin-2 homolog is a broader spectrum anti-microbial acting in response to the Aplanochytrium cells, while the royalisin homolog may be more involved with normal anti-microbial processes as its expression is higher in the un-exposed sea fans. Wound repair Cnidarians have a high capacity for regeneration and wound repair, including re-modeling of tissues after physical damage or infection by pathogens [ Hydra : (Bosch, 2007 ), (Augustin and Bosch, 2010 ); Porites : (Palmer et al., 2011 )]. In Hydra, macromolecules (laminins, collagens, heparin sulfate proteoglycans and fibronectin-like molecules) of the mesoglea (extracellular matrix) are key for regeneration, and wound repair is regulated by metalloproteinases (Bosch, 2007 ). Matrix metalloproteinases are a major group of enzymes that regulate the cell matrix composition, and ample evidence exists for the role of matrix metalloproteinases (Matrix metalloproteinase-14) in normal and pathological processes (Massova et al., 1998 ). In response to Aplanochytrium , a metalloproteinase was up-regulated. Additionally, the peroxidasin gene, which codes for a protein that acts both in extracellular matrix organization (associated with phagocytosis and defense) and hydrogen catabolic processes (i.e. catalyzing hydrogen peroxide) [ Drosophila ; (Nelson et al., 1994 )] [ Xenopus tropicalis ; (Tindall et al., 2005 )], was up-regulated."
} | 3,947 |
27141848 | PMC4855234 | pmc | 2,614 | {
"abstract": "Microalgae have emerged as a promising source for biofuel production. Massive oil and starch accumulation in microalgae is possible, but occurs mostly when biomass growth is impaired. The molecular networks underlying the negative correlation between growth and reserve formation are not known. Thus isolation of strains capable of accumulating carbon reserves during optimal growth would be highly desirable. To this end, we screened an insertional mutant library of Chlamydomonas reinhardtii for alterations in oil content. A mutant accumulating five times more oil and twice more starch than wild-type during optimal growth was isolated and named c onstitutive o il a ccumulator 1 ( coa1 ). Growth in photobioreactors under highly controlled conditions revealed that the increase in oil and starch content in coa1 was dependent on light intensity. Genetic analysis and DNA hybridization pointed to a single insertional event responsible for the phenotype. Whole genome re-sequencing identified in coa1 a >200 kb deletion on chromosome 14 containing 41 genes. This study demonstrates that, 1), the generation of algal strains accumulating higher reserve amount without compromising biomass accumulation is feasible; 2), light is an important parameter in phenotypic analysis; and 3), a chromosomal region (Quantitative Trait Locus) acts as suppressor of carbon reserve accumulation during optimal growth.",
"discussion": "Results and Discussion Isolation of a Mutant with Higher Oil and Starch Content during Optimal Growth As part of our effort to dissect lipid metabolic pathways and identify novel factors involved in carbon reserve formation, an insertional mutant library has been generated as described previously 26 . The parental line used here is the wild-type CC124. This mutant library was then screened for alterations in oil content during optimal growth conditions, according to the screening method described previously 27 . Overall, after screening 7,000 independent transformants by Nile red coupled to Flow cytometry, a mutant (initially named 6D8) showing higher level of Nile red fluorescence than WT under normal growth condition was isolated. Higher Nile red fluorescence is generally used as a probe for increased cellular oil content, and indeed, TAG quantification confirmed that the mutant 6D8 made 5 times more oil than WT under nitrogen-replete conditions ( Fig. 1A ). In addition to an increase in oil content, an increased number of lipid droplet (LDs) was observed in the mutant cells, as revealed by staining with two independent lipophilic dyes i.e. Bodipy ( Fig. 1C,D ) or Nile red ( Fig. 1E,F ). Furthermore, the mutant 6D8 accumulated 1.5 times more oil than WT after being starved for nitrogen for 3 days ( Fig. 1B ). Based on these findings, the mutant 6D8 was re-named as c onstitutive o il a ccumulator 1 ( coa1 ). No significant difference could be detected in the major membrane lipids ( Supplemental Figure S1A,B ). Additionally, we also observed that during the recovery phase following a period of nitrogen starvation, the mutant was also impaired in oil remobilization ( Supplemental Figure S2 ). This result could imply that the observed net increase in oil content in the mutant during optimal growth could be, at least partly, due to a defect in oil breakdown. But this and other possibilities should be tested in the future with labelling studies to measure the rate of oil turnover during optimal growth. Furthermore, the coa1 mutant made almost twice more starch than its parental strain CC124 during normal growth ( Fig. 2A ), an increase from ~25 μg mm −3 to over ~50 μg mm −3 . It is well known that the wild-type strains of C. reinhardtii store starch as a major form of polysaccharide reserve 20 , for reasons of simplicity, we have referred to polysaccharide reserves as starch throughout this paper. However it is worth noting here that the additional accumulation of polysaccharides observed in the mutant coa1 could be starch or any other type of polysaccharide made of α-glucans, or a mixture of both. Under the same cultivation condition, coa1 mutant contained less chlorophyll than WT ( Fig. 2B ). The parallel increase in intracellular oil and starch observed in the coa1 mutant suggests that a common mechanism underlies regulation of these two storage compounds. This regulatory mechanism would be affected in the mutant, resulting in an increased carbon flux towards reserve formation. The coa1 Mutant Forms Palmelloid Structures but Shows Similar Growth Rate as WT During cell counting, the coa1 mutant was found to form aggregates, showing much bigger particles in diameter than WT ( Fig. 3A ). The aggregated clusters were also observed under a microscope ( Fig. 3B ). Such a phenotype has been previously described in Chlamydomonas mutant strains, and these are referred to as palmelloid colonies 28 . Formation of palmelloid colonies seem to be common 29 , and has been suggested as part of microalgal self-defense against stressors 30 . In an attempt to avoid cell aggregation, strategies were employed to assess whether this phenotype was due to growth conditions, or whether it was intrinsic to the mutant. Colonies were picked and re-plated successively in order to recover a single cellular organization, and also floating cells were recovered from the culture medium by allowing cells to settle at room temperature without shaking for an hour. These isolated cells were transferred to new cultures, thus selecting only those individual cells that are lighter and motile with active flagella, however the growth phenotype persisted. Due to the difficulty in accurately counting clustered cell aggregates, cellular volume has been used for cellular quantification rather than cell numbers. On a total cellular volume basis, similar growth rates were observed between the coa1 mutant and WT ( Fig. 3C ). The High-Oil and High-Starch Phenotype of the coa1 Mutant is Dependent on Light When comparing oil contents of wild-type and mutant cells grown in flasks, we sometimes observed big variations in measurements despite the fact that cells were apparently cultivated under similar conditions. We then hypothesized that such variations may be related to a light effect, as the light absorbed by algal cells is greatly reduced during the time course of a batch culture performed in flasks, due to a shading effect as cell density gets higher 31 . In order to test this hypothesis, we cultivated the mutant and WT in well-controlled conditions of illumination, by using photobioreactors (PBRs) operated as turbidostats under photoautotrophic conditions. In this experimental set-up, cell density was assessed by measuring the turbidity (OD 880nm ) and was maintained at a constant value (OD 880nm = 0.4 corresponding to 2 million cells mL −1 ) by step-wise injection of fresh culture medium. Using this system, photoautotrophic cultures can be maintained at physiological steady states under different light regimes. It should be mentioned here that because PBRs are radially illuminated and cell density maintained at a relatively low level, the light required in this set-up to observe physiological effects of the same magnitude as in flasks are much lower (a factor of 4 is observed: i.e. 40 μmol photons m −2 s −1 in the PBR set-up being approximately equivalent to ~160 μmol photons m −2 s −1 in shake flask culture). Increasing light intensity from 40 to 120 μmol photons m −2 s −1 had very little effect on intracellular oil levels in the WT strain, but led to an increase in oil content from 0.5 to 2.5 μg mm −3 in mutant cells ( Fig. 4A ). Intracellular starch increased in both WT and the coa1 mutant with increasing light intensity, but the increase was more pronounced in the mutant ( Fig. 4B ). Such an effect of light on starch accumulation has been previously reported for different species, including duckweed 32 , Scenedesmus sp. 33 and Chlorella sp. 31 , and also in Chlamydomonas reinhardtii 34 , however a physiological explanation has not been put forward. When cells were grown under low light, the chlorophyll content was about 50% lower in the coa1 mutant than in the WT ( Fig. 4C ). However, the efficiency of the photosynthetic machinery was only partially affected in coa1 , as shown by photosystem II yields measured under various light intensities by means of chlorophyll fluorescence ( Supplemental Figures S3 ). As generally observed during high light acclimation 35 , the chlorophyll content of both strains decreased ( Fig. 4C ), reaching a similar value under high light. It is well known that when algae or plants are grown under high light (HL) intensity, photosystems develop smaller antennae sizes and contain lower amounts of Chlorophyll b (Chl b ) thus resulting in an increase in the chlorophyll a / b (Chl a / b ) ratio 36 . An increase in the Chl a / b was observed in both strains under HL, the Chl a / b ratio being however higher in the coa1 mutant than in the WT ( Fig. 5A ). It is important to note that when cells were grown in the dark both strains displayed similar Chl a / b ratios ( Supplemental Figure S4 ) showing that this effect is related to light acclimation. Altogether, these data suggest that the coa1 mutant is affected in its ability to regulate the antenna size and the chlorophyll content in response to HL conditions. This interpretation is supported by the immunoblot analysis which shows lower amounts of the light harvesting complex (LHCII) antenna protein LHCB5 in the coa1 in comparison to the WT, with LHCB5 decreasing in response to HL acclimation in both strains ( Supplemental Figure S5 ). The increased starch and the decreased chlorophyll content observed in the mutant together suggest that the coa1 mutant is somehow more sensitive to light as we have observed repeatedly, i.e. behaving under lower light similarly to WT under high light. Moreover, light is found to trigger higher oil accumulation in the mutant under increasing light intensity, whereas oil content increased only slightly in the WT under the light range tested. The coa1 mutant therefore provides a means to probe the link between light intensity and reserve formation, a phenomenon which has sometimes been observed in the literature 37 but has rarely been explored at a mechanistic level. The Phenotype of the coa1 Mutant Results from a Single Insertional Event Insertional mutagenesis can lead to insertion of several copies of DNA cassettes randomly in a genome 38 39 . As a first step to determine whether the coa1 phenotype is genetically linked to the insertion of the paromomycin cassette, we performed a DNA/DNA hybridization analysis (Southern blot). After digesting the coa1 genomic DNA with several restriction enzymes independently, a single hybridizing band was observed in all cases using a probe specific to the DNA cassette harboring paromomycin gene AphVIII . This suggested that there is only one insertion of AphVIII cassette in the coa1 genome ( Fig. 6A ). As a second step, we performed genetic backcrosses between coa1 ( mt − ) and the wild-type strain CC125 ( mt + ) which is the mt − strain mostly closely related to the coa1 ’s progenitor (CC124). From the analysis of 3 complete tetrads ( Fig. 6B ) and 9 incomplete tetrads ( Supplemental Figure S6A ), we found that all paromomycin-resistant strains accumulated higher oil amount than paromomycin-sensitive strains and showed persistent palmelloid colony formation. Chlorophyll content in the three complete tetrads also showed the same segregation as cellular oil content, i.e. those progenies sensitive to paromomycin also contained less chlorophyll, which was already obvious from visual inspection of the color of the culture ( Supplemental Figure S6B ). No strain issued from this cross harbored the constitutive TAG accumulating phenotype in the absence of paromomycin resistance, thus indicating a genetic link between the oil phenotype and the single insertion of the antibiotic cassette. Whole Genome Re-Sequencing Identifies a Large Deletion in Chromosome 14 Although Southern blot analysis revealed a single insertional event occurred in the coa1 mutant ( Fig. 6A ), two distant sites of insertion were identified on chromosome 14 by sequencing flanking regions of the insertion (one at 69,200 bp and the other at 315,218 bp) using Genome Walker technique. In order to understand how a single insertion event could lead to the identification of two distant sites, we re-sequenced the whole genome of Chlamydomonas . The genome sequencing generated 34257132 clean reads, with 90 bp per read and has a calculated coverage of 23.7x the whole Chlamydomonas genome ( Table 1 ). Genomic reads were paired using FASTQ joiner 40 and mapped to the C. reinhardtii genome 41 (version 4.3) using the BAM tools package available on the Galaxy project website ( https://main.g2.bx.psu.edu/ ). Global and local coverage was determined by analysis of BAM output by GATK (Genome Analysis Tool Kit). The genomic region between 69,200 bp and 315,218 bp compared to the reference Chlamydomonas genome version 4.3 revealed low read coverage in the region of the insertion/deletion (mean coverage depth of 1.6, compared to a mean coverage depth of 25.4 for the entire chromosome 14). Peaks of high read coverage in the region between 69,200 bp and 317,163 bp on the reference genome were identified as highly repetitive motifs found elsewhere in the Chlamydomonas genome. Furthermore, reads mapped to the 3′ and 5′ end of the gene cassette, and reads paired to those which mapped to the cassette revealed that the genomic loci adjacent to the gene cassette had sequence identity to 69,200 bp and 315,218 bp on chromosome 14. Taken together, the whole genome re-sequencing analysis and flanking genomic DNA sequencing by Genome Walker revealed that a single complete antisense AphVIII cassette was inserted in a 3′ –>5′ orientation between the 69,200 bp and 317,163 bp on chromosome 14, resulting in a substantial genomic deletion of >200 kb in chromosome 14 between the coordinates 69,266 and 317,163 ( Fig. 6C ). Chromosomal deletions caused by insertional mutagenesis in C. reinhardtii have previously been reported 17 35 . Within the >200 kb region, 41 putative protein-coding genes have been found ( Table 2 ). The phenotype associated with coa1 could thus be attributed to either the result of one of the genes removed in the deleted locus of the genome, or a combinatorial effect of multiple genes. This and other possibilities are discussed in the next section. Suppressors of Carbon Reserve Accumulation Within Chromosome_14:69,266.317,163 The link between the lesion on chromosome 14 and the observed increased level of TAG and starch under high light conditions suggest that carbon reserve formation is repressed in optimal growth in wild-type strains. In the absence of this chromosomic region (the coa1 mutant), the cellular homeostasis of carbon reserves is disturbed, thus creating strains that over-accumulate oil and starch simultaneous to growth. Furthermore, it is found that one or more of the genes encoded on the same region may be key in the orchestration of the light response in C. reinhardtii . The reason Chlamydomonas exhibits an increased proportion of TAG and starch during high light growth remains poorly defined, and may remain difficult to analyze without the access to genome editing in Chlamydomonas , however the generation of an indexed genome-wide mutant library for C. reinhardtii ( http://jonikaslab.dpb.carnegiescience.edu/chlamy-mutant-library ) 42 may aid in more rapidly elucidating which of these genes encode key functions in regulation of TAG homeostasis during high light. While there is little information known about the proteins in the locus of interest ( Table 2 ), some proteins do deserve particular note due to their potential involvement in the observed phenotype. For example, the presence of a lipase-like protein, a putative lipid transfer protein or a Coenzyme A binding protein in this region could perhaps contribute to the observed oil content phenotype in coa1 mutant. The presence of a Myb domain-containing putative DNA binding protein is another example. Homologs of these proteins have been identified as potential key players in the carbon concentration mechanism (CCM) as transcription factors involved in the response to the physiological pressure of low carbon growth 43 . A gene encoding a chlorophyllide b reductase (Cre14.g608800) is also located in this chromosomic region. Chlorophyllide b reductase catalyzes the first step in the degradation of chlorophyll b in the chlorophyll cycle 44 . Its deletion in the coa1 mutant may have perturbed the chlorophyll cycle thus explaining the altered chlorophyll content and chlorophyll a / b ratio observed in the mutant ( Fig. 5B ). Increase in chlorophyll a / b ratio is an integral feature of cells’ acclimation to high light conditions 45 . Although molecular mechanisms remain to be elucidated, the mutant coa1 displays several phenotypic features (such as decreased chlorophyll content, increased chlorophyll a / b ratio and increased intracellular starch), indicating a higher sensitivity to light that may contribute to an increased overall flux in cellular carbon and an increase in oil content. Outlook One of the major obstacles to economically feasible production of algal fuel is the requirement of nitrogen starvation or other stress conditions for higher reserve accumulation 2 . Here we isolated a mutant of Chlamydomonas , coa1 , which accumulates five times more oil and twice more starch than the WT during optimal growth. To our knowledge, this is the first report of such a mutant accumulating higher oil amount in parallel to optimal growth for the green alga C. reinhardtii . Through the study of the mutant coa1 , we also pointed out a missing link underlying the regulation of reserve accumulation under high light conditions. Molecular characterization of the coa1 mutant identified a QTL responsible for the observed phenotype. Detailed examination of the genes, via cross-references to published transcriptomic datasets ( Table 2 ) 34 , encoded in the missing region should help understanding the molecular mechanisms that led to increased amount of carbon reserves in the mutant, and should provide molecular tools for uncoupling lipid accumulation from impairment in cell growth. This report in conjunction with the availability of genome-wide indexed mutant libraries ( http://jonikaslab.dpb.carnegiescience.edu/chlamy-mutant-library ) 42 , and systems analysis of cells’ response to various stresses 10 13 34 , should make it faster to assign the coa1 phenotype to particular gene(s) located within this region on chromosome 14. Thus the isolation of the coa1 mutant demonstrates that mutants over-accumulating carbon reserves under non-stress conditions can be isolated and further provides a list of candidate genes involved in suppressing carbon reserve accumulation during optimal growth."
} | 4,789 |
35055092 | PMC8776115 | pmc | 2,616 | {
"abstract": "This paper describes the use of silk protein, including fibroin and sericin, from an alkaline solution of Ca(OH) 2 for the clean degumming of silk, which is neutralized by sulfuric acid to create calcium salt precipitation. The whole sericin (WS) can not only be recycled, but completely degummed silk fibroin (SF) is also obtained in this process. The inner layers of sericin (ILS) were also prepared from the degummed silk in boiling water by 120 °C water treatment. When the three silk proteins (SPs) were individually grafted with glycidyl methacrylate (GMA), three grafted silk proteins (G-SF, G-WS, G-ILS) were obtained. After adding I2959 (a photoinitiator), the SP bioinks were prepared with phosphate buffer (PBS) and subsequently bioprinted into various SP scaffolds with a 3D network structure. The compressive strength of the SF/ILS (20%) scaffold added to G-ILS was 45% higher than that of the SF scaffold alone. The thermal decomposition temperatures of the SF/WS (10%) and SF/ILS (20%) scaffolds, mainly composed of a β-sheet structures, were 3 °C and 2 °C higher than that of the SF scaffold alone, respectively. The swelling properties and resistance to protease hydrolysis of the SP scaffolds containing sericin were improved. The bovine insulin release rates reached 61% and 56% after 5 days. The L929 cells adhered, stretched, and proliferated well on the SP composite scaffold. Thus, the SP bioinks obtained could be used to print different types of SP composite scaffolds adapted to a variety of applications, including cells, drugs, tissues, etc. The techniques described here provide potential new applications for the recycling and utilization of sericin, which is a waste product of silk processing.",
"conclusion": "5. Conclusions This study reports the development of a green method for degumming both SF fibers and whole sericin samples, as well as a process for obtaining inner sericin samples using a high-temperature and high-pressure method. These three proteins were grafted with glycidyl methacrylate to obtain three different grafted silk proteins: G-SF, G-WS, and G-ILS. A variety of SP inks was prepared with PBS and a photoinitiator and used to bioprint a variety of SF scaffolds and SP composite scaffolds. The experimental results showed that the SF grafting reaction obtained by degumming 2.5% GMA and 0.05% Ca(OH) 2 for 10 min resulted in the most optimal SP composite scaffold, incorporating grafted whole sericin that significantly increased the maximum compressive strain. The compressive strength and maximum compressive strain of the SP composite scaffolds containing G-ILS were also increased. In addition, both the G-WS and G-ILS sericins were able to increase average pore size and porosity. The three SP scaffolds, namely SF alone, SF/WS (10%), and SF/ILS (20%) were composed of mainly β-sheet structures, which delayed the hydrolysis of neutral protease, as well as the rate of insulin release after 5 days, and reached 52% and 62%, respectively. Fluorescence microscopy observation showed that L-929 cells adhered and grew well on the SP scaffolds and demonstrated good cell compatibility.",
"discussion": "3. Discussion As a by-product of the silk textile industry, sericin is widely available, though it is often regarded as a waste product of traditional silk processing. The side chain of sericin contains more –NH 2 and –COOH functional groups than that of silk fibroin, which facilitates the synthesis of new products through chemical crosslinking. However, there are few reports on the applications of sericin in the preparation of bioinks. Sericin, especially partially degraded sericin, has good biocompatibility and biodegradability, comparable to that of silk fibroin. It has excellent biological properties, including antioxidant, anti-inflammatory, and antibacterial activity, as well as whitening effects and glucosidase inhibition [ 55 ]. Sericin has also been found to promote cell adhesion, growth, and migration [ 56 ]. Therefore, partially degraded sericin was incorporated into the SF scaffold base of an SF bioink in order to ensure that the SP composite scaffolds not only possessed certain mechanical properties, but also properties that promoted cell adhesion, growth, and proliferation. The SF bioink printing scaffolds and silk fibroin composite scaffolds have certain mechanical properties, as well as a porous network structure, which are conducive to the transportation of bioactive substances and the discharge of cell metabolites. Furthermore, they improve cell adhesion and growth, an important requirement for medical tissue engineering. For example, SF, WS, and ILS bioinks can be bioprinted into corresponding SP scaffolds by using single ink or composite inks in accordance with specific medical application requirements. After the SF ink is mixed with the relevant cells, the holes removed from bone tumors, for example, could be filled by injection, followed by the introduction of ultraviolet radiation through optical fibers to fix the bioink and accelerate the proliferation and repair of cells. SF could be used as a wound dressing or artificial skin to repair tissue, along with the use of related growth factors and anti-inflammatory drugs, rather than a photoinitiator. In addition, traditional oral or injected medications typically result in short-term drug concentrations in human blood that are substantially higher than that needed for treatment, which may increase side effects or lead to a decrease in efficacy. SP scaffolds have potential applications as drug carriers. An SP scaffold could release drugs gradually through diffusion and penetration, thereby prolonging the efficacy and flexibly controlling the drug release site. Scaffolds should be biocompatible, so as to integrate with the tissue around the implant site in order to avoid rejection. Sericin and silk fibroin are natural animal proteins, and biological scaffolds will be gradually degraded by proteases in vivo. The degradation products are small peptides or amino acids, which have no toxic effects and can be absorbed and utilized by the human body. This process could provide suitable space for new tissue and release the relevant growth factors. Moreover, degradation of the materials in vivo would mean that no additional surgery would be required, which would provide an additional benefit to patients. Some studies have shown that during the degradation process, sericin shows increased antioxidant activity and thereby can inhibit cellular damage due to oxidative stress damage as well as apoptosis. Therefore, silk protein scaffolding implanted in vivo, even while undergoing degradation, not only shows higher antioxidant activity, but also promotes cell adhesion and growth, in addition to cell proliferation, among other characteristics. The use of silk protein scaffolds as a novel medical biomaterial provides a starting point for exploring potential applications for sericin and silk fibroin. However, in vivo verification of the efficacy of silk fibroin scaffolds needs further research, and the scope of potential applications requires further discussion."
} | 1,778 |
31640553 | PMC6805351 | pmc | 2,617 | {
"abstract": "Background Escherichia coli C forms more robust biofilms than other laboratory strains. Biofilm formation and cell aggregation under a high shear force depend on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. Results Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for lipopolysaccharide (LPS) synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the − 35 region of P4 promoter and blocks the transcription from the sigma 32 and sigma 70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene. And finally, E. coli C encodes an additional sigma 70 subunit driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions provided insights into understanding this regulatory pathway in E. coli . Conclusions Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains of E. coli grown for decades in vitro have evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain of E. coli C produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence of this classic strain, which provides for a base level of characterization and makes it useful for many biofilm-based applications.",
"conclusion": "Conclusions Biofilms are the most prevalent form of bacterial life [ 9 , 30 ] and as such have drawn significant attention from the scientific community over the past quarter century. However, only in 2018 did the number of biofilm related articles reach 24,000, based on a Google Scholar search. As in all other fields, biofilm research needs to develop and follow standard protocols and methods that can be used in different laboratories and give comparable results. Unfortunately, a standardized methodological approach to biofilm models has not been adopted, leading to a large disparity among testing conditions. This has made it almost impossible to compare data across multiple laboratories, leaving large gaps in the evidence [ 64 ]. In our work, we described and characterized biofilm formation in the classic laboratory strain, E. coli C [ 2 , 65 ]. We have used that strain in our biofilm-related research for almost a decade and we would like to share it with the biofilm community and propose to use it as a model organism in E. coli -based biofilm-related research.",
"discussion": "Discussion E. coli is the most common bacterial research model organism. Out of the five strains used only the E. coli C genome has not been sequenced. Here, we sequenced and analyzed the E. coli C genome and revealed its specific features that lead to enhanced biofilm formation. Recently, a new E. coli strain C genome has been submitted to the GenBank database (CP029371.1). Homology search revealed that this strain was not closely related to our strain. However, the sequence homology search of GenBank available E. coli genomes revealed that two isolates, WG5 (CP024090.1) and NTCT122 (LT906474.1), showed identical csrA promoter regions. Strain WG5 is in fact an E. coli C derivative resistant to nalidixic acid [ 59 , 60 ]. This E. coli C, also known as strain CN, is publicly available in the ATCC (ATCC number 700078). We found that our sequence is very similar to the WG5 sequence, although the inverted 300 kb region between 107 and 407 kb was not present in WG5. Also some of the insertion sequences were not present in the WG5 genome. These findings again revealed a role of different mobile elements in genome rearrangements and evolution. As the bacterial genome undergoes a constant evolution and adaptation [ 61 ] and bacterial mobile elements are the most common mechanism of those processes [ 62 , 63 ], one may ask why in this particular strain, unlike the other laboratory strains, the selection toward planktonic cells did not take place. There is no simple answer; however, we can speculate that as this strain is used for proliferation of bacteriophages the fact that phages kill planktonic cells might reduce the selection toward free floating cells. The second hypothesis is that for bacteriophage research using the E. coli C, the ATCC recommends low-salt (0.5% NaCl) or no salt Nutrient (#139) broth medium. As we showed, the low-salt medium reduced bacterial stress and most likely reduced the level of genome rearrangements, keeping the natural properties for biofilm formation characteristic for the wild-type strains in this laboratory E. coli C strain."
} | 1,347 |
28084217 | null | s2 | 2,618 | {
"abstract": "One goal of neuromorphic engineering is to create 'realistic' robotic systems that interact with the physical world by adopting neuromechanical principles from biology. Critical to this is the methodology to implement the spinal circuitry responsible for the behavior of afferented muscles. At its core, muscle afferentation is the closed-loop behavior arising from the interactions among populations of muscle spindle afferents, alpha and gamma motoneurons, and muscle fibers to enable useful behaviors. We used programmable very- large-scale-circuit (VLSI) hardware to implement simple models of spiking neurons, skeletal muscles, muscle spindle proprioceptors, alpha-motoneuron recruitment, gamma motoneuron control of spindle sensitivity, and the monosynaptic circuitry connecting them. This multi-scale system of populations of spiking neurons emulated the physiological properties of a pair of antagonistic afferented mammalian muscles (each simulated by 1024 alpha- and gamma-motoneurones) acting on a joint via long tendons. This integrated system was able to maintain a joint angle, and reproduced stretch reflex responses even when driving the nonlinear biomechanics of an actual cadaveric finger. Moreover, this system allowed us to explore numerous values and combinations of gamma-static and gamma-dynamic gains when driving a robotic finger, some of which replicated some human pathological conditions. Lastly, we explored the behavioral consequences of adopting three alternative models of isometric muscle force production. We found that the dynamic responses to rate-coded spike trains produce force ramps that can be very sensitive to tendon elasticity, especially at high force output. Our methodology produced, to our knowledge, the first example of an autonomous, multi-scale, neuromorphic, neuromechanical system capable of creating realistic reflex behavior in cadaveric fingers. This research platform allows us to explore the mechanisms behind healthy and pathological sensorimotor function in the physical world by building them from first principles, and it is a precursor to neuromorphic robotic systems."
} | 533 |
21625450 | PMC3098866 | pmc | 2,619 | {
"abstract": "Background An important function of many complex networks is to inhibit or promote the transmission of disease, resources, or information between individuals. However, little is known about how the temporal dynamics of individual-level interactions affect these networks and constrain their function. Ant colonies are a model comparative system for understanding general principles linking individual-level interactions to network-level functions because interactions among individuals enable integration of multiple sources of information to collectively make decisions, and allocate tasks and resources. Methodology/Findings Here we show how the temporal and spatial dynamics of such individual interactions provide upper bounds to rates of colony-level information flow in the ant Temnothorax rugatulus . We develop a general framework for analyzing dynamic networks and a mathematical model that predicts how information flow scales with individual mobility and group size. Conclusions/Significance Using thousands of time-stamped interactions between uniquely marked ants in four colonies of a range of sizes, we demonstrate that observed maximum rates of information flow are always slower than predicted, and are constrained by regulation of individual mobility and contact rate. By accounting for the ordering and timing of interactions, we can resolve important difficulties with network sampling frequency and duration, enabling a broader understanding of interaction network functioning across systems and scales.",
"introduction": "Introduction An important function of many complex networks (e.g. HIV infections, power grids, mobile phone calls) is to inhibit or promote the transmission of disease, resources, or information between individuals [1] , [2] . These interactions are often critical to determining individual and group-level functions. While social network analysis has provided a powerful framework for understanding the structure of these interaction networks, it is less useful for understanding the temporal dynamics of these networks. With few exceptions [3] , [4] , [5] , it has been hard to study empirical flows of resources or information between individuals, or to compare these dynamics across systems and scales. Our knowledge of dynamic biological interaction networks is particularly poor, perhaps due to the difficulties inherent to quantifying or manipulating large natural systems [6] , [7] . Ant colonies are a model comparative system for understanding general principles linking individual-level interactions to group-level functions. Local interactions between individuals via direct antennal contact are known to be functionally important [8] . While empirical knowledge of these interactions networks is limited [9] , [10] , interactions among individuals enable integration of multiple sources of information to collectively make decisions [11] , [12] , and allocate tasks and resources [13] , [14] and modulation of activity level [15] and energy usage [16] . Previous theoretical network models have linked individual behavior to colony-level oscillations in activity [17] , [18] and information flow [19] , and previous empirical work has demonstrated how individual mobility [20] and spatial fidelity [21] can influence colony-level functions like decision-making [22] . Nevertheless, a comprehensive and detailed picture of how individual-level interactions influence group-level information flow remains lacking. We developed two broadly applicable tools to understand network dynamics: first, a diffusion model to predict bounds to rates of information flow in groups of different size; and second, a ‘time ordered network’ framework for empirically tracing potential pathways of information flow through dynamic interaction networks. We used the ant Temnothorax rugatulus to test the hypothesis that empirical bounds to information flow would reach a theoretical bound based on the mobility of individuals. Colonies of T. rugatulus can be kept in artificial transparent nests that closely mimic natural conditions. In four colonies of a range of sizes (n = 6–90 individuals) we obtained complete time-stamped records of all interactions between all individuals for approximately 1800-second intervals. We uniquely identified ants by marking each with colored paints. Interactions, defined here as antenna-body contact between individuals, can convey chemical or tactile information and are a proxy for communication ( Fig. 1A ). To understand long-term dynamics, each colony was filmed at two time points separated by approximately three weeks ( Table S1 ). To assess mobility of individuals, in half of these filmings we also recorded the position of every individual at every interaction. Using these data we uniformly rejected our hypothesis but were able to determine important mechanisms that limited information flow in these ant colonies. 10.1371/journal.pone.0020298.g001 Figure 1 Ant interaction networks. \n a ) We used marked colonies of the ant Temnothorax rugatulus to study the structure and dynamics of interaction networks. Interactions are a proxy for chemical or tactile communication and are defined as contact between the antenna of one ant and the body of another ant. b ) Time-ordered networks enable inference about group-level information flow and causality from individual behavior. Individuals are linked to themselves in time and to other individuals during interactions; lines that travel horizontally or upward between nodes represent pathways for information flow. c ) Time-aggregated networks can be recovered from time-ordered networks by accumulating data along the time axis. d ) An empirical time-ordered network from a colony with 69 individuals, drawn as in b. e ) The same data represented as a time-aggregated network. Time-aggregated networks inherently hide more the fundamental dynamic processes.",
"discussion": "Discussion Our results call for a deeper general understanding of the adaptive significance of different network structures. Variable rates of information flow may control efficient group function [8] , [10] . Fast local flow can be adaptive for many common tasks (in ants, including brood care, resource distribution, and grooming) that can be negotiated quickly between individuals in local neighborhoods [30] . The results of these interactions may not be relevant to individuals in other locations, improving task performance in species with spatial fidelity [21] . Some ant species are known to regulate individual contact rate, sometimes by local feedback processes [8] , [10] , [17] . Slow global flow may limit interaction rates to maximize time available for task completion and reduce the potential for the disease spread [5] . However, network structure and information flow should change under stressful conditions when information must be globally propagated (e.g. famine relief [14] or nest destruction in ants). The temporary emergence of some highly interactive individuals, akin to ‘hubs’ in time-aggregated networks, may play an important role in these situations. Extensions of our model to consider systematic individual variation in interaction rate, and feedbacks between interactions, may be fruitful. For example, in some species of ants, colony-level oscillations in activity may be contingent upon worker interactions with brood [17] . Additionally, we are aware of very few studies [2] , [14] that empirically trace the propagation of a known signal or resource under variable conditions. Comparative studies across species and systems using temporal methods are needed to explore the adaptive costs and benefits of network dynamics in different environments [31] . We have provided strong bounds to information flow in ant networks that are set by constrained mobility and regulation of interactions between unspecialized individuals. These results provide a unique perspective on the organization of ant colonies of a range of sizes and contrast strongly with the common ‘scale-free’ nature of many human systems, challenging notions of structural universality in self-organized networks. A dynamic approach using our framework and model will provide important insights into the link between individual behaviors and group function in other biological networks like food webs [32] plants and pollinators [33] and pathogens [34] . Understanding how and why the dynamics of ant networks are different from those of human communication [1] , disease [2] or proximity networks [3] will shed light upon general principles that control information flow and network evolution across systems."
} | 2,162 |
28093073 | PMC5240273 | pmc | 2,620 | {
"abstract": "Methane emissions from ruminal fermentation contribute significantly to total anthropological greenhouse gas (GHG) emissions. New meta-omics technologies are beginning to revolutionise our understanding of the rumen microbial community structure, metabolic potential and metabolic activity. Here we explore these developments in relation to GHG emissions. Microbial rumen community analyses based on small subunit ribosomal RNA sequence analysis are not yet predictive of methane emissions from individual animals or treatments. Few metagenomics studies have been directly related to GHG emissions. In these studies, the main genes that differed in abundance between high and low methane emitters included archaeal genes involved in methanogenesis, with others that were not apparently related to methane metabolism. Unlike the taxonomic analysis up to now, the gene sets from metagenomes may have predictive value. Furthermore, metagenomic analysis predicts metabolic function better than only a taxonomic description, because different taxa share genes with the same function. Metatranscriptomics, the study of mRNA transcript abundance, should help to understand the dynamic of microbial activity rather than the gene abundance; to date, only one study has related the expression levels of methanogenic genes to methane emissions, where gene abundance failed to do so. Metaproteomics describes the proteins present in the ecosystem, and is therefore arguably a better indication of microbial metabolism. Both two-dimensional polyacrylamide gel electrophoresis and shotgun peptide sequencing methods have been used for ruminal analysis. In our unpublished studies, both methods showed an abundance of archaeal methanogenic enzymes, but neither was able to discriminate high and low emitters. Metabolomics can take several forms that appear to have predictive value for methane emissions; ruminal metabolites, milk fatty acid profiles, faecal long-chain alcohols and urinary metabolites have all shown promising results. Rumen microbial amino acid metabolism lies at the root of excessive nitrogen emissions from ruminants, yet only indirect inferences for nitrogen emissions can be drawn from meta-omics studies published so far. Annotation of meta-omics data depends on databases that are generally weak in rumen microbial entries. The Hungate 1000 project and Global Rumen Census initiatives are therefore essential to improve the interpretation of sequence/metabolic information.",
"conclusion": "Conclusions Current -omics technologies can provide detailed information about the animal genome, the ruminal metagenome and their respective functional activities from the metatranscriptome and metaproteome. Comparative analysis using these technologies allows us to characterise the interaction between the animal and its rumen microbiota. At the present time, it is mainly the power and potential of metagenomics, metatranscriptomics and metaproteomics that are being investigated, with fewer studies investigating their application to problems associated with animal production. Furthermore, integrating the results of various meta-omics analyses remains a challenge. Improving the data present in public databases to include progressively more information on rumen microbial species is a priority. Indeed, research groups around the world are joining forces to meet these challenges. A much larger knowledge base for rumen microbial genomics will allow these methods to become more robust for the detection of relevant species as well as for a correct identification and quantification of microbial genes and proteins directly related to rumen metabolic pathways, which could have an important role in the improvement of livestock productions and breeding programmes. Some progress has been made with methane emissions. However, an arguably more acceptable strategy, particularly to the livestock producer, would be to focus on the efficiency of feed utilisation rather than methane itself. The equally important issue of N emissions has received too little attention."
} | 1,014 |
26199945 | PMC4496466 | pmc | 2,622 | {
"abstract": "Cyanobacteria are widely distributed Gram-negative bacteria with a long evolutionary history and the only prokaryotes that perform plant-like oxygenic photosynthesis. Cyanobacteria possess several advantages as hosts for biotechnological applications, including simple growth requirements, ease of genetic manipulation, and attractive platforms for carbon neutral production process. The use of photosynthetic cyanobacteria to directly convert carbon dioxide to biofuels is an emerging area of interest. Equipped with the ability to degrade environmental pollutants and remove heavy metals, cyanobacteria are promising tools for bioremediation and wastewater treatment. Cyanobacteria are characterized by the ability to produce a spectrum of bioactive compounds with antibacterial, antifungal, antiviral, and antialgal properties that are of pharmaceutical and agricultural significance. Several strains of cyanobacteria are also sources of high-value chemicals, for example, pigments, vitamins, and enzymes. Recent advances in biotechnological approaches have facilitated researches directed towards maximizing the production of desired products in cyanobacteria and realizing the potential of these bacteria for various industrial applications. In this review, the potential of cyanobacteria as sources of energy, bioactive compounds, high-value chemicals, and tools for aquatic bioremediation and recent progress in engineering cyanobacteria for these bioindustrial applications are discussed.",
"conclusion": "7. Conclusion Owing to their simple growth requirements, ease of genetic manipulation, and ability to capture solar energy and fix atmospheric carbon dioxide directly into industrial products, the potentials of cyanobacteria for various biotechnological applications have been well recognized. However, the application of cyanobacterial cultures for large-scale synthesis of products of interest is technologically challenging. Efficient and cost-effective photosynthetic bioreactors need to be developed to achieve maximum productivity in large-scale cyanobacterial cultures with minimum operation costs. With the advances in genetic and metabolic engineering approaches as well as development of suitable cultivation systems, we can harness the photosynthetic efficiency of cyanobacteria to provide green paths for the synthesis of industrial products.",
"introduction": "1. Introduction Cyanobacteria, also referred to as blue-green algae, are the oldest photosynthetic organisms on earth that originated approximately 2.6–3.5 billion years ago [ 1 ]. Indeed, the origin of photosynthetic organelle in eukaryotes is thought to have possibly arisen by the process of endosymbiosis between a phagotrophic host and a cyanobacterium [ 2 ]. Cyanobacteria are morphologically diverse and exist in different forms including unicellular, filamentous, planktonic or benthic, and colonial (coccoid) ones [ 3 , 4 ]. They are by far the most widespread occurring photosynthetic organisms. They can thrive in a wide range of ecological habitats, ranging from marine, freshwater, to terrestrial environments. Cyanobacteria are also well known for their ability to perform different modes of metabolism and the capacity to switch rapidly from one mode to another [ 5 ]. All cyanobacteria are capable of oxygenic photosynthesis but some cyanobacterial species can switch to sulfide-dependent anoxygenic photosynthesis [ 6 ]. In dark or under anoxic conditions, cyanobacteria can perform fermentations for energy generation [ 7 ]. Some filamentous cyanobacteria have evolved specialized cells known as heterocysts to carry out nitrogen fixation [ 8 ]. At present, many bioindustrial processes rely on the fermentations of heterotrophic bacteria to produce various fine chemicals such as vitamins, enzymes, and amino acids. Nevertheless, the economic viability of these production schemes is limited by the cost of carbon substrates used in the fermentation processes. Cyanobacteria, endowed with photosynthesis system to fix carbon dioxide into reduced form, are ideal biosynthetic machinery for sustainable production of various chemicals and biofuels. Unlike heterotrophic bacteria, cyanobacteria require only sunlight, carbon dioxide, water, and minimal nutrients for growth, eliminating the cost of carbon sources and complex growth media. Sunlight is the most readily available and inexpensive resource on earth and the use of cyanobacteria for the production of fine chemicals and biofuels from solar energy offers a greener path for the synthesis process. Equipped with superior photosynthesis capabilities, cyanobacteria have higher photosynthesis and biomass production rates compared to plants and can convert up to 3–9% of the solar energy into biomass compared to ≤0.25–3% achieved by crops, for example, corn, sugar cane [ 9 ]. They also require less land area for cultivation than terrestrial plant, reducing the competition for arable land with crops intended for human consumption. Cyanobacteria utilize carbon dioxide, a type of greenhouse gases, during photosynthesis and help to achieve a carbon neutral production process. Being prokaryotes, cyanobacteria possess relatively simple genetic background that eases manipulation [ 10 ]. In addition, the residual cyanobacteria biomasses that are left over after high-value products extraction can be used as animal feed or converted into organic fertilizer. Considering the aforementioned inherent merits of cyanobacteria, they are one of the attractive candidates for use in diverse biotechnological application. With the recent advances in genetic and metabolic engineering technologies and the availability of more than 300 cyanobacterial genome sequences, there is significant progress in research directed towards realizing the full potential of these photosynthetic bacteria. Cyanobacteria have gained considerable attention in recent years for their possible use in agriculture, nutraceuticals, effluent treatment, and the production of biofuels, various secondary metabolites including vitamins, toxins, and enzymes. In this paper, the recent progress in developing cyanobacteria for various potential applications in biotechnology is discussed."
} | 1,545 |
28861523 | PMC5566838 | pmc | 2,623 | {
"abstract": "Microbial methane consumption is a critical component of the global carbon cycle, with wide-ranging implications for climate regulation and hydrocarbon exploitation. Nonetheless, quantifying methane metabolism typically involves logistically challenging methods and/or specialized equipment; these impediments have limited our understanding of methane fluxes and reservoirs in natural systems, making effective management difficult. Here, we offer an easily implementable, precise method using monodeuterated methane (CH 3 D) that advances three specific aims. First, it allows users to directly compare methane consumption rates between different experimental treatments of the same inoculum. Second, by empirically linking the CH 3 D procedure with the well-established 14 C radiocarbon approach, we determine absolute scaling factors that facilitate rate measurements for several aerobic and anaerobic systems of interest. Third, CH 3 D represents a helpful tool in evaluating the relationship between methane activation and full oxidation in methanotrophic metabolisms. The procedural advantages, consistency, and novel research questions enabled by the CH 3 D method should prove useful in a wide range of culture-based and environmental microbial systems to further elucidate methane metabolism dynamics.",
"conclusion": "Conclusions. The ability to accurately measure methane consumption and oxidation rates—both comparatively and in absolute values—is an important component of methanotrophic studies. Such measurements frequently depend on radiotracers or measurements of chemical species that are related to, but not directly indicative of, methane metabolism. The monodeuterated-methane technique presented here represents a novel approach to investigate methane oxidation rates, notable for its logistical ease and straightforward sampling procedures. We have demonstrated that the D/H ratio is a reliable proxy for methane oxidation activity when subjected to ground truth experiments on a sample-specific basis with the well-established 14 CH 4 method; in several applications, methane consumption values calculated via the CH 3 D method were directly proportional to 14 C radiolabel-derived methane oxidation rates. Values of the proportionality constant differ based on the experimental system, likely dictated by environmental variables and the relative proportions of aerobic and anaerobic methanotrophic metabolisms, though additional experiments to determine the nature of the putative mixing line are needed. By providing a way to measure how hydrogen atoms are mobilized and processed, deuterated methane represents a promising approach to help researchers disentangle several aspects of methane-associated metabolisms. Methane biogeochemistry is a dynamic field of study with implications for carbon cycling, microbial ecology, and climate dynamics, though experimental challenges have slowed our understanding of methane-based biological reactions. With the CH 3 D approach as an added tool in the arsenal of rate-based examinations, a broader understanding of the intricacies of methane metabolism, as well as its role in environmental and anthropogenic systems, is within reach.",
"introduction": "INTRODUCTION Methane-consuming microbial processes represent an important component of biogeochemical cycles in natural freshwater and marine environments, as well as in human-impacted systems. In terrestrial soils, methane production in rice fields, anoxic wetlands, and thawing permafrost supports methanotrophic communities ( 1 – 4 ). In marine settings, an estimated 85 Tg of methane per year, derived from biogenic and thermogenic sources, enters the subseafloor, the vast majority of which is anaerobically consumed in anoxic sediments ( 5 ). Much of what remains is taken up in microoxic or oxic zones of the sediment or water column by aerobic methanotrophic microorganisms ( 6 ). Methanotrophy is also of interest in a range of human-impacted contexts, including groundwater ( 7 , 8 ), wastewater treatment plants ( 9 ), landfills ( 10 ), shale gas ( 11 ), coalbed harvesting ( 12 ), and oil spills ( 13 ). In addition to its climatic and economic implications, the biochemical details of the methanotrophic process have stimulated many investigations. The sulfate-linked anaerobic oxidation of methane (AOM; reaction 1) has proven particularly enigmatic; this process typically involves a mutualistic relationship between anaerobic methanotrophic (ANME) archaea and sulfate-reducing bacteria (SRB) ( 14 – 16 ), although nitrate ( 17 , 18 ) and, potentially, metals such as iron and manganese ( 19 – 21 ) can serve as alternative electron acceptors for some ANME lineages. Methane is oxidized aerobically (reaction 2) by members of the classes Gammaproteobacteria (e.g., type I and type X) and Alphaproteobacteria (type II); verrucomicrobial representatives can perform aerobic methanotrophy under extremely acidic conditions ( 22 , 23 ). Methane is converted to methanol, which is further oxidized to formaldehyde; assimilatory pathways branching at this point can incorporate carbon into central metabolism through the ribulose monophosphate (RuMP) cycle (type I and type X methanotrophs) or the serine cycle (type II).\n (reaction 1) CH 2 + SO 4 2 − → HCO 3 − + HS − + H 2 O \n (reaction 2) CH 4 + 2 O 2 → HCO 3 − + H 2 O + H + Methanotrophy is both a biogeochemically relevant activity that modulates the global climate and a poorly understood biochemical process; given this dual role, there is substantial interest in measuring its rate and in understanding elemental flows through metabolic pathways. The oxidation of methane in environmental samples has traditionally been studied using a few techniques. Numerical models incorporating environmental sediment profiles of sulfate and methane concentrations can be used to back-calculate methane consumption rates ( 24 ). 13 CH 4 can be used to probe rates under controlled conditions ( 25 – 28 ), but the presence of natural 13 C in marine dissolved inorganic carbon (DIC) pools requires long incubations as well as accurate measurements of isotopically resolved concentrations of reactants and products ( 29 ). Gas chromatography (GC) quantification of dissolved ( 30 – 32 ) or headspace ( 33 , 34 ) methane concentrations has also been demonstrated as a rate measurement tool, though low concentrations can hamper reproducibility and exacerbate background contamination issues, particularly in field-based settings ( 35 ). Perhaps the most sensitive approach uses radiolabeled 14 CH 4 to track the oxidation of methane-associated carbon to inorganic carbon species ( 36 , 37 ). Tritiated methane was introduced for water column aerobic methane oxidation measurements due to its higher activity per radionuclide ( 6 , 38 ). Logistical challenges and health and safety regulations led Pack et al. ( 29 ) to develop an accelerator mass spectrometry detection method that requires 10 3 to 10 5 less radiolabel than previous 14 C and 3 H approaches, though the analytical procedure remains labor-intensive. Despite the range of methods available, measurement of microbial methane utilization rates remains cumbersome, and a precise, safe, and easily enacted approach would be a welcome contribution for a diverse array of researchers. Nearly all of the aforementioned approaches are carbon based; a hydrogen-based tracer offers a complementary approach to investigations of methane biochemical dynamics. Here we introduce a novel method for biologically mediated methanotrophy rate measurement that utilizes monodeuterated methane (CH 3 D) as a substrate and measures the D/H ratio of the aqueous solution. This approach offers several advantages for prospective users: it does not require the logistical, safety, and administrative hurdles associated with radiotracers such as 14 CH 4 and [ 3 H]CH 4 , it compares favorably in terms of equipment cost and portability, and it provides an additional analytical option that enables hydrogen stable isotope-based measurement of methane activation that is complementary to carbon-based stable isotope ( 13 C) or radiocarbon ( 14 C) methods. As a proof of concept, we apply the monodeuterated-methane approach to pressurized methane seep sediment incubations in order to test the role of an important environmental variable on methanotrophic rates under nontraditional empirical conditions.",
"discussion": "RESULTS AND DISCUSSION Comparison of CH 3 D and 14 CH 4 rates in aerobic methanotroph cultures. D/H ratios were acquired and corresponding values of methane consumption were calculated at eight points during the Methylosinus trichosporium growth curve and seven points of the Methyloprofundus sedimenti growth curve. Three measurements of 14 C distributions were acquired for each strain, targeting exponential and stationary phases ( Fig. 1 ). The type II alphaproteobacterial methanotroph M. trichosporium exhibited methane consumption rates more than an order of magnitude greater than those of M. sedimenti (gammaproteobacterial type I methanotroph), yet the scaling factor relating the CH 3 D- and 14 CH 4 -derived rates was remarkably consistent in both cases. Scaling factors were calculated for both exponential growth and stationary phase, using data points from both CH 3 D and 14 CH 4 experiments. The M. trichosporium rate value calculated from the CH 3 D experimental treatment point (47.5 h, 4.16 × 10 4 nmol of methane consumed) was compared with the rate determined from the 14 CH 4 experimental treatment point (47.5 h, 2.78 × 10 4 nmol of methane consumed), yielding a scaling factor of 1.5 for exponential-phase growth. Similarly, data from the experimental treatment point at 140 h (5.27 × 10 4 nmol of methane, CH 3 D) and 166.5 h (4.24 × 10 4 nmol of methane, 14 CH 4 ) were used for M. trichosporium ’s stationary-phase scaling factor. Equivalent values were determined for M. sedimenti using the following data points: 7.07 × 10 3 nmol of methane after 140 h with CH 3 D and 3.35 × 10 3 nmol of methane after 102 h with 14 CH 4 for the exponential growth phase, and 7.53 × 10 3 nmol of methane after 476 h with CH 3 D and 4.30 × 10 3 nmol of methane after 432 h with 14 CH 4 for the stationary phase ( Fig. 1 ). It should be noted that simultaneous sampling of CH 3 D and 14 CH 4 experiments was not always possible, as they were conducted at different institutions. Nonetheless, the optical density at 600 nm (OD 600 ) and rate-based growth curves indicate that all sampling occurred within the designated growth phase ( Fig. 1 and see Fig. S1 in the supplemental material). 10.1128/mSphereDirect.00309-17.2 FIG S1 OD 600 growth curves for cultures of the type II methanotroph M. trichosporium (a) and the type I methanotroph M. sedimenti (b). Error bars show standard errors for three biological replicates. Download FIG S1, PPTX file, 0.1 MB . Copyright © 2017 Marlow et al. 2017 Marlow et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . FIG 1 Amount of methane consumed over time for cultures of the type II methanotroph M. trichosporium (a) and the type I methanotroph M. sedimenti (b) using C corr (values were derived from the CH 3 D method [circles]) and the 14 CH 4 method (diamonds), calculated as discussed in the text. 14 CH 4 -derived data convey values of methane consumption and full oxidation, while CH 3 D-derived data provide a measure of methane activation. Error bars show standard errors for three biological replicates, except for the 14 CH 4 killed control ( n = 1). Obscured data points exhibited values between −60 and 110 nmol for the results in panel a and between 0 and 60 nmol for the results in panel b. In this way, the ratios of methane consumption rates derived from the CH 3 D method (using equations 1 to 7 [see Materials and Methods]) and the 14 CH 4 method (using equation 8) can be compared. This value is herein referred to as the D/ 14 C tracer ratio. This ratio can be used to evaluate the consistency of the monodeuterated-methane method compared with the well-established 14 CH 4 approach and as a potent investigatory tool to probe the relationship between partial and complete metabolism of methane. D/ 14 C tracer ratio values for aerobic methanotroph cultures tested in this study are shown in Table 1 ; their consistency is a promising indicator of the utility of the monodeuterated-methane approach. By dividing the methane activation rates derived from D/H values ( R CH 3 D [see “Rate measurements derived from CH 3 D addition” below]) by 1.5, an estimate of full-oxidation methanotrophy—that is, the complete biological oxidation of methane to carbon dioxide—can be attained. TABLE 1 D/ 14 C tracer ratios for the experimental treatments addressed in this study a Sample tested Ratio in: Exponential phase Stationary phase Oxic incubations Anoxic incubations Aerobic methanotroph cultures M. trichosporium 1.5 1.48 M. sedimenti 1.54 1.59 \n Methane seep sediments and carbonates A.Sed-5128 1.62 2.05 L.Sed-5043 1.71 2.01 A.Carb-5305 1.65 1.96 A.Carb-5152 1.63 2.08 L.Carb-5028 1.69 1.86 a Cultures of the aerobic methanotrophs M. trichosporium and M. sedimenti were tested alongside environmental samples, sediments (Sed) and carbonate rocks (Carb), from Hydrate Ridge methane seeps. “A” refers to sites of active seepage, while “L” indicates locations of low seepage activity, where clear signs of contemporary methane seepage were absent. (See the text for additional sampling details.) Comparison of CH 3 D and 14 CH 4 rate measurements in environmental methane seep samples. Methane consumption rates under oxic ( Fig. 2a ) and anoxic ( Fig. 2b ) microcosm incubation conditions, derived from both CH 3 D and 14 CH 4 measurements, are provided for five different sample types from marine methane seeps (active sediment, low-activity sediment, active porous carbonate, active massive carbonate, and low-activity massive carbonate) and were calculated from data collected after 4 days (oxic) or 8 days (anoxic) of incubation. FIG 2 Methanotrophy in oxic (a) and anoxic (b) incubations of active and low-activity seep sediment and carbonate rocks ( n = 3 in all cases). Values compare rates of methane consumption and full oxidation derived from 14 CH 4 measurements (blue) and rates of methane activation derived from the CH 3 D approach (green, R CH 3 D values). Values are reflective of rock and initial sediment volumes (not including added water). Rates derived from triplicate A.Sed-5128 killed-control incubations were subtracted from all samples. Standard error bars are provided. The D/ 14 C tracer ratio was 1.66 ± 0.02 standard error (SE) for the oxic and 1.99 ± 0.04 SE for the anoxic incubations ( Table 1 ). These relatively consistent values across physical substrate type (sediment and carbonates of various lithologies) and collection site activity level (active and low activity) suggest an underlying metabolic basis of the D/ 14 C tracer ratio that is unperturbed by physicochemical factors or relative activity levels. Understanding the D/ 14 C tracer ratio. The CH 3 D and 14 CH 4 approaches quantify distinct aspects of methanotrophy: methane activation and complete conversion to CO 2 , respectively. The 14 CH 4 technique quantifies the amount of 14 C (initially supplied as methane) that is fully oxidized and persists as soluble species (HCO 3 − ) or acid-labile precipitation products (CaCO 3 ). The CH 3 D protocol, on the other hand, reports the extent to which methane-derived hydrogen atoms are detected in water. Abiotic exchange between methane- and water-associated hydrogen atoms is not expected. Indeed, D/H ratios in killed-control experiments remained stable (e.g., exhibiting a value of 1.40 × 10 −4 ± 3.1 × 10 −8 SE at time zero [ T 0 ] and 1.40 × 10 −4 ± 2.9 × 10 −8 SE at 140 days [ T 140 ] during experimentation with M. trichosporium [data are incorporated into Fig. 1a ]). The activation of methane thereby indicates enzymatic functionalization, but the ultimate fate of each hydrogen atom during methane oxidation is not known. The flow of methane-derived hydrogen atoms through anaerobic and aerobic methanotrophic metabolisms was examined in an attempt to predictively evaluate the consequence of monodeuterated-methane reactions. Previously published reports were used to compile Fig. 3 ( 39 – 41 ) and Fig. 4 ( 42 ), which trace anaerobic and aerobic methane metabolisms, respectively, with a specific focus on hydrogen atoms. In this context, our observations of relatively consistent but distinct D/ 14 C tracer ratios for anaerobic and aerobic methanotrophy ( Table 1 ) likely reflect different aspects of the two metabolic pathways. In AOM, metabolite back-flux ( 43 ) may increase the D/H ratio; in aerobic methanotrophy, biomass growth represents a substantial carbon and hydrogen shunt. FIG 3 Schematic diagram demonstrating the potential fate of methane-associated hydrogen atoms in the reverse-methanogenesis pathway. Hydrogen atoms are distinguished by color and superscript number, and potential exchanges with inter- and intracellular water are shown; asterisks represent location-specific ambiguity. Potentially detectable methane-derived hydrogen atoms (four, occurring throughout the oxidation pathway) and carbon atoms (one, requiring full oxidation) are highlighted in orange and purple boxes, respectively. Shorter back-flux arrows reflect the observation that all enzymes ( 85 ) and the entire pathway ( 43 ) have been shown to be reversible. For figure simplicity, not all cofactors or isotopically distinct back-flux products are shown. Enzyme abbreviations are in black-lined boxes, and the extended dashed line represents the cell membrane. Fd ox , oxidized ferredoxin; Fd red , reduced ferredoxin; MF, methanofuran; H4MPT, tetrahydromethanopterin. The D/ 14 C tracer ratio in anaerobic methanotrophy. AOM is depicted in Fig. 3 via the reverse-methanogenesis pathway, which is believed to be enacted by anaerobic methanotrophic archaea based on genetic ( 41 , 44 , 45 ) and proteomic ( 46 , 47 ) data. In this metabolic process, methyl-coenzyme M reductase (Mcr) activates methane and generates methyl-coenzyme M (methyl-CoM). A tetrahydromethanopterin molecule supplants CoM, and subsequent carbon oxidation steps release hydrogen atoms into the medium. Ultimately, the number of methane-derived hydrogen atoms that enter water-exchangeable products determines the physiological interpretation of aqueous D/H ratios. For example, if just one methane-derived hydrogen enters an intermediate and is freely exchangeable with water, then observed water-based deuterium must be multiplied by 4 (to account for methane’s hydrogen-carbon stoichiometry [see equation 5 in Materials and Methods]) and the appropriate primary isotope effect (not evaluated here) to arrive at the actual quantity of activated methane molecules. In this context, the experimental D/ 14 C tracer ratio values may provide useful insight. A D/ 14 C tracer ratio of 2 for the reverse-methanogenesis pathway suggests that for every methane molecule that is fully oxidized to CO 2 , two hydrogen atoms enter water-exchangeable intermediates. However, the back-reaction of enzymatic processes ( 48 ) may lead to heightened D/H ratios in the absence of full carbon oxidation. For example, upon the activation of methane by Mcr, HS-coenzyme B (HS-CoB) and CH 3 -S-CoM form, with the thiol hydrogen exchanging with water-bound hydrogen. If the initially formed S-bound hydrogen is deuterium, this atom then exchanges with 1 H from water. Upon Mcr back-reaction, CH 4 is formed and the aqueous deuterium causes a heightened D/H ratio despite a lack of complete methane oxidation ( Fig. 3 ). We analyzed the remaining headspace of seep sediment incubations for the formation of CH 4 from CH 3 D via 1 H-nuclear magnetic resonance (NMR) spectroscopy. Over the course of 58 days in triplicate active-sediment 5128 (A.Sed-5128) incubations prepared with exclusively CH 3 D headspace, CH 4 in the headspace increased from 0.33% ± 0.02% SE to 4.48% ± 0.27% SE. If this demonstrated reversibility reflects only the back-reaction of Mcr, then the CH 4 increase (4.15%) must be multiplied by 4 (=16.6%) to reflect the actual percentage of headspace methane that was re-formed by Mcr; if the reversibility reflects back-reaction of the entire pathway, then no scaling factor is needed. Full methane oxidation rates measured via 14 CH 4 in different replicates of the same inoculum ( Fig. 2b ) revealed that 4.1% of the available methane was observed in the fully oxidized state (i.e., as 14 C-labeled dissolved inorganic carbon [DIC]) during the 58-day incubation and thus did not participate in the back-reaction. An estimated 95.9% of the initial methane remained at the time of NMR measurement, meaning that the amount of initial CH 3 D that may have re-formed as CH 4 through a partial or complete back-reaction is between 3.98 and 15.92%. For clarity, these calculations neglect isotope effects and activity by methanogens, the latter of which was highly endergonic given the lack of added hydrogen or acetate. These factors can be explored through further experimentation. Reversibility can be evaluated in future stable isotope work by (i) including a [ 13 C]DIC source in the water and measuring 13 CH 4 and/or (ii) utilizing commercially available multiply deuterated methane as the initial headspace and quantifying all possible isotopologues. Nonetheless, even the upper bound of partially and reversibly oxidized CH 3 D suggests that the majority of the D/H change is attributable to reactions indicative of net methane consumption, if not complete oxidation. The D/ 14 C tracer ratio in aerobic methanotrophy. In aerobic methanotrophic cultures, a D/ 14 C tracer ratio of ~1.5 was observed, suggesting that on average, 2.67 of the four methane-derived hydrogen atoms likely enter water-exchangeable products during the course of a full-oxidation pathway. M. trichosporium is a type II methanotroph, a member of the Alphaproteobacteria that uses the serine pathway for carbon assimilation; M. sedimenti is a gammaproteobacterial type I methanotroph that uses the RuMP carbon assimilation pathway ( 49 ). The pathway data presented in Fig. 4 suggest that all methane-bound hydrogens are water exchangeable during the catabolic oxidation of methane to carbon dioxide. Thus, to achieve a D/ 14 C tracer ratio less than 4, a substantial proportion of methane-derived formaldehyde would need to proceed down the assimilatory pathway, a requirement that was likely met given the cultures’ increase in cell density ( Fig. S1 ). Intriguingly, the D/ 14 C tracer ratios were similar for the two cultured organisms despite their distinct metabolic pathways; a similar phenomenon of consistent carbon conversion efficiency was recently observed among distinct aerobic methanotroph communities in English riverbeds ( 50 ). Previous studies of aerobic methanotrophy compared rates derived from radiolabeled carbon ( 14 C)- and hydrogen ( 3 H)-based approaches, yielding unpredictable ratios spanning multiple orders of magnitude ( 29 , 51 ). These findings were attributed to discrepancies in incubation temperatures and metabolic priming effects between methods, highlighting the need for consistent experimental parameters. FIG 4 Schematic diagram demonstrating the potential fate of methane-associated hydrogen atoms in the aerobic methanotrophy pathway. Hydrogen atoms are distinguished by color and superscript number; asterisks represent location-specific ambiguity. Potentially detectable methane-derived hydrogen atoms and carbon atoms are highlighted in orange and purple boxes, respectively. Mmo enzymes are not believed to perform reversible reactions. FDH, formate dehydrogenase; CytC ox , oxidized cytochrome c ; CytC red , reduced cytochrome c ; MDH, methanol dehydrogenase; pMMO, particulate methane monooxygenase; sMMO, soluble methane monooxygenase. The oxic incubations of methane seep sediment produced a D/ 14 C tracer ratio of 1.66 ± 0.02 SE. Given that the known modes of biological methane oxidation—type I and type II aerobic methanotrophy and reverse-methanogenesis anaerobic methanotrophy—bound this observed value, it appears likely that the oxic sediment incubations supported a mixture of both aerobic and anaerobic methane oxidation processes. Aerobic methane oxidation likely dominated, based on the ~7 × 10 4 -Pa partial pressure of O 2 and the proximity of the D/ 14 C tracer ratio to that of the aerobic methanotrophic cultures, but anoxic niches likely remained or developed in the incubation bottles. Specialized application of monodeuterated methane: examining methane activation under pressure. To demonstrate the utility of the CH 3 D rate measurement approach in nontraditional empirical contexts, we sought to evaluate the influence of in situ pressure on methanotrophic rates of Hydrate Ridge seep sediment microbial communities. Material collected for microbiological studies of AOM is frequently obtained from marine settings of various depths that are subjected to distinct and substantial pressure regimes ( 52 ). Pressure is not always rigorously incorporated into microcosm experiments, though evidence suggests that it can be an important determinant of methanotrophic rates ( 53 – 56 ). In addition, some procedural aspects of the 14 CH 4 protocol, including headspace sampling and full-volume transfer, are not established for use with Mylar bags, which lack gas-tight sampling ports, making the monodeuterated-methane approach an appealing alternative in this context. Parallel seep sediment incubations were subjected to 0.1 MPa (atmospheric pressure) and 9.0 MPa (equivalent to an ~900-m depth). Nitrogen in the form of ammonium (500 μM NH 4 Cl) or the amino acid glycine (500 μM) was added to assess whether distinct nitrogen sources influenced AOM rates. Methane consumption rates derived from heightened D/H ratios are shown in Fig. 5 . A significant increase in methane consumption was observed under both live conditions at high pressure, corresponding to sediment incubated with glycine (samples 1a and 1b) and ammonium chloride (samples 2a and 2b). Neither live controls lacking CH 3 D (samples 3a and 3b) nor autoclaved, killed controls (samples 4a and 4b) showed activation of CH 3 D (see Table S1 for sample setup details). The simulation of in situ Hydrate Ridge pressures led to a 79.5% (±6.5% SE) increase in relative methane consumption rates. Incubation with 500 μM glycine rather than ammonium at high and low pressures resulted in small but consistent rate increases of 12% ± 4.1% SE, potentially reflecting the energetic and biosynthetic distinction between exogenous amino acids and unprocessed fixed nitrogen. 10.1128/mSphereDirect.00309-17.4 TABLE S1 Experimental setup for methane seep sediment pressurized rate measurement incubations. The samples ran for 38 days at 4°C, and each sample was contained in a sealed Mylar bag. Pressure values indicate absolute pressure exerted on the incubated Mylar bags. Download TABLE S1, DOCX file, 0.01 MB . Copyright © 2017 Marlow et al. 2017 Marlow et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . FIG 5 Pressure experiment results showing methane consumption rates derived from aqueous D/H values, with standard error bars, of seep sediment samples following 38-day incubations with CH 3 D at 9.0 MPa (gray bars, “b” samples) or 0.1 MPa (green bars, “a” samples). Additional details on sample treatments can be found in Table S1 . Values are reflective of rock and initial sediment volumes (not including added water). Previous reports have found a wide range of different pressure-related effects. In a sulfate-coupled AOM bioreactor, pressures were varied from 1 to 8 MPa, and sulfide production approximately tripled ( 55 ). Compared with treatment at 0.101 MPa, a 10.1-MPa bioreactor with sediment from Eckernförde Bay demonstrated a cessation of methanogenesis, a 4-fold increase in methane oxidation rates, and high relative abundances of ANME-2a/b and ANME-2c ( 56 ). A continuous incubation system with Black Sea microbial mats at 16 MPa measured a 10- to 15-fold increase in methane-dependent sulfide generation compared with ambient pressure ( 57 ). Methane partial pressures of 1.1 MPa led to a 5-fold increase in sulfate reduction rates relative to ambient atmospheric pressure with Hydrate Ridge sediments demonstrating methane-dependent sulfate reduction ( 31 ). With methane seep sediment from the Japan Trench, however, methane-driven sulfate reduction rates did not correlate with changing pressure ( 58 ). Nauhaus et al. ( 54 ) suggested that the pressure-induced rate increases are due more to heightened methane solubility and bioavailability than to physiological effects or biomolecular reordering. Bowles et al. ( 53 ) presented a very different perspective by showing a 6- to 10-fold AOM rate increase at 10 MPa when methane concentrations were held constant. Deconvolving these two influences and how they depend on community composition or physicochemical parameters is feasible with pressure chamber experiments utilizing monodeuterated methane. Intriguingly, in combination with a carbon-based isotopic probe, aqueous D/H measurements might be used to evaluate predictions that AOM at heightened pressure exhibits decreased back-fluxes ( 39 , 59 ) and, if so, whether the barrier occurs after complete or partial oxidation. More broadly, understanding the relative contributions of environmental and physiological effects to methane oxidation will help constrain methane fluxes across a large envelope of the planet’s methanotrophically active zones. Using monodeuterated methane in experimental investigations. Based on 14 CH 4 ground truth experiments with aerobic methanotrophic cultures, oxic seep sediment, and anoxic seep sediment, as well as the proof-of-concept pressurized experiments, we believe that the monodeuterated-methane approach to methane oxidation rate measurement is a useful addition to a biogeochemist’s tool set ( Table 2 ). Compared with radiolabel approaches ( 14 CH 4 , [ 3 H]CH 4 , 35 SO 4 2− ), the method requires less safety-oriented planning and is procedurally simpler, more affordable, and less susceptible to hydrogen-associated isotope fractionation effects (relative to 3 H). Our results also suggest that the monodeuterated-methane technique appears to be a more precise method based on standard error calculations ( Fig. 1 and 2 ; Table S2 ). Direct comparisons of environmental incubations are complicated by the microheterogeneity of seep settings ( 60 , 61 ), as well as the fact that different aliquots of the same initial material were used in our experiments. Analysis of culture-based and seep substrate experiments reveals that standard errors from CH 3 D-derived values were between 1.56 times lower ( M. trichosporium cultures) and 4.76 times lower (anoxic seep substrate incubations) than those derived from 14 CH 4 -based values ( Table S2 ). 10.1128/mSphereDirect.00309-17.5 TABLE S2 Standard errors from experiments comparing CH 3 D- and 14 CH 4 -derived measurements. For aerobic methanotroph cultures (data shown in Fig. 1 ), experimental treatments were compared, and ratios of standard errors were compared between the closest available time points (designated by color shading). For seep sediment substrates (data shown in Fig. 2 ), endpoint rates were used for comparison; the SE for CH 3 D/SE for 14 CH 4 ratios do not change if overall quantities rather than rates are used. Mean ratios of the standard errors derived from the two methods are provided as well for the different experiments. The reciprocal of these ratios indicates how much more precise the numerator method is than the denominator method. For example, for anoxic seep substrate incubations, the CH 3 D approach is 4.76 times more precise (1/0.21) than the 14 CH 4 method. Download TABLE S2, DOCX file, 0.1 MB . Copyright © 2017 Marlow et al. 2017 Marlow et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . TABLE 2 Brief summary of the features and potential challenges associated with some of the most prominent methods of experimental methane rate assessment Method Feature(s) Challenges Methane concn measurements Directly measures net methane consumption or production Low sensitivity; limited information on metabolic end product \n [ 14 C]CH 4 High sensitivity; tracks carbon atoms and can quantify full methane oxidation; applicable to intact sediment cores; allows high-throughput sampling in the field Radiolabel faces health and safety regulations; processing samples is procedurally time-intensive \n 13 CH 4 Tracks carbon atoms and can quantify anabolic and catabolic processes, including full methane oxidation Naturally occurring dissolved inorganic carbon pools can complicate experiments; not yet tested for intact sediment cores \n [ 3 H]CH 4 High sensitivity; high specific activity; tracks hydrogen atoms; allows high-throughput sampling in the field Radiolabel faces health and safety regulations; not practicable for sediment systems; inconsistent relationship with carbon-based rate measurements \n CH 3 D Measures methane activation; tracks hydrogen atoms to enable a better understanding of methane metabolism; logistically and procedurally straightforward; high measurement precision Hydrogen atom dynamics in methane metabolisms are not fully known; not yet tested for intact sediment cores Because the monodeuterated-methane method focuses on methane-bound hydrogen atoms, it offers information about methanotrophic systems that is different from and yet complementary to that offered by carbon-based techniques like 13 C stable-isotope tracking or quantification of methane or bicarbonate. While this distinction complicates the interpretation of isolated D/H ratios, it can offer additional information for analysis of methane-derived intermediates in relevant metabolisms. Given these caveats, we recommend three applications for monodeuterated methane in methane oxidation rate measurement studies. First, the approach can be employed in a strictly comparative context using an analogous inoculum exposed to a range of different conditions, as demonstrated with the pressure-based sediment incubations presented above. Promising applications include evaluating the effect of different conditions such as temperature ranges, chemical concentrations, or energetic landscapes on seep sediment methane-oxidizing rates. Comparative analysis of rates at different seep sites would also be useful, provided anaerobic or aerobic methanotrophic processes could be isolated. Second, by performing side-by-side monodeuterated-methane and radiocarbon tests, a sample-specific D/ 14 C tracer ratio can be determined, and estimated rates of complete methane oxidation can then be assessed in subsequent experiments on aliquots of the same initial sample material using CH 3 D. Conducting such paired studies under additional environmental or lab-based conditions would help clarify the universality of the ratios presented here. In particular, maintaining consistent headspace proportions and ensuring full equilibration between phases in cultures and incubations would eliminate two potential sources of uncertainty. Mohr et al. ( 62 ) showed that more than an hour of continuous shaking was needed to approach full equilibrium dissolution of N 2 gas and that nitrogen fixation rates had traditionally been underestimated as a result. If similar kinetics govern methane solubility, shorter incubations might have artificially low D/ 14 C tracer ratios, though such patterns were not observed between exponential and stationary phases of aerobic methanotroph culture experiments ( Table 1 ). Although initial dissolved methane concentrations were equivalent between the CH 3 D and 14 CH 4 experiments, the larger overall quantity of methane available to CH 3 D incubations with headspace may have enabled a more exergonic methane-oxidizing metabolism as the experiments progressed. Further interrogation of these variables would help to clarify their relative importance while providing a robust framework for application of the CH 3 D technique to each user’s experimental system. In addition, experiments with the intra-aerobic pathway of “Candidatus Methylomirabilis oxyfera” ( 63 , 64 ) or nitrate- or metal-reducing methanotrophic metabolisms ( 18 , 20 , 21 ) would be valuable contributions, as would the extension of the approach to other experimental setups, such as intact sediment cores. We also encourage side-by-side comparisons with other rate measurement approaches, including [ 3 H]CH 4 radiotracer and methane concentration assessments, to develop additional pairwise conversion factors and better constrain carbon and hydrogen metabolism in methane-based biological reactions. Finally, the use of monodeuterated methane as an analytical tool, alongside additional methods, such as carbon- or sulfur-tracking procedures, would enable the examination of anabolic and catabolic processes in methane-based metabolisms using multiple types of atoms. In particular, the D/ 14 C tracer ratios presented here reveal intriguing and seemingly systematic relationships between carbon and hydrogen anabolic and catabolic partitioning across distinct physiologies, yet an underlying theoretical framework regarding the fate of methane-bound hydrogen atoms remains outstanding. In anaerobic methanotrophic systems, back-reaction rates and equilibrium constants might be evaluated by (i) including a 13 CO 2 source in the water and measuring 13 CH 4 , (ii) using 13 CH 4 in the headspace and quantifying its dilution by 12 CH 4 produced during back-reaction ( 28 , 65 ), or (iii) adding multiply deuterated methane as the initial headspace and measuring all possible isotopologues via NMR or high-resolution mass spectrometry. Tracking sulfur and oxygen isotopic distributions of sulfate can characterize the back-flux of sulfate reduction ( 59 , 66 ); linking this process with methane oxidation and back-reaction would provide insight into the close metabolic coupling between ANME and SRB. For aerobic methanotrophs, evaluating D/ 14 C tracer ratios under more clearly defined growth and maintenance phases would elucidate distinct values associated with catabolic, RuMP, and serine pathways, enabling future use of that parameter as an arbiter of relative anabolic and catabolic activities. Furthermore, additional environmental variables can be tested to gain insight into distinct redox pathways and dynamics of reversibility. For example, under low sulfate concentrations, back-flux of the AOM reaction increases as methane and carbon dioxide approach carbon isotopic equilibrium ( 67 ), and a higher D/ 14 C tracer ratio might be expected. In this context, the D/ 14 C tracer ratio could be further developed as a measure of microbially mediated isotopic equilibration. Conclusions. The ability to accurately measure methane consumption and oxidation rates—both comparatively and in absolute values—is an important component of methanotrophic studies. Such measurements frequently depend on radiotracers or measurements of chemical species that are related to, but not directly indicative of, methane metabolism. The monodeuterated-methane technique presented here represents a novel approach to investigate methane oxidation rates, notable for its logistical ease and straightforward sampling procedures. We have demonstrated that the D/H ratio is a reliable proxy for methane oxidation activity when subjected to ground truth experiments on a sample-specific basis with the well-established 14 CH 4 method; in several applications, methane consumption values calculated via the CH 3 D method were directly proportional to 14 C radiolabel-derived methane oxidation rates. Values of the proportionality constant differ based on the experimental system, likely dictated by environmental variables and the relative proportions of aerobic and anaerobic methanotrophic metabolisms, though additional experiments to determine the nature of the putative mixing line are needed. By providing a way to measure how hydrogen atoms are mobilized and processed, deuterated methane represents a promising approach to help researchers disentangle several aspects of methane-associated metabolisms. Methane biogeochemistry is a dynamic field of study with implications for carbon cycling, microbial ecology, and climate dynamics, though experimental challenges have slowed our understanding of methane-based biological reactions. With the CH 3 D approach as an added tool in the arsenal of rate-based examinations, a broader understanding of the intricacies of methane metabolism, as well as its role in environmental and anthropogenic systems, is within reach."
} | 10,314 |
28225593 | null | s2 | 2,624 | {
"abstract": "Hierarchical organization of macromolecules through self-assembly is a prominent feature in biological systems. Synthetic fabrication of such structures provides materials with emergent functions. Here, we report the fabrication of self-assembled superstructures through coengineering of recombinant proteins and nanoparticles. These structures feature a highly sophisticated level of multilayered hierarchical organization of the components: individual proteins and nanoparticles coassemble to form discrete assemblies that collapse to form granules, which then further self-organize to generate superstructures with sizes of hundreds of nanometers. The components within these superstructures are dynamic and spatially reorganize in response to environmental influences. The precise control over the molecular organization of building blocks imparted by this protein-nanoparticle coengineering strategy provides a method for creating hierarchical hybrid materials."
} | 241 |
25791378 | null | s2 | 2,625 | {
"abstract": "The production of biogas (methane) by an anaerobic digestion is an important facet to renewable energy, but is subject to instability due to the sensitivity of strictly anaerobic methanogenic archaea (methanogens) to environmental perturbations, such as oxygen. An understanding of the oxidant-sensing mechanisms used by methanogens may lead to the development of more oxidant tolerant (i.e., stable) methanogen strains. MsvR is a redox-sensitive transcriptional regulator that is found exclusively in methanogens. We show here that oxidation of MsvR from Methanosarcina acetivorans (MaMsvR) with hydrogen peroxide oxidizes cysteine thiols, which inactivates MaMsvR binding to its own promoter (P(msvR)). Incubation of oxidized MaMsvR with the M. acetivorans thioredoxin system (NADPH, MaTrxR, and MaTrx7) results in reduction of the cysteines back to thiols and activation of P msvR binding. These data confirm that cysteines are critical for the thiol-disulfide regulation of P(msvR) binding by MaMsvR and support a role for the M. acetivorans thioredoxin system in the in vivo activation of MaMsvR. The results support the feasibility of using MaMsvR and P(msvR), along with the Methanosarcina genetic system, to design methanogen strains with oxidant-regulated gene expression systems, which may aid in stabilizing anaerobic digestion."
} | 334 |
21790934 | PMC3187870 | pmc | 2,626 | {
"abstract": "The concept of alternative stable states has long been a dominant framework for studying the influence of historical contingency in community assembly. This concept focuses on stable states, yet many real communities are kept in a transient state by disturbance, and the utility of predictions for stable states in explaining transient states remains unclear. Using a simple model of plant community assembly, we show that the conditions under which historical contingency affects community assembly can differ greatly for stable versus transient states. Differences arise because the contribution of such factors as mortality rate, environmental heterogeneity and plant-soil feedback to historical contingency changes as community assembly proceeds. We also show that transient states can last for a long time relative to immigration rate and generation time. These results argue for a conceptual shift of focus from alternative stable states to alternative transient states for understanding historical contingency in community assembly.",
"conclusion": "Conclusion The alternative stable states concept has greatly contributed to improving our understanding of the role of historical contingency in community assembly. However, uncritical applications of the concept may have misled us in understanding communities because, as we have shown here, the assumptions necessary to make alternative stable states relevant to explanation of transient states can be easily violated. Our results argue for a conceptual shift of attention from a narrow focus on alternative stable states to a more inclusive focus on both alternative stable states and alternative transient states. Specifically, rather than studying determinants of final variability in species composition (e.g. feedback strength, f ), it will be more informative to also investigate those of initial variability (e.g. habitat heterogeneity, h ) and the rate at which the initial level of variability tends toward the final level (e.g. mortality rate, m ). We believe these efforts will allow ecologists to more tightly integrate two closely related, but historically separated subfields of community ecology – community-assembly research, which has focused on final states, and succession research, which has focused on temporal changes – for a better understanding of the influence of historical contingency in community assembly and its consequences for species diversity and ecosystem functioning.",
"introduction": "Introduction It is increasingly recognised that the species composition and diversity of ecological communities can be greatly influenced by the history of community assembly. Growing evidence indicates that the effect of biotic interactions on species abundances may depend on the order and timing of species immigration during community assembly, the phenomenon known as priority effect (e.g. Schoener 1976 ; Drake 1991 ; Almany 2003 ). The extent of historical contingency due to priority effect is difficult to quantify because immigration history is impossible to reconstruct in sufficient detail for most natural communities. Nevertheless, theory suggests that biotic historical effects can be substantial ( Gilpin & Case 1976 ; Drake 1990 ; Law 1999 ; Fukami 2004b ; Steiner & Leibold 2004 ), with profound implications for understanding and conserving species diversity. For example, priority effect can cause unexpectedly high variability in community structure, or high beta diversity sensu Whittaker (1960 , 1972) , among similar sites ( Fukami 2004b ; Chase 2010 ). Further, if historical contingency is important, restoring native diversity in degraded sites may require specific sequences of species removal and introduction to be successful ( Fukami et al. 2005 ; Young et al. 2005 ; Suding & Hobbs 2009 ; Kardol & Wardle 2010 ). In this light, much research has been directed toward identifying the environmental factors that determine the importance of assembly history, such as habitat productivity ( Steiner & Leibold 2004 ), ecosystem size ( Fukami 2004a ), disturbance frequency ( Jiang & Patel 2008 ) and environmental heterogeneity ( Shurin et al. 2004 ; Van Nes & Scheffer 2005 ). In the effort to understand the role of historical contingency in community assembly, the concept of alternative stable states (also known as multiple stable points, multiple stable equilibria, alternative attractors, multiple domains of attraction and other similar terms) has played a dominant role as the guiding theoretical framework (e.g. Lewontin 1969 ; Sutherland 1974 ; May 1977 ; Peterson 1984 ; Drake 1991 ; Petraitis & Dudgeon 1999 ; Beisner et al. 2003 ; Schröder et al. 2005 ; Suding & Hobbs 2009 ). According to this concept, there can be more than one final stable state of species composition that assembling communities may approach depending on immigration history, even under the same environmental conditions and the same species pool. Once a community reaches a stable state, it cannot move to another unless heavily disturbed ( Lewontin 1969 ; Gilpin & Case 1976 ; Law 1999 ). This concept places a special emphasis on the analysis of stable states, not necessarily because stable states characterise natural communities, but primarily because of mathematical tractability ( DeAngelis & Waterhouse 1987 ; Hastings 2004 , 2010 ). As long recognised since at least Cowles (1899) , many real communities are in a transient, not stable, state, because disturbance keeps communities from reaching a stable state (reviewed in Pickett & White 1985 ). Despite this mismatch between theory and reality, theoretical predictions about alternative stable states can be useful in understanding real communities if two further assumptions are met. One assumption (hereafter assumption 1) is that, even if natural communities are not in a stable state, theoretically predicted stable states help to explain transient communities ( Chase & Leibold 2003 ; Didham et al. 2005 ; Schröder et al. 2005 ). In other words, transient and stable states do not differ qualitatively with respect to the conditions that make assembly history important to community structure, as measured by the level of beta diversity generated by priority effect. A second assumption (hereafter assumption 2) is that, even if assumption 1 is not always true, the transient states to which assumption 1 does not apply are so short-lived that any discrepancy between stable and transient states is of minor importance. These assumptions are, however, only tacitly implied in most studies thus far. Given the central role that the concept of alternative stable states has played in community assembly research, surprisingly little is known about the validity of these assumptions. In this paper, we examine their validity using a simple simulation model of plant community assembly. Our results suggest that both assumptions may be easily violated. The aim of this paper is not to downplay the well-appreciated importance of studying stable states, but rather to highlight the underappreciated importance of studying transient states. For example, we show that the environmental conditions under which community assembly is particularly sensitive to historical contingency can be understood only by studying transient states directly because it is often not possible to infer transient states from stable states. More generally, we seek to provide new perspectives on community assembly in order to stimulate more research on alternative transient states, which we believe will help to advance the understanding of historical contingency in community assembly and its effect on species diversity. We define alternative transient states as follows: communities are in alternative transient states when they have not reached a stable state, but vary in structure (e.g. species composition and diversity) and/or function (e.g. total biomass and carbon flux) because of variable immigration history and other stochastic processes, even though they have assembled under the same environmental conditions, have received the same set of species multiple times, and have undergone population dynamics over multiple generations of the species involved. This definition ensures that alternative transient states do not include obvious cases in which communities vary in composition simply because they vary in environmental conditions or species pool or because they are at an early stage of assembly where species composition is inevitably variable. Thus, our definition of alternative transient states is identical to that of alternative stable states proposed by Connell & Sousa (1983) and further articulated by Chase (2003) , except that communities exhibiting alternative transient states have not reached a stable state, whereas those in alternative stable states have. Here, a community is considered stable when the locally coexisting species are permanent members of the community and are resistant to colonisation by any additional species in the region ( Law 1999 ). In the following sections, after describing the main model employed, we will present results that indicate that assumptions 1 and 2 can easily be violated. We will then discuss implications of the violated assumptions for understanding how the importance of historical contingency varies along environmental gradients. Because any theoretical prediction needs to be evaluated by empirical evidence, we will also discuss empirical data relevant to our simulation results. We will end by suggesting several future research directions for further improving our understanding of alternative transient states."
} | 2,411 |
23868125 | PMC3715667 | pmc | 2,627 | {
"abstract": "Caloramator celer strain JW/YL-NZ35 is a Gram-positive thermophilic, alkalitolerant, and strictly anaerobic bacterium capable of producing hydrogen and ethanol under extreme conditions. The draft genome sequence presented here will provide valuable information to further explore the physiology of this species and its potential for biofuel production."
} | 88 |
33892498 | PMC8220308 | pmc | 2,630 | {
"abstract": "Abstract The identification of the asgard archaea has fueled speculations regarding the nature of the archaeal host in eukaryogenesis and its level of complexity prior to endosymbiosis. Here, we analyzed the coding capacity of 150 eukaryotes, 1,000 bacteria, and 226 archaea, including the only cultured member of the asgard archaea. Clustering methods that consistently recover endosymbiotic contributions to eukaryotic genomes recover an asgard archaeal-unique contribution of a mere 0.3% to protein families present in the last eukaryotic common ancestor, while simultaneously suggesting that this group’s diversity rivals that of all other archaea combined. The number of homologs shared exclusively between asgard archaea and eukaryotes is only 27 on average. This tiny asgard archaeal-unique contribution to the root of eukaryotic protein families questions claims that archaea evolved complexity prior to eukaryogenesis. Genomic and cellular complexity remains a eukaryote-specific feature and is best understood as the archaeal host’s solution to housing an endosymbiont.",
"introduction": "Introduction Four billion years of prokaryotic evolution has only once resulted in the emergence of highly compartmentalized cells and eventually macroscopic body plans: following the origin of eukaryotes through endosymbiosis. The difference between pro- and eukaryotic biology is evident and the lack of intermediates between the two types of cells places endosymbiosis at the event horizon of eukaryogenesis. The analysis of core eukaryotic features such as the nucleus, mitochondria, sex and meiosis, compartmentalization and dynamic membrane trafficking, and virtually all of the associated protein families, consistently point to their presence in the last eukaryotic common ancestor (LECA) ( Fritz-Laylin et al. 2010 ; Koonin et al. 2013 ; Koumandou et al. 2013 ; Garg and Martin 2016 ). We possess a reasonable understanding of the basic cellular features and coding capacity of LECA, owing to the growing number of genome sequences spanning all of eukaryotic diversity. All eukaryotes stem from a single ancestor that in terms of cellular and genomic complexity rivaled those of extant eukaryotic supergroups ( Fritz-Laylin et al. 2010 ; Koonin et al. 2013 ; Koumandou et al. 2013 ). There is general consensus that LECA was a product of the integration of an alphaproteobacterium into an archaeal host following endosymbiosis ( Lane 2011 ; Blackstone 2013 ; Martin et al. 2015 ; Dacks et al. 2016 ; Spang et al. 2019 ). Through the description of the asgard archaea, current debates once again concern the cellular complexity of the host that came to house the endosymbiont and what contribution the mitochondrion could have played in establishing the eukaryotic cell ( Dacks et al. 2016 ; Martin et al. 2017 ). Asgard archaea, a novel phylum assembled from metagenome data, are viewed as bridging the gap between pro- and eukaryotic cells, because they encode proteins homologous to eukaryotic ones that are involved in intracellular vesicle trafficking and the regulation of actin cytoskeleton dynamics ( Spang et al. 2015 ; Zaremba-Niedzwiedzka et al. 2017 ; Neveu et al. 2020 ). The cellular complexity of the host cell that acquired the alphaproteobacterial endosymbiont has been a matter of speculation ever since the realization that endosymbiosis was pivotal in the transition to eukaryotic life. Modern models of eukaryogenesis differ regarding the timing of mitochondrial acquisition, the extent of the cellular complexity of the host, and the selective reasons provided for explaining the presence, function, and emergence of eukaryotic traits prior or ensuing endosymbiosis ( O’Malley 2010 ; Martin et al. 2015 ; Gould et al. 2016 ; Tria et al. 2021 ; Vosseberg et al. 2020 ). Understanding the steps of eukaryogenesis is a demanding intellectual challenge that explores the past of life and one of its most radical transitions. It holds the key to understanding the steps toward cellular complexity, the timing of mitochondrial entry, and what limits prokaryotes to frequently evolve eukaryote-like complexity. Was eukaryogenesis really a matter of luck ( Booth and Doolittle 2015 ) and how important was the benefit provided by the mitochondrion to the host cell ( Lane and Martin 2010 ; Lynch and Marinov 2015 ; Lane and Martin 2016 ) or the possible availability of altering terminal electron acceptors ( Speijer 2017 )? Any model that views endosymbiosis as some kind of terminal coincidence on the evolutionary roadmap to the eukaryotic domain of life needs to explain the singularity that is eukaryogenesis and the lack of comparable complexity among prokaryotes. A consistent motivation for speculating on the archaeal host cell’s grade of complexity is trying to understand whether the host cell was phagocytotic or not ( Cavalier-Smith 1987 ; Yutin et al. 2009 ; Martijn and Ettema 2013 ; Martin et al. 2017 ), and one that would offer an explanation for the mode of endosymbiont entry. This is complicated by the description of a phagocytosis-like process in a planctomycete ( Shiratori et al. 2019 ) and the conflicting evidence for intracellular prokaryotic endosymbionts in the absence of phagocytosis ( Embley and Finlay 1993 ; Fenchel and Bernard 1993 ; Schmid 2003 ; Duplessis et al. 2004 ; Zientz et al. 2004 ; Thacker 2005 ; Woyke et al. 2006 ; Husnik et al. 2013 ). Some of the asgard archaea encode actin-regulating profilins ( Akıl and Robinson 2018 ), small Rab-like GTPases ( Surkont and Pereira-Leal 2016 ), and prototypic SNARE proteins ( Neveu et al. 2020 ), but they are not phagocytotic ( Burns et al. 2018 ). Phagocytosis might have evolved multiple times independently ( Yutin et al. 2009 ; Mills 2020 ) and is a mode of feeding, which is incompatible with the syntrophic foundation that underpins eukaryogenesis ( Martin and Müller 1998 ; Vellai et al. 1998 ; Martin et al. 2015 ; Spang et al. 2019 ; Imachi et al. 2020 ). A sole focus on this single eukaryotic trait might distract and furthermore discounts the complexity of the transition that was involved. What is certain is that images of an asgard archaeon, Candidatus Prometheoarchaeum syntrophicum MK-D1, reveal cells with typical archaeal morphology, half a micron in diameter, with obligate syntrophy, and devoid of any endomembrane system ( Imachi et al. 2020 ). Here, we clustered the available genomes of 150 eukaryotes, 1,000 bacteria, and 226 archaea (including asgard archaea metagenomic assemblies, and for comparison the complete genome of the cultured Candidatus P. syntrophicum strain MK-D1) in order to evaluate the asgard archaeal-unique contribution to eukaryogenesis that is understood as support for early cellular complexity in asgard archaea.",
"discussion": "Discussion The identification of asgard archaea from deep-sediment metagenome data ( Seitz et al. 2016 ; Zaremba-Niedzwiedzka et al. 2017 ) provides valuable new information from which to re-evaluate key issues surrounding the tree of life and the emergence of its eukaryotic branch. To begin with, the iconic tree that introduced three aboriginal lineages ( Woese and Fox 1977 ) might require a revision. Phylogenomic analysis of asgard archaea provides evidence for a two-domains tree and the emergence of the host cell lineage of eukaryogenesis from within the archaeal domain ( Cox et al. 2008 ; McInerney et al. 2014 ; Hug et al. 2016 ; Williams et al. 2020 ) with some skepticism, however, remaining ( Forterre 2015 ; Da Cunha et al. 2018 ; Liu et al. 2021 ). Parallel to the discovery of the asgard archaea were immediate speculations regarding their cellular complexity ( Dacks et al. 2016 ; Pittis and Gabaldón 2016 ; Rout and Field 2017 ; Akıl and Robinson 2018 ; Zachar et al. 2018 ; Neveu et al. 2020 ) and a faith of having identified the missing link between pro- and eukaryotic biology based on the identification of a few eukaryote signature proteins (ESPs), which we find to be 27 on average. In light of these numbers, the potential of these archaea to display eukaryote-like cell complexity is hard to maintain. Our analysis confirms a patchy distribution of ESPs among asgard archaea ( Dacks et al. 2016 ; Klinger et al. 2016 ; Zaremba-Niedzwiedzka et al. 2017 ; Bulzu et al. 2019 ; Imachi et al. 2020 ; Inoue et al. 2020 ; Liu et al. 2021 ) ( fig. 1 ). Although of course the archaeal host brought in 1,000s of genes, the unique contribution of this lineage to the eukaryotic protein families is substantially less than what one might infer from the original metagenome reports and subsequent interpretations ( Dacks et al. 2016 ; Pittis and Gabaldón 2016 ; Rout and Field 2017 ; Akıl and Robinson 2018 ; Zachar et al. 2018 ; Neveu et al. 2020 ; Vosseberg et al. 2020 ). The irregular gene distribution ( fig. 2 a ) might reflect differential gene loss upon the segregation of the common ancestor of asgard archaea and the archaeal host cell lineage ( Eme et al. 2017 ). Considering the role of pangenomes in the transformation of prokaryotic lineages and the conquering of ecological niches ( McInerney et al. 2017 ), pangenomes offer a complementary explanation to the differential loss of genes. The archaeal ancestor of eukaryotes might have tapped a gene pool more extensively than the sister lineages leading to extant asgard archaea. The notion that asgard archaeal contributions to eukaryotes were higher only to be eventually replaced by bacterial (endosymbiotic) contributions might be brought up ( Pittis and Gabaldón 2016 ; Eme et al. 2017 ; Vosseberg et al. 2020 ). The absence of extant archaeal relatives with similar distributions of ESPs indicates that archaea neither have the necessity nor the selective pressure for maintaining ESPs in the absence of an endosymbiont. Any theory that hinges upon a larger presence of ESPs in archaea (or bacteria) prior to mitochondrial endosymbiosis ignores the complete lack of such an accumulated number of ESPs in extant prokaryotes to a degree that even remotely matches that of any given eukaryotic lineage. The absence of ESPs in the archaeal host lineage could be attributed to the combined effect of selection and extinction, rendering the unique combination of ESPs of such a hypothetical host lineage only advantageous for a brief period of time. Although feasible, the acknowledgment of such mechanisms in evolution then also applies to the endosymbiont. For the mitochondrion, however, the presence of bacterial genes that do not clearly branch with alphaproteobacteria are often interpreted as LGT events ( Pittis and Gabaldón 2016 ; Eme et al. 2017 ; Vosseberg et al. 2020 ). Any considerations on the environmental conditions and evolutionary pressures that promoted the evolution and origin of ESPs, must also be equally considered for any genes that are currently thought to be recent independent gene transfer events from prokaryotes to eukaryotes and not of endosymbiotic (i.e., alphaproteobacterial) origin. Our protein family clustering method, which readily detects the mitochondrial contribution (and the cyanobacterial contributions in the case of the Archaeplastida; supplementary fig. 2, Supplementary Material online) failed to detect a comparable asgard archaeal-unique contribution. A stacked bar diagram puts gene family cluster contribution in each lineage into a global perspective ( fig. 3 a ; supplementary fig 1, Supplementary Material online). There is a small proportion of eukaryotic homologs (E, mustard yellow) visible, for example, in the Candidatus P. syntrophicum MK-D1, but it is substantially smaller in comparison to the eukaryote-bacteria (EB, green) specific homologs evident in eukaryotes (and bacteria vice versa) that reflects the mitochondrial contribution to eukaryogenesis ( Brueckner and Martin 2020 ). Neglecting the surprisingly low number of asgard archaeal-unique homologs to eukaryotic genomes, our analysis demonstrates that asgard archaea are among the most genetically diverse group of archaea when comparing it to the genus Methanococci and the phylum Crenarchaeota ( fig. 2 ). The odd length distributions of the asgard archaeal proteins with no homology ( fig. 3 b ) are strange as well, because protein length across pro- and eukaryotes is usually well conserved ( Xu et al. 2006 ). This could hint at an assembly and/or binning issue, which was also observed regarding the anomalous phylogenetic behavior of their ribosomal proteins and concatenated gene trees ( Da Cunha et al. 2018 ; Garg et al. 2021 ). If not, it is a biological phenomenon absent in other sequenced prokaryotes that requires explaining. Considering the amount of data gathered in just a few years ( Klinger et al. 2016 ; Villanueva et al. 2017 ; Bulzu et al. 2019 ; Imachi et al. 2020 ; Inoue et al. 2020 ; Liu et al. 2021 ), it is surprising asgard archaea have escaped identification for so long. Their habitats had been sampled before, so it is likely that the method used and maybe an obligate dependency on syntrophy hindered culturing, except for one imposing exception ( Imachi et al. 2020 ). Dedicated phylogenomic efforts are necessary to resolve their taxonomic classification, whereas only culturing can picture their cell morphology. Analyses of asgard archaeal ESPs show they have the potential to function similar to their eukaryotic homologs in a eukaryotic system ( Klinger et al. 2016 ; Rout and Field 2017 ; Akıl and Robinson 2018 ; Neveu et al. 2020 ), but cross-kingdom inferences have their limits ( Dey et al. 2016 ). The analysis of archaeal small GTPases ( Surkont and Pereira-Leal 2016 ) and homologs of ESCRT proteins, the CDVs ( Lindås et al. 2008 ; Lindås and Bernander 2013 ; Caspi and Dekker 2018 ), serve as examples. One needs to interpret asgard archaeal ESPs in their prokaryotic context and in cells lacking an endosymbiont. The images of an asgardarchaeon, those of Candidatus P. syntrophicum MK-D1 and its dependency on a bacterial partner ( Imachi et al. 2020 ), define the current standard from which to plot eukaryogenesis and the steps leading to eukaryotic cell and genome complexity. The identification of the asgard archaea and the culturing of one representative represent an important milestone in micro- and evolutionary biology. Their phylogenetic analysis echoes two previously predicted outcomes: 1) eukaryotes to branch from within archaea, solidifying the two-domains tree of life, and 2) that the closer we zoom in on the two prokaryotic partners from which eukaryotes evolved, the higher the number of otherwise eukaryote-typical genes we identify in prokaryotes. The description of Candidatus P. syntrophicum MK-D1 ( Imachi et al. 2020 ) reminds us to not conflate genotypic with phenotypic complexity. This predicts that future asgard archaea we see cultured will lack eukaryotic traits, too, and most, if not all, will depend on syntrophy. Our analysis also predicts that much of asgard archaeal diversity remains to be described and will require further taxonomic sorting, but that the gap between the pro- and eukaryotic protein families will remain decisive and to change little. Placing the endosymbiotic event and the energetic benefit offered by mitochondria to fuel the transition early in eukaryogenesis, explains the lack of physical evidence for eukaryote-like complexity in asgard archaea despite them encoding ESPs. It offers a comprehensive full-service theory for the singularity that is the origin of the eukaryotic cell that mitochondria-late models fail to provide."
} | 3,896 |
33552017 | PMC7854539 | pmc | 2,631 | {
"abstract": "Although microbial communities of anaerobic bioreactors have been extensively studied using DNA-based tools, there are still several knowledge gaps regarding the microbiology of the process, in particular integration of all generated data is still limited. One understudied core phylum within anaerobic bioreactors is the phylum Chloroflexi, despite being one of the most abundant groups in anaerobic reactors. In order to address the abundance, diversity and phylogeny of this group in full-scale methanogenic reactors globally distributed, a compilation of 16S ribosomal RNA gene sequence data from 62 full-scale methanogenic reactors studied worldwide, fed either with wastewater treatment anaerobic reactors (WTARs) or solid-waste treatment anaerobic reactors (STARs), was performed. One of the barriers to overcome was comparing data generated using different primer sets and different sequencing platforms. The sequence analysis revealed that the average abundance of Chloroflexi in WTARs was higher than in STARs. Four genera belonging to the Anaerolineae class dominated both WTARs and STARs but the core populations were different. According to the phylogenetic analysis, most of the sequences formed clusters with no cultured representatives. The Anaerolineae class was more abundant in reactors with granular biomass than in reactors with disperse biomass supporting the hypothesis that Anaerolineae play an important role in granule formation and structure due to their filamentous morphology. Cross-study comparisons can be fruitfully used to understand the complexity of the anaerobic digestion process. However, more efforts are needed to standardize protocols and report metadata information.",
"conclusion": "Conclusion and Future Directions From this meta-analysis, we managed to identify that four genera (midas_g_467, midas_g_156, Bellilinea and Leptolinea ) belonging to Anaerolineae class dominated WTARs and STARs with different core populations. All the species observed had no cultured representatives, limiting our knowledge. From the few isolated species belonged from the genera detected, we could hypothesize that the growth of Anaerolineae could be favored by fermentable substrates and by the syntrophic association with methanogenic archaea. In addition, Anaerolineae was more abundant in reactors with granular biomass (WTARs), than in those in which the microbial biomass typically does not exhibit granulation but have instead dispersed biomass growth (STARs), suggesting that Anaerolineae members play an important role in the granule structure due to their filamentous morphology and/or adhesiveness properties. Despite the extensive research that has been done on microbial communities in anaerobic reactors there is no consensus about experimental protocols, bioinformatics analyses or operational data provided, which makes it difficult to perform global comparisons of microbiome studies. We think that more efforts are needed in each study to provide information on key operational parameters associated with each reactor, since it is fundamental to infer potential roles of lineages of interest. Cross-study comparisons can be fruitfully used to understand the complexity of the anaerobic digestion process. The rapidly advancing fields of metagenomics and metatranscriptomics will provide unique insights into the patterns of microbial activity in anaerobic digestion systems, even for species which have not yet been isolated in pure culture.",
"introduction": "Introduction Anaerobic digestion is an efficient biological process widely applied to treat solid organic waste and wastewater, where the organic matter can be converted into a renewable energy source known as biogas. As it is a mixture containing mainly methane (CH 4 ) and carbon dioxide (CO 2 ), it can be used as a replacement for fossil fuels to generate heat or electricity ( Angenent et al., 2004 ; Verstraete et al., 2005 ; Appels et al., 2011 ). Chloroflexi has been reported as one of the most predominant phylum present in solid waste and wastewater treatment systems ( Shu et al., 2015 ; Petriglieri et al., 2018 ; Bovio et al., 2019 ). In particular, the Anaerolineae class has been identified as one of the core microbial populations in full-scale anaerobic reactors ( Nelson et al., 2011 ; St-Pierre and Wright, 2014 ; Bovio et al., 2019 ). From 12 isolated strains within the Anaerolineae class, five have been isolated from wastewater treatment anaerobic reactors (WTARs) and one from solid-waste treatment anaerobic reactors (STARs) ( Sekiguchi et al., 2003 ; Yamada et al., 2006 , 2007a ; Sun et al., 2016 ). All isolated species share similar phenotypic traits such as filamentous morphology, strict anaerobic growth, and the ability to ferment carbohydrates or amino acids. However, the ecological role of the Anaerolineae remains uncertain due to the scarcity of isolates and annotated genome sequences. In recent years, efforts to assemble genomes from metagenomes have increased due to the difficulty to obtain Chloroflexi strains in pure culture. Genomes belonging to Anaerolineales ( Candidatus Brevefilum fermentans CAMBI-1), Ardenticatenales ( Candidatus Promineofilum), and Caldilineales ( Candidatus Amarolinea aalborgensis) orders have been assembled from shotgun metagenomic sequencing data obtained from aerobic and anaerobic reactors ( McIlroy et al., 2017 , 2016 ; Andersen et al., 2018 ). These genomes showed similar potential metabolism compared to isolated members. It has been suggested that the common prevalence of Anaerolineae class in WTARs and STARs might be due to their advantageous cellular adhesiveness, their potential as cellulose degraders and/or because of being anaerobic syntrophs ( Xia et al., 2016 ). For example, some Anaerolineae species require syntrophic association with hydrogenotrophic methanogens for efficient growth ( Sekiguchi et al., 2001 ; Yamada et al., 2005 , 2006 ). The two most common types of anaerobic digestion systems currently in use are up-flow anaerobic sludge blanket (UASB) reactors and continuously stirred tank reactors (CSTR). In UASB systems, and variations such as expanded granular sludge bed (EGSB) and internal circulation (IC) reactors, the active microbial biomass form compact anaerobic granules which settle against the hydraulic up-flow inside the reactor, preventing biomass washout ( Skiadas et al., 2003 ). Contrastingly, in CSTR systems, generally used for solid waste anaerobic treatment, the active microbial biomass typically does not exhibit granulation but grow in suspension and is constantly removed from the system ( Hofman-Bang et al., 2003 ; Klocke et al., 2007 ). It has been hypothesized that Chloroflexi are relevant for the granule skeleton formation in WTARs as they grow as filaments and therefore play an important role in sludge sedimentation ( Yamada et al., 2005 ). On the other hand, Chloroflexi has been reported to be occasionally involved in bulking episodes in WTARs, caused by their overgrowth, generating biomass washout ( Sekiguchi et al., 2001 , 2015 ; Yamada et al., 2007b ; Borzacconi et al., 2008 ; Li et al., 2008 ). Meanwhile, it has been suggested that, in STARs some of these Chloroflexi derive and migrate from the aerobic activated sludge community when they are coupled together, although many were identified as being exclusive to the digester ( Kirkegaard et al., 2017 ; Petriglieri et al., 2018 ). The phylum Chloroflexi has been studied in STARs and WTARs mainly by molecular methods in separate studies. However, a comparison of Chloroflexi diversity and abundance between full scale STARs and WTARs has still not been investigated. During the last decade, the amount of studies using amplicon sequencing to analyze the microbial community in STARs and WTARs have been in constant growth, but the experimental approaches or data analysis differ widely. This strongly limits our ability to compare among studies and draw general conclusions regarding their diversity or the identification of important taxa. The recent ecosystem-specific Microbial Database for Activated Sludge (MiDAS) 16S rRNA gene amplicon sequencing-based survey, facilitates the understanding of wastewater treatment ecosystem diversity and function ( Nierychlo et al., 2020 ). Data analysis appears to be an important area where further improvements and unification of experimental procedures are necessary. The main objective of this study was to answer the following questions: Is there a particular Chloroflexi population predominant in all anaerobic reactors or are there several groups? Is there a selection of different groups between STARs and WTARs? To address these questions, we conducted a meta-analysis of publicly available microbial datasets generated by high-throughput sequencing in 62 full scale anaerobic reactors treating 29 different solid wastes or wastewaters.",
"discussion": "Discussion Chloroflexi bacteria are widely distributed on Earth and particularly abundant in engineered systems such as solid waste and wastewater treatment plants. With the aim of better describing the abundance and diversity of this phylum, we performed a meta-analysis of the publicly available diversity datasets (i.e., 16S rRNA metabarcoding) in 62 full scale anaerobic reactors. Anaerolineae Class Dominate the Chloroflexi Microbial Communities in the Anaerobic Reactors Studied Despite the bias generated from using different hypervariable regions we found that the Anaerolineae class largely dominated in both STARs and WTARs which was consistent with previous DNA based surveys ( Rivière et al., 2009 ; Nelson et al., 2011 ; Bovio et al., 2019 ). The dominance of Anaerolineae in both types of systems might be explained through several hypotheses. First, most of the isolated strains of Anaerolineae present the potential to degrade cellulose, carbohydrates, and/or proteins anaerobically playing an important role as primary and secondary fermenters, having relevance in the bottlenecking hydrolysis step of anaerobic digestion ( Sekiguchi et al., 2001 , 2003 ; Yamada et al., 2005 , 2006 , 2007a ; Narihiro et al., 2015 ). Thus, the fact that these systems in general treat different fermentable compounds might be favorable for the growth of Anaerolineae. A second explanation to the predominance of Anaerolineae could be that their growth is significantly stimulated when co-cultivated with methanogens ( Sekiguchi et al., 2001 , 2003 ; Yamada et al., 2005 , 2006 , 2007a ; Narihiro et al., 2015 ) indicating some potential for synergistic relationships. If the Anaerolineae group was a major donor of acetate or hydrogen to methanogens ( Narihiro et al., 2015 ; McIlroy et al., 2017 ; Zamorano-López et al., 2020 ), the association in a “spaghetti-like” structure could promote the metabolites transfer flux between both groups, resulting in high methane production rates. In addition, the adhesiveness properties of Methanosaeta ( Wiegant, 1986 ; Angenent et al., 2004 ; Zheng et al., 2006 ; Li et al., 2015 ) and members of Anaerolineae ( Xia et al., 2016 ) could facilitate the metabolites transfer flux improving its dominance in these systems. Interestingly, Anaerolineae had higher relative abundance in WTARs, which generally have granular biomass, than in STARs, in which the microbial biomass typically does not exhibit granulation but has instead dispersed biomass growth ( Hofman-Bang et al., 2003 ; Klocke et al., 2007 ). In a previous study, we also determined that UASB reactors with granular biomass presented higher abundance of Anaerolineae than start-up UASB reactors with disperse biomass ( Bovio et al., 2019 ). This might be explained by the fact that the filamentous morphology of Anaerolineae contribute to the granule formation in WTARs ( Sekiguchi et al., 2001 ; Yamada et al., 2005 ; Satoh et al., 2007 ) presenting more abundance in reactors with granules than in those with dispersed biomass. Another explanation could be the fact that the higher flow velocity used in WTARs compared to STARs, may select organisms which can adhere to each other to form well-settling granular sludge, favoring the growth of Anaerolineae in these systems over other microorganisms ( Xia et al., 2016 ). Four Genera Within Anaerolineae Predominate in the Full Scale Anaerobic Reactors Studied We found that the predominant genera and species in STARs and WTARs were the same, but changes in their relative abundances occurred. Four genera were predominant, two of them were closely related to previously described genera ( Bellilinea and Leptolinea ) and two of them did not harbor any cultured member and were named by the MIDAS database as midas_g_467 and midas_g_156. As for the isolated members of Anaerolineae, species belonging to the genus Leptolinea and Bellilinea present filamentous morphology and ferment carbohydrates and proteins ( Yamada et al., 2006 , 2007a ). However, midas_g_467 and midas_g_156 previously named genus T78 in MiDAS database version 2 ( Nierychlo et al., 2020 ), does not have representative cultured members. Despite this, some possible physiological features and ecological roles have been suggested for these genera in anaerobic reactors, which could explain the predominance of genus T78 ( Kirkegaard et al., 2017 ; Zhu et al., 2017 ; Petriglieri et al., 2018 ; Jiang et al., 2020 ). Some of these previous reports suggest that their filamentous morphology and particular position within flocs or granules, might indicate that they play a role in maintaining floc structure ( Petriglieri et al., 2018 ) as well as the granular structure ( Zhu et al., 2017 ). It has also been reported that genus T78 could play an important role in hydrolysis and fermentation steps in the anaerobic digestion process ( Yuan et al., 2015 ; Jiang et al., 2019 ). T78 also could metabolize alcohols and carbohydrates through syntrophic interactions ( Praveckova et al., 2016 ). In the literature we found that two species belonging to the midas_g_467 correlated positively with different operational parameters: being total ammonia nitrogen, biogas yield, temperature, and organic loading rate (OLR) ( Jiang et al., 2020 ). B. fermentans CAMBI-1 (LT859958) was the most closely related metagenome-assembled genome of the T78 cluster. The annotation of this genome suggested that members of the phylotype are important fermenters in mesophilic anaerobic digestion systems ( McIlroy et al., 2017 ). Also, they are co-localized with the filamentous Archaea Methanosaeta spp. which suggests a potential undetermined synergistic relationship ( McIlroy et al., 2017 ). Both, the potential fermenting metabolism as well as the potential syntrophism with archaea could explain the dominance of the T78 genus in anaerobic reactors. Our correlation analysis between Euryarchaeota and Chloroflexi supports this hypothesis. Hence, our main hypothesis about the predominance of the same four genera in both systems, relies on their capacity to undergo fermentative and hydrolytic metabolism. Further studies are needed to elucidate the function of these uncultured bacteria, which seem to have different responses to different conditions. On the other hand, STARs and WTARs showed different core microbiomes of Chloroflexi members. In STARs the Chloroflexi core was less diverse comprising only midas_g_467 genus (4 OTUs) while in WTARs it was composed by at least midas_g_156, midas_g_467, midas_g_2702, Leptolinea , and Bellilinea genus (18 OTUs). This result was in accordance with the differences observed for the Shannon diversity index. The existence of a core microbiome based on the type of reactor (STARs/WTARs) despite of the heterogeneity of wastewater/solids and environmental factors (e.g., pH, temperature) may suggest that this core could be functionally important in each system. Specific core of microorganism between STARS and WTARs has been detected previously ( Calusinska et al., 2018 ) but regarding Chloroflexi, no previous reports were found. A possible explanation for the different Chloroflexi cores found could be related to the biomass structure found in each type of system. Further exploration of this hypothesis is needed which would contribute to a better understanding of the role of Chloroflexi in the structure of granules and flocs and their metabolic role. Systematic studies examining multiple anaerobic reactor designs with greater depth of coverage and using platforms that generate long-reads or primer-free alternatives should help further define the “core microbiome” of Chloroflexi populations in methanogenic reactors. However, it is important to keep in mind that further recovery of isolated members or interpretative genomes is required to disclose the ecological importance of Anaerolineae as a core population in the anaerobic digestion process. Challenges and Limitations of Comparing Sequences Retrieved From Databases Generated With Different Primer Sets and Sequencing Platforms One of the bottlenecks in comparing microbial profiles is that different studies use different 16S rRNA gene regions generating an important bias. In the present study, we found an underestimation of Chloroflexi populations using primers targeting regions V1–V2 and V5–V9. An example was the underestimation of Anaerolineae class when V1–V2 regions were used recovering only a 5% of the total population. Meanwhile, primers targeting the V3–V5 region in general better reflected the abundances at all taxonomic levels. Previous studies investigating the influence of different hypervariable regions of 16S rRNA gene over the total community in activated sludge identified no perfect region concerning abundant functional genera, except for some regions with less bias, i.e., V1 and V2 ( Cai et al., 2013 ; Guo and Zhang, 2013 ). However, ( Albertsen et al., 2015 ) recommended primers targeting the V1–V3 regions which better reflected the abundances of Chloroflexi in activated sludge. More studies are needed to identify the best taxonomic profiling effectiveness between different hypervariable regions of the 16S rRNA gene over Chloroflexi populations in anaerobic reactors. As a general rule, drawing conclusions based only on one sequencing region should be avoided due to the potential false negative results, additionally amplicon sequencing of the 16S rRNA gene often limits resolution at genus level. We need primer-free alternatives to get the entire picture of the microbial diversity in anaerobic reactors ( Karst et al., 2018 ). Using the full-length sequence has the potential to become a tool for more precise microbial community profiling that better allows global comparisons of microbiome studies and should potentially increase the accuracy and the resolution of closely related taxa. We highlight the effort to build the MiDAS reference database for microbes in wastewater treatment systems since it allows comparing uncultured microorganisms in different studies ( Nierychlo et al., 2020 ). Regarding the bias of different sequencing platforms, alpha diversity is significantly affected by both sequence length and depth ( Singh et al., 2015 ). Platforms such as pyrosequencing which were discontinued, had less sequencing depth. To be able to compare samples with different sequencing depths, it is necessary to perform normalization at the lowest value of reads per sample. This leads to a loss of information in the less dominant groups which is an important limitation that has to be taken into account. This problem becomes relevant when comparing data from studies performed in the past (using outdated sequencing platforms) with data obtained more recently where the technology allows a much deeper sequencing. Moreover, it has been reported that Ion Torrent as well as Pyrosequencing platforms had comparatively higher error rates than Illumina platform ( Siqueira et al., 2012 ; Salipante et al., 2014 ). Although it has been reported that while primer choice considerably influences quantitative abundance estimations, the sequencing platform has relatively minor effects when matched primers are used ( Singh et al., 2015 ). Singh et al. (2015) reported that beta diversity metrics are surprisingly robust to both primer and sequencing platform biases. New developments in single-cell genomics and metagenomics have in recent years provided new insights into the ecology and evolution of many novel uncultured microorganisms ( Albertsen et al., 2013 ; Sekiguchi et al., 2015 ; Dam et al., 2020 ). The genomes have enabled the construction of metabolic models that attempt to explain the physiology of these organisms in detail. The genome-based models form the basis for more extensive investigations, such as in situ single-cell characterization, metatranscriptomics, and proteomics ( Koch et al., 2014 ). Importance of Standardized Metadata Repositories for Full Scale Methanogenic Reactors A second issue on performing a meta-analysis is the lack of standardized information on key operational parameters. It is important to have more reactor performance data associated with each reactor, such as compositions of influent waste/wastewater stream, COD loading/removal rate, pH, and temperature. These parameters should be fully used to infer potential roles of lineages of interest and should also be included. In our analysis it was not possible to correlate the microbial community with operational parameters data because in several articles this metadata was incomplete. For this reason, it would be essential to supply the operational parameters together with the raw data in each new study. In this sense, the creation of metadata repositories specific for anaerobic reactors including the 16S rRNA gene datasets must be a great advance. As far as we know there are some initiatives in other ecosystems as the Microbial Antarctic Resource Systems (MARS) 2 in Antarctic ecosystems."
} | 5,481 |
36133603 | PMC9418055 | pmc | 2,638 | {
"abstract": "A bio-based piezoelectric egg shell membrane (ESM) is used for energy harvesting applications in the form of two and three-component nanohybrids. A bio-waste piezo-filler in a piezoelectric polymer matrix was designed through an induced β-phase nucleation in the matrix using an organically modified two-dimensional nanoclay. Structural alteration (α to β-phase) in the presence of the nanoparticles was also manifested by morphological changes over spherulite to a needle-like morphology; thus, these nanohybrid materials are suitable for energy harvesting applications. ESM-based nanogenerators were fabricated with local ordering of piezo phases, as revealed via atomic force microscopy, leading to the generation of mostly electroactive phases in the whole nanohybrid. The voltage outputs from the optimized device were measured to be ∼56 and 144 V in single and multiple stacks (five), respectively, with corresponding power densities of 55 μW cm −2 and 100 μW cm −2 . The efficiency of the device was verified using a variety of body movements, e.g. bending, twisting, walking, and foot tapping, causing mechanical energy dissipation, which eventually transformed into energy storage. The underlying mechanism of high conversion of energy is explained by the synergistically induced piezo-phase in the polymer matrix together with the floppy piezo-filler. The mechanical stability, durability and repeated energy conversion of the hybrid device make it a robust nanogenerator. The biocompatibility of the nanogenerator was verified through cellular studies, demonstrating its appropriate use in powering biomedical devices/implants.",
"conclusion": "4. Conclusion This work demonstrates a nanohybrid nanogenerator based on abundantly available bio-waste egg shell membrane (ESM) as an efficient energy harvester. Two/three-component hybrids/nanohybrids have been synthesized by embedding nanoclay and a nanoclay-ESM combination in PVDF through a solution route. ESM does not alter the structure of PVDF, while nanoclay in the presence of ESM almost completely converts the matrix polymer into piezoelectric phase. The structural conversion was confirmed through XRD and FTIR studies and was also supported by morphological studies using optical and scanning electron microscopy. The local ordering of piezo-phase was revealed through AFM studies. Devices were fabricated using piezo-in-nonpiezo and piezo-in-piezo phases of hybrids/nanohybrids with various ESM contents. The optimized nanogenerator displays an open circuit voltage of 56 V and a maximum power density of 55 μW cm −2 under finger pressing; meanwhile, upon increasing the stack number (five), the output voltage improves to 144 V with a maximum power density of 100 μW cm −2 . The performance of the device was also shown to be efficient under different types of mechanical stress and human motions, such as bending, twisting, foot tapping, and walking, which indicates its sensitive nature towards minimal load application and significantly higher power output. The underlying mechanism of the energy storing capability of the nanogenerator has been established to be the very high piezoelectric phase induced by ESM and nanoclay. The nanogenerator has the potential to light LEDs through the finger tapping mode of stress application. The device can be installed under shoe soles, trade mills, floors and any other daily used devices to power up through human activities. The nanohybrid was found to be fully biocompatible, as evident from cellular studies (cell viability, cell adhesion and cell proliferation). Because the device is biocompatible, it has the potential to be used as an energy source for self-powered implants, e-health care monitoring and in vitro / in vivo diagnostics.",
"introduction": "1. Introduction With the decrease in available fuel and increase in environmental pollution from fossil fuel burning, it is urgent to find alternative green energy resources. 1–4 Various alternative sources of energy have been developed, and energy harvesting is one of the efficient alternatives to reuse it in other form. Energy harvesting from mechanical waste energy is quite effective because it is ecological, sustainable and readily accessible. 5,6 Mechanical energy from finger tapping, heel imparting, jogging, bending, joint movements, air flow, water flow, breathing and talking have been used for energy harvesting by several researchers. 7–11 Self-powered devices or nanogenerators harvest the mechanical energy from different sources. To date, many approaches have been proposed based on triboelectric 12 and piezoelectric 13,14 nanogenerators. Triboelectric nanogenerators have high energy conversion efficiency and high output voltage; 5,15 however, they possess drawbacks such as low durability and warping problems due to their large size. Due to these problems, researchers are currently focusing on piezoelectric nanogenerators with mechanical durability, high performance and better sensitivity. 6,7,16 Numerous materials have been used for piezoelectric nanogenerators, such as PZT, ZnO, BaTiO 3 , Na/KNbO 3 and ZnSnO 3 nanoparticles 17–20 as well as certain polymers, such as poly(vinylidene fluoride) (PVDF), 21 its copolymers with hexafluoropropylene [P(VDF-HFP)] 22 and trifluoroethylene [P(VDF-TrFE)], 23 and poly(vinyl acetate) (PVAc). 24 Many of these materials have limitations, such as brittleness, toxicity, non-biodegradability/non-biocompatibility and associated complex synthesis and fabrication processes. Although, polymers also have advantages, such as toughness, non-toxicity and biocompatibility; this benefits implants and biomedical health monitoring systems, which require devices to be biocompatible. Biocompatible self-powered piezoelectric nanogenerators focus on common human body motions, particularly, in in vivo conditions, for health monitoring and safety purposes. 25,26 Naturally abundant piezoelectric materials are suitable for the design of bio-medical nanogenerators or sensors, which can operate without any adverse effects on living systems. Natural piezoelectric materials such as hydroxyapatite, 27 collagen fibrils, 28,29 cellulose 30 and chitin 31 are suitable choices for designing biocompatible piezoelectric nanogenerators. Bio-based piezoelectric materials are also used as energy harvesting materials. These materials have advantages such as non-toxicity and biocompatibility along with environmentally friendly behavior. A virus-based piezoelectric nanogenerator was fabricated; however, it showed a very low power density. 26 The power generated using prawn shell 31 and fish scale 32 as bio-waste was found to be 0.76 and 1.14 μW cm −2 , respectively, while bio-waste onion skin has been reported for electricity generation with a meager power density of 1.7 μW cm −2 . 33 Here, we used an egg shell membrane (ESM) as a piezoelectric filler for preparing nanohybrids of PVDF and two-dimensional layered silicate (nanoclay) for energy harvesting. ESM also has many advantages: (i) it is inexpensive and readily available for use as industrial and household waste; (ii) it is non-toxic and environmentally friendly; (iii) it is composed of many proteins and amino acids having functional groups present on their surfaces, resulting in better functionalization capability; and (iv) it has weak chemical bonds, so it can be readily modified using carbonization and dissolution. Due to these advantages, ESM has been used in many fields, such as chemical, electrical, environmental and biomedical engineering. 34 By adding ESM as a second filler in the nanohybrid, its energy harvesting capability was enhanced significantly along with its biocompatible nature, making it suitable for powering biomedical devices, including implants.",
"discussion": "3. Results and discussion 3.1. Induced structure and morphology The X-ray diffraction (XRD) patterns of pure PVDF and its nanohybrids with ESM (40 wt%) and nanoclay are shown in Fig. 1a . Pure α-phase peaks appear at the 17.6° (100), 18.3° (020) and 19.9° (110) planes in pure PVDF, 22,36,37 and similar peak positions were observed in the P-ESM hybrid. Moreover, the crystalline β-phase peak at ∼20.5° (corresponding to the 200/110 planes) was noted in the spectrum of the PC-ESM nanohybrid. There is a clear phase change from α to β in the presence of nanoclay (nanoclay-induced phase transformation), arising from the intimate interactions between the nanoclay and the PVDF chains, 11,38 while ESM alone was unable to induce β-phase in the polymer. It should be mentioned that ESM shows ∼40% crystalline phase (ESI Fig. S1 † ) and is known to be piezoelectric. 39 The amounts of piezoelectric β-phase in the hybrids were calculated through the deconvolution of the XRD peaks ( Fig. 1b ), and the percentage β-phase fractions of various nanohybrids as a function of the ESM content are shown in Fig. 1c . PC shows a minimum piezoelectric β-phase (32%); meanwhile, a similar quantity of nanoclay induced large amounts of β-phase in the three-component nanohybrids (82% β-phase using 40% ESM), with a gradual enhancement in the piezoelectric phase with ESM content. It is worth mentioning that ESM alone cannot induce piezoelectricity in the PVDF matrix, while in the presence of 2-D nanoclay, high piezoelectricity in induced in the matrix polymer. The gradual change in the structure of the nanohybrids is presented in ESI Fig. S2a and b. † The structural alterations were also verified using FTIR spectroscopy with the presence of peaks at 836, 880 and 1167 cm −1 , assigned to β-phase, in the nanohybrids (PC, PC-ESM) against the pure α-peaks at 761, 797, 869, 974 and 1146 cm −1 in PVDF and P-ESM ( Fig. 1d ). 11,40 It is interesting to note that the α-phase peak intensity of pure PVDF considerably decreased in P-ESM, although there was no β-phase peak; this indicates decreased crystallinity in P-ESM, presumably due to the greater interaction between PVDF and ESM. The interaction was further visualized from the presence of carbonyl, amide and –OH groups in ESM (ESI Fig. S3 † ), which is responsible for greater interactions through hydrogen bonding and dipole–dipole interactions. However, the presence of ESM enhances the piezoelectric β-phase significantly in the three-component nanohybrids, and therefore is suitable for energy harvesting applications. Fig. 1 (a) XRD patterns of pure PVDF, P-ESM40 and PC-ESM40 nanohybrids, showing the changes in their structure; (b) deconvolution of the XRD pattern of PC-ESM40 for the calculation of the phase fraction; (c) plot of β-phase fraction with ESM content, showing higher β-phase in PC-ESM. The dashed line indicates β-phase for PC and the vertical arrow shows the increase in the piezo-phase content. (d) FTIR spectra of pure PVDF, P-ESM40 and PC-ESM40 nanohybrids indicating the peak positions of the different phases. Structural changes are often associated with the transformation in surface morphology. The polarized optical microscope images of pure PVDF and the nanohybrids are shown in Fig. 2a . Pure PVDF shows a spherulitic pattern, indicative of α-phase, with an average diameter of 200 μm; meanwhile, tiny spherulite (50 μm) along with a fibrous morphology is observed in P-ESM in the presence of 40 wt% ESM. In contrast, a mesh-like morphology is evident in PC-ESM, which is a clear indication of β-phase in the presence of nanoclay and ESM. It should be mentioned that a fibrous morphology in observed in pure ESM (ESI Fig. S4 † ). A similar change of morphology was also observed through SEM; large and small spherulites are evident in P and P-ESM, respectively, while the needle-like β-phase morphology is obvious in PC-ESM in the presence of nanoclay and ESM ( Fig. 2b ). However, the fibrous morphology of ESM along with β-nucleating 2-D layered silicate aids the growth of piezoelectric β-phase in large quantities. The enhancement of the piezoelectric phase in the three-component nanohybrids was also verified through piezo force microscopy. The changes in the piezo-response and phases indicate the presence of varying quantities of piezo phase domains upon the application of a DC voltage of 10 V ( Fig. 2c ). The phase angle corresponding to the line profiles of three different specimens clearly demonstrates significant changes in the phase behavior of PC-ESM compared to that of pure PVDF or the P-ESM hybrid ( Fig. 2d ). The changes in other piezo-responses are presented in ESI Fig. S5. † It is worth mentioning that the application of potential causes the development of strain in the piezo specimens (because PVDF and ESM both have negative piezoelectric coefficients), as is evident from the considerable differences in the profiles before and after applying potential to the samples (ESI Fig. S6 † ). However, a larger piezo domain is evident in PC-ESM vis-à-vis pure PVDF or the P-ESM hybrid. In this juncture, it should be mentioned that ESM (piezo filler) distributed in the PVDF matrix cannot induce piezo phase in polymer matrix; meanwhile, it induces a large quantity of piezo phase in association with another 2-D nanoclay and converts most of the matrix into piezo phase, as shown in the cartoon in Fig. 2e , commensurate with the XRD and FTIR results; this leads to the development of two types of piezoelectric materials, with piezo filler in a non-piezo matrix (P-ESM) and piezo filler in a piezo matrix (PC-ESM), which are expected to exhibit very different energy harvesting behaviors. Fig. 2 (a) Polarized optical images of the indicated specimens showing spherulite in P and P-ESM and the absence of spherulite in PC-ESM. (b) Scanning electron microscope images of pure PVDF and the P-ESM and PC-ESM nanohybrids. (c) Piezo force microscopic images of pure PVDF and the P-ESM40 and PC-ESM40 nanohybrids showing better phase development in PC-ESM. (d) Phase profiles of the indicated specimens obtained from the PFM images. (e) Cartoon showing the dispersion of piezoelectric filler (grey) in the non-piezo (green) and induced piezo matrix (orange) in the absence and presence of nanoclay, respectively. 3.2. Energy harvesting using nanohybrids Because the nanohybrids exhibits very high piezoelectric phase, they should demonstrate energy harvesting capability. Nanogenerators were fabricated for energy harvesting using a suitable layer-by-layer assembly of an active piezoelectric material electrode and a stable coating, as shown in Fig. 3a . A rectangular-shaped electroactive hybrid material electrode was attached with aluminum foil on both sides, followed by wrapping of the whole device with poly(dimethyl siloxane). Fig. 3b shows the open circuit voltage (OCV) arising from the devices made of pure PVDF and the nanohybrids with ESM and nanoclay plus ESM under finger tapping with a frequency of ∼5 Hz. The three-component nanohybrid (PC-ESM) exhibits the maximum peak-to-peak open circuit voltage of 56 V compared to the meager 4 and 33 V shown by the devices using pure PVDF and P-ESM, respectively. The estimated force from finger tapping is calculated to be around 40 kPa (details are given in the ESI, Note S1 † ). The device containing the two-component hybrid (P-ESM) shows strong ESM content variation under similar strain application using finger tapping ( Fig. 3c ). The OCV increased systematically with ESM content and reached 35 V using 40 wt% ESM. Interestingly, the three-component nanohybrids (PC-ESM) displayed higher OCVs compared to the two-component hybrid (P-ESM) with similar ESM loading; 56 V was obtained using 40 wt% ESM ( Fig. 3d ), presumably due to the higher amount of piezoelectric β-phase in the nanohybrid vis-à-vis P-ESM. Fig. 3 (a) Layer assembly steps for the fabrication of the nanogenerator devices using pure PVDF and the nanohybrids; (b) open circuit voltage from the devices using pure PVDF, P-ESM and PC-ESM nanohybrids with 40 wt% ESM. (c) Output OCVs from the devices fabricated using the indicated ESM contents in P-ESM. (d) Output OCVs from the devices fabricated using the indicated ESM contents in PC-ESM. (e) Variations in power density with resistance in devices fabricated with pure PVDF, P-ESM and PC-ESM nanohybrids with 40 wt% ESM content. (f) Power density variations with ESM content from the devices made with P-ESM and PC-ESM nanohybrids. The dashed line indicates the power output value of the device made with PC (PVDF and nanoclay). (g) Open circuit voltages from the devices fabricated with the PC-ESM (40 wt% ESM content) nanogenerator with increasing number of stacks as indicated. (h) Voltage output from the indicated unimorphs using PVDF and its nanohybrids. (i) The development of charges under two different types of mechanical force (applied and retractive force) leading to the formation of an alternating voltage – working mechanism for charge generation upon application of stress. (j) Schematics showing local ordering in the different nanohybrids, demonstrating the induced structure in the presence of ESM alone and the combined effects of ESM and nanoclay. (k) Power output from the device as a function of ESM content in the PC-ESM nanohybrid, demonstrating the synergism of nanoclay and ESM, which exhibits significantly high output. Now, it is pertinent to quantify the performance of the nanogenerator on the basis of output power density. The output power is calculated by measuring the voltage at different resistances because the power depends on the external load applied on the system. The open circuit voltage increases initially with increasing resistance and then saturates (ESI Fig. S7 † ). The power density is calculated using the equation: where V is the voltage across the resistance R and A is the area of the active device. Interestingly, the maximum power density of the PC-ESM40 nanohybrid is 55 μW cm −2 ( Fig. 3e ), which is the highest power output value obtained using bio-waste material for energy harvesting to date. As per our knowledge, the values of output voltage and power from different bio-based nanogenerators are presented in ESI Table S1 † and compared with this work; PC-ESM definitely exhibits the highest power output to date. Fig. 3f shows the enhancement in power density from the devices using both the nanohybrids with increasing ESM content, exhibiting systematic improvement of the power density; meanwhile, the PC-ESM devices show significantly higher power output vis-à-vis P-ESM, mainly because of the higher piezoelectric content (β-phase) in the presence of both ESM and nanoclay. The increase in power density of the nanohybrids is significant compared to the PVDF-nanoclay composite alone (indicated by the dashed line in Fig. 3f ). To check the commercial viability of the device, multiple devices (2, 3 and 5 units) were assembled in series, and their corresponding output voltages are presented in Fig. 3g under similar finger tapping as before; a gradual increase of output voltage can be observed with increasing number of stacks, with very high (144 V) output and 100 μW cm −2 for 5 units of the device stacked in series. Thus, the output voltage of the device can be amplified to the desired value by assembling multiple devices and it is thus more feasible for use in large scale industrial applications. Fig. 3h shows the signals (output voltage) from different nanogenerator unimorphs under a single compression and releasing cycle. The starting and end points of the voltage generation are indicated by down arrows, suggesting a high response time (100 ms) for the device made with PC-ESM against 30 ms for PC. It should be mentioned that the response time indicates the duration of power generation from the device under mechanical stress. Another intriguing feature of the nanohybrid device is that there is a considerable time gap between the compress and release modes of power generation which is higher for PC-ESM than for P-ESM; this phenomenon is not present for PC. The working principle of bio-based piezoelectric materials is still under investigation because their unusual nature cannot be explained by classical piezoelectricity theory, which is based on the ideal/perfect crystalline structure. 41 The possible working mechanism of the voltage generation is explained in Fig. 3i by considering the physical changes (rotation of dipole) upon application of mechanical stress in the compression and release modes. Due to the floppy nature of ESM, as evident from its SEM image, the PC-ESM and P-ESM hybrids are slow relaxation systems; thus, they require relatively longer times to rotate dipoles under the two modes of force (compression and release) compared to the compact system in PC, where no noticeable time lag is observed between the two modes. In PVDF and the nanoclay hybrid (PC), the piezoelectricity is explained through epitaxial crystallization of the PVDF on the surface of the 2-D nanoclay, which leads to the transformation of its α-phase into electroactive β-phase; the extent of conversion dictates the piezoelectric phase. ESM has a porous structure, so it is soft and flexible; this eventually leads to more displacement than the normal compact structure experiences under a fixed load, giving rise to high piezoelectricity in ESM. 4 ESM contains collagens type I, V, X and different proteins, such as osteopontin, keratin, proteoglycans and glycoproteins. The piezoelectricity in ESM is due to the combined effects of the collagen and the proteins. The polarization and piezoelectricity in type I collagen fibrils has been established to be due to the existence of N and C terminal telopeptides and C6 symmetry in the crystalline chains. 29 Collagen fibers and different proteins are present in ESM. External compressive stress on the oriented collagen structure induces high internal friction among the hydrogen-bonded α-helices. 42 Therefore, the deformation of the triple helical structure aids the creation of dipole moments in ESM under stress. Similar mechanisms have been observed in M13 bacteriophage, 26 fish scale 34 and fish swimming bladder. 43 ESM contains many collagen micro-fibrils which generate electric dipole moments upon the application of mechanical stress. 42 Hence, under applied stress, charges develop on the top and bottom of ESM due to breaking of the symmetry present in the collagen moieties. 29,44 The addition of ESM to PVDF or to the PVDF-nanoclay matrix converts the system into piezo phase in the non-piezo matrix and piezo phase in the piezo matrix, respectively. The enhancement of the piezoelectric response in the nanohybrids is directly related to the mutual electromechanical interactions among the fibers (needle-like β-phase in the matrix and ESM fibrils) under external applied stress. 29,44 There are different types of proteins in ESM; hence, there is a possibility of interconnections between them, either through hydrogen bonding or van der Waals interactions, which disrupt under mechanical stress, leading to enhanced piezoelectricity in the nanohybrids. In addition to this, the porosity of ESM plays an important role because a porous structure experiences more displacement than a compact structure under similar applied stress. 4,45 However, the potential difference, caused by the orientation of the electric dipole, results in electron flow from one direction to another; meanwhile, the electrons flow in the reverse direction upon release of the stress, causing alternating current in the circuit. 46 Based on the varying nature of the interactions in various hybrids/nanohybrids, the location and domain sizes are presented in the form of a cartoon in Fig. 3j . Pure PVDF crystallizes only in α-phase and does not show any piezoelectricity; meanwhile, nanoclay induces β-phase in PVDF (PC), whose overall crystallinity is lower, and thereby exhibits meager piezoelectricity. The introduction of electroactive ESM in pure PVDF (P-ESM; piezo phase in a non-piezo matrix) shows an α-phase PVDF matrix where piezo filler is dispersed; this system exhibits moderately high piezoelectricity in the hybrids with higher ESM content. On the other hand, good interactive three-component systems (PC-ESM) induce β-phase in the PVDF matrix along with piezo filler; this creates a near-ideal system where the whole materials exhibit piezo phase (except for a small amount of amorphous phase present in the system) showing layered orientation of three different components, namely β-phase on the surface of the 2-D nanoclay along with a layer of ESM. Currently, it is apparent that ESM increases the piezoelectricity to a limited extent only, while nanoclay and ESM enhance the piezoelectricity and subsequent power generation to a much greater value; this raises the possibility of synergism. The output power from the devices has been plotted as a function of ESM content in PC-ESM, showing strong synergism (significantly above the values predicted by the linear mixture rule, as shown by the dotted line in the figure) ( Fig. 3k ). This synergism is insignificant in P-ESM, presumably due to less interaction between PVDF and ESM (ESI Fig. S8 † ). However, a very high output voltage of 56 V was achieved from the nanohybrid, with a maximum power density of 55 μW cm −2 from a single piezoelectric device which increased to 144 V of OCV and 100 μW cm −2 from a stack containing 5 units of the device. 3.3. Practical applications of nanogenerators In the preceding section, very high power generation is demonstrated using the three-component nanohybrid by applying the finger tapping method. The harvesting ability of the devices with different human activities as waste mechanical energy sources can also be verified to understand the efficacy of the energy harvesting. Fig. 4a shows the voltage output upon twisting the nanogenerator (PC-ESM), which produces a peak-to-peak output voltage of ∼4 V. The inset images show the force application mode and zooming of the output voltage. On bending ( Fig. 4b ) and coin dropping ( Fig. 4c ) (INR 5-rupee coin), the device produces ∼5.6 and 8 V, respectively; although the weight of the coin is lower, the impact of the freefalling coin is greater, which justifies the higher output. For more realistic application, the output voltage from the nanogenerator on walking is shown in Fig. 4d as ∼9.5 V; on foot tapping, the output voltage was measured to be ∼10 V ( Fig. 4e ), which can also light up LEDs. The hand slapping method was found to be more effective, with an output voltage of ∼50 V; this is significantly higher than the other modes, primarily due to the greater impact and higher contact area ( Fig. 4f ). However, various body motions could also generate significant voltage for harvesting energy. The respective power densities of these human activities have also been calculated and are presented in ESI Fig. S9; † the corresponding videos are shown in ESI Video S1. † The mechanical stability and durability tests of the device are shown in Fig. 4g , which clearly demonstrates good performance of the device even after repeated use. Now, it is pertinent to understand whether the device is able to store charge in a capacitor. The device was attached to a capacitor (1 μF) through a rectifier circuit and also connected to a digital oscilloscope to read the corresponding charging and discharging voltages from the device. The hand tapping method clearly demonstrates charging of the capacitor and reaches a plateau, followed by discharging under a suitable external load ( Fig. 4h ). The response times for the charging and discharging phenomena were found to be 30 and 35 s, respectively. Repeated charging and discharging of the capacitor is shown in ESI Fig. S10. † However, the nanogenerator made with bio-waste and nanoparticles embedded in PVDF could produce sufficient energy to be stored and utilized later, demonstrating the efficacy of the bio-inspired device for energy harvesting purposes. Using the nanogenerator, LEDs could be lit by waste mechanical stress. The video is shown in ESI Video S2. † From these results, it is clear that our bio-based piezoelectric nanogenerator is efficient for realistic applications. Fig. 4 Demonstration of practical applications of the nanogenerator to harvest energy by applying normal human movements, e.g. (a) twisting; (b) bending; (c) coin dropping; (d) walking; (e) foot tapping; and (f) hand slapping. (g) Mechanical and durability tests of the device after obtaining the device performance for a sufficiently long time, showing very similar output voltages; and (h) the ability of the power output from the device made with PC-ESM40 to charge a capacitor, followed by the discharging kinetics. 3.4. Biocompatibility study Cytocompatibility is one of the essential parameters for the biomedical application of any material. The cytocompatibility of a material is quantified in terms of cell viability, and the MTT assay is performed to check the viable cells in the presence of different samples. Yellow tetrazolium salt is converted to red formazan derivatives by the mitochondria of viable cells through intercellular reduction in the MTT assay. The cell viability of HeLa cells in a 96-well culture plate without any material is taken as a control, and the cell viability in the presence of a sample is expressed with respect to the control. Fig. 5a shows that the cell viabilities of pure PVDF, ESM, P-ESM40 and PC-ESM40 are ∼100%, and no significant changes in cell viability were observed after 48 and 72 hours. This result suggests that the developed materials help the cells to proliferate and are cytocompatible in nature. It should be mentioned that the cell viability of PC-ESM40 after 72 hours is greater than those of ESM and P-ESM40. Further, the cell adhesion over the surface of the samples was studied in terms of morphological investigation of the cells. Cell adhesion is the preliminary step which decides the subsequent steps, such as cell proliferation. The phase contrast images ( Fig. 5b ) show an almost spread morphology of the cells on the surface of the samples; this indicates that the cells are nicely adhered to the samples, which helps the cells to proliferate further with time, as observed in the cytotoxicity measurements. The viability of HeLa cells was further confirmed through fluorescence imaging of the HeLa cells in the presence of pure PVDF, ESM, P-ESM40 and PC-ESM40 ( Fig. 5c ). 47 A combination of acridine orange (AO) and ethidium bromide (EtBr) was used to stain the cells. The acridine orange binds with DNA to show green fluorescence, while the ethidium bromide shows red fluorescence. Live cells only show AO fluorescence, while apoptotic cells show both AO and EtBr fluorescence. Fig. 5c shows the number densities of HeLa cells, which are almost equal for all the samples at a particular time of cell proliferation, including the control; the cell density increases gradually with time. Hence, the developed nanohybrid materials help the cells to proliferate; thus, these materials can safely be used for biomedical applications, especially for implant devices which can generate energy utilizing waste biomechanical performance. Fig. 5 (a) Cell viability as a function of time comparing pure PVDF and its hybrids; (b) phase contrast images of cells adhered on the indicated samples; and (c) fluorescence images of pure PVDF and its hybrid/nanohybrids showing their biocompatibility through cell proliferation."
} | 7,864 |
39511174 | PMC11543930 | pmc | 2,641 | {
"abstract": "Microbial formation and oxidation of volatile alkanes in anoxic environments significantly impacts biogeochemical cycles on Earth. The discovery of archaea oxidizing volatile alkanes via deeply branching methyl-coenzyme M reductase variants, dubbed alkyl-CoM reductases (ACR), prompted the hypothesis of archaea-catalysed alkane formation in nature (alkanogenesis). A combination of metabolic modelling, anaerobic physiology assays, and isotope labeling of Candidatus Syntrophoarchaeum archaea catalyzing the anaerobic oxidation of butane (AOB) show a back flux of CO 2 to butane, demonstrating reversibility of the entire AOB pathway. Back fluxes correlate with thermodynamics and kinetics of the archaeal catabolic system. AOB reversibility supports a biological formation of butane, and generally of higher volatile alkanes, helping to explain the presence of isotopically light alkanes and deeply branching ACR genes in sedimentary basins isolated from gas reservoirs.",
"introduction": "Introduction Biological formation and oxidation of volatile alkanes, particularly methane, have a major impact on biogeochemical cycles on Earth. Carried out by methanogenic archaea, methanogenesis is one of the major processes of biomass degradation in deep, anoxic sediment horizons, generating vast amounts of methane, a potent greenhouse gas 1 . Most of the formed methane, along with methane released from thermogenic reservoirs, is oxidized already in anoxic sediments. The anaerobic oxidation of methane (AOM) is carried out by anaerobic methanotrophic archaea (ANME) essentially via the reverse methanogenesis pathway 2 . The key enzyme of both pathways is methyl-coenzyme M reductase (MCR), which catalyzes both the release of methane in methanogenesis, and the initial step of methane oxidation in AOM 3 . The pathways of methanogenesis and AOM operate close to thermodynamic equilibrium, and are fully reversible, with demonstrated back fluxes of reaction products into the substrate pools 2 , 4 , 5 . Recent findings showed that ANME-related archaea can oxidize higher, C 2+ alkanes using biochemical mechanisms similar to those driving the AOM 6 – 10 . The anaerobic oxidation of C 2+ alkanes (AOAlk) is apparently initiated by deeply branching MCR variants, dubbed alkyl-coenzyme M reductases (ACR) 6 , 8 , 11 . ACRs are structurally similar to MCR, and similar enzymatic mechanisms are expected. Notable differences include amino acids substitutions leading to wider catalytic chambers suited to accommodate substrates bulkier than methane 3 , 12 , 13 . A potential reversibility of AOAlk, suggested by enzyme similarities and shared pathway modules with AOM 6 , is supported by the unspecific conversion of ethyl-CoM to ethane by MCRs of methanogens, and by back flux studies with an archaea-SRB consortium catalyzing the anaerobic oxidation of ethane (AOE) 7 , 14 . Here we show that the entire archaeal pathway for the anaerobic oxidation of butane (AOB) is reversible by measuring a steady back flux of carbon from the CO 2 to the butane pools during net AOB. Experiments were done with a thermophilic AOB enrichment culture, culture Butane50, obtained from Guaymas Basin hydrothermal vent sediments 8 . This culture consists of two archaeal species, Candidatus Syntrophoarchaeum butanivorans and Ca . S. caldarius, which form tightly-packed aggregates with partner sulfate-reducing bacteria (SRB) 8 . Oxidation of butane was assumed to be carried out by the more abundant Ca . S. butanivorans, while the role of Ca . S. caldarius was unclear. The partner SRB were apparently scavenging reducing equivalents generated during butane oxidation, which are used to reduce sulfate to sulfide. The net reaction resembles AOB by anaerobic bacteria which couple butane oxidation to CO 2 and sulfate reduction to sulfide in the same cell, with similar net stoichiometries and energy yields 15 , 16 .",
"discussion": "Discussion The back flux assay results allow two main observations. First, the extent of the AOB back reaction in Ca . Syntrophoarchaeum cultures was low. Lower back fluxes are expected for the anaerobic oxidation of higher alkanes, as the oxidation reaction becomes more exergonic with increasing alkane chain length (Supplementary Note 7 and Supplementary Tables 4 and 5 ). We consider that back fluxes as measured here are likely to have a minor impact on isotopic signatures of butane in gas reservoir samples. Second, Ca . Syntrophoarchaeum cultures apparently do not catalyze an isotope exchange between CO 2 and butane in the absence of net AOB, unlike AOM, and potentially AOE, where forward and back fluxes continue even in the absence of net oxidation 7 , 24 . We reasoned that the observed back flux dynamics displayed by Ca . Syntrophoarchaeum reflected integrated outcomes of energetics and kinetics effects underlying the AOB pathway. According to the flux force theorem 25 , 26 , the shift of AOB free energy towards more positive values (shift trend towards 0) predicts a monotonic increase of AOB back flux (Eqs. 19 and 20 ), but does not explain the observed sharp decrease of the back flux in the later experimental stages (Fig. 4 ). The latter could be likely explained by kinetic bottlenecks along the pathway, governed by shrinking pools of pathway intermediates and of reducing equivalents, including those mediating the transfer of electrons to the partner SRB. When cultures are under sulfate or butane limitation (for example, at late growth stages when most butane has been oxidized), less energy is available to drive the endergonic AOB reactions, leading to reduced cell capacity to replenish intermediate pools and to regenerate XH 2 . The resulting intermediates or XH 2 shortages limits enzyme kinetics, lowering the magnitude of AOB back fluxes. Particularly affected could be the energy coupled reactions like the conversion of butyl-CoM to butyryl-CoA. Overall, our results show that the entire AOB pathway is essentially reversible, suggesting that given a supply of electron donors with low redox potential 27 archaea could catalyze the formation of higher alkanes via ACR-based pathways. In an attempt to identify or emulate the potential electron donor, we supplied sulfate-free cultures of Ca . Syntrophoarchaeum with H 2 , sulfide, zero-valent sulfur compounds, and reduced artificial electron carriers (AQDS). These failed to stimulate a reverse AOB pathway, leaving open the question of the identity of XH 2 . Similarly, the nature of X could not be resolved by metagenomic analyses (Supplementary Note 1 ). Generally, the archaeal AOAlk pathways are considered to have evolved by metabolic modular extension of AOM, and evolutionary adaptations of MCRs to accommodate substrates bulkier than methane 6 , 12 . All known constituent metabolic modules, including the Wolfe, Wood-Ljungdahl, and beta-oxidation pathways, are known to be reversible 28 , 29 . The reactions converting acyl-CoA to alkyl-CoM are presently unknown (Supplementary Note 2 and Supplementary Data 1 - 5 ). In a hypothetical alkanogenesis scenario, one of the critical steps would be the release of alkane from alkyl-CoM. For this we calculated Δ G 0′ ranging from –19 ± 10 kJ mol −1 (ethyl-CoM → ethane) to −16.9 ± 10 kJ mol −1 (butyl-CoM → butane), comparable with the release of methane from methyl-CoM (−30 ± 10 kJ mol −1 , Supplementary Note 8 ). Like in methanogenesis, energy conservation during the hypothetical alkanogenesis could be for example coupled to CoM alkylation and cycling of the CoM-S-S-CoB heterodisulfide 30 . An ACR-dependent alkanogenesis offers an alternative microbiological and mechanistic explanation to the proposed biological origin of C 2+ volatile alkanes. Ethane and propane depleted in 13 C relative to thermogenic gasses have been frequently detected in marine gas hydrates, sedimentary basins, and deep marine sediments, often in deeply buried sediments isolated from gas reservoirs 31 – 35 . In addition, formation of ethane has been observed in sediment slurries, with experimental evidence suggesting an involvement of archaea 35 . To date, the involved microorganisms and underlying biochemical mechanisms have not been identified. Here we propose that such alkanes are formed by archaea harboring ACR-dependent pathways. Moreover, our results expand the range of C 2+ alkanes that could be biologically formed to include butane. Butane is less abundant in biogenic gas samples, and reports of its isotopic composition are scarce 36 . To test if Ca . Syntrophoarchaeum and other alkane-oxidizing archaea (ALOX) 37 occur in environments harboring traces of volatile alkanes or 13 C-depleted alkanes, we performed a global taxonomy survey of ACR-encoding archaea and of AcrA sequences. We retrieved Ca . Syntrophoarchaeum primarily from hot springs, cold seeps, and hydrothermal vents, while other ACR-encoding genera, like Bathyarchaeota 38 , are apparently found in a broader range of aquatic and sedimentary ecosystems (Fig. 6 ). The environmental distribution of AcrA fragments followed closely the biogeographic patterns of ACR-encoding genera (Fig. 6 ). Although in most cases the presence of ALOX could not be directly correlated with alkane geochemistry, environments like hot springs apparently host both ACR genes (Fig. 6 ) 11 , 39 and traces of C 2+ alkanes including butane 40 . Such energy-limited chemolithotrophic geothermal settings may be hot spots of both ALOX archaea and of genuine alkanogenic archaea in nature. Fig. 6 Global biogeography of ACR-encoding archaea and of AcrA sequences. A Maximum likelihood tree of AcrA protein sequences. AcrA’s from experimentally validated anaerobic multicarbon alkane oxidizing archaea are highlighted with red squares. AcrA’s from Ca . Syntrophoarchaeum and other ACR-encoding genera are marked with red and cyan squares. The transparent circles reflect the number of AcrA fragments from IMG/M database that are placed to the corresponding branches by RAxML EPA. Clades of methanogenic archaea are collapsed for visualization purposes. Scale bar = substitutions per site. B Boxplot of relative abundance of Ca . Syntrophoarchaeum (red) and other ACR-encoding genera (cyan) shown in the AcrA phylogeny. C Biogeographic distribution of Ca . Syntrophoarchaeum (circles) and other ACR-encoding genera (triangles) across diverse biomes. D Biogeographic distribution of AcrA sequences retrieved from environmental samples. Sequences were retrieved from assembled metagenomes in the JGI IMG/M database. The phylogenetic placement of fragments is shown in A . The fragments that are annotated with C-terminus (PF02249) and N-terminus (PF02745) domains of McrA are shown with circles and triangles, respectively. Source data are provided as a Source Data file."
} | 2,699 |
36135659 | PMC9504979 | pmc | 2,642 | {
"abstract": "The mutualistic interactions between mycorrhizae and plants first occurred along with the terrestrialization of plants. The majority of vascular plants are in symbiosis with mycorrhizal fungi. Due to their importance to the economy and ecology, arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi emerge as the most popular ones. However, the mechanism underlying the beneficial function of ECM fungi is not as clear as AM fungi. Here, the interaction between Parametarhizium hingganense , a novel fungal species isolated from forest litter, and mung bean ( Vigna radiata ) was studied. P. hingganense demonstrated P solubilization ability in vitro. Treatment of P. hingganense on the seeds resulted in promoted growth with enhanced P content. The hyphae of green fluorescence protein (GFP)-tagged P. hingganense were found to surround the roots and develop between cells, suggesting the establishment of an ectomycorrhizal symbiosis. Upon symbiosis with P. hingganense , the levels of jasmonic acid (JA) and gibberellin (GA 1 ) and total phenolic and flavonoid content elevated. Meanwhile, damping off caused by Rhizoctonia solani in mycorrhizal plants was alleviated. Taken together, the above findings suggested that symbiosis with P. hingganense conferred growth promotion and priming of defense responses to host plants which should be associated with facilitated P uptake and increased JA and GA 1 levels.",
"conclusion": "5. Conclusions Here, we presented that P. hingganense , a new fungal species of Parametarhizum from forest litter, established ectomycorrhizal symbiosis with the crop (mung bean). Promoted growth with elevated Pi occurred in mycorrhizal plants, and enhanced JA and GA 1 were detected in mycorrhizal roots. Although defense hormones, such as SA and ABA, were repressed, total phenolic and flavonoid content increased due to P. hingganense colonization. The improved tolerance of mung bean plants to disease caused by R. solani further supported the supposition that induced defense responses were activated upon symbiosis with P. hingganense . The underlying mechanism of ECM fungi-induced defense response has not been elucidated yet. Most of the evidence was gathered from the interaction between ECM fungi and host trees. Therefore, our findings demonstrated an alternative strategy for investigation of the beneficial functions of ECM fungi. On the other hand, the beneficial effects of P. hingganense on the host plant provide a culturable fungal strain to use as a potential biofertilizer and biocontrol agent.",
"introduction": "1. Introduction Mutualistic symbiosis with mycorrhizal fungi occurred along with plant terrestrialization [ 1 ]. There are four major types of mycorrhizal association: arbuscular mycorrhizal (AM), ectomycorrhizal (ECM), ericoid mycorrhizal, and orchid mycorrhizal [ 2 ]. Most of the vascular plants are in symbioses with AM fungi (72%), and only 2%, 1.5%, and 10% are associated with ECM, ericoid mycorrhizal, and orchid mycorrhizal fungi, respectively [ 3 ]. Amongst mycorrhiza-forming fungi, AM and ECM are intensively studied. The hyphae of both AM and ECM fungi colonize the surface of the root [ 2 ]. However, inside the plant tissues, AM fungal hyphae penetrate into the living cells and form highly branched arbuscules, while ECM fungal hyphae develop only between epidermal cells [ 2 ]. Fossil records reveal that associations of AM fungi with plants are ancient events that can be traced back to at least 450 million years ago with a single origin, while ECM fungi associations occurred in the Cretaceous and evolved independently with multiple origins [ 3 ]. Growing evidence shows that mycorrhizal symbiosis improves the performance of host plants, mainly through growth promotion and elevated plant resistance to biotic and abiotic stresses [ 2 , 4 , 5 ]. First, both AM and ECM fungi can deliver nutrients to the host plants, particularly P which is one of the least mobile plant macronutrients in soil [ 6 , 7 ]. Generally, the availability of P in soil is extremely low [ 6 ]. In AM fungi, high-affinity inorganic phosphate (Pi) transporters locate on the extraradical hyphae and in the colonized cortical cells, which function in the uptake of Pi from soil and translocation into the host plants [ 2 , 5 ]. In contrast, only a few Pi transporters are characterized in ECM fungi [ 8 ]. However, ECM fungi influence Pi uptake through enzyme production and organic acid excretion, facilitating the utilization of insoluble Pi and organic phosphate, which are not preferred by AM fungi [ 6 , 7 ]. Second, upon mycorrhizal symbiosis, endogenous levels of phytohormones are affected, including growth hormones (auxin and gibberellins (GAs)) and defense hormones (jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA)). Auxin appears to function throughout the lifespan of plants, from embryogenesis to senescence [ 9 ]. GAs are essential to plant growth and development, including seed germination, stem elongation, leaf expansion, flower initiation, and flower and fruit development [ 10 ]. Furthermore, sufficient GA signaling positively regulates AM fungal hyphal branching and facilitates AM fungi colonization on the host plant [ 11 ]. DELLA proteins play central roles during AM symbiosis development and in the cross-talk among GA, ABA, and Indole-3-acetic acid (IAA) signaling pathways [ 12 ]. Despite the function of SA and JA signaling in mycorrhiza development being ambiguous, available evidence shows that both AM and ECM fungi primed defense responses to stresses are associated with the activation of SA and JA signaling pathways, although most of the findings are generated from AM symbiosis [ 12 , 13 , 14 , 15 ]. In contrast to the diverse plant species (most of them are crops) and fungal strains involved in AM symbiosis, limited plant species appear in research into ECM–plant interaction, and the interaction between poplar and Laccaria bicolor is extensively investigated [ 4 ]. However, unraveling the mechanism underlying the ECM-induced defense responses is progressing [ 4 ]. Rhizoctonia solani is a soil-borne phytopathogen with a wide range of hosts and worldwide distribution. It can cause sheath blight on rice [ 16 ], seed rot and root lesions on radish [ 17 ], black scurf and stem canker on potato [ 18 ], damping off on cotton [ 17 ], root rot and damping off on mung bean [ 19 , 20 ], etc. The R. solani complex can be further divided into anastomosis groups (AGs), and 13 AGs have been recognized [ 18 , 21 ]. For example, AG-1 can cause seed and hypocotyl rot, as well as damping off of many plant species such as rice and mung bean [ 16 , 17 ]. It has been reported that R. solani can cause a serious disease on mung bean and yield loss up to 57% [ 20 ]. Parametarhizium , a fungal genus in the Clavicipitaceae family which was originally isolated from forest litter and erected in 2021, includes P. hingganense and P. changbaiense , both of which possess anti-insect activities [ 22 ]. Fungi in Clavicipitaceae show diverse lifestyles including soil saprotrophs, plant pathogens, plant symbionts, and invertebrate parasites such as Epichloë spp. (symbionts of grasses), ergot fungi ( Claviceps spp.) that parasitize ears of cereals, and Metarhizium spp. (parasites of insects and plant symbionts) [ 23 , 24 , 25 ]. Recently, it was found that Metarhizum spp. could be plant symbionts to promote plant growth and enhance plant resistance to disease [ 26 ]. Parametarhizium as a newfound genus from forest litter is phylogenetically close to the genus Metarhizium . Consistent with Metarhizum , Parametarhizium spp. are also entomopathogenic fungi [ 22 ]. However, it remains unknown whether Parametarhizium spp. are plant symbionts and what the influences of Parametarhizium spp. on the performance of plants are during the plant– Parametarhizium spp. interactions. Here, we demonstrated that P. hingganense established an ectomycorrhizal symbiosis with mung bean roots. P. hingganense colonization significantly promoted the growth of mung beans with elevated Pi content and altered phytohormone levels. Furthermore, mycorrhizal symbiosis conferred resistance to disease caused by R. solani . Collectively, our findings provide evidence of the influence of a new fungal species from forest litter, P. hingganense , on host crop plants and an alternative biocontrol agent to promote plant growth and enhance plant disease resistance.",
"discussion": "4. Discussion Plants have benefitted from the mutualistic relationship with fungi since the occurrence of terrestrialization [ 42 ]. Both ectomycorrhiza and arbuscular mycorrhiza possess the capacity to boost the growth of plants and impart tolerance to biotic and abiotic stresses [ 4 , 5 , 8 ]. Given that the primary hosts of ECM fungi are trees, applications of ECM symbionts are mainly focused on forest ecosystems [ 43 ]. In this study, the beneficial functions of P. hingganense belonging to the novel Parametarhizum genus were explored. P. hingganense colonized on the root surface and intercellularly developed between the adjacent cells. The establishment of the symbiotic association with P. hingganense promoted the growth of mung bean plants and imparted tolerance to the damping off induced by R. solani . The genus Parametarhizum was newly discovered and phylogenetically closely related to the genus Metarhizum , which has been wildly used as a pesticide [ 26 ]. Meanwhile, many Metarhizum spp. could colonize the plants and increase growth [ 36 , 44 , 45 ]. For instance, GFP-tagged M. anisopliae was found on the surface and interior of wheat roots [ 36 ]. When M. anisopliae was used as a seed coating agent, it promoted the growth of wheat shoots and single-spike weight, as well as exhibited biocontrol ability against Fusarium graminearum [ 36 ]. M. anisopliae could also promote the growth of soybeans and alleviate oxidative stress induced by high salinity [ 46 ]. Similarly, red fluorescence proteins expressing M. robertsii were observed to form a network surrounding the Arabidopsis root and promoted lateral root growth and root hair development [ 47 ]. Likewise, Parametarhizum was capable of killing three farmland pests: Monolepta hieroglyphica , Callosobruchus chinensis , and Rhopalosiphum maidis [ 22 ]. Furthermore, our results showed that P. hingganense exhibited moderate anti-fungal activity and formed a symbiotic relationship with mung bean plants. These findings suggested that the capacities of Parametarhizum might resemble the members of its sister group. However, the colonization of Metarhizium species on plants was demonstrated to be extraradical and inside the root tissue [ 36 ]; whether the hyphae penetrate the cell or develop intercellularly was unknown. Given that GFP-tagged P. hingganense was located both intercellularly and on the surface of the root, the symbiotic relationship between P. hingganense and mung bean plants was more similar to the ectomycorrhizal interaction. In addition, most reported ectomycorrhizal fungi are associated with trees in forests. The forest litter source of P. hingganense further supported an ectomycorrhizal symbiont formed by P. hingganense and its associated plant. Enhanced phosphorus uptake is a conserved strategy for mycorrhiza, including ectomycorrhiza and arbuscular mycorrhiza, to promote plant growth [ 5 , 7 ]. However, the performance of mycorrhizal fungi in phosphorus utilization is generally regulated by phosphate bioavailability. The root endophyte Colletotrichum tofieldiae only exhibited growth promotion activity on Arabidopsis under limited amounts of Pi [ 48 ]. Glomus intraradices also only contributed to the phosphorus uptake of Solidago canadensis under phosphorus-deficient conditions [ 49 ]. Furthermore, fertilization reduced mycorrhizal colonization in six coexisting arbuscular mycorrhizal temperate tree species [ 50 ]. During the process of AM fungi colonization, low phosphorus levels in the plants triggers the biosynthesis of strigolactones which will be recognized by AM fungi and lead to the development of AM symbiosis [ 51 ]. In contrast, the underlying mechanism of ECM fungi colonization is largely unknown. Research on ECM fungi L. bicolor -poplar symbiosis revealed the relevance of L. bicolor -produced lipochitooligosaccharides in the establishment of ECM association [ 52 ]. In the present study, P. hingganense exhibited in vitro phosphate solubilization ability, and the symbiosis with P. hingganense led to an increment of Pi content in the host plant, implying that the growth promoting ability of P. hingganense should be independent of conditions with phosphorus deficiency. The functional mechanism of P. hingganense in phosphorus uptake awaits further investigation. Despite growth hormones playing crucial roles in mycorrhizal plants, growth promotion is not always accompanied by altered growth hormone levels. Pochonia chlamydosporia , another close relative of P. hingganense , promotes growth, accelerates floral transition, and enhances yield without changes in GAs and auxin [ 53 ]. Here, IAA was absent in P. hingganense liquid culture, indicating IAA production was negative for P. hingganense . This is further supported by the fact that the IAA level in P. hingganense -associated mung bean plants decreased, indicating growth promotion of P. hingganense should be independent of the IAA signaling pathway. Nonetheless, it is worth mentioning that GA 1 in P. hingganense -associated roots significantly increased. Although GAs are essential regulators for plant development [ 10 ], the roles of GAs in plant-fungi symbiosis were mostly focused on the colonization of mycorrhizal fungi [ 54 ]. The complex mechanisms have been well characterized in plant–AM fungi symbiosis [ 12 , 51 ]. GAs acted positively in AM fungi hyphal branching, and negatively in AM fungi entry into the cells [ 11 ]. Despite the elevated content of GAs being repeatedly reported in mycorrhizal plants, the growth promotion effect of GAs does not always accompany the reported results. During the development of tobacco– Glomus intraradices symbiosis, levels of GA 1 , GA 8 , GA 19 , and GA 20 were elevated but GA-related growth effect was lacking [ 55 ]. Yet, the endogenous levels of GA 1 and GA 4 in soybean seedlings co-cultivated with GAs-producing endophytic Porostereum spadiceum under salinity stress were enhanced and accompanied by higher shoot growth and increased biomass [ 56 ]. Consistently, altered GAs content occurred in the symbiosis of poplar with L. bicolor , which led to increased GA 6 and GA 19 but decreased GA 4 [ 13 ]. In the present study, given that increased GA 1 in P. hingganense -associated roots was accompanied by obvious growth promotion, GAs could be involved not only in the colonization of P. hingganense , but also in contributions to growth promotion. Enhanced resistance to various stresses is another hallmark of mycorrhizal plants [ 14 ]. JA and SA signaling pathways are two key components of mycorrhiza-induced resistance [ 4 ]. Here, only JA was induced in P. hingganense -associated roots, while SA was repressed. However, resistance to damping off caused by necrotrophic R. solani was evident in P. hingganense -colonized plants. Despite the influence of JA on AM fungi colonization being controversial, which could be positive, neutral, or negative, the priming function of the JA pathway in the defense system of the host plant is noticeable, especially in the resistance against necrotrophic pathogens [ 4 , 12 ]. The protective effect of ECM fungi colonization was mainly represented by the resistance of host trees to herbivores [ 4 ]. Transcriptomic and metabolomic network analysis unraveled the influence of L. bicolor association on the JA pathway, which in turn mitigated damage from the poplar leaf beetle Chrysomela populi [ 57 ]. Therefore, the JA signaling pathway is essential to the defense response of both AM- and ECM-associated plants. In this study, the fact that defense hormones SA and ABA were suppressed in both P. hingganense -colonized aerial parts and roots further supported the idea that JA could play an important role in P. hingganense -induced local defense response. In addition, P. hingganense -induced disease resistance may also result from enriched phenolic and flavonoid content in whole plants, both of which are antioxidants that scavenge excess ROS induced by stresses [ 58 ]. This elevation could function in eliminating ROS generated by P. hingganense colonization as well, given that no significant difference in MDA content was observed in non-mycorrhizal and mycorrhizal plants. Together, the increment of JA content and antioxidants (phenolic compounds and flavonoids) in P. hingganense -colonized plants could strengthen the defense system and lead to tolerance to pathogen infection."
} | 4,247 |
37427028 | PMC10323739 | pmc | 2,643 | {
"abstract": "As the global human population continues to grow, the demand for food rises accordingly. Unfortunately, anthropogenic activities, climate change, and the release of gases from the utilization of synthetic fertilizers and pesticides are causing detrimental effects on sustainable food production and agroecosystems. Despite these challenges, there remain underutilized opportunities for sustainable food production. This review discusses the advantages and benefits of utilizing microbes in food production. Microbes can be used as alternative food sources to directly supply nutrients for both humans and livestock. Additionally, microbes offer higher flexibility and diversity in facilitating crop productivity and agri-food production. Microbes function as natural nitrogen fixators, mineral solubilizers, nano-mineral synthesizers, and plant growth regulator inducers, all of which promote plant growth. They are also active organisms in degrading organic materials and remediating heavy metals and pollution in soils, as well as soil-water binders. In addition, microbes that occupy the plant rhizosphere release biochemicals that have nontoxic effects on the host and the environment. These biochemicals could act as biocides in controlling agricultural pests, pathogens, and diseases. Therefore, it is important to consider the use of microbes for sustainable food production.",
"conclusion": "Conclusions Microbes are one the important natural resources that should be utilized for sustainable food production. Microbes can be directly taken as human food and feed supplements for livestock. In agri-food production, microbes can be utilized as natural fertilizers, improving soil health, and biocides to protect crops from pathogens and diseases. Extensive utilization of microbes can reduce the dependence on synthetic fertilizers and pesticides for sustainable food production.",
"introduction": "Introduction The global demand for food is increasing in tandem with the growth of the human population. Agriculture is the primary source of our food supply, but its efficiency is closely linked to the climate. Climate change leads to long-term and seasonal variations in weather conditions, waterlogging, freshwater availability, increases in air temperature, and declines in relative humidity, as well as extreme rainfall events and seawater intrusion in arid lands (Miron et al., 2023 ). Agricultural lands located near coastal areas are highly diverse, fragile, and vulnerable. Furthermore, high rainfall and floods during the wet season, soil salinization during the dry season, acidity, high organic matter, and nutritional toxicities or deficiencies throughout the year contribute to low and unstable agricultural productivity. According to Mukhopadhyay et al. ( 2021 ), over 954 million hectares of land in 120 countries are affected by salt, with these salt-affected soils covering 20% of cultivated and 33% of irrigated lands, and contributing to an 8% loss of productivity. According to Mukhopadhyay et al. ( 2021 ), the rate of increase in salt-affected areas is expected to accelerate by 2050. Salinization would affect approximately 600 million people living in the coastal zone, who are primarily involved in agriculture and aquaculture activities (Ismail et al., 2022 ). Changes in weather conditions and the emergence of new diseases can have adverse effects on agricultural growth and production (Corwin, 2020). Over the past two decades, newly developed rice varieties that are tolerant to salinity and/or floods have been deployed in affected areas, resulting in remarkable impacts (Bhowmick et al., 2020 ). These varieties have provided opportunities for designing better stress- and variety-specific management options and have given farmers more confidence to invest in input use and good crop husbandry (Imhoff-Kunsch et al., 2019 ; Bhowmick et al., 2020 ). Enhancing the productivity and profitability of ricebased cropping systems in these coastal areas, with assured quality management services through proper harvest, postharvest processing, and value addition, will significantly improve smallholder farmers' livelihoods. Significant capital investment, large-scale adaptations to relevant technologies, and knowledge of know-how have demonstrated positive impacts in managing floods and salt intrusion, and in maximizing the agricultural lands in the potential coastal zone for agri-food production. Site-specific reclamation management strategies such as amendments, irrigation and drainage, and the use of environmental tolerance of planting materials and microbes are the components of climate-smart agriculture that can ensure food security (Jat et al., 2019 ; Bhowmick et al., 2020 ). Figure 1 illustrates the potential application of microbes in food production. Fig. 1 The potential application of microbes for sustainable food production"
} | 1,212 |
39505912 | PMC11542040 | pmc | 2,644 | {
"abstract": "While the ruminant gut archaeome regulates the gut microbiota and hydrogen balance, it is also a major producer of the greenhouse gas methane. However, ruminant gut archaeome diversity within the gastrointestinal tract (GIT) of ruminant animals worldwide remains largely underexplored. Here, we construct a catalogue of 998 unique archaeal genomes recovered from the GITs of ruminants, utilizing 2270 metagenomic samples across 10 different ruminant species. Most of the archaeal genomes (669/998 = 67.03%) belong to Methanobacteriaceae and Methanomethylophilaceae (198/998 = 19.84%). We recover 47/279 previously undescribed archaeal genomes at the strain level with completeness of >80% and contamination of <5%. We also investigate the archaeal gut biogeography across various ruminants and demonstrate that archaeal compositional similarities vary significantly by breed and gut location. The catalogue contains 42,691 protein clusters, and the clustering and methanogenic pathway analysis reveal strain- and host-specific dependencies among ruminant animals. We also find that archaea potentially carry antibiotic and metal resistance genes, mobile genetic elements, virulence factors, quorum sensors, and complex archaeal viromes. Overall, this catalogue is a substantial repository for ruminant archaeal recourses, providing potential for advancing our understanding of archaeal ecology and discovering strategies to regulate methane production in ruminants.",
"introduction": "Introduction The continuous rise in greenhouse gas (GHG) emissions has triggered rapid changes in global climate patterns, resulting in the frequent occurrence of extreme weather events such as droughts, floods, and heatwaves. These changes profoundly impact the global ecosystem and human livelihoods 1 . Methane (CH 4 ), which ranks second only to carbon dioxide in terms of greenhouse gases on Earth, has a global warming potential approximately 82.5 times greater than that of carbon dioxide over a 20-year period. Hence, it is recognized as a major contributor to the greenhouse effect 2 , 3 . Ruminants contribute significantly to methane emissions, making them a focal point of research. CH 4 emissions from ruminants constitute 16% of global greenhouse gas emissions and 30%-32% of global anthropogenic CH 4 emissions 4 , 5 . In addition, CH 4 emissions represent a substantial energy loss to animals, ranging from 2 to 12% of gross energy intake 6 . Consequently, mitigating CH 4 emissions from ruminants is crucial in the context of sustainable agricultural practices and global climate change mitigation efforts 7 . CH 4 emitted from ruminants is produced primarily by archaea in the gastrointestinal tract (GIT), especially in the rumen 8 . Therefore, a systematic analysis of the composition and function of archaea in the GIT of ruminant animals is crucial for providing background information. Archaea play a key role in syntrophic metabolism by consuming the end products of biomass fermentation from other microbiota, thus maintaining the hydrogen balance of the GIT 9 . Archaea can utilize compounds such as CO 2 , CO, ethanol, formate, acetate or methyl compounds to form CH 4 through three major pathways: hydrogenotrophic, methylotrophic, and aceticlastic. Each trophic type necessitates distinct functions in groups of archaea. Researchers have emphasized that the hydrogenotrophic pathway is responsible for more than 80% of rumen CH 4 production 10 – 12 . Methanobrevibacter spp., which are hydrogenotrophic methanogens, have emerged as the predominant genus involved in rumen methanogenesis 12 , 13 . Additionally, Methanosarcinales , Methanosphaera , and Methanomethylophilaceae are also involved in reducing methylamine and methanol to CH 4 14 – 16 . Methanosarcinales is notable for its ability to effectively dissolve acetate into carbon dioxide and methane 17 . Yaks, as native ruminants, have been identified as ‘low methane’ emitters 18 , 19 . The variations in CH 4 emissions among different ruminants may be attributed to the distinct composition of methanogens in the rumen 19 – 22 . Although numerous researchers have explored the function and composition of the primary methanogens in the GIT of ruminants 2 , 9 , 11 , 23 , comprehensive and global analyses of the systematic abundance of different ruminant gut archaea are lacking. In this work, we establish a catalogue of the ruminant GIT archaeome by collecting and assembling 2,270 ruminant gut metagenomic samples, including previously assembled and cultured archaeal genomes. The catalogue comprises a total of 998 dereplicated archaeal genomes (99.9% Average Nucleotide Identity-ANI similarity), and 42,691 protein clusters and 216/556 strains (99% ANI similarity) that were previously undescribed. Furthermore, our study extends to the gut biogeography of the archaeome across various ruminants, revealing host- and gut-segment-dependent characteristics of the archaeal composition. This catalogue expands the taxonomic and functional variation of the ruminant gut archaeome, and also holds the potential to advance our understanding of archaeal ecology in ruminants.",
"discussion": "Discussion Although extensive research has been conducted on the rumen and intestinal microbial communities of ruminants, with a primary focus on bacterial populations 29 , 57 – 60 , systematic analyses of the ruminant gut archaeome remain insufficient. This study provides insights into the biology of the ruminant gastrointestinal tract (GIT) archaeome by assembling and cataloging 998 nonredundant archaeal genomes. Initial associations between the diversity and function of ruminant gut-associated archaea of different breeds and enterotypes were established. However, some intestinal segments excluding the rumen from many geographic locations, such as regions in America, Africa, Oceania, and Russia, are inadequately sampled. Further efforts should aim to increase the analysis of the gut microbiota in these areas or other environments. Since a small subset of genomes (21/998 = 2%) has been obtained from cultured archaeal representatives 29 , 59 , it is crucial to obtain more cultured isolates from the ruminant gut to better understand the ecology, evolution, and function of archaeal genomes. Advancements in high-throughput cultivation methods, machine learning, and Raman microspectroscopy technologies can accelerate the isolation of many isolates on demand, overcoming the limitations of traditional labor-intensive methods 61 – 63 . Moreover, the dataset of archaeal genomes can serve as a reference and a starting point for targeted cultivation of new members of the ruminant gut archaeome. Additionally, the use of long-read metagenomics sequencing techniques, such as Oxford Nanopore Technology (ONT) and PacBio, has the advantage of longer read lengths (capable of obtaining 10 kb to 1 Mb fragments), which can aid in assembling and recovering more complete MAGs 64 . The use of specialized methods to reduce host contamination, increase cell lysis, and improve DNA extraction might also reduce the amount of information available on archaea from different gut biogeographies 65 , 66 . Devices used to sample regions of the human intestinal tract might also be adapted to easily sample wild ruminant animals that cannot be slaughtered 67 . In summary, the use of adapted technologies might improve and enable profound capture of more diverse ruminant gut archaeomes. The observed percentage of archaea in the ruminant gut microbiome varied by breed and gut biogeography, ranging from 0.14% to 1.76% in the rumens of different ruminants (Fig. 3g ). This finding was similar to the average percentages reported for human gut archaea (~1.2%) 25 . However, similar to that in humans, the abundance of methanogenic archaea in ruminants is highly variable and positively correlated with methane exhalation 25 . Interestingly, low-methane emitters, such as yaks, present different archaeal compositions in the rumen. The Methanomethylophilaceae enterotype dominates the landscape of the archaeal community composition according to the definition of enterotypes 26 , 32 . This finding agrees with studies on methane emissions from yak 18 , 36 and the comparison of methanogen diversity between yak ( Bos grunniens ) and cattle ( Bos taurus ) 68 . The rumen archaea of camels also contain the Methanomethylophilaceae enterotype, which was also observed in camels that produce less methane than in those of ruminants such as cattle, sheep and goats 69 . However, the mechanism underlying the archaeal enterotypes and activity of high- and low-methane emitters remains largely unclear, revealing a potential avenue for reducing methane emissions from ruminants. Further research is essential to elucidate the intricacy of biochemical processes, such as the transfer and utilization of major methyl substrates and dissolved hydrogen, as well as microbial interactions involving bacteria, archaea, viruses, fungi, and protozoa. Advanced techniques such as metagenomics, metatranscriptomics, and stable isotope probing can offer valuable insights into the metabolic activities and functional roles of methylotrophic methanogens in the context of ruminant digestion. Moreover, the rumen, as one of the most efficient anaerobic fiber fermentation systems involving archaea, holds tremendous potential for the development of biomass energy 70 . The presented establishment of ruminant gut-associated archaeal genomes, along with a catalogue of 1.6 million predicted proteins, provides foundational information and serves as a specialized data source for addressing major questions in future research. Understanding the shaping of the diversity and function of the archaeome by ruminant host and archaeome-host interactions, potentially through microRNA secretion by host cells, is crucial for breeding ruminants with low CH 4 emissions and guides microbiome-informed breeding strategies 11 . The ruminant gut microbiome, particularly in the rumen, functions through trophic-like levels to ferment the diet and generate nutrients to meet the host’s requirements 11 . Further research is warranted to investigate how the archaeome, as the third sublevel, interacts with various microorganisms (bacteria, viruses, fungi, and protozoa) at other trophic-like levels, ultimately influencing the host’s digestion and utilization efficiency of feed. The ruminant gut archaeome potentially carries virulence and resistance genes, with a high prevalence of tet -type category ARGs that confer resistance to tetracycline. Considering archaea, conducting global-scale studies is imperative to evaluate the risks associated with gene transfer, environmental impact, food safety, and human health implications 41 , 42 . The archaeal virome also encodes auxiliary metabolic genes (AMGs), particularly those related to methane metabolism (Supplementary Fig. 4 ). However, exploring the process and extent to which the archaeal virome influences methane emissions in ruminants and the potential for targeted reduction of methane emissions by focusing on specific archaea requires further research 1 , 55 . In summary, we successfully delineated the landscape of the archaeome in the GIT of ruminant animals by assembling 2270 ruminant gastrointestinal metagenomes and incorporating previously assembled and isolated archaeal genomes. We confirmed two distinct enterotypes in ruminant GIT archaea, namely, the Methanobacteriaceae enterotype and the Methanomethylophilaceae enterotype, with their composition and dominant methanogenesis pathways showing variations on the basis of breed and gut biogeography. The viral community infecting archaea in the GIT of ruminants carries many methanogenesis genes, offering the potential to regulate archaea to reduce methane emissions. These findings provide comprehensive insights into the ecology and functionality of the gut archaeome in ruminant animals, laying a solid foundation for further exploration in mitigating greenhouse gas emissions through precise regulation of archaea. However, the limitations of this study include the lack of additional culture and in-depth mechanistic validation of relevant methanogens 8 and the lack of incorporation of third-generation sequencing data to improve the quality and recovery rate of the assembly. In further studies combining culturomics and the long sequence lengths of third-generation sequencing, complete genomes and MAGs with higher quality could be obtained. This would contribute to gaining deeper insights into higher-resolution strains, allowing for a more precise understanding of their genetic diversity, functional capabilities, and ecological roles in the gut of ruminants."
} | 3,181 |
31772294 | PMC6879535 | pmc | 2,645 | {
"abstract": "The repertoire of redox-active enzymes produced by the marine fungus Peniophora sp. CBMAI 1063, a laccase hyper-producer strain, was characterized by omics analyses. The genome revealed 309 Carbohydrate-Active Enzymes (CAZymes) genes, including 48 predicted genes related to the modification and degradation of lignin, whith 303 being transcribed under cultivation in optimized saline conditions for laccase production. The secretome confirmed that the fungus can produce a versatile ligninolytic enzyme cocktail. It secretes 56 CAZymes, including 11 oxidative enzymes classified as members of auxiliary activity families (AAs), comprising two laccases, Pnh_Lac1 and Pnh_Lac2, the first is the major secretory protein of the fungi. The Pnh_Lac1-mediator system was able to promote the depolymerization of lignin fragments and polymeric lignin removal from pretreated sugarcane bagasse, confirming viability of this fungus enzymatic system for lignocellulose-based bioproducts applications.",
"conclusion": "Conclusions For the first time, the genomic and secretomic analyses of marine-derived Peniophora sp. CBMAI 1063 revealed an important repertoire of valuable extracellular CAZymes, especially lignin and polyphenols-degrading enzymes. Interestingly, this marine fungus presents a higher number of unique orthologous gene clusters compared to the other two genomes from Peniophora species, demonstrating its singularity. In addition, our findings revealed that Peniophora sp. CBMAI 1063 has the ability to secrete a powerful set of oxidative enzymes of biotechnological interest, mainly the laccase Pnh_Lac1 which could be of interest in lignin modification and depolymerization strategies, bioconversion in industries and bioremediation.",
"introduction": "Introduction Marine-derived fungal species have been considered attractive producers of ligninolytic, hemicellulolytic and other industrial enzymes, presenting different properties compared to terrestrial enzymes, such as high salt tolerance and thermostability 1 . The white-rot basidiomycete Peniophora sp. CBMAI 1063, isolated from the seawater sponge Amphimedon viridis , has been reported as a producer of oxidative enzymes under saline and non-saline conditions, and recently a transcriptome analysis revealed several sequences encoding for putative laccases 2 , 3 . Fungi from the Basidiomycota phylum are considered the most efficient lignin degraders, and according to their lifestyle and ability to degrade polymeric constituents, they can be classified as white-rot or brown-rot fungi 4 . White-rot fungi simultaneously attack lignin, hemicellulose and cellulose, the main components of the plant cell wall, and their genomes generally have more genes encoding for oxidative enzymes when compared to brown-rot and other groups of basidiomycetes 5 , 6 . Lignin degradation/modification in white-rot fungi is generally performed via the action of enzymes such as laccases and peroxidases while in brown-rot fungi it is driven by Fenton reactions (Fe 2+ + H 2 O 2 → Fe 3+ + HO • + OH − and Fe 3+ + H 2 O 2 → Fe 2+ + HOO• + H + ). Laccase and peroxidases genes are abundant in white-rot fungi genomes and reduced or absent in brown-rot fungi 5 , 7 . The degradation and modification of aromatic compounds by white-rot fungi involves several enzymes classified as Auxiliary Activity (AA) families in the CAZy database 7 . The AA1 family contains laccases, which can be classified in three subfamilies AA1_1, AA1_2 and AA1_3. The subfamily AA1_1 has members of the ‘true’ laccases EC 1.10.3.2 or the blue copper oxidases, which are able to oxidize a wide range of aromatic compounds, including lignin, with an oxygen molecule as the final electron acceptor 8 . The AA2 family includes lignin peroxidases, manganese peroxidases and versatile peroxidases, which all use hydrogen peroxide as a cofactor for lignin degradation 7 . Moreover, enzymes from families AA4 (vanillyl-alcohol oxidases), AA5 (glyoxal oxidases/alcohol oxidases) and AA6 (1,4-benzoquinone reductases) are also correlated with the oxidation or reduction of phenolic compounds derived from lignin 9 , 10 . Other AA families are also reported to potentially drive lignin degradation and modification through Fenton reactions, such as AA3, AA7 and AA8, since the majority of their members can generate H 2 O 2 as a by-product 7 , 11 . In this report, we unveiled the repertoire of genes encoding for ligninolytic enzymes from the genome of Peniophora sp. CBMAI 1063, allied with the transcriptome and secretome analyses of the fungus growth in an optimized cultivation media for laccase production, which was formulated without any complex lignocellulosic component. Although the omics analyses were focused on the Peniophora sp. CBMAI 1063 oxidative enzyme system, cellulose and hemicellulose-degrading enzymes were identified from the secretome analysis as well. Furthermore, the major laccase secreted by Peniophora sp. CBMAI 1063 was identified by mass spectrometry. Structural characteristics of the protein and its potential application to promote lignin modification and degradation were explored.",
"discussion": "Results and Discussion The Peniophora sp. CBMAI 1063 genome content is distinct to other related species The genome of the marine-derived Peniophora sp. CBMAI 1063 was sequenced using Illumina sequencing, with 165X coverage and reaching 93% genome completeness. The resulting draft genome assembly of Peniophora sp. CBMAI 1063, access number PRJEB28379, is 47.9 Mb in length with an N50 of 155.8 Kb, and average G + C content of 55% (Table S1 ). The genome size is similar to the other two Peniophora genomes available on the JGI Mycocosm portal: 48.4 Mb for Peniophora sp. CONTA (Lopni1) and 46.0 Mb for Peniophora aff. cinerea (Ricme1), both of which are plant pathogens. The majority of the predicted genes are common among the three strains analyzed, however a significant number of genes were found to be specific to each strain (Fig. 1 ). Among these genes, 457 were found to be specific to CBMAI 1063, 187 to Lopni1, and 164 to Ricme1 strains. The two plant-pathogen fungi strains, Lopni1 and Ricme1, shared 2791 orthologous genes (Fig. 1 ). The number of orthologous genes shared between Peniophora sp. CBMAI 1063 with Lopni1 or Ricme1 is significantly lower, only 370 and 381, respectively. The phylogenetic analysis using a set of 92 single-copy gene markers and 37 genomes from Basidiomycetes phylum (Fig. 2 ) indicated that Peniophora sp. CBMAI 1063 clustered together with the other two Peniophora species previously sequenced by JGI, forming a monophyletic clade in the Russulales order. Figure 1 A Venn diagram showing the distribution of orthologous gene clusters across the marine-derived Peniophora sp. CBMAI 1063, Peniophora sp. CONTA (Lopni1) and Peniophora aff. cinerea (Ricme1) ( A ) and the total number of orthologous gene clusters of each organism ( B ). Figure 2 Phylogenetic tree of the Agaricomycetes class based on 34 fungi genomes distributed among the Basidiomycota phylum. A set of 92 single copy gene markers was used to perform the phylogenetic analysis. Bootstrap values for 1000 replicates are show in the branches. The Peniophora sp. CBMAI 1063 ligninolytic enzyme content The genome of Peniophora sp. CBMAI 1063 encodes 17,714 predicted/putative genes, including 11,827 clusters of orthologous genes. A comparison of the predicted genes against the Carbohydrate-Active Enzymes database 7 (CAZy) identified 310 predicted coding genes related to ligninolytic and carbohydrate-active enzymes (Fig. 3 ). Among the predicted genes encoding for the degradation/modification of lignin and the derived phenolic compounds, Peniophora sp. CBMAI 1063 exhibited 18 genes from family AA1 (laccases), 17 genes from AA2 (peroxidases), 3 genes from AA4 (vanillyl-alcohol oxidases), 7 genes from AA5 (glyoxal and alchool oxidases) and 3 from AA6 (1,4-benzoquinone reductase). Among those genes, 17 laccases AA1, 16 peroxidases AA2 and 7 glyoxal/alcohol oxidases AA5 are predicted to possess a secretion signal while all genes of AA4 and AA6 lack this signal. Figure 3 Transcriptome and genome profile of the marine-derived Peniophora sp. CBMAI 1063. ( A ) Transcriptome profile represented as log 10 of TPM (Transcripts Per Million). ( B ) Genome profile represented as the number of predicted genes encoding CAZymes among the different classes of enzymes according to the CAZy database ( http://www.cazy.org/ ). A total of 41 coding genes of protein family AA3 (glucose oxidase, glucose dehydrogenases and cellobiose dehydrogenases) along with 16 genes of AA9 (LPMOs), 27 genes of AA7 (glucooligosaccharide oxidases) and 1 gene of AA8 (cellobiose dehydrogenases) were present in the genome of Peniophora sp. CBMAI 1063 (Fig. 3 ). The LPMOs from family AA9 have been found only in fungi and they are often co-expressed with sugar oxidases, such as those from the families AA3, AA7 and AA8 43 . All AA9 genes exhibit secretion signals while for the AA3-coding genes the signal peptide is absent. Furthermore, several genes encoding glycoside hydrolases (GH) were identified, including cellobiohydrolases from families GH6 and GH7, xylanases from GH10 and GH11, and pectinases from family GH28. Protein-coding genes for carbohydrate esterases (CE) families CE4, CE8 and CE16, and CBM families 20 and 21 (Fig. 3 ) were also identified. A high number of putative genes encoding for family GH5 (21 genes) and GH43 (19 genes), whose characterized members are typically endoglucanases and xylosidases, respectively, were found in the Peniophora sp. CBMAI 1063 repertoire of CAZymes. Interestingly, the fungus contains putative/predicted GH9-encoding genes, a family of endoglucanases found in anaerobic bacteria producing cellulosomes, plants and termites 44 . The lignocellulolytic capabilities of marine fungi associated with algae, sponges, and mangrove habitats have previously been highlighted in other studies 1 , 45 , although the number of available sequenced genomes is still restricted. The high content of lignocellulosic materials from terrestrial sources that enter the ocean, as well as the symbiotic relationships with other organisms, justify the presence of genes encoding putative CAZymes in marine fungi, which may be found in high number compared to their terrestrial plant-degrading counterparts 46 . Furthermore, the oceans are the largest source of biogenic organohalogens containing chlorine or bromine, which are biosynthesized by myriad seaweeds, sponges, corals, tunicates, bacteria, and other marine life 47 . In particular, the function of organohalogens in sponges is presumably to prevent feeding by fish and fouling by barnacles, bacteria, and fungi 48 . Since pyrroles, indoles, phenols, and tyrosines are commonly found to be halogenated in sponges, it is not a surprise that associated bacteria, microalgae or fungi are adapted to biosynthesize specific metabolites 49 . This work does not intend to elucidate fungal-sponge relationships by genomic comparisons, however this wide repertoire of CAZymes, in particular oxidoreductive enzymes, may be important to enable Peniophora sp. CBMAI 1063 to live in close association with its sponge host ( Amphimedon viridis) in a marine environment. Transcript encoding extracellular AA family members were expressed by Peniophora sp. CBMAI 1063 during growth in optimized conditions Otero et al . 3 described a preliminary global transcriptome analysis of Peniophora sp. CBMAI 1063 cultivated in a medium optimized for laccase production for 7 days in Erlenmeyer flask. Herein, the former RNA-Seq data set was used to validate the genome analysis, and also to depict the set of other partner genes encoding for lignin degradation and modifications enzymes. Additionally, the transcriptome data assisted in understanding not only the ligninolytic system, but also the cellulolytic genes co-expressed in media, which was formulated without a complex carbon source, i.e. lignocellulosic biomass. From the 310 predicted genes related to lignin modification and degradation and CAZymes, 303 were expressed in the optimized media (Fig. 3 ). The genes encoding ligninolytic enzymes from families AA1, AA2, AA4, AA5 and AA6 were abundant in the transcriptome (Fig. 3 ). Peniophora sp. CBMAI 1063 exhibited high laccase activity in the optimized cultivation media 1 , 2 , corroborating with the present analyses, which identified the complete set of 18 genes expressed as predicted for the family AA1 (Fig. 3 ). The AA1 family includes multicopper oxidases, including laccases, ferroxidases and laccase-like multicopper oxidases. Among all genes predicted as AA1 in the fungus, 15721.t1, g1591.t1, and g714.t1 were the most abundant transcripts found, considering the TPM of 53.2, 46.2 and 26.7, respectively. Concerning the family AA2, the genes encoding for peroxidases g10529.t1, g14863.t1, and g8820.t1 were the most abundant (Fig. 3 ). The gene of highest TPM among all CAZymes was the AA3 gene (g15979.t1). This transcript encodes a cytoplasmatic protein containing a glucose-methanol-choline (GMC) oxidoreductase domain, which is related to hydrogen peroxide generation and may act as a co-factor for peroxidases 50 . LPMOs from family AA9 and their electron donor protein partners, as well as the genes coding for AA3, AA7, and AA8, were also present in the transcriptome. Among the GH families involved in cellulose, hemicellulose and pectin degradation, genes coding for the families GH1, GH3, GH5, GH7, GH10, GH11, GH43 and GH51 were identified along with the transcription of CE genes from several families (CE4, CE12, CE5, CE16) (Fig. 3 ). Secretomic analysis A wide variety of oxidoreductases and CAZymes related to the degradation of plant cell wall polymers are present in Basidiomycota species 51 . However, depending on the species and lifestyles, the repertoire of enzymes and their gene numbers can differ significantly 52 . In this work the Peniophora sp. CBMAI 1063 secretome was obtained when cultivated under optimized conditions for laccase production in bioreactor 2 . Additionally, the FDR based on use of a randomized decoy was 0.03%, indicating that the database employed was of high quality for mass spectrometry-based proteomics analysis. Collectively, 126 proteins were identified in the secretome, of which 56 were classified as CAZymes, 57 were non-CAZymes and 13 were hypothetical proteins (Fig. 4 , Table S2 ). As predicted by SignalP v.4.0 34 and Yloc 35 , the majority of the enzymes identified (67%) in the secretome exhibited signal peptides. Among the ligninolytic enzymes, two laccases were identified which both presented predicted signal peptides. Although g15721.t1 presented the highest TPM value among all laccases in the transcriptome, the laccase g1591.t1 exhibited high spectrum counts (255 peptides) in comparison with all CAZymes identified in the secretome (Table 1 ). The laccase g1591.t1, herein named Pnh_Lac1, was identified with 12 unique peptides matches, covering 41% of the protein sequence. Four enzymes from family AA5 (related to glyoxal oxidases) were also identified in the secretome, all of which presented signal peptides. Peptides for peroxidases from family AA2, vanillyl-alcohol oxidases from AA4 and 1,4-benzoquinone reductase from AA6 were not found in the secretome, despite their relative abundance according to the genome and transcriptome data. However, this result should be considered expected since they lack peptide signals. Figure 4 Distribution of proteins identified in the Peniophora sp. CBMAI 1063 secretome obtained after cultivation under saline conditions by mass spectrometry analysis (LC-MS/MS). Non-hypothetical proteins were classified into non-CAZymes and CAZymes groups. Table 1 Predicted lignin-active enzymes identified in the secretome of Peniophora sp. CBMAI 1063 cultivated in a bioreactor under saline conditions. Accession Number Molecular Weight a Amino acid length a dbCAN b PFAM c Description Signal Peptide d Location e Unique peptides Spectrum counts Lignin-Active Enzymes identified in the Secretome g1591.t1 58 kDa 546 AA1 PF00394 PF07731 PF07732 Multicopper oxidase YES SP 12 255 g17194.t1 58 kDa 540 AA1 PF00394 PF07732 PF07731 Multicopper oxidase YES SP 3 2 g5706.t1 71 kDa 671 AA5 PF07250 PF09118 Glyoxal oxidase N- terminus YES SP 2 1 g5707.t1 81 kDa 769 AA5 PF07250 PF09118 Glyoxal oxidase N-terminus YES SP 3 1 g5709.t1 84 kDa 799 AA5 PF07250 PF09118 Glyoxal oxidase N-terminus YES SP 2 1 g9556.t1 59 kDa 554 AA5 PF07250 PF09118 Glyoxal oxidase N-terminus YES SP 4 5 g13672.t1 60 kDa 567 AA7 PF01565 PF08031 FAD binding domain berberine YES SP 5 6 g17067.t1 51 kDa 485 AA7 PF01565 FAD binding domain YES SP 3 5 g7475.t1 59 kDa 548 AA7 PF01565 PF08031 FAD binding domain berberine YES SP 3 1 a Molecular Weight and a Amino acid length determined by LC-MS/MS. The results were processed by Mascot v.2.3.01 engine (Matrix Science Ltd.) software against the genome sequencing database of Peniophora sp. CBMAI and Scaffold – Proteome Software (version Scaffold_4.3.2 20140225). b Web server and database for automated carbohydrate-active enzyme annotation generated based on the family classification from CAZy database: AA –Auxiliary Activity. c Protein Family Domain analysis. d The presence of a signal peptide of secreted proteins predicted by SignalP v.4.0. e The subcellular localization of proteins predicted by YLoc (Interpretable Subcellular Localization Prediction): SP – secreted pathway; C – cytoplasm; M – mitochondrial location. Among the cellulolytic and hemicellulolytic-active enzymes, the secretome contained 38 proteins classified in different families of GHs, which are involved in cellulose and hemicellulose breakdown (Tables 2 – 4 ). Enzymes from families GH6 and GH7 were absent in the secretome. Two GH5 were identified, where this family contains cellulase and hemicellulose activities. Five GHs exhibited CBMs: GH18 with CBM5, GH72 with CBM43, GH43 with CBM35, GH72 with CBM43 and GH15 with CBM20. Carbohydrate esterases (CE) from family 4 and 8, involved in deacetylation of hemicelluloses, and a polysaccharide lyase (PL) from family 22 (oligogalacturonate lyase) were also identified (Tables 3 and 5 ). Interestingly, a GH78 family whose characterized members are typically α-l-Rhamnosidases [E.C. 3.2.1.40] was identified along with a pectin-active enzymes group. These enzymes specifically cleave terminal α-l-rhamnose from a wide range of natural products and have important biotechnological applications in the food and pharmaceutical industries 53 . Table 2 Predicted cellulose-active enzymes identified in the secretome of Peniophora sp. CBMAI 1063 cultivated in a bioreactor under saline conditions. Accession Number Molecular Weight a Amino acid length a dbCAN b PFAM c Description Signal Peptide d Location e Unique peptides Spectrum counts Cellulose-Active Enzymes g13682.t1 71 kDa 640 AA3 PF05199 PF00732 Glucose-methanol-choline oxidoreductase NO C 18 19 g16244.t1 62 kDa 585 AA3 PF00732 PF05199 Glucose-methanol-choline oxidoreductase NO SP 6 7 g6504.t1 26 kDa 242 AA9 PF03443 Glycoside hydrolase family 61 YES SP 3 71 g6425.t1 35 kDa 348 AA16 PF03067 Lytic polysaccharide monooxygenase YES SP 2 1 g11705.t1 81 kDa 760 GH3 PF01915 PF00933 PF14310 Glycoside hydrolase family 3 YES SP 9 18 g15376.t1 93 kDa 879 GH3 PF01915 PF00933 PF14310 Glycoside hydrolase family 3 YES SP 2 1 g1658.t1 37 kDa 353 GH5 PF00150 Glycoside hydrolase family 5 YES SP 2 2 g5589.t1 49 kDa 452 GH5 PF00150 Glycoside hydrolase family 5 YES SP 4 7 a Molecular Weight and a Amino acid length determined by LC-MS/MS. The results were processed by Mascot v.2.3.01 engine (Matrix Science Ltd.) software against the genome sequencing database of Peniophora sp. CBMAI and Scaffold – Proteome Software (version Scaffold_4.3.2 20140225). b Web server and database for automated carbohydrate-active enzyme annotation generated based on the family classification from CAZy database: GH- Glycoside Hydrolases; AA- Auxiliary Activities. c Protein Family Domain analysis. d The presence of a signal peptide of secreted proteins predicted by SignalP v.4.0. e The subcellular localization of proteins predicted by YLoc (Interpretable Subcellular Localization Prediction): SP – secreted pathway; C – cytoplasm; M – mitochondrial location. Table 3 Predicted hemicellulose-active enzymes identified in the secretome of Peniophora sp. CBMAI 1063 cultivated in a bioreactor under saline conditions. Accession Number Molecular Weight a Amino acid length a dbCAN b PFAM c Description Signal Peptide d Location e Unique peptides Spectrum counts Hemicellulose-Active Enzymes g14451.t1 46 kDa 429 CE3 PF13472 PF00657 GDSL-like lipase/acyl hydrolase YES SP 7 44 g10047.t1 55 kDa 513 CE4 Carbohydrate esterase YES SP 3 5 g4642.t1 38 kDa 354 CE4 PF01522 Polysaccharide deacetylase YES SP 6 14 g11401.t1 37 kDa 345 GH10 PF00331 Glycoside hydrolase family 10 YES SP 4 8 g11177.t1 59 kDa 540 GH125 PF06824 Protein of unknown function NO SP 5 4 g14331.t1 30 kDa 277 GH128 PF11790 Glycoside hydrolase catalytic core YES SP 2 6 g7518.t1 34 kDa 318 GH16 Glycoside hydrolases family 16 YES SP 4 18 g7519.t1 33 kDa 313 GH16 Glycoside hydrolases family 16 YES SP 6 66 g16729.t1 57 kDa 524 GH30 PF02055 Glycoside hydrolase family 30 YES SP 3 3 g3624.t1 53 kDa 499 GH30 PF14587 O-Glycoside hydrolase family 30 YES SP 10 57 g14525.t1 36 kDa 341 GH43 PF04616 Glycoside hydrolases family 43 YES SP 2 2 g4370.t1 33 kDa 321 GH43 PF04616 Glycoside hydrolases family 43 YES SP 4 54 g5765.t1 33 kDa 310 GH43 PF04616 Glycoside hydrolases family 43 YES SP 5 55 g9420.t1 35 kDa 329 GH43 PF04616 Glycoside hydrolases family 43 YES SP 2 13 g15433.t1 47 kDa 444 GH43 CBM35 PF04616 Glycoside hydrolases family 43 YES SP 2 2 g3571.t1 69 kDa 638 GH51 PF06964 α-L-arabinofuranosidase YES SP 10 33 g14175.t1 58 kDa 564 GH72 CBM43 PF03198 PF07983 Glucanosyltransferase X8 domain YES SP 4 3 g11973.t1 40 kDa 375 GH76 PF03663 Glycoside hydrolase family 76 YES SP 5 8 g4124.t1 40 kDa 370 GH76 PF03663 Glycoside hydrolase family 76 YES SP 4 5 g11336.t1 91 kDa 827 GH92 PF07971 Glycoside hydrolase family 92 YES SP 13 14 g1967.t1 67 kDa 606 GH92 PF07971 Glycoside hydrolase family 92 NO SP 10 10 g5807.t1 88 kDa 812 GH92 PF07971 Glycoside hydrolase family 92 YES SP 13 15 g5441.t1 186 kDa 1691 GH95 PF14498 Glycoside hydrolase family 95 NO SP 2 1 a Molecular Weight and a Amino acid length determined by LC-MS/MS. The results were processed by Mascot v.2.3.01 engine (Matrix Science Ltd.) software against the genome sequencing database of Peniophora sp. CBMAI and Scaffold – Proteome Software (version Scaffold_4.3.2 20140225). b Web server and database for automated carbohydrate-active enzyme annotation generated based on the family classification from CAZy database: GH - Glycoside Hydrolases; CE - Carbohydrate Esterases; CBM: Carbohydrate-binding module. c Protein Family Domain analysis. d The presence of a signal peptide of secreted proteins predicted by SignalP v.4.0. e The subcellular localization of proteins predicted by YLoc (Interpretable Subcellular Localization Prediction): SP – secreted pathway; C – cytoplasm; M – mitochondrial location. Table 4 Predicted chitin, starch & other carbohydrate-active enzymes identified in the secretome of Peniophora sp. CBMAI 1063 cultivated in a bioreactor under saline conditions. Accession Number Molecular Weight a Amino acid length a dbCAN b PFAM c Description Signal Peptide d Location e Unique peptides Spectrum counts Chitin, Starch & Others Carbohydrate-Active Enzymes g13668.t1 61 kDa 580 CBM20 GH15 PF00723 PF00686 Glycoside hydrolases family 15 YES SP 20 197 g7315.t1 82 kDa 769 CBM5 PF17168 PF16335 PF08760 PF02839 Carbohydrate binding domain YES SP 10 15 g7861.t1 50 kDa 474 GH13 PF00128 PF02806 Alpha amylase YES SP 5 18 g11641.t1 97 kDa 890 GH31 PF01055 PF16863 PF13802 Glycoside hydrolases family 31 YES SP 3 4 g15819.t1 109 kDa 997 GH31 PF01055 PF16863 Glycoside hydrolases family 31 NO SP 12 9 g15820.t1 104 kDa 944 GH31 PF01055 PF16863 Glycoside hydrolases family 31 YES SP 6 6 g11828.t1 60 kDa 547 GH32 PF00251 Glycoside hydrolases family 32 NO SP 8 35 g11153.t1 49 kDa 460 GH88 PF07470 Glycoside hydrolase Family 88 YES SP 3 3 g11942.t1 45 kDa 418 GH18 PF00704 Glycoside hydrolases family 18 YES SP 2 12 g11425.t1 49 kDa 472 GH18; CBM5 PF02839 Carbohydrate binding domain YES SP 2 5 g6083.t1 136 kDa 1254 GH18; CBM5 PF00009 PF03764 PF14492 PF00679 PF00704 PF02839 PF03144 Elongation factor Tu GTP binding domain18 YES M 7 23 g10368.t1 60 kDa 560 GH20 PF00728 PF14845 Glycoside hydrolase family 20 YES SP 5 2 g15989.t1 134 kDa 1230 GH20 PF00728 PF14845 PF02838 Glycoside hydrolase family 20 YES SP 7 9 a Molecular Weight and a Amino acid length determined by LC-MS/MS. The results were processed by Mascot v.2.3.01 engine (Matrix Science Ltd.) software against the genome sequencing database of Peniophora sp. CBMAI and Scaffold – Proteome Software (version Scaffold_4.3.2 20140225). b Web server and database for automated carbohydrate-active enzyme annotation generated based on the family classification from CAZy database: GH - Glycoside Hydrolases; CBM- Carbohydrate-binding module. c Protein Family Domain analysis. d The presence of a signal peptide of secreted proteins predicted by SignalP v.4.0. e The subcellular localization of proteins predicted by YLoc (Interpretable Subcellular Localization Prediction): SP – secreted pathway; C – cytoplasm; M – mitochondrial location. Table 5 Predicted pectin-active enzymes identified in the secretome of Peniophora sp. CBMAI 1063 cultivated in a bioreactor under saline conditions. Accession Number Molecular Weight a Amino acid length a dbCAN b PFAM c Description Signal Peptide d Location e Unique peptides Spectrum counts Pectin-Active Enzymes g8265.t1 35 kDa 330 CE8 PF01095 Pectin esterase YES SP 2 2 g8087.t1 70 kDa 665 GH78 PF05592 Bacterial alpha-L-rhamnosidase YES SP 7 30 g10469.t1 74 kDa 673 PL22 PF07676 WD40-like beta propeller repeat YES SP 2 1 g1538.t1 45 kDa 413 GH28 PF00295 Glycoside hydrolase family 28 YES SP 2 1 a Molecular Weight and a Amino acid length determined by LC-MS/MS. The results were processed by Mascot v.2.3.01 engine (Matrix Science Ltd.) software against the genome sequencing database of Peniophora sp. CBMAI and Scaffold – Proteome Software (version Scaffold_4.3.2 20140225). b Web server and database for automated carbohydrate-active enzyme annotation generated based on the family classification from CAZy database: GH- Glycoside Hydrolases; PL- Polysaccharide Lyases; CE- Carbohydrate Esterases. c Protein Family Domain analysis. d The presence of a signal peptide of secreted proteins predicted by SignalP v.4.0. e The subcellular localization of proteins predicted by YLoc (Interpretable Subcellular Localization Prediction): SP – secreted pathway; C – cytoplasm; M – mitochondrial location. Omics data integration Omics approaches can provide hypotheses regarding function for the large number of genes predicted from genome sequences. In this study, an integrative analysis of genomic, transcriptomic and proteomic data was performed for Peniophora sp. CBMAI 1063 Although the cultivation medium had the same composition, an important point to take in account is that the transcriptomic analysis was performed at the seventh day of small-scale cultivation (200 mL) while the secretome analysis was at the fifth day of cultivation in bioreactor scale (5 L). Moreover, it is well documented in the literature the poor correlation between transcriptomic and proteomic data due to several factors, including pre and pos translational processes 54 , 55 . Even though, omic data integration was performed in this work in order to explore the biotechnological potential of Peniophora sp. CBMAI 1063 under its optimized cultivation medium. Thus, according to our omics data, of 18 genes encoding for laccases from family AA1 were found in the genome, only 2 proteins were identified as secreted in the condition analyzed (Figs. 3 and 4 and S1 ). Post-transcriptional, translational and degradation regulation events regarding the gene Pnh_Lac1 may be involved to explain the differences between gene expression and protein secretion under the conditions evaluated. Conversely, the fungus preferentially secrets Pnh_Lac1 in the optimized media (Fig. S1 ). Family members from AA4 were not identified in the proteomic data, although 3 genes were identified in the genome and in the transcriptome (Figs. 3 and S2 ). This was similar to the observations for AA2, AA6 and AA8 family members, where the transcripts were expressed but the proteins were not identified in the secretome (Figs. 3 and 4 ). Regarding the family AA5, 4 proteins were found to be secreted from the 7 encoding genes identified in the genome (Figs. 3 and S2 ). Assigned to a glyoxal oxidase family domain according to PFAM, AA5 members are copper-containing enzymes that mainly oxidize aldehydes generated during lignin and carbohydrates degradation 56 . Although genes encoding glyoxal oxidases enzyme are widely distributed among white-rot fungi and symbiotic fungi, the number of characterized enzymes is still restricted 10 . The presence of these proteins in the secretome suggests that the fungus possesses a strong ligninolytic capacity. In this context, further studies of the AA5 enzymes and their role in Peniophora sp. CBMAI 1063 are extremely relevant. Furthermore, among the 27 genes encoding AA7 proteins, only 3 were found to be secreted. According to the secretome data, in the case of the carbohydrate-binding module (CBM), predicted genes for CBM5, CBM20, CBM 21 and CBM 42 were found; while the transcriptomic data exhibited 4 transcripts for CBM21 and one for CBM20 (Figs. 3 and 4 ). Among all GH-coding genes identified, GH5 and GH43 exhibited the highest number of predicted genes (Fig. 3 ), and 2 and 5 proteins were identified in the secretome, respectively (Fig. 3 ). Homology and structural insights of the major laccase (Pnh_Lac1) secreted by Peniophora sp. CBMAI 1063 According to a sequence search using BLASTp, the most similar proteins to Pnh_Lac1 are multicopper oxidases from Peniophora sp . (87% identity to KZV66389.1 and 66% identity to KZV69698.1) and laccases from P. lycii (AWC08468.1) and Meripilus giganteus (CBV46340.1), both showing 61% identity. The laccase signature composed of four ungapped sequence segments L1–L4 57 was in Pnh_Lac1 (Fig. S3 ). Segments L1-L4 contain the amino acids that bind to the copper centers: H64 and H66 from L1; H109 and H111 from L2; H390, H393 and H395 from L3; and H448 and H452 from L4 (Fig. 5B ). Figure 5 3D structure of Pnh_Lac1 generated by homology modeling. General structure of laccases, consisting of three cupredoxin domains ( A ) Zoom on the copper ion centers surrounded by the fungal laccase signature segments L1 (blue), L2 (green), L3 (magenta) and L4 (orange). The ion coordination is performed by the histidines presented in the L1-L4 regions ( B ). Performing a sequence search in the Protein Data Bank (PDB), the Pnh_Lac1 shows between 55–60% identity with different laccases from subfamily AA1_1. Due to the highly conserved structure of this family, the three-dimensional model of Pnh_Lac1 could be generated by homology modelling (Fig. 5 ), which was considered reliable according to the C-score value of 0.7 (range from −5 to 2) 37 . The enzyme presents typical folding of laccases, consisting of three cupredoxin domains with a mononuclear copper center located in domain 3, and a trinuclear center between domains 1 and 3 58 (Fig. 5A ). Collectively, these results, including the sequence alignment of the conserved regions, as well as the structural alignment of Pnh_Lac1 with other members of family AA1_1, are important to support the gene prediction data (Figs. 3 and S4 ) described in the present work. Pnh_Lac1 effects on lignin depolymerization and enzymatic digestibility of steam-exploded sugarcane bagasse The biochemical properties of purified Pnh_Lac1 were previously characterized by Mainardi et al . 2 : presenting 986.0 and 30.8 U mg −1 , using ABTS and SGD as substrates, respectively, and optimum activity at pH 5.0 and 30 °C. To illustrate a potential biotechnological application, the purified Pnh_Lac1 was employed in combination with a synthetic mediator (ABTS) to promote the depolymerization of a lignin isolated from sugarcane bagasse by alkaline pretreatment 39 . The soluble fraction after incubation with LMS was analyzed by UV-light absorbance and GPC. An increase of 50% in UV-light absorbance at 280 nm was observed compared to the control experiment without Pnh_Lac1 (Fig. 6a ). According to Arzola et al . 59 , spectra changes detected between 260 and 270 nm (in LMS-treated samples) indicate the introduction of new functional groups in the phenylpropane unit or aromatic rings of lignin fragments (Fig. 6a ), resulting in auxochrome groups, new nonconjugated hydroxyl groups in the side-chain phenylpropane unit or new Cα–Cβ double bonds. Figure 6 UV-light absorbance ( a ) and GPC chromatograms ( b ) showing the spectroscopic profile and molecular weight distribution of the lignin derived products obtained after the incubation of lignin extracted from SCB with purified Pnh_Lac1 and ABTS as the mediator (LMS); Enzymatic hydrolysis of LMS-treated SCB (grey bars) or non-treated SCB (white bars) performed with commercial cocktails at low dosage. The y-axis shows the cellulose conversion in percentage of the maximum theoretical cellulose conversion after 72 h at 50 °C. Error bars represent the standard errors of the means of triplicate experiments ( c ); GPC chromatograms showing the molecular weight distribution of the solubilized lignin-derived products and polyphenolics from LMS-treated SCB. Red lines (in figures a , b , d ) represent experiments with LMS and black lines refer to control experiments without Pnh_Lac1. Spectral changes in the visible region were also detected, especially at 480 nm. Chromogen groups are commonly introduced after laccase activity, which is related to the reddish color appearance, as can be observed in Fig. 6a . The GPC analysis shows that soluble low molecular weight lignin derived compounds (<500 Da) were generated after LMS treatment (Fig. 6b ), indicated by the arrows. The intense peak corresponding to high molecular weight (HMW) lignin (~4 kDa) appeared thinner when compared to the untreated sample, which is indicative of polymer depolymerization. The results indicate that Pnh_Lac1, when combined with a mediator, was able to promote lignin solubilization and depolymerization. This enzyme is therefore should be of biotechnological interest especially for the bioconversion of industrial lignins streams, including those generated from lignocellulose processing biorefineries where utilization is hampered by its chemically heterogeneous nature, low solubility and reactivity 38 . Previously works have shown that LMS treatments can improve the enzymatic hydrolysis of lignocellulose materials, based on lignin solubilization, depolymerization and modification mechanisms 60 . However, there is also evidence that LMS can have detrimental effects on enzymatic hydrolysis yields, as a result of enzymes inhibition, oxygen competition and grafting of phenoxy radicals 39 , 61 . We are therefore led to believe that positive or negative effects can depend on singularities of the laccase employed and assay conditions. In this sense, we evaluated the ability of the LMS based on Pnh_Lac1 and ABTS to remove lignin from pretreated SCB, which could improve the subsequent saccharification of lignocellulosic material. The SCB was first treated with LMS and then with two different commercial cocktails. Increases of 1.9% and 11.0% were observed in saccharification yield using Accellerase ® 1500 and Cellic TM Ctec2, respectively, compared to the untreated samples (Fig. 6c ). The GPC analysis of the soluble fraction after LMS treatment confirmed that the lignin fragments were partially degraded, leading to its solubilization into the supernatant (Fig. 6d ). The new peaks detected in the LMS-treated sample correspond to soluble low molecular weight phenolic compounds, between 400–200 Da (indicated by arrows) (Fig. 6 ). The HMW lignin fragments from 4–1.7 kDa were found in lower intensity in the chromatogram. Interesting, as can be observed in the LMS-treated supernatant, the peak corresponding to low molecular weight phenolic compounds (around 100 Da) is absent (Fig. 6 ). The LPMO-containing cellulose cocktail Cellic™ Ctec2 has better performance than traditional enzyme cocktails mainly consisted of hydrolytic cellulases for biomass saccharification 62 . In addition, it has been demonstrated that soluble lignin derived compounds act as electron donors to LPMOs, boosting its catalytic efficiency 39 , 63 . Thus, the higher saccharification yield using Cellic™ Ctec2 for pretreated SCB may indicate that the soluble low molecular weight phenolic compounds generated by the LMS based on Pnh_Lac1 and ABTS enhanced the LPMO activity. Collectively, our results demonstrate that Pnh_Lac1 could be applied be applied in bioconversion technologies, based on promoting lignin depolymerization and solubilization from the lignocellulosic substrate, thus facilitating the action of cellulolytic enzymes. However, further studies are necessary to combine the laccase with different mediators and higher enzyme loadings, which could improve the effectiveness of the LMS pre-treatment."
} | 9,437 |
29255804 | PMC5733113 | pmc | 2,650 | {
"abstract": "Maximizing output power density with design parameters.",
"conclusion": "CONCLUSIONS Triboelectric generators make use of the surface charge maintained in an insulating material during the repeated contact and separation of a dissimilar material. Electrostatic induction due to the separation of two charged surfaces builds a potential that drives a current through the load. Power generation is more effective when the time-varying RC product of the generator better matches the mechanical motion frequency during the cycle. A larger 1/ C device is beneficial for good matching but reduces the current level. Generally, the power output characteristic is more stable in the good matching regime (high 1/ C device ), which could be achieved by connecting a series capacitor to the circuit. The device figure of merit ( Eq. 8 ) determines the maximum output power density obtainable from a generator at steady state. Here, the only material-related parameter is surface charge density, which defines the material figure of merit. Parasitic capacitance reduces the power generation effectiveness. Enlarging the surface area of the dielectric-metal interface can minimize power reduction due to parasitic capacitances. The fundamentals of triboelectric generation presented in this paper could lead to designs of more sophisticated applications and help to establish triboelectric generators as an attractive energy-harvesting source.",
"introduction": "INTRODUCTION Rubbing two different materials is a charge-generating method as old as the discovery of electricity itself. Attempts to produce electricity from this triboelectric method has led to modern instruments such as the Van de Graaff generator ( 1 ), which is for sourcing an extremely high voltage. Today, with the renewed interest in energy harvesting for distributed power sources, triboelectric generators are gaining interest as an alternative source. Many variants of application schemes have been demonstrated or conceptualized recently ( 2 – 17 ) and described as triboelectric nanogenerators ( 18 ) in pioneering work, leading to deepened interest in understanding their power and efficiency ( 19 – 25 ). Nevertheless, power optimization schemes are still premature where, often, only the load resistance is adjusted with lack of a general principle for full optimization. Furthermore, the impact of device imperfections ( 23 ) tends to be discussed in less detail despite its significant influence on device operation. Here, we concisely model the contact-separation mode generator and reveal that the output power could be increased substantially by optimizing the capacitance in addition to the load resistance. Matching the resistance-capacitance ( RC ) product to the characteristic frequency of the triboelectric process is essential to use the mechanical motion and effectively convert it into electrical power. An added capacitor is necessary to stabilize the RC product because the total capacitance varies during the cycle. Adjusting resistance alone leads to poor matching conditions and, thus, low power. We also model how parasitic capacitance, which is practically inevitable, affects the performance of the device. We show that proper adjustments to minimize the ineffectiveness caused by parasitic capacitance could also lead to a substantial increase in output power. We anticipate that, with the power improvement strategies learned from our model analysis, triboelectric generators could be a competitive energy-harvesting source.",
"discussion": "DISCUSSION Comparison to experiments Our model shows good agreement with the experimental data reported by Niu et al . ( 19 ). We compare the power generation curve with respect to load resistance. For the data set where the device capacitance was not varied, the corresponding model curve is a vertical cross section of Fig. 3 (also see fig. S3). It is seen in Fig. 5A that the curve shapes from three different driving speeds all fit well to a fixed curve shape given by 1/ C * = 0.07. Fig. 5 Model fitting to experimental data from Niu et al . ( 19 ). ( A ) Power change with respect to load resistance. Experimental data sets consist of driving conditions with average speeds of 0.08 m/s (black circles), 0.04 m/s (blue circles), and 0.02 m/s (red circles). Solid lines are from the ideal model by assuming a fixed 1/ C * = 0.07, which determines the shape of the curve. The scaling of the model curve to real dimensions was determined by fitting due to the lack of experimental details in the study of Niu et al. ( 19 ). ( B ) The change of maximum power (left axis) and optimum resistance (for a fixed device capacitance; right axis) with respect to the average driving speed. The curves represent the expected scaling behavior of maximum power ( ∝ v ¯ , Eq. 7 ) and optimum load resistance (∝ 1/ω for fixed x max ;, Eq. 3 ) from the ideal model. Because the maximum power conditions for the three different driving speeds in the data set are expected to be described with identical dimensionless parameters (that is, a set of 1/ C * and R *), the maximum power and optimal load resistance in real dimensions are expected to scale with respect to average speed as compared in Fig. 5B . The small deviations are thought to be associated with parasitic capacitance, which was not possible to be accounted for with the given information. Parasitic capacitance seems to explain the power scaling observed with respect to an increase in x max . Niu et al . ( 19 ) increased x max by five times, which increases the FOM device by the same factor. However, the observed power increase was limited to a factor of 2.3. Note that for a given parasitic capacitance, C par * scales with x max ( Eq. 11 ); that is, the influence of parasitic capacitance increases. As a result, the benefit of increased x max is diminished. Regarding the material figure of merit, several experimental reports have demonstrated the significance of σ 2 ( 28 – 31 ). For quantitative assessment of the scaling behavior with respect to σ 2 , power output at the load resistance should be measured while taking into account the 1/ C * of the generator. Conversion efficiency Because the power production in a triboelectric generator is purely by electrostatic induction, the energy conversion efficiency of this process itself is 100%. It can be confirmed (see the Supplementary Materials) with our model that the power output is exactly equal to the net work done by the mechanical motion, which is true, in general, even for nonoptimal R * and 1/ C * and a finite C par * . Loss of efficiency originates from other imperfections rather than from the electrostatic conversion process or purely capacitive leakage. For example, the surface charge on the dielectric layer could leak through the film, which is equivalent to resistive loss. This type of loss can be minimized by keeping the dielectric film resistance much larger than the load resistor. Frictional losses regarding the mechanical motion or the charge exchange process (during the contact of two surfaces) are other sources of losses that could be important. Practical requirements in implementing the generator could also lead to reduced efficiency. The 100% conversion efficiency of the electrostatic induction is by considering the net mechanical work, including both positive (during separation of charged surfaces) and negative (while joining the charged surfaces back together) work input. In applications where negative work could not be meaningful, the relevant efficiency would have to be calculated with only the positive work input. This alternative definition gives 45 % efficiency for the maximum power condition (see the Supplementary Materials). Another example is when the load is not simply a load resistor. Then, there would be losses associated with power conditioning ( 32 ) to convert the output into a “useful” form."
} | 1,969 |
38637578 | PMC11026409 | pmc | 2,651 | {
"abstract": "Trait-based frameworks are promising tools to understand the functional consequences of community shifts in response to environmental change. The applicability of these tools to soil microbes is limited by a lack of functional trait data and a focus on categorical traits. To address this gap for an important group of soil microorganisms, we identify trade-offs underlying a fungal economics spectrum based on a large trait collection in 28 saprobic fungal isolates, derived from a common grassland soil and grown in culture plates. In this dataset, ecologically relevant trait variation is best captured by a three-dimensional fungal economics space. The primary explanatory axis represents a dense-fast continuum, resembling dominant life-history trade-offs in other taxa. A second significant axis reflects mycelial flexibility, and a third one carbon acquisition traits. All three axes correlate with traits involved in soil carbon cycling. Since stress tolerance and fundamental niche gradients are primarily related to the dense-fast continuum, traits of the 2nd (carbon-use efficiency) and especially the 3rd (decomposition) orthogonal axes are independent of tested environmental stressors. These findings suggest a fungal economics space which can now be tested at broader scales.",
"introduction": "Introduction Soils are exposed to multiple anthropogenic pressures and changing environmental conditions that threaten microbial functions essential for biogeochemical processes maintaining soil sustainability 1 . Soil microbial communities are affected by environmental change—with a plethora of sequencing studies revealing shifts in microbial community structure following changes in environmental conditions 2 . Yet, the much needed projection of community shifts onto microbial functions lags behind 3 , 4 . Achieving such functional predictability and mechanistic understanding in soil microbial ecology represents one of the frontiers in this field. In particular, predicting the microbial cycling of soil organic carbon (C) requires in-depth knowledge about microbial functions 5 , 6 . There is an ongoing debate whether and how predictability in complex soil microbial communities can be achieved 7 , which is especially relevant in the context of rapidly advancing soil biogeochemical models 5 . A most promising tool in this context is given by trait-based frameworks, which directly couple community composition to function 8 . These frameworks proved to be powerful in global models implementing plant traits, and there is considerable effort now to establish such frameworks in soil microbiology 3 , 9 . The strength of functional trait-based approaches lies in the simple concept of constrained resource allocation possibilities—organisms cannot be good at everything 10 . Consequently, trade-offs allow to predict that species with certain trait expressions will not express functions at the other end of a trait continuum 11 . Frameworks like the widely applied Grime´s C-S-R (competitor-stress tolerant-ruderal) triangle expand on this simple idea and sort species into respective life history strategies 12 , 13 . This concept has been recently applied to microbial soil C cycling functions - the Y-A-S (yield-acquisition-stress tolerant) or similar C-S-O (competitors-stress tolerators-opportunists) framework 6 , 14 . These simple frameworks are regarded as promising tools to implement microbial traits in soil C models 5 , 15 , since they directly connect environmental stressors to C cycling properties: If traits indicative of stress tolerance trade-off with C acquisition rates, this enables the prediction of a decrease in C mineralization in response to respective stressors at the community level (stress is defined here as an external constraint limiting fungal growth 12 , anthropogenic stress refers to external growth constraints induced or intensified by human activities). In practice, though, these simple categories fail to capture the continuous nature of trait expressions along independent dimensions, are highly subjective in their interpretation and lack objective quantifiable trait matrices. Due to these drawbacks, in plant ecology these categorical frameworks have been replaced a while ago by the idea of an economics spectrum 16 , 17 . Based on relatively few traits, plants can be directly sorted on a main trait axis along the spectrum of conservative to acquisitive traits, also referred to as the slow-fast continuum 18 . The main axes of the plant economics spectrum explain life-history strategies 19 , relate to environmental parameters 20 and are relevant predictors in global models 21 . Following this powerful approach, it has been suggested to similarly transition to a trait continuum in microbial ecology rather than focusing on inconclusive functional groups 22 . Microbial ecology clearly lags behind in trait-based ecology. This is no surprise given the tremendous diversity within soil microbial communities and the challenging methodological impracticalities preventing in vivo measurements of many traits of individual microbes. Consequently, experimental evidence on functional (multivariate) trait axes remains scarce 23 – 26 . Rapidly evolving soil metagenomics allows the analyses of microbial traits at larger scales 27 . However, genomic information only provides a glimpse on the potential of an organism, not its actual phenotype 22 . More importantly, these data provide little information on primary trade-offs in resource investment in microorganisms. To make genomic data applicable, such functional trade-offs must be established first in the organisms itself. Advanced experimental insights into a broader spectrum of microbial functional traits and their integration in an environmental context are needed 3 , 9 . At this stage, existing trait frameworks on soil microbes are only based on conceptual considerations of microbial physiology, lacking data to support them 6 , 28 – 30 . Major concepts are simply derived from macro-ecological theory, which may explain their limitations to predict soil C dynamics 31 , 32 . Thus, in a first step we may monitor correlations of functional traits in microbial isolates; in a way comparable with the establishment of main trait axes in small sets of plant species under common garden experiments that took place more than 20 years ago 16 , 33 . We here address these challenges and provide insights into relevant functional traits in saprobic soil fungi. Saprobic fungi in soil represent a highly diverse microbial guild that drives C and nutrient turnover, contributes large portions of soil microbial biomass 34 , 35 and activity 36 and plays a major role in soil C sequestration 37 , 38 . Although this group exhibits a common phenotype including filamentous growth (though not exclusively) and osmotrophy, fungal natural history records reveal a broad phylogenetic diversity of soil saprobic fungi, as well as a wide variety of growth forms. Such diverse growth forms are indicative of distinct resource allocation strategies that may have significant impacts on C and nutrient cycling dynamics currently unrecognized in microbial ecology. An economics spectrum for saprobic fungi would represent a major milestone in microbial ecology. Relevant functional traits driving such a spectrum must capture the function and ecology of fungal mycelia developing in soil, characterized by exploration (exploring and connecting different resource patches) and exploitation (intensively exploiting resources with densely branched mycelia and enzymes produced) 39 , 40 . Saprobic fungi must invest resources to explore the heterogeneous soil environment, both with new hyphae formed as well as asexual spores produced, which enable fast dispersal and colonization of new resource patches 29 , 41 . In return, energy and nutrients become available for growth as simple sugars (and nutrients), or complex C sources demanding various levels of enzymatic capacity. In this respect, relevant functional traits include mycelial architecture and composition, mycelial strategies indicative of exploration vs. exploitation (including internal resource recycling and translocation within hyphae), asexual spore production, and resource uptake strategies 13 , 42 , 43 . While several authors have in theory suggested many traits fitting those criteria 44 , 45 , in practice too few trait combinations have been measured to establish general trait axes in soil saprobic fungi 25 , 46 , 47 . Therefore, we made a first attempt to establish primary trait axes of a fungal economic spectrum/space in soil saprobic fungi. We included morphological, physiological and life-history variables from a phylogenetically diverse set of fungi isolated at spatial scales where they are likely to interact (Fig. 1 ; traits were assessed in vitro). Since saprobic soil fungi interact at a much smaller scale than plants, this set of 28 saprobic fungal isolates originating from the same grassland soil (Fig. 1 , Table S3 ) provides an appropriate test group. Primary trade-offs in resource investment (e.g., acquisitive vs. conservative) should act on a local scale of species interactions, and ideally expand to larger scales 33 . The environmental relevance of the derived economics space was further tested by correlations with fundamental niche dimensions. In this study we tested whether (i) the primary fungal economics spectrum will reflect successional patterns observed in other organism groups, i.e., a slow-fast spectrum 18 , 48 , (ii) fast growth strategies will be supported by efficient hyphal strategies and rapid asexual sporulation and (iii) the main axes of the fungal economics space will relate to stress tolerance and be a good predictor for fundamental niche spaces of fungal isolates. Although our trait measurements come from artificial lab conditions (experiments were conducted in petri dishes), we believe that they represent a meaningful first step to infer the resource allocation strategies of saprobic soil fungi. Fig. 1 Visual illustration of the comprehensive fungal trait collection analyzed in this study. Trait abbreviations used throughout the manuscript are added in brackets (detailed explanation of traits can be found in Table S1 ). Some of the trait data used here represent functional traits not previously measured in saprobic fungi, others have been published in previous studies (see Table S2 ). C carbon, N nitrogen, P phosphorus, S sulfur, Mg magnesium, K potassium, X nutrient, PC principal component axis, PLFA phospholipid fatty acids, PDA potato dextrose agar, WA water agar, Cu copper, mg milligram.",
"discussion": "Results and discussion Functional traits forming the fungal economics spectrum We assembled a large dataset to describe a fungal economics spectrum for 28 (potentially) co-occurring soil fungal isolates, including several traits that have been assessed for the first time in fungi (to the best of our knowledge; Fig. 1 , Table S1 ). Despite the strength of this large trait collection, it would not be expedient to include every available trait, also due to the complex correlation structure among them (Fig. S 1 ). Instead, functional traits capturing most variation and being ecologically significant in soil were to be selected. This has been a successful strategy in plants 16 ; though even in plant ecology—despite extensive trait databases—it is a long-lasting ongoing endeavor to define these traits 18 , 49 , 50 . Here, we defined primary trait axes starting with a randomized principal component analysis (PCA) method. This approach revealed a consistent first principal component (PC) axis capturing a continuous spectrum of slow to fast growing fungal isolates (Fig. S 2 , S 3 ). While slow fungi were characterized by dense highly-branched mycelia, fast fungal isolates showed high mycelial extension rates, i.e., investment to rapid tip growth. This primary axis likely represents a physiological trade-off based on the simple nature of mycelial development (characterized by tip growth, branching and negative autotropism 39 ), but also reflects the fungal strategies of exploitation versus exploration in soil. The correlation (PC loadings) of further functional traits with this 1st PC axis revealed an overall trait continuum from slow growing (dense) fungi with high melanin contents and larger enzymatic capacities to fast growing competitive fungi, characterized by high sporulation, yield, and stress tolerance (Fig. 2 ). The low average loadings of many traits on this 1st axis further indicated the significance of additional relevant functional trait axes. Fig. 2 Alignment of all fungal traits with the primary dense-fast spectrum in saprobic fungi based on a randomized principal component analysis (PCA) approach. Bars display average loadings of individual functional traits on PC1 (principal component axis 1) based on 10,000 PCA repeats with random inclusions of 10 traits, in addition to extension and density. Abbreviations from left to right: enz_cel enzymatic activity of cellobiohydrolase, soil_aggreg soil aggregate formation ability, enz_C_div C enzyme diversity, asegurl ability to switch to explorative growth under resource limitations, WA_explor investment to exploration, enz_lac enzymatic activity of laccase, leaf_decomp leaf litter decomposition, decomp PC1 1st PC axis of leaf and wood decomposition ability, complC_use complex carbon use, wood_decomp wood decomposition, water_cont water content, stoich_C_X PC1 1st PC axis of stoichiometric C:X (nutrient) ratios, stoich_N_X PC1 1st PC axis of stoichiometric N:X (nitrogen: nutrient) ratios, stoich_flex stoichiometric flexibility, C_cont fungal C content, PLFAc total PLFA concentration, enz_pho enzymatic activity of phosphatase, hyphal_diam hyphal diameter, hydrop mycelial hydrophobicity, CUE carbon-use efficiency, biomass complC fungal biomass on complex C sources, biomass st fungal biomass under standard conditions, cu_str stress tolerance to copper, spore_RRx relative change in spore production under nutrient scarcity, enz_leu enzymatic activity of leucine aminopeptidase, stoich_N_X PC2 2nd PC axis of stoichiometric N:X (nitrogen: nutrient) ratios, drought_str stress tolerance to drought; DNAc: DNA concentration, spore_abund spore abundance, biomass opt fungal biomass under optimal conditions, fungic_str stress tolerance to fungicide, stress_tol PC1 1st PC axes of stress tolerance (copper, drought, fungicide), stress_tol av average stress tolerance (copper, drought, fungicide), comp_glu competitive ability under glucose supply. In order to analyze the distinct patterns of a complete fungal economics space in more detail, we then selected traits with high functional and ecological significance for saprobic fungal growth in soil (Fig. 3 , see methods section). The prior reduction of trait variables did not reduce the number of PC axes being significant (Fig. S 4 ), making us confident that selected traits captured the fungal economics space well. In this final PCA (Fig. S 5 ), the loadings of functional traits on PC axes clustered as distinct ecological groups of trait spectra (Fig. 3a ). The first PC axis—representing the largest variation present in the data (29%)—roughly reflected the dense-fast continuum described above (Figs. 2 and 3a , S 5 ). Additionally, two further axes were statistically significant: The second PC axis was mainly formed by traits related to mycelial strategies, while the third axes showed high loadings of C acquisition traits (Fig. 3a ). These patterns led to distinct ecological clusters of trait variables (Fig. 3a ), coinciding with stronger correlations among these trait variables (Fig. S 1 ). Fig. 3 Visualization of the main three-dimensional fungal economics space in saprobic fungal isolates based on functional traits. a Heatmap visualization of the main principal component analysis (PCA, Fig. S 5 ), with loadings of individual traits on PC axes being displayed, as well as their groupings (shown as correlogram based on hierarchical cluster analyses of loadings). The column width corresponds to the eigenvalues of PC axes (insignificant axes are shown in gray scales), asterisks indicate significant loadings of traits on respective axes (based on PCAtest 93 ). Red colors indicate negative, blue colors positive loadings, with color intensity referring to respective loading values. The clustering groups of traits reflect their loadings on varimax rotated components (RC), highlighted by gray squares. Fungal economics space defined by PCA followed by varimax rotation, showing the rotated components (RC) RC1 and 2 ( b ) and RC2 and 3 ( c ). Arrows represent eigenvectors of traits on RC axes, dots individual isolates (shapes of dots indicate phylogenetic placement). For details on functional traits see Fig. 1 and Table S1 ; abbreviations are given in Figs. 1 and 2 ). In order to maximize the interpretation of functional trait axes representative of the fungal economics space we implemented a varimax rotation (a simple rotation maintaining the orthogonal (linearly uncorrelated) structure of PC axes; Fig. 3 , Fig. S 5 ). This led to three RC (rotated component) axes, each reflecting distinct ecological trait spectra. The first axis (RC1 dense-fast) represented a clear slow-fast continuum (Fig. 3b ), here referred to as the dense-fast spectrum: A continuum of slow growing (dense and highly branched, i.e., exploitative) fungi to fast growing, competitive, extensively sporulating fungal isolates (explorative) 39 . The dense side of the spectrum was further characterized by high melanin contents, as well as an ability to switch to explorative growth under resource limitation which to our knowledge is a novel trait (we refer to it with the abbreviation ‘asegurl’; Fig. S 6 ). The fast side coincided with high stress tolerance, here defined by a low growth reduction in response to different stressors (i.e., copper, drought and fungicides). A second axis (RC2 flexibility) emerged from traits representing mycelial growth strategies, i.e., internal resource recycling and flexible growth, as well as stoichiometric flexibility in response to resource limitations. These variables relevant for efficient mycelial growth were assessed as a functional trait in saprobic fungi; contrary to our hypothesis, though, efficient growth strategies were not correlated with the fast spectrum (Fig. 3b ). Instead, this axis was related to efficient biomass production and high carbon-use efficiency (CUE). And finally, the third axis (RC3 C acquisition) primarily covered C acquisition traits (Fig. 3c ), including enzymatic capacities and the ability to use more complex C sources, which also positively correlated with litter decomposition ability 51 . Since these first three axes were statistically significant and together defined the main functional trait space of fungal isolates tested here, we believe they provide robust support to define the three-dimensional fungal economics space in our set of fungi. Fungal isolates were evenly distributed throughout this trait space without forming separate functional groups (Fig. 3 , as found also in previous fungal trait studies 40 , 52 ), which supports the idea of continuous spectra rather than distinct life-history categories. The significance of functional trait axes for soil processes Even though our data clearly confirm distinct continuous axes forming an economics space present in soil saprobic fungi, it is still interesting to note that these main axes correlate to life-history strategies proposed by classical life-history frameworks like C-S-R, or especially its counterpart for soil microbes, the Y-A-S (yield-acquisition-stress tolerance) 6 , 12 . The first axis (RC1 dense-fast) observed here involves stress tolerance (S), the second axis (RC2 flexibility) yield (Y), and the third axis (RC3 C acquisition) acquisition traits (A). However, contrary to the basic idea of these trait frameworks, described strategies did not form trade-offs, but were found on orthogonal (independent) trait axes. This result contradicts some basic assumptions underlying currently discussed life-history frameworks in soil 14 : A lack of direct trade-offs among these strategies (especially C cycling traits with stress responses) would reduce its predictive value in trait applications (see discussion about resilience below). Classical life-history frameworks are particularly relevant in soil, because they directly link microbial traits to soil processes. Similarly, each functional trait axis described here corresponds to relevant soil processes (Fig. 3 ), for example melanin contents and soil aggregation ability loaded on the dense side of the first axis (RC1 dense-fast). Melanin contents reduce decomposition rates of fungal necromass, and may play a significant role in soil organic C sequestration 53 , while soil aggregation is integral for soil health and C stabilization 54 . The second axis of the fungal economics space (RC2 flexibility) was mainly characterized by flexible mycelia (in terms of C:N ratios, but also explorative growth) and internal resource recycling capacities. These traits correlated with higher yields—fungal biomass and also CUE (Fig. 3 )—likely caused by efficient growth due to internal recycling 42 , but also by a potentially larger subsequent fraction of inactive/dead C-enriched hyphae that increase CUE values 55 , 56 . Regarding C acquisition/mineralization traits, we anticipated that they would be captured with the dense-fast spectrum—showing trade-offs with stress tolerance or fast growing early successional traits 52 , 57 . However, even though slow/dense isolates seem to have high enzymatic capacity (Figs. 2 , 3a ), this does not necessarily translate to higher mineralization rates. Litter decomposition only correlated with the third axis of the fungal economics space (RC3 C acquisition, Fig. 3c ), best predicted by the ability of fungi to use more complex C sources 51 . Stress tolerance traits are supposed to directly link soil process related traits to environmental change. We found stress tolerance to be associated with the fast (explorative) side of the first axis (RC1 dense-fast). Ecologically, the rapid colonization of new resource patches by fast (early successional) fungi exposes them to a wide range of environmental conditions. Being tolerant to environmental stressors and having wider niche breadths (Fig. 4c ) will be beneficial for this strategy. Across other organism groups stress tolerance has also been associated with the fast side of the economics spectrum 58 – 60 . However, we here studied stress tolerance defined as the ability to maintain growth under sublethal conditions/moderate stress. Longevity and survival of hyphae are likely captured by the slow/dense side of the spectrum—if fungi follow the universal pattern found in other organism groups that slow growth correlates with longevity 48 , 60 . Indeed, high melanin contents (costly hyphae, Fig. 3 , S 1 ) on the dense side of the gradient support this hypothesis: Melanin is a complex biomolecule relevant for survival in extreme habitats 61 . Analyses of melanin contents in a large collection of basidiomycete fungi by ref. 62 confirmed these patterns: A similar trade-off in melanin production with fungal growth rates as well as with genes associated with stress tolerance was observed. Fig. 4 Visualization of the fungal fundamental niches and their correlations with functional traits. a Position of fungal optimal growth within the three-dimensional fundamental niche space. Dots represent individual isolates, shapes reflect phylogenetic placement (round: Ascomycota, square: Basidiomycota, triangle: Mucoromycota, diamond: Mortierellomycota). b Niche breadth of individual fungal isolates in the three-dimensional fundamental niche space. Edge lengths of the boxes correspond to respective niche breadths within each fundamental niche gradient, defined as >25% of maximum growth. Fungal isolates (individual graphs) are sorted by the scores of isolates on RC1 (rotated component 1, Fig. 3b ), dark colors correspond to low values (dense mycelial growth), light gray colors to high values (fast mycelial growth). c Direct correlations of RC axes and individual functional traits with niche traits. Dot size and color correspond to correlation coefficients r obtained by Pearson´s or Spearman rank correlations—only significant values are displayed ( P < 0.05). Crosses indicate significant predictors for the position of fungal optima within the three-dimensional fundamental niche matrix (permutation multivariate analyses of variances (RC axes) and stepwise model selection based on redundancy analyses (individual functional traits)). Effects of fundamental niche gradients on the relative distribution of RC scores, calculated as arithmetic means of RC scores weighted by the relative abundance of fungal isolates along the fundamental niche gradients of C:N supply ( d ), temperature ( e ), and water availability ( f ) (Fig. S 7 ). Lines represent outputs from generalized additive models. Water potential was modeled only to −1.2 MPa, below this value data were driven by two isolates only. For trait abbreviations see Figs. 1 and 2 ; niche matrix: optima in the three-dimensional niche matrix; N_opt, temp_opt, water_opt: isolate-specific optima in C:N (N), temperature (temp) and water availability gradients; N_breadth, temp_breadth, water_breadth: isolate-specific niche breadth in respective gradients; niche_cube: three-dimensional niche space for individual isolates (Table S1 ). Fungal trait distribution in fundamental niche spaces To further explore the ecological significance of the fungal economics space, we correlated functional trait axes with the fundamental niche position and breadth of individual isolates. Niche breadth and tolerance to suboptimal growth conditions have been previously used as a surrogate for stress tolerance 47 , but more importantly provide insights into potential functional shifts in fungal communities to changing environmental conditions 20 . Beside certain individual functional traits, the first functional trait axis (RC1 dense-fast) of the fungal economics space significantly correlated to fundamental niche optima and breadths (Fig. 4c ). The position of fungal isolates along the first axis of the fungal economics space not only correlated with their position in the fundamental niche space, but also with a fungal optimum at lower water potentials (dryer conditions), associated with wider niche breadths in this gradient. By contrast, fast-growing fungal isolates showed a narrower niche breadth in respect to nitrogen availability (Fig. 4c ). The second axis (RC2 flexibility) was positively correlated with optima at low N supply and wide niche breadths along the C:N gradient, while the third axis (RC3 C acquisition) showed no significant correlation with any niche traits (Fig. 4c ). To effectively describe the correlation of trait axes to fundamental niche gradients, we modeled the relative abundances of individual isolates at different fundamental niche positions (Fig. S 7 ) and calculated respective weighted average RC scores along fundamental niche axes (isolate scores on the three RC axes of the fungal economics space (Fig. 3 ) under different conditions (Fig. 4d–f )). This modeling approach revealed that the relative abundance of isolates with positive scores on the dense-fast spectrum was greater under high N supply (low C:N), and slightly increased at temperatures deviating from the optimum (average fungal optimum at 25 °C (Fig. S 7 )). Noteworthy, scores on the second axis (RC2 flexibility) most strongly shifted along fundamental niche (resource) gradients: Fungal isolates with high scores on RC2 showed relatively less biomass reduction under low resource supply, i.e., high C:N and low water potential. Flexible mycelial strategies thus sustained growth under nitrogen and water limitations. By contrast, the scores indicative of C acquisition traits (RC3) responded least to fundamental niche gradients. Proposed classification of saprobic fungi in terms of life-history strategies Even though the traits involved in the fungal economics space are specific to saprobic filamentous fungi, it does mirror life history strategies established in other organism groups. In summary, the primary axis of the fungal economics space represents a trade-off between the strategy of fast growing fungi competitive with simple sugars, versus slow growing, potentially long lived exploitative fungi with the ability to use more complex C sources. The extremes of this spectrum resemble some classical functional categories like Guerilla/Phalanx or copiotrophy/oligotrophy 22 , 40 , but the nature of the primary trait axis indeed better fits to the concept of the slow-fast or trait economics spectrum described in plants and animals 16 , 18 , 60 , 63 . The concept of the economics spectrum primarily describes a trade-off between the cost of structure and rate of resource return, which leads to a successional differentiation 17 , 18 : Slow growth, structural investment, longevity and slow but prolonged resource return (late successional) versus fast growth, short-lived and quick resource return (early successional). In plants, this reflects the interpretation of the leaf or fine root economics spectrum along a conservative-acquisitive spectrum (slow vs. fast return). In the proposed first axis of the fungal economics spectrum, a similar pattern is associated with slow growing dense fungi with higher investment to structural components (e.g., melanin) and enzyme capacities (late successional in a soil context), whereas fast growth is linked to quick resource return by the use of simple sugars, “cheap” structures (no melanin, no other evidence so far) and intense sporulation to allow rapid colonization of new patches (early successional). Such a differentiation must be understood in light of the heterogeneous nature of soil, with high spatial and temporal fluctuations leading to rapid succession of small-scale resource patches. Previous studies describe a dominance-tolerance trade-off in soil fungi 45 , 47 , and sort fungal species according to their competitive ability and stress tolerance into life-history strategies 13 , 24 , 29 , 64 . However, competitive ability measured in artificial media (comp_glu) 47 only expresses fungal dominance in the presence of simple sugars, which allows fungi to quickly colonize productive environments (early successional) and outcompete other species 22 (by fast resource uptake and/or the production of defense compounds). Under more complex C substrate conditions though, high enzymatic capacities may characterize superior competitors (late successional) 6 , 46 . Consequently, fungi at both ends of the dense-fast spectrum can be competitive, just at different successional stages (Fig. 5a ). Similarly, stress tolerance can be widely defined as a tolerance (maintenance of biomass production) to unfavorable conditions, but is used in practice interchangeably for environmental stressors and resource limiting conditions 6 , 27 . While fast growing isolates seem to be more tolerant to moderate anthropogenic stressors, fungi with dense mycelium likely can survive extreme conditions, and better cope with stress caused by a lack of rich C or nitrogen sources (Fig. 4d ). Mycelial flexibility (RC2), on the other hand, helps to maintain growth under nutrient or water limitations (due to the ability to recycle internal resources and flexibly adjust mycelial strategies to resource conditions). In conclusion, the primary trade-offs observed in this study seem to be driven by niche differentiation among fungal isolates in a successional context: The three-dimensional fungal economics space, and the relative importance of axes in terms of variability explained, indicate that successional trade-offs are the primary driver of the evolution of fungal life-history strategies—a universal pattern across organism groups 18 . Further trait variability in resource or carbon use efficiency (2nd and 3rd axes) seem to be secondary factors leading to further niche differentiation and diversification in soil saprobic fungi. Fig. 5 Conceptual overview of the three-dimensional fungal economics space and its significance in an environmental context. a The fungal economics space based on the fungal isolates tested here, with characteristics of the dense-fast spectrum displayed. Dots visualize the position of fungal isolates in the three-dimensional PCA (principal component analysis) space. Drawings show deduced mycelial structures (and spore production) at the extremes of the continuum. b Illustration of the proposed mechanism of functional resilience due to the orthogonal nature of main functional trait axes and their correlation to ecological strategies relevant in soil. Dots indicate a hypothetical modeled community within boundaries of the fungal economics space and the abundances of individual species/isolates in response to a moderate anthropogenic stressor (dot sizes correspond to abundances, red colors indicate negative responses to stressors). Notably, there is no shift in abundances along the second and third axis, which are independent of stress responses. Predicted responses in soil processes are indicated by arrows (round symbols = lack of response). SOC soil organic carbon, CUE carbon-use efficiency. Ecological significance of the fungal economics space The underlying assumption of models and theories addressing the role of microbial life-history strategies on C cycling rely on trade-offs among ecologically relevant traits 6 , 27 , 29 . By contrast, we observed that some major ecological strategies maintained “orthogonal” relationships with each other, which was manifested through uncorrelated PC axes (Fig. 5 ). We are aware that these established trait axes are based on a limited number of isolates, derived from one site only (a compromise to achieve in depth trait analyses) and future studies including more species are necessary to validate its generalizability. Still, our results indicate that variation in traits relevant for C dynamics in soil are partly uncorrelated to environmental stress responses. In our data, stress tolerance was primarily associated with the first axis. Consequently, the axes of the fungal economics spectrum representing C acquisition, as well as mycelial flexibility (mainly correlated to yield and CUE) are independent of certain stressors (Fig. 5b ). No functional shifts in these parameters of C cycling would be expected despite apparent community shifts—potentially explaining the often observed functional resilience in soil 65 . Resilience in this context refers to functional stability despite microbial community shifts under environmental change, a phenomenon that in the past led to the idea of functional redundancy 65 , 66 . In this novel mechanism described, though, fungal isolates are not functionally redundant/similar 65 (redundancy is defined as the “ability of one microbial taxon to carry out a process at the same rate as another under the same environmental conditions” 67 ). Instead, stress-tolerant fungi are as good or as bad in C cycling functions as fungi negatively affected by the stressor (Fig. 5b ). This type of resilience applies to some stressors tested in this study, as well as to environmental growth conditions that shifted scores on the first axis of the fungal economics spectrum (Figs. 3 , 4 ), and the functional traits assessed. A multitude of other relevant functions of soil fungi have not even been measured in this study; only the diversity of functional trait expressions among isolates mirrors their diverse functional roles in soil (Fig. S 9c ), with all of them potentially relevant in the maintenance of sustainable soils 27 , 68 . This high diversity and especially uniqueness of fungal isolates, in both functional traits (Fig. S 9c ) and fundamental niches (Fig. 4a, b ), undermines the idea that losses of microbial species have no immediate functional consequences (as suggested by the concept of functional redundancy 69 ). This may be the case in the short-term for specific functions under stable environmental conditions 70 , 71 . However, the large number of functional traits assessed here gave insights into the full diversity in functional trait spaces covered. Each isolate did not only occupy a separate position in the trait space (Fig. 3 ), but also showed extreme values in one or few of the traits analyzed (Fig. S 9c ). In parallel, isolate-specific growth optima were evenly and widely distributed throughout the fundamental niche space, and each isolate occupied its very own niche space (Fig. 4a, b ). The soil environment can be seen as a heterogeneous, temporally and spatially fluctuating mosaic with seemingly endless combinations of niches, which apparently translates to a large diversity of functional and niche traits in these co-occurring fungi contributing to microbial co-existence, where each species fills a unique niche. Critical insights for future studies By measuring a large and comprehensive set of traits, we were able to define a fungal economics space in tested fungal isolates and establish potential shifts in functional diversity along environmental gradients. Functional traits specific to filamentous fungal growth in heterogeneous soils defined the main three axes of the fungal economics space, namely dense versus fast growth, mycelial flexibility and C acquisition. In support of our hypotheses, the primary axis of the three-dimensional fungal economics space revealed a slow-fast spectrum in fungi similar to other organism groups, and reflected potential successional patterns of saprobic fungi growing in complex soils. Mycelial flexibility and also C acquisition traits, both discussed as being highly relevant for fungal growth and its effects on soil processes, were largely independent of this primary axis. Consequently, proposed microbial life-history strategies did not form classical trade-offs, but were found on independent orthogonal trait axes, questioning the predictive power implied by current life-history frameworks applied to soil. To critically examine the general validity of the fungal economics space described here, we suggest testing these patterns with broader taxonomic and geographic coverage, as it has been done in the past for plants 16 . Future studies may focus on a few primary traits representative of main trait axes (Fig. 3 ). Following the establishment of main trade-offs in phenotypic functional traits here, further analyses may also take advantage of -omics data 22 , 64 . Recent progress in metagenomics reveals a promising path to connect community composition to function, though the significance of these data in respect to phenotypic trait expressions must be further analyzed 27 , 59 . To keep the concepts relevant for soil biogeochemical models, the range of functional guilds included should be ecologically meaningful: We here worked with saprobic fungi that contribute to C cycling in soil to varying degrees, reflecting the functional (and phylogenetic) diversity covered in fungal sequencing studies in grasslands or agricultural soils 72 . In this set of soil fungi, the functional diversity along with fundamental niche differentiation reflects the complex dynamics acting on soil microorganisms. Unlike in classical successional litterbag or wood decomposition studies, the soil habitat is shaped by constant input and turnover of small heterogeneous resource patches coupled with small-scale variations in environmental conditions. This diversity results in rapid shifts among successional stages, and likely drives the observed trait spectrum, its continuous nature, and the unique niches of co-occurring soil fungal isolates. To make these findings applicable to soil science, relations among functional strategies must also be resolved for bacteria 6 . This work may provide interesting starting points: Since soil bacteria are exposed to the same complex soil environment, similar patterns may be expected 28 , especially since the main slow-fast spectrum appears universal across different organism groups 22 , 60 . Interactions among bacteria and fungi (as well as other trophic groups) will likely further influence the trait expressions and trade-offs described. This dynamic is further nuanced by the intricate complexity inherent in heterogeneous soil environments, which will be important to implement in the application of life-history frameworks in soil microbial communities. Technically, many functional traits can only be assessed in vitro, which makes this design valuable to establish physiological trait trade-offs in mycelia. It is also likely that physiologically inherent growth patterns will translate to isolate-specific dynamics in soil, especially regarding the successionally driven primary axis of the proposed fungal economics space. Still, evaluating the validity of key traits in real soil systems must be incorporated in future experimental designs, e.g., using artificial/sterile soil systems or soil-pore like microchips 73 , 74 in combination with metagenomic and –transcriptomic soil analyses. In light of the complexity present in soil, the relatively clear fungal economics space we find here is still intriguing. Soil biogeochemical models are advancing rapidly and start to include microbial functional groups, making such insights into functional dynamics in microbial communities most relevant 5 , 6 . We hope the fungal economics space will inspire microbial ecologists to invest more into the definition of relevant functional strategies in soil, and more precisely define microbial functions that can (or cannot) be predicted based on trade-offs. These results also add an interesting mechanism of functional resilience in soil, which potentially provides a relevant buffering effect towards certain global change factors 66 . Still, the uniqueness of the functional niche space that we found also highlights potential consequences of microbial diversity loss for long-term soil sustainability."
} | 10,588 |
30483277 | PMC6240842 | pmc | 2,652 | {
"abstract": "Plant root symbiosis with Arbuscular mycorrhizal (AM) fungi improves uptake of water and mineral nutrients, improving plant development under stressful conditions. Unraveling the unified transcriptomic signature of a successful colonization provides a better understanding of symbiosis. We developed a framework for finding the transcriptomic signature of Arbuscular mycorrhiza colonization and its regulating transcription factors in roots of Medicago truncatula . Expression profiles of roots in response to AM species were collected from four separate studies and were combined by direct merging meta-analysis. Batch effect, the major concern in expression meta-analysis, was reduced by three normalization steps: Robust Multi-array Average algorithm, Z-standardization, and quartiling normalization. Then, expression profile of 33685 genes in 18 root samples of Medicago as numerical features, as well as study ID and Arbuscular mycorrhiza type as categorical features, were mined by seven models: RELIEF, UNCERTAINTY, GINI INDEX, Chi Squared, RULE, INFO GAIN, and INFO GAIN RATIO. In total, 73 genes selected by machine learning models were up-regulated in response to AM (Z-value difference > 0.5). Feature weighting models also documented that this signature is independent from study (batch) effect. The AM inoculation signature obtained was able to differentiate efficiently between AM inoculated and non-inoculated samples. The AP2 domain class transcription factor, GRAS family transcription factors, and cyclin-dependent kinase were among the highly expressed meta-genes identified in the signature. We found high correspondence between the AM colonization signature obtained in this study and independent RNA-seq experiments on AM colonization, validating the repeatability of the colonization signature. Promoter analysis of upregulated genes in the transcriptomic signature led to the key regulators of AM colonization, including the essential transcription factors for endosymbiosis establishment and development such as NF-YA factors. The approach developed in this study offers three distinct novel features: (I) it improves direct merging meta-analysis by integrating supervised machine learning models and normalization steps to reduce study-specific batch effects; (II) seven attribute weighting models assessed the suitability of each gene for the transcriptomic signature which contributes to robustness of the signature (III) the approach is justifiable, easy to apply, and useful in practice. Our integrative framework of meta-analysis, promoter analysis, and machine learning provides a foundation to reveal the transcriptomic signature and regulatory circuits governing Arbuscular mycorrhizal symbiosis and is transferable to the other biological settings.",
"conclusion": "Conclusion In this study, we developed a new approach for reducing heterogenicity between experiments (batch effect) and direct merging meta-analysis by combining meta-analysis, multi-step normalization, and supervised attribute weighting models. We employed this approach to obtain a unified transcriptomic signature of successful AM colonization in roots of Medicago truncatula . The genes of identified in the signature, derived by integration of meta-analysis with supervised attribute weighting models, were strongly up-regulated in all AM symbioses and probably correspond to the end targets of the symbiotic programme. The identified meta-genes of successful AM colonization discriminated efficiently between AM inoculated and non-inoculated samples. Furthermore, the developed signature showed high performance in distinguishing AM-colonized roots from non-inoculated ones in an independent RNA-seq experiment. Important protein classes such as the AP2 domain class transcription factor (MTR_6g029180), GRAS family transcription factors (MTR_1g069725 and MTR_2g089100), and cyclin-dependent kinase (MTR_1g098300) were highly upregulated during AM successful colonization. The developed direct merging-based meta-analysis, by combining meta-analysis, multi-step normalization, and supervised attribute weighting models, provides the possibility of data collection from different experiments even when a treatment or a control is missing in one or more of the experiments. We suggest that the promoters of meta-genes identified in the transcriptomic signature of AM colonization may have the power to unravel key transcription factors as master regulators of AM symbiosis. Analysis of promoter regions of the top upregulated meta-genes in the AM-successful colonization signature in this study identified enriched transcription factor binding sites and led us to possible master regulators that form the transcriptome expression pattern. These included AP2 domain class transcription factors, CCAAT-binding family transcription factors, SEF transcription factors, and response to fungus ASRC transcription factors. Further functional characterization of these transcription factors is needed to understand their precise role in AM symbioses. This study provides a framework for an improved understanding of the dynamics of successful AM colonization in establishing microsymbionts. It offers a new approach for related investigations into the other symbiosis systems.",
"introduction": "Introduction Arbuscular mycorrhiza (AM) fungal symbiosis expands the surface area of plant root, allowing for better absorption of substances such as phosphorus, ammonium, and zinc from soil. This symbiosis supports plant development, particularly under nutrient deficiency and other stressful conditions. Specific genetic programs activated by AM inoculation lead to successful microsymbiont colonization and functional symbiosis. Most studies in AM symbiosis are limited to the investigation of a single gene or a cluster of similar genes. Genes such as DMI1, DMI2, NFP, NSP1 (Oláh et al., 2005 ), MtBcp1 (Hohnjec et al., 2005 ), ENOD11 (Genre et al., 2005 ), MIG1 (Heck et al., 2016 ), RAM1 (Rich et al., 2017 ), nfr1, nfr5 , l ys11 (Rasmussen et al., 2016 ), and NIN (Guillotin et al., 2016 ) are reported to play roles in the formation of mycorrhizal symbiosis. The regulatory mechanisms underpinning AM symbiosis in plants are poorly understood. The GRAS transcription factor family contains the best known regulators of AM symbiosis. The function of ATA/RAM1 , a member of this family, in reprogramming AM symbiosis has been established (Rich et al., 2017 ). It has been suggested that RAM1 controls the expression of many essential AM-related genes such as STR, STR2, RAM2 , and PT4 (Rich et al., 2017 ). Another member of the GRAS transcription factor family, MIG1 , interacts with DELLA1 and the root GA signaling pathway to regulate cortical cell expansion in developing AM symbiosis (Heck et al., 2016 ). The role of small RNAs, such as miR171 in establishment of AM symbiosis has also been investigated recently (Couzigou et al., 2017 ). Successful AM colonization is vital to establish symbiosis and improve phosphorous and water uptake. The AM type, as well as many, environmental and genetic factors affect the intensity, timing, and the success of AM colonization. Cross-comparison of successful colonization between different AM types in a range of experiments by meta-analysis provides the opportunity to move toward understanding the genetic basis of endosymbiosis (Tromas et al., 2012 ), the conserved transcriptomic program that can reflect successful AM colonization and establishment. Those genes can unravel the functional groups that may play key roles in the establishment and functioning of the three AM symbioses. The transcriptomic signature of AM colonization can be further employed for: (1) increasing AM efficiency by application of chemical and environmental treatments, (2) monitoring successful/unsuccessful AM colonization, and (3) finding the upstream regulatory mechanisms and regulators such as transcription factors and microRNAs that control AM colonization and symbiosis. However, no attempt has been made to identify the unified transcriptomic signature of AM symbiosis. The term of “Unified transcriptomic signature” or “biosignature” refers to robust transcript responses that can monitor the successful AM colonization. Overlaps observed in transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi (Hohnjec et al., 2005 ) support the possibility of achieving a unified transcriptomic signature of AM colonization to provide an insight into the genetic program activated during AM. The emerging field of meta-analysis may solve the issue of merging different experiments to identify a unique biosignature of Medicago root response to AM inoculation. Cross-species meta-analysis of transcriptomic data has received increased attention in recent years due to the advances in pattern discovery and meta-analysis models (Tromas et al., 2012 ; Farhadian et al., 2018b ). Meta-analysis enables the combination of expression datasets and is highly advantageous in increasing statistical power to detect biological phenomena from studies with a restricted sample size (Johnson et al., 2007 ). The biosignature of AM inoculation obtained may be utilized to further computational systems biology analysis, such as promoter analysis, common regulator discovery, and common target discovery, in order to lead us to the key regulators and targets of the AM symbiosis pathway. Different statistical methods have been developed for meta-analysis of expression data such as combining effect sizes, combining ranks, combining p -values, vote counting, and direct merging (DM) (Borenstein et al., 2009 , 2010 ; Campain and Yang, 2010 ; Chang et al., 2013 ; Sharifi et al., 2018 ). Within meta-analysis approaches, DM analysis of expression data or genomic variant data of different studies is an attractive meta-analysis method to increase statistical power and lead to a robust transcriptomic or genomic signature (Tseng et al., 2012 ). DM, as a meta-analysis approach, has been used in web-tools such as INMEX (Xia et al., 2013 , 2015 ), A-MADMAN (Bisognin et al., 2009 ), WGAS (Dai et al., 2007 ), and GEOSS (Bisognin et al., 2009 ) for integrative meta-analysis of expression data. DM-based meta-analysis provides the possibility of data collection from different experiments, even when a treatment or a control is missing in one or more experiments. This contributes to a higher statistical power of meta-analysis. The major concern about the DM approach is heterogenicity across studies. The success of the DM approach depends on normalization across studies to reduce non-biological experimental variation as well as biological variations unrelated to treatment (also called batch effects or study effects) (Johnson et al., 2007 ; Tseng et al., 2012 ). Collection of arrays from similar platforms across all studies (mainly Affymetrix) and pre-processing of the CEL expression files by model-based robust multi-array (RMA) normalization (Irizarry et al., 2003 ) have been suggested to decrease heterogenicity across all studies (Lee et al., 2008 ; Sims et al., 2008 ; Tseng et al., 2012 ). However, it has been debated that RMA is not strong enough to remove batch effects (Guerra and Goldstein, 2009 ). To sufficiently reduce batch effects for accurate DM, additional normalization techniques such as empirical Bayes methods (Johnson et al., 2007 ), cross-platform normalization (Shabalin et al., 2008 ), weighted distance weighted discrimination (Qiao et al., 2010 ), enrichment-based meta-analysis, and Ratio adjustment and calibration scheme (Cheng et al., 2009 ) have been used. Recent advances in application of supervised machine learning models in transcriptomic studies have opened a new venue to engage data mining models in decreasing batch effects and integration of different studies (Pashaiasl et al., 2016a , b ). Supervised machine learning has brought new possibilities to predictive studies (Bakhtiarizadeh et al., 2014a ; Ebrahimi et al., 2014 ; Zinati et al., 2014 ; Kargarfard et al., 2015 ; Pashaiasl et al., 2016a , b ). The capability to simultaneously analyse both categorical and numerical features, power to analyse large data, and various predictive algorithms with diverse statistical backgrounds are distinguished features of supervised machine learning models (Shekoofa et al., 2014 ; Ebrahimi et al., 2015 ; Jamali et al., 2016 ). The possibility to include the categorical variables in predictive models can outstandingly decrease the heterogenicity across studies as the batch effects (Shekoofa et al., 2014 ). For example, in this study, the different experiments or types of AM can be added as variables and analyzed in the predictive model of the AM transcriptomic signature. This possibility is highly limited in traditional multivariate or regression models. Due to the central role of colonization in establishing a microsymbiont, we developed a framework for finding the transcriptomic signature of successful AM colonization on roots of Medicago truncatula by integration of meta-analysis and machine learning (attribute weighting) models. Special attention was paid to reducing the batch effects by utilizing normalization methods and finding reliable gene candidates by machine learning models. The genes discovered in the transcriptomic signature were further used as the input of promoter analysis to identify the transcription factors which regulate the signature.",
"discussion": "Discussion Finding a biosignature/predictors based on a single transcriptomic experiment is a major challenge due to a large prediction error caused by a large number of independent predictors (genes) and a restricted number of observations (replications) (Baseri et al., 2011 ). Also of concern is the repeatability of a selected subset of a gene derived from a single experiment/condition. Inter-species analysis of a range of experiments by meta-analysis and machine learning techniques is able to deal with theseshortcomings, leading to the generation of a robust and repeatable biosignature (Farhadian et al., 2018b ). Meta-analysis has received increased attention in recent years because of its remarkable potential to increase the statistical power and generalizability of single study analysis (Farhadian et al., 2018a ; Sharifi et al., 2018 ). Meta-analysis not only reinforces the findings of the individual studies, but is also may identify new undetected outcomes/patterns in single studies as meta-analysis considers the direction/trend of variables in each experiment to find the consistent, robust and repeatable patterns in all experiments (Sharifi et al., 2018 ). The inter-species DE-based meta-analysis employed in this study had more samples and stronger statistical power and was successful in achieving a statistically-reliable transcriptomic biosignature of successful AM inoculation, independent from the study. In addition, the biosignature was repeatable and discriminative when a new and independent RNA-seq experiment was used for its validation. Due to the availability of Medicago truncatula transcriptomic data (as a model plant), the meta-analysis was solely performed on this plant resulting in the identification of a robust and high performance transcriptomic signature of AM colonization. However, in non-model plants with the subsequent generation of new transcriptomic data, it will be necessary the identified Medicago truncatula -derived transcriptomic signature of AM colonization will need further examination. In DE-based meta-analysis, it is crucial to adjust for batch effects before combining expression datasets. Heterogenicity (batch effects) is the major concern in meta-analysis of expression data (Leek and Storey, 2007 ; Ramasamy et al., 2008 ). In this study, we developed a new approach for reducing batch effects and direct merging meta-analysis by combination of meta-analysis, multi-step normalization, and supervised attribute weighting models. We observed that quartiling outperforms the scaling approach in reducing the batch effect. Heterogenicity-reducing based on the quartiling approach has been used extensively for knowledge discovery and pattern recognition in large data analysis, particularly in integrated classification and association-rule mining (CBA) algorithm (Kargarfard et al., 2015 , 2016 ). As an example, CBA analysis of quartiled protein and DNA measurements was able to find a biosignature for increased host range and the emergence of an outbreak in influenza (Kargarfard et al., 2016 ). Supervised machine learning has brought new possibilities to predictive studies (Bakhtiarizadeh et al., 2014a ; Ebrahimi et al., 2014 ; Zinati et al., 2014 ; Ebrahimie et al., 2018b ). Supervised attribute weighting (feature selection) algorithms are techniques for reducing the variables and identifying a subset of highly relevant ones in order to improve the efficiency of classification algorithms (Rosario and Thangadurai, 2015 ). The capability to simultaneously analyse both categorical and numerical features, power to analyse large data, and the ability to produce various predictive algorithms with diverse statistical backgrounds are distinguished features of supervised machine learning models (Ebrahimie et al., 2011 ; Shekoofa et al., 2014 ). The possibility to include the categorical variables in predictive models can remarkably decrease the heterogenicity across studies as the batch effects and other non-biological experimental variation were incorporated in the models (Shekoofa et al., 2014 ). In this study, different experiments or types of AM were added as variables and analyse in the predictive model that resulted in remarkable control of batch effect. This possibility is very limited in traditional multivariate or regression models. The identified meta-genes of successful AM colonization, derived by integration of meta-analysis with supervised attribute weighting models, was able to discriminate efficiently between AM-inoculated and non-inoculated samples. As a validation analysis, the developed signature showed high performance in distinguishing AM-colonized roots from non-inoculated ones in an independent RNA-seq experiment. Recently, integration of supervised machine learning algorithms with meta-analysis has been used to identify a mastitis bio-signature and early prediction of its occurrence (Ebrahimie et al., 2018a ; Sharifi et al., 2018 ). The developed integrative approach in this study, comprising multi-step normalization, direct-merging meta-analysis, and supervised attribute weighting models, is platform-independent approach. By subsequent generation of more RNA-seq data, the developed pipeline may be employed for biosignature discovery in RNA-seq transcriptomic data, integration of microarray and transcriptomic data as is possible using some other NGS platforms, such as ChIP-Seq and SNP to perform meta-analysis on significant peaks in ChIP-Seq experiments and frequency of SNPs in genome-wide experiments. The core 73 upregulated genes in the developed transcriptomic biosignature contain novel regulators of AM colonization including two transcription factors from the GRAS family (MTR_1g069725, MTR_2g089100), one transcription factor from AP2 domain class (MTR_6g029180), and one Zinc finger protein. It has been documented that the GRAS-type transcription factors, such as NSP1 (Nodulation Signaling Pathway1) and NSP2, play essential signaling functions in promoting both Rhizobium nodulation and mycorrhizal colonization (Kaló et al., 2005 ; Smit et al., 2005 ; Liu et al., 2011 ; Gobbato et al., 2012 ). Another transcription discovered factor, MTR_6g029180, has an AP2 domain in this structure. Interestingly, it has been reported that ERF transcription factors with a highly conserved AP2 DNA-binding domain are necessary for nodulation and symbiosis (Middleton et al., 2007 ). Cyclin-dependent kinase (MTR_1g098300) was another highly upregulated gene in the signature of successful AM colonization in this study. Mycorrhizal colonization is classified as postembryonic development of plant organs that need a constant interplay between the cell cycle and developmental programs (Kondorosi and Kondorosi, 2004 ). Cyclin-dependent kinase controls cell cycle and plays the key role in endoreduplication and activation of the anaphase-promoting complex during symbiotic cell development (Kondorosi and Kondorosi, 2004 ). The discovery of the essential transcription factors of successful mycorrhizal colonization and symbiosis in the developed biosignature highlights the robustness and applicability of meta-analysis in the AM colonization signature discovery and the importance of the developed transcriptomic signature. The biosignature obtained here provides a platform for increasing the efficiency of AM inoculation in future by finding accelerator AM colonization agents, such as small molecules/chemicals, and manipulating the expression of key genes in the biosignature. The reasons that some previously-reported AM-associated genes were not identified in the AM meta-signature might be: (1) there are other genes with higher and more repeatable expression in response to AM induction and colonization which are, as a result, selected. These new candidates have higher preference over some of the previously-known biomarkers of AM symbiosis, (2) some AM markers might interact with the type of AM and consequently these will not appear in cross-species meta-analysis, and (3) some AM markers may interact with a specific condition or timing of AM symbiosis. As example, mycorrhiza-specific phosphate transporter seems to be more closely related to P homeostasis rather than colonization as the phosphate transporter mediates early root responses to phosphate status in non-mycorrhizal roots (Volpe et al., 2016 ). Reinforcing the importance of the existence of AP2 transcription factors in the upregulated transcriptomic signature of AM colonization, promoter analysis demonstrated that the P$FLO2 transcription factor matrix family, with the AP2 domain structure and ethylene-responsive element-binding, had the highest number of promoter binding sites of all 20 highly upregulated genes in the AM inoculation signature. Floral homeotic protein APETALA 2, a member of P$FLO2 matrix family, has a documented role in the control of flower and seed development (Jofuku et al., 1994 ). Strong induction of APETALA 2 in developing nodules of Medicago truncatula has been observed and suggested as a potential regulator of the symbiotic program (El Yahyaoui et al., 2004 ). Another enriched transcription factor matrix family was the P$TOEF matrix family that contains the AP2 domain in its structure and is involved in early activation/response (Table 4 , Supplementary Table 6 ). GO analysis showed that these are involved in organ morphogenesis. P$CAAT was another potential master regulator of the identified AM colonization signature that contains CCAAT-binding family transcription factors. It has been documented that CCAAT-binding family transcription factors are essential for endosymbiosis establishment and development (Diédhiou and Diouf, 2018 ). Laser microdissection has documented the expression of CAAT-Box transcription factor in AM, correlated with fungal contact and spread (Hogekamp et al., 2011 ). Two members of this CCAAT-binding family, NF-YA1a and NF-YA1b , are positive regulators of AM colonization in soybean (Schaarschmidt et al., 2013 ). Before the present study, most of the known CCAAT-binding family transcription factors had been reported to be involved in nodulation (Marsh et al., 2007 ; Soyano et al., 2013 ). Functional genetic studies of symbiotic genes in Medicago truncatula indicate a role for a CCAAT-box transcription factor in rhizobial infection (Cousins, 2016 ). Analytical approaches based on literature mining have suggested association between a number of potential microRNAs (particularly microRNA169 and microRNA156) and microRNA-regulated transcription factors, which may be involved in the coordinated regulation of nitrogen and phosphorous starvation responses in soybean and NF-YA3 and NF-YA8 are targets of microRNA169 (Dehcheshmeh, 2013 ; Chiasson et al., 2014 ). A MYB transcription factor belonging to P$MYBL matrix family was also enriched on promoter region of the identified signature of AM colonization. It has been demonstrated that a transcriptional program for arbuscule degeneration during AM symbiosis is regulated by MYB1 (Floss et al., 2017 ). At the regulatory level, promoter analysis of co-expressed genes has demonstrated high potential in identifying key enriched transcription factors, finding undiscovered roles of genes (Deihimi et al., 2012 ), developing the functional genomics catalog of activated transcription factors during a phenomenon (Mahdi et al., 2013 ; Zinati et al., 2014 ), and discovery of transcriptional regulatory networks (Bakhtiarizadeh et al., 2013 , 2014b ). It has been also shown that number and diversity of differential cis-regulatory elements on promoter regions are strong predictors of gene function and level of expression under different conditions (Babgohari et al., 2014 ; Shamloo-Dashtpagerdi et al., 2015 ). This has resulted in developing new indicators of gene importance not based on the gene sequence but on the promoter region. In our previous study, we developed a novel pairwise comparison method for in silico discovery of statistically significant cis-regulatory elements in eukaryotic promoter regions (Shamloo-Dashtpagerdi et al., 2015 ). Transcription factors have interactions with DNA to regulate gene expression in cells (Pomerantz et al., 2015 ). In future studies, genome-wide mapping of binding sites of the identified transcription factors [GRAS family transcription factor (MTR_1g069725, MTR_2g089100), AP2 domain transcription factor (MTR_6g029180), and CCAAT-binding transcription factors] by CHIP-seq techniques may unravel the cistrome of successful AM colonization in symbiosis establishment."
} | 6,542 |
33912358 | null | s2 | 2,654 | {
"abstract": "Hydrogels are frequently used biomaterials due to their similarity in hydration and structure to biological tissues. However, their utility is limited by poor mechanical properties, namely, a lack of strength and stiffness that mimic that of tissues, particularly load-bearing tissues. Thus, numerous recent strategies have sought to enhance and tune these properties in hydrogels, including interpenetrating networks (IPNs), macromolecular cross-linking, composites, thermal conditioning, polyampholytes, and dual cross-linking. Individually, these approaches have achieved hydrogels with either high strength ("
} | 153 |
31491923 | PMC6770704 | pmc | 2,655 | {
"abstract": "Metal hyperaccumulating plants should have extremely efficient defense mechanisms, enabling growth and development in a polluted environment. Brassica species are known to display hyperaccumulation capability. Brassica juncea (Indiana mustard) v. Malopolska plants were exposed to trace elements, i.e., cadmium (Cd), copper (Cu), lead (Pb), and zinc (Zn), at a concentration of 50 μM and were then harvested after 96 h for analysis. We observed a high index of tolerance (IT), higher than 90%, for all B. juncea plants treated with the four metals, and we showed that Cd, Cu, Pb, and Zn accumulation was higher in the above-ground parts than in the roots. We estimated the metal effects on the generation of reactive oxygen species (ROS) and the levels of protein oxidation, as well as on the activity and gene expression of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The obtained results indicate that organo-specific ROS generation was higher in plants exposed to essential metal elements (i.e., Cu and Zn), compared with non-essential ones (i.e., Cd and Pb), in conjunction with SOD, CAT, and APX activity and expression at the level of encoding mRNAs and existing proteins. In addition to the potential usefulness of B. juncea in the phytoremediation process, the data provide important information concerning plant response to the presence of trace metals.",
"conclusion": "5. Conclusions This study was conducted to determine the interactive role of Pb, Cu, Cd, and Zn in metal uptake, plant growth, and the antioxidative system of B. juncea . Plants accumulated high amounts of trace metals, i.e., more than 40% in the roots, and in the above-ground parts, the values for Cu, Cd, Zn, and Pb were 58%, 55%, 52%, and 38%, respectively. The results suggest that B. juncea var. Malopolska is a good hyperaccumulator of trace metals, especially Cu, Cd, and Zn, and can be useful in phytoremediation. The presence of metals resulted in a considerable reduction in B. juncea biomass; the highest reduction was observed in plants treated with Cu and Cd. Despite the visible influence of trace metals on plant morphology, the IT coefficient was high and exceeded 90%, indicating the high resistance of B. juncea plants. Trace metals lead to the production of ROS, which causes an imbalance in the redox state in the plant cells and increases the level of oxidized proteins. We noticed that under the conditions of oxidative stress, the antioxidant system was activated: SOD, CAT, and APX. We observed that the presence of metals influenced the increase in the activity of antioxidant enzymes, while no significant differences were observed in the levels of CuZnSOD and MnSOD transcripts and proteins. The results obtained indicate that B. juncea var. Malopolska has efficient defense mechanisms to cope with different metals.",
"introduction": "1. Introduction Trace metal element contamination in soils is one of the world’s major environmental problems, posing significant risks to human health, as well as to ecosystems ([ 1 ]). Metals such as zinc (Zn), iron (Fe), and copper (Cu) are essential micronutrients required for a wide range of physiological processes in all plant organs, and the processes are based on the activities of various metal-dependent enzymes and proteins. However, they can also be toxic at elevated levels. Metals such as arsenic (As), mercury (Hg), cadmium (Cd), and lead (Pd) are nonessential and potentially highly toxic [ 2 ]. Trace metal element toxicity includes changes in the chlorophyll concentration in leaves and damage of the photosynthetic apparatus, inhibition of transpiration, and destruction of carbohydrate metabolism, as well as nutrition and oxidative stress, which collectively affect plant development and growth [ 3 , 4 , 5 , 6 , 7 ]. Biological organisms are incapable of degrading metals, so they persist in their body parts and environment, leading to health hazards [ 8 ]. Metal accumulation and other abiotic stresses cause excess reactive oxygen species (ROS) generation, leading to oxidative stress [ 7 ]. Plant cells are equipped with enzymatic mechanisms to eliminate or reduce oxidative damage that occurs under metal accumulation. The antioxidative defense system includes superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), which are regarded as responsible for maintaining the balance between ROS production and scavenging [ 9 ]. The Brassicaceae family includes many genera abundant in metallophytes, such as Thlaspi , Brassica, and Arabidopsis . They accumulate a wide range of heavy metals, especially Zn, Cd, nickel (Ni), thallium (Tl), chromium (Cr), and selenium (Se) [ 10 ]. The term hyperaccumulator is used for plants that accumulate 1000 mg per kg of dry matter of any above-ground tissue when grown in their natural habitat [ 11 , 12 ]. As of 2013, approximately 500 metal hyperaccumulator plant species were described [ 13 , 14 ], and the number is increasing. B. juncea exhibits some traits of a metal hyperaccumulator—this species can take up significant quantities of Pb, Cd [ 15 , 16 ], Cr, Cu, Ni, Pb, and Zn [ 10 , 17 ], although its translocation ability is not as efficient as shown for other known hyperaccumulators. Metal hyperaccumulating plants should have extremely efficient defense mechanisms, enabling growth and development in a polluted environment. Therefore, the objective of the present study was to estimate the contribution of the B. juncea (v. Malopolska) enzymatic antioxidant system to combating the oxidative stress induced by essential (Cu, Zn) and non-essential (Pb, Cd) metal elements to allow survival under adverse environmental conditions. The analysis included trace metal accumulation, level of stress parameters, and antioxidant enzyme activity, as well as estimation of encoding mRNA and enzyme protein levels.",
"discussion": "3. Discussion Trace metals are one of the most important abiotic stress factors affecting the natural environment. As a result of anthropogenic activities, we can observe their increasing levels from year-to-year. Metal toxicity results in effects at physiological and cellular levels, leading to distorted metabolism, including plant metabolism [ 18 ]. Abiotic stresses, including the presence of trace metals in soil, are estimated to be the main cause of global crop yield reduction of ca. 70% and thus are considered a great constraint to crop production. This situation has worsened due to disturbed equilibrium between crop production and human population growth. Therefore, it is especially important to understand plant responses to such stress factors. This also applies to trace metals [ 12 ]. In the present study, this was clearly visible in the growth of plant biomass, which significantly decreased during the culture in the presence of heavy metals. Copper and zinc ions are essential for the normal growth and development of all organisms but can be toxic to plants at excessive levels. Lead and cadmium are nonessential elements and are toxic to plants even at low levels [ 8 ]. Essential and nonessential trace elements, when exceeding the threshold limits, can cause different physiological, morphological, and genetic plant anomalies, including reduced growth, mutations, and increased mortality [ 8 ]. Therefore, plants suitable for phytoremediation are, at present, of great importance. In our study, we noticed that in the case of B. juncea v. Malopolska, all the mentioned metals used at 50 μM concentration displayed moderate phytotoxic properties. The biomass increments ranged between 96 mg for Pb-treated plants and 61 mg for Cu-treated plants, and the values were approximately 7% and 41% lower, respectively, than those in control plants. Several studies have shown that high concentrations of trace metals in the soil cause plant growth impairment [ 9 , 19 ]). In Sesbania drummondii , a reduction in seedling biomass was caused by Pb—21%, Cu—46.3%, Ni—31.5% and Zn—25.2% [ 20 ]. The inhibition of shoot growth by trace metals may be due to a decrease in photosynthesis, as trace metals disturb mineral nutrition and water balance, change hormonal status, and affect membrane structure and permeability [ 21 ]. Trace metals might cause an inhibition of root growth that alters water balance and nutrient absorption [ 12 ] and decrease calcium uptake in root tips, leading to a decrease in cell division or cell elongation [ 9 , 22 , 23 ]. According to Marshner [ 23 ], Cd-induced mineral stress can reduce plant dry weight accumulation. Other authors have shown a negative influence of Pb [ 24 ], Cu [ 25 ], Cd [ 26 ], and Zn [ 20 ]. Despite the inhibitory effect caused by trace metals on the growth of the biomass of B. juncea, we observed a high IT amounting to approximately 90% resistance of the plants to trace metals. The bioaccumulation of trace metals is different for various plant species, reflected by their growth, reproduction, occurrence, and survival in metal-contaminated soil, because the mechanisms of elemental uptake by plants are not the same for all species. The capacity of plants to take up trace metals is different for different metals, and the same trace metal can be accumulated at different ratios in different plant species [ 27 ]. Metal bioavailability is also affected by the presence of organic compounds of that metal in plants [ 8 ]. The ICP-MS results we obtained indicate that the accumulation of trace metals was higher in above-ground parts than in roots, especially for cadmium, lead, and zinc. The metal concentrations followed an order of Pb > Cu > Zn > Cd in roots, Zn > Cu > Pb > Cd in the stem, and Zn > Cu > Cd > Pb in leaves [ 28 ]. Based on the obtained results, it can be concluded that B. juncea is a hyperaccumulator of Cd, Zn, and Pb. Cherif and co-authors [ 29 ] reported that Zn induced a decrease in Cd uptake and a simultaneous increase in Zn accumulation, indicating a strong competition between these two metals for the same membrane transporters. In our earlier study [ 28 ] in B. juncea plants treated with a binary combination of metals, namely, PbCu, PbCd, PbZn, CuZn, CuCd, and ZnCd, at a concentration of 25 μM of each, a synergistic response between Zn and Pb was observed, resulting in an increased accumulation of the two metals. The accumulation results obtained for plants treated with Cu are different from those of other researchers. Purakayastha and others [ 30 ] showed that Cu is accumulated mainly in above-ground parts of B. juncea . This difference may result from different exposure durations of the plant to the metal, other metal concentrations, and different plant ages at the time of analysis of the collected metal. Quaritacci et al. [ 31 ] reported that B. juncea was identified as a species able to take up and accumulate metals in its above-ground parts, such as Cd, Cu, Ni, Zn, Pb, and Se. It has been observed that this species concentrated Cu, Pb, and Zn in its above-ground parts in amounts much higher than those detected in the metal soluble fractions present in a soil contaminated by acidic water and pyritic slurry [ 31 ]. The accumulation of trace metals in organs is dangerous for plants. In an earlier study [ 32 ], we confirmed that plants are not adequately protected by the detoxification system because trace metals penetrate in areas with high metabolic activity, such as the cytoplasm, mitochondria, or cell membrane. The occurrence of oxidation stress conditions in B. juncea treated with the trace metals Pb, Cu, Cd, and Zn was confirmed by the increase in the level of oxidized proteins in the roots (approximately 7–12%) and above-ground parts (approximately 13%). Several metals, including Cd, Pb, and Hg, have been shown to cause protein oxidation by depletion of protein thiol groups [ 33 ]. ROS cause protein modifications through the formation of carbonyl groups at certain amino acid residues. Such modifications were caused by the presence of heavy metals, e.g., cadmium [ 34 ], mercury lead, aluminum, zinc, copper, cobalt, nickel, and chromium [ 35 ]. ROS also act as signaling molecules involved in the regulation of many key physiological processes, such as root hair growth, stomatal movement, cell growth, and cell differentiation, when finely tuned and regulated by an antioxidative defense system [ 12 ]. We showed an increase in the level of ROS compared to control plants in all plants treated with heavy metals. The O 2 .− rate after 2 h of culture was 2 times higher than that observed in plants grown under control conditions. The high level of O 2 .− was the highest between 24 to 72 h of the treatment depending on the research variant. The highest value of O 2 − was measured in plants treated with Zn, while the highest H 2 O 2 values were observed in plants treated with Cu and Cd. Similar results were obtained by other researchers. Markovska et al. [ 36 ] showed a 10-fold higher level of H 2 O 2 in the leaves of B. juncea after 5 days of treatment with Cd ions at a concentration of 50 μM. Wang et al. [ 37 ] observed the highest levels of H 2 O 2 in B. juncea roots treated with Cu ions for 4 days. In our research, the highest level of H 2 O 2 was obtained after 4 days in plants treated with single metals. The reduction of O 2 .− and the H 2 O 2 content in roots and above-ground parts of plants treated with trace metals during the cultivation period suggests that some antioxidative enzymes would work effectively in the removal of ROS. To detect ROS in plant cells, we used incubation with fluorescent labels such as 2′7′-difluoroscein and dihydroethidium and imaging under confocal microscopy. We observed increased generation of O 2 .− and H 2 O 2 in the roots of B. juncea treated with trace metals—especially Cd and Zn for O 2 .− , and Cu, Cd, and Zn for H 2 O 2 . The increase in ROS production in metal-treated plants was precisely associated with changes in the activity of antioxidant enzymes. We always observed the induction of antioxidant enzyme activity in B. juncea roots and leaves, although there were no significant differences between the used metals. We observed increasing activity of antioxidant enzymes, i.e., 20–158% for SOD, 15–147% for CAT, and 6–68% for APX. The highest activity of SOD in both roots and shoots was observed in plants treated with Zn and Cu. The first line of defense against ROS-mediated toxicity is through SOD, which catalyzes the dismutation of superoxide anions to H 2 O 2 and O 2 . The stimulation of SOD activity has also been reported in several plants exposed to Pb, Cu, Cd, Zn, Ni, and as ions [ 20 , 25 , 38 , 39 ]. We noticed that in the roots of B. juncea, the most induced activity of CAT was for Zn, compared with Cd in the above-ground parts. APX was definitely lower than catalase, especially in the above-ground parts, which means that this enzyme complements CAT catalytic activity. APX activity was significantly elevated in the metal-treated plants, which suggests its role in the detoxification of H 2 O 2 . Enhanced CAT and APX activity has been observed in various plant species after application of trace metals: Pb, Cu, Cd, Zn, Ni, and As [ 20 , 25 , 38 , 39 , 40 ]. APX may be responsible for controlling the levels of H 2 O 2 as signal molecules, and the CAT function is to remove large amounts of oxygen during oxidative stress. APX may be responsible for controlling the levels of H 2 O 2 as signal molecules, and the CAT function is to remove large amounts of oxygen during oxidative stress [ 41 ]. Mohamed et al. [ 42 ] showed in B. juncea that the higher activity of antioxidant enzymes offers a greater detoxification efficiency, which provides better plant resistance against trace metal-induced oxidative stress. Yadav and co-authors [ 25 ] reported increases in the activities of antioxidant enzymes: SOD by 16.2%, DHAR by 27–58%, GR by 35.74%, GST and GPX by 19.19%, and APX by 42.75% in B. juncea plants treated with 0.0005 M Cu. The authors indicated that brassinostereoids can regulate the activity of the antioxidant system and help in scavenging overproduced ROS, and can provide tolerance by inducing the expression of regulatory genes such as respiratory burst oxidase homologue, mitogen-activated protein kinase-1, and mitogen-activated protein kinase 3, as well as activating genes involved in antioxidative defense and responses [ 25 ]. Other authors [ 12 ] have noted that brassinosteroids are a group of hormones that regulate ion uptake in plant cells and reduce trace metal accumulation in plants. An exogenous application of brassinosteroids is widely used to improve crop yield, as well as stress tolerance, in various plant species. We previously demonstrated an increase in the activity of the antioxidant system at the physiological and biochemical levels. The next step was to determine whether trace metals influence the transcription level of genes encoding suitable defense proteins. ROS concentration at an appropriate level can promote plant development and reinforce resistance to stressors by modulating the expression of a set of genes and redox signaling pathways [ 12 ]. In our research, we observed differences in the expression induction depending on the exposure time and the metal used. We observed an increase in the level of the gene coding for CuZnSOD in plants treated with copper, zinc, and lead. The highest level of expression was obtained after 4 h in roots and 8 h in above-ground parts. Romero-Puertas and co-authors [ 34 ] noted a drastic reduction in the expression of genes coding for CuZnSOD and no changes in MnSOD in Pisum sativum under conditions of stress caused by the presence of Cd. Their results showed a reduction in CuZnSOD levels in the presence of Cd, while in our study, we did not observe significant differences in the level of transcript for plants treated with this metal in relation to control plants. We observed the induction of gene expression encoding MnSOD in B. juncea roots after 8 h of exposure to Zn and Pb ions, compared with lead ions in above-ground parts. Other authors did not observe any changes or a low expression of genes coding for SOD, e.g., Fidlago et al. [ 43 ] showed no differences in MnSOD-related mRNA accumulation in leaves and roots, but CuZnSOD-related transcripts decreased in leaves but did not change in roots in Cd-treated Solanum nigrum L. Others authors [ 44 ] indicated that Cd stress induced an upregulated expression of FeSOD, MnSOD, Chl CuZnSOD, Cyt CuZnSOD, APX, GPX, GR, and POD at 4–24 h after treatment began for Lolium perenne L., and their results suggested that the gene transcript profile was related to the enzyme activity under Cd stress. Romero-Puertas et al. [ 34 ] indicated two groups of genes in pea plants treated with Cd. First, some elements of the signal transduction cascade accentuated or attenuated the Cd effect on CAT, MDHAR, and CuZnSOD mRNA expression. The second was formed by the genes MnSOD, APX, and GR that were not affected by these modulators during the Cd treatment because their expression was not modified compared to control plants. The effect of Cd on the expression of CuZnSOD was reversed by a nitric oxide (NO) . scavenger, indicating that NO . must be a key element in the regulation of this SOD, showing the existence of a relationship between an increase in ROS production and NO. NO-dependent downregulation was also observed for MnSOD, while the opposite effect was found for APX and GR. This suggests that protein phosphorylation is involved in the response to Cd stress [ 34 ]. Bernard and co-authors [ 45 ] indicate that molecular analysis (gene expression) is the first level of integration of environmental stressors, and it is supposed to respond to stressors earlier than biochemical markers. Our results from Western blotting indicate that the presence of trace metals does not increase the synthesis of the proteins CuZnSOD and MnSOD in the organs of B. juncea plants, but induces an increase in their activity."
} | 5,018 |
25338507 | PMC4206842 | pmc | 2,658 | {
"abstract": "Quasi-periodic structures of natural biomaterial membranes have great potentials to serve as resonance cavities to generate ecological friendly optoelectronic devices with low cost. To achieve the first attempt for the illustration of the underlying principle, the Pieris canidia butterfly wing was embedded with ZnO nanoparticles. Quite interestingly, it is found that the bio-inspired quasi-single-mode random laser can be achieved by the assistance of the skeleton of the membrane, in which ZnO nanoparticles act as emitting gain media. Such unique characteristics can be interpreted well by the Fabry-Perot resonance existing in the window-like quasi-periodic structure of butterfly wing. Due to the inherently promising flexibility of butterfly wing membrane, the laser action can still be maintained during the bending process. Our demonstrated approach not only indicates that the natural biological structures can provide effective scattering feedbacks but also pave a new avenue towards designing bio-controlled photonic devices.",
"discussion": "Discussion To further understand the quasi-single-mode laser action derived from ZnO NPs/butterfly wing composite, let us examine the underlying structure of butterfly wing. The insets of Figs. 5(a) and (b) show the SEM images of butterfly wing and ZnO NRs, respectively. It is seen that the skeleton of butterfly wing shows a quasi-periodic structure. We further consider the Fabry-Perot (FP) resonance due to the window-like quasi-periodic structure of butterfly wing as the dominant factor to assist the achievement of quasi-single-mode laser action. Each single pore, or “micro window” of butterfly wing provides well-organized two-end facets for the formation of the FP resonance. The FP effect can be described by the following equation 27 : where λ is the resonant wavelength (~385 nm), n is the refractive index (~1), L is the resonant cavity length, and M is a positive integer. After calculating with the M integer of 5, the resultant L is of about 0.96 μm, which is in good agreement with the size scale of each micro window of the butterfly wing. Furthermore, the resonant mode spacing can be derived by the formula 27 28 : Considering the cavity length of around 1 μm, the resonant mode spacing is estimated to have the magnitude of about 74 nm. As a consequence, only one single Fabry-Perot mode can be observed in the UV emission range of ZnO NPs. In addition to the dominant single-mode laser, the rest peaks can be attributed to the mechanism of random lasing generated by multiple light scattering in random cavities formed by ZnO NPs and butterfly wing microstructure. By using the information of the wavelength difference (Δλ) derived from the two nearest lasing peaks, the transport mean free path (L) of a light in the composite system can be further calculated by L = λ 2 /2nΔλ 29 , and the approximate value is about 82.3 μm. In Figs. 3(a) and (b) , it is seen that the lasing peak positions slightly fluctuate under different pumping and excitation energy. The underlying reason might be attributed to the quasi-periodic structure of butterfly wing, in which, each of the micro window between lamellae appears nearly the same but slightly different size. The fluctuating sizes of the micro windows thus result in different FP cavity length L and leads to the unfixed single-mode lasing peak. In Fig. 6 , we have performed an enlarged SEM image of the ZnO NPs/butterfly wing composite. The upper inset denotes the schematics illustration of a Fabry-Perot (FP) cavity provided by the butterfly wing microstructure. Inside the cavity, the emission derived from ZnO NPs can be multiply scattered back and forth (denoted by red arrows), leading to the amplification and generation of the single-mode laser action. And the pictured cavity length is in good agreement with the theoretical calculation (~0.96 μm) based on equation (1) . The yellow-circled region indicates the ZnO nanoparticles lying on and between the porous network of butterfly wing scale. In addition to the FP mechanism, the randomly distributed ZnO NPs existing among the wing structure can also lead to the occurrence of random lasing 26 . Furthermore, due to the quasi-periodic structure of butterfly wing, the coupling among FP cavity modes is also efficient to selectively enhance the peak intensity of some particular eigenmodes. Therefore, with the coupling among FP resonances and closed-loop paths arising from scattering caused by ZnO NPs, a quasi-single-mode random laser can be expected and obtained. Finally, we extended our work to a unique and facile potential practical application by placing the ZnO NPs/butterfly wing composite onto a mechanically flexible PET substrate. The bending measurements were performed and shown in Fig. 7 . The device was bended to a curvature radius of around 1.5 cm. The lasing spectra of flat and bending state (R ~ 1.5 cm) were shown in Figs. 7(a) and (b) , respectively. The insets show the illustrations of the flat and bending devices. The excitation is fixed at an energy of 85 μJ. Interestingly, for both of the experimental conditions, the quasi-single-mode laser can be well maintained and clearly observed. However, during the bending process, the bending device exhibits a broadened background spectrum and less lasing efficiency. In summary, we have successfully made the first demonstration of a flexible quasi-single-mode random laser based on ZnO NPs/butterfly wing composite. Such unique lasing properties can be well interpreted in terms of both of the random lasing mechanism and Fabry-Perot resonance existing in the window-like quasi-periodic structure of Pieris canidia butterfly wing scales and ZnO NPs. In addition, the inherited soft of butterfly wings can provide excellent flexibility and the laser action can be sustained even under the bending state. Our results not only indicate that the natural butterfly wing can be an attractive candidate to realize bio-inspired photonic lasing properties, but also motivate the research towards studying various types of biological materials and structures for ecological friendly optoelectronic devices with low cost."
} | 1,543 |
34433669 | PMC8536335 | pmc | 2,659 | {
"abstract": "Significance Designing neuromorphic hardware for cryoelectronics is an important area of research as the field of computing paradigms beyond complementary metal-oxide-semiconductor (CMOS) progresses. Superconductivity and metal−insulator transitions are two of the most celebrated emergent, collective properties found in quantum materials such as strongly correlated oxides. Here, we present simulations of artificial neural networks that can be designed by combining superconducting devices (e.g. Josephson junctions) with Mott metal−insulator transition−based tunable resistor devices. Our simulations show that 1) neurons and synapses can be seamlessly created, 2) their functions can be tuned via learning, and 3) controlling disorder by incorporating light ions enables exponential multiplicity of states. The results open up directions for incorporating emergent behavior seen in condensed matter into hardware design for artificial intelligence.",
"conclusion": "Outlook and Conclusions A key aspect of any hardware neural network design is the connectivity between elements. The cuprate-based loop-junction devices we describe are submicron scale and can easily be fabricated on thin films, allowing for a vast array of them. Importantly, the unique aspect of utilizing similar fluxon dynamics with simple geometrical variations of superconducting loop structures to achieve both neuron and synapse operation offers a new kind of flexibility. For instance, the storage of flux quanta in superconducting loops with Josephson junctions brings about aspects such as plasticity that is required for synapses, while the oscillatory properties of these loops in response to spiking inputs resemble properties that are needed in neurons. Furthermore, the two material platforms we present could be connected by growing an island of thin film of nickelate over the cuprate film with all the loops already patterned. Since the hydrogen is not known to affect the properties of the cuprate films, the whole device can then be implanted with hydrogen, which only affects the nickelate overlayer. Then, the two films can be wire bonded through many possible entry paths across the networks, allowing for highly flexible and complex network design patterns. Combining the two systems opens up a particularly interesting possibility to realize a key aspect of biological brains: the ability to operate memories that span different time scales. While cuprate synapses exhibit dynamic and volatile memory with ease in “learning” and “forgetting,” the nickelate synapses exhibit long-term nonvolatile memory. Therefore, the voltage spikes produced in the cuprate system can interact with the nickelate synapse arrays to update their long-term memory configurations. In summary, our proposed arrays of devices can be implemented into networks with flexible connectivity platforms. In conclusion, we present simulations of artificial neural network components combining superconductivity and metal-insulator transitions in complex oxides, two of the most spectacular examples of emergent physics in condensed matter. By creating electronic and structural disorder induced by light ions, one can design individual devices that mimic neurons and synapses in the brain. These devices can easily be combined into coupled networks using well-established lithography methods, and then the number of response states increases, making them potential candidates for neuromorphic cryoelectronics. Moreover, our key finding demonstrates that a randomly spaced network of devices shows an exponential number of collective states that begins to mimic the emergent behavior of features found in living organisms. We emphasize that the use of light ions to modify the electronic response properties of oxides can be applied to several families of strongly correlated materials, thus paving the way for a multitude of studies that can be performed in the search for a new computational paradigm."
} | 987 |
25645243 | null | s2 | 2,660 | {
"abstract": "In hot deserts, plants cope with aridity, high temperatures, and nutrient-poor soils with morphological and biochemical adaptations that encompass intimate microbial symbioses. Whereas the root microbiomes of arid-land plants have received increasing attention, factors influencing assemblages of symbionts in aboveground tissues have not been evaluated for many woody plants that flourish in desert environments. We evaluated the diversity, host affiliations, and distributions of endophytic fungi associated with photosynthetic tissues of desert trees and shrubs, focusing on nonsucculent woody plants in the species-rich Sonoran Desert. To inform our strength of inference, we evaluated the effects of two different nutrient media, incubation temperatures, and collection seasons on the apparent structure of endophyte assemblages. Analysis of >22,000 tissue segments revealed that endophytes were isolated four times more frequently from photosynthetic stems than leaves. Isolation frequency was lower than expected given the latitude of the study region and varied among species a function of sampling site and abiotic factors. However, endophytes were very species-rich and phylogenetically diverse, consistent with less arid sites of a similar latitudinal position. Community composition differed among host species, but not as a function of tissue type, sampling site, sampling month, or exposure. Estimates of abundance, diversity, and composition were not influenced by isolation medium or incubation temperature. Phylogenetic analyses of the most commonly isolated genus (Preussia) revealed multiple evolutionary origins of desert-plant endophytism and little phylogenetic structure with regard to seasonality, tissue preference, or optimal temperatures and nutrients for growth in vitro. Together, these results provide insight into endophytic symbioses in desert-plant communities and can be used to optimize strategies for capturing endophyte biodiversity at regional scales."
} | 497 |
24936704 | PMC4108209 | pmc | 2,661 | {
"abstract": "The noncoded aromatic 3,4-dihydroxy- l -phenylalanine (DOPA) amino acid has a pivotal role in the remarkable adhesive properties displayed by marine mussels. These properties have inspired the design of adhesive chemical entities through various synthetic approaches. DOPA-containing bioinspired polymers have a broad functional appeal beyond adhesion due to the diverse chemical interactions presented by the catechol moieties. Here, we harnessed the molecular self-assembly abilities of very short peptide motifs to develop analogous DOPA-containing supramolecular polymers. The DOPA-containing DOPA–DOPA and Fmoc–DOPA–DOPA building blocks were designed by substituting the phenylalanines in the well-studied diphenylalanine self-assembling motif and its 9-fluorenylmethoxycarbonyl (Fmoc)-protected derivative. These peptides self-organized into fibrillar nanoassemblies, displaying high density of catechol functional groups. Furthermore, the Fmoc–DOPA–DOPA peptide was found to act as a low molecular weight hydrogelator, forming self-supporting hydrogel which was rheologically characterized. We studied these assemblies using electron microscopy and explored their applicative potential by examining their ability to spontaneously reduce metal cations into elementary metal. By applying ionic silver to the hydrogel, we observed efficient reduction into silver nanoparticles and the remarkable seamless metallic coating of the assemblies. Similar redox abilities were observed with the DOPA–DOPA assemblies. In an effort to impart adhesiveness to the obtained assemblies, we incorporated lysine (Lys) into the Fmoc–DOPA–DOPA building block. The assemblies of Fmoc–DOPA–DOPA–Lys were capable of gluing together glass surfaces, and their adhesion properties were investigated using atomic force microscopy. Taken together, a class of DOPA-containing self-assembling peptides was designed. These nanoassemblies display unique properties and can serve as multifunctional platforms for various biotechnological applications.",
"conclusion": "Conclusions In summary, we have shown that the substitution of phenylalanine with DOPA in the known self-assembling peptide motif FF yields self-assembling peptides that are able to form ordered supramolecular nanostructures substantially decorated with catechol functional groups. Due to the intrinsic properties of the catechol group, the obtained supramolecular structures can be used as multifunctional platforms for various technological applications. Upon the incorporation of additional lysine residue containing ε-amine, significant adhesion was obtained, possibly due to electrostatic interactions between the protonated amine and negatively charged oxide surface. The remarkable seamless silver deposition reflects the tendency of the dense catechol array to facilitate coating rather than adhesion. The properties of this deposition are unique, as compared to any known electroless metal coating of biological or polymer nanoassemblies and should prove very useful in the templating of inorganic materials on organic surfaces at the nanoscale for various applications.",
"discussion": "Results and Discussion The designed DOPA–DOPA and Fmoc–DOPA–DOPA peptides were examined under different conditions and were found to self-assemble into ordered nanostructures in the presence of ethanol and water ( Figure 1 b–g ). Macroscopically, the Fmoc–DOPA–DOPA peptide formed a self-supporting hydrogel ( Figure 2 ). To gain a better insight into the molecular organization and morphology of the formed structures, electron microscopy was employed. Transmission electron microscopy (TEM) analysis of both peptides revealed the formation of a tangled fibrous network composed of flexible, elongated fibrillar structures. We observed the existence of twisted multistrand fibers alongside single fibrils. The DOPA–DOPA dipeptide assembled into fibers with a cross section ranging from 20 to 50 nm ( Figure 1 b,c ), while Fmoc–DOPA–DOPA formed narrower fibers, varying in width from approximately 4 to 30 nm ( Figure 1 d–f ). To characterize the morphology of the Fmoc–DOPA–DOPA hydrogel under humid conditions, environmental scanning electron microscopy (E-SEM) was carried out. Upon gradual dehydration of the sample, a network of supramolecular substructures was observed ( Figure 1 g ). Figure 2 Rheological and structural properties of the Fmoc–DOPA–DOPA hydrogelator. Strain sweep (a) and frequency sweep (b) characterization of 5 mg/mL in situ -formed hydrogel at 25 °C. (c) Gelation kinetics of Fmoc–DOPA–DOPA at different concentrations at 25 °C. (d) Gelation kinetics of 5 mg/mL Fmoc–DOPA–DOPA at different temperatures. (e) Kinetics of absorbance at 405 nm at two concentrations and macroscopic visualization of the preparation. (f) HR-SEM micrographs of the turbid peptide solution immediately after inducing the assembly process (left and center panels) and of the semitransparent gel after 2 h of incubation (right panel). The hydrogel formed by the LMW Fmoc–DOPA–DOPA peptide was further characterized ( Figure 2 ). The viscoelastic properties of the gel were assessed using rheological measurements. Oscillatory strain (0.01–100%) and frequency sweep (0.01–100 Hz) tests were conducted to determine the linear viscoelastic regime ( Figure 2 a,b ). These tests revealed that, at the linear region, the storage modulus ( G ′) of the hydrogel is more than 1 order of magnitude larger than the loss modulus ( G ″), a rheological behavior that is characteristic of elastic hydrogels. As shown in Figure 2 c , the plateau storage modulus of the hydrogel was found to be modulated with a high dynamic range of ∼20 Pa to ∼5 kPa, corresponding to the final concentration of the peptide. Furthermore, the gelation kinetics was found to be temperature-dependent ( Figure 2 d ), as gelation was highly decelerated at 4 °C compared to 25 or 37 °C. At higher temperatures (25 or 37 °C), the storage moduli of the hydrogels were approximately 40-fold higher than the storage modulus of hydrogels formed at 4 °C. The gelation process of Fmoc–DOPA–DOPA was also accompanied by a change in the optical properties of the sample, transforming from a turbid viscous solution to a semitransparent hydrogel ( Figure 2 e ). When the 2.5 mg/mL sample was observed macroscopically, the solution cleared within minutes, corresponding to the formation of the hydrogel ( Figure 2 e ). In contrast, in the case of the 5 mg/mL sample, although the solution cleared within minutes, gelation occurred after longer periods of time ( Figure 2 e ). This interesting macroscopic observation is not fully understood; typical self-assembly processes have a higher rate as the concentration increases. This contrasting finding suggests that, at high concentrations, initial disordered aggregation is followed by internal reorganization, which facilitates the subsequent macroscopic transition. This hypothesis is consistent with high-resolution structural examination. High-resolution SEM (HR-SEM) analysis of the 5 mg/mL samples taken minutes or hours after the initiation of the assembly process ( Figure 2 f ) indicated that large aggregates are present at the initial stage when the solution is turbid, whereas after several hours, ordered structures with much smaller diameter appear, corresponding to a considerably clearer solution. This observation is also in line with a previous hypothesis put forward by our group linking the optical transition from turbid to transparent in Fmoc-protected hydrogel preparation to the structural organization over time. 27 This restructuring, from irregular aggregates with dimensions similar to or higher than the wavelengths in the visible spectrum to ordered structures with final diameters much lower than these wavelengths, results in the change of the scattering properties of the solution. It should be mentioned that a similar optical phenomenon was also reported with regards to the hydrogelation process of another LMW Fmoc-containing hydrogelator. 28 The increase in turbidity was explained in light of a phase separation process, leading to the formation of unstable spherical assemblies. The subsequent clearing of the solution and hydrogelation were correlated with the formation of a fibrous network at the expanse of the spherical assemblies. Following the characterization of DOPA–DOPA and Fmoc–DOPA–DOPA peptides, we decided to explore the functional properties of the catecholic groups and investigate the redox properties of the assemblies by monitoring the reduction of ionic silver. The catechol group of the DOPA residues is redox-active, allowing the spontaneous reduction of metal cations into solid metal. 11 This property was previously utilized to prepare gold and silver nanoparticles from gold chloride or silver nitrate. 29 − 31 Recently, catechol redox chemistry was also utilized to form polymer-coated metal nanoparticles and mussel-inspired silver-releasing antibacterial hydrogels. 32 , 33 We decided to follow this approach and examine whether the obtained assemblies can spontaneously reduce ionic silver to silver nanoparticles. In addition, this reaction was used to determine the availability and directionality of the functional DOPA moieties in the assembled structures. DOPA groups that are exposed to the solution are expected to react with ionic silver to form silver nanoparticles or clusters that are detectable by electron microscopy. Moreover, the reduction of silver is accompanied by a change in color due to strong absorbance at approximately 410 nm that can be easily observed macroscopically. The addition of silver nitrate solution to a preprepared Fmoc–DOPA–DOPA hydrogel led to a change in the hydrogel color from semitransparent to dark brown over a period of several hours to a few days ( Figure 3 a ). UV–vis spectra taken 5 days after silver nitrate was added to the hydrogel revealed an increased absorbance above 300 nm. The broad-band increase in absorption in this area could be due to scattering by silver nanoparticles and DOPA oxidation. It should be noted that this change in color did not occur immediately as reported in other systems; 32 , 33 instead, the kinetics were rather slow. Two possible mechanisms, perhaps co-occurring, could account for this difference. First, the diffusion rate of ionic silver is much lower at the gel phase compared with the solution phase. Indeed, when gel formation occurred in the presence of ionic silver, significantly faster reduction of silver was observed (data not shown). Second, silver reduction by Fmoc–DOPA–DOPA was performed under acidic pH, with the peptide dissolved in water only (pH 5.5). Under these conditions, the silver reduction potential is lower as compared to similar reactions reported in the literature that were carried out under neutral or alkaline pH. When dissolving the peptide in water with the pH adjusted to neutral (pH 7.0) or alkaline (pH 8.0) using diluted NaOH solution, the rate of reduction was indeed higher (data not shown), due to a higher reduction potential of the silver or to the possible existence of solvent-exposed Fmoc–DOPA–DOPA monomers; such monomers exist in buffered neutral or alkaline solution, in which self-assembly and hydrogelation of the peptide are not observed. Figure 3 Silver reduction by preprepared Fmoc–DOPA–DOPA hydrogel. (a) Macroscopic visualization and UV–vis spectra of assemblies at 5 mg/mL taken after 5 days of incubation. (b) TEM micrographs of the formation of silver particles after 1 day of incubation of assemblies at 2.5 mg/mL (bottom panels) and a control gel with no addition of silver nitrate (top panel). (c) TEM micrographs of the assemblies at 5 mg/mL after 3 days of incubation. The arrows indicate noncoated peptide assemblies. In all micrographs, negative staining was not applied. The ionic silver reduction process was further monitored by TEM. We observed a slow, gradual transition from the formation of local silver nanoparticle nuclei to the formation of a continuous silver layer ( Figure 3 b ). Distinct formation of silver nanoparticles was observed after short incubation or at a low concentration of the peptide. Under those conditions, most of the fibrillar network was still not coated. However, after longer incubation at high peptide concentration, seamless coating of the peptide assemblies was observed in parts of the preparation ( Figure 3 c ). In some cases, we could observe both coated and noncoated areas on the same fibril ( Supporting Information Figure S1). Such uniform and continuous coating is unique and is most likely the result of both the slow reduction kinetics as discussed above and the high density of catechol groups presented by the assemblies. Silver reduction was also observed with the addition of ionic silver to DOPA–DOPA peptide assemblies (Figure S2). This redox activity demonstrated by the self-assembling DOPA–DOPA structures could be utilized to produce silver-facilitated antimicrobial materials as well as for additional biotechnological applications. 34 − 36 To explore additional functionality, we examined the adhesive properties of the assemblies formed by the two DOPA-containing peptides. Although these assemblies were found to possess inherent redox activity due to their decoration with catechol functional groups, no adhesive properties were macroscopically observable. When considering this finding, it should be noted that the role of DOPA in MAPs and its specific contribution to MAPs adhesion are not fully understood. DOPA could exhibit several adhesion-related functionalities based on the oxidative state of the catechol moiety. The oxidized and non-oxidized catechol functionalities of DOPA are assumed to facilitate cohesion and adhesion of the MAPs, respectively, and it was evident that to a certain extent cohesion is necessary for adhesion. 2 A common belief is that interfacial adhesion to substrates is established by interactions between the non-oxidized catechol form of DOPA and the functional groups at the surface of the solid substrates. 4 , 37 This may take place given the non-oxidized catechol is able to form (i) hydrogen bonds with hydrophilic polymers, (ii) complexes with metal ions, metal oxide, and silicon dioxide which are all present in mineral surfaces, and (iii) π–π interactions with aromatic surfaces. 38 − 41 This chemical interplay clearly complicates any possible interpretation of the lack of adhesive properties presented by the investigated peptides. Recent evidence suggested that oxidation of DOPA residues to DOPA-quinone or DOPA-semiquinone can lead to intermolecular cross-linking of the MAPs, either with other DOPA residues by a radical mechanism or with the ε-amino group of lysine (Lys) residues by Michael addition mechanism giving rise to solidification of the adhesive. 4 Indeed, lysine is a common residue in MAPs, and the DOPA–Lysine motif was used for the design of adhesive polymers. 42 , 43 Due to the suggested significant contribution of the ε-amino group to the cohesive properties, we hypothesized that the incorporation of lysine residues into the DOPA-containing peptide assemblies would contribute to cohesion and thus indirectly improve adhesion. Moreover, lysine residues may also contribute to adhesion via ionic bonding to negatively charged surfaces. Therefore, we designed the Fmoc–DOPA–DOPA–Lys-protected tripeptide. Upon examination of the protected tripeptide under the conditions applied to the two peptides studied initially, the Fmoc–DOPA–DOPA–Lys peptide was also found to self-assemble into well-ordered fibrillar structures that show some degree of resemblance to the fibers obtained by Fmoc–DOPA–DOPA ( Figure 4 a ). However, in contrast to the fibers formed by the latter, the fibers assembled by the Fmoc–DOPA–DOPA–Lys were narrower, with an approximated width of less than 10 nm. Moreover, the fine fibers were only formed by dissolving the peptide to higher concentrations (1.25 versus 0.5 wt %). Fmoc–DOPA–DOPA–Lys was also found to self-assemble into ordered nanostructures in the presence of dimethyl sulfoxide (DMSO) and water, forming assemblies with high structural similarity to the Fmoc–DOPA–DOPA structures ( Figure 4 a ). Figure 4 Characterization of Fmoc–DOPA–DOPA–Lys assemblies. (a) Chemical structure and TEM analysis of 1.25 wt % (17.2 mM) Fmoc–DOPA–DOPA–Lys assemblies prepared in either 12.5% ethanol or 12.5% DMSO. (b) Adhesion force map and corresponding histogram of 1.25 wt % Fmoc–DOPA–DOPA–Lys prepared in ethanol and water. (c) Adhesion force map and corresponding histogram of 1.25 wt % Fmoc–DOPA–DOPA–Lys prepared in DMSO and water. (d) AFM images of the exposed area of the bottom (left and center panels) and top (right) glass surfaces after peeling two glass slides that were adhered overnight by an aliquot of 1.25 wt % Fmoc–DOPA–DOPA–Lys in 12.5% ethanol. (e) AFM images of the exposed area of the bottom (left and center panels) and top (right panel) glass surfaces after peeling two glass slides that were adhered overnight by a preparation of 1.25 wt % Fmoc–DOPA–DOPA–Lys in 12.5% DMSO. Macroscopically, we observed that this tripeptide forms viscoelastic glue capable of adhering two glass slides. To quantify the adhesive forces of the tripeptide sample to the glass surfaces, atomic force microscopy (AFM) was used. Specifically, we assessed the adhesion of the structures to a silicon oxide (SiO 2 ) colloidal probe by employing force–distance measurements. This type of measurement allows the determination of the attractive forces between the AFM probe and the contacted surface, where this force is represented by the minimum value of the force–distance curve. 44 To estimate the adhesion of the self-assembled structures to silicon oxide, peptide solution was deposited on a glass slide and several force–distance curves were measured at different locations. We also calculated the average adhesion of the peptides in a broader area by adhesion force maps ( Figure 4 b,c ). Our results revealed that the examined tripeptide possesses the ability to adhere to silicone oxide, in accordance with our macroscopic observation. In comparison to the very low adhesion of the AFM probe to bare glass, a glass surface covered with Fmoc–DOPA–DOPA–Lys tripeptide assemblies displayed significant adhesive forces ( Figure 4 b,c ). The adhesion of the tip to bare glass was less than 10 nN, whereas adhesion to the Fmoc–DOPA–DOPA–Lys preparation to the glass was calculated to be more than 300 nN when prepared in DMSO and 214 nN (mean force) when prepared in ethanol. To compare the adhesion of the Fmoc–DOPA–DOPA–Lys assemblies to the Fmoc–DOPA–DOPA assemblies, force maps were measured for the latter when prepared in ethanol at the same molar concentration (17.2 mM). The obtained histograms reveal that more counts corresponding to forces above 200 pN were present in the preparation of the protected tripeptide as compared to the dipeptide one (Figure S3). Similar behavior was also observed under lower concentration (12.9 mM, Figure S4). As noted above, under both ethanol and DMSO conditions, the Fmoc–DOPA–DOPA–Lys sample displayed macroscopic adhesive properties, capable of gluing together two glass slides. It should be noted that this gluing phenomenon, in the presence of DMSO, showed recovery behavior—after separating the glass slides by peeling, they were able to be rejoined. Interestingly, this was not the case when ethanol was used for the preparation of Fmoc–DOPA–DOPA–Lys solutions; when this procedure was repeated with an ethanol-prepared tripeptide sample, the sample lost its adhesive properties after peel forces were applied. To understand the basis for the recovery differences between the DMSO and ethanol tripeptide samples, after the two glass slides were glued together, we peeled off the top glass slide and examined the exposed area. AFM analysis of Fmoc–DOPA–DOPA–Lys in ethanol after peeling exhibited unidirectional fine fibrous structures ( Figure 4 d ). In contrast, AFM analysis of Fmoc–DOPA–DOPA–Lys in DMSO after peeling revealed the presence of twisted spheres that were unidirectionally retracted ( Figure 4 e ). It therefore seems that the existence of the larger patches of peptide assemblies have a role in the re-adhesion process."
} | 5,079 |
24397404 | PMC3897908 | pmc | 2,664 | {
"abstract": "Background Over the recent years the production of Ehrlich pathway derived chemicals was shown in a variety of hosts such as Escherichia coli , Corynebacterium glutamicum , and yeast. Exemplarily the production of isobutyric acid was demonstrated in Escherichia coli with remarkable titers and yields. However, these examples suffer from byproduct formation due to the fermentative growth mode of the respective organism. We aim at establishing a new aerobic, chassis for the synthesis of isobutyric acid and other interesting metabolites using Pseudomonas sp. strain VLB120, an obligate aerobe organism, as host strain. Results The overexpression of kivd , coding for a 2-ketoacid decarboxylase from Lactococcus lactis in Ps . sp. strain VLB120 enabled for the production of isobutyric acid and isobutanol via the valine synthesis route (Ehrlich pathway). This indicates the existence of chromosomally encoded alcohol and aldehyde dehydrogenases catalyzing the reduction and oxidation of isobutyraldehyde. In addition we showed that the strain possesses a complete pathway for isobutyric acid metabolization, channeling the compound via isobutyryl-CoA into valine degradation. Three key issues were addressed to allow and optimize isobutyric acid synthesis: i) minimizing isobutyric acid degradation by host intrinsic enzymes, ii) construction of suitable expression systems and iii) streamlining of central carbon metabolism finally leading to production of up to 26.8 ± 1.5 mM isobutyric acid with a carbon yield of 0.12 ± 0.01 g g glc -1 . Conclusion The combination of an increased flux towards isobutyric acid using a tailor-made expression system and the prevention of precursor and product degradation allowed efficient production of isobutyric acid in Ps . sp. strain VLB120. This will be the basis for the development of a continuous reaction process for this bulk chemicals.",
"conclusion": "Conclusion The combination of an increased flux towards isobutyric acid using a tailor-made expression system and the prevention of precursor and product degradation allowed efficient production of isobutyric acid in Ps . sp. strain VLB120. This work experimentally verifies the genome derived metabolic network structure. A true platform organism was designed, which has the ability to produce an even wider product spectrum directly from glucose by only slight changes on the level of cell metabolism. In general, studies investigating Pseudomonas for the fermentative production of chemicals are few [ 19 ]. But clear benefits like low/no byproduct formation during glucose fermentation, high tolerance towards toxic substances [ 12 ], improved NAD(P)H regeneration rates under stress conditions [ 21 ], and a diverse gene repertoire for the processing of organic molecules underline the potential of Pseudomonads for industrial applications. The strain engineering of this novel organism sets the stage for the development of aerobic biofilm based processes for the continuous production of isobutyric acid and other secondary metabolites. Apart from increasing product titers, the long-term catalyst robustness is a most important but often neglected issue in host engineering, which will be a key focus in future studies regarding this newly introduced organism.",
"discussion": "Discussion In the present study, different variants of recombinant Ps. sp. strain VLB120 have been constructed by means of directed and random mutagenesis, which are capable of producing significant amounts of C4-precursors from glucose in aerobic fermentations with the focus on isobutyric acid synthesis. Isobutyraldehyde metabolism in Ps. sp. strain VLB120 Overexpressing simply one gene encoding for a branched chain 2-ketoacid decarboxylase (KivD) in Ps . sp. strain VLB120, enabled this organism to produce isobutyric acid, isobutanol, and 3-methyl-1-butanol, directly from glucose. Isobutyraldehyde was not detected in the fermentation broth, indicating direct intracellular conversion of this compound. Based on genome analysis, 32 aldehyde dehydrogenases (Additional file 1 : Table S2), and about 19 alcohol dehydrogenases (Additional file 1 : Table S3) have been identified in Ps. sp. strain VLB120 capable to either reduce or oxidize isobutyraldehyde. Among these enzymes are ALDHs homologs known to be highly active towards isobutyraldehyde (e.g. PVLB12825, a 42% homolog to padA [ 7 ] of E .coli K12). In addition, the strain harbors ADHs known to be able to catalyze the reduction of isobutyraldehyde (e.g. PVLB10545, a 86% homolog to adhP [ 41 ] of E. coli K12). However, gene deletions of these candidate genes did not significantly improve product titers (data not shown). Rodriguez et al. [ 41 ], aiming to overproduce isobutyraldehyde in E. coli BW25113, identified eight ADHs in the genome of E. coli BW25113 and observed that the final product titer could only be increased by combining multiple ADH deletions. While in E. coli BW25113 the degradation of isobutyric acid is not described [ 7 ], the present work shows that Ps. sp. strain VLB120 harbors a complete pathway for isobutyric acid utilization (Figure 1 ), which enabled Ps. sp. strain VLB120 to grow on isobutanol, isobutyraldehyde and isobutyric acid (Table 2 ). The existence of an isobutyric acid degradation pathway was already described for P. putida , Candida rugosa , Yarrowia lipolytica and Desulfococcus multivorans and used in mutant strains of the first three species for the synthesis of 3-hydroxyisobutyric acid using isobutyric acid as precursor [ 36 , 42 - 45 ]. NTG-random mutagenesis to prevent isobutyric acid degradation To prevent undesired isobutyric acid degradation, several mutants were created by random mutagenesis. The apparent correlation between isobutyric acid and 3-hydroxyisobutyric acid accumulation (in mutants B57, B83 and D67) indicates the mmsAB operon (PVLB_03765, PVLB_03770) [ 46 ] coding for methylmalonate-semialdehyde-dehydrogenase and 3–hydroxyisobutyrate dehydrogenase to be involved in the reaction of isobutyric acid to isobutyryl-CoA. Exemplarily, the respective DNA loci were sequenced in mutants B83 and C18. The DNA sequencing revealed, that mmsB is altered in variant B83. A transition (G/C → A/T) of nucleotide 413 occurred, confirming that this special transition is favored using NTG mutagenesis [ 47 ]. On the protein level, this mutation leads to an exchange of the strictly conserved residue Gly137 with asparagine [ 48 ] and thereby explains the non-active 3-hydroxyisobutyirc acid dehydrogenase in mutant B83. Mutant C18 shows no alteration in the mmsAB operon. In this case, most probably a broad substrate range acetyl-CoA-synthetase or a global regulator involved in isobutyric acid degradation was affected by NTG mutagenesis [ 45 , 49 ]. Fermentative production of isobutyric acid in engineered Ps. sp. strain VLB120 The efficient fermentative production of isobutyric acid requires the overexpression of the genes of the valine synthesis pathway ( alsS , ilvC , ilvD ), a keto acid-decarboxylase ( kivd ) and a highly expressed gene for an aldehyde dehydrogenase (ALDH). While ilvC , ilvD and the ALDH gene are expressed homologously, alsS and kivd are derived from other species and show an altered codon usage, which often results in lower expression levels [ 50 ]. In order to maximize expression levels and overcome promoter related problems like catabolite repression by various carbon sources [ 51 ], the so far used P alk promoter was replaced by a T7 RNA polymerase based system. Using eGFP fluorescence as a fast and easily detectable read-out for expression (Additional file 1 : Figure S2), the performance of this system was evaluated confirming recently published results for P. putida KT2440 T7 [ 52 ]. In addition, enzyme activity assays of single pathway genes proved successful overexpression in Ps. sp. strain VLB120 T7 (Table 3 ). Overexpression of the 2-KIV synthesis pathway genes and kivd in the T7 variants of Ps. sp. strain VLB120 resulted in drastically increased isobutyric acid titers (Figure 4 / Table 1 ). In addition, the variant C18 T7 showed an increased stability of the formed product, which confirms the results of the 2-KIV biotransformations (Figure 2 ) under fermentative conditions. During glucose utilization, gluconate accumulated in Ps. sp. strain VLB120. This phenomenon is well understood and described in Pseudomonas species, which are known to metabolize glucose exclusively via the Entner-Doudoroff pathway, using 6-phosphogluconate as key intermediate [ 53 ]. Ps. sp. strain VLB120 possesses a glucose dehydrogenase (PVLB_05240), but is lacking a gluconate dehydrogenase, which prevents 2-ketogluconate formation. The incomplete conversion of gluconate in mutant strain C18 T7 is comparable to the behavior of Pseudomonas putida KT2440 during poly-hydroxyalkonate synthesis observed by Poblete-Castro et al. [ 54 ]. In their work, they enhanced the production of PHAs by the deletion of glucose dehydrogenase without affecting the specific growth rate. One may speculate, that this deletion leads to an increased flux towards pyruvate and thus more precursors for isobutyric acid synthesis are available. Optimization of isobutyric acid production by the deletion of competing pathways To prevent unproductive 2-KIV depletion via isobutyryl-CoA or various amino acid synthesis pathways (Figure 1 ), the influence of the genes bkd , ilvE , leuA, pycAB and panB was investigated. The strongest effect on isobutyric acid titers was measured for the mutants Δ panB and Δ bkd. By deleting subunit A of the branched chain α-keto acid dehydrogenase complex ( bkd ), growth on 2-KIV could be completely inhibited in Ps. sp. strain VLB120. Growth could be restored by overexpressing the decarboxylase gene kivd , channeling 2-KIV over isobutyraldehyde (Figure 1 ). The mutant strain shows drastically increased product concentrations of isobutyric acid and isobutanol during 2-KIV biotransformations, while during growth on glucose (fermentation) this effect seems to be insignificant, as the production rate of Ps . sp VLB120Δ bkd C18-T7 harboring Δ bkd is comparable to Ps. sp VLB120 . Under fermentative conditions comprising high glucose und only low 2-KIV concentrations the activation of 2-KIV to isobutyryl-CoA seems to be only a minor carbon sink. Similar observations have been reported by Lu et al. [ 55 ] for Ralstonia eutropha. Deletion of ilvE helped to prevent undesired carbon loss, this confirms similar results reported for C. glutamicum and R. eutropha [ 55 - 57 ]. Deletion of panB had the strongest impact on carbon yield for isobutyric acid synthesis. panB is the first gene of pantothenic acid synthesis pathway, and in addition a precursor for CoA [ 58 ]. The deletion of panB seems to increase 2-KIV availability, as also described for C. glutamicum [ 59 , 60 ], and thereby results in an increased carbon yield of 0.12 ± 0.01 g g -1 , which is about 25% of the theoretical maximum. panB deletion leads to mutants unable to grow on minimal medium (most probably connected to the disability to synthesize CoA). For E. coli BW25113 10 g L -1 on a 5 mL scale using 40 g L -1 glucose has been reported [ 7 ] while for Ps. sp. strain VLB120 2 g L -1 have been observed using only 20 g L -1 carbon source. Experiments have been conducted in shake flasks with glucose excess where gluconate temporarily accumulated resulting in a pH drop to pH 6.8 which was even more pronounced during isobutyric acid formation. To prevent this pH drop, hampering catalyst robustness, glucose needs to be limited and the pH controlled. It is to be expected, that in such a controlled environment (bioreactor) higher final product titers and better carbon yields will be reached. Isobutyric acid production and beyond In consequence, the application of the demonstrated design principles, the use of the genome sequence of Ps . sp. strain VLB120 and the established metabolic engineering tools, will give access to a far broader product spectrum. Based on the engineered, highly active 2-KIV synthesis pathway the production of isobutanol, 3-methyl-1-butanol, 3-hydroxyisobutyric acid, isobutyraldehyde, valine and D-pantothenate would be feasible by slight pathway modifications. Beside the synthesis of isobutyric acid, we were already able to detect the Ehrlich pathway products isobutanol and 3-methyl-1-butanol, which are interesting bulk chemicals [ 33 , 61 ]. More over 3-hydroxyisobutytric acid was accumulated in the not optimized mutant B83 with remarkable titers, being highly valuable synthons for the fine chemical industry [ 62 ]."
} | 3,178 |
37332719 | PMC10272611 | pmc | 2,665 | {
"abstract": "Arbuscular mycorrhizal fungi (AMF) play a key role in terrestrial ecosystems, while the ecological restoration application of AMF in mining areas has been progressively gaining attention. This study simulated a low nitrogen (N) environment in copper tailings mining soil to explore inoculative effects of four AMF species on the eco-physiological characteristics of Imperata cylindrica , and provided plant-microbial symbiote with excellent resistance to copper tailings. Results show that N, soil type, AMF species, and associated interactions significantly affected ammonium ( \n NH 4 + \n ), nitrate nitrogen ( \n NO 3 − \n ), and total nitrogen (TN) content and photosynthetic characteristics of I. cylindrica . Additionally, interactions between soil type and AMF species significantly affected the biomass, plant height, and tiller number of I. cylindrica . Rhizophagus irregularis and Glomus claroideun significantly increased TN and \n NH 4 + \n content in the belowground components I. cylindrica in non-mineralized sand. Moreover, the inoculation of these two fungi species significantly increased belowground \n NH 4 + \n content in mineralized sand. The net photosynthetic rate positively correlated to aboveground total carbon (TC) and TN content under the high N and non-mineralized sand treatment. Moreover, Glomus claroideun and Glomus etunicatum inoculation significantly increased both net photosynthetic and water utilization rates, while F. mosseae inoculation significantly increased the transpiration rate under the low N treatment. Additionally, aboveground total sulfur (TS) content positively correlated to the intercellular carbon dioxide (CO 2 ) concentration, stomatal conductance, and the transpiration rate under the low N sand treatment. Furthermore, G. claroideun , G. etunicatum , and F. mosseae inoculation significantly increased aboveground \n NH 4 + \n and belowground TC content of I. cylindrica , while G. etunicatum significantly increased belowground \n NH 4 + \n content. Average membership function values of all physiological and ecological I. cylindrica indexes infected with AMF species were higher compared to the control group, while corresponding values of I. cylindrica inoculated with G. claroideun were highest overall. Finally, comprehensive evaluation coefficients were highest under both the low N and high N mineralized sand treatments. This study provides information on microbial resources and plant-microbe symbionts in a copper tailings area, while aiming to improve current nutrient-poor soil conditions and ecological restoration efficiency in copper tailings areas.",
"introduction": "1 Introduction Arbuscular mycorrhizal fungi (AMF) are the most widely distributed endophytic and mycorrhiza fungal group and the key microbes which affect terrestrial ecosystems ( Gao et al., 2022 ). Studies have shown that different AMF communities can utilize different soil spatial resources, leading to host plant resource niche differentiation ( Li, 2021 ). AMF can also improve plant stress resistance, which effectively enhances plant resistance to disease, drought, waterlogging, salt and alkali content, heavy metals, weeds, and high and low temperatures ( Rivero et al., 2018 ; Li et al., 2019 ; Wang et al., 2020 ; Han et al., 2022 ; Zhu et al., 2022 ). Moreover, AMF promotes the nutrient absorption and water-use efficiency of host plants, improves their photosynthetic and osmoregulatory capacity, and contributes to improvements of their antioxidant capacity and drought resistance ( Estrada et al., 2013 ; Han et al., 2022 ). Furthermore, AMF can directly or indirectly improve the stress resistance of host plants in many aspects. For example, AMF can improve plant water absorption, which would otherwise be difficult for root systems to absorb through their mycelial networks, while improving the overall water and nutritional status of plants, being instrumental in their nutritional status under stress. Through means of regulating soil microecology in the rhizosphere via improvements in soil organic matter (SOM) and microbial levels ( Kong, 2021 ), exogenous mycelia can promote water absorption and regulate the transmission of plant root chemical signals ( Green et al., 1998 ). This subsequently promotes the rapid transmission of water and nutrients to aboveground plant components, reduces stomatal conductance and transpiration rates, improves the photosynthetic capacity of plants, and regulates the osmotic capacity of plants to better cope with drought ( Zhu et al., 2015 ). Moreover, damage to the cytoplasmic membrane can be alleviated by regulating the ion balance of plant cells under stress ( Cao et al., 2015 ). Symbiosis between plant roots and AMF can help improve plant nitrogen (N) and phosphorus (P) absorption efficiency ( Zhou et al., 2021 ). AMF can symbiotically secrete various enzymes ( Saia et al., 2014 ) and organic acids ( Tawaraya et al., 2006 ) with plants to promote availability of P and N (as well as other nutrient) in soil. Moreover, AMF can help symbionts to form mycelial networks and bridges between plants ( Whitfield, 2007 ). The vast surface area of mycelia can also effectively improve plant and soil interactions and promote root activity ( Balogh-Brunstad et al., 2008 ). Mycelial bridges can directly transfer N that will subsequently be directly absorbed into the host root system. This can also affect N redistribution ( Govindarajulu et al., 2005 ). Additionally, AMF can alter species composition and productivity under N application practices while increasing the relative abundance and aboveground biomass of plants ( Zhang et al., 2016 ). The role that AMF play is important for host plant photosynthetic processes ( Xu, 2021 ). Studies have shown that AMF inoculation can effectively improve the photosynthetic capacity and carbon (C) assimilation efficiency of plants under drought stress ( Metwally et al., 2019 ; Ye et al., 2022 ). Additionally, AMF can significantly increase the net photosynthetic rate of host plants, increase dry matter accumulation in plants, and enhanced plant drought resistance. Currently, it remains unclear how AMF affect photosynthetic plant processes ( Xu, 2017 ). According to Huang et al. (2011) , AMF mainly improves the nutrient absorption of host plants, which in turn helps promote the accumulation of sufficient amounts of N and P for effective photosynthesis. Additionally, Ludwig-Miiller (2010) reported that AMF inoculation may affect hormone (i.e., abscisic acid [ABA]) levels of host plants that regulate stomatal conductance, thus impacting photosynthetic efficiency ( Ludwig-Miiller, 2010 ). Additionally, AMF species differ regarding their effect on eco-physiological host characteristics. One study found that Rhizoglomus aggregatum , Glomus etunicatum , Glomus claroideun , and Funneliformis constrictus can improve plant growth and photosynthesis ( Wang et al., 2022 ). Among these, R. aggregatum plays a dominant role in promoting seedling height and G. etunicatum and G. claroideun play a dominant role in promoting root regeneration. Moreover, R. aggregatum , G. etunicatum , and G. claroideun can maximize the net photosynthetic rates of plants. On the other hand, F. mosseae can effectively alleviate a decline in the photosynthetic capacity of host plants under stress conditions ( Wang et al., 2022 ). Technological-based AMF approaches used in the ecological restoration of mining areas have gradually been gaining attention in recent years due to their low cost and high efficiency ( Bi and Xie, 2021 ). For example, AMF can be used to increase vegetation survival rates while improving land reclamation efficiency ( Druille et al., 2013 ; Hao et al., 2014 ). AMF not only have a positive effect on plant nutrient absorption and enzyme activities, but also can enhance the stability of soil aggregates, improve soil permeability and water retention, and boost overall soil quality ( Yang et al., 2016 ; Choi et al., 2018 ). The Zhongtiao Mountains copper mining region, Shanxi Province, is North China’s largest, producing 7 million tons of copper annually. It is the largest non-coal underground mining area in China. This mining region produces vast amounts of copper tailings, resulting in severe pollution and damage to the local ecological environment. Previous studies have reported that nutrient levels are low in copper tailings ore. Imperata cylindrica is the dominant grass species in this region, and may form a symbiotic relationship with AMF during phytoremediation ( Jia et al., 2022 ). Based on this hypothesis, we simulated the low N conditions of this copper tailings region to explore how four different AMF inoculation species types will affect the eco-physiological characteristics of I. cylindrica . For this study, we screened out plant-microbial symbiont strains to improve resistance in copper tailings areas, to enhance the status quo of nutrient scarcity, and to increase the efficiency of ecological restoration in copper tailings areas.",
"discussion": "4 Discussion and conclusions AMF play a crucial role in plant nutrient absorption and stress resistance ( Zhang, 2013 ). Our study found that AMF species significantly affected belowground and aboveground biomass, tiller number, plant height, and mycorrhizal infection rates of I. cylindrica , which was consistent with a previous study ( Huang, 2020 ). This may be because the extraradical mycelium network of AMF can penetrate areas inaccessible to plant roots, subsequently expanding the area of nutrient absorption. Additionally, the extraradical mycelium network can connect to the cortex of plants to form arbuscular structures ( Ge et al., 2020 ), which is advantageous when water and mineral nutrients are transferred via plant shoots for purposes of growth and metabolism, promoting biomass accumulation ( Ren et al., 2014 ; Zhang et al., 2018 ; Teng et al., 2020 ). The mycorrhizal infection rate can reflect symbiotic intensity between AMF and host plants ( Qin, 2022 ), while infection rates will directly affect the ability of AMF to obtain C from host plants for its own growth requirements, thus affecting spore germination and hyphal growth ( Cai, 2017 ). This study found that the spore density of the GC inoculant was significantly higher under the HN treatment in mineralized sand compared to the corresponding LN treatment, indicating that AMF inoculation was conducive to the germination and growth of fungal spores. Moreover, the N content of soil also affected AMF growth. This is consistent with results from a previous study ( Cai, 2017 ). AMF infection rates will differ under different environmental factors, such as the available mineral nutrients, organic matter content, and soil pH in different regions. Studies have shown that AMF inoculation can significantly increase mycorrhizal infection rates, that excessively high N applications are not conducive to mycorrhizal infection, and that more significant root mycorrhizal infection rates will occur under LN levels. Additionally, mycorrhizal infections will vary among different plant species and different N application levels ( Wang, 2012 ). Being one of the three essential elements limiting plant growth and development, N is a key chemical element of plant organic matter ( Cai, 2017 ), while its availability will be affected by soil type, N form type, etc. ( Liu et al., 2019 ). AMF species not only absorb \n NH 4 + \n and \n NO 3 − \n from the surrounding environment and transfer them to host plants ( Hodge et al., 2001 ), they also accelerate organic matter decomposition and improve plant N absorption by secreting enzymes from extraradical hypha. This study found that N content, soil type, AMF infection type, and associated interactions significantly affected the \n NH 4 + \n , \n NO 3 − \n , and TN content of I. cylindrica . Hawkins et al. (2000) reported that \n 15 NH 4 + \n absorption (per unit weight) by FM mycelia was significantly higher than that of \n 15 NO 3 − \n , with a value greater by a factor of 15. The \n NH 4 + \n absorption rate (per unit weight) by mycelia was higher compared to that of \n NO 3 − \n . Similarly, in non-mineralized sand the GC inoculant significantly increased the \n NH 4 + \n content in belowground components of I. cylindrica in this study, while the GC and RI inocula in mineralized sand also significantly increased the \n NH 4 + \n content in belowground components of I. cylindrica . Using mineralized sand as a substrate, \n NO 3 − \n content in belowground components of I. cylindrica significantly increased in the GC inoculant under the HN treatment, while the \n NO 3 − \n content in belowground components of I. cylindrica significantly increased in the GE inoculant under the LN treatment. The reason behind differences in AMF absorption between these two inorganic N forms could be that \n NH 4 + \n requires less energy for absorption and assimilation compared to \n NO 3 − \n . The absorption process of the latter is as follows: it first reduces to NH 3 and then enters into the GS/GOGAT pathway, requiring both energy consumption and reductase participation ( Wu and Ca, 2022 ). However, \n NH 4 + \n can directly enter the GS/GOGAT pathway under conditions of low energy consumption. For \n NO 3 − \n , via the plant root diffusion process (i.e., where it is absorbed [ Sun et al., 2005 ]), absorption is more difficult due to mycorrhizal associations. This is because of its high mobility. On the other hand, \n NH 4 + \n mobility is less robust, forming in the soil within the \n NH 4 + \n enrichment region ( Smith, 2010 ), making it easier for roots to absorb \n NH 4 + \n outside the hyphae. Photosynthesis is the fundamental means by which plants synthesize organic matter and obtain energy ( Zhu et al., 2010 ). In this study, we found that N, soil type, AMF infection, and associated interactions significantly affected the net photosynthetic rate, the intercellular CO 2 concentration, the transpiration rate, and the water vapor pressure deficit of I. cylindrica . Studies have found that AMF inoculation can also significantly increase chlorophyll content in plant leaves ( Sannazzaro et al., 2006 ; Sheng et al., 2008 ; Zhu et al., 2010 ; Liu et al., 2011 ). Results from this study showed that AMF inoculation under the LN treatment significantly increased the chlorophyll a content of I. cylindrica . This may be because AMF inoculation helps I. cylindrica to obtain the water and nutrients necessary for metabolic photosynthetic processes to take place in belowground components, subsequently promoting chlorophyll synthesis and enhancing the photosynthetic capacity of plant leaves. Additionally, the net photosynthetic rate directly reflects the assimilation capacity of leaves (per unit area), which is an important indicator in measuring the photosynthetic capacity of plants ( Hu et al., 2020 ). Plants provide the AMF photosynthate that most benefits them, and AMF also tends to provide soil nutrients to plants that deliver the most photosynthate for their usage ( Kiers et al., 2011 ). Studies have also shown that AMF symbiosis can promote photosynthetic rates, transpiration rates, and a means for host plants to uptake water ( Gavito et al., 2019 ; Puschel et al., 2020 ), which can improve the photosynthetic capacity of plants, although still regulated by environmental conditions and available nutrient elements. Similarly, the average membership function values of each I. cylindrica index inoculated with AMF were higher compared to the control. For non-mineralized sand, the net photosynthetic rate of I. cylindrica inoculated with GC and GE under the HN treatment significantly increased, while the net photosynthetic rate positively correlated with aboveground TC and TN content. The transpiration rate of I. cylindrica inoculated with FM under the LN treatment increased significantly. This may be because N enhances the enzyme activities associated with the photosynthetic electron transport chain while promoting photosynthesis, and P is an important enzyme component that is necessary for plant photosynthesis and ATP synthesis. AMF inoculation promotes N and P absorption and utilization in I. cylindrica , subsequently promoting plant photosynthesis ( Evans and Von Caemmerer, 1996 ; Wu and Zhao, 2010 ; Wang et al., 2016 ). Moreover, S plays a key role in the synthesis and metabolism of photosynthetic pigments and proteases ( Shao, 2004 ). In this study, AMF inoculation significantly increased the S content in aboveground I. cylindrica components, and this significantly and positively correlated with Ci, Gs, and Tr in LN mineralized sand, which was beneficial to the synthesis of various plant proteins, chlorophyll and carotenoid content, and stress resistance. In conclusion, different AMF inoculation had significant effects on the eco-physiological characteristics of I. cylindrica under differing soil nitrogen conditions. AMF strains can improve plant physiological characteristics to varying degrees."
} | 4,298 |
26146457 | PMC4394151 | pmc | 2,666 | {
"abstract": "Graphical abstract",
"introduction": "1 Introduction An emerging technology for the treatment of a variety of reducible pollutants is the utilization of palladium metal (Pd(0)) catalysts [1] . Heterogeneous Pd(0) catalysts are able to dissociatively absorb reactive hydrogen, which can drive hydrogenation reactions with adsorbed target compounds [2–4] . Although molecular hydrogen (H 2 ) has been the most extensively used electron donor during Pd(0)-mediated catalysis, it is poorly soluble in water and other more soluble forms of electron donor, typically simple organic acids such as formate (HCOO − ), have been employed as an alternative [5] . This catalytic approach to contaminant remediation has been demonstrated to be effective towards a variety of key contaminants, including chlorinated hydrocarbons [6–10] , nitrobenzene [11] , nitrate [12,13] and Cr(VI) [14–19] . A wide variety of Pd(0) catalysts have been developed, typically supported upon a carrier particle or in combination with a promoter metal to improve recoverability and increase reactivity [20,21] . Microbial synthesis techniques have also been employed, through the direct enzymatic reduction of a Pd(II) solution by bacterial cells, to form biomass supported Pd(0) [22–26] . Further to this, a novel whole-cell mediated method was developed; using a model Fe(III)-reducing bacterium to reduce an Fe(III) oxyhydroxide, producing nano-scale magnetite with a narrow size distribution with controllable reactivity and particle size [27] . The biogenic nano-magnetite was then used to abiotically reduce aqueous Pd(II) to create magnetically recoverable magnetite supported Pd(0) nanoparticles [28] . This novel nano-scale heterostructure was used initially to catalyze organic coupling reactions [28] , and subsequently to treat Cr(VI) in neutral pH test solutions [17] . A sizable Cr(VI) contamination problem has resulted from the poorly regulated disposal of chromite ore processing residue (COPR), as a waste product of the “high lime” chromite ore processing technique [29,30] . Upon saturation with water, COPR yields a highly alkaline (pH 10–12.5) leachate which, due to the relatively high solubility of most Cr(VI) minerals, can yield high concentrations of aqueous Cr(VI) [31,32] . Specifically in Glasgow, UK, >2 million tons of COPR was disposed of, leading to extensive contamination of ground and surface waters with Cr(VI) at concentrations up to 100 mg L −1 \n [33,34] . Cr(VI) typically forms soluble oxyanions [35,36] , which are regarded as toxic and potential carcinogens [37] . As a result, an upper limit of 0.05 mg L −1 Cr(VI) in drinking water has been set by the World Health Organization [38] . The reduced Cr(III) state, in contrast, is regarded as non-toxic and far less soluble, forming a range of stable oxides and (oxy) hydroxides [39] . The reductive stabilization of the toxic Cr(VI) to non-toxic Cr(III), is therefore the aim of most remediation strategies [40] . However, remediation of COPR related Cr(VI) has proven problematic, due to the large quantities of materials involved, and the adverse alkaline pH that often impacts on the efficiency of conventional chemical treatments [32,41,42] . A recent study employing biogenic nano-scale magnetite and nano-scale zero valent iron highlighted the potential for nano-particle treatment of COPR and its groundwater [43] . Significantly the electron donating capacity of these particles was limited by the supply of reactive Fe, and passivation of the reactive surface by the reduced Cr(III) and groundwater chemical components. Using Pd(0) functionalized nano-scale biomagnetite (Pd-BnM) [28] , this study aims to extend our understanding of catalytic Cr(VI) reduction to the environmentally relevant alkaline pH range, and to assess its applicability to the treatment of COPR leachates. As formate has been previously proposed as an alternative electron donor to H 2 gas for pollutant reduction [5,16] , the performance of both electron donors was assessed in experiments using model alkaline Cr(VI) solutions and a COPR leachate. Inactivation of the catalyst is also of great concern when considering catalyst applications, therefore this was investigated after reaction with the model and COPR solutions, using a variety of spectroscopic and nano-imaging techniques.",
"discussion": "4 Discussion 4.1 Removal of aqueous Cr(VI) – model solutions Heterogeneous catalysts are widely reported to react with substrates via an initial adsorption of the electron donor to an active Pd(0) site, followed by heterolytic or homolytic fission, charging the Pd(0) with reactive H• [2,59] . In the case of Cr(VI) reduction, this is then followed by the co-adsorption of the Cr(VI) anion and reaction with H•. As the speciation of the Pd-BnM surface, Cr(VI) oxyanions and the formate are pH dependent, a complex relationship is likely to exist between their electrostatic interactions over the pH range. Under acidic conditions, where the H 2 electron donor is most efficient (pH 2) and the formate system highly variable, with both most inhibited (pH 2) and optimal removal observed (pH 4), the surface of the Pd-BnM is likely to be more electro-positively charged, while the dominant Cr(VI) anion is likely to be the negative HCrO 4 − \n [60] . The increased attraction of the positive surface and negative Cr(VI) anion potentially accounts for the increased reaction rate in the H 2 experiment under acidic conditions. The complex behavior when employing formate is potentially due to the speciation of the formate, which is in equilibrium with formic acid (p K A = 3.75) [5] , with inhibition coinciding with the increased dominance of formic acid. At near neutral and moving to the environmentally relevant alkaline pH conditions, deprotonated CrO 4 2− and HCOO − will dominate, while the Pd-BnM surface is likely to be increasingly electro-negatively charged; where previous studies using synthetic Pd(0) on magnetite recorded a point of zero charge (pzc) of 7 [61] . Despite these conditions which are expected to favor the repulsion of similarly negatively charged Pd-BnM surface and reactant anions, in the high pH range tested, no obvious increasing loss in reactivity was observed. This is potentially a result of a concurrent increase in dispersion, due to electrostatic repulsion between Pd-BnM particles, which will help to maximize the reactive surface of the particles. This is all the more significant due to the observed generation of alkalinity which occurs by the liberation of OH − during the reduction of the Cr(VI) anions [39] , where, however, the formate does appear to act as a buffer limiting pH change. The k obs kinetic data for Cr(VI) reduction and removal from model Cr(VI) solution, with increasing Pd-BnM loadings, indicates that catalyst concentration exerts a strong control. As the reaction is mediated by Pd(0) content, and its availability as a reactive surface, increasing Pd-BnM loadings would enable more efficient coupling of H• to Cr(VI), increasing reaction rates. The non-linear relationship observed between Pd-BnM loading and k obs , for both electron donors, most likely represents a reactive surface limited system in the lowest Pd-BnM loading experiments. It is also highly likely that the reductive precipitation of Cr, implicated in investigations in to catalyst inactivation, limits k obs values by further decreasing reactive surface area. Magnetite, used as the support for the catalyst in this study, has previously been investigated in regards to its reactivity towards Cr(VI), via its surface Fe(II) content [17,57,62] . However the maximum levels of Cr(VI) removal from model solutions by the Pd-BnM presented here, using both formate or H 2 , are far greater than stoichiometrically possible using un-functionalized magnetite, ∼75 mg Cr(VI) g −1 magnetite assuming complete consumption of the electrons available by Fe(II). The biogenic magnetite, employed as the carrier particle in this study, has previously been used to treat pH 12 Cr(VI) solutions, and recorded a removal of 32 mg Cr(VI) g −1 magnetite due to passivation of the particles reactive surface [43] . In addition, other studies have also found the functionalization by Pd(0) greatly increased the potential Cr(VI) removal compared to magnetite [17] . It should also be noted that a no electron donor control was performed and is presented in S.I. Fig S1, this showed minimal removal of Cr(VI) by Pd-BnM, at the catalyst concentration used, when compared to experiments conducted in the presence of the electron donor formate. Inactivation of catalysts by a variety of co-solutes has been demonstrated previously for the treatment of halogenated solvents [61] , although inactivation by Cr has received little attention. The inactivation of the Pd-BnM in model solutions is possibly due to the accumulation of the reduced Cr(III) phase upon the surface, noted in TEM-EDX and XPS analysis. This phase formed over the surface of the Pd-BnM and is likely to act as an insulating layer, limiting surface mediated contact between the Cr(VI) or electron donor and the reactive Pd-BnM surface. This process is likely to be similar to the passivation reported for magnetite and zero valent iron (ZVI) treatment of Cr(VI) [58,63,64] . Upon analysis of the reacted Cr(III) phase on the Pd-BnM, the Cr K edge XANES spectra bear more resemblance to those previously reported for Cr(OH) 3 and CrOOH [65,66] , lacking the edge feature of the spinel FeCr 2 O 4 standard presented here. Further to this, EXAFS analyses and subsequent fitting of data from the samples of Pd-BnM formate and H 2 reacted with model Cr(VI) solutions, indicated that the same Cr phase forms irrespective of the electron donor used. The fitted Cr-O shell (1.97–1.98 Å) is consistent with a Cr(III) octahedral co-ordination, where Cr(VI) typically forms a tetrahedral co-ordination at shorter interatomic distances of 1.67–1.69 Å [54,58,67] . Considering the TEM-EDX maps which indicate overgrowth of the Fe surface with a discreet Cr phase, the two outer shells (3.01 and 3.60–3.61 Å) are likely to be Cr-Cr. The first Cr-Cr/Fe shell (3.01 Å) is consistent with the edge sharing distances reported for polymeric CrOOH polymorphs at 3.00–3.06 Å [57,68–70] . Significantly the fitted spectra lack the corner sharing Cr-Cr shell, at ∼3.98 Å [54,68] , common to ɣ-CrOOH. The second fitted Cr-Cr/Fe shell (3.60–3.61 Å) has been interpreted previously as a double corner sharing path between adsorbed Cr(III) and Fe(III) hydroxides [70,71] . It is also pertinent that the fitted spectra lack the larger Cr-Cr atomic distance shells associated with chromite [58] . This supports TEM observations and previous studies [17] , which suggest that the majority of Cr in such systems is in a non-magnetic surface phase, as opposed to incorporated into a spinel structure [72] . 4.2 Removal of Cr(VI) – COPR leachate The chemical composition of the COPR leachate employed here reflects the cementitous nature of the COPR [73] ; with a highly alkaline pH and containing aqueous Cr, Ca, Si and CO 3 2− \n [31,34,74] . The presence of co-solutes has been implicated previously in inactivation and inhibitory processes during treatment by Pd(0) catalysts [61,75–77] . By comparison of Cr removal data obtained from Pd-BnM/formate treating COPR and model solutions, a significant inhibition of removal with the COPR was observed. We suggest that this inhibition is related to the presence of the co-solutes Ca 2+ and CO 3 2− . Upon addition of formate to the COPR system, the complete removal of CO 3 2− and the partial removal of Ca indicate precipitation of carbonate species, e.g. CaCO 3 . Although assuming a 1:1 stoichiometric ratio of Ca 2+ to CO 3 2− , it cannot fully account for the loss of CO 3 2− . This behavior is potentially responsible for the loss of catalytic activity in the COPR Pd-BnM/formate experiments by blocking catalyst interactions by surface precipitation. Such precipitation processes have been noted previously to lead to a decrease in reaction rates and a loss in Cr(VI) removal capacity in ZVI permeable barriers [78,79] . The impact of Ca 2+ and CO 3 2− was explored further in experiments using model Cr(VI) solutions, via the addition of the major leachate co-solutes detailed in S.I. Text S2. These experiments implicated the presence of CaCO 3 in the inhibition of Cr(VI) removal, while Ca 2+ or CO 3 2− in isolation did not, S.I. Fig. S6, S7 and S8 and S.I. Table S4. In contrast, we noted significant promotion, in comparison to model solutions, of k obs values at higher Pd-BnM loadings in the H 2 COPR experiment. Here changes in the solution chemistry suggest a limited role for precipitation of carbonate species. The co-solute experiment, again detailed in S.I. Text S2, implicated the Ca 2+ cation in promotion of Cr(VI) removal rates, interestingly in the absence of CO 3 2− the formate system also exhibited a promotion effect, S.I. Fig. S6–S8 and S.I. Table S4. The causes of this Ca 2+ -mediated promotion are however unclear. This promotion effect in the COPR experiment did not extend to the lower Pd-BnM loadings, where a slowing of the rate over the experiment was also noted. At lower Pd-BnM loadings this is interpreted as a reactive surface limitation effect, with increasing passivation of the surface with the Cr, Ca and Si, as seen in TEM-EDX maps and XPS data. The maximum levels of Cr(VI) removal from COPR with the Pd-BnM/H 2 treatment, represent far greater removals than previously reported for micron-scale and nano-scale zero valent iron (ZVI) from COPR groundwater, of 1 and 73 mg Cr(VI) g −1 Fe(0), respectively [80] . The removals are also far in excess of those previously reported for the unfunctionalized biogenic magnetite, which were able to remove 24 mg Cr(VI) g −1 magnetite from COPR groundwater [43] . The greater removal reported here (352 mg Cr(VI) g −1 Pd-BnM) is a result of the sustained catalytic reactivity in the presence of the electron donor H 2 , as opposed to the finite electron source Fe(0) of the ZVI or the Fe(II) of magnetite. However catalyst inactivation, after removing appreciable quantities of Cr(VI), was noted for all treatments in both model and COPR solutions. The increased complexity of the COPR solution is inferred to be responsible for the decrease in total Cr(VI) removals by the Pd-BnM. As previously discussed, when employing formate, this is evident as a loss of catalytic activity, potentially mediated by CaCO 3 precipitation. The ∼50% decrease in maximum Cr(VI) removal, compared to the model solution, during the Pd-BnM/H 2 experiment, is also likely to be a result of the more complex chemistry of the COPR. As seen from TEM-EDX maps and XPS data there is a significant presence of Ca and Si on the surfaces, likely to increase the passivation of the surface, again, as previously seen in both magnetite and ZVI systems [43,74] . It should be noted that these experiments, performed with limited Pd-BnM and an excess of both Cr(VI) and co-solutes, reflect a limited surface system where the co-solutes, with a contribution from Cr(VI), are able to passivate the surface. This experimental set up was chosen to imitate a sustained reaction scenario where Cr(VI) and co-solutes would be re-supplied until inactivation of the catalyst. It is however unclear if an increased Pd-BnM surface was employed, which is able to attenuate the non-target co-solutes, would still maintain a reactive Pd(0) surface leading to sustained Cr(VI) removal. In conclusion, pH was found to have a major control over the efficiency of aqueous Cr(VI) removal when formate was used as an electron donor for Pd-BnM-mediated metal reduction, while the system was less sensitive to pH effects when H 2 was used as the electron donor. At environmentally relevant alkaline pH conditions, challenged with model Cr(VI) solutions, electron donors coupled with Pd-BnM were able to remove aqueous Cr(VI) efficiently by reduction to Cr(III). In time, this led to catalyst inactivation, most likely due to the formation of an insulating surface CrOOH phase. In the more complex solution chemistry of the COPR leachates, significant inhibition was noted in the presence of formate, while Cr(VI) removal rates were enhanced in the H 2 experiment. Cr(VI) removal in model solutions, with combinations of co-solutes implicated the formation of CaCO 3 in inhibition in the formate experiment, while the presence of Ca 2+ (in the absence of carbonate) resulted in promotion of the catalytic reaction. The higher solute loading in COPR leachates significantly decreased the maximum Cr(VI) removal possible by the Pd-BnM/H 2 experiment, inferred to be the result of the co-solutes Ca and Si occupying significant proportions of the catalyst surface, while completely inhibiting catalytic activity in the formate experiment. The data published in this study illustrate the clear potential of biotechnologically engineered Pd(0)-bearing nanocatalysts for the remediation of Cr(VI) from contaminated waters at environmentally relevant alkaline conditions. Although catalyst inactivation was noted, the quantities of Cr(VI) removal, prior to loss of reactivity, from the model alkaline solutions and COPR leachates (in the Pd-BnM/H 2 experiment) are far greater than those reported under similar conditions using conventional nZVI treatments [80] . The findings of this study also highlight the importance of the electron donor used, with superior performance using H 2 , compared to the formate-driven experiments, where significant inhibition was noted in COPR leachates. The reactive life time of the catalyst, and potential for re-activation, are of importance when considering the cost and effectiveness of catalysts for contaminant remediation. Although several catalyst water treatments have reached field scale application [81–84] , all have targeted organic contaminants which, unlike the reductive precipitation of Cr(VI), do not generate products directly implicated in catalyst deactivation. These studies did however note a loss of efficiency of the catalyst upon long term deployment, with various reactivation treatments used to regenerate the catalyst. The relatively high expense of Pd, where it makes up ∼2% by mass of the Pd-BnM particles, means further investigations on reactivation of the inactivated catalyst, where its magnetic properties are likely to aid its retrieval, are warranted."
} | 4,637 |
39660099 | PMC7617206 | pmc | 2,667 | {
"abstract": "Photosynthetic microalgae are an attractive source of food, fuel, or nutraceuticals, but commercial production of microalgae is limited by low spatial efficiency. In the present study we developed a simple photosynthetic hydrogel system that cultivates the green microalga, Marinichlorella kaistiae KAS603, together with a novel strain of the bacteria, Erythrobacter sp. We tested the performance of the co-culture in the hydrogel using a combination of chlorophyll- a fluorimetry, microsensing, and bio-optical measurements. Our results showed that growth rates in algal–bacterial hydrogels were about threefold enhanced compared to hydrogels with algae alone. Chlorophyll- a fluorimetry–based light curves found that electron transport rates were enhanced about 20% for algal–bacterial hydrogels compared to algal hydrogels for intermediate irradiance levels. We also show that the living hydrogel is stable under different environmental conditions and when exposed to natural seawater. Our study provides a potential bio-inspired solution for problems that limit the space-efficient cultivation of microalgae for biotechnological applications.",
"conclusion": "Conclusions This study developed a simple hydrogel system for microalgal cultivation in co-culture with a novel strain of Erythrobacter sp. Our findings demonstrate enhanced photosynthetic activity and growth rates of microalgae in co-culture when immobilized in our hydrogel system. We further show that our gelatin-based hydrogel is easy to fabricate, requires low maintenance, and remains stable when the co-culture is exposed to natural contaminants. Our study suggests that co-cultivation in hydrogels of microalgae with Erythrobacter sp. enhances microalgal growth and density, and could potentially reduce the need for costly antibiotics. We conclude that hydrogel algal–bacterial co-culture is a simple, bio-inspired approach that can be further developed to solve some problems that currently limit microalgal cultivation. These improvements compared to conventional cultivation methods demonstrate potential practical applications of our findings toward more efficient micro-algal cultivation.",
"introduction": "Introduction Microscopic photosynthesizing algae produce a range of high value products including lipids and pigments ( Borowitzka 2013 ). In addition, algal biomass is of great interest for use as feedstocks in aquaculture and for the generation of biofuels ( Villarruel-Lopez et al. 2017 ; Khan et al. 2018 ). However, commercial large-scale production of microalgae is still limited by low spatial efficiency and associated high production and processing costs (e.g., Borowitzka and Vonshak 2017 ). Algal cultivation techniques can generally be divided into open pond systems, closed photobioreactors, and biofilm-based systems ( Posten 2009 ). Open pond systems cultivate algae in raceway ponds and have low maintenance cost but generate only limited biomass per area ( Tan et al. 2020 ). Photobioreactor systems allow for controlled conditions of irradiance, gas flux and temperature, and yield higher algal growth efficiencies, but have high operation and maintenance costs ( Lee 2001 ; Tan et al. 2020 ). Biofilm-based systems cultivate algae as surface-attached biofilms rather than in liquid suspensions. Algal biofilm cultivation can lead to reduced operation costs due to limited water and energy use, as well as improved algal harvesting efficiencies ( Ozkan et al. 2012 ; Berner et al. 2015 ). Biofilm systems also demonstrate greater CO 2 utilization efficiency and reduced harvesting cost ( Blanken et al. 2017 ; Roostaei et al. 2018 ). These systems, however, are also constrained, often relying on sophisticated artificial architectures to compete with the efficiency of natural systems and are much harder to scale up. More recently, algae have also been cultivated while immobilized in hydrogels ( Berner et al. 2015 ). Hydrogel immobilization enables reduced water usage during algal cultivation and provides a potential physical barrier against bacterial infections ( Brenner et al. 2008 ; Covarrubias et al. 2012 ). 3D bioprinting has been used to create different hydrogel structures growing a range of microalgal strains ( Krujatz et al. 2015 ; Lode et al. 2015 ; Wangpraseurt et al. 2020 ). To optimize light propagation in hydrogels with high microalgal densities, coral-inspired biomaterials have recently been developed ( Wangpraseurt et al. 2020 ). However, the cultivation of microalgae in hydrogel-based systems still requires further development regarding the exchange of gases and metabolites that are essential for microalgal growth ( Podola et al. 2017 ). To overcome diffusion limitation in attached cultivation systems, previous efforts have included the development of porous substrate-based bioreactors that make use of a porous membrane to deliver nutrients and promote gas exchange, while the surface of the biofilm is in direct contact with the ambient gas phase ( Podola et al. 2017 ). In nature, benthic photosynthetic symbiotic organisms (e.g., corals, anemones) have faced similar challenges as photosynthesis in thick tissues can theoretically become limited by the diffusion-limited provision of HCO 3 − from the ambient water phase ( Schrameyer et al. 2014 ). However, it has been shown that coral animal and bacterial respiration promote photosynthesis of their symbiotic microalgae, suggesting that the coral host provides essential metabolites and nutrients locally to the microalgae (e.g., Kuhl et al. 1996 ; Schrameyer et al. 2014 ). In corals, the microbial community performs critical functions for the coral holobiont including pathogen protection, sulfur, and nitrogen cycling as well as beneficial modulations of the host microhabitat ( Rosenberg et al. 2009 ; Ceh et al. 2013 ; Krediet et al. 2013 ). Benefits of bacterial communities for an algal host have been documented in free-living algae as well (e.g., Kazamia et al. 2012 ). Some bacteria can provide a local supply of essential nutrient compounds required by the algae, including nitrogen, inorganic carbon, vitamin B 12 (cobalamin), and growth-promoting hormones ( Kouzuma and Watanabe 2015 ). For example, one study estimated that 50% of algal species are cobalamin auxotrophs, implying a reliance on bacterial-produced cobalamin ( Croft et al. 2005 ). More generally, symbiotic relationships between microalgae and bacteria often employ a mutually beneficial exchange of carbon and nitrogen ( Thompson et al. 2012 ; de-Bashan et al. 2016 ). Experiments working with the microalgae Chlorella in co-culture with a known growth-promoting bacteria in alginate beads demonstrated enhanced growth which can be utilized for biotechnological applications ( Gonzalez and Bashan 2000 ). Likewise, Chlorella minutissima was co-cultured with Escherichia coli under mixotrophic conditions and resulted in enhanced production of biofuel precursors ( Higgins and VanderGheynst 2014 ). Accordingly, there is a growing interest in exploiting the potential of algal–bacterial co-cultures for algal biotechnology ( Lian et al. 2021 ; Sánchez-Zurano et al. 2020 ; Padmaperuma et al. 2018 ; Meyer and Nai 2018 ). Here, we aimed to develop a simple gelatin-based hydrogel system by combining microalgae and bacteria for spaceefficient microalgal cultivation. We hypothesized that cocultivation of algae and bacteria would result in improved growth and performance of the algae in hydrogels. For this, we chose the green microalga Marinichlorella kaistiae KAS603 and screened 14 marine bacterial strains for beneficial effects on algal biomass. Based on these results, we further measured the bio-optical properties and photosynthetic performance of a synthetic co-culture between M. kaistiae KAS603 and a novel strain of Erythrobacter sp. We also aimed to evaluate the beneficial effects of the Erythrobacter strain on a range of microalgae covering coccolithophorids, red algae, and other species of green microalgae. Finally, the mechanical stability of our hydrogel system was tested under different environmental conditions.",
"discussion": "Results and discussion Here, we developed a simple hydrogel system for the spaceefficient co-culture of microalgae. We found that a novel strain of Erythrobacter sp. (SIO_La6, Fig. 2 ) isolated from Southern California coastal waters (off Scripps Pier) has beneficial effects on growth and photosynthetic performance of microalgae immobilized in hydrogels. Cell density differences between treatments Microalgal cell density was on average 2.3-fold enhanced for M. kaistiae KAS603 gels co-cultured with SIO_La6 (mean = 2.85 × 10 7 cells mL −1 , SD = 5.94 × 10 6 , n = 5) compared to monoculture gels (1.18 x10 7 cells mL −1 , SD = 4.06 × 10 6 , n = 5) after 72 h of cultivation (unpaired t test, p < 0.01, Fig. 3a ). The cell doubling time was 16.75 h for co-cultures compared to 33.11 h for monocultures ( Fig. 3 ). The beneficial effects of co-culture with Erythrobacter sp. SIO_La6 were also evident in liquid culture, although the relative growth-stimulating effect was 15% higher in hydrogel cultivation ( Supplementary Fig. 2 ). In a stagnant hydrogel, gas exchange is likely to become a limiting growth factor, while such limitation is unlikely to occur in a liquid mixed culture. Thus, the relative enhancement for hydrogel cultures could suggest that bacterial colonies stimulate gas exchange and provide nutrients and/or growth-promoting hormones locally within the hydrogel. Indeed, bacteria observed during confocal microscopy were observed forming aggregates around algal cells ( Supplementary Fig. 3 ). Likewise, it is known that different Erythrobacter strains induce aggregation of different diatom species ( Tran et al. 2020 ). Previous research into immobilized algae-bacteria co-cultures have observed similar formations of aggregates and biofilms, which resulted in improved growth and stability ( de-Bashan et al. 2011 , 2016 ). This proximity, in a gel compared to liquid culture, may facilitate and/or stabilize the interactions between the algae and bacteria for provision of photosynthate from the algae and in return growth-enhancing micronutrients (e.g., vitamins) and gases (e.g., CO 2 ) from bacteria ( Kazamia et al. 2012 ; Paerl et al. 2015 ; Higgins et al. 2016 ; Helliwell 2017 ). Following the successful tests with M. kaistiae KAS603, other common microalgae were tested in co-culture with SIO_La6. The bacterial co-culture enhanced microalgal growth for three of the five microalgal strains compared to monoculture controls ( Fig. 3b ). Cell densities after 3 days of cultivation were at least twofold higher for the coccolithophorid alga P. carterae and the red alga P. cruentum when grown in co-culture hydrogels ( Fig. 3b ). Interestingly, cultures that did not perform well in co-culture (e.g., Micromonas sp. and A. carterae ) also showed limited growth when encapsulated in the gelatin-based hydrogel in monoculture, suggesting that hydrogel immobilization interfered with the growth dynamics of these algae ( Fig. 3b ). This suggests that Micromonas sp. and A. carterae might not be suitable candidates for biotechnological applications using hydrogel immobilization. Understanding the metabolic and molecular mechanisms underlying this beneficial interaction is a complex task that would require potential metabolomic and proteomic approaches (see, e.g., Kazamia et al. 2016 ; Helliwell et al. 2018 ) which was beyond the scope of the present study. However, it is noteworthy that we found growth-enhancing effects of Erythrobacter SIO_LA6 on vitamin B 12 –independent algae ( M. kaistiae KAS603) and vitamin B 12 –dependent algae ( P. carterae , Croft et al. 2005 ). This suggests that the beneficial effects are unlikely due to vitamin production by Erythrobacter SIO_LA6 and rather related to other benefits (e.g., growth hormones or gas exchange). Co-culture effects on microalgal photosynthesis and bio-optics Compared to M. kaistiae KAS603 monocultures, O 2 microsensor measurements in co-cultures indicated 4.9-fold enhancements of net photosynthesis at high light (550 μmol photons m −2 s −1 ) irradiance regimes ( Fig. 4a ). In addition, co-cultures exhibited about 4.3-fold greater rates of dark respiration ( Fig. 4a ). Variable chlorophyll- a fluorimetry measurements showed significant enhancements in the maximum quantum yield of PSII (F v /F m ) for co-culture hydrogels compared to monoculture hydrogels during 7 days of growth (mean = 0.603, SD = 0.022 vs. mean = 0.535, SD = 0.004, respectively; Fig. 4b , unpaired t test p = 0.0339). F v /F m is a key parameter used to assess the healthiness of photosynthesizing microalgae (e.g., Baker 2008 ) and thus suggests that algae in co-culture displayed superior photosynthetic capacities. Likewise, relative electron transport rates showed clear differences in key photosynthetic parameters including α and ETR max ( Fig. 4d–f , Table 1 ). For instance, at day 3 ETR max was about 71.6% higher for cocultures versus monocultures ( Fig. 4d–f , Table 1 ). Although areal net photosynthetic ( P n ) rates were strongly enhanced in co-culture, these differences were also affected by the greater algal growth in co-culture ( Fig. 3 ). However, normalizing P n rates to the differences in biomass still suggests an approximate doubling in net photosynthesis in co-culture versus monoculture (compare Figs. 3a and 4a). As Erythrobacter spp. are anoxygenic phototrophic bacteria and thus does not produce O 2 ( Koblizek et al. 2003 ), such differences strongly suggest cell-specific enhancements of photosynthetic activity by M. kaistiae KAS603 in the presence of Erythrobacter. It is important to note that these measurements include respiratory activity by the bacteria, further strengthening the argument of enhanced algal photosynthesis in co-culture. PAM measurements can detect potential electron transport by Eyrythrobacter sp. ( Chandaravithoon et al. 2020 ); however, we did not find any measurable quantum yield of PSII from SIO_LA6 in monoculture (F v / F m = 0, data not shown). In addition, diffuse reflectance measurements did not show characteristic absorption peaks of bacteriochlorophyll a at ~ 750 nm ( Fig. 5 , Yurkov and Beatty 1998 ), suggesting that pigment synthesis and photosynthetic electron transport might be low by this Erythrobacter strain. In turn, reflectance in the nearinfrared region (~ 750 nm) was about 2.5-fold enhanced which could be indicative of the production of lightscattering microbial extracellular polymeric substances (EPS; Flemming and Wingender 2001 ). Such EPS has previously been shown to scatter light and could potentially enhance the internal actinic irradiance intensity which would further promote photosynthesis ( Decho et al. 2003 ; Fisher et al. 2019 ). Clearly, there are various potential mechanisms underlying the enhanced photosynthetic performance of the co-culture hydrogels and a detailed understanding of the mechanisms was beyond the scope of this first study. However, taken together, our results indicate that Erythrobacter sp. SIO_La6 enhances M. kaistiae KAS603 photosynthesis ( Table 1 ) which could explain the enhanced algal biomass in co-culture. Contamination resistance in hydrogels A potential key problem in cultivating microalgae in hydrogels is that most biopolymers are readily degraded by various bacterial communities ( Pathak et al. 2017 ). We hypothesized that co-cultivation might provide protection from such degradation by occupying microbial habitats within the hydrogel and potentially producing antibiotics. Such concept is analogous to the role of the microbial community in the coral mucus, which protects from opportunistic microbes ( Shnit-Orland and Kushmaro 2009 ). Following exposure to natural seawater, co-culture gels remained viable and no visible degradation of the gelatin matrix was noticeable even after 7 days of cultivation ( Fig. 6a–e ). However, monocultures showed clear degradation and liquefaction of the polymer matrix within 24 h ( Fig. 6a–e ). Likewise, previous experiments using Chlorella–bacteria co-cultures in alginate beads found reduced contamination by foreign bacteria from the environment and concluded that co-cultured bacteria provide a physical barrier ( Covarrubias et al. 2012 ). Here, it is likely that DOC produced by the algae might enhance virulence factors (present in SIO_La6 genomes, J. Dinasquet personal communication) and toxin production as observed in other Erythrobacter species in the presence of algal DOC ( Cárdenas et al. 2018 ). This induced pathogenicity might have antagonistic effects against environmental contaminants. Although the mechanisms warrant further investigation, these initial results suggest protective effects of our synthetic co-culture hydrogel from external microbes. Thus, co-cultivation with Erythrobacter SIO_LA6 stabilizes the biopolymer matrix and reduces the chance for bacterial degradation. This could therefore reduce the need for costly measures to prevent invasion by adventitious bacteria or other predators that might be attracted by the breakdown products. Given that surface-associated/biofilm-based cultivation methods are increasing in various algal biotechnological applications, our study potentially provides a simple and cheap cultivation system with minimal maintenance requirements. This approach can be further developed as a viable bio-inspired alternative to costly antibiotic treatments that are currently used in such cultivation approaches ( Berner et al. 2015 )."
} | 4,418 |
39660099 | PMC7617206 | pmc | 2,667 | {
"abstract": "Photosynthetic microalgae are an attractive source of food, fuel, or nutraceuticals, but commercial production of microalgae is limited by low spatial efficiency. In the present study we developed a simple photosynthetic hydrogel system that cultivates the green microalga, Marinichlorella kaistiae KAS603, together with a novel strain of the bacteria, Erythrobacter sp. We tested the performance of the co-culture in the hydrogel using a combination of chlorophyll- a fluorimetry, microsensing, and bio-optical measurements. Our results showed that growth rates in algal–bacterial hydrogels were about threefold enhanced compared to hydrogels with algae alone. Chlorophyll- a fluorimetry–based light curves found that electron transport rates were enhanced about 20% for algal–bacterial hydrogels compared to algal hydrogels for intermediate irradiance levels. We also show that the living hydrogel is stable under different environmental conditions and when exposed to natural seawater. Our study provides a potential bio-inspired solution for problems that limit the space-efficient cultivation of microalgae for biotechnological applications.",
"conclusion": "Conclusions This study developed a simple hydrogel system for microalgal cultivation in co-culture with a novel strain of Erythrobacter sp. Our findings demonstrate enhanced photosynthetic activity and growth rates of microalgae in co-culture when immobilized in our hydrogel system. We further show that our gelatin-based hydrogel is easy to fabricate, requires low maintenance, and remains stable when the co-culture is exposed to natural contaminants. Our study suggests that co-cultivation in hydrogels of microalgae with Erythrobacter sp. enhances microalgal growth and density, and could potentially reduce the need for costly antibiotics. We conclude that hydrogel algal–bacterial co-culture is a simple, bio-inspired approach that can be further developed to solve some problems that currently limit microalgal cultivation. These improvements compared to conventional cultivation methods demonstrate potential practical applications of our findings toward more efficient micro-algal cultivation.",
"introduction": "Introduction Microscopic photosynthesizing algae produce a range of high value products including lipids and pigments ( Borowitzka 2013 ). In addition, algal biomass is of great interest for use as feedstocks in aquaculture and for the generation of biofuels ( Villarruel-Lopez et al. 2017 ; Khan et al. 2018 ). However, commercial large-scale production of microalgae is still limited by low spatial efficiency and associated high production and processing costs (e.g., Borowitzka and Vonshak 2017 ). Algal cultivation techniques can generally be divided into open pond systems, closed photobioreactors, and biofilm-based systems ( Posten 2009 ). Open pond systems cultivate algae in raceway ponds and have low maintenance cost but generate only limited biomass per area ( Tan et al. 2020 ). Photobioreactor systems allow for controlled conditions of irradiance, gas flux and temperature, and yield higher algal growth efficiencies, but have high operation and maintenance costs ( Lee 2001 ; Tan et al. 2020 ). Biofilm-based systems cultivate algae as surface-attached biofilms rather than in liquid suspensions. Algal biofilm cultivation can lead to reduced operation costs due to limited water and energy use, as well as improved algal harvesting efficiencies ( Ozkan et al. 2012 ; Berner et al. 2015 ). Biofilm systems also demonstrate greater CO 2 utilization efficiency and reduced harvesting cost ( Blanken et al. 2017 ; Roostaei et al. 2018 ). These systems, however, are also constrained, often relying on sophisticated artificial architectures to compete with the efficiency of natural systems and are much harder to scale up. More recently, algae have also been cultivated while immobilized in hydrogels ( Berner et al. 2015 ). Hydrogel immobilization enables reduced water usage during algal cultivation and provides a potential physical barrier against bacterial infections ( Brenner et al. 2008 ; Covarrubias et al. 2012 ). 3D bioprinting has been used to create different hydrogel structures growing a range of microalgal strains ( Krujatz et al. 2015 ; Lode et al. 2015 ; Wangpraseurt et al. 2020 ). To optimize light propagation in hydrogels with high microalgal densities, coral-inspired biomaterials have recently been developed ( Wangpraseurt et al. 2020 ). However, the cultivation of microalgae in hydrogel-based systems still requires further development regarding the exchange of gases and metabolites that are essential for microalgal growth ( Podola et al. 2017 ). To overcome diffusion limitation in attached cultivation systems, previous efforts have included the development of porous substrate-based bioreactors that make use of a porous membrane to deliver nutrients and promote gas exchange, while the surface of the biofilm is in direct contact with the ambient gas phase ( Podola et al. 2017 ). In nature, benthic photosynthetic symbiotic organisms (e.g., corals, anemones) have faced similar challenges as photosynthesis in thick tissues can theoretically become limited by the diffusion-limited provision of HCO 3 − from the ambient water phase ( Schrameyer et al. 2014 ). However, it has been shown that coral animal and bacterial respiration promote photosynthesis of their symbiotic microalgae, suggesting that the coral host provides essential metabolites and nutrients locally to the microalgae (e.g., Kuhl et al. 1996 ; Schrameyer et al. 2014 ). In corals, the microbial community performs critical functions for the coral holobiont including pathogen protection, sulfur, and nitrogen cycling as well as beneficial modulations of the host microhabitat ( Rosenberg et al. 2009 ; Ceh et al. 2013 ; Krediet et al. 2013 ). Benefits of bacterial communities for an algal host have been documented in free-living algae as well (e.g., Kazamia et al. 2012 ). Some bacteria can provide a local supply of essential nutrient compounds required by the algae, including nitrogen, inorganic carbon, vitamin B 12 (cobalamin), and growth-promoting hormones ( Kouzuma and Watanabe 2015 ). For example, one study estimated that 50% of algal species are cobalamin auxotrophs, implying a reliance on bacterial-produced cobalamin ( Croft et al. 2005 ). More generally, symbiotic relationships between microalgae and bacteria often employ a mutually beneficial exchange of carbon and nitrogen ( Thompson et al. 2012 ; de-Bashan et al. 2016 ). Experiments working with the microalgae Chlorella in co-culture with a known growth-promoting bacteria in alginate beads demonstrated enhanced growth which can be utilized for biotechnological applications ( Gonzalez and Bashan 2000 ). Likewise, Chlorella minutissima was co-cultured with Escherichia coli under mixotrophic conditions and resulted in enhanced production of biofuel precursors ( Higgins and VanderGheynst 2014 ). Accordingly, there is a growing interest in exploiting the potential of algal–bacterial co-cultures for algal biotechnology ( Lian et al. 2021 ; Sánchez-Zurano et al. 2020 ; Padmaperuma et al. 2018 ; Meyer and Nai 2018 ). Here, we aimed to develop a simple gelatin-based hydrogel system by combining microalgae and bacteria for spaceefficient microalgal cultivation. We hypothesized that cocultivation of algae and bacteria would result in improved growth and performance of the algae in hydrogels. For this, we chose the green microalga Marinichlorella kaistiae KAS603 and screened 14 marine bacterial strains for beneficial effects on algal biomass. Based on these results, we further measured the bio-optical properties and photosynthetic performance of a synthetic co-culture between M. kaistiae KAS603 and a novel strain of Erythrobacter sp. We also aimed to evaluate the beneficial effects of the Erythrobacter strain on a range of microalgae covering coccolithophorids, red algae, and other species of green microalgae. Finally, the mechanical stability of our hydrogel system was tested under different environmental conditions.",
"discussion": "Results and discussion Here, we developed a simple hydrogel system for the spaceefficient co-culture of microalgae. We found that a novel strain of Erythrobacter sp. (SIO_La6, Fig. 2 ) isolated from Southern California coastal waters (off Scripps Pier) has beneficial effects on growth and photosynthetic performance of microalgae immobilized in hydrogels. Cell density differences between treatments Microalgal cell density was on average 2.3-fold enhanced for M. kaistiae KAS603 gels co-cultured with SIO_La6 (mean = 2.85 × 10 7 cells mL −1 , SD = 5.94 × 10 6 , n = 5) compared to monoculture gels (1.18 x10 7 cells mL −1 , SD = 4.06 × 10 6 , n = 5) after 72 h of cultivation (unpaired t test, p < 0.01, Fig. 3a ). The cell doubling time was 16.75 h for co-cultures compared to 33.11 h for monocultures ( Fig. 3 ). The beneficial effects of co-culture with Erythrobacter sp. SIO_La6 were also evident in liquid culture, although the relative growth-stimulating effect was 15% higher in hydrogel cultivation ( Supplementary Fig. 2 ). In a stagnant hydrogel, gas exchange is likely to become a limiting growth factor, while such limitation is unlikely to occur in a liquid mixed culture. Thus, the relative enhancement for hydrogel cultures could suggest that bacterial colonies stimulate gas exchange and provide nutrients and/or growth-promoting hormones locally within the hydrogel. Indeed, bacteria observed during confocal microscopy were observed forming aggregates around algal cells ( Supplementary Fig. 3 ). Likewise, it is known that different Erythrobacter strains induce aggregation of different diatom species ( Tran et al. 2020 ). Previous research into immobilized algae-bacteria co-cultures have observed similar formations of aggregates and biofilms, which resulted in improved growth and stability ( de-Bashan et al. 2011 , 2016 ). This proximity, in a gel compared to liquid culture, may facilitate and/or stabilize the interactions between the algae and bacteria for provision of photosynthate from the algae and in return growth-enhancing micronutrients (e.g., vitamins) and gases (e.g., CO 2 ) from bacteria ( Kazamia et al. 2012 ; Paerl et al. 2015 ; Higgins et al. 2016 ; Helliwell 2017 ). Following the successful tests with M. kaistiae KAS603, other common microalgae were tested in co-culture with SIO_La6. The bacterial co-culture enhanced microalgal growth for three of the five microalgal strains compared to monoculture controls ( Fig. 3b ). Cell densities after 3 days of cultivation were at least twofold higher for the coccolithophorid alga P. carterae and the red alga P. cruentum when grown in co-culture hydrogels ( Fig. 3b ). Interestingly, cultures that did not perform well in co-culture (e.g., Micromonas sp. and A. carterae ) also showed limited growth when encapsulated in the gelatin-based hydrogel in monoculture, suggesting that hydrogel immobilization interfered with the growth dynamics of these algae ( Fig. 3b ). This suggests that Micromonas sp. and A. carterae might not be suitable candidates for biotechnological applications using hydrogel immobilization. Understanding the metabolic and molecular mechanisms underlying this beneficial interaction is a complex task that would require potential metabolomic and proteomic approaches (see, e.g., Kazamia et al. 2016 ; Helliwell et al. 2018 ) which was beyond the scope of the present study. However, it is noteworthy that we found growth-enhancing effects of Erythrobacter SIO_LA6 on vitamin B 12 –independent algae ( M. kaistiae KAS603) and vitamin B 12 –dependent algae ( P. carterae , Croft et al. 2005 ). This suggests that the beneficial effects are unlikely due to vitamin production by Erythrobacter SIO_LA6 and rather related to other benefits (e.g., growth hormones or gas exchange). Co-culture effects on microalgal photosynthesis and bio-optics Compared to M. kaistiae KAS603 monocultures, O 2 microsensor measurements in co-cultures indicated 4.9-fold enhancements of net photosynthesis at high light (550 μmol photons m −2 s −1 ) irradiance regimes ( Fig. 4a ). In addition, co-cultures exhibited about 4.3-fold greater rates of dark respiration ( Fig. 4a ). Variable chlorophyll- a fluorimetry measurements showed significant enhancements in the maximum quantum yield of PSII (F v /F m ) for co-culture hydrogels compared to monoculture hydrogels during 7 days of growth (mean = 0.603, SD = 0.022 vs. mean = 0.535, SD = 0.004, respectively; Fig. 4b , unpaired t test p = 0.0339). F v /F m is a key parameter used to assess the healthiness of photosynthesizing microalgae (e.g., Baker 2008 ) and thus suggests that algae in co-culture displayed superior photosynthetic capacities. Likewise, relative electron transport rates showed clear differences in key photosynthetic parameters including α and ETR max ( Fig. 4d–f , Table 1 ). For instance, at day 3 ETR max was about 71.6% higher for cocultures versus monocultures ( Fig. 4d–f , Table 1 ). Although areal net photosynthetic ( P n ) rates were strongly enhanced in co-culture, these differences were also affected by the greater algal growth in co-culture ( Fig. 3 ). However, normalizing P n rates to the differences in biomass still suggests an approximate doubling in net photosynthesis in co-culture versus monoculture (compare Figs. 3a and 4a). As Erythrobacter spp. are anoxygenic phototrophic bacteria and thus does not produce O 2 ( Koblizek et al. 2003 ), such differences strongly suggest cell-specific enhancements of photosynthetic activity by M. kaistiae KAS603 in the presence of Erythrobacter. It is important to note that these measurements include respiratory activity by the bacteria, further strengthening the argument of enhanced algal photosynthesis in co-culture. PAM measurements can detect potential electron transport by Eyrythrobacter sp. ( Chandaravithoon et al. 2020 ); however, we did not find any measurable quantum yield of PSII from SIO_LA6 in monoculture (F v / F m = 0, data not shown). In addition, diffuse reflectance measurements did not show characteristic absorption peaks of bacteriochlorophyll a at ~ 750 nm ( Fig. 5 , Yurkov and Beatty 1998 ), suggesting that pigment synthesis and photosynthetic electron transport might be low by this Erythrobacter strain. In turn, reflectance in the nearinfrared region (~ 750 nm) was about 2.5-fold enhanced which could be indicative of the production of lightscattering microbial extracellular polymeric substances (EPS; Flemming and Wingender 2001 ). Such EPS has previously been shown to scatter light and could potentially enhance the internal actinic irradiance intensity which would further promote photosynthesis ( Decho et al. 2003 ; Fisher et al. 2019 ). Clearly, there are various potential mechanisms underlying the enhanced photosynthetic performance of the co-culture hydrogels and a detailed understanding of the mechanisms was beyond the scope of this first study. However, taken together, our results indicate that Erythrobacter sp. SIO_La6 enhances M. kaistiae KAS603 photosynthesis ( Table 1 ) which could explain the enhanced algal biomass in co-culture. Contamination resistance in hydrogels A potential key problem in cultivating microalgae in hydrogels is that most biopolymers are readily degraded by various bacterial communities ( Pathak et al. 2017 ). We hypothesized that co-cultivation might provide protection from such degradation by occupying microbial habitats within the hydrogel and potentially producing antibiotics. Such concept is analogous to the role of the microbial community in the coral mucus, which protects from opportunistic microbes ( Shnit-Orland and Kushmaro 2009 ). Following exposure to natural seawater, co-culture gels remained viable and no visible degradation of the gelatin matrix was noticeable even after 7 days of cultivation ( Fig. 6a–e ). However, monocultures showed clear degradation and liquefaction of the polymer matrix within 24 h ( Fig. 6a–e ). Likewise, previous experiments using Chlorella–bacteria co-cultures in alginate beads found reduced contamination by foreign bacteria from the environment and concluded that co-cultured bacteria provide a physical barrier ( Covarrubias et al. 2012 ). Here, it is likely that DOC produced by the algae might enhance virulence factors (present in SIO_La6 genomes, J. Dinasquet personal communication) and toxin production as observed in other Erythrobacter species in the presence of algal DOC ( Cárdenas et al. 2018 ). This induced pathogenicity might have antagonistic effects against environmental contaminants. Although the mechanisms warrant further investigation, these initial results suggest protective effects of our synthetic co-culture hydrogel from external microbes. Thus, co-cultivation with Erythrobacter SIO_LA6 stabilizes the biopolymer matrix and reduces the chance for bacterial degradation. This could therefore reduce the need for costly measures to prevent invasion by adventitious bacteria or other predators that might be attracted by the breakdown products. Given that surface-associated/biofilm-based cultivation methods are increasing in various algal biotechnological applications, our study potentially provides a simple and cheap cultivation system with minimal maintenance requirements. This approach can be further developed as a viable bio-inspired alternative to costly antibiotic treatments that are currently used in such cultivation approaches ( Berner et al. 2015 )."
} | 4,418 |
39660099 | PMC7617206 | pmc | 2,668 | {
"abstract": "Photosynthetic microalgae are an attractive source of food, fuel, or nutraceuticals, but commercial production of microalgae is limited by low spatial efficiency. In the present study we developed a simple photosynthetic hydrogel system that cultivates the green microalga, Marinichlorella kaistiae KAS603, together with a novel strain of the bacteria, Erythrobacter sp. We tested the performance of the co-culture in the hydrogel using a combination of chlorophyll- a fluorimetry, microsensing, and bio-optical measurements. Our results showed that growth rates in algal–bacterial hydrogels were about threefold enhanced compared to hydrogels with algae alone. Chlorophyll- a fluorimetry–based light curves found that electron transport rates were enhanced about 20% for algal–bacterial hydrogels compared to algal hydrogels for intermediate irradiance levels. We also show that the living hydrogel is stable under different environmental conditions and when exposed to natural seawater. Our study provides a potential bio-inspired solution for problems that limit the space-efficient cultivation of microalgae for biotechnological applications.",
"conclusion": "Conclusions This study developed a simple hydrogel system for microalgal cultivation in co-culture with a novel strain of Erythrobacter sp. Our findings demonstrate enhanced photosynthetic activity and growth rates of microalgae in co-culture when immobilized in our hydrogel system. We further show that our gelatin-based hydrogel is easy to fabricate, requires low maintenance, and remains stable when the co-culture is exposed to natural contaminants. Our study suggests that co-cultivation in hydrogels of microalgae with Erythrobacter sp. enhances microalgal growth and density, and could potentially reduce the need for costly antibiotics. We conclude that hydrogel algal–bacterial co-culture is a simple, bio-inspired approach that can be further developed to solve some problems that currently limit microalgal cultivation. These improvements compared to conventional cultivation methods demonstrate potential practical applications of our findings toward more efficient micro-algal cultivation.",
"introduction": "Introduction Microscopic photosynthesizing algae produce a range of high value products including lipids and pigments ( Borowitzka 2013 ). In addition, algal biomass is of great interest for use as feedstocks in aquaculture and for the generation of biofuels ( Villarruel-Lopez et al. 2017 ; Khan et al. 2018 ). However, commercial large-scale production of microalgae is still limited by low spatial efficiency and associated high production and processing costs (e.g., Borowitzka and Vonshak 2017 ). Algal cultivation techniques can generally be divided into open pond systems, closed photobioreactors, and biofilm-based systems ( Posten 2009 ). Open pond systems cultivate algae in raceway ponds and have low maintenance cost but generate only limited biomass per area ( Tan et al. 2020 ). Photobioreactor systems allow for controlled conditions of irradiance, gas flux and temperature, and yield higher algal growth efficiencies, but have high operation and maintenance costs ( Lee 2001 ; Tan et al. 2020 ). Biofilm-based systems cultivate algae as surface-attached biofilms rather than in liquid suspensions. Algal biofilm cultivation can lead to reduced operation costs due to limited water and energy use, as well as improved algal harvesting efficiencies ( Ozkan et al. 2012 ; Berner et al. 2015 ). Biofilm systems also demonstrate greater CO 2 utilization efficiency and reduced harvesting cost ( Blanken et al. 2017 ; Roostaei et al. 2018 ). These systems, however, are also constrained, often relying on sophisticated artificial architectures to compete with the efficiency of natural systems and are much harder to scale up. More recently, algae have also been cultivated while immobilized in hydrogels ( Berner et al. 2015 ). Hydrogel immobilization enables reduced water usage during algal cultivation and provides a potential physical barrier against bacterial infections ( Brenner et al. 2008 ; Covarrubias et al. 2012 ). 3D bioprinting has been used to create different hydrogel structures growing a range of microalgal strains ( Krujatz et al. 2015 ; Lode et al. 2015 ; Wangpraseurt et al. 2020 ). To optimize light propagation in hydrogels with high microalgal densities, coral-inspired biomaterials have recently been developed ( Wangpraseurt et al. 2020 ). However, the cultivation of microalgae in hydrogel-based systems still requires further development regarding the exchange of gases and metabolites that are essential for microalgal growth ( Podola et al. 2017 ). To overcome diffusion limitation in attached cultivation systems, previous efforts have included the development of porous substrate-based bioreactors that make use of a porous membrane to deliver nutrients and promote gas exchange, while the surface of the biofilm is in direct contact with the ambient gas phase ( Podola et al. 2017 ). In nature, benthic photosynthetic symbiotic organisms (e.g., corals, anemones) have faced similar challenges as photosynthesis in thick tissues can theoretically become limited by the diffusion-limited provision of HCO 3 − from the ambient water phase ( Schrameyer et al. 2014 ). However, it has been shown that coral animal and bacterial respiration promote photosynthesis of their symbiotic microalgae, suggesting that the coral host provides essential metabolites and nutrients locally to the microalgae (e.g., Kuhl et al. 1996 ; Schrameyer et al. 2014 ). In corals, the microbial community performs critical functions for the coral holobiont including pathogen protection, sulfur, and nitrogen cycling as well as beneficial modulations of the host microhabitat ( Rosenberg et al. 2009 ; Ceh et al. 2013 ; Krediet et al. 2013 ). Benefits of bacterial communities for an algal host have been documented in free-living algae as well (e.g., Kazamia et al. 2012 ). Some bacteria can provide a local supply of essential nutrient compounds required by the algae, including nitrogen, inorganic carbon, vitamin B 12 (cobalamin), and growth-promoting hormones ( Kouzuma and Watanabe 2015 ). For example, one study estimated that 50% of algal species are cobalamin auxotrophs, implying a reliance on bacterial-produced cobalamin ( Croft et al. 2005 ). More generally, symbiotic relationships between microalgae and bacteria often employ a mutually beneficial exchange of carbon and nitrogen ( Thompson et al. 2012 ; de-Bashan et al. 2016 ). Experiments working with the microalgae Chlorella in co-culture with a known growth-promoting bacteria in alginate beads demonstrated enhanced growth which can be utilized for biotechnological applications ( Gonzalez and Bashan 2000 ). Likewise, Chlorella minutissima was co-cultured with Escherichia coli under mixotrophic conditions and resulted in enhanced production of biofuel precursors ( Higgins and VanderGheynst 2014 ). Accordingly, there is a growing interest in exploiting the potential of algal–bacterial co-cultures for algal biotechnology ( Lian et al. 2021 ; Sánchez-Zurano et al. 2020 ; Padmaperuma et al. 2018 ; Meyer and Nai 2018 ). Here, we aimed to develop a simple gelatin-based hydrogel system by combining microalgae and bacteria for spaceefficient microalgal cultivation. We hypothesized that cocultivation of algae and bacteria would result in improved growth and performance of the algae in hydrogels. For this, we chose the green microalga Marinichlorella kaistiae KAS603 and screened 14 marine bacterial strains for beneficial effects on algal biomass. Based on these results, we further measured the bio-optical properties and photosynthetic performance of a synthetic co-culture between M. kaistiae KAS603 and a novel strain of Erythrobacter sp. We also aimed to evaluate the beneficial effects of the Erythrobacter strain on a range of microalgae covering coccolithophorids, red algae, and other species of green microalgae. Finally, the mechanical stability of our hydrogel system was tested under different environmental conditions.",
"discussion": "Results and discussion Here, we developed a simple hydrogel system for the spaceefficient co-culture of microalgae. We found that a novel strain of Erythrobacter sp. (SIO_La6, Fig. 2 ) isolated from Southern California coastal waters (off Scripps Pier) has beneficial effects on growth and photosynthetic performance of microalgae immobilized in hydrogels. Cell density differences between treatments Microalgal cell density was on average 2.3-fold enhanced for M. kaistiae KAS603 gels co-cultured with SIO_La6 (mean = 2.85 × 10 7 cells mL −1 , SD = 5.94 × 10 6 , n = 5) compared to monoculture gels (1.18 x10 7 cells mL −1 , SD = 4.06 × 10 6 , n = 5) after 72 h of cultivation (unpaired t test, p < 0.01, Fig. 3a ). The cell doubling time was 16.75 h for co-cultures compared to 33.11 h for monocultures ( Fig. 3 ). The beneficial effects of co-culture with Erythrobacter sp. SIO_La6 were also evident in liquid culture, although the relative growth-stimulating effect was 15% higher in hydrogel cultivation ( Supplementary Fig. 2 ). In a stagnant hydrogel, gas exchange is likely to become a limiting growth factor, while such limitation is unlikely to occur in a liquid mixed culture. Thus, the relative enhancement for hydrogel cultures could suggest that bacterial colonies stimulate gas exchange and provide nutrients and/or growth-promoting hormones locally within the hydrogel. Indeed, bacteria observed during confocal microscopy were observed forming aggregates around algal cells ( Supplementary Fig. 3 ). Likewise, it is known that different Erythrobacter strains induce aggregation of different diatom species ( Tran et al. 2020 ). Previous research into immobilized algae-bacteria co-cultures have observed similar formations of aggregates and biofilms, which resulted in improved growth and stability ( de-Bashan et al. 2011 , 2016 ). This proximity, in a gel compared to liquid culture, may facilitate and/or stabilize the interactions between the algae and bacteria for provision of photosynthate from the algae and in return growth-enhancing micronutrients (e.g., vitamins) and gases (e.g., CO 2 ) from bacteria ( Kazamia et al. 2012 ; Paerl et al. 2015 ; Higgins et al. 2016 ; Helliwell 2017 ). Following the successful tests with M. kaistiae KAS603, other common microalgae were tested in co-culture with SIO_La6. The bacterial co-culture enhanced microalgal growth for three of the five microalgal strains compared to monoculture controls ( Fig. 3b ). Cell densities after 3 days of cultivation were at least twofold higher for the coccolithophorid alga P. carterae and the red alga P. cruentum when grown in co-culture hydrogels ( Fig. 3b ). Interestingly, cultures that did not perform well in co-culture (e.g., Micromonas sp. and A. carterae ) also showed limited growth when encapsulated in the gelatin-based hydrogel in monoculture, suggesting that hydrogel immobilization interfered with the growth dynamics of these algae ( Fig. 3b ). This suggests that Micromonas sp. and A. carterae might not be suitable candidates for biotechnological applications using hydrogel immobilization. Understanding the metabolic and molecular mechanisms underlying this beneficial interaction is a complex task that would require potential metabolomic and proteomic approaches (see, e.g., Kazamia et al. 2016 ; Helliwell et al. 2018 ) which was beyond the scope of the present study. However, it is noteworthy that we found growth-enhancing effects of Erythrobacter SIO_LA6 on vitamin B 12 –independent algae ( M. kaistiae KAS603) and vitamin B 12 –dependent algae ( P. carterae , Croft et al. 2005 ). This suggests that the beneficial effects are unlikely due to vitamin production by Erythrobacter SIO_LA6 and rather related to other benefits (e.g., growth hormones or gas exchange). Co-culture effects on microalgal photosynthesis and bio-optics Compared to M. kaistiae KAS603 monocultures, O 2 microsensor measurements in co-cultures indicated 4.9-fold enhancements of net photosynthesis at high light (550 μmol photons m −2 s −1 ) irradiance regimes ( Fig. 4a ). In addition, co-cultures exhibited about 4.3-fold greater rates of dark respiration ( Fig. 4a ). Variable chlorophyll- a fluorimetry measurements showed significant enhancements in the maximum quantum yield of PSII (F v /F m ) for co-culture hydrogels compared to monoculture hydrogels during 7 days of growth (mean = 0.603, SD = 0.022 vs. mean = 0.535, SD = 0.004, respectively; Fig. 4b , unpaired t test p = 0.0339). F v /F m is a key parameter used to assess the healthiness of photosynthesizing microalgae (e.g., Baker 2008 ) and thus suggests that algae in co-culture displayed superior photosynthetic capacities. Likewise, relative electron transport rates showed clear differences in key photosynthetic parameters including α and ETR max ( Fig. 4d–f , Table 1 ). For instance, at day 3 ETR max was about 71.6% higher for cocultures versus monocultures ( Fig. 4d–f , Table 1 ). Although areal net photosynthetic ( P n ) rates were strongly enhanced in co-culture, these differences were also affected by the greater algal growth in co-culture ( Fig. 3 ). However, normalizing P n rates to the differences in biomass still suggests an approximate doubling in net photosynthesis in co-culture versus monoculture (compare Figs. 3a and 4a). As Erythrobacter spp. are anoxygenic phototrophic bacteria and thus does not produce O 2 ( Koblizek et al. 2003 ), such differences strongly suggest cell-specific enhancements of photosynthetic activity by M. kaistiae KAS603 in the presence of Erythrobacter. It is important to note that these measurements include respiratory activity by the bacteria, further strengthening the argument of enhanced algal photosynthesis in co-culture. PAM measurements can detect potential electron transport by Eyrythrobacter sp. ( Chandaravithoon et al. 2020 ); however, we did not find any measurable quantum yield of PSII from SIO_LA6 in monoculture (F v / F m = 0, data not shown). In addition, diffuse reflectance measurements did not show characteristic absorption peaks of bacteriochlorophyll a at ~ 750 nm ( Fig. 5 , Yurkov and Beatty 1998 ), suggesting that pigment synthesis and photosynthetic electron transport might be low by this Erythrobacter strain. In turn, reflectance in the nearinfrared region (~ 750 nm) was about 2.5-fold enhanced which could be indicative of the production of lightscattering microbial extracellular polymeric substances (EPS; Flemming and Wingender 2001 ). Such EPS has previously been shown to scatter light and could potentially enhance the internal actinic irradiance intensity which would further promote photosynthesis ( Decho et al. 2003 ; Fisher et al. 2019 ). Clearly, there are various potential mechanisms underlying the enhanced photosynthetic performance of the co-culture hydrogels and a detailed understanding of the mechanisms was beyond the scope of this first study. However, taken together, our results indicate that Erythrobacter sp. SIO_La6 enhances M. kaistiae KAS603 photosynthesis ( Table 1 ) which could explain the enhanced algal biomass in co-culture. Contamination resistance in hydrogels A potential key problem in cultivating microalgae in hydrogels is that most biopolymers are readily degraded by various bacterial communities ( Pathak et al. 2017 ). We hypothesized that co-cultivation might provide protection from such degradation by occupying microbial habitats within the hydrogel and potentially producing antibiotics. Such concept is analogous to the role of the microbial community in the coral mucus, which protects from opportunistic microbes ( Shnit-Orland and Kushmaro 2009 ). Following exposure to natural seawater, co-culture gels remained viable and no visible degradation of the gelatin matrix was noticeable even after 7 days of cultivation ( Fig. 6a–e ). However, monocultures showed clear degradation and liquefaction of the polymer matrix within 24 h ( Fig. 6a–e ). Likewise, previous experiments using Chlorella–bacteria co-cultures in alginate beads found reduced contamination by foreign bacteria from the environment and concluded that co-cultured bacteria provide a physical barrier ( Covarrubias et al. 2012 ). Here, it is likely that DOC produced by the algae might enhance virulence factors (present in SIO_La6 genomes, J. Dinasquet personal communication) and toxin production as observed in other Erythrobacter species in the presence of algal DOC ( Cárdenas et al. 2018 ). This induced pathogenicity might have antagonistic effects against environmental contaminants. Although the mechanisms warrant further investigation, these initial results suggest protective effects of our synthetic co-culture hydrogel from external microbes. Thus, co-cultivation with Erythrobacter SIO_LA6 stabilizes the biopolymer matrix and reduces the chance for bacterial degradation. This could therefore reduce the need for costly measures to prevent invasion by adventitious bacteria or other predators that might be attracted by the breakdown products. Given that surface-associated/biofilm-based cultivation methods are increasing in various algal biotechnological applications, our study potentially provides a simple and cheap cultivation system with minimal maintenance requirements. This approach can be further developed as a viable bio-inspired alternative to costly antibiotic treatments that are currently used in such cultivation approaches ( Berner et al. 2015 )."
} | 4,418 |
39660099 | PMC7617206 | pmc | 2,668 | {
"abstract": "Photosynthetic microalgae are an attractive source of food, fuel, or nutraceuticals, but commercial production of microalgae is limited by low spatial efficiency. In the present study we developed a simple photosynthetic hydrogel system that cultivates the green microalga, Marinichlorella kaistiae KAS603, together with a novel strain of the bacteria, Erythrobacter sp. We tested the performance of the co-culture in the hydrogel using a combination of chlorophyll- a fluorimetry, microsensing, and bio-optical measurements. Our results showed that growth rates in algal–bacterial hydrogels were about threefold enhanced compared to hydrogels with algae alone. Chlorophyll- a fluorimetry–based light curves found that electron transport rates were enhanced about 20% for algal–bacterial hydrogels compared to algal hydrogels for intermediate irradiance levels. We also show that the living hydrogel is stable under different environmental conditions and when exposed to natural seawater. Our study provides a potential bio-inspired solution for problems that limit the space-efficient cultivation of microalgae for biotechnological applications.",
"conclusion": "Conclusions This study developed a simple hydrogel system for microalgal cultivation in co-culture with a novel strain of Erythrobacter sp. Our findings demonstrate enhanced photosynthetic activity and growth rates of microalgae in co-culture when immobilized in our hydrogel system. We further show that our gelatin-based hydrogel is easy to fabricate, requires low maintenance, and remains stable when the co-culture is exposed to natural contaminants. Our study suggests that co-cultivation in hydrogels of microalgae with Erythrobacter sp. enhances microalgal growth and density, and could potentially reduce the need for costly antibiotics. We conclude that hydrogel algal–bacterial co-culture is a simple, bio-inspired approach that can be further developed to solve some problems that currently limit microalgal cultivation. These improvements compared to conventional cultivation methods demonstrate potential practical applications of our findings toward more efficient micro-algal cultivation.",
"introduction": "Introduction Microscopic photosynthesizing algae produce a range of high value products including lipids and pigments ( Borowitzka 2013 ). In addition, algal biomass is of great interest for use as feedstocks in aquaculture and for the generation of biofuels ( Villarruel-Lopez et al. 2017 ; Khan et al. 2018 ). However, commercial large-scale production of microalgae is still limited by low spatial efficiency and associated high production and processing costs (e.g., Borowitzka and Vonshak 2017 ). Algal cultivation techniques can generally be divided into open pond systems, closed photobioreactors, and biofilm-based systems ( Posten 2009 ). Open pond systems cultivate algae in raceway ponds and have low maintenance cost but generate only limited biomass per area ( Tan et al. 2020 ). Photobioreactor systems allow for controlled conditions of irradiance, gas flux and temperature, and yield higher algal growth efficiencies, but have high operation and maintenance costs ( Lee 2001 ; Tan et al. 2020 ). Biofilm-based systems cultivate algae as surface-attached biofilms rather than in liquid suspensions. Algal biofilm cultivation can lead to reduced operation costs due to limited water and energy use, as well as improved algal harvesting efficiencies ( Ozkan et al. 2012 ; Berner et al. 2015 ). Biofilm systems also demonstrate greater CO 2 utilization efficiency and reduced harvesting cost ( Blanken et al. 2017 ; Roostaei et al. 2018 ). These systems, however, are also constrained, often relying on sophisticated artificial architectures to compete with the efficiency of natural systems and are much harder to scale up. More recently, algae have also been cultivated while immobilized in hydrogels ( Berner et al. 2015 ). Hydrogel immobilization enables reduced water usage during algal cultivation and provides a potential physical barrier against bacterial infections ( Brenner et al. 2008 ; Covarrubias et al. 2012 ). 3D bioprinting has been used to create different hydrogel structures growing a range of microalgal strains ( Krujatz et al. 2015 ; Lode et al. 2015 ; Wangpraseurt et al. 2020 ). To optimize light propagation in hydrogels with high microalgal densities, coral-inspired biomaterials have recently been developed ( Wangpraseurt et al. 2020 ). However, the cultivation of microalgae in hydrogel-based systems still requires further development regarding the exchange of gases and metabolites that are essential for microalgal growth ( Podola et al. 2017 ). To overcome diffusion limitation in attached cultivation systems, previous efforts have included the development of porous substrate-based bioreactors that make use of a porous membrane to deliver nutrients and promote gas exchange, while the surface of the biofilm is in direct contact with the ambient gas phase ( Podola et al. 2017 ). In nature, benthic photosynthetic symbiotic organisms (e.g., corals, anemones) have faced similar challenges as photosynthesis in thick tissues can theoretically become limited by the diffusion-limited provision of HCO 3 − from the ambient water phase ( Schrameyer et al. 2014 ). However, it has been shown that coral animal and bacterial respiration promote photosynthesis of their symbiotic microalgae, suggesting that the coral host provides essential metabolites and nutrients locally to the microalgae (e.g., Kuhl et al. 1996 ; Schrameyer et al. 2014 ). In corals, the microbial community performs critical functions for the coral holobiont including pathogen protection, sulfur, and nitrogen cycling as well as beneficial modulations of the host microhabitat ( Rosenberg et al. 2009 ; Ceh et al. 2013 ; Krediet et al. 2013 ). Benefits of bacterial communities for an algal host have been documented in free-living algae as well (e.g., Kazamia et al. 2012 ). Some bacteria can provide a local supply of essential nutrient compounds required by the algae, including nitrogen, inorganic carbon, vitamin B 12 (cobalamin), and growth-promoting hormones ( Kouzuma and Watanabe 2015 ). For example, one study estimated that 50% of algal species are cobalamin auxotrophs, implying a reliance on bacterial-produced cobalamin ( Croft et al. 2005 ). More generally, symbiotic relationships between microalgae and bacteria often employ a mutually beneficial exchange of carbon and nitrogen ( Thompson et al. 2012 ; de-Bashan et al. 2016 ). Experiments working with the microalgae Chlorella in co-culture with a known growth-promoting bacteria in alginate beads demonstrated enhanced growth which can be utilized for biotechnological applications ( Gonzalez and Bashan 2000 ). Likewise, Chlorella minutissima was co-cultured with Escherichia coli under mixotrophic conditions and resulted in enhanced production of biofuel precursors ( Higgins and VanderGheynst 2014 ). Accordingly, there is a growing interest in exploiting the potential of algal–bacterial co-cultures for algal biotechnology ( Lian et al. 2021 ; Sánchez-Zurano et al. 2020 ; Padmaperuma et al. 2018 ; Meyer and Nai 2018 ). Here, we aimed to develop a simple gelatin-based hydrogel system by combining microalgae and bacteria for spaceefficient microalgal cultivation. We hypothesized that cocultivation of algae and bacteria would result in improved growth and performance of the algae in hydrogels. For this, we chose the green microalga Marinichlorella kaistiae KAS603 and screened 14 marine bacterial strains for beneficial effects on algal biomass. Based on these results, we further measured the bio-optical properties and photosynthetic performance of a synthetic co-culture between M. kaistiae KAS603 and a novel strain of Erythrobacter sp. We also aimed to evaluate the beneficial effects of the Erythrobacter strain on a range of microalgae covering coccolithophorids, red algae, and other species of green microalgae. Finally, the mechanical stability of our hydrogel system was tested under different environmental conditions.",
"discussion": "Results and discussion Here, we developed a simple hydrogel system for the spaceefficient co-culture of microalgae. We found that a novel strain of Erythrobacter sp. (SIO_La6, Fig. 2 ) isolated from Southern California coastal waters (off Scripps Pier) has beneficial effects on growth and photosynthetic performance of microalgae immobilized in hydrogels. Cell density differences between treatments Microalgal cell density was on average 2.3-fold enhanced for M. kaistiae KAS603 gels co-cultured with SIO_La6 (mean = 2.85 × 10 7 cells mL −1 , SD = 5.94 × 10 6 , n = 5) compared to monoculture gels (1.18 x10 7 cells mL −1 , SD = 4.06 × 10 6 , n = 5) after 72 h of cultivation (unpaired t test, p < 0.01, Fig. 3a ). The cell doubling time was 16.75 h for co-cultures compared to 33.11 h for monocultures ( Fig. 3 ). The beneficial effects of co-culture with Erythrobacter sp. SIO_La6 were also evident in liquid culture, although the relative growth-stimulating effect was 15% higher in hydrogel cultivation ( Supplementary Fig. 2 ). In a stagnant hydrogel, gas exchange is likely to become a limiting growth factor, while such limitation is unlikely to occur in a liquid mixed culture. Thus, the relative enhancement for hydrogel cultures could suggest that bacterial colonies stimulate gas exchange and provide nutrients and/or growth-promoting hormones locally within the hydrogel. Indeed, bacteria observed during confocal microscopy were observed forming aggregates around algal cells ( Supplementary Fig. 3 ). Likewise, it is known that different Erythrobacter strains induce aggregation of different diatom species ( Tran et al. 2020 ). Previous research into immobilized algae-bacteria co-cultures have observed similar formations of aggregates and biofilms, which resulted in improved growth and stability ( de-Bashan et al. 2011 , 2016 ). This proximity, in a gel compared to liquid culture, may facilitate and/or stabilize the interactions between the algae and bacteria for provision of photosynthate from the algae and in return growth-enhancing micronutrients (e.g., vitamins) and gases (e.g., CO 2 ) from bacteria ( Kazamia et al. 2012 ; Paerl et al. 2015 ; Higgins et al. 2016 ; Helliwell 2017 ). Following the successful tests with M. kaistiae KAS603, other common microalgae were tested in co-culture with SIO_La6. The bacterial co-culture enhanced microalgal growth for three of the five microalgal strains compared to monoculture controls ( Fig. 3b ). Cell densities after 3 days of cultivation were at least twofold higher for the coccolithophorid alga P. carterae and the red alga P. cruentum when grown in co-culture hydrogels ( Fig. 3b ). Interestingly, cultures that did not perform well in co-culture (e.g., Micromonas sp. and A. carterae ) also showed limited growth when encapsulated in the gelatin-based hydrogel in monoculture, suggesting that hydrogel immobilization interfered with the growth dynamics of these algae ( Fig. 3b ). This suggests that Micromonas sp. and A. carterae might not be suitable candidates for biotechnological applications using hydrogel immobilization. Understanding the metabolic and molecular mechanisms underlying this beneficial interaction is a complex task that would require potential metabolomic and proteomic approaches (see, e.g., Kazamia et al. 2016 ; Helliwell et al. 2018 ) which was beyond the scope of the present study. However, it is noteworthy that we found growth-enhancing effects of Erythrobacter SIO_LA6 on vitamin B 12 –independent algae ( M. kaistiae KAS603) and vitamin B 12 –dependent algae ( P. carterae , Croft et al. 2005 ). This suggests that the beneficial effects are unlikely due to vitamin production by Erythrobacter SIO_LA6 and rather related to other benefits (e.g., growth hormones or gas exchange). Co-culture effects on microalgal photosynthesis and bio-optics Compared to M. kaistiae KAS603 monocultures, O 2 microsensor measurements in co-cultures indicated 4.9-fold enhancements of net photosynthesis at high light (550 μmol photons m −2 s −1 ) irradiance regimes ( Fig. 4a ). In addition, co-cultures exhibited about 4.3-fold greater rates of dark respiration ( Fig. 4a ). Variable chlorophyll- a fluorimetry measurements showed significant enhancements in the maximum quantum yield of PSII (F v /F m ) for co-culture hydrogels compared to monoculture hydrogels during 7 days of growth (mean = 0.603, SD = 0.022 vs. mean = 0.535, SD = 0.004, respectively; Fig. 4b , unpaired t test p = 0.0339). F v /F m is a key parameter used to assess the healthiness of photosynthesizing microalgae (e.g., Baker 2008 ) and thus suggests that algae in co-culture displayed superior photosynthetic capacities. Likewise, relative electron transport rates showed clear differences in key photosynthetic parameters including α and ETR max ( Fig. 4d–f , Table 1 ). For instance, at day 3 ETR max was about 71.6% higher for cocultures versus monocultures ( Fig. 4d–f , Table 1 ). Although areal net photosynthetic ( P n ) rates were strongly enhanced in co-culture, these differences were also affected by the greater algal growth in co-culture ( Fig. 3 ). However, normalizing P n rates to the differences in biomass still suggests an approximate doubling in net photosynthesis in co-culture versus monoculture (compare Figs. 3a and 4a). As Erythrobacter spp. are anoxygenic phototrophic bacteria and thus does not produce O 2 ( Koblizek et al. 2003 ), such differences strongly suggest cell-specific enhancements of photosynthetic activity by M. kaistiae KAS603 in the presence of Erythrobacter. It is important to note that these measurements include respiratory activity by the bacteria, further strengthening the argument of enhanced algal photosynthesis in co-culture. PAM measurements can detect potential electron transport by Eyrythrobacter sp. ( Chandaravithoon et al. 2020 ); however, we did not find any measurable quantum yield of PSII from SIO_LA6 in monoculture (F v / F m = 0, data not shown). In addition, diffuse reflectance measurements did not show characteristic absorption peaks of bacteriochlorophyll a at ~ 750 nm ( Fig. 5 , Yurkov and Beatty 1998 ), suggesting that pigment synthesis and photosynthetic electron transport might be low by this Erythrobacter strain. In turn, reflectance in the nearinfrared region (~ 750 nm) was about 2.5-fold enhanced which could be indicative of the production of lightscattering microbial extracellular polymeric substances (EPS; Flemming and Wingender 2001 ). Such EPS has previously been shown to scatter light and could potentially enhance the internal actinic irradiance intensity which would further promote photosynthesis ( Decho et al. 2003 ; Fisher et al. 2019 ). Clearly, there are various potential mechanisms underlying the enhanced photosynthetic performance of the co-culture hydrogels and a detailed understanding of the mechanisms was beyond the scope of this first study. However, taken together, our results indicate that Erythrobacter sp. SIO_La6 enhances M. kaistiae KAS603 photosynthesis ( Table 1 ) which could explain the enhanced algal biomass in co-culture. Contamination resistance in hydrogels A potential key problem in cultivating microalgae in hydrogels is that most biopolymers are readily degraded by various bacterial communities ( Pathak et al. 2017 ). We hypothesized that co-cultivation might provide protection from such degradation by occupying microbial habitats within the hydrogel and potentially producing antibiotics. Such concept is analogous to the role of the microbial community in the coral mucus, which protects from opportunistic microbes ( Shnit-Orland and Kushmaro 2009 ). Following exposure to natural seawater, co-culture gels remained viable and no visible degradation of the gelatin matrix was noticeable even after 7 days of cultivation ( Fig. 6a–e ). However, monocultures showed clear degradation and liquefaction of the polymer matrix within 24 h ( Fig. 6a–e ). Likewise, previous experiments using Chlorella–bacteria co-cultures in alginate beads found reduced contamination by foreign bacteria from the environment and concluded that co-cultured bacteria provide a physical barrier ( Covarrubias et al. 2012 ). Here, it is likely that DOC produced by the algae might enhance virulence factors (present in SIO_La6 genomes, J. Dinasquet personal communication) and toxin production as observed in other Erythrobacter species in the presence of algal DOC ( Cárdenas et al. 2018 ). This induced pathogenicity might have antagonistic effects against environmental contaminants. Although the mechanisms warrant further investigation, these initial results suggest protective effects of our synthetic co-culture hydrogel from external microbes. Thus, co-cultivation with Erythrobacter SIO_LA6 stabilizes the biopolymer matrix and reduces the chance for bacterial degradation. This could therefore reduce the need for costly measures to prevent invasion by adventitious bacteria or other predators that might be attracted by the breakdown products. Given that surface-associated/biofilm-based cultivation methods are increasing in various algal biotechnological applications, our study potentially provides a simple and cheap cultivation system with minimal maintenance requirements. This approach can be further developed as a viable bio-inspired alternative to costly antibiotic treatments that are currently used in such cultivation approaches ( Berner et al. 2015 )."
} | 4,418 |
35345437 | PMC8941415 | pmc | 2,669 | {
"abstract": "Classic computational models of collective motion suggest that simple local averaging rules can promote many observed group-level patterns. Recent studies, however, suggest that rules simpler than local averaging may be at play in real organisms; for example, fish stochastically align towards only one randomly chosen neighbour and yet the schools are highly polarized. Here, we ask—how do organisms maintain group cohesion? Using a spatially explicit model, inspired from empirical investigations, we show that group cohesion can be achieved in finite groups even when organisms randomly choose only one neighbour to interact with. Cohesion is maintained even in the absence of local averaging that requires interactions with many neighbours. Furthermore, we show that choosing a neighbour randomly is a better way to achieve cohesion than interacting with just its closest neighbour. To understand how cohesion emerges from these random pairwise interactions, we turn to a graph-theoretic analysis of the underlying dynamic interaction networks. We find that randomness in choosing a neighbour gives rise to well-connected networks that essentially cause the groups to stay cohesive. We compare our findings with the canonical averaging models (analogous to the Vicsek model). In summary, we argue that randomness in the choice of interacting neighbours plays a crucial role in achieving cohesion.",
"conclusion": "4 . Concluding remarks In this study, using a spatially explicit agent-based model, we show that group-level cohesion can emerge when organisms move towards just one other randomly chosen nearby organism. We show that a random choice of the neighbour, rather than a fixed neighbour such as the nearest individual, considerably improves the group cohesion. Cohesion emerges even with such simple stochastic pairwise interactions because choosing a neighbour randomly creates a well-connected long-ranged interaction network. We show that the connectedness of the interaction network correlates well with the cohesivity of the mobile group. Constructing the interaction network was possible because we had complete access to all information pertaining to the interactions, their time-stamps and organisms-indices, owing to the theoretical nature of the work. In an experimental setting, it would be challenging to estimate the underlying network structure from data of organismal motion. In a recent study, a ray casting approach was used to identify a network based on the vision of individual fish [ 48 ]. This network had an edge connecting an organism to every other organism in its perceivable neighbourhood; not specific to attraction interactions (or any other). We believe that re-constructing the hidden interaction networks from movement data would be an exciting future direction for research. However, do organisms really choose a random neighbour to interact with? A random neighbour could be chosen in many ways. For instance, an organism could prefer a faster-moving individual to interact with, over other slower-moving ones. Also, because organisms may move at different speeds during the course of their motion, which change continuously owing to spontaneous activity and collisions, this ‘faster-individual’ may be found anywhere within a neighbourhood of a certain size. Lei and co-workers argue that fish choose to interact with a few of their most-influential neighbours [ 17 ]. However, since the ‘influence’ a neighbour has on a fish is a function of its proximity, relative positions and orientations, which change continuously as fish move in a school [ 16 ], the most-influential fish could essentially take any position within the school at a given time: from the nearest neighbour to the farthest one. We speculate that choosing the most influential neighbour could be similar to choosing a fish randomly from a neighbourhood of size K . In summary, our study shows that when an organism randomly chooses another to interact with, irrespective of specific mechanisms, it results in an interaction network that is well connected, giving rise to considerable group cohesion in small to intermediate group sizes. However, we expect large systems of collective motion to exhibit dynamic fission and fusion; since the topological neighbourhood an organism should perceive to maintain group cohesion will be much larger than what is biologically feasible. We welcome further research on empirically motivated and parametrized models of collective motion that account for stochastic decision-making of organisms with an emphasis on group cohesion, fission–fusion group dynamics and explore the functional significance of the role of heterogeneity between individuals in the group.",
"introduction": "1 . Introduction Organisms that live in social groups often exhibit collective motion, which is important for achieving functions like foraging, navigation, evasion from predation, etc. [ 1 – 4 ]. To explain the highly coordinated motion of such animal groups, classic models of collective motion assume that an agent moves along the average direction of motion of all neighbours that are within a short metric distance around it [ 5 ]. Subsequent models extend on these ideas to incorporate cohesion [ 6 – 8 ]; they assume that agents also move towards an average direction determined by the location of all nearby individuals. Broadly, theory and computational studies predict that non-trivial group-level phenomena can emerge even when organisms follow such simple rules that depend on the states of their neighbours [ 5 , 6 , 9 – 15 ]. Surprisingly, recent empirical studies [ 16 – 20 ] show that organisms interact through rules that are probably much simpler than averaging information of several individuals; in fact, each organism may interact with just a single randomly chosen neighbour (termed stochastic pairwise interaction) to achieve high levels of group polarization [ 18 ]. This order, counterintuitively, can emerge from sampling biases in the interactions owing to the finite size of the group. Consequently, once the group is in a polarized state, it continues to reside in that state for a long time [ 21 ]. To maintain group polarization, group cohesion is a must. However, the mechanisms that keep the group cohesive–in particular the role of stochastic decision making—are not as well explored. Traditionally, to explain group cohesion, computational models assume that organisms interact with all individuals within a fixed metric distance [ 6 , 22 , 23 ]. However, several empirical investigations [ 24 – 26 ] have shown that organisms, in fact, interact with a select few, referred to as topological neighbours, and are not strictly limited by metric distances (say, seven nearest neighbours in the case of starling flocks [ 24 ]). They argue that such topological interactions provide substantially better cohesion than metric distance-based rules. In some fish schools, interaction with the nearest one appears to be sufficient in producing the observed cohesion [ 27 ]. In another species [ 17 ], fish appear to choose the most influential one among their neighbours to maintain cohesion. In echolocating organisms like bats, it is challenging to detect the neighbours as the returning echoes are faint and are probably masked by the loud calls of their neighbours. Consequently, in large groups, bats may only detect one neighbour at a time [ 28 ]; yet roosting bats manage to maintain cohesion even in large mobile groups. While we do expect species-specific behavioural rules at fine scales, one broad question arises at this point: how does group cohesion depend on the choice of neighbours? In this context, we note that real organisms’ behaviours are probably stochastic. While computational models do include an element of randomness for the motion of organisms, they typically ignore this in the context of choice of interacting neighbours (but see [ 29 , 30 ]). Here, we reveal the surprising role of randomness in the choice of neighbours in maintaining group cohesion. In this article, we investigate how cohesion emerges from stochastic attraction interactions using a spatially explicit agent-based model. We study mobile groups made up of individuals that interact with just one neighbour at a time. We explore a class of interaction models that differ only in the way the organism chooses its neighbour to interact with—based on randomness in choice of neighbours. To understand how local interactions lead to cohesion at the group level, we reconstruct the underlying interaction network and employ a graph-theoretic analysis to study the properties of the network in light of how it is linked to the group’s ability to stay cohesive. We compare our findings with the canonical equivalents of the averaging interactions to explain how simpler interactions are sufficient in achieving cohesion.",
"discussion": "3 . Results and discussion 3.1 . Random choice of interacting neighbour, even with one individual, promotes group cohesion When the number of individuals are small, which correspond to group sizes in many simple experiments of collective motion [ 16 , 27 , 32 ], individuals form groups and remain reasonably cohesive even when the interaction is of the near-neighbour-type, i.e. organisms align and attract with only the nearest neighbour ( K = 1, k = 1; see figure 2 a for N = 3, 5). However, as the size of the group increases, interacting with just the nearest neighbour is no longer sufficient. Groups begin to break into smaller clusters of size 2 or 3 where the interactions between the organisms are confined to within the cluster. These clusters eventually drift away (see inset corresponding to K = 1 of figure 2 a ).\n Figure 2 . High levels of cohesion are achieved in mobile groups even when organisms interact with just one neighbour (pairwise interaction) randomly chosen from a nearby neighbourhood of size K . ( a ) Number of clusters N c a group breaks into, as a function of the neighbourhood of the organism K , for different group sizes. As K increases, N c → 1. Inset: snapshots from simulation for size N = 30 and K = {1, 10}. ( b ) Cohesion parameter C increases with neighbourhood size ( K ); inset shows that cohesion reaches the maximum value when the neighbourhood is around 30% of the group size across groups of several sizes. The hashed region is K / N < 0.3 where the change in C is significant. See the electronic supplementary material, video-1 for visualizations of a moving group for different values of K . With increasing size of the topological neighbourhood while still interacting with only one random neighbour, i.e. larger K but still with k = 1, cohesivity of the groups ( C ) increases, taking values close to 1. This indicates that organisms reside in one tightly knit cluster stably throughout all time (see figure 2 b ; also see the electronic supplementary material, video 1 for visualization). This is likely because the number of unique individuals the organisms interact with increases with K . By contrast, when organisms interact only with their nearest neighbour, it is likely that they are interacting with the same neighbour repeatedly (for more information, see the electronic supplementary material, S4). We find the number of neighbours K required to achieve the same level of cohesion scale with group size N . Simply put, the proportion of the topological neighbours required to achieve a given level of group cohesion is independent of the group size N . We find the cohesion parameter to saturate when the ratio of the topological neighbourhood to the total group size reaches a threshold of approximately 0.3 (hashed region in figure 2 b ). We find that this threshold ratio reduces when organisms’ speed ( s 0 ) reduces, or with increasing rates of attraction ( r a ) and alignment ( r p ) (see the electronic supplementary material, S2). 3.2 . Attraction interaction network reveals why cohesion emerges To understand how cohesion emerges even from simple stochastic pairwise interactions, we turn to a graph-theoretic analysis of the underlying attraction interaction network between organisms in a group. We emphasize that we focus on attraction interactions rather than alignment interactions since our study is centred around how organisms maintain cohesion. While it is true that local directional alignment alone can also cause some degree of attraction, a major determining cause of group cohesion is the local attraction (see the electronic supplementary material, S5). In our analysis, individual organisms can be considered as nodes, and a directed edge can be constructed from organisms i to j , whenever i exhibits an attraction interaction towards j . Since organisms interact asynchronously in our model, these edges are formed at distinct instants of time. Hence, to construct a graph that faithfully represents the underlying interaction network, we observe the different connections that arise between individuals over a time window t w . To choose an appropriate time scale t w for the analysis, we use the length and velocity scales in the system corresponding to the motion of organism required to break free from its associated cluster: ε (maximum distance between organisms belonging to the same cluster) and s 0 (desired speed of an individual). The time scale is then defined as 3.1 t w = ϵ s 0 . Notice that if t w ≫ 1, then we would (at least in some cases) expect a network that is dense or fully connected since each organism would have interacted many numbers of times and if t w ≪ 1, the network would be sparse. Both these extremes would not represent the ‘correct’ interaction network responsible for cohesion in a mobile group. Figure 3 a illustrates how the attraction interaction network is constructed over a time period t w . The network that emerges owing to the interactions is directed in nature; i.e. i → j does not imply j → i since each individual randomly and asynchronously chooses a neighbour to interact. We argue that this underlying directed–network encodes information pertaining to the group’s cohesiveness. Although there are studies that investigate network properties in collective motion models [ 38 , 43 , 44 ], as far as we know, there are no off-the-shelf measures to characterize the interaction network to probe into why a group stays cohesive or breaks apart. In this section, we explore the correspondence between the properties of the network and the emergence of cohesion.\n Figure 3 . Analysing the interaction network helps in understanding the emergence of cohesion in mobile groups (illustrated with the schematics of fish schools). ( a ) Schematic outlining how an attraction interaction network is constructed, over a time window of t w , with the knowledge of when an interaction occurred, between which of the organisms and at what time. ( b ) The interaction network is analysed to identify the sub-groups based on the reachability A ~ . In the example shown, there are two sub-groups (1-3-2) and (4-5) (marked with different colours). The dotted line extending between the two sub-groups only connects them one way. See the electronic supplementary material, video 2 for visualization of the network structure and the cohesivity of the mobile group. ( c ) C and N p are plotted as a function of K for the group of size N = 30. Inset: N p is plotted as function of C for different group sizes. The black dotted line marks the diagonal. It is reasonable to expect a well-connected network to represent a cohesive group and a sparsely connected one to represent a non-cohesive group. A simple measure that characterizes how well a network is connected can be computed from an adjacency matrix A , where each element ( i , j ) of A takes the value 1 when there is an edge connecting i to j . From A , the average connections for a node can be computed—which is simply the average number of neighbours an organism interacts with within time t w . Note here that the directed nature of the graph results in an A that is asymmetric. However, for cohesion to emerge, organisms need not necessarily interact with every other organism in the group. An organism interacting with just a few of its immediate neighbours could result in a chain of events that lead to cohesion. To include this feature that arises not just from primary (or immediate) but from connections that are secondary, tertiary, etc., we compute the reachability matrix A ~ , where an element takes the value 1 when there is a path from i to j , in the directed graph. To connect this property of the network to the group cohesivity, we divide a group into sub-groups based on A ~ . A sub-group, in this context, is defined as a set of all organisms that have a path from and to every other organism in that sub-group. Then, we compute a network parameter N p that is the size of the largest sub-group (normalized by the size of the group), averaged over time and several realizations (see figure 3 b for illustration; see appendix C for details on numerical computation and the electronic supplementary material, video 2 for a visualization of the network structure and corresponding group cohesion.). We find that N p increases with the neighbourhood size K , in a manner qualitatively similar to the cohesion parameter C (see figure 3 c ; also see the electronic supplementary material, S6, where the network parameter is shown to describe the qualitative trends in C \\ v e r s u s K consistently for different levels of group cohesion). When K increases, interactions between organisms result in a network that is well connected, i.e. there is a path from every organism to almost every other in the group, even when K ≃ 0.3 × N . This informs us that when organisms select individuals to interact with at random from a considerable topological neighbourhood, an opportunity is created for the group to stay cohesive. However, an interaction network created need not always materialize into a cohesive group. An organism can, in principle, interact with another organism in a cluster far away (in space) to create a well-connected network since the interactions in our model are topological. However, other interactions like spontaneous turning, alignment or collisions, can break the network before it can cause the two clusters to come together. For this reason, we find N p to reach a high value (≃1) faster than C for most cases (points over the diagonal in the inset of figure 3 c ). However, when group sizes are small ( N ≤ 5) or when organisms break into small clusters (for the case of K = 1), we find N p to be lower than C (points under the diagonal in the inset of figure 3 c ). These points refer to cases where the organisms are cohesive, but interactions are sparse, giving rise to a not fully connected network. Here, a considerable number of organisms reside in the periphery of the clusters that do not have visible neighbours to interact with and hence get isolated from the rest of their neighbours in the calculation of the network parameter. Hence, these clusters have a lower value for N p even though they are spatially in proximity to their local neighbours (electronic supplementary material, S6). 3.3 . Cohesion owing to averaging interactions In canonical models for flocking, like the Vicsek model for alignment, an agent often averages the information from a neighbourhood to find its direction of movement. Here we compare the group cohesivity achieved through stochastic pairwise interactions with that of the averaging-type interactions. We recall that while stochastic–pairwise interactions are achieved by setting k = 1 and K > 1, we obtain the topological averaging interaction (like the Vicsek model for alignment) by setting k = K . We find averaging interactions also achieve cohesion, with cohesion parameter C increasing rapidly with the size of the neighbourhood, K ( figure 4 ). We emphasize that while focal agents interact with all neighbours K in the averaging-type interactions (because k = K ), stochastic pairwise interactions permit interaction with only one neighbour ( k = 1). Averaging interaction, by definition, consumes information from all the interacting K neighbours, while a pairwise interaction takes in information from only one of its K neighbours at a time. Thus, it is not surprising that cohesion is achieved more rapidly in the local-averaging type interaction. This is in line with the results in the literature [ 6 , 26 ].\n Figure 4 . Emergence of cohesion owing to pairwise interactions ( k = 1) compared to averaging-type interactions ( k = K ) for a group of size N = 30. When K is large, both interactions lead to cohesive mobile groups: hashed region. To achieve the same level of cohesion, a group interacting pairwise should have K = K p neighbours while a group interacting via averaging-interactions need only K = K a < K p neighbours (horizontal line at C = 0.75 ). See the electronic supplementary material, video 3 for visualization. Interestingly, beyond a certain value of neighbourhood size K , both the averaging and the pairwise interactions produce similar (maximum) cohesion. Hence, organisms interacting via these two different interaction types will not have any additional advantage with regards to cohesivity. However, if we compared the neighbourhood sizes required to achieve a given value of cohesion (say, 0.75), then we observe that the averaging interaction can achieve that level of cohesion with less number of neighbours K a than a pairwise interaction K p (see horizontal line at C = 0.75 in figure 4 ; also see the electronic supplementary material, video 3 for visualization). From the viewpoint of the organism’s cognitive capacity, the choice is between: (i) assimilating information from all neighbours in a small neighbourhood of size K a and averaging them, or (ii) assimilating information of one neighbour from a larger set of K p neighbours. While it is known that the cognitive load required to track a large number of neighbours is high [ 45 – 47 ]; we do not yet know, which of these two processes have a smaller cognitive load. However, one could safely assume that an organism capable of integrating information from multiple sources and limited by its ability to observe only a small part of its neighbourhood would prefer method (i) over method (ii), while a different organism that finds integrating information together difficult would choose (ii) to achieve the same level of cohesion. An important question may arise at this point: are these two kinds of interactions, averaging and pairwise, truly distinct? More specifically: is it possible to produce an ‘averaging-interaction’ by merely applying pairwise interactions multiple times over different neighbours? This is an interesting question with important consequences from the point of inferring the behavioural rules in organisms. In earlier work from our group [ 18 , 21 ], in the context of alignment, we show that pairwise copying (agent interacting with just one other) produces different jump moments in comparison to any higher order interaction (either three-agent interaction or averaging) which gives rise to distinct mean-field models (stochastic differential equations (SDE)) for the polarization order parameter ( m ). The fluctuations in the order parameter produced by pairwise copying can be explained with the following SDE: d m / d t = [ − a m ] + [ N − 1 c ( 1 − | m | ) 2 + a ] η ( t ) , where a and c represent the rate of random turn and rate at which agents copy a randomly chosen neighbour, respectively and η ( t ) is Gaussian white noise. Here, the order emerges owing to the multiplicative noise term (that which multiplies the noise η ( t )). While a higher order alignment interaction, occurring at rate h , which is an equivalent to averaging, can be modelled with the following SDE: d m / d t = [ − a m + h ( 1 − | m | 2 ) m ] + [ N − 1 ( c + h ) ( 1 − | m | ) 2 + a ] η ( t ) . Here, order emerges owing to the deterministic part of the SDE. In these studies, we clearly demonstrate that these two types of interactions can be differentiated in empirical investigations by extracting the governing SDE from data. These results reveal the inherent qualitative differences between a pairwise and an averaging interaction: a pairwise interaction is symmetric and does not guarantee a net increase in the order parameter and so order emerges only because of noise, while a higher-order interaction like that of the averaging can guarantee net increase in order and can deterministically hold the system at an ordered state. We speculate that the same principles can be used to argue that the pairwise and averaged attraction interactions are distinct. We welcome further work in this area to identify measurable metrics that will differentiate a cohesive mobile group exhibiting averaging attraction interactions from one that interacts via pairwise attraction interactions."
} | 6,283 |
27324572 | PMC4958695 | pmc | 2,670 | {
"abstract": "Cable bacteria are long, multicellular filaments that can conduct electric currents over centimeter-scale distances. All cable bacteria identified to date belong to the deltaproteobacterial family Desulfobulbaceae and have not been isolated in pure culture yet. Their taxonomic delineation and exact phylogeny is uncertain, as most studies so far have reported only short partial 16S rRNA sequences or have relied on identification by a combination of filament morphology and 16S rRNA-targeted fluorescence in situ hybridization with a Desulfobulbaceae -specific probe. In this study, nearly full-length 16S rRNA gene sequences of 16 individual cable bacteria filaments from freshwater, salt marsh, and marine sites of four geographic locations are presented. These sequences formed a distinct, monophyletic sister clade to the genus Desulfobulbus and could be divided into six coherent, species-level clusters, arranged as two genus-level groups. The same grouping was retrieved by phylogenetic analysis of full or partial dsr AB genes encoding the dissimilatory sulfite reductase. Based on these results, it is proposed to accommodate cable bacteria within two novel candidate genera: the mostly marine “ Candidatus Electrothrix”, with four candidate species, and the mostly freshwater “ Candidatus Electronema”, with two candidate species. This taxonomic framework can be used to assign environmental sequences confidently to the cable bacteria clade, even without morphological information. Database searches revealed 185 16S rRNA gene sequences that affiliated within the clade formed by the proposed cable bacteria genera, of which 120 sequences could be assigned to one of the six candidate species, while the remaining 65 sequences indicated the existence of up to five additional species.",
"introduction": "Introduction The term “cable bacteria” is collectively used for long, multicellular filamentous bacteria affiliated to the deltaproteobacterial family Desulfobulbaceae that can mediate electric currents over centimeter-scale distances in marine, freshwater, and salt-marsh sediments [16] , [20] , [36] , [39] , [41] , [44] . Cable bacteria have been proposed to perform electrogenic sulfur oxidation via long-distance electron transport. Thereby they electrically couple the oxidation of sulfide in anoxic layers with the reduction of oxygen at the sediment surface [29] , [31] . This unique type of microbial metabolism creates distinct geochemical signals that can be detected by depth profiling of O 2 , pH, H 2 S, and electric potential [8] , [29] , [35] , and drastically changes the geochemistry of cable bacteria-populated sediments [24] , [28] , [37] . Cable bacteria have so far evaded cultivation in axenic culture, although all specimens reported to date are morphologically conspicuous, cm-long filaments with distinct longitudinal ridges on their surface [31] and belong to an apparently monophyletic sister lineage of the genus Desulfobulbus \n [31] , [36] . However, their taxonomic delineation and diversity are uncertain, as most studies have only reported short partial sequences that covered different regions of the 16S rRNA gene [16] , [21] , [23] , [46] . Molecular identification of cable bacteria therefore currently relies on matching short environmental sequences with high sequence identity (>97%) to “confirmed” cable bacteria sequences ( i.e. 16S rRNA gene sequences obtained from single filaments) [31] , [36] , [39] . Alternatively, filament morphology and the general Desulfobulbaceae -specific, 16S rRNA-targeted probe DSB706 [18] are combined to detect and quantify cable bacteria by fluorescence in situ hybridization (FISH) without further identification [20] , [41] , [44] . The aim of the current study was to establish a robust phylogenetic framework for cable bacteria, which could be used for their taxonomic delineation and the reliable identification of cable bacteria environmental sequences. Therefore, individual filaments of cable bacteria were picked from freshwater and marine sediments for whole genome amplification, sequencing, and assembly of nearly full-length 16S rRNA and dsr AB gene sequences in order to complement the 16S rRNA phylogeny with that of a faster evolving marker gene [25] . The data were used to reconstruct a robust cable bacteria phylogeny, which, together with the available morphological, physiological and ecological information, led to the proposal of six candidate species within two novel candidate sister genera. Using this taxonomic framework, cable bacterial 16S rRNA gene sequences were identified in public databases, and a comprehensive overview of their environmental diversity and distribution is presented together with the design of novel oligonucleotide probes for their specific detection.",
"discussion": "Results and discussion Refined phylogeny of cable bacteria Picking of individual cable bacteria filaments with subsequent whole genome amplification and 16S rRNA sequence reconstruction enabled direct correlation of sequence information with the phenotypic property of cable bacteria and their environmental origin, in line with earlier studies of large sulfur bacteria [19] , [26] , [38] . All 16 full-length 16S rRNA gene sequences retrieved from single filaments in this study clustered together as a distinct, monophyletic sister group to the genus Desulfobulbus within the family Desulfobulbaceae ( Fig. 1 A). This cable bacteria clade displayed two major, genus-level branches with a 16S rRNA gene sequence identity of <94.5% ( Table 1 ), which is the cut-off for discriminating different genera [51] . One branch contained the twelve sequences originating from marine and salt marsh sites, whereas the other comprised the four freshwater sequences, separated into two distinct species-level clusters, below the 98.7% 16S rRNA gene sequence identity threshold proposed for discriminating different species [42] . Overall, the analysis thus reproduced the cable bacteria phylogeny reported previously [36] . However, due to the full-length sequences, it was also possible to differentiate four species that had a <98.7% 16S rRNA gene sequence identity ( Table 1 ) and were clearly phylogenetically distinct ( Fig. 1 A) in the hitherto unresolved marine group [16] , [21] , [23] , [31] , [39] , [46] . The same grouping was retrieved by phylogenetic analysis of DsrAB amino acid sequences ( Fig. 1 B). The dsr A and dsr B genes encode the alpha and beta subunits of the dissimilatory sulfite reductase and are widely used functional and phylogenetic marker genes for sulfate-reducing and sulfide oxidizing-prokaryotes [25] , [47] . The identities of the dsr AB genes between 16S rRNA-defined species were ≤92%, <82% between members of the two cable bacteria genera, and <80% between cable bacteria and the genus Desulfobulbus ( Table 2 , Table S4 ). These values were within the 90–92% dsr AB identity range proposed for species delineation [15] , [25] , or the 75–82% identity range for genus delineation inferable from the data set of Müller et al. [25] , and thus supported the 16S rRNA-based analysis. Proposal of novel candidate taxa Based on these results, it is proposed to accommodate cable bacteria within two novel candidate genera: the preferably marine “ Candidatus Electrothrix”, with four novel candidate species, “ Ca. E. marina”, “E. aarhusiensis”, “E. communis”, and “E. japonica”, and the preferably freshwater “ Candidatus Electronema”, with two novel candidate species, “ Ca. E. nielsenii”, and “E. palustris”. The proposed genera and species can be distinguished based on their 16S rRNA and dsr AB gene sequences ( Fig. 1 ; Table 1 , Table 2 ), and can also be identified in situ by FISH with a suite of newly designed oligonucleotide probes ( Table 3 ; Fig. 2 A–D). In contrast, cell diameter was not a good predictor for taxonomic affiliation, due to the considerable size variation among cable bacteria of the same species [39] and even within a single filament ( Fig. 2 E). Cable bacteria diameters ranged from 0.4 to 8 μm, with an average cell length of 3 μm [16] , [21] , [23] , [31] , [39] , [46] ( Fig. 2 ). The total length of cable bacteria is difficult to determine, as they often form bundles and break when handling sediment samples. However, filaments up to 1.5 cm long have been recorded [31] ( Fig. S1 ), suggesting that cable bacteria are long enough to span the entire suboxic zone. Unifying morphological characteristics for all members of the candidate genera Electrothrix and Electronema are continuous ridges that run along the entire length of a filament [31] . Depending on cell diameters, single filaments contain 15–71 ridges with diameters of 30–100 nm [21] , [31] that are detectable by TEM, SEM, and AFM ( Fig. 2 F–H). Cells within a filament share a common outer membrane and periplasm [31] . Cable bacteria filaments are unbranched and do not store elemental sulfur in globules, as is characteristic for many other large sulfur oxidizing bacteria [27] , [50] , but polyphosphate inclusions have been observed in some cable bacteria cells [44] . They show gliding motility [5] and conduct electrons from sulfide to oxygen [31] and, at least in some cases, nitrate/nitrite [23] . Environmental distribution of cable bacteria The robust phylogenetic and taxonomic framework for cable bacteria described above enabled a more informed database search in order to assess the environmental diversity and distribution of the candidate genera Electrothrix and Electronema. In total, 185 16S rRNA gene sequences were retrieved that affiliated with these cable bacteria ( Fig. 3 ; Table S3 ); of these, 120 sequences could be assigned to the six candidate species, while the remaining 65 sequences indicated the existence of up to five additional species ( Fig. 3 ; Table S3 ). The clear phylogenetic demarcation of the “ Ca . Electrothrix/Electronema” cable bacteria clade also allowed a re-evaluation of the cable bacteria diversity suggested in earlier studies. Notably, this analysis revealed that nine of the twelve Desulfobulbaceae sequence types detected in a marine nitrate-enrichment [23] , and all Desulfobulbus relatives detected at an anode in freshwater sediment [10] , [39] affiliated outside the cable bacteria lineage, indicating most likely that they were not cable bacteria. Members of the “ Ca . Electrothrix/Electronema” cable bacteria lineage were found in a wide range of marine, brackish, salt lake, and freshwater habitats ( Fig. 3 ), confirming the global distribution of cable bacteria reported earlier [21] . Except for the salinity-based genus-level divide reported earlier (“ Ca . Electrothrix” apparently prefers marine and saline habitats, “ Ca . Electronema” freshwater habitats) [36] , no habitat-specific distribution patterns of the six candidate species were observed. However, there was an overrepresentation of deep sea- and hydrothermal vent-derived cable bacteria sequences outside the six candidate species ( Fig. 3 , Table S3 ), suggesting a promising environment for the description of novel cable bacteria species. A somewhat surprising result, given the cable bacteria's global distribution [21] ( Fig. 3 ) and periodically high in situ biomass [41] , [44] , was the overall low number and biased distribution of cable bacteria sequences in public databases: reads were mostly obtained from next-generation sequencing projects ( i.e. when thousands to millions of reads were analyzed), when Desulfobulbaceae -specific primers had been used, or when RNA had been extracted ( Table S3 ). This may indicate a general problem with cell lysis and DNA extraction of cable bacteria, probably due to the rigid outer structures [31] ( Fig. 2 F–H), which needs to be considered when searching for cable bacteria in the environment. In conclusion, the taxonomic framework presented here can be used to identify environmental sequences as cable bacteria, even without morphological information, and the newly developed FISH probes allow, in combination with the Desulfobulbaceae -specific probe DSB706, quick assignment of cable bacteria to one of the described candidate species. Etymology of the Candidatus taxa “ Candidatus Electrothrix” E.lec’tro.thrix. Gr. neut.n. electron , amber (which is the origin of the term electric); Gr. fem. n. thrix , hair; N.Gr. electric hair. Multicellular filaments, up to several centimeters in length, with 15–71 characteristic longitudinal ridges and shared periplasm across cells; electron-conducting; typically spanning the suboxic zone in surface sediments; individual cells are 0.4–8 μm × 3 μm in size; polyphosphate inclusions; no sulfur inclusions; gliding motility; marine, including salt marsh and salt lake inhabiting. “ Candidatus Electronema” E.lec.tro.ne’ma. Gr. neut.n. electron , amber; Gr. neut. n. nema , thread; N.Gr. electric thread. Multicellular filaments, up to several centimeters in length, with 15–58 characteristic longitudinal ridges and shared periplasm across cells; electron-conducting; typically spanning the suboxic zone in surface sediments; individual cells are 0.4–3 μm × 3 μm in size; no sulfur inclusions; gliding motility; mostly freshwater-inhabiting. “ Candidatus Electrothrix marina” ma.ri’na. L.fem.adj. marina of or belonging to the sea, marine, referring to its marine habitat. Marine members of the candidate genus Electrothrix; distinguishable by their 16S rRNA and dsr AB sequences; identifiable by specific probe EXma430 (5′-TTTCTTCCCTTCTGACAGGGTTT-3′); accession numbers KR912340-42 (16S rRNA), KU844005-10 ( dsr AB). “ Candidatus Electrothrix aarhusiensis” aar.hu.si.en'sis. N.L.adj. aarhusiensis from Aarhus, referring to the place of the first discovery of cable bacteria. Mostly coastal and intertidal members of the candidate genus Electrothrix; distinguishable by their 16S rRNA and dsr AB sequences; identifiable by specific probe EXaa1016 (5′-CTCTCAAAGAGAGCACTTCCCTA-3′); accession numbers KR912338 (16S rRNA), KU844014, KU844015 ( dsr AB). “ Candidatus Electrothrix japonica” ja.po’ni. ca. N.L.fem.adj. japonica Japanese, referring to its discovery in Tokyo Bay, Japan. Coastal, intertidal, and marine members of the candidate genus Electrothrix; distinguishable by their 16S rRNA and dsr AB sequences; can be detected together with Ca. E. communis by specific probe EXco1271 (5′-GCTTTCAGGGATTTGCGCCT-3′); accession numbers KR912349 (16S rRNA), KU844019, KU844020 ( dsr AB). “ Candidatus Electrothrix communis” com.mu’nis. L.fem.adj. communis common, widespread, referring to its widespread distribution in diverse habitats. Ubiquitous members of the candidate genus Electrothrix; distinguishable by their 16S rRNA and dsr AB sequences; can be detected together with Ca. E. japonica by specific probe EXco1271 (5′-GCTTTCAGGGATTTGCGCCT-3′); accession numbers KR912339; KR912343-48 (16S rRNA), KU844004; KU844016-18; KU844021-28 ( dsr AB). “ Candidatus Electronema nielsenii” niel.se’ni.i. N.L. gen.n. nielsenii of Nielsen, named in honor of Lars Peter Nielsen, the Danish microbial ecologist who started the cable bacteria studies by discovering electric currents in the seafloor; the species has been extracted from his backyard. Members of the candidate genus Electronema; creeks, streams, and freshwater ponds; distinguishable by their 16S rRNA sequences; identifiable by specific probe ENni1437 (5′-CCCGAAGGTCCGCCCAGCT-3′); accession numbers KP728462, KP728465 (16S rRNA). “ Candidatus Electronema palustris” pa.lus’tris. L. fem. adj. palustris , marshy, swampy, wetland-inhabiting. Mostly freshwater members of the candidate genus Electronema; distinguishable by their 16S rRNA and dsr AB sequences; identifiable by specific probe ENpa1421 (5′-CCAGCTGCTTCTGGTGCAATCG-3′); accession numbers KP728463, KP728464 (16S rRNA), KU844011-13 ( dsr AB)."
} | 3,991 |
27077867 | PMC4851038 | pmc | 2,671 | {
"abstract": "In this paper, two different piezoelectric transducers—a ceramic piezoelectric, lead zirconate titanate (PZT), and a polymeric piezoelectric, polyvinylidene fluoride (PVDF)—were compared in terms of energy that could be harvested during locomotion activities. The transducers were placed into a tight suit in proximity of the main body joints. Initial testing was performed by placing the transducers on the neck, shoulder, elbow, wrist, hip, knee and ankle; then, five locomotion activities—walking, walking up and down stairs, jogging and running—were chosen for the tests. The values of the power output measured during the five activities were in the range 6 µW–74 µW using both transducers for each joint.",
"conclusion": "6. Conclusions In this paper, two piezoelectric transducers were placed inside a tight wearable suit in proximity to the main human body joints, neck, shoulder, elbow, wrist, hip, knee and ankle, respectively, in order to harvest energy generated by common body movements in the form of casual walking, walking down and up stairs, jogging and running. In order to work at its best, it is very important for a BMEH system using the piezoelectric transducers to ensure as close contact as possible between the transducers and the skin; therefore, a special manufactured body suit was produced to be worn during the activities executed in this work. When examining the user wearability of the two transducers, PVDF technology is more adequate than soft PZT, because the value of its folding parameter is higher: as a result, it is more comfortable for the user and can better adhere to body movements. When examining the power output measured during the five common activities, the values of the power output are in the range of 2–46 µW/cm 3 for a single transducer for a joint, while using both transducers for a joint, the values of the power output are in the range of 6 µW–74 µW, thus confirming the possibility to include these harvesters into more general systems for long-term monitoring. In order to continue the work on the development of a system for BMEH, PVDF material has produced the best results. The soft PZT technology produced higher values of power output, but its lack of comfort makes it difficult to be worn in long-term activities, to adequately follow the body segment movements.",
"introduction": "1. Introduction Body motion energy harvesting (BMEH) means recovering energy from body movement. BMEH has been the object of study by researchers from around the world for the past twenty years. Starner [ 1 ], who is considered one of the first researchers who studied energy harvesting from human motion, explored the possibility of recovering the energy produced by body movements, during everyday activities. He pointed out that human beings produce the highest amount of energy during walking. Thus, Shenck and Paradiso [ 2 ] developed an energy harvesting system, mounted on the shoes, that enables one to power a wide range of body-worn devices. Then, Gonzalez et al. [ 3 ] and Niu et al. [ 4 ] analyzed the feasibility of using the energy harvested from the human body to power wearable sensors, which include the functions of data processing and wireless communication [ 5 , 6 ]. Following the same logic, Rome et al. [ 7 ] developed a suspended-load backpack that converts mechanical energy of the vertical movement of the carried load into electrical energy. In addition, Donelan et al. [ 8 ] developed a biomechanical energy harvester mounted on the knee that provides power generation at the end of the swing phase, thus assisting deceleration of the knee joint during walking and jogging. Mitcheson et al. [ 9 ] reviewed the principles in a motion-driven miniature energy harvester to introduce the basis for a wearable and comfortable body-worn energy harvesting system. Vullers et al. [ 10 ] published a review dealing with the techniques of micropower energy harvesting in order to confirm the theory, earlier discussed by Huang et al. [ 11 ] and Hanson et al. [ 12 ], about the wireless body sensor network (WBSN), according to which reliable energy harvesting has now become a reality for human conditions monitoring. Nowadays, BMEH systems are becoming fundamental for sports [ 13 ], medical [ 14 ] and military [ 15 ] applications. The research trend is to develop smart clothes with incorporated BMEH systems. Stoppa and Chiolerio [ 16 ] presented a paper on recent progress in the field of smart textiles, and Misra et al. [ 17 ] presented a paper on flexible technologies that enable ultra-long battery lifetime and user comfort. Despite a large body of literature focusing on the development and testing of BMEH systems, to the authors’ knowledge there is a notable lack of studies targeting the optimization of harvester kinds, and their placement, to be used in everyday life activities. To fill this gap, in this work, piezoelectric transducers placed in a tight suit were tested and compared to find out the best location where the BMEH system can be placed, to maximize energy production in everyday life. To this end, individual body movements of single body joints were tested, and then five different locomotion activities were chosen to test the sensors in real-life conditions. The positions of the BMEH systems were chosen taking into account the wearability of the transducers and the user comfort during exercises, with the perspective of incorporating them into smart clothing.",
"discussion": "5. Discussion As it can be clearly seen from the tests shown in the previous section, the power output measured at each joint is sufficient enough to consider both transducers suitable to be used as energy harvesters for BMEH applications. When comparing the performed tests, it resulted that soft PZT technology is more efficient than PVDF in terms of generated power output; however, PVDF technology is more comfortable in terms of user wearability. The results obtained in this work are in line with the results of the current wearable fabrics for BMEH found in the present scientific literature and reported below as follows: Zhang et al. [ 22 ] developed a fabric nanogenerator able to produce 10.02 nW when it is attached on an elbow pad and bent by human arms. Yang and Yun [ 35 ] prepared three fabrics in the form of a band for wearing on elbow joint, measuring 0.21 mW for a bending velocity of 5 rad/s. Dhakar et al. [ 36 ] presented a triboelectric nanogenerator able to generate voltages up to 90 V with a mild finger touch, and Yang et al. [ 37 ] developed a flexible triboelectric nanogenerator for energy harvesting from various types of mechanical motions, able to deliver an open-circuit voltage of 700 V and a short-circuit current of 75 µA. Pu et al . [ 38 ] developed a textile triboelectric nanogenerator able to generate 20, 2 and 0.8 µA rectified output currents by foot pressing, arm swinging and elbow bending, respectively. Yun et al. [ 39 ] presented a very flexible harvester design that can elastically stretch to 1.6-times its normal length, allowing it to be used on a large range of motion body areas. Li et al. [ 40 ] developed a power shirt based on triboelectrification and the electrostatic induction effect, able to achieve a maximum peak power density of 4.65 µW/cm 2 , and Wu et al. [ 41 ] produced a wearable nanogenerator able to produce 6 V of output voltage and 45 nA of output current. The results of the power output harvested from the transducers in this paper represent the power output generated by the five locomotion activities and, thus, may represent an added value to the results found in the current scientific literature, which represent values of the power output generated only from individual body movements."
} | 1,930 |
34964290 | PMC8650569 | pmc | 2,672 | {
"abstract": "Abstract Interspecific interactions within biofilms determine relative species abundance, growth dynamics, community resilience, and success or failure of invasion by an extraneous organism. However, deciphering interspecific interactions and assessing their contribution to biofilm properties and function remain a challenge. Here, we describe the constitution of a model biofilm composed of four bacterial species belonging to four different genera ( Rhodocyclus sp., Pseudomonas fluorescens, Kocuria varians , and Bacillus cereus ), derived from a biofilm isolated from an industrial milk pasteurization unit. We demonstrate that the growth dynamics and equilibrium composition of this biofilm are highly reproducible. Based on its equilibrium composition, we show that the establishment of this four‐species biofilm is highly robust against initial, transient perturbations but less so towards continuous perturbations. By comparing biofilms formed from different numbers and combinations of the constituent species and by fitting a growth model to the experimental data, we reveal a network of dynamic, positive, and negative interactions that determine the final composition of the biofilm. Furthermore, we reveal that the molecular determinant of one negative interaction is the thiocillin I synthesized by the B. cereus strain, and demonstrate its importance for species distribution and its impact on robustness by mutational analysis of the biofilm ecosystem.",
"conclusion": "5 CONCLUSIONS Emergent properties of bacterial communities grown as biofilms, driven by social interactions, have huge implications for research and practical knowledge in such contexts as human health, food safety, rhizosphere role in plant growth, or even bioremediation. One approach to understanding these social interactions is to create and study artificial biofilm consortia in the laboratory. However, very few studies report such reconstructions of multispecies biofilm and elucidate the interspecies interaction networks that take place within. Moreover, the molecular determinant of these interactions and the analysis of their impact on the biofilm ecosystem properties have been reported in only a few studies. Here, we not only deciphered the active network of interactions that shapes a four‐species biofilm community and determine its robustness but also identified the molecular determinant of one of these interactions and revealed how it impacts the structure and properties of this community.",
"introduction": "1 INTRODUCTION In contrast to typical laboratory conditions of growth in liquid culture, bacteria in natural environments and those contaminating hospitals, or industrial and food‐processing procedures are more often found in multicellular surface‐associated communities known as biofilms (Costerton et al., 1987 ; Flemming et al., 2016 ; Hall‐Stoodley et al., 2004 ). Such biofilms are generally complex communities harboring numerous bacterial species in close spatial proximity (Elias & Banin, 2012 ). Diverse physical and social interactions between species take place in these communities. They are considered to determine not only the structure and spatial organization of the biofilm but also its global functions by modulating gene expression in the different species (Bridier et al., 2017 ; Burmølle et al., 2014 ; Liu et al., 2016 ; Rendueles & Ghigo, 2012 ). The physiology of each microbial species in complex, multispecies biofilms might be distinct from that in monospecific biofilms (L. B. S. Hansen et al., 2017 ; Liu et al., 2019 ). Moreover, multispecies biofilms exhibit emergent properties such as increased tolerance against antimicrobial agents (Bridier et al., 2012 ; Burmølle et al., 2006 ; Schwering et al., 2013 ; Yan & Bassler, 2019 ), synergistic degradation of toxic compounds (Breugelmans et al., 2008 ; Perera et al., 2019 ; Yoshida et al., 2009 ), stronger defense against protozoan grazing (Koh et al., 2012 ; Raghupathi et al., 2018 ), increased virulence in infection (Pastar et al., 2013 ; Wang et al., 2020 ) and protection against the action of biocides (Sanchez‐Vizuete, Le Coq, et al., 2015 ; Sanchez‐Vizuete, Orgaz, et al., 2015 ; Yan & Bassler, 2019 ). Studies on multispecies biofilms have also reported enhanced stress resistance, productivity, or biomass production (Burmølle et al., 2006 ; Lee et al., 2014 ; Liu et al., 2019 ; Ren et al., 2015 ), and, importantly, “community‐intrinsic properties” (Madsen et al., 2018 ) emerging from the social interactions between members of the biofilm and which may be important for its interaction with its environment. It is thus important to decipher these interactions, positive or negative, at the molecular and biochemical levels to better understand the ecological and evolutionary factors that drive community function in natural or engineered systems (Rice et al., 2016 ; Ziesack et al., 2019 ). However, the number and types of interactions within multispecies biofilms are expected to grow very rapidly with the number of species present in the biofilm (Røder et al., 2016 ). Characterization of the interactions in complex biofilms and their underlying molecular mechanisms remains a challenge, as well as the evaluation of the importance of these interactions for the overall robustness of the structure and functionalities of these biofilms (Røder et al., 2020 ). Here, we constructed a biofilm community from four species isolated from a biofilm consortium contaminating a milk processing plant, to analyze interspecies interactions and robustness to environmental stresses. This multispecies biofilm was highly reproducible, allowing us to use it as a model to study the dynamic interactions that take place between the species during its development. We were thus able to test the resistance of this complex biofilm and its formation process towards different continuous or transient perturbations. Then we identified the molecular determinant and assessed the contribution of one major interspecies interaction to the overall robustness of the biofilm.",
"discussion": "4 DISCUSSION 4.1 A model for multispecies biofilm with low complexity Naturally occurring biofilms are multispecies ecosystems that constitute attractive opportunities to study interspecies interactions and community reactions to changes in their environment. Unfortunately, their complexity, combined with the difficulty of implementing controlled changes, limits their use in such studies. The model described in this study, developed from a selection of species from a biofilm isolated in a food industry setting, is of reduced complexity but demonstrates the positive and negative interactions characteristic of more complex biofilms. Because of its relative simplicity, it remains tractable in terms of biological analysis and mathematical modeling at the species level and can be used to provide answers to basic questions concerning the molecular mechanisms of interspecies interactions in microbial communities. The relative simplicity of the system may also play a part in its experimental reproducibility, this being a prerequisite for a laboratory model, while lack of reproducibility is a frequent problem in mixed biofilm studies (Røder et al., 2016 ). The quantity of biofilm and the relative proportions of each community member stabilized by 72 h (Figure 1 ). The reproducibility of the results (in particular the size of each bacterial population along time) and the approximate number of generations in each population during the experiment are incompatible with the hypothesis of mutations leading to more or less fitted subpopulations that would explain the changes in population size through time. Furthermore, one test of the biofilm dynamics under standard conditions performed using isolates from the 72 h biofilm of a previous experiment gave similar results as with stock isolates. Notably, cell counts of the minority species in the biofilm were stable as much as were the dominant species, demonstrating that they were not simply disadvantaged in the consortium, but attained an equilibrium, where positive and negative interactions, cell growth, and cell loss from the biofilm balanced each other. 4.2 Dynamic positive and negative interactions during growth of the biofilm The final species distribution in the biofilm differs greatly from that at the initial stages of colonization: after the attachment phase, the biomass of two species, P. fluorescens and Rhodocyclus , strongly increased whereas that of the two other, B. cereus and K. varians , remained fairly constant (Figure 1 ). The three most abundant species ( P. fluorescens, Rhodocyclus , and B. cereus ) reached similar biomasses in one‐species and four‐species biofilms, ruling out the simple competition as the major force in defining the final species composition and indicating that species abundances were not limited by the carrying capacity of the substratum. The total cell numbers in the four‐species biofilm were higher than that of a biofilm composed of any one of the species alone, but they were not larger than the sum of those of the one‐species biofilms. As such, we do not see general interspecific cooperation in the mature, steady‐state biofilm, an observation in agreement with the results of Foster and Bell ( 2012 ) and in contrast to the conclusions of Ren et al. ( 2015 ). The absence of apparent general cooperation in the present biofilm could be related to its modest richness, with only four different bacterial species, and/or to the fact that these precise strains may not have cohabited for a long time: At the most 20 weeks (Mettler & Carpentier, 1997 ) for the three species isolated from an industrial device, as part of a larger community of microorganisms and under very different conditions from those of the experimental biofilm. Except for B. cereus , the different species demonstrated notably different growth kinetics in four‐species biofilms compared to growth as single‐species, and each showed a different reaction to the growth in a mixed‐species community (Figure 5 ). P. fluorescens development was delayed in the four‐species biofilm while its final abundance was unchanged. The biofilm community interacted negatively with K. varians throughout its development and had a major impact on its final abundance, while it had a positive effect on Rhodocyclus at the attachment and initial growth phases. The striking effect in the four‐species biofilm on the adhesion and/or early growth of Rhodocyclus in the absence of changes in its final abundance (Figure 5a ) suggests physical interactions aiding in attachment to the matrix, for example by modification of the substratum properties, or by epiphytic growth of one species on another. Growth of B. cereus was neither stimulated nor inhibited in four‐species biofilm, and maintained a constant, subdominant cell density in the biofilms throughout the experimental time frame, being able to grow to this level even when seeded at very low density (Figure 4 ). In addition, the final equilibrium populations at 72 h of the other species were not altered a 1000‐fold reduction of the B. cereus inoculum, suggesting that the equilibrium is maintained by interspecies interactions and is not a function of the history of the system, where, for example, space on the substratum might be irreversibly colonized by a species initially present as a high proportion of the total population. It thus appears that the development and the final steady‐state of the biofilm is determined by a network of dynamic positive and negative interactions between the four species. 4.3 Molecular mechanism of interference competition by growth inhibition We found that the negative effect on K. varians in the four‐species biofilm was due to the production of thiocillin by B. cereus and that this negative interaction was partly mitigated by the presence of P. fluorescens and Rhodocyclus . This bacteriocin is active against Gram‐positive bacteria, and B. cereus was found to inhibit the growth of all Staphylococcus tested, explaining the failure of these strains to form a mixed‐species biofilm (Table 1 ). The bactericidal activity of thiocillin has been reported to be associated mainly with the cell fraction (Wieland Brown et al., 2009 ), presumably because of thiocillin's poor solubility. Similarly, the inhibitory activity of B. cereus on K. varians was seen in conditions of growth at a close distance (on agar plates, Figure 7 , or in biofilm, compare Figures 4 and 8 ), but not in a four‐species planktonic coculture (compare Figure 3b,d ). The coexistence of antagonistic strains in stable, or in cyclically evolving, proportions is predicted by theory for a range of interaction parameters (Chesson, 2000 ; Czárán et al., 2002 ; Hassell et al., 1994 ) and has been demonstrated in laboratory experiments (Kerr et al., 2002 ). Notably, these latter authors showed that antagonistic strains can coexist if interactions and dispersal occur at a local scale, coexistence under these conditions being associated with structured spatial organization of the strains. This biofilm structuration, which can protect sensitive species from the antagonistic effects of others (Kim et al., 2011 ), is also favored by positive interactions (Breugelmans et al., 2008 ; S. K. Hansen et al., 2007 ), such as the observed stimulation of Rhodocyclus adhesion by the biofilm community (Figure 5d ). Nevertheless, the mechanism of mitigation by P. fluorescens and Rhodocyclus of the inhibitory action of B. cereus on K. varians may involve nonspecific protection by the physical presence of these two species—for example, physical separation and/or adsorption of the thiocillin molecule at the bacterial surface. 4.4 Interspecies interactions and robustness of the biofilm community The establishment of the four‐species biofilm was resistant in the face of the transient perturbations that we tested, but the species composition changed in response to continuous perturbations, where the environment was permanently modified. We then looked more closely at the effect on biofilm community stability of one of the interspecies interactions, in particular, the negative effect of B. cereus on the growth of K. varians . As described above, the substitution of the B. cereus wild‐type by the thiocillin mutant strain altered the final composition of the biofilm, permitting increased growth of K. varians which became codominant together with Rhodocyclus and P. fluorescens . Neither the wild‐type nor the mutant biofilm was robust to strong continuous perturbations (compare Figures 4 with 8 ). Moreover, the mutant biofilm appeared to be less resistant than the wild‐type to transient perturbations, suggesting that in this model the major negative interaction may play a role in robustness that is only partially compensated by other stabilizing interactions (Burmølle et al., 2006 ; Lee et al., 2014 ). This is in agreement with the results of a study by Thompson et al. ( 2020 ) concerning a community composed of bacteria isolated from a potable water distribution system, where the authors detected redundant interspecies interaction effects. The interaction between one species and either one of two others had a positive effect on the biomass of their model biofilm, and this effect was more marked in the absence of a fourth, whose presence independently compensated for the loss of the interactions. Redundant interactions were also brought to light in our model biofilm but it must be remembered that the bacteria in the biofilm have had little time to coevolve together—probably 15–20 generations—and that the major negative interaction, though it may be important in this model, is not part of an evolved ecosystem. Growth of the biofilm in the undiluted medium resulted in considerably higher numbers of B. cereus (Figure 4 ), possibly due to a capacity for exploiting the increased nutrient availability, to changed interactions in the biofilm community, or a combination of the two effects. In contrast, cell density of the B. cereus Δ tclE‐H was decreased in rich medium (Figure 8 ), both in relation to growth under standard conditions and to the wild‐type in the undiluted medium. These observations suggest that the differential reaction of the wild‐type and mutant biofilms to growth in the undiluted medium is at least partly explained by a modification of interspecies interactions other than competition."
} | 4,138 |
37275577 | PMC10238267 | pmc | 2,674 | {
"abstract": "Microbial bioelectrochemical system (BES) is a promising sustainable technology for the electrical energy recovery and the treatment of recalcitrant and toxic pollutants. In microbial BESs, the conversion of harmful pollutants into harmless products can be catalyzed by microorganisms at the anode (Type I BES), chemical catalysts at the cathode (Type II BES) or microorganisms at the cathode (Type III BES). The application of synthetic biology in microbial BES can improve its pollutant removing capability. Synthetic biology techniques can promote EET kinetics, which is helpful for microbial anodic electro-respiration, expediting pollutant removing not only at the anode but also at the cathode. They offer tools to promote biofilm development on the electrode, enabling more microorganisms residing on the electrode for subsequent catalytic reactions, and to overexpress the pollutant removing-related genes directly in microorganisms, contributing to the pollutant decomposition. In this work, based on the summarized aspects mentioned above, we describe the major synthetic biology strategies in designing and improving the pollutant removing capabilities of microbial BES. Lastly, we discuss challenges and perspectives for future studies in the area.",
"conclusion": "3 Conclusions and perspectives Microbial BES possesses great promise for the efficient treatment of recalcitrant and toxic pollutants. The application of synthetic biology in microbial BES can improve its pollutant removing capability. Synthetic biology techniques can promote EET kinetics, which is helpful for microbial anodic electro-respiration, expediting pollutant removing not only at the anode but also at the cathode. Furthermore, synthetic biology techniques offer opportunities to promote biofilm development on the electrode, enabling more microorganisms residing on the electrode for subsequent catalytic reactions. Moreover, the direct overexpression of the pollutant removing-related enzymes in microorganisms by synthetic biology tools contributes to the pollutant decomposition. However, there exist a series of challenges to be addressed before the practical application of synthetic biology techniques in microbial BES. For instance, the bidirectional EET kinetics of bioelectrodes are still too sluggish and need to be further improved. The. Pollutant degradation pathway should be rationally designed and the intracellular metabolism ought to be systematically optimized. In addition, developing more efficient synthetic biology tools to genetically engineer gene targets in those non-model microbes is still a challenging work. Moreover, for those electro-inert microorganisms, they cannot directly convey electrons to or uptake electrons from the electrode owing to the lack of EET system, which constrains their application in microbial BES accordingly. Besides, given that the treatment of contaminants by microbial BES may eventually be carried out in an open environment, the spread of artificially engineered microorganisms into the natural ecology and the corresponding horizontal gene transfer into the environment must be considered seriously. In future, synthetic biology may work with other techniques such as materials science to solve problems mentioned above. For instance, novel materials can be developed to immobilize the genetically engineered microorganism in microbial BES to prevent them from leaking into the environment.",
"introduction": "1 Introduction Microbial bioelectrochemical system (BES) is widely considered as a promising sustainable technology for not only the electrical power recovery but also the wastewater treatment. In microbial BES, the microbial oxidation of organic matter takes place on the anode, yielding electrons and protons. The electrons released from electroactive microorganisms during metabolism are conveyed to the anode, which serves as the solid electron acceptor during the microbial electro-respiration process. Afterwards, these electrons are transported to the cathode through an external circuit, during which the chemical energy stored in the chemical bonds of organic matter is converted into the electrical energy [ 1 , 2 ]. In this process, the difference in redox potentials between the oxidation reaction in the anode and the reduction reaction in the cathode drives these electrons to flow spontaneously from the lower potential to the higher potential. In addition, when an external voltage is applied to that system, the electrons move in reverse order as compared with the direction mentioned-above and accordingly, the electricity is converted into the chemical energy ( Fig. 1 a). When microorganisms catalyze reactions on the anode, the electrode is called bioanode and when microorganisms catalyze reactions on the cathode, the electrode is then called biocathode [ 3 , 4 ]. Fig. 1 (a) Schematic diagram of a typical microbial BES showing catalytic processes at the anode and the cathode surfaces and corresponding catalysts involved. The electricity either can be harvested through the external circuit or should be input into the system for the reaction to proceed. AEM, anion exchange membrane; CEM, cation exchange membrane. (b) Type I microbial BES device: The conversion of harmful pollutants into harmless products is microbially catalyzed by microorganisms at the anode. (c) Type II microbial BES device: The conversion of harmful pollutants into harmless products is chemically catalyzed at the cathode. (d) Type III microbial BES device: The conversion of harmful pollutants into harmless products is microbially catalyzed by microorganisms at the cathode. Fig. 1 Apart from the energy recovery, microbial BES are also able to be applied to the treatment of recalcitrant and toxic pollutants. Based on where the pollutant removing reaction occurs and who catalyzes the reaction accordingly, microbial BESs can be classified into three categories. Type I: The conversion of harmful pollutants into harmless products is microbially catalyzed by microorganisms at the anode ( Fig. 1 b). Type II: The conversion of harmful pollutants into harmless products is chemically catalyzed at the cathode ( Fig. 1 c). Type III: The conversion of harmful pollutants into harmless products is microbially catalyzed by microorganisms at the cathode ( Fig. 1 d). All these types share one common element: the bioanode. That is, the anodic substrate oxidation is catalyzed by microorganisms in all these types. Thus, by means of the electrochemical device, the microbial substrate metabolism and the electron generation processes are separated so as to enable the more flexible and well-controlled use of these electrons for subsequent application such as the pollutant removing."
} | 1,677 |
22629347 | PMC3357441 | pmc | 2,676 | {
"abstract": "Background The diversity of plants and arbuscular mycorrhizal fungi (AMF) has been experimentally shown to alter plant and AMF productivity. However, little is known about how plant and AMF diversity interact to shape their respective productivity. Methodology/Principal Findings We co-manipulated the diversity of both AMF and plant communities in two greenhouse studies to determine whether the productivity of each trophic group is mainly influenced by plant or AMF diversity, respectively, and whether there is any interaction between plant and fungal diversity. In both experiments we compared the productivity of three different plant species monocultures, or their respective 3-species mixtures. Similarly, in both studies these plant treatments were crossed with an AMF diversity gradient that ranged from zero (non-mycorrhizal controls) to a maximum of three and five taxonomically distinct AMF taxa, respectively. We found that within both trophic groups productivity was significantly influenced by taxon identity, and increased with taxon richness. These main effects of AMF and plant diversity on their respective productivities did not depend on each other, even though we detected significant individual taxon effects across trophic groups. Conclusions/Significance Our results indicate that similar ecological processes regulate diversity-productivity relationships within trophic groups. However, productivity-diversity relationships are not necessarily correlated across interacting trophic levels, leading to asymmetries and possible biotic feedbacks. Thus, biotic interactions within and across trophic groups should be considered in predictive models of community assembly.",
"introduction": "Introduction An important goal in community ecology is to understand the relationship between biodiversity and ecosystem functioning. Much of this research began with seminal studies that sparked a lasting research interest on the ecosystem-level consequences of local biodiversity [1] , [2] . Many studies have focused on diversity-productivity relationships in plants, because they are the main primary producers at the base of food webs. Plants are also amenable to field and laboratory manipulations, and general results of such studies show that ecosystem productivity often asymptotically increases with plant diversity; but also see [3] . These positive relationships are typically explained by a sampling effect or by functional complementarity among coexisting species [4] , [5] . In addition, functional and phylogenetic diversity have also been shown to affect ecosystem productivity [6] , [7] . It is clear though that ecosystem productivity is not solely a function of plant community structure. Other trophic groups such as decomposers, pathogens, pollinators, herbivores and microbial symbionts may alter plant community structure and ecosystem functioning and contribute to productivity [8] – [12] . In the present study we focus on the interaction between plants and arbuscular mycorrhizal fungi (AMF), soil-dwelling symbionts that associate with the roots of most terrestrial plants [13] . The presence and diversity of AMF can influence plant diversity and productivity [14] – [16] . Mycorrhizal symbionts may receive significant amounts of photosynthates from their host plants, making them also an important component of the terrestrial carbon cycle [17] . Plant diversity was reported to influence AMF community structure and increase AMF abundance [18] – [20] . Conversely, plant diversity-productivity relationships were significantly altered by the presence of different AMF [21] or by AMF diversity [15] , [16] . Thus far, manipulations of AMF and plant diversity have been done separately, which is why we have little understanding as to whether AMF and plant diversity are independently affecting community structure and ecosystem productivity. The main goal of the present study was to determine the effect of AMF and plant diversity, and their interaction on AMF and plant productivity. We experimentally co-manipulated initial plant and AMF community structure by establishing different plant monocultures and their respective plant mixtures in two complementary greenhouse experiments. We inoculated each host treatment by either morphologically distinct single AMF morphotypes or their mixture (Experiment 1), or by two different AMF mixtures, each comprised four genetically distinct AMF (either distinct or the same morphotypes), or both of these treatments combined (Experiment 2). At the end of the experiments we assessed both plant and AMF productivity by the total plant shoot biomass and the total extraradical fungal volume (EFV), respectively. We found positive diversity-productivity relationships within both trophic groups, without evidence for a significant interaction between AMF and plant diversity. The increase in AMF productivity in AMF mixtures was largely independent of plant productivity and went beyond what could be explained by a sampling effect. Different plant monocultures significantly altered EVF and AMF spore communities, whereas within plant mixtures AMF productivity appeared to be driven by the dominant plant species. Our results show that the interactions within and between trophic groups influence community structure and productivity of above- and belowground communities.",
"discussion": "Discussion Our study shows that AMF productivity was influenced by host community composition, supporting previous reports of significant host plant effects on AMF abundance [18] , [20] . In our experiments, AMF productivity consistently increased with AMF richness, but was not influenced by plant productivity or plant species richness. Since the productivity of AMF mixtures was overall greater than of that of its individually measured constituents, the observed positive relationship between AMF diversity and productivity may not only be explained by a sampling effect [33] , complementing recent findings of AMF diversity effects on plant productivity [34] , [35] . Our results also show that different plant species affected individual AMF abundances (spore production), corroborating that host plants can induce significant AMF community changes [19] , [36] , [37] . In light of rapid global environmental change, there is an urgent need to improve our understanding of the fundamental processes that determine the abundance and distribution of organisms and the functioning of ecosystems. Many studies indicate that changes in climate, the spread of invasive species, or changes in land-use can have profound effects on ecosystem function. However, no single study can quantify all possible processes that impact individual species. For plant communities, some mechanisms, especially those involving belowground interactions, often remain unquantified. This is problematic, especially in light of recent data indicating that soil biota feedbacks may be more important plant community determinants than previously thought [38] – [40] . Regarding AMF, single and multi-isolate effects on plant growth are relatively well documented [15] , [16] , [24] , [28] – [30] , [34] , [35] , [41] , [42] . Much less, however, is known about how plant species and their assemblages influence AMF productivity. Our results corroborate that both host community type and AMF diversity jointly drive AMF productivity [16] , [18] , [21] , [42] . Even though individual AMF morphotypes did indeed have altered growth and symbiotic function when associating with different plant monocultures, we detected no significant interaction between AMF and host diversity on AMF and plant productivity. Positive diversity-productivity relationships typically arise from sampling effects or through functional niche complementarity. In the latter case, synergistic productivity effects are caused by functional dissimilarities among species due to a more efficient capture of available resources. An important novelty of our study is that we manipulated the diversities of two interacting trophic groups. The results of our two experiments provide evidence of both processes, especially in regards to AMF productivity. In Experiment 1, the most productive monoculture ( G. mosseae ) also dominated the AMF mixtures, but these mixtures were overall more productive than G. mosseae, particularly in terms of root colonization rates and extraradical hyphal production. Since host roots and soil are the main carbon and mineral nutrient sources for AM fungi, respectively, increasing AMF diversity resulted in a denser colonization of intra- and extraradical habitats by AMF, i.e. a seemingly more efficient resource capture. Two additional observations drew our attention in Experiment 1. S. heterogama increased spore production in AMF mixtures relative to its monocultures, suggesting that interactions among AMF are not necessarily competitive and may include facilitation. Secondly, of all AMF monocultures, A. morrowiae produced the highest amounts of extraradical hyphae, but only in symbiosis with A. millefolium . Since the growth traits of these clonal fungi are also fitness traits [22] , [32] , these findings support that the success of different individual AMF structures depends on the biotic environment. In a similar set-up as in Experiment 2, inoculation of unsterilized field soil by G. irregulare from ROCs decreased the diversity of native AMF inside host roots [28] . This suppressive effect may have been due to the strong inoculum potential of ROCs at the onset of the experiment. In our Experiment 2 no similar suppressive effect was observed, indicating that potential differences in inoculum potential among morphotypes at the start of the experiment did not cause any systematic competitive exclusion. Thus, future research should increasingly focus on the challenging topic of how different AMF (and their abundances) interact and how such interactions depend and feedback on community structure or other environmental factors. In our experiments we used AMF morphotypes of different families, for which one may expect a higher degree of functional complementarity [7] . It is well documented, for instance, that AMF of the Gigasporacaea family tend to have higher extraradical hyphal lengths and lower or delayed intraradical root colonization than Glomus spp., which tend to produce more spores [7] , [29] , [43] . We assessed the spore formation of different morphotypes and our findings are consistent with Glomus spp. producing more spores than AMF of the Gigasporaceae. In Experiment 2, however, Gigasporaceae morphotypes represented a considerable proportion of the total AMF spore volume due to their relatively large spore sizes. Gigasporaceae AMF reportedly have a delayed root colonization compared to other AMF [43] , possibly because their spores are the most important infective units (propagules) after disturbance; other AMF regrow from spores as well as other propagules such as dried colonized root or hyphal fragments [44] . Finally, in our experiments all AMF taxa persisted in AMF mixtures and no morphotype was consistently excluded, which is in line with a recent study that found the highest realized AMF species richness in phylogenetically overdispersed AMF communities [7] . In comparison, we used phylogenetically similarly dispersed (although species-poorer) AMF mixtures, which may explain why we did not detect such a strong phylogenetic signal. In summary our study provides new insights into how functional complementarity of different AMF explains, at least in part, the enhanced AMF productivity or co-existence in diverse AMF assemblages. At the level of trophic groups, however, such functional differences seemingly “evened out” with increasing diversity: Overall both AMF and plant productivity were mainly determined by the diversity at the two trophic levels, with no interaction among them. We also found that whole-pot evapotranspiration rates in Experiment 1 were affected by AMF inoculation treatments. Similar effects in the field could potentially create localized soil moisture gradients that may also affect subsequent community assembly. Thus, our study also provides a basis for more eco-physiologically based studies, to test how AMF may impact the water use efficiency of vegetation or different host species. In natural communities multiple mechanisms operate at different temporal and spatial scales to shape species distributions. Even though AMF have the potential to alter plant communities and whole ecosystem properties [16] , [17] , [45] , little is known about key factors affecting their own growth, fitness, and dispersal in space and time [46] , [47] . Variations of our approach should be applicable to more realistic microcosm or field experiments. A key challenge for future studies is to determine the relative importance of different types of interactions ( e.g. , mycorrhizal symbioses, pathogens, competition, predation) for shaping succession dynamics, community structure and ecosystem functioning [45] , [48] – [51] . While recent findings suggest that even climatic origin of AMF may affect plant growth [29] , most current vegetation models do not incorporate biotic feedbacks, adding uncertainty to our understanding of how communities assemble in nature."
} | 3,335 |
35155840 | PMC8816652 | pmc | 2,677 | {
"abstract": "Microbial cell factories (bacteria and fungi) are the leading producers of beneficial natural products such as lycopene, carotene, herbal medicine, and biodiesel etc. These microorganisms are considered efficient due to their effective bioprocessing strategy (monoculture- and consortial-based approach) under distinct processing conditions. Meanwhile, the advancement in genetic and process optimization techniques leads to enhanced biosynthesis of natural products that are known functional ingredients with numerous applications in the food, cosmetic and medical industries. Natural consortia and monoculture thrive in nature in a small proportion, such as wastewater, food products, and soils. In similitude to natural consortia, it is possible to engineer artificial microbial consortia and program their behaviours via synthetic biology tools. Therefore, this review summarizes the optimization of genetic and physicochemical parameters of the microbial system for improved production of natural products. Also, this review presents a brief history of natural consortium and describes the functional properties of monocultures. This review focuses on synthetic biology tools that enable new approaches to design synthetic consortia; and highlights the syntropic interactions that determine the performance and stability of synthetic consortia. In particular, the effect of processing conditions and advanced genetic techniques to improve the productibility of both monoculture and consortial based systems have been greatly emphasized. In this context, possible strategies are also discussed to give an insight into microbial engineering for improved production of natural products in the future. In summary, it is concluded that the coupling of genomic modifications with optimum physicochemical factors would be promising for producing a robust microbial cell factory that shall contribute to the increased production of natural products.",
"conclusion": "9 Concluding remarks and future perspectives This review emphasizes the significance of monoculture- and consortium-based bioprocessing approaches for the development of natural products that have gained popularity in many food and non-food industries because of their high yields, ease of optimization, economic feasibility, robust growth on inexpensive media, and stability. More importantly, these microbial-derived natural products are currently grabbing attention due to the rapid development of controllable consortia and monoculture through advanced genetic and process optimization techniques. In particular, we briefly foreground some of the practical implementation of microbial consortium and a monoculture and some of the directions for future research that offer the potential of enhanced production yields and reduced processing costs. Potential directions include (i) development of inexpensive gene-chip assay and high-throughput screening tools that cause directed evolution in multiple communities (ii) in-depth understanding of microbial interactions (in case of a synthetic consortium), and metabolic networks to develop rational metabolic engineering design (iii) Advancement in omics approaches (such as transcriptomics and metabolomics) appears to be an effective way to quantify biochemical changes and metabolic mechanisms, as well as advances in metagenomics positively contributes to enhanced understanding of immensely diverse microbial sources, such as rivers, lakes, sub-seafloor sites, and ice cores. Ultimately, the scientific community will utilize advanced biotechnological data and computational models to achieve optimum natural products production via stable monoculture and consortium."
} | 916 |
19116616 | null | s2 | 2,678 | {
"abstract": "Systems analysis of metabolic and growth functions in microbial organisms is rapidly developing and maturing. Such studies are enabled by reconstruction, at the genomic scale, of the biochemical reaction networks that underlie cellular processes. The network reconstruction process is organism specific and is based on an annotated genome sequence, high-throughput network-wide data sets and bibliomic data on the detailed properties of individual network components. Here we describe the process that is currently used to achieve comprehensive network reconstructions and discuss how these reconstructions are curated and validated. This review should aid the growing number of researchers who are carrying out reconstructions for particular target organisms."
} | 190 |
30356284 | PMC6200223 | pmc | 2,680 | {
"abstract": "Understanding which factors enhance or mitigate the impact of high temperatures on corals is crucial to predict the severity of coral bleaching worldwide. On the one hand, global warming is usually associated with high ultraviolet radiation levels (UVR), and surface water nutrient depletion due to stratification. On the other hand, eutrophication of coastal reefs increases levels of inorganic nutrients and decreases UVR, so that the effect of different combinations of these stressors on corals is unknown. In this study, we assessed the individual and crossed effects of high temperature, UVR and nutrient level on the key performance variables of the reef building coral Pocillopora damicornis . We found that seawater warming was the major stressor, which induced bleaching and impaired coral photosynthesis and calcification in all nutrient and UVR conditions. The strength of this effect however, was nutrient dependent. Corals maintained in nutrient-depleted conditions experienced the highest decrease in net photosynthesis under thermal stress, while nutrient enrichment (3 μM NO 3 - and 1 μM PO 4 + ) slightly limited the negative impact of temperature through enhanced protein content, photosynthesis and respiration rates. UVR exposure had only an effect on total nitrogen release rates, which significantly decreased under normal growth conditions and tended to decrease also under thermal stress. This result suggests that increased level of UVR will lead to significant changes in the nutrient biogeochemistry of surface reef waters. Overall, our results show that environmental factors have different and interactive effects on each of the coral’s physiological parameters, requiring multifactorial approaches to predict the future of coral reefs.",
"introduction": "Introduction Anthropogenic climate change has emerged as a serious global-scale threat to the structure, functions and viability of coral reef ecosystems [ 1 , 2 , 3 ]. In particular, reefs in shallow waters are facing increases in seawater temperature, ultraviolet radiation (UVR) levels and storm events frequency [ 4 , 5 ]. Beyond their direct effects on reefs, such environmental changes are responsible for variations in nutrient concentrations. For instance, heat waves enhance nutrient depletion through water column stratification [ 6 ], while storms can induce water-column mixing and nutrient upwelling [ 7 ]. On top of these global changes, coastal reefs suffer from local pressure, such as overfishing, sedimentation and eutrophication (increased supply in organic and inorganic nutrients) [ 8 ]. Although healthy, thriving coral reefs do occur in a broad range of environmental conditions and nutrient availabilities [ 9 , 10 ], alterations of the surface water conditions have often led to the decline of scleractinian corals, the major reef builders [ 2 , 9 , 11 ]. Over the last decade, we observed an increase in the frequency and the intensity of coral bleaching event ( i . e . massive loss of symbionts or photosynthetic pigments from the host tissue), responsible for high coral mortality [ 11 , 12 ]. Coral bleaching, the most pressing issue for tropical coral reefs on a global scale [ 12 ], is often associated with high temperature anomaly events. Local conditions may also influence coral resilience to elevated temperatures by exacerbating or mitigating its detrimental effects [ 13 , 14 ]. Because of the complexity of coral response to each factor and the multiple interactions that exist between them (additive, synergistic or even antagonistic interactions [ 15 ], the combined effects of these factors on coral physiology are not yet well understood. In particular, several UVR and nutrient levels can occur in a reef during a thermal stress event. Nutrient level can vary with the eutrophication status of the reef, but also with rainfalls, floods, or local biological activity depleting nutrients from surface waters [ 16 , 17 ], while UVR level reaching a coral colony depends on the local shading, the surface orientation, the cloud cover or the depth at which corals grow [ 18 , 19 ]. Results from previous studies showed different responses to the combination of thermal stress with UVR [ 20 , 21 , 22 , 23 , 24 , 25 ] or with nutrient enrichment [ 14 , 26 , 27 ], depending on the environmental context, as well as the quality and severity of the stress. For example, different species of nitrogen (e.g., nitrate, ammonium, urea), may have very different effects on individual compartments of the coral holobiont, which may ultimately affect overall holobiont functioning [ 26 , 28 ]. All together, the results obtained call for further studies on the individual and interactive effects of these factors to better forecast the ecological consequences of global warming on reef corals. Corals are also known to release large amounts of organic matter (OM) in particulate and dissolved forms into the surrounding seawater [ 29 ], and release rates can be largely impacted by environmental changes. As OM is a main nutritional source for reef microorganisms [ 30 ], modifications in OM quality and/or quantity can affect reef water biochemistry, microbial community composition and activity and may ultimately even trigger virulence in reef-associated microbes [ 31 , 32 ]. Despite this key role of OM, very little information is available on species-specific OM release rates under thermal stress [ 33 , 34 ], UVR exposure and nutrient availability [ 35 ]. In this study, we describe the individual and combined effects of thermal stress, UVR exposure and nutrient (nitrate, phosphate) enrichment/depletion on the key performance variables of the reef-building coral Pocillopora damicornis . The first aim was to test whether the combination of multiple stressors (i.e. UVR and nutrient levels) reduces coral resistance to temperature- induced bleaching. The second aim was to investigate the effect of multiple environmental parameters (temperature, UVR and nutrients) on the release rates of OM. Interactions of multiple stressors, and the resulting cumulative impacts, have been identified as a research priority by management bodies and researchers (summarized in [ 15 ]). The results obtained will help assessing whether local conservation efforts (such as reduction in urban runoff) could significantly enhance the ability of corals to withstand the effects of climate change and could avoid the emergence of coral diseases.",
"discussion": "Discussion We have examined here the response of the common coral species Pocillopora damicornis to the combined effects of three stressors (seawater warming, nutrient enrichment/depletion and UVR exposure), to improve coral reef management. Overall, this study established seawater warming as one of the major stressors impacting the key performance variables of P . damicornis . The strength of this effect however, is determined by a range of local factors. We have indeed identified increased negative effects of high temperature on certain features of the coral holobiont when combined to nutrient starvation or UVR exposure. Altogether, our results demonstrate that seawater warming will have different effects on shallow and deep corals, which are exposed to different UVR and trophic conditions. Overall, heat stress led to a significant impairment of the gross and net productivity of P . damicornis , with a similar decrease in all UVR and nutrient level conditions. In other words, nutrient levels, or UVR exposure, did not increase the severity of bleaching above back ground levels, because temperature had a large impact alone. Two other studies, which have analysed the bleaching severity at different distances from nutrient sources, found no significant impact of nutrient levels on bleaching, in agreement with our observations [ 3 , 52 ]. It was however observed that high nutrient levels did, to some degree, prolonged bleaching in some coral species, and had a distinct effect on coral microbiomes that was independent of temperature [ 52 ]. Many other studies have linked seawater eutrophication with a higher bleaching susceptibility of different coral species [ 11 , 53 , 54 , 55 ]. Similarly, coral response to UVR exposure was shown to be species specific and dependent on the duration and/or intensity of the stress (summarized in [ 56 ]). All together, these observations suggest that the link between local stressors (such as UVR and nutrients) and temperature is more complicated than originally thought, and may depend, among others, on the severity of the thermal stress. Severe and prolonged warming will mask the effects of local stressors. However, at more moderate levels of heat stress, local environmental conditions, such as nutrient level and UVR exposure can modulate the effect of heat on coral physiology. Nutrient levels vary in nature with the water quality, the upwellings, the stratification of the water column, the seasonality or the presence of fish and bird colonies on the surrounding islands [ 57 , 58 , 59 ]. Reefs are generally oligotrophic, with ca. 0.5 μM total nitrogen (N) and 0.2 μM reactive phosphorus (P) [ 60 , 61 ], which is equivalent to our control conditions. During the stratification of the water column, levels of nutrients can decrease to less than 0.2 μM N and un-measurable concentrations of P. However, in many eutrophicated areas, total N can increase to ca. 2–4 μM and even ca. 20 μM, while P vary between 0.3 to 1.5 μM (summarized in [ 61 ]). These large changes in nutrient levels may modulate the effect of thermal stress on the main physiological traits of corals. Our results assign a crucial importance to nutrient starvation for coral health. Colonies of P . damicornis reared in the very low nutrient condition showed a higher bleaching susceptibility, with significant lower symbiont density, chlorophyll content, and rates of photosynthesis compared to nutrient-enriched corals. This impact of nutrient starvation has been observed in few previous studies [ 62 , 63 ], and support the importance of maintaining a minimum level of nutrients, especially nitrogen and phosphorus, in seawater. Overall, reefs experiencing overfishing, or nutrient shortage due to important water stratification should be particularly susceptible to bleaching during warming episodes. On the contrary to nutrient-starved colonies, those supplemented with NO 3 - and PO 4 + maintained slightly higher chl a and protein content as well as higher photosynthetic rates during thermal stress compared to control colonies. Although it has been shown that nutrient recycling from fish catabolism, or seawater nutrient enrichment can alleviate the negative effects of environmental stress on coral’s photosynthetic capacity [ 14 , 64 , 65 , 66 ], many studies have also shown detrimental effects of nutrient enrichment on coral health [ 11 , 53 , 54 , 55 ]. All together, these observations suggest that the beneficial effect of nutrient supplementation depends on the concentrations and exposure time to the nutrients, on the N:P ratios [ 27 , 67 ] or on intrinsic physiological characteristics of corals, such as symbiont type or density in coral tissue [ 68 ]. Analysis of recent studies points to a relationship between nutrient impact and symbiont density, with a detrimental effect for coral species with symbiont density higher than 10 6 symbionts cm -2 [ 25 , 69 ], and a beneficial effect for corals with lower symbiont densities, such as in this study [ 14 , 64 ]. However, correlations between nutrient and temperature need to be further investigated to fully understand their combined effects on coral health. Apart from nutrient starvation, exposure to UVR worsened the thermal-induced damages on photosynthesis, but only for P . damicornis colonies maintained under control conditions. This is in agreement with several studies finding synergistic effect of temperature and UVR increases on coral bleaching and mortality [ 15 , 22 , 36 , 70 , 71 ] and with the general observation that UVR exposure is generally linked to a reduction of the temperature threshold of coral bleaching [ 15 , 23 , 72 ]. However some studies measured no effect, or even a mitigating effect of UV on thermal-stress induced bleaching [ 22 , 73 ], and the same was observed in this study with corals incubated in depleted and enriched nutrient conditions. All together, these observations suggest that drawing broad conclusions about the combined effects of UVR and temperature on coral bleaching still poses a considerable challenge. Several factors can interact, such as the pre-exposure or acclimation to the stressors [ 74 , 75 ]; the species genotype [ 76 ], or even the nutrient level in seawater (this study) and can mitigate or enhance the bleaching susceptibility of corals exposed to the combined UVR-temperature stress. Concerning the organic matter fluxes, there was no effect of nutrients and/or temperature on the carbon and nitrogen release rates. A literature review demonstrates that there is no clear relationship between temperature and OM release rates in corals. Some studies observed increased OM release in heat stressed or bleached corals [ 33 , 77 , 78 ], while others measured no release [ 77 ], or even an uptake of dissolved organic matter [ 79 ]. However, UVR significantly changed the quality of the organic matter (OM) released by P . damicornis , since OM was significantly depleted in nitrogen under UVR exposure. Several factors could have resulted in this observation, such as the impairment in the coral capacity to take up nitrogen, or a different utilization/translocation of nitrogen within the symbiosis, leading to a higher retention in host tissue or symbionts. Although this has never been investigated in corals, and will need more in depth experiments with different coral species, several studies on phytoplankton observed a positive correlation between total nitrogen release and nitrogen uptake [ 80 , 81 , 82 ], as well as a reduction in nitrogen uptake under UVR [ 67 ]. Such reduction was attributed to membrane damage and to the inhibition of the enzymes involved in nitrogen metabolism [ 83 , 84 , 85 ]. If this UVR effect is repeatedly observed in corals, elevation in UVR levels on reefs is thus expected to generate products with high C:N molar ratio, i.e. enriched in carbon and depleted in nitrogen. During OM regeneration, bacteria will likely rapidly take up the small amounts of nitrogen available, leading to a depletion (or possibly starvation) of bioavailable nitrogen in surface reef waters. These preliminary observations suggest that future UVR and temperature-induced changes in the quality of the organic matter released by corals, combined with other changes in microbial nutrient recycling, will induce dramatic changes in the seawater C:N:P ratios in surface reef waters. These changes can lead to imbalance nutrient ratios that will have considerable impact on the ecology of coral reefs from the micro to the macro scale. However, more data are needed to fully understand the impact of environmental changes on the biogeochemical cycles. For instance, N 2 fixation by corals and other benthic functional groups, such as algal turfs [ 86 ] may increase in the future under heat exposure, following an increase in diazotroph community size and activity [ 87 ]. Elevated N 2 fixation rates are already observed in summer compared to winter [ 88 , 89 ]. This increase N 2 fixation may counterbalance the decreased nitrogen availability through coral OM production and bacterial remineralization. One of the most significant challenges now for the scientific community is to identify the factors that promote or worsen coral’s resilience to global warming. This is because interactions among several stressors, where the effect of one is dependent on the magnitude of another, are very common in nature [ 90 ]. The results obtained in this study, confirm those obtained in a recent meta-analysis [ 3 ], and show that seawater warming is the main factor impacting coral health. Therefore, during severe heat-stress, other factors such as UVR exposure, nutrient availability and water quality affords little resistance to bleaching. These factors can however become important during mild episodes of heat stress, or for the recovery of corals after warming episodes. Our study also highlights a significant effect of UVR and high temperature on the quality and degradation of the organic matter released by corals into reef waters. Overall, both UVR and seawater warming will induce changes in the seawater C:N:P ratios in surface reef waters, which in turn will have considerable impact on the ecology of coral reefs."
} | 4,185 |
35258342 | PMC9040813 | pmc | 2,681 | {
"abstract": "ABSTRACT Methylomicrobium album BG8 is an aerobic methanotrophic bacterium with promising features as a microbial cell factory for the conversion of methane to value-added chemicals. However, the lack of a genome-scale metabolic model (GEM) of M. album BG8 has hindered the development of systems biology and metabolic engineering of this methanotroph. To fill this gap, a high-quality GEM was constructed to facilitate a system-level understanding of the biochemistry of M. album BG8. Flux balance analysis, constrained with time-series data derived from experiments with various levels of methane, oxygen, and biomass, was used to investigate the metabolic states that promote the production of biomass and the excretion of carbon dioxide, formate, and acetate. The experimental and modeling results indicated that M. album BG8 requires a ratio of ∼1.5:1 between the oxygen- and methane-specific uptake rates for optimal growth. Integrative modeling revealed that at ratios of >2:1 oxygen-to-methane uptake flux, carbon dioxide and formate were the preferred excreted compounds, while at ratios of <1.5:1 acetate accounted for a larger fraction of the total excreted flux. Our results showed a coupling between biomass production and the excretion of carbon dioxide that was linked to the ratio between the oxygen- and methane-specific uptake rates. In contrast, acetate excretion was experimentally detected during exponential growth only when the initial biomass concentration was increased. A relatively lower growth rate was also observed when acetate was produced in the exponential phase, suggesting a trade-off between biomass and acetate production. IMPORTANCE A genome-scale metabolic model (GEM) is an integrative platform that enables the incorporation of a wide range of experimental data. It is used to reveal system-level metabolism and, thus, clarify the link between the genotype and phenotype. The lack of a GEM for Methylomicrobium album BG8, an aerobic methane-oxidizing bacterium, has hindered its use in environmental and industrial biotechnology applications. The diverse metabolic states indicated by the GEM developed in this study demonstrate the versatility in the methane metabolic processes used by this strain. The integrative GEM presented here will aid the implementation of the design-build-test-learn paradigm in the metabolic engineering of M. album BG8. This advance will facilitate the development of a robust methane bioconversion platform and help to mitigate methane emissions from environmental systems.",
"introduction": "INTRODUCTION Anthropogenic activities have led to significant increases in atmospheric methane ( 1 ), which contributes to climate change and perturbs the global carbon cycle ( 2 ). Nevertheless, methane derived from renewable sources is an attractive substrate in the production of value-added products ( 3 – 5 ), and methane conversion processes represent a promising trend in bioindustry ( 6 , 7 ). Methanotrophic bacteria utilize methane as their source of carbon and energy, and these microorganisms have become increasingly important in the biomanufacturing of valuable chemical compounds ( 4 , 8 – 10 ). Although methanotrophic species are metabolically active under both aerobic and anaerobic conditions ( 11 – 14 ), considerations of cost, sustainability, and environmental impact have led to a preference for aerobic methane-oxidizing bacteria in large-scale biorefining applications ( 15 , 16 ). Methylomicrobium album BG8 (formerly known as Methylobacter albus , Methylomonas albus , or Methylomonas alba ) is an obligate aerobic, Gram-negative, gammaproteobacterial methanotroph that uses methane or methanol as its sole source of carbon and energy ( 17 ). A DNA-DNA hybridization study revealed high levels of similarity between its genome and those of Methylomicrobium agile ATCC 35068 (99.16%), Methylotuvimicrobium alcaliphilum 20Z (75.69%), and Methylotuvimicrobium buryatense 5G (76.64%) ( 18 ). Recent phylogenomic analyses based on the average amino acid identity and average nucleotide identity have shown that M. album BG8 is also closely related to Methylomicrobium (formerly Methylosarcina ) lacus LW14 ( 19 ). M. album BG8 has been isolated from swampy soils and freshwater sediments ( 18 , 20 ) and has been widely studied due to its importance in environmental microbiology for bioremediation of different environmental pollutants ( 21 – 23 ). Through recent physiological and omics analyses, M. album BG8 has also been identified as a promising microbial cell factory ( 24 , 25 ) for applications in the methane-based biotechnology industry ( 26 ). The current metabolic characterization of methanotrophic bacteria suggests there are three main groups ( 27 ). Methanotrophs in group I ( Gammaproteobacteria ) utilize the ribulose monophosphate (RuMP) cycle to metabolize formaldehyde derived from methane oxidation, group II ( Alphaproteobacteria ) members direct the carbon flux resulting from the oxidation of methane to formate to a complete serine cycle, and group III ( Verrucomicrobia ) members possess a complete Calvin-Benson-Bassham cycle for carbon dioxide utilization. The metabolic engineering of methanotrophs has enabled advances in the biotechnology of methane conversion to produce succinate ( 28 ), 3-hydroxypropionic acid ( 29 ), 2,3-butanediol ( 30 ), putrescine ( 9 ), α-humulene ( 31 ), cadaverine ( 32 ), lysine ( 32 ), shinorine ( 33 ), and acetoin ( 33 ). These laboratory achievements have been aided by the results of genome-scale metabolic model (GEM)-based simulations, which are used to enhance the system-level understanding of methanotrophy. Thus far, GEMs of nine methanotrophic species have been constructed, including three group I species [ Methylotuvimicrobium buryatense 5G(B1) ( 34 ), Methylotuvimicrobium alcaliphilum 20Z ( 35 ), and Methylococcus capsulatus Bath ( 36 , 37 )] and six group II species ( Methylocystis hirsuta [ 38 ], Methylocystis sp. strain SC2 [ 38 ], Methylocystis sp. strain SB2 [ 38 ], Methylocystis parvus OBBP [ 39 ], Methylocella silvestris [ 40 ], and Methylosinus trichosporium OB3b [ 41 ]). Although some of these GEMs have been validated using growth yields, methane- and oxygen-specific uptake rates ( 38 ), transcriptomics data ( 34 ), or enzyme kinetics ( 35 ), most do not contain other integrated experimental data. Despite the potential of M. album BG8 as a tool in environmental and industrial biotechnology, no GEM of this methanotroph has been developed; therefore, an integrative system-level understanding of methanotrophy in this strain remains lacking. In this study, a high-quality GEM of M. album BG8 was constructed by stringently following well-established systems biology protocols ( 42 , 43 ). Furthermore, an integrative modeling framework was applied, wherein experimental time-series growth and compound uptake and excretion data collected under different initial methane and oxygen headspace percentages and biomass concentrations were integrated with the initial GEM to construct parametrized GEMs. Subsequently, the metabolic states that promote biomass production and carbon dioxide, formate, and acetate excretion were identified through a flux balance analysis (FBA). The study findings provide novel insight into the metabolic versatility of M. album BG8 and highlight the associations between biomass production and organic compound excretion.",
"discussion": "DISCUSSION The aim of this study was to elucidate the system-level metabolism of M. album BG8 using an integrative systems biology framework. The integration of time-series growth and compound uptake and excretion data into a newly developed, high-quality GEM yielded novel insights into the metabolic mechanisms of this methanotrophic bacterial strain. Furthermore, the experimental data collected in this study enabled the construction of parametrized GEMs, and the application of an FBA enabled the system-level understanding of the metabolism of M. album BG8 under different initial concentrations of oxygen, methane, and biomass. The conversion factor of 0.26 gDCW liter −1 derived between the OD 600 and DCW of M. album BG8 in our study differs from a previously reported conversion factor of 0.33 gDCW liter −1 ( 51 ). Although a linear regression of our data with a y intercept of −0.044 yielded a conversion factor of 0.32 gDCW liter −1 (see Fig. S1 in the supplemental material), this model was not used due to a lower adjusted R 2 and a less significant P value than those of our selected model. These results highlight the need for caution regarding potential variability in the conversion factor between OD 600 and DCW when studying M. album BG8. However, the Monod model was used to estimate growth kinetics as a function of the initial oxygen headspace percentage, and the calculated μ max value (0.11 h −1 ) is similar to other reported growth rates for M. album BG8 (e.g., 0.10 h −1 to 0.18 h −1 with different concentrations of methane [ 24 , 51 ], 0.09 h −1 to 0.13 h −1 with different concentrations of chloromethane [ 52 ], and 0.11 h −1 with methanol [ 53 ]). However, the Monod model assumed that oxygen was the only limiting substrate, which may not be true in a real-life scenario. We identified oxygen as the key driver of methane oxidation by M. album BG8, as it exerted strong effects on both biomass production and organic compound excretion. In our analysis, optimal growth could be sustained at a ratio of ∼1.5:1 between the specific uptake rates of oxygen and methane. In agreement with our results, a ratio of ∼1.5:1 between oxygen and methane uptake rates has also been found by FBA to be optimal for growth for the methanotroph Methylomicrobium buryatense 5GB1 after careful correction of pathways in the GEM ( 54 ). Biomass production was maximized under culture conditions of 20% methane and 20% oxygen, with a yield of 0.021 gDCW mmol-CH 4 −1 . This was the only culture indicated by the parametrized GEM to have a completely active TCA cycle and to exhibit a low mean level of absolute flux throughout its metabolic network. We speculate that these results of integrative M. album BG8 modeling indicate an optimal methanotrophic state for the allocation of molecular and metabolic resources in which optimal biomass production is preserved and enzyme use is minimized. Future work is required to further explore this notion. These results also complement the findings of recent work in which M. album BG8 was identified as the bacterium with the highest biomass yield (nearly double that of the others tested) among a group of industrially relevant methanotrophs ( 25 ). Intriguingly, the GEM showed the excretion of methanol as a by-product of growth under the condition with the lowest initial oxygen availability (5%). The GEM was parametrized (based on the experimental data) with an uptake rate bounded at 1.52 to 3.16 mmol h −1 gDCW −1 for methane and an uptake rate bounded at 0.05 to 2.24 mmol h −1 gDCW −1 for oxygen. A possible explanation for this methanol-excreting phenotype from the FBA is that methanol works as an additional control for the metabolic branching at low oxygen concentrations, leading to tetrahydrofuran-based oxidation of formaldehyde in addition to metabolic flux through the EDD variant of the RuMP cycle and partial serine pathways. Hence, instead of actual methanol release to the extracellular environment (which was not detected in the medium), M. album BG8 could be using the mentioned pathways to more efficiently oxidize methanol to gain biomass. A recent transcriptomics and metabolomics study of M. album BG8 grown on methanol ( 55 ) yielded a phenotype that is similar to the condition with low availability of oxygen. Increased transcription of genes related to carbon metabolism through the RuMP EDD variant and pentose phosphate pathways as well as formaldehyde detoxification through the glutathione dependent pathway, of which formate is the product, were observed ( 55 ). Taking the reported transcriptomics and metabolomics data ( 55 ) together with the GEM and FBA results presented here, they suggest that M. album BG8 favors methanol oxidation over methane as oxygen availability diminishes. Although carbon dioxide and formate are usually excreted during aerobic methanotrophy ( 27 ), these metabolites have recently become valuable by-products ( 56 , 57 ) and are used as carbon sources in synthetically constructed methanotrophic modular microbial consortia to produce value-added compounds ( 58 ). In our study, the highest carbon dioxide yield (0.39 mmol mmol-CH 4 −1 ) was achieved under culture conditions of 20% methane and 20% oxygen, and the highest formate yield (0.025 mmol mmol-CH 4 −1 ) was obtained under conditions of 20% methane and 25% oxygen. In contrast to a recent report ( 25 ), formate excretion was detected in all of the cultures in this study. The phenotype with the highest formate yield exhibited the highest mean absolute flux through its metabolic network and the second highest flux through the H 4 MPT formaldehyde oxidation pathway. In other words, by actively using the complete H 4 MPT pathway, this phenotype can generate two reducing equivalents [NAD(P)H]. Based on these results, developers of metabolic engineering strategies that aim to optimize carbon dioxide and/or formate excretion by M. album BG8 during methane metabolism may consider directing carbon flux toward the H 4 MPT pathway. Nevertheless, future attempts to optimize formate production by M. album BG8 should consider the achievement of significantly larger formate yields by other methanotrophs grown on methanol instead of methane ( 59 – 61 ). Although a previous study investigated the effects of methane and methanol on formate excretion by M. album BG8, the authors did not detect formate under the tested conditions ( 25 ), likely because the samples collected in the reported experiments corresponded to the early stages of M. album BG8 growth in our experimental setup. Acetate is a key precursor in high-value chemical production ( 62 ). Therefore, the biotechnological potential of M. album BG8 in the conversion of methane to valuable compounds depends on the ability to produce a high acetate yield. Among the different culture conditions in this study, the highest acetate yield (0.072 mmol mmol-CH 4 −1 ) was unexpectedly obtained under the conditions of 20% methane, 20% oxygen, and a relatively high initial biomass concentration ( Fig. 7A ). This culture of M. album BG8 excreted acetate during the exponential phase ( Fig. S3 ) but resulted in a relatively lower growth rate at this phase ( Fig. 7B ), indicating a trade-off between biomass production and acetate excretion ( Fig. 7 ) that was further supported by the model FBA ( Fig. 8C ). However, the molecular and metabolic mechanisms behind this trade-off remain unclear and need to be further investigated. Previous reports have described acetate excretion by other gammaproteobacterial methanotrophs under prolonged oxygen starvation ( 63 ) and oxygen-limited growth ( 64 ). Our experimental results agree with those findings, as acetate excretion was detected in all cultures in which oxygen was present at a very low concentration or completely depleted. Similarly, the FBA showed a high acetate excretion flux in M. album BG8 at a low (<1.5:1) oxygen-to-methane uptake flux ratio ( Fig. 6C ). Overall, we expect that the metabolic characterization of acetate excretion presented here will support future attempts to increase acetate yields and achieve the metabolic reprogramming of acetyl-CoA conversion in M. album BG8. 10.1128/msystems.00073-22.3 FIG S3 Time-series-normalized concentration (divided by the maximum concentration per compound) profiles of biomass production, oxygen and methane consumption, and acetate excretion for the experiment with 20% methane, 20% oxygen, and a relatively high initial biomass concentration (C20_O20_ODH). Download FIG S3, PDF file, 0.1 MB . Copyright © 2022 Villada et al. 2022 Villada et al. https://creativecommons.org/licenses/by/4.0/ This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . Both the oxygen-to-methane headspace ratio and specific uptake ratio have been explored intensively in methanotrophy studies ( 59 , 63 , 65 – 68 ), as these variables are thought to control the differential activation of pathways required for the production of biomass, generation of energy, and induction of fermentation-like metabolism ( 63 , 64 ). The M. album BG8 genome encodes homologs of enzymes found in the EDD and EMP RuMP cycle variants in M. alcaliphilum 20Z and M. buryatense 5G(B1) ( 64 ). However, M. album BG8 lacks 3 of the 18 key enzymes [i.e., phosphate acetyltransferase, d -fructose 6-phosphate phosphoketolase, and NAD(P)-dependent malic enzyme] required for fermentation-like metabolism in M. alcaliphilum 20Z ( 64 ) and M. buryatense 5G(B1) ( 63 ) at low oxygen-to-methane concentration ratios. Nevertheless, the M. album BG8 cultures exhibited a fermentation-like metabolism with high acetate excretion and low carbon dioxide and formate excretion mainly during the stationary phase when the oxygen-to-methane concentration ratio was very low. However, in terms of energetic investigations with the current version of the GEM, it is important to note that the growth-associated and non-growth-associated ATP maintenance have not been specifically determined for M. album BG8. Thus, future work is required to experimentally determine these parameters for M. album BG8, as has been described elsewhere ( 69 , 70 ). Finally, we identified a nonlinear relationship between the initial oxygen-to-methane headspace ratio and the oxygen-to-methane uptake flux ratio in batch cultures of M. album BG8, which will facilitate control of the oxygen-to-methane uptake flux ratio in batch systems. In summary, the experimental results and the FBA presented in this study elucidate some of the characteristics of M. album BG8. However, many other aspects of metabolism in this strain remain to be explored in detail. The newly developed GEM of M. album BG8 is a valuable tool that will aid future investigations of this important methanotroph in the context of methane conversion applications in biomanufacturing or methane emission mitigation in environments that experience dynamic fluxes of methane and oxygen."
} | 4,640 |
35704862 | PMC10123809 | pmc | 2,682 | {
"abstract": "The concept of creating all-mechanical soft microrobotic systems has great potential to address outstanding challenges in biomedical applications, and introduce more sustainable and multifunctional products. To this end, magnetic fields and light have been extensively studied as potential energy sources. On the other hand, coupling the response of materials to pressure waves has been overlooked despite the abundant use of acoustics in nature and engineering solutions. In this study, we show that programmed commands can be contained on 3D nanoprinted polymer systems with the introduction of selectively excited air bubbles and rationally designed compliant mechanisms. A repertoire of micromechanical systems is engineered using experimentally validated computational models that consider the effects of primary and secondary pressure fields on entrapped air bubbles and the surrounding fluid. Coupling the dynamics of bubble oscillators reveals rich acoustofluidic interactions that can be programmed in space and time. We prescribe kinematics by harnessing the forces generated through these interactions to deform structural elements, which can be remotely reconfigured on demand with the incorporation of mechanical switches. These basic actuation and analog control modules will serve as the building blocks for the development of a novel class of micromechanical systems powered and programmed by acoustic signals.",
"conclusion": "Conclusions We introduced a suite of mechanical microsystems with relatively basic design yet complex motion. Precise control over geometric parameters facilitated detailed analysis of forces and fluid–structure interactions. We made two important discoveries. First, we showed that a single bubble can provide multiple-DOF motion along prescribed trajectories with high fidelity through the application of frequency- and amplitude-modulated acoustic signals. Second, as a result of higher-order interactions, forces between oscillating bubbles emerge, and their amplitude and direction can be programmed by tuning the excitation frequency. The realization of the former required printing of capsules with multiple orifices at different sizes, a calibration process, and dynamic control. Direct laser writing has been used to develop wireless soft microactuators powered by magnetic fields 63 and photothermal effects. 64 , 65 Acoustic actuation is appealing for in vivo biomedical applications, specifically to remotely steer ultraflexible microcatheters 66 and control the release of biologics from miniaturized wireless implants. 67 Ultrasound is minimally invasive, acoustic microactuators are compatible with magnetic resonance imaging, generating acoustic waves does not require sophisticated and expensive equipment, and the whole device is fully biodegradable. We developed an open-loop control scheme that drives a mechanical system to follow prescribed trajectories by applying a complex signal, based on the model and calibration. The position errors between the programmed and executed motion are not negligible, which may stem from multiple sources. We assumed that the 3D printed cantilever beam has isotropic stiffness. However, the material stiffness depends on the degree of polymerization, which might show spatial variations along the vertical axis. Moreover, the controller assumes small deflections although the beam deflected beyond the limit defined by the theoretical linear model. Finally, at relatively large deflections, the beam started to tilt out-of-plane and rotate around its axis while we only considered in-plane translation. We focused our attention on geometric arrangements of entrapped bubbles, as a means to program the soft robotic system. Leveraging frequency-selective bubble excitation, we could operate systems in different modes at different frequencies. Considering the interactions among neighboring actuators would advance the programming framework. An actuator may be attracted to other actuators from multiple directions, realizing a sophisticated motion that highly depends on the excitation frequency and time-varying spacing among bubbles. We have also incorporated acoustically actuated bistable mechanisms as onboard control units to introduce the capability to operate in different modes under the same input signal. This last functionality represents the first step toward reconfigurable mechanical systems that operate analogously to mechatronic systems. To this end, reversible switching of several bistable structures will be instrumental. Moving forward, bistable beams could be replaced by 3D architected materials that display sensing, pattern analysis, and multistability. 68–70 The fabrication of such complex structures is feasible with two-photon lithography, and local actuation initiated by entrapped bubbles will reveal unprecedented reconfigurability and multifunctionality.",
"introduction": "Introduction Soft materials can be programmed to change their physical properties such as shape and stiffness on-the-fly by the externally applied light, heat, mechanical forces, or magnetic fields. 1–5 Such material formulations have great potential, particularly at small scales, to achieve functionalities that are unattainable by conventional mechatronic systems. 6–13 Notably, simple mechanical structures fabricated from magnetorheological or liquid crystal elastomers displayed a virtually unlimited number of degrees of freedom (DOF), as a result of either the spatial complexity of the magnetization profile 14–16 or the use of structured illumination. 17 , 18 Moreover, rationally designed flexible structures, also known as mechanical metamaterials, can realize programmable digital logic. 19–29 This route for physical intelligence has already been pursued by living organisms, 30–32 and extensively studied for the development of autonomous soft robots. 33–36 Acoustofluidics has several unique properties that can reveal the full potential of programmable soft matter. Air bubbles are efficient transducers that generate forces through interactions with the external pressure waves, the surrounding fluid, and with each other. 37–45 Therefore, these interactions can be programmed by modulating the acoustic wave as well as the geometry and the spatial distribution of bubbles. The generated forces are determined by primary and secondary acoustic effects that are highly nonlinear. Thus, a detailed understanding of the underlying physics is instrumental to using air bubbles for controlling deformable elements. To address this unmet challenge, we combined analytical modeling, finite element simulations, and experimental analysis that involved detailed characterization of acoustic pressure, bubble oscillations, and forces generated by bubbles with coupled dynamics. In this study, we present an integrated design, fabrication, and control methodology that transforms monolithically printed flexible structures into programmable soft robotic systems. The key innovation is the spatial patterning of polymer capsules that stably contain individual air bubbles along ultraflexible beams using direct laser writing ( Fig. 1a , Supplementary Fig. S1 and Supplementary Movie S1 ). By tuning the geometry of the capsules and the architecture of the material, we precisely controlled the acoustofluidic interactions ( Fig. 1b, c ). We demonstrate the compatibility of our acousto-active systems with mechanical logic by constructing actuated bistable mechanisms. FIG. 1. Conceptual illustration of the presented methodology. (a) Actuator modules (i.e., capsules with entrapped air bubbles) are nanoprinted using two-photon polymerization technique. Two different acoustic actuation scenarios are systematically studied. (b) A single actuator module is excited at the natural frequency to deform ultraflexible structures. (c) Pairs of identical or distinct actuator modules are excited at various frequencies to generate attractive and repulsive forces.",
"discussion": "Results and Discussion The basic actuator module consists of a 3D printed cylindrical capsule with a single circular orifice ( Fig. 2a ). A similar manufacturing technique and geometry has been recently used to generate polymer microparticles with liquid-phase core. 46 We printed all the structures as a monolithic piece from a single biocompatible soft polymer, trimethylolpropane ethoxylate triacrylate (TPETA), 47 using two-photon polymerization ( Fig. 2b ). A detailed description of the experimental methods and control system, including the peripheral electronics is provided in Supplementary Note S1 and pictured in Supplementary Figure S9 . Within this capsule, the bubble is physically isolated from the surrounding fluid except at the side of the opening ( Fig. 2c ). Acoustic excitation of a single bubble submerged in liquid generates microstreaming. 48 By entrapping bubbles inside cavities, their oscillations are constrained to regions defined by the orifices. FIG. 2. Design, fabrication, and operation of acoustic actuator modules. (a) The natural oscillation frequency of entrapped microbubbles were set by tuning the volume of the cavity, V C , and the radius of the orifice, R . (b) Scanning electron microscopy image of a partially printed actuator module shows the cavity and the orifice. (c) Brightfield images of an actuator module when the air bubble is entrapped inside the cavity. (d) Streamlines around an acoustic module are visualized using fluorescent tracer particles. (e) A computed contour plot showing the dependence of the entrapped microbubble natural frequency on the orifice size ( R ) and cavity volume ( V C ). The black dashed lines indicate the volume and radii that correspond to the selected geometric parameters. The red and magenta curves denote the operating frequencies of 170 and 85 kHz, respectively. (f) Frequency-selective powering of multiple actuator modules. The first natural frequencies of the two modules are set by prescribing different orifice sizes. The blue arrow points to the actuator module that is activated at the specified frequencies, f 1 and f 2 . Scale bars, 20 μm. In this configuration, acoustic streaming generated counter-rotating vortices and a jet localized in the center, which was captured using high-speed recordings of tracer particles around the actuator module ( Fig. 2d ). Once an actuator is connected to a cantilever beam, it is expected to generate thrust normal to the orifice's plane, thereby deforming the beam. By carefully tuning the acoustic frequency, different vibration modes of the coupled fluid-structure system can be excited. 49 The vibration modes are manifested by the unique deformation patterns of the bubble at the interface. Nevertheless, we focused our attention on the first mode because, assuming a uniform distribution, the projection of the impinging pressure is maximal. Large deformation and complex motion can be generated with slender structural elements, including beams, plates, and shells. 50–52 We have recently developed an analytical model that calculates the natural frequencies and corresponding vibration modes of bubbles entrapped inside arbitrarily shaped cavities with multiple circular orifices. 49 In brief, we extended a previously published model 53 to accommodate multiple orifices on the same cavity, incorporate all the possible mode shapes, and consider the geometry adjacent to the orifice. We derived the model by formulating the potential and kinetic energy in the system. The contribution to the kinetic energy comes from the fluid motion while the gas compressibility and interface deformation determine the potential energy. The acoustic wavelength is much larger than the capsule's largest dimension for the frequencies studied in this work. Thus, the instantaneous pressure in the gas can be considered uniform and, as a result, the interior shape of the capsule has a negligible effect. The spherical shape was chosen to maximize the gas volume, increase the bubble's stability, and simplify the printing procedure. We recorded the power spectrum of the ultrasound transducer using a hydrophone to determine the frequency bands at which the actuators could be effectively powered ( Supplementary Fig. S2 ). Using our model, we designed the geometry (i.e., volume and orifice radii) so that the first natural frequencies of the entrapped bubbles are within these bands ( Fig. 2e ). For the chosen parameters, variations in the bubble orifice radius, R , have a greater influence on the natural frequency than the internal volume, V c , because R influences both the potential and kinetic energy of the system. We printed two capsules with orifice radii of 7.75 and 13 μm, respectively ( Supplementary Table S1 ). Entrapped bubbles were actuated selectively at their estimated natural frequencies of either 85 or 170 kHz, which led to a sequential deformation of the beams ( Fig. 2f and Supplementary Movie S2 ). Actuators with orifices close to the substrate are not ideal for the quantification of forces because the entrapped bubbles may interact with the substrate's surface. 44 , 45 To minimize such perturbations, we printed the beams vertically, raising the actuator modules 50 μm above the substrate ( Fig. 3a ). Laser scanning confocal microscope images of fluorescently labeled samples verified that the structures were printed according to the CAD design ( Supplementary Fig. S3 ). In this configuration, the beam bending could be followed from the actuator's in-plane displacement ( Fig. 3b ), which increased quadratically with the input voltage ( Fig. 3c ). The bending modulus was calculated from the material's Young's modulus, E = 13 MPa, 47 and the dimensions of the beam were measured using electron microscopy ( Supplementary Fig. S3 ). The generated force was then estimated using linear beam theory ( Supplementary Note S2 ). FIG. 3. Mechanical characterization of the actuator module and the pressure field. (a) Schematic illustration of the bending of a flexible cantilever beam that is printed vertically to minimize the interactions of the actuator module with the bottom surface. (b) Brightfield images showing the displacement of the actuator module, which is denoted as δ . δ is used to measure the deflection of the beam. Scale bar, 20 μm. (c) The displacement increases quadratically with the voltage applied to the acoustic transducer. (d) The thrust calculated using a linear analytical model and a FEM model are in excellent agreement. (e) The acoustic pressure increases linearly with the voltage applied to the transducer. Pressure measurements were performed using a hydrophone. (f) The deflection of the entrapped bubble measured at the center of the orifice linearly increases with the acoustic pressure. The deflection was measured using a laser vibrometer. FEM, finite element method. Although the linear theory was used, the displacement is a nonlinear function of the applied force because the part of the beam corresponding to the location of the actuator was taken as rigid ( Supplementary Fig. S10 ). The tip displacement, δ, is given as:\n (1) δ = L P 6 E I L 3 l + 2 L + 6 l 2 l + L 4 + L 2 2 l + L 2 P 2 E 2 I 2 \n where L is the length of the beam, l is the length of half the actuator, E is Young's modulus, I is the second moment of inertia of the cross-section and P is the applied force. Nonlinear finite element method (FEM) simulations (i.e., large deformations) using linear elastic material ( E = 13 MPa and Poisson's ratio is taken as 0.45) were in perfect agreement with the analytical model, thus confirming that the linearity assumption was acceptable for the given range of deflections ( Fig. 3d ). The analytical model was used to calculate the total force acting on a deforming beam from the recorded deflection. We observed a linear relationship between the voltage applied to the transducer and the resultant acoustic pressure measured by a hydrophone ( Fig. 3e ), as predicted by the theory. 54 The acoustic energy density in the workspace quadratically increases with increasing pressure, 55 and so does the streaming velocity around the bubble. 44 Sequentially increasing the input voltage resulted in a new configuration within milliseconds. Furthermore, when the input voltage was turned off, the beam immediately returned to its original position, confirming that the actuation was in the elastic range. We characterized the oscillation amplitude at the bubble's first natural frequency using a custom-built experimental platform ( Supplementary Fig. S4 ). The data showed that the deflection amplitude at the air–fluid interface increases linearly for the range of pressure we applied ( Fig. 3f ). An actuator module with multiple DOF would drastically increase the dexterity and form factor of printed structures. We postulated that a capsule, which contains multiple orifices of different sizes, would serve this purpose. To test this idea, we printed an actuator module in the shape of an equilateral triangular prism with three orifices, one at the center of each face ( Fig. 4a ). The actuator module was connected atop a 50-μm-long and 2-μm-diameter cantilever beam. The sizes of the orifices (15, 9.5, and 7 μm) were chosen in a way that corresponding natural frequencies were well spaced and within the effective range of the transducer ( Fig. 4b ). Simulation results have shown that primarily one interface is deformed at each mode ( Fig. 4c ). FIG. 4. Design, calibration, and control for multi DOF motion. (a) An illustration of the actuator module with multiple orifices. The ceiling is removed for visualization. The different orifices are numbered starting from the largest to the smallest. The radii are 15, 9.5, and 7.5 μm, respectively. (b) A modal mobility plot, showing the first three modes of the system excited with uniform pressure. Dashed lines highlight the natural frequencies. (c) An illustration of the first three normal vibration modes. Deformation of the interfaces is scaled according to the amplitude of calculated deflection. (d) Brightfield images of a device showing the deflection as a response to excitation at the chosen frequencies. (e) A system is driven with the same signal twice to follow a rectangular trajectory. The rectangle in red dashed lines shows the desired trajectory. (f) A system is driven to follow two circular trajectories of different sizes. Circles in red dashed lines show the desired trajectories. (g) An actuator module moving along the letters of EPFL. Data points are shown by blue dots and prescribed trajectories are shown by red continuous lines . Scale bar, 20 μm. DOF, degrees of freedom; EPFL, Ecole Polytechnique Fédérale de Lausanne. We excited the system at multiple frequencies simultaneously to control the position. The force is unidirectional, therefore, at least three frequencies are required to fully control the 2D position. Ideally, these frequencies are the ones where the largest displacement is obtained, which are expected to be the bubble's natural frequencies. However, the transducer has its own dynamical response, and the input pressure varies with the excitation frequency ( Supplementary Fig. S2 ). We addressed this issue by experimentally calibrating the response of the system. We recorded the actuator motion while sweeping the frequency from 40 to 160 kHz at a constant input voltage. Using a subpixel resolution image processing algorithm, 56 we extracted the planar position of the module ( Supplementary Fig. S5a ). We then selected three frequencies where the largest deflections in different axes were recorded. Figure 4d shows the undeformed and deformed states of the system at the chosen frequencies. Next, we measured the actuator's deflection at each frequency and built the vectors spanning the planar displacement field. We implemented an open-loop controller that leverages the superposition principle and excites the system with a signal comprising three harmonic terms, which correspond to the aforementioned vectors. We controlled two DOF with three forces, thus, there are infinite combinations. We solved a constrained linear least-squares problem, 57 where we minimized the voltage signal amplitude. We performed a series of experiments to evaluate the control strategy. First, we programmed a calibrated device to repeatedly follow a rectangular trajectory, slightly offset from the origin where we had better control over the motion ( Fig. 4e ). Second, we programmed several circular trajectories with different radii and offsets ( Supplementary Movie S3 ). The position error increased with the distance from the origin ( Fig. 4f ). Third, we used the calibration parameters of one device to control another with the same design ( Supplementary Fig. S5b ). The trajectories of the two devices were close, suggesting that calibration might be performed once and used repeatedly for different devices. As a final demonstration, we designed four trajectories prescribing the initials Ecole Polytechnique Fédérale de Lausanne to show that control was not limited to specific geometries ( Fig. 4g ). Acoustically excited bubbles interact with each other through the surrounding fluid when they reside in close proximity. The total force acting on a bubble is the result of the exciting primary pressure field, and higher-order fields emanating from neighboring bubbles that also act as acoustic sources. 38 , 58 The distances between bubbles are significantly smaller than the acoustic wavelength in all our prototypes. Therefore, we can assume that the acoustic radiation forces caused by the primary field do not affect the relative displacement of the actuators. The secondary forces that act on the coupled actuators are thrust, drag-induced acoustic streaming, and secondary acoustic radiation force (also known as secondary acoustic radiation force or secondary Bjerknes force). Identical bubbles are expected to generate the same acoustic streaming; thus, the generated thrust would push the bubbles away from each other. On the other hand, the magnitude of the acoustic radiation force, which primarily acts to pull the bubbles toward each other, depends on the distance between them. 38 , 58 The total force, F B , acting on an untethered bubble is\n (2) F B = F R + F A S + F d \n where F R denotes the acoustic radiation force, F AS denotes thrust generated by streaming, and F d is the drag force acting on a bubble due to the streaming generated by the adjacent bubble. In the following analysis, we simplify the kinematics by assuming that actuators move along a virtual line connecting their centers. To quantify the total force generated by interacting bubbles, we printed two adjacent cantilever beams with identical actuators that were faced toward each other ( Supplementary Table S1 ). The initial distance between the actuators, d i , was systematically varied to study the effect of spacing. At equilibrium, | F B | in Equation (2) equals the elastic force applied by the cantilever beam, denoted by P in Equation (1). We observed two distinct regimes in the dynamics of the coupled actuators ( Supplementary Movie S4 ). When the initial distance between the actuators was smaller than a critical distance, d c , the radiation forces dominated the thrust generated by acoustic streaming ( Fig. 5a ). For the given actuator design, this critical distance was 50 μm (i.e., d c = 50 μm). As a result, the beams bent toward each other until the bubbles made contact ( Fig. 5b ). With increasing d i , the magnitude of the acoustic radiation force decreased, emphasizing the contribution of streaming forces ( Fig. 5c ). For d i > d c , the sign of the total force switched, where the beams started to move away from each other ( Fig. 5d ). FIG. 5. Characterization of acoustic radiation forces between two actuator modules. (a) An illustration showing the relative magnitude of the three forces acting on the actuator module; drag force ( F d ), acoustic streaming force ( F AS ), and acoustic radiation force ( F R ) for the case where the initial distance ( d i ) between the actuator modules is shorter than the critical distance ( d c ). In this case, F R is larger than the sum of the other two forces. (b) Representative microscope images from an experiment where the actuator modules were attracted to each other and, as a result, the beams bent toward each other. (c) An illustration showing the relative magnitude of the three forces acting on the actuator module for the case where d i is longer than d c . In this case, F AS and F d dominate the radiation force. (d) Microscope images from an experiment where the actuator modules moved away from each other and, as a result, the beams bent in opposite directions. (e) The equilibrium distance between acoustically actuated beams as a function of input voltage. The beams were printed with different spacing. In all the experiments, the frequency was tuned to 125 kHz. (f) The theoretical bode plot. The top panel depicts the normalized oscillation magnitude, where the natural frequencies are highlighted by black dashed lines . The blue and orange curves in the lower panel show the phase response of the small and big bubbles, respectively. The yellow curve depicts the phase difference. In the gray region , the phase difference results in a repulsive force while in the white regions the interbubble forces are attractive. (g) A schematics showing the frequency-dependent motion of actuator modules with different cavity sizes. The in-phase and out-of-phase vibrations of the entrapped bubble determine the magnitude of the radiation forces. (h) Representative brightfield images showing the frequency-dependent deformation of coupled beams. Scale bars, 20 μm. Figure 5e summarizes the nonlinear responses of coupled actuators with respect to the initial distance and the input voltage. The further away the actuators were from each other at rest, the more the beam deflection resembled that of the isolated single beam ( Fig. 3c ). Based on this empirical observation, we hypothesized that the dynamics could be captured by an analytical model where all forces are proportional to the input voltage squared. 59 We assumed that F d did not depend on the distance between the actuators, therefore | F AS | = | F d | at all times. This assumption is reasonable as the distance between the bubbles was always comparable to the bubble size, which was significantly shorter than the acoustic wavelength. We also assumed that F R inversely depends on the distance squared. 38 , 58 The force balance equation, Equation (2), is then rewritten as:\n (3) F B = 2 γ − α d − 2 V 2 \n where F B > 0 indicates bubble repulsion, 37 \n α and γ are functions of the excitation frequency and geometry, and d is the distance between the bubbles' vibrating surface. We fitted α = 174.5 nN V −2 μm −2 , and γ = 0.027 nN V −2 at 125 kHz to the empirical data shown in Figure 5e . The model could capture the dynamics represented in the experimental data ( Supplementary Fig. S6 ). This analytical model has been used to design the prototypes presented in the rest of the article. Our results have shown that radiation forces between identical actuator modules do not change direction for a given spacing. Actuators with different orifice sizes displayed more complex interactions. We discovered that depending on the excitation frequency, actuators with the same initial distance attracted or repelled each other. An analogous phenomenon was observed between acoustically excited free-floating spherical bubbles with different radii. 38 , 58 Different bubbles oscillate with a different amplitude and phase for the same impinging pressure wave ( Fig. 5f ). The relative oscillation phase dictates the direction of the radiation force. When the bubbles oscillate in a relative phase of less than a quarter of a period, the force is attractive, but if the phase differs by more than a quarter but less than three-quarters of a period, the force is repulsive. Therefore, identical bubbles tend to attract each other, and nonidentical bubbles can either attract or repel each other, depending on the excitation frequency ( Fig. 5g ). In our experiments, actuator modules with cavity radii of 25 and 17.5 μm and orifice radii of 10 μm, attracted or repelled one another at 85 and 125 kHz, respectively ( Fig. 5h and Supplementary Movie S5 ). We designed a flextensional mechanism that leverages this frequency-dependent behavior to manifest multiple distinct deformation patterns on the same system ( Supplementary Fig. S7 and Supplementary Movie S6 ). Both couples of arms simultaneously closed at one frequency, and one couple opened while the other closed at another frequency. The frequencies at which the arms would open or close were determined by the relative phase of oscillations, which was modified by capsule geometry. Both operation modes were independent of the input signal amplitude, therefore, the angle between the arms could be tuned with the applied voltage. So far, we focused on a paradigm where actuator modules were patterned on different structures. In this arrangement, a rapid increase in radiation forces with decreasing distance limits the range of motion that the actuators can generate. To extend the interval at which the structure bends in a graded fashion, we constrained the actuators' motion. To this end, we connected two actuators with a truss so that they were not allowed to come very close to each other ( Fig. 6a ). As expected, the arms progressively bent out of plane for a large range of input voltage ( Supplementary Movie S7 ). To report the deformation, we recorded the displacement of the actuators along the y -axis ( Fig. 6b ). Although the arms bent under the dominant radiation forces, the displacement curve did not follow the highly nonlinear trend presented in Figure 5e . We built a FEM model of the mechanism based on the CAD design and the material properties ( Fig. 6c, d ). The radiation force magnitude and its direction are expected to change as the arms bend due to the relative position of the bubbles. FIG. 6. Mechanical programming of soft micromachines. (a) Out-of-plane deformation mechanism. Two pairs of identical actuator modules are printed on both arms. The displacement, δ , is used to measure the bending of the arms. (b) \n δ Increases monotonically with increasing voltage until a threshold at which the deformation ceases. The radiation forces generated by the entrapped bubbles are insufficient to bend the structure further. The actuator modules are constrained in their motion so that they do not snap with each other. (c) A CAD model and free body diagram of the out-of-plane deformation mechanism. (d) The deformed configuration of the out-of-plane mechanism as computed by the FEM model. (e) A linear microactuator constructed with a flexible spring mechanism. The top actuator module is fixed to the ground while the bottom one is free to move. The motion of the bottom actuator module is constrained in the y -axis by the spring mechanism. (f) The distance between the actuator modules gradually decreases with the input voltage, where beyond 20 μm increasing the voltage further abruptly closes the gap. Simulation results closely match the experimental data. (g) A 3D FEM model of the machine showing the stresses acting on the mechanism. The same model is used to calculate the stiffness of the spring. Scale bars, 50 μm. We simplified the model by assuming that the radiation force always acted to pull the actuators toward each other, and its magnitude was proportional to the thrust. We took the thrust calculated for a single actuator module ( Fig. 3 ) as input and estimated the acoustic radiation force for the applied voltage values by fitting the empirical data shown in Figure 6b . In this study, the acoustic radiation force was taken as F R = − β V 2 d − 2 following Equation (2), where β was estimated as 745.6 nN V −2 μm −2 (Supplementary Note S3 and Supplementary Fig. S11 ). In the experiments, at relatively high voltage values, further increment did not cause further deformation ( Fig. 6b ). The experimentally observed plateau may be due to the drag force applied to the moving actuators by the anchored ones. The out-of-axis bending is a classic example for unimorph actuators where we control the angular displacement. By simply connecting a microbubble pair with an ultraflexible spring mechanism, we developed a linear microactuator ( Fig. 6e ). Acoustic forces were primarily uniaxial and, as expected, we did not observe out-of-axis deflection during operation ( Supplementary Movie S8 ). Attractive radiation forces between the two microbubbles caused the gap to narrow. The deflection of the spring followed an almost linear trend with respect to applied voltage until the bubbles were 20 μm apart, at which point the radiation forces overcame the structural stiffness leading to a fast closure of the mechanism ( Fig. 6f ), similar to the pull-in instability observed in microelectromechanical systems devices. 60 When the excitation signal was turned off, the acoustic forces vanished, and the actuator returned to its initial position. We calculated the magnitude of forces that correspond to the input voltage using Equation (3) and applied these forces to an FEM model. The model captured the behavior of the experimentally recorded spring deflection, verifying that we have a reliable way of calculating forces generated by the acoustic actuator modules ( Fig. 6f, g ). We calculated the stiffness of the spring as 0.7563 nN/μm by computing the derivative of the displacement with respect to the total force ( Supplementary Fig. S8 ). Constrained elastic beams can exhibit complex mechanical responses depending on the geometry, degree of confinement, and boundary conditions. Previous work has shown that such mechanisms instantiate embodied logic and programmable functionality in soft machines. 19 , 33 , 61 , 62 We designed beams to present a snap-through instability so that application of a relatively small thrust would be sufficient to cross the energy barrier and trigger rapid and large deformation toward a second stable configuration. Key geometric parameters for the beam design are the inclination angle of the beam, θ , and the slenderness ratio, w ∕ L , where w and L denote the width and length of the beam, respectively ( Fig. 7a ). The mechanism was driven by a single actuator module that operated in acoustic streaming mode ( Fig. 7b ). The modules stayed indefinitely in both stable states, and the thrust generated by the actuator module was high enough to pass the energy barrier. FIG. 7. Mechanical reprogramming of soft micromachines through elastic instabilities. (a) The design of a bistable mechanism with a snap through. The inclination angle, θ , is modulated to construct mechanisms with varying energy barriers. (b) Representative microscope images showing the switching of a bistable mechanism from one stable state to another upon acoustic excitation. The mechanism stays indefinitely in both stable states unless it is excited to switch. (c) The acoustic pressure required to pass the energy barrier and switch the mechanism for prototypes with varying θ . (d) The energy landscape of bistable mechanisms with different θ . (e) Reversible actuation of bistable mechanisms. Two actuator modules with identical cavity sizes and distinct orifice sizes are connected on the same unit for frequency selective actuation in opposite directions. (f) A bistable mechanism was used as a control module. A third separate actuator module deformed a cantilever beam in the clockwise direction when the control module is kept at one stable state. Switching the control module to the left completely changes the force balance on the third actuator module. The radiation forces generated between the control module and the third actuator module reversed the motion and deformed the beam in the counterclockwise direction. Scale bars, 50 μm. For a fixed beam length and cross-sectional profile, as θ increases, the input pressure required to switch the mechanism state is expected to increase while the deformed position becomes more stable. 20 We fabricated three different prototypes that only differ in inclination angle ( θ = 30°, 45°, and 60°) to validate the theoretical predictions. We observed a monotonic increase in the pressure at which the beam managed to switch states ( Fig. 7c ). We used a 2D FEM model to obtain the double-well potential energy landscape for the same design parameters ( Fig. 7d ). The simulation results showed that the strain energy quadruples when θ is increased from 30° to 70°, and the module displacement doubles. We calculated the force required to switch the mechanism at different θ from the empirical data using Equation (3), where we only considered the thrust generated by a single actuator module (i.e., F B = 0 . 027 V 2 ). Comparing these values with the simulated force showed that the switching occurred at lower levels than predicted ( Supplementary Table S2 ). The rationale behind this discrepancy is that the actuator module did not move along a straight line as simulated, and, instead, moved in 3D by following a more favorable energy landscape. We harnessed frequency-selective thrust generation to realize reversible actuation for the bistable mechanism ( Fig. 7e ). We kept the bubble size constant and tuned the actuators' orifice size to be able to activate them at distinct frequencies, 85 and 170 kHz ( Fig. 2e ). Connecting two actuator modules with a single beam to the anchor point proved to be undesirable. To stabilize the structure and ensure reliable operation, we extended the mechanism by adding support structures. The resulting mechanism could be switched repeatedly at the same amplitude and excitation frequency ( Supplementary Movie S9 ). As a final demonstration, we programmed the motion of a continuously bending cantilever beam using an actuated bistable mechanism, which we refer to as the control module ( Fig. 7f -left). Here, triggering the control module changes the direction of bending by introducing radiation forces to a system otherwise driven solely by acoustic streaming ( Supplementary Movie S10 ). To trigger the controller and actuate the beam at different frequencies, we engineered capsules with different geometries. At 125 kHz, the beam bent clockwise while the control module stayed idle ( Fig. 7f -middle). Exciting the system at 85 kHz activated the bubble located on the left of the control module, moving the right one closer to the actuator connected to the beam. The control module stayed in this stable state when the system was turned off, as expected. Then, exciting the system again at 125 kHz bent the beam counterclockwise due to the attractive radiation forces generated between the two neighboring bubbles ( Fig. 7f -right). In this prototype, the control module must be reset manually because the radiation forces are stronger than the thrust. However, by tuning the bubbles' geometry, we can introduce a third frequency at which the control module would be reset."
} | 9,852 |
24466058 | PMC3899235 | pmc | 2,683 | {
"abstract": "The photosynthetic cyanobacterium, Synechocystis sp. strain 6803, is a potential platform for the production of various chemicals and biofuels. In this study, direct photosynthetic production of a biopolymer, polyhydroxyalkanoate (PHA), in genetically engineered Synechocystis sp. achieved as high as 14 wt%. This is the highest production reported in Synechocystis sp. under photoautotrophic cultivation conditions without the addition of a carbon source. The addition of acetate increased PHA accumulation to 41 wt%, and this value is comparable to the highest production obtained with cyanobacteria. Transcriptome analysis by RNA-seq coupled with real-time PCR was performed to understand the global changes in transcript levels of cells subjected to conditions suitable for photoautotrophic PHA biosynthesis. There was lower expression of most PHA synthesis-related genes in recombinant Synechocystis sp. with higher PHA accumulation suggesting that the concentration of these enzymes is not the limiting factor to achieving high PHA accumulation. In order to cope with the higher PHA production, cells may utilize enhanced photosynthesis to drive the product formation. Results from this study suggest that the total flux of carbon is the possible driving force for the biosynthesis of PHA and the polymerizing enzyme, PHA synthase, is not the only critical factor affecting PHA-synthesis. Knowledge of the regulation or control points of the biopolymer production pathways will facilitate the further use of cyanobacteria for biotechnological applications.",
"introduction": "Introduction Cyanobacteria are believed to be one of the oldest groups of photosynthetic organisms on Earth and played a significant role in the development of the oxygenic atmosphere we breathe today [1] . In modern day, cyanobacteria continue to play a pivotal role in global carbon recycling, the nitrogen cycle and most importantly, the maintenance of the composition of the atmosphere [2] , [3] . Cyanobacteria are considered to be ideal producers of various fine chemicals and biofuels because they fix carbon dioxide into biomass using solar energy. Fluctuations of nutrient concentrations constantly occur in natural environments and microorganisms respond to nutrient starvation by accumulating various carbon and energy storage compounds [4] . The study of these storage polymers, particularly polyhydroxyalkanoate (PHA), has gained considerable interest in recent years in an attempt to address the waste disposal problems caused by petrochemical plastics [5] . At present, the major biological processes utilized for industrial production of PHA are fermentations of heterotrophic bacteria. Nevertheless, the economic viability of PHA as a commodity polymer is limited by high production costs due to costly carbon substrates and requirements during the fermentation processes. Substantial effort has been devoted to investigating PHA production processes that are more cost-effective [6] . An interesting and promising approach is the use of photosynthetic cyanobacteria as the host for PHA production. The cyanobacteria, as ‘microbial factories’, can fix carbon dioxide from the atmosphere into high molecular weight PHA directly via photosynthesis. Besides being photoautotrophic, cyanobacteria require minimal nutrients for growth, eliminating the cost of carbon sources and complex growth media [7] . Thus, the application of cyanobacteria offers the potential of a cost-competitive and sustainable approach for the production of this environmentally friendly polymer. The presence of PHA in cyanobacteria was first described by Carr whom analyzed PHA in Chloroglea fritschii based on acid hydrolysis of poly(3-hydroxybutyrate), P(3HB), to crotonic acid followed by UV spectroscopic measurement of the hydrolysis product [8] . Since then, much research has demonstrated the presence of PHA in several other cyanobacteria including Aphanothece sp. [9] , Oscillatoria limosa \n [10] , some species of the genus Spirulina \n [11] , [12] and the thermophilic strain Synechococcus sp. MA19 [13] . So far, cyanobacteria are characterized by their ability to produce PHA containing only 3-hydroxybutyrate (3HB) and/or 3-hydroxyvalerate (3HV) monomers [9] , [10] , [14] . Although there are many reports on the occurrence of PHA in cyanobacteria, most of these studies explored the physiology and fermentation aspects of PHA accumulation in cyanobacteria. The biochemical and molecular basis of PHA synthesis in cyanobacteria are not well understood. The model cyanobacterium Synechocystis sp. strain PCC 6803 is considered as a promising candidate for various biotechnological productions because of the availability of its genome sequence information [15] and the ease of genetic manipulation of this strain due to its naturally transformable feature [16] . In this study, Synechocystis sp. was metabolically engineered by increasing the flux of intermediates to PHA biosynthesis and introducing a PHA synthase with higher activity. RNA-seq analysis was carried out to examine the differential expression involved in the global biological processes and metabolic pathways during the improved photoautotrophic production of PHA. This information will facilitate the potential use of cyanobacteria for the sustainable production of this ‘green’ polymer.",
"discussion": "Discussion Current limitation of direct photosynthetic production using cyanobacteria is the relatively low PHA content obtained. In this study, it was encouraging to obtain 14 wt% of P(3HB) from direct photosynthetic fixing of carbon dioxide without the addition of an external carbon source. Although cyanobacteria have simple nutrient requirements, the addition of 0.4%(w/v) acetate was found to increase P(3HB) content up to 41 wt% under air-exchange limiting conditions. Previous studies suggested that enhanced P(3HB) accumulation was the result of direct metabolism of acetate for PHA synthesis by employing an existing pathway operating in cyanobacteria [7] , [21] . The provision of exogenous carbon was found to have a positive impact on PHA accumulation albeit at concentrations that were 10- to 20-fold lower than those required by heterotrophic bacteria. Recently, the development of new photobioreactors for mass cultivation of cyanobacteria is in progress and these findings will greatly aid the use of cyanobacteria for potential industrial applications [29] , [30] . Early studies indicate that the PHA biosynthetic genes of Synechocystis sp. 6803 do not co-localise together to form an operon [31] , [32] . Instead, the PHA synthase of Synechocystis sp. consisting of phaC and phaE subunits are linked in the genome and co-expressed. On the other hand, the β-ketothiolase and acetoacetyl-CoA reductase of Synechocystis sp. do not map close to the PHA synthase locus but are probably clustered together and constitute an operon in a different section of the genome. The expression levels of these two genes were surprisingly lower in the recombinant Synechocystis sp. strains C Cs A Cn B Cn and C Cs NphT7B Cn that had higher PHA production potential compared to strain pTKP2013V that accumulated a lower content of PHA. These results suggest that the endogenous PHA biosynthetic pathway operating in Synechocystis sp. did not have a significant impact on the PHA-synthesizing abilities of strains C Cs A Cn B Cn and C Cs NphT7B Cn . The Chromobacterium sp. PHA synthase and C . necator acetoacetyl-CoA reductase that were introduced into the genome as an operon showed similar lower expression in strain C Cs NphT7B Cn . The observation that the expression levels of most of the PHA biosynthetic genes were lower in strain C Cs NphT7B Cn suggests that the concentration of these enzymes is not the limiting factor in achieving higher PHA accumulation. Based on the results presented here, the transcription of genes encoding enzymes involved in PHA biosynthesis is highly regulated and may be affected by the PHA content in the cells ( Fig. 3 ). When the PHA accumulated by the cells has exceeded a certain threshold level, adequate levels of the enzymes may already be present to meet the biosynthetic demand. Thus, the PHA granule itself or some other sensing factors may exert negative feedback on the expression of these enzymes. However, the expression levels of the enzyme catalyzing the last step of PHA biosynthesis, Synechocystis sp. PHA synthase, remained grossly constant in all recombinant Synechocystis sp. because negative feedback regulations are likely exerted in the upper part of the pathway. 10.1371/journal.pone.0086368.g003 Figure 3 The scheme shows the regulation of PHA synthesis-related gene expression in recombinant Synechocystis sp. Previous genetic studies have focused on the engineering of various bacteria or plant hosts for PHA production, but less is known about the global transcriptional changes of the recombinant host under a PHA-synthesizing environment. A comprehensive view of the cyanobacterial transcriptome during cultivation under conditions favorable for PHA synthesis was generated using RNA-seq analysis. One particularly interesting observation is the up-regulation of photosynthetic activity in recombinants Synechocystis sp. with higher PHA-synthesizing potential ( Fig. 4 A and B). In recent years, there has been tremendous interest in strategies to improve photosynthetic activity in crops [33] , [34] . It has been suggested that an increase in photosynthetic activity will improve the yield of crops and provide a potential solution to future food shortages [35] . In this context, the increase of photosynthetic activity in cyanobacteria may explain the higher PHA accumulation observed in recombinant Synechocystis sp. strains C Cs NphT7B Cn and C Cs A Cn B Cn . 10.1371/journal.pone.0086368.g004 Figure 4 The scheme shows the cellular changes in recombinant Synechocystis sp. strains (a) C Cs A Cn B Cn and C Cs NphT7B Cn (compared with pTKP2031V) (b) C Cs NphT7B Cn (compared with C Cs A Cn B Cn ) under photoautotrophic PHA biosynthesis conditions. Only a selection of cellular changes is shown. The genes or pathways that are up-regulated are in red; the downregulated ones are in green. Black dashed lines indicate the engineered route. AP, allophycocyanin; PC/PEC, phycocyanin/phycoerythrocyanin; Cytb 6 /f, cytochrome b6/f complex; PQ, plastoquinone; FNR, ferredoxin-NADP(+) reductase; Pc, plastocyanin; PSI, photosystem I; PSII, photosystem II; Ndh, NADH dehydrogenase; Glc-6-P, glucose-6-phosphate; Fru-6-P, fructose-6-phosphate; Fru-1,6-bp; fructose-1,6-biphosphate; Glycerate-1,3-P 2 , 1,3-biphosphoglycerate; 3-P-Glycerate, 3-phosphoglycerate; Ru-1,5-bisP, ribulose-1,5-biphosphate; PEP, phosphoenolpyruvate. The gene encoding one of the most important enzymes in carbon fixation, the ribulose-1,5 biphosphate carboxylase/oxygenase (RuBisCo) large subunit ( rbcL ), was up-regulated in both C Cs NphT7B Cn and C Cs A Cn B Cn . RuBisCo is a biologically important enzyme that catalyzes the first step of the reaction that converts atmospheric carbon dioxide into organic carbon [36] . Besides RuBisCo, genes encoding proteins involved in different aspects of photosynthesis and electron transport chain were significantly induced in both C Cs NphT7B Cn and C Cs A Cn B Cn. In particular, the induction of photosynthesis and electron transport chain-related genes was most prominent in strain C Cs NphT7B Cn with the highest PHA accumulation, suggesting the possible correlation of photosynthetic activity with PHA content. The Synechocystis sp. cells may utilize enhanced photosynthesis, carbon fixation and electron transport chain activities as a means to provide precursors that are necessary to drive the production of PHA. The increased photosynthetic production of PHA reveals that similar metabolic engineering approaches can be applied to the production of biofuels or chemicals using this versatile organism. As cyanobacteria and plants share similar photosynthetic machinery, it is likely that the strategy can be extended in future efforts to improve PHA production in higher plants. In living cells, catabolic reactions that produce energy and anabolic biosynthetic reactions are regulated to maintain a balance of supply and demand. To cope with the higher PHA production demand, carbon dioxide fixing was enhanced to replenish the pool of carbon that was lost to PHA formation. Concomitant with the increase in photosynthetic activity, the flow of newly fixed carbon dioxide into biosynthetic reactions other than PHA was reduced. Genes encoding metabolism of cofactors and vitamins as well as protein metabolic process were found to be down-regulated in strains C Cs NphT7B Cn and C Cs A Cn B Cn. The reduced growth of recombinant Synechocystis sp. under nutrient-deficient cultivation conditions may account for the depression of these metabolic processes. These cellular anabolic reactions were regulated to maintain the balance of resources in cells. The expression levels of genes involved in the tricarboxylic acid cycle (TCA) were shown to be down-regulated in strains C Cs NphT7B Cn and C Cs A Cn B Cn . These observations agree well with previous finding that reported on the repressed of the TCA cycle genes in C . necator H16 during PHA production [37] . RNA-seq transcriptome analysis reveals that the heterologous expression of PHA synthesis-related genes in Synechocystis sp. affect not only the regulation of PHA biosynthesis but also the preceding pathways that are involved in the provision of precursors for this biosynthesis. The direct photosynthetic production of 14 wt% of P(3HB) from strain C Cs NphT7B Cn is the highest value achieved for Synechocystis sp. 6803 so far. This work suggests the use of carbon flux as a possible driving force for the biosynthesis of intracellular inclusions e.g. PHA. Future work can be done to confirm this finding by enhancing carbon fixation in cyanobacteria through engineering or overexpressing the enzymes involved in the process."
} | 3,529 |
39811608 | PMC11732554 | pmc | 2,684 | {
"abstract": "Protein-based biomaterials are in high demand due to their high biocompatibility, non-toxicity, and biodegradability. In this study, we explore the bacterial E. coli secreted protein A (EspA), which self-assembles into long extracellular filaments, as a potential building block for new protein-based biomaterials. We investigated the morphological and mechanical properties of EspA filaments and how protein engineering can modify them. Our study include three types of filaments: natural EspA filaments, full-length recombinant EspA filaments, and truncated recombinant EspA filaments lacking a third of the original codon region. The recombinant EspA proteins formed curly, thin filaments with higher longitudinal elasticity (shorter persistence length) compared to the natural, linear filaments. Additionally, the recombinant filaments had a radial elastic modulus about an order of magnitude lower than the natural filaments. The truncated recombinant filaments had a higher radial modulus than the full-length ones, and unlike the purely elastic natural filaments, recombinant filaments were less compliant with the applied force that penetrated them. These differences underscore the potential to modulate EspA filament properties through protein sequence mutations. Our findings suggest EspA as a fundamental element for developing a new biomaterial with a hierarchical structure, enabling the fabrication of macroscopic substances from self-assembled EspA-modulated filaments.",
"conclusion": "4 Conclusions In this study, we examined the morphological and mechanical properties of EspA filaments by AFM image height analysis and nanoindentation. We compared three types of filaments: natural T3SS EspA filaments that were severed from live bacteria, self-assembled recombinant full-length filaments, and truncated EspA filaments. We observed that the two recombinant EspA proteins spontaneously assembled to form elongated spiral thin filamentous structures, which are in sharp contrast to the natural arrangement of the linear thick EspA filament of the T3SS complex. Specifically, while the two recombinant EspA filaments share a similar overall structure, their mechanical characteristics exhibit significant disparities. The longitudinal elasticity of the NF filaments, reflected by their persistence length distributions, is considerably higher than those of the R and RT filaments, by about an order of magnitude, which means that it is substantially stiffer. The RT filaments are stiffer by 2–3 times than the R filaments. The R filaments measured a range of elastic radial moduli close to those measured in the NF filaments, and are about an order of magnitude lower compared to the radial elastic moduli of the RT filaments. The elastic radial modulus offers localized insights into the helical structure of the filaments, while the longitudinal elasticity reflects the overall consistency of the EspA filament material. This suggests that the truncation of 58 residues from the N-terminal of EspA maintained some structural elements while somewhat increasing the interaction within the RT filaments (in accord with its stiffening). Additionally, the recombinant filaments displayed rupture events that were not observed in the natural filaments. The differences we report here between the three types of filaments showcase the capacity to fine-tune and improve the mechanical properties of the filaments through protein engineering. The observed relationship between specific protein alterations and resulting structural-mechanical changes suggests the potential for rational design in protein engineering. This capacity opens new possibilities for tailoring a new brand of material made from structured protein filaments, with EspA as its building block that will exhibit a three-fold hierarchical response, spanning from the single EspA protein through the assembled filament and up to a potential fabricated matrix. However, the precise mechanisms governing these structure-function relationships require further investigation to fully elucidate the principles that could guide targeted modifications of protein assembly and mechanical properties. Accordingly, numerical simulations can help decouple the effects of helical structure and protein assembly mechanics. Experimental characterizations as presented in this study are influenced by material geometry and modulus, reflecting the protein assembly. Coarse-grained models effectively bridge atomistic behavior and experimental techniques like AFM, enabling exploration of structural and mechanical properties at relevant scales [ 50 ]. Such multiscale approach offers valuable insights into the hierarchical structure and mechanical behavior of EspA filaments. Lastly, it will be interesting to investigate how filaments with altered mechanical properties influence the infection capability and translocation activity of the pathogenic strain in future studies.",
"introduction": "1 Introduction Protein-based biomaterials represent a rapidly growing field that integrates principles from physics, engineering, biology, and chemistry to create innovative solutions to biomedical and biotechnological challenges [ [1] , [2] , [3] , [4] , [5] , [6] , [7] , [8] ]. These protein-based biomaterials are superior to synthetic materials as they are biocompatible, offer broad versatility of biochemical and biophysical properties and structural diversity, can mimic tissue behavior, are biodegradable under physiological conditions, and are more sustainable for the environment [ 9 , 10 ]. As such, these materials can be employed for various biomedical applications (drug delivery, medical devices, sensors, tissue engineering, wound healing, etc.). Over the last two decades, a class of proteins known to spontaneously self-assemble to form large structures has been integrated into various biomaterials [ 8 , [11] , [12] , [13] , [14] ]. This class of proteins gained significant attention due to their ability to form hierarchical self-assemblies, ranging from nano- to meso-scale. The self-assembled proteins usually oligomerize to form a sizeable multi-subunit macromolecule upon environmental triggers (pH, temperature, light, etc.) and can be shaped into parallel sheets, fibrous structures, net-like structures, twisted helices, cyclic structures, and amorphous aggregates. In the context of these materials, we investigate the properties of E. coli secreted protein A (EspA) that exhibits spontaneous self-assembly, resulting in the formation of long elongated filamentous structures. EspA, in its native function, forms a long hollow tubular filament through which it transports virulence proteins during infection. The filament is part of a large protein complex called type 3 secretion system (T3SS). The T3SS is a “syringe-like” structure, where the EspA filament constitutes its “needle” part that forms a physical bridge between the bacterium and the host cell. Apart from E. coli , T3SSs are found in many harmful bacterial pathogens, such as Salmonella , Yersinia , and Shigella [ [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] ]. The inherent self-assembly capability of EspA indicates its potential applicability for biomaterial development. The employment of EspA as a potential building block of a new biomaterial has several significant advantages, originating from it being a bacterial protein that is naturally secreted to the extracellular environment. Thus, its manipulation, expression, and purification are relatively simple, and it can be produced on large scales. Additionally, considering its biological role, it is expected to induce a low immune response. In this study, we use atomic force microscopy (AFM) to study the morphological and mechanical properties of the EspA filaments formed by the recombinant full-length protein (termed recombinant - R) and compared them to the natural EspA filaments (termed natural filament - NF) obtained from T3SS-expressing live bacteria [ 23 ]. To explore the possibility of producing filaments with altered properties, we created three truncated designs of the recombinant EspA protein and examined their ability to preserve the filamentous structure. We found that only EspA deleted for residues 1–58 (EspA 59-192 ) formed long filaments similar to the recombinant full-length EspA, while the EspA designs deleted for the C-terminus of the protein, EspA 1-59 and EspA 1-148 , lost this ability. Moreover, we found that while the NF obtained a straight “antenna-like” structure, the recombinant filaments displayed a spiral shape. Our measurements revealed a significant increase in the longitudinal elasticity of the recombinant filaments compared to the natural filaments, translating to two orders of magnitude decrease in persistence length. Interestingly, the truncated recombinant filaments exhibit higher radial elastic modulus than their full-length counterparts. These findings pave the way for using filaments formed by EspA or its modified versions as building blocks for innovative biomaterials.",
"discussion": "3 Results and discussion 3.1 Design of the recombinant and truncated recombinant filaments The native T3SS EspA filament consists of recurring units characterized by a central core of alpha helices enveloped by folded domains, forming a structure reminiscent of a bouquet (PDB code 7K7K [ 26 ]). This arrangement results in a hollow core with a polar surface that facilitates the passage of the unfolded or partially folded T3SS proteins. Although it is established that EspA undergoes self-oligomerization to generate long filaments, the specific protein region responsible for this organization remains unidentified. To investigate the capability of a truncated EspA protein to form filaments, we generated three designs of truncated EspA, based on its structure with its chaperone, CesA [ 16 ]; encoding residues 1–59, 1–148, and 59–192 ( Fig. 1 ). The full-length and truncated EspA proteins were expressed, purified, and examined for their ability to create large complexes using size exclusion chromatography (SEC). We observed that full-length EspA eluted at an exclusion volume of 8–9 mL, corresponding to very large molecular weight complexes ( Fig. 1 ), while the truncated EspA 1-59 and EspA 1-148 , which lack the C-terminus of the EspA protein, eluted at an exclusion volume of 14–15 mL, corresponding to complexes with much smaller size of around 75–158 kDa ( Fig. 1 ). Unexpectedly, EspA 59-192 , which lacks the 58 N-terminal residues of the EspA sequence, was eluted at an exclusion volume of 8–9 mL, similar to the full-length protein ( Fig. 1 ). Fig. 1 Self-assembly of EspA full-length and truncated designs. a. Schemes of EspA full-length, EspA 1-59 , EspA 1-148 , and EspA 59-192 are presented. b. Full-length and truncated EspA-His proteins were purified, subjected to SEC (Superdex 200 10/300 GL), and monitored at a wavelength of 280 nm (absorbance in arbitrary units - AU) to follow protein elution. SEC analyses were performed by monitoring UV as a function of the eluted volume. Markers at the top of the SEC profile indicate the positions of the standards: Ferritin (440 kDa); Aldolase (158 kDa); Conalbumin (75 kDa); Carbonic Anhydrase (29 kDa); and Ribonuclease (13.7 kDa). Fig. 1 3.2 Filaments morphology To determine whether there is a correlation between the SEC results and the proteins' ability to create fibrous structures, we imaged the purified EspA proteins using AFM. While filamentous structures were visualized for the EspA 59-192 truncated protein (hereafter referred to as recombinant truncated or RT), we could not detect similar structures for the purified EspA 1-59 (data not shown) and EspA 1-148 proteins ( Fig. 2 ). These results suggested that the C-terminus of EspA is crucial for forming large filamentous structures, while the N-terminus, comprising about one-third of the coding region, is dispensable for oligomerization and filament formation. Moreover, this indicates that the strong, inherent tendency of EspA to oligomerize and create long filaments is driven by specific domains/motifs along the protein sequence. This enables modifications to the EspA protein while preserving its filament-forming ability. Furthermore, the correlation we observed between the SEC results and the formation of filamentous fibers implies that SEC can serve as an effective method to determine which recombinant EspA designs (truncated or mutants) are prone to form filaments. Fig. 2 AFM images of purified recombinant EspA 1-148 and EspA 59-192 (RT) proteins. Fig. 2 To assess the filamentous structure of the recombinant EspA proteins, we purified EspA natural filaments (NF, black) by detaching them from bacterial surfaces and visualize them along with purified recombinant full-length EspA (R, light blue) and truncated EspA (RT, purple), using AFM ( Fig. 3 - in two magnifications). Despite identical protein composition, the images demonstrated a striking morphological difference between the natural filaments (NF) and the recombinant filaments (R and RT filaments). The natural filaments ( Fig. 3 a) display their well-characterized “antenna-like” straight formation, which is well documented in the literature [ 15 , 16 , 22 , 23 ]. Contrary, the R ( Fig. 3 b) and RT ( Fig. 3 c) filaments display curly features, where their general contour is twisted around their central axis, and they can form curved (and circular) filamentous structures. Fig. 3 AFM images and height analysis of EspA filaments. a. EspA natural filaments (NF). b. Recombinant (R) EspA self-assembled filaments. c. Recombinant truncated (RT) EspA self-assembled filaments. The upper and middle panels show filament images on large and small scales, respectively. The lower panels show the corresponding probability densities of the filaments' heights. Fig. 3 To provide an explanation of the curvy nature of the RT form, we removed the 58 N-terminal residues from each EspA subunit within the published filament structure. This resulted in noticeable gaps between the monomers, primarily visible on the external surface, that might grant the monomers increased flexibility relative to one another ( Fig. 4 ). While this is a simplified interpretation of the structural data, which assumes that truncated EspA filaments form a similar oligomerized filamentous structure as the native protein, it does not reflect the final folded structure of the filament formed by the truncated protein. Yet, it could potentially provide an explanation for the distinct characteristics of the truncated filament compared to the natural filament. Fig. 4 Structure and electrostatic surface representations of full-length (upper panel - PDB code 7K7K ) and truncated (lower panel) EspA filament. Each color represents a different EspA subunit. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 4 However, since the recombinant EspA has a similar sequence as the EspA of the natural “antenna-like” filaments, an enhanced flexibility between the monomers cannot explain the curvy filaments observed for the recombinant EspA protein, which were also previously reported to oligomerize and form filamentous curvature structures spontaneously [ 16 ]. Therefore, we propose that the natural filaments removed from live bacteria probably contain additional T3SS proteins, such as the inner rod, or other biological components that support the formation of straight helical filaments. The probability density functions (PDFs) of the filament's heights are shown in the lower panels of Fig. 3 . The data was evaluated from 114 NF filaments, 55 R filaments, and 63 RT filaments. The NF population displayed a relatively narrow distributed single peak with a median of h NF = 11.52 nm, with interquartile range (IQR) varying between 11.06 and 11.93 nm ( Fig. 3 a lower histogram, black). The h NF value aligns with the reported outer diameter of the T3SS EspA filament [ 15 , 22 , 23 ]. The two recombinant filaments showed bi-modal distributions, which are associated with the curvature twists along their curly contour. The characteristic high and low height values (medians) of the R population are h R, \n high = 9.20 nm (IQR: 8.47–10.12 nm) and h R, \n low = 5.21 nm (IQR: 4.64–5.72 nm), and for the RT populations h RT, \n high = 9.61 nm (IQR: 8.78–10.48 nm) and h R, \n low = 3.60 nm (IQR: 3.19–3.90 nm). We hypothesize that the lower height values (∼5.2 nm in R and ∼3.6 nm in RT) represent the thickness of the self-assembled filaments while the elevated values (around ∼9.5 nm in R and 9.6 nm in RT) probe the height of the upper part of the loop in the twisted filament configuration (the curled segment that is suspended above the surface). In other words, unlike typical tubular structures, the R and RT filaments exhibit alternating high and low heights, indicating an open helical ribbon formation. The lower height measurement corresponds to the ribbon's thickness. Interestingly, the diameters of the truncated filaments are smaller by more than half compared to the external diameter of the NF EspA filaments in their natural T3SS configuration. We speculate that the hollow tubular structure, shown to exist in the natural EspA structure [ 22 ], is considerably reduced in the R filaments and possibly does not exist in the RT filaments, both having more compressed structures. This means that the R and RT filaments may assume a cylindrical micellar formation, where the inner tunnel of the NF is not fully preserved. Alternatively, it is possible that the natural filaments that were removed from live bacteria were filled with T3SS substrates that extrude the inner core and extend the filaments' size. 3.3 Mechanical properties of the filament 3.3.1 Filament contour and persistence lengths We used FentoScan software to assess the contour and persistence lengths of the filaments from the AFM images. Although these two characteristics are given in terms of length, the contour length represents the overall end-to-end length of the filament, and the persistence length describes mechanical compliance (will be discussed in the next section). The NF filament population had a median length of L NF = 338 nm (IQR: 260–499 nm). The length distribution of the NF populations, shown on the upper panel in Fig. 5 , which ranges from 104 nm short filaments up to 1,097 nm long filaments, closely resembles the length distribution reported for EspA filaments that were pulled, which still attached to the bacterium surface [ 23 ]. Fig. 5 AFM images with length and persistence length analysis of EspA filaments. Each row shows an exemplary AFM image of a specific filament population (NF in the first row, R in the second row, and RT in the third row, with scale bars in each image). To the right of each filament population is its probability densities of the corresponding (contour) length and persistence length calculated from all the measured images. Fig. 5 The median lengths of the recombinant filaments display shorter lengths compared to the NF population, with L R = 103 (IQR: 69–183 nm) and L RT = 142 nm (IQR: 100–225 nm). This indicates that the spontaneous arrangement of the recombinant EspA proteins creates structures with shorter lengths (as shown on the two lower middle panels in Fig. 5 ) relative to the natural EspA filaments. Considering the spiral configuration of the recombinant filaments, it is possible that applying pulling force could elongate them, potentially aligning them with the length of the natural filaments. The persistence length provides a measure to characterize how flexible or stiff a tiny thread-like molecule or filament is. It describes how well the average direction of the molecule aligns with its overall end-to-end (contour) length. The median persistence length of the NF population was found to be l p,NF = 2,632 nm (IQR: 1,836–3,888 nm), showing a striking resemblance to the values reported for T3SS EspA filament of l p ∼2,400 nm, which was measured using force spectroscopy, a completely different experimental technique [ 23 ]. Even more remarkable are the measured persistence length values of the recombinant filaments, with median values of l p,R = 18.2 nm (IQR: 12.9–23.1 nm) and l p,RT = 51.8 nm (IQR: 17.3–135.4 nm). These values are two orders of magnitude smaller than that of the natural filaments. These values emerge from the spiral, curved shape of the filaments, which alludes to a considerably higher longitudinal elasticity than the straight and rigid natural filaments. Comparing the persistence length distributions among the recombinant filament populations revealed that while both distributions are of the same order of magnitude, the RT population is about twice stiffer than the R population (two lower right panels in Fig. 5 ). This signifies how the truncation of the EspA protein affects the mechanical properties of the filament as a whole. The RT filaments exhibit a similar morphology as the R filaments, but an alteration in their building blocks enhances the longitudinal stiffness of the assembled super-structure. 3.3.2 Nanoindentation and rupture force To further characterize the mechanical properties of the three filament populations, we performed AFM-based force-spectroscopy nanoindentation measurements. These measurements can provide valuable mechanical information through the interactions of a sharp AFM tip with the filaments, as illustrated in Fig. 6 . AFM-based nanoindentation measurements have proven to be highly efficient and provide high-resolution data on many nanoscale biological systems [ [27] , [28] , [29] ], such as on the mechanical properties and stability of single cells [ [30] , [31] , [32] , [33] , [34] ], viruses [ [35] , [36] , [37] , [38] ], thin filaments [ 23 , [39] , [40] , [41] , [42] ], etc. Fig. 6 Illustrations of nanoindentation measurements of exemplary FD traces, with forward traces shown in red and backward traces in blue. a. Elastic deformation (observed mainly in the NF population). b. Rupture events following the elastic deformation (observed only on R and RT filaments). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 6 Two types of force-distance (FD) curves were recorded: the red FD curve of the approaching/forward motion of the cantilever tip and the blue FD of the retraction/backward motion. The NF population displayed simple elastic deformation, where the approach and the retract curves almost entirely overlap ( Fig. 6 a). The elastic compression (expansion in the retract FD trace) regime spans over a separation distance, Δ x E , that spans from x contact , the initial point where the tip engages in contact with the sample, up to a point where the extent of the compression begins to grow more rapidly (the force propagation rate increases rapidly). This region is used for the evaluation of the elasticity of the filaments in their radial direction, characterized by its local radial elastic modulus, E R . The R and RT populations displayed more complex behavior that involved rupture events at some force F after the initial elastic compression ( Fig. 6 b). These rupture events are manifested as a discontinuity in the increase of the force, which is followed by a drop in the force (perforation). These rupture events signify the entry of the cantilever tip into the filaments, with the force peak representing the force needed to surpass the interaction between two adjacent EspA proteins within the filament or potentially the rupture of one of the EspA subunits within the filament. After the elastic response and the rupture events, the tip presses against the surface, where the force increases with no change in the indentation depth. When a rupture event occurs, a hysteresis (area colored in gray) is observed between the forward and backward traches. This hysteresis signifies the plastic deformation of the filament and represents the dissipated work during the recovery process, denoted as W diss . 3.3.3 Elastic modulus Nanoindentation of soft, thin materials on stiffer surfaces poses not only an experimental challenge but also a challenge in terms of its interpretation with a suitable model [ 43 , 44 ]. When the deformation of the AFM probe is much smaller than the thickness of the soft material sample, information on its elasticity can be extracted from FD curves by fitting them to the Hertz model [ 45 ] with the appropriate geometrical correction for a probe with a conical geometry [ 27 ]. Here, these standard models do not accurately capture the thin filament properties that lay on an underlying hard surface, which can significantly affect the calculated elastic modulus. To account for this effect, we used a model that expands the Hertz model with a correction for the finite thickness of the filaments [ 46 ]: (1) F = 8 3 π tan ( α ) E R δ 2 [ 1 + 0.721 χ + 0.650 χ 2 + 0.491 χ 3 + 0.225 χ 4 ] where E R is the elastic modulus at the contact, δ = x – x contact is the separation distance coordinate. The compression factor is given by χ = [Δ x E /Δ x ]tan( α ), in which Δ x is the indentation depth, or sample thickness, taken as the distance between x contact and the final point at the trace, and α is the half-opening angle of the indenter, taken here as 18 ° . This model was fitted to the elastic region of the compression across Δ x E . Validation of this model across indentation depths (40 % to <10 % and below) displays an inverse relationship between sample thickness and bottom effects [ 46 ]. This methodology proved valuable for analyzing diverse thin biological specimens [ 23 , 33 , [47] , [48] , [49] ]. Equation (1) was fitted to the following number of distinct data points in the middle of the filaments: N NF = 74, N R = 102, and N RT = 179. The traces that were selected displayed up to 40 % indentation. Fig. 7 shows the radial elastic moduli for all three filament populations. The PDFs of the radial elasticity of all filament types showed a long tail distribution, with characteristic values of E NF = 0.163 MPa (IQR: 0.080–0.357 MPa), E R = 0.277 MPa (IQR: 0.164–0.400 MPa), and E RT = 2.082 MPa (IQR: 1.211–3.094 MPa). While the elastic moduli of the R filaments distribute in a similar fashion to NF, the RT filaments are stiffer by an order of magnitude. Fig. 7 Elastic moduli PDFs of a. NF, b. R, and c. RT filaments were obtained by fitting Eq. (1) to the nanoindentation FD curves. d. Box and whisker representation of the fitted radial elastic moduli for NF (black empty circles), R (light blue empty squares), and RT (purple empty triangles) filaments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 7 Given the similar elastic moduli we found for the natural filament and the recombinant filament, we focused on determining the properties of the self-assembled R and RT filaments. These filament populations displayed rupture force events, which influenced the overall force response, resulting in hysteresis ( Fig. 6 b). The rupture force ( Fig. 8 a) showed higher median values for the RT filaments with F R = 151 pN (IQR: 98–272) compared to F RT = 199 pN (IQR: 150–330). Yet, considering the distribution range of the rupture forces of R and RT, this difference may not necessarily indicate that the RT filament is more resilient than the R filament. Fig. 8 Box and whisker representation of the rupture force (a) and dissipated energy (b) of the recombinant (light blue squares) and recombinant truncated (purple triangles) filaments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 8 When a rupture occurs, the retraction curve exhibits the following behavior: An initial rapid reduction in the force occurs, with no change in the indentation depth. This means that the tip and the filament are still in contact. This is followed by a moderate lowering of the force across the deformation length of the filament until it coincides, in most cases, with the early (elastic) indentation stage of the approach curve. The region between zero displacement and the point where the approach and retract curves overlap (before the contact point) is where the filament expands and gently applies force on the deflecting cantilever tip during retraction. It's important to highlight that this recovery doesn't always signify the healing of the rupture or damage caused to the filament by the tip. This hysteresis area can provide an estimation of the dissipated work during the rupture and recovery of the filament, W diss = ∫( F approach – F retract ) dx between x contact and Δ x . Comparing the calculated W diss of R and RT ( Fig. 8 b) shows no distinct difference with W diss, \n R = 315 kT (IQR: 202–514 kT), and W diss, \n RT = 308 kT (IQR: 198–449 kT). Taken together, although the elastic moduli of the RT filaments were considerably larger than the R filaments, we did not detect any substantial differences in their recovery force patterns – not in terms of their ability to withstand local application of force and not in their dissipative behavior."
} | 7,350 |
22313382 | null | s2 | 2,685 | {
"abstract": "The aim of the study was to determine the extent and mechanism of influence on silica condensation that is presented by a range of known silicifying recombinant chimeras (R5: SSKKSGSYSGSKGSKRRIL; A1: SGSKGSKRRIL; and Si4-1: MSPHPHPRHHHT and repeats thereof) attached at the N-terminus end of a 15-mer repeat of the 32 amino acid consensus sequence of the major ampullate dragline Spindroin 1 (Masp1) Nephila clavipes spider silk sequence ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](15)X). The influence of the silk/chimera ratio was explored through the adjustment of the type and number of silicifying domains (denoted X above), and the results were compared with their non-chimeric counterparts and the silk from Bombyx mori. The effect of pH (3-9) on reactivity was also explored. Optimum conditions for rate and control of silica deposition were determined, and the solution properties of the silks were explored to determine their mode(s) of action. For the silica-silk-chimera materials formed there is a relationship between the solution properties of the chimeric proteins (ability to carry charge), the pH of reaction, and the solid state materials that are generated. The region of colloidal instability correlates with the pH range observed for morphological control and coincides with the pH range for the highest silica condensation rates. With this information it should be possible to predict how chimeric or chemically modified proteins will affect structure and morphology of materials produced under controlled conditions and extend the range of composite materials for a wide spectrum of uses in the biomedical and technology fields."
} | 411 |
33203914 | PMC7672225 | pmc | 2,687 | {
"abstract": "Corals are dependent upon lipids as energy reserves to mount a metabolic response to biotic and abiotic challenges. This study profiled lipids, fatty acids, and microbial communities of healthy and white syndrome (WS) diseased colonies of Acropora hyacinthus sampled from reefs in Western Australia, the Great Barrier Reef, and Palmyra Atoll. Total lipid levels varied significantly among locations, though a consistent stepwise decrease from healthy tissues from healthy colonies (HH) to healthy tissue on WS-diseased colonies (HD; i.e. preceding the lesion boundary) to diseased tissue on diseased colonies (DD; i.e. lesion front) was observed, demonstrating a reduction in energy reserves. Lipids in HH tissues were comprised of high energy lipid classes, while HD and DD tissues contained greater proportions of structural lipids. Bacterial profiling through 16S rRNA gene sequencing and histology showed no bacterial taxa linked to WS causation. However, the relative abundance of Rhodobacteraceae-affiliated sequences increased in DD tissues, suggesting opportunistic proliferation of these taxa. While the cause of WS remains inconclusive, this study demonstrates that the lipid profiles of HD tissues was more similar to DD tissues than to HH tissues, reflecting a colony-wide systemic effect and provides insight into the metabolic immune response of WS-infected Indo-Pacific corals.",
"introduction": "Introduction Coral reefs globally are under pressure from both local anthropogenic impacts and global climate factors 1 , 2 . These cumulative stressors are linked with increasing disease outbreaks that contribute to coral cover decline 3 – 7 . White syndromes (WSs) are a macroscopic grouping of prevalent coral diseases based on gross lesion characteristics that are reported across the Indo-Pacific, particularly affecting the dominant reef-forming family Acroporidae 8 – 11 . Visually, WSs manifest as a distinct lesion forming between affected and unaffected tissues. While coral tissue ahead of the lesion appears healthy, tissue at the lesion boundary is necrotic and actively sloughing away, revealing the bare white skeleton beneath 8 , 12 , 13 . These lesions can progressively migrate across a coral colony, resulting in either partial or whole colony mortality 8 , 14 . The underlying factors leading to the onset of WS disease lesions are unknown, although it is likely that multiple modes of pathogenesis manifest similarly as slow or rapid tissue loss in corals. Hence, like many coral diseases, a number of biotic and abiotic factors are linked with WSs 8 , 10 , 15 . For example, complex synergistic effects between environmental and host factors contribute to disease onset, with outbreaks often correlated with warm seawater anomalies and high coral density 16 – 18 . In addition, biological agents including vibrios 19 , ciliates 20 , 21 , viruses 22 , parasites, and helminths 23 , as well as cellular apoptosis 24 have been linked with WS disease causation. The presence of Vibrio \n sp . or other microbes suggests that such taxa may cause disease, or that they opportunistically proliferate in hosts with compromised health. Corals have a suite of defences in their immune repertoire, including physical barriers (e.g. mucus, melanin deposits) 25 – 27 , molecular pattern recognition 28 , 29 , secretion of antimicrobial macromolecules 30 , and cellular and enzymatic responses (e.g. phagocytosis, prophenoloxidase, reactive oxygen species) 31 – 33 . When these defences fail and infection takes over, lesions and tissue mortality may manifest through molecular and cellular signatures of apoptosis 24 , 34 , 35 . For most coral species, the speed at which recovery occurs is critical to survival, since lesion progression rate is directly related to tissue mortality. As such, fast healing may preclude settlement and overgrowth by competing organisms or loss of physiological integration of the colony 12 , 36 – 38 . However, the upregulation of immunity is an energetically expensive process and the ability of corals to resist, respond and ultimately recover from disease-induced lesions is largely dependent on physiological traits that confer resilience, such as high energy reserves or beneficial microbial communities 39 – 41 . Traditionally, total or ‘crude’ lipid concentration has served as a proxy for coral energy reserves, and is used to infer coral health status 42 , 43 . In the marine environment, lipids provide highly dense forms of energy, with around one-third more energy relative to proteins or carbohydrates 44 . Lipids are a major component of the coral proximate composition (10–40% of dry biomass) and their constituent classes and fatty acids provide important structural and energy storage functions 45 , 46 . Lipid stores in invertebrates are also involved in the regulation of innate immune homeostasis 47 . When a coral strays from homeostasis, its response is dependent on the colony’s physiological competence, thus total lipid content has been correlated with a coral’s ability to respond to stressors 48 , 49 . For example, large lipid stores can mitigate the detrimental effects of ocean warming and acidification 48 , 49 . Conversely, depletion of lipid reserves can increase susceptibility to disease and mortality 50 , 51 . Further, gene expression studies have shown that diseased coral tissues upregulate pathways associated with innate immunity, tissue repair, and lipid and carbohydrate metabolism, suggesting higher usage of stored lipids in diseased versus healthy coral tissue 31 . Importantly, numerous experiments have shown that healthy corals preferentially direct energetic resources (e.g. metabolites and photoassimilates) toward physically-induced lesions for regeneration 12 , 31 , 52 , 53 . However, these same compounds are preferentially transferred away from disease-induced lesions 31 , including WS lesions 54 . This is consistent with the absence of tissue regeneration observed at degenerative WS lesion borders 18 , 54 . Assuming that corals possess finite energy reserves available for life functions 55 , 56 , the preferential translocation of energy reserves away from WS lesions may represent a ‘shutdown’ response of the colony to the rapid expansion of necrotic tissues to prevent further resource loss 54 , thereby protecting the remaining colony 57 . Examining lipid profiles between diseased and healthy tissues on the same coral colony would provide valuable insight into the transfer and partitioning of compounds around the colony as part of the overall immune response to WSs. Coral-associated microorganisms including protozoa, fungi, bacteria, archaea, and viruses (collectively termed the microbiome) also contribute to host functioning and fitness 6 , 58 , 59 , and thus coral resilience is tightly linked with its associated microbial community. It is likely that the coral microbiome is involved in coral immunity either directly through production of antimicrobial compounds 60 or indirectly through niche exclusion of opportunistic or pathogenic organisms 61 , 62 . While some microbial taxa are directly linked to disease onset 63 , it has also been proposed that changes to the coral host’s normal microbial community composition (i.e. dysbiosis) can induce disease or disease-like signs 63 – 65 . Host immune processes can also be involved in the establishment and maintenance of stable microbial communities during stress or infection 66 , 67 , thus potentially draining host resources such as lipid reserves. As such, comparison of microbial communities between healthy and diseased coral tissues can give further insight into the holobiont response to disease and may establish a link between coral physiology and its microbiome. The present study profiled lipids, lipid classes, fatty acids, and the microbial communities of healthy tissue from colonies of healthy Acropora hyacinthus (HH), seemingly healthy tissue on WS-diseased colonies (HD; i.e. preceding the lesion boundary) and diseased tissue on WS-diseased colonies (DD; i.e. lesion front) from three locations across the Indo-Pacific: Western Australia (WA), the Great Barrier Reef (GBR), and Palmyra Atoll (PA). This work aimed to identify patterns that might indicate a holobiont-wide systemic effect of WSs, as well as providing insight into the immune response process. Microbial communities were also profiled to identify changes in holobiont community structure, with the patterns compared with previously published data from WS lesions from the Great Barrier Reef 68 .",
"discussion": "Discussion Examining WSs from corals distributed across dispersed geographical sites of the Indo-Pacific can help identify patterns that are consistent between lesions and further clarify aspects of the host response to disease. In this study, we provide insight into the energetic reserves and microbial ecology of three tissue types of WSs sampled from plating acroporid colonies from WA, PA, and the GBR. At the gross colony level, all lesions displayed similar disease signs of macroscopic loss of tissue across a broad front resulting in necrotic tissue and exposing irregular bands of white skeleton. Total lipid concentrations displayed a stepwise decrease from HH to HD to DD samples, with healthy samples being characterised by a high proportion of high energy storage lipids. Microbial communities associated with tissues of healthy and diseased areas were distinct when compared between health state and region, supporting previous studies that show corals undergo changes in microbial community composition when affected by WS, and this change is characterised by relative increases in Rhodobacteraceae-affiliated sequences 68 , 96 , 97 . Cnidarian immune responses to disease are, in general, thought to involve the production of antimicrobial peptides and reactive oxygen species to kill bacteria, antioxidants to reduce self-harm, and the accumulation of melanin to prevent the spread of infection 27 , 98 , 99 . Each of these processes are energetically expensive and will lead to a reduction of stored reserves. As such, the stepwise decrease in total lipid concentrations from HH to HD to DD tissues, regardless of location, may indicate catabolism of lipid reserves for energy to combat disease progression through such immune responses. Alternatively, diminished lipid reserves in diseased tissue may also reflect intracolonial transfer of important energy reserves away from lesion sites towards healthy sites. The coral animal is a physiologically integrated collection of individual polyps connected through a shared gastrovascular system, which allows both partitioning and sharing of resources. Lipids have been shown to be transferred from branch bases to tips to contribute to colony growth and calcification 52 , 100 , and at colony edges, lipids and fatty acids are lowest, suggesting catabolism of these compounds to support tissue synthesis 101 . Lipid translocation in response to WSs would have the combined effect of shutting down the diseased tissue to prevent further resource loss, whilst also fortifying healthy tissues with additional energetic resources 12 . It should also be considered that the capacity to replenish energy reserves via autotrophy or heterotrophy by coral polyps near the lesion interface would be severely reduced, further decreasing lipid concentrations. Neither can we exclude lipid depletion as a result of consumption by opportunistic microbes proliferating in diseased tissues. A surprising observation in this study was the marked differences in crude lipid quantity between sites, regardless of health state. Of note was that the lowest lipid concentration in WA corals (DD tissue) was comparable to that of healthy tissues (HH) from the GBR. Lipid concentrations are often used as a proxy for coral health. However, if we consider the high concentration of lipids observed in healthy WA samples as a standard, it suggests that no level is sufficiently high to prevent infection and progression of WS disease. In addition, these results support the growing consensus that crude lipid concentrations alone do not provide an accurate proxy for overall coral health. Indeed, lipid concentrations among healthy corals are known to differ in line with a range of geographical and physicochemical factors 102 . Instead, examining patterns among individual lipid classes and fatty acids, as well as ratios between major groups such as storage and structural lipids, can provide more accurate insight to coral health status. Furthermore, future work should incorporate measures of local conditions (e.g. temperature, pH, dissolved oxygen and nutrients, available prey items) as well as other measures of coral health (i.e. Symbiodiniaceae density) to investigate correlations with health metrics. In the present study, the opacity of the crude lipid results between sites was resolved upon examination of the constituent lipid classes and fatty acids. Trends in lipid classes between HH, HD, and DD tissues were consistent across sites, with storage lipids being lowest in HD and DD tissue, regardless of the total lipid concentration. Storage lipids such as wax esters, triacylglycerols and free fatty acids are high energy compounds that can be rapidly catabolised for ATP production to fuel basal metabolism and energetically-expensive processes during times of stress 103 . For example, colonies of Porites compressa and Montipora capitata were shown to preferentially catabolise storage lipids when subjected to bleaching conditions 104 . Free fatty acids were particularly depleted in DD samples, indicating that these tissues were consuming lipid reserves even more quickly through a sequential metabolism by the coral host 105 . The shift toward structural lipids in HD and DD tissues was driven by decreases in triacylglycerol and concurrent increases in phosphatidylcholine and phosphatidylethanolamine. These two phospholipids are major components of biological membranes, and are not thought to be involved in host metabolism 106 . Thus, the predominance of structural lipids in HD and DD corals likely reflects their indispensable roles in cell membrane structure and function. Notably, HD tissues were more similar to DD than to HH tissues in both total lipid concentrations and patterns of storage and structural lipids, demonstrating that WS incites a colony-wide systemic response, with apparently healthy tissues responding to the disease before polyps display visual signs. The mechanisms of this response are as yet unknown, but may include the prophenoloxidase-activating system, as this immune function has also been shown to be suppressed in visually healthy tissues of WS-infected colonies 107 . Resource partitioning may also vary with colony size and distance from the lesion front. HD tissues were collected ~ 10 cm away from the lesion and therefore potentially influenced more than tissues further from the lesion. Future studies should sample over a distance gradient away from the lesion front and combine time-series sampling of apparently healthy colonies prior to and during WS infection to elucidate how lipid levels relate to disease susceptibility. Consistent with the lipid class results, the fatty acids that were most strongly associated with HH tissues were high energy compounds such as 14:0, 16:0, 18:1n-9, 20:5n-3 and 22:6n-3. In contrast, the fatty acids influencing the separation of the DD tissues were indispensable, membrane-bound compounds such as 18:0, and compounds associated with wound-healing and inflammatory responses, namely 22:5n-3 and 20:4n-6. Arachidonic acid (ARA; 20:4n-6) is the main component of membrane phospholipids and is also one of the primary precursor molecules for the biosynthesis of eicosanoids. Eicosanoids are a complex family of signalling molecules that play a critical role in regulating physiological processes relating to homeostasis and inflammation, such as the production of cytokines and migration of phagocytic cells 108 , 109 . Docosapentaenoic acid (DPA; 22:5n-3) is an intermediary product between eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), with evidence suggesting it is involved in wound-healing through the migration of cells in vertebrates 110 . However, the role of 22:5n-3 in invertebrate immunity is not yet known. While both 20:4n-6 and 22:5n-3 were shown to influence the separation of the DD tissues from HH tissues using multivariate analyses, the quantitative data also showed they were largely depleted in DD tissues, along with another prominent eicosanoid precursor, eicosapentaenoic acid (20:5n-3), providing evidence that they were oxidised to eicosanoids in diseased sites and catabolised to fuel the immune response. Furthermore, the maintenance of high levels of PC and PE in diseased tissue coupled with low levels of 20:4n-6 and 20:5n-3 suggest that the depletion of 20:4n-6 and 20:5n-3 is caused by breakdown and usage of these fatty acids rather than impaired membrane structure. However, their catabolism for other metabolic processes cannot be completely discounted at this point in time, with further concomitant investigations into circulating eicosanoid levels required to shed light on this topic. Previous histological analyses of samples from the GBR 68 reported extensive necrosis and tissue degeneration in disease lesions, and comparison of these samples with additional samples from WA and PA confirmed these patterns. For example, only some WS lesion tissues displayed the presence of ciliate and fungal cells and few signs of tissue necrosis, swelling, abnormalities or microbial colonization were apparent in the healthy tissues preceding the lesion front (~ 1 mm in front of lesion). While we cannot categorically rule out the role of microbial communities or viruses in disease onset and/or progression based on histological evidence alone, the low densities of ciliates and fungi across the disease tissue samples from the three sites suggests that they are unlikely to be causative agents of WS at these sites, and rather are secondary invaders following initial infection. While previous work has implicated ciliate histophagy as a form of secondary pathogenesis following bacterial challenge, which is required to produce the tissue loss patterns characteristic of WS 21 , we would expect to identify ciliates in 100% of samples if this were the case. However, histological and FISH approaches may not be adequate to detect all microbial entities present, and other diagnostic techniques would more fully resolve the microbial community dynamics of WS. Helminths were present in even lower abundances but were also present in healthy tissue, and are therefore also unlikely to be epidemiologically relevant, but rather opportunistic or parasitic settlers within compromised coral tissue. Aligning with previous work on GBR samples 68 , FISH analysis confirmed the presence of bacteria within all WA disease lesions, while no bacteria were visualised in healthy tissue (with the exception of coral-associated microbial aggregates; CAMAs 95 ), even within 1 mm of the lesion boundary. Bacterial signal was much higher in the compromised, necrosing tissue behind the lesion front, supporting the hypothesis that bacteria are secondary, opportunistic colonisers rather than drivers of disease. Vibrio bacteria were detected in a small proportion of lesions but were visualised in much lower abundance than total bacteria, and thus are also unlikely to drive WS causation. Our results contrast with previous studies that identify Vibrio bacteria 111 , ciliates 20 , 21 , viruses 22 , and helminths 23 as potential causative agents, highlighting that the cause of WSs are likely multifaceted and thus adding to the evidence that WSs encompasses various distinct aetiologies. Though there was no single dominant bacterial taxon associated with disease lesions from the disparate sampling sites to provide any indication of a specific bacterial agent linked to WS causation, there were differences in microbial community composition between healthy and diseased tissues. Healthy tissue from healthy colonies (HH) and healthy tissue from diseased colonies (HD) showed no consistent differences in microbial community composition. However, both these tissue types were distinct from diseased tissue (DD), which had consistently higher within-sample bacterial community diversity than healthy tissues. While the differences in within-sample diversity observed here were not statistically distinguishable, fluctuations in diversity have been proposed to underlie biological and ecological stability (i.e. the Anna Karenina principle) 112 . Hence, a decrease in microbiome diversity can be linked with negative impacts on the host 107 , 112 , 113 . However, an increase in diversity also represents potential opportunistic colonization and dysbiosis 64 , especially following host tissue mortality as seen in WSs. The sequences recovered from the disease lesions across the three sampling sites indicate this is the case with compromised and necrosing diseased tissues likely supporting a diverse and variable opportunistic bacterial community. Diseased tissue was characterised by relative increases in Cyanobacteria and Bacteroidetes, and was dominated by Proteobacteria with increased abundance of Alphaproteobacteria (particularly Rhodobacteraceae) compared to healthy tissue. These observations were consistent across the three sampling sites and align with previous findings that a distinct microbiome occurs at the lesion front, characterized by a positive differential abundance of Rhodobacteraceae-affiliated sequences 68 , 96 , 97 , 114 . The family Rhodobacteraceae is emerging as a potential indicator of compromised coral health, with reports of elevated levels in WSs 68 , 96 , 97 , 114 as well as several other coral diseases 115 – 118 . In this study, the significant increase in Rhodobacteraceae in diseased tissue across locations supports previous work that proposes this group to be associated with compromised health. Rhodobacteraceae-affiliated sequences represented nearly 20% of all disease sample sequences, while representing 12% of HH colony sequences and only 3% of HD colony sequences. However, the decrease in Rhodobacteraceae between HH colonies and HD colonies suggests the relationship of this family of bacteria to disease is not straightforward. Indeed, this group has been frequently identified as core members of the coral microbiome 119 – 121 , but is also implicated in a variety of stress responses 96 , 122 – 124 . It is likely, therefore, that this group comprises a combination of commensal, as well as opportunistic and potentially pathogenic members. It is possible that the observed decrease in Rhodobacteraceae from HH to HD tissue represents a loss of commensal community members, with the subsequent increase in DD tissue as opportunistic colonization of compromised tissue occurs. Similarly, Bacteroidetes have been implicated in diseased sponges and corals 125 , 126 including white plague infected colonies in Brazil 127 – 129 , but also have been associated with secondary colonisation following tissue mortality 125 . Bacteroidetes preferentially consume high molecular weight organic matter 130 , and so it is likely that necrotic tissues provide a varied source of nutrients and may drive the increase in Bacteroidetes abundance in diseased tissue 126 . In this study, however, a large proportion of Bacteroidetes ASVs failed to classify below Class level and thus no specific taxa can be pinpointed, and thus it remains unclear if these are associated with diseased tissue or skeletal overgrowth. Interestingly, a complete loss of sequences associated to the genus Halomonas (Gammaproteobacteria) was observed between healthy to diseased tissue. Halomonas spp. have been implicated in the metabolism of DMSP and its breakdown product acrylic acid, which may generate antimicrobial compounds such as TDA, and thus Halomonas has been proposed as a potential member of a probiotic consortia for microbiome engineering 131 – 133 . Future investigations into these taxa, including how their abundance changes over the course of disease onset and their role in metabolism, may be of particular interest especially in the context of reef restoration initiatives 131 . In conclusion, the underlying causation of white syndromes of acroporid corals of the Indo-Pacific remains elusive. However, here we show that the coral energetic response is similar across locations and is characterized by a stepwise loss of high energy storage lipids from healthy to diseased tissue. This agrees with previous studies which have profiled coral responses to other kinds of stress (such as bleaching and ocean acidification), whereby corals utilize their energy reserves to launch an immune response. Visually healthy tissue from diseased colonies (HD) had lipid patterns more similar to diseased tissue (DD) than to healthy tissue from visually unaffected colonies (HH). The similarities in lipid patterns between HD and DD tissues are signatures of a systemic response, suggesting that WS depletes resources across an entire colony through polyp connective tissue, with lipids likely being catabolised to launch an immune response. Importantly, we have demonstrated that examining the qualitative aspects of lipids in diseased corals is required to gain comprehensive insight into their health. Several studies have relied on total lipid quantification alone as a measure of health 49 , 103 , 134 , 135 . Here we demonstrate that the total lipid in diseased corals from WA is akin to that of healthy corals from the GBR, and thus no minimum lipid level can be used to characterize coral health across locations. Therefore, while high lipid stores have been shown to mitigate adverse effects of warming and acidification, the same does not appear to be true for disease. Further studies are needed to define local standards of lipid biomarkers for coral health, specifically how these relate to autotrophic and heterotrophic energy acquisition. Investigation into fatty acids also revealed low levels of ARA and EPA in diseased tissue, suggesting oxidation of these compounds to eicosanoids may be occurring to combat infection. The role of specific fatty acids in coral immune response is poorly understood and merits further research. We detected increased abundance of Rhodobacteraceae-associated sequences, which aligns with previous studies identifying this group as colonising compromised coral tissues, though its role in pathogenesis is unknown. The loss in relative abundance of Halomonas from healthy tissues provides further support for the role of these microbes in coral health. To further elucidate the interaction of environment, host energetics and microbes in WS onset and progression, high resolution time-series sampling may aid in identifying the contributing factors that ultimately manifest as lesions on acroporid coral colonies across Indo-Pacific reefs."
} | 6,773 |
33287204 | PMC7761717 | pmc | 2,688 | {
"abstract": "Bacteria are the driving force of the microbial fuel cell (MFC) technology, which benefits from their natural ability to degrade organic matter and generate electricity. The development of an efficient anodic biofilm has a significant impact on the power performance of this technology so it is essential to understand the effects of the inoculum nature on the anodic bacterial diversity and establish its relationship with the power performance of the system. Thus, this work aims at analysing the impact of 3 different types of inoculum: (i) stored urine, (ii) sludge and (iii) effluent from a working MFC, on the microbial community of the anodic biofilm and therefore on the power performance of urine-fed ceramic MFCs. The results showed that MFCs inoculated with sludge outperformed the rest and reached a maximum power output of 40.38 mW·m −2 anode (1.21 mW). The power performance of these systems increased over time whereas the power output by MFCs inoculated either with stored urine or effluent decreased after day 30. These results are directly related to the establishment and adaptation of the microbial community on the anode during the assay. Results showed the direct relationship between the bacterial community composition, originating from the different inocula, and power generation within the MFCs.",
"conclusion": "4. Conclusions The current study highlights the impact of the inoculum type on the microbial anodic community in urine-fed MFCs and consequently, on power performance. To this end, the MFCs were inoculated with stored urine, sludge and effluent from a working MFC and then fed continuously with neat urine (in triplicate). The results were compared in terms of microbial anodic community and power performance, as well as long-term functionality. Among the different conditions tested, the inoculation of MFCs with sludge seems to promote a more electroactive biofilm, which results in higher values of power output. MFCs inoculated with sludge outperformed the rest, reaching a maximum power output of 40.38 mW·m −2 anode (1.21 mW). Results of the microbial community analysis begin to shed some light on the synergistic activities of bacterial populations, whilst metabolising a complex substrate such as urine and transferring electrons as part of anaerobic respiration.",
"introduction": "1. Introduction The effects of global warming on everyday life, along with the fossil fuel depletion have fuelled the search for alternative, clean energy technologies. Microbial fuel cells (MFCs) emerge as an environmentally friendly technology where bacteria drive the electricity production from the chemical energy stored in a specific substrate [ 1 , 2 ]. These devices consist of an anodic compartment, where bacteria degrade the organic matter, releasing cations, electrons and other metabolic by-products; this half-cell is physically separated from the cathode chamber, by a semi-permeable membrane. Electrons flow from the anode, through an external circuit, to the cathode, where they recombine with the incoming cations (e.g., protons diffusing through the semi-permeable membrane) to complete the reaction and close the circuit. While in the anodic chamber, oxidation of the organic matter takes place, in the cathode an oxidant, e.g., oxygen, is reduced to produce water. However, the oxygen reduction reaction (ORR) on the cathode is one of the limiting factors of this technology because of the need for a catalyst to accelerate the process [ 3 , 4 ]. So far, great efforts have been made in terms of material science to find a suitable catalyst, which balances high catalytic activity, good long-term stability and low cost [ 5 , 6 , 7 , 8 , 9 ]. Platinum group metals (PGMs) are widely used due to their high catalytic activity. However, their high cost and poisoning limitations make them unsuitable for use in scaled-up systems. Carbonaceous materials such as activated carbon or metal-free catalysts represent a real alternative to PGMs due to their low cost and longevity, which enable real-world implementation of this technology [ 8 , 10 , 11 ]. In addition to the advancements in designing new and efficient catalysts, the modification of the anode surface has been reported as a suitable way to improve the energy generation from MFCs. Activated carbon is a cost-effective material that increases the specific surface area, which facilitates bacterial attachment and development of a viable electroactive biofilm [ 12 ]. Other materials as well as techniques have also resulted in increasing specific anode area and consequently the power performance of the MFCs. One of these techniques is the modification of the anode with a wide variety of conductive polymers such as polyaniline (PANI) [ 13 ], polypyrrole (PPy) [ 14 ] or poly(3,4-ethylenedioxythiophene)-polystyrenesulfonate (PEDOT-PSS) [ 15 ]. Another important element of MFCs is the membrane, which separates the anode and the cathode. Commercial polymer-based membranes, e.g., Nafion or Ultrex, have been commonly used due to their high ionic conductivity and good performance. However, their high cost and moderate long-term performance have steered research into low-cost alternatives, which enable the commercialisation of MFCs [ 16 ]. Ceramics represent a broad range of materials with meaningful advantages over conventional polymer-based membranes, for instance, their natural availability, cost-effectiveness and robustness, which reduces the maintenance needed in potential scaled-up systems [ 17 , 18 ]. The recent use of MFCs in remote location opts for low-cost materials with low environmental impact. Another benefit of using ceramic materials is that their properties, such as porosity or ionic conductivity, can be tailored by changing the kilning temperature or doping the raw material [ 19 , 20 , 21 ]. One of the unique advantages of MFCs is the use of different types of waste as feedstock for microbes, whereby contaminants are broken down for the production of electricity [ 22 , 23 ]. So far, a wide range of waste materials have been used as substrates, e.g., waste from the brewery, dairy or oil refinery industries, food processing wastewater and domestic wastewater, among others [ 24 , 25 , 26 ]. More recently, the use of neat urine as feedstock in different kinds of bioelectrochemical systems has gained a lot of interest due to its abundance, buffering capacity and high chemical oxygen demand [ 27 , 28 , 29 ]. This natural waste product has been successfully used for ammonium recovery, powering lights, charging smartphones or powering a microcomputer through MFCs [ 30 , 31 ]. For MFCs, it is well known that the concentration, organic matter loading and bacterial diversity of the inoculum as well as feedstock, affect the power performance. The use of complex substrates with high organic loading usually helps in stablishing a diverse and electrochemically active microbial community on the anode. The feed-rate and shear-stress are also important parameters that need to be optimised in continuously fed systems, as is the temperature, since all these can affect the behaviour of the anodic microbial communities. In terms of pH stability, it is important to use a feedstock with natural buffering capacity, such as urine, in order to avoid adding external pH buffer solutions, which increases cost and complicates maintenance. Thus, the choice of an appropriate inoculum and suitable operating conditions, significantly influences the performance of MFCs [ 32 , 33 ]. At present, research is focused on moving the MFC technology from the laboratory to the field. The real implementation of this technology still poses a challenge especially when the deployment takes place in rural places with low or limited development. As the anodic biofilm dictates power output, it is vital to understand the ecological behaviour of the anodic microbial community under different operating conditions [ 34 ]. The influence of factors such as the substrate [ 35 , 36 ], external resistance [ 37 ] or anode materials [ 38 ], on the microbial community have already been reported. According to previous work, the variation of the operating conditions, substrate or electrode materials strongly affects the anodic microbial community and, therefore, the power performance of MFCs. In order to promote the real implementation of this technology, it is important to explore the behaviour of the microbial diversity under different operating conditions, especially in the start-up phase, which still poses a challenge for the functioning of the system. The enrichment of the bacterial community of the anodic biofilm is a strong indication of process functionality. However, despite the plethora of bacteria present in MFCs, only those that are capable of electroactive metabolism would directly contribute to electricity generation. As such, bacterial population or diversity increases in MFCs do not often translate to increased power generation. Nevertheless, a synergistic approach to electricity generation has been reported where different bacterial strains work in symbiosis to bring about the degradation of various contaminants thereby providing the needed substrates for the electroactive strains [ 36 ]. According to this approach, this work aims to investigate in-depth the effect of using three different types of inocula based on human urine on the anodic microbial community of MFCs and, therefore, its correlation with the power performance of the overall system. All tests were performed in triplicate with a total number of 9 MFCs run simultaneously in continuous mode for 90 days. The results were analysed in terms of microbial diversity and power output but also stability and functionality for long operating processes."
} | 2,435 |
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