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32434837 | PMC7380569 | pmc | 8,615 | {
"abstract": "Over the last century, humans have substantially altered nitrogen and phosphorus cycling. Use of synthetic fertilizer and burning of fossil fuels and biomass have increased nitrogen and phosphorus deposition, which results in unintended fertilization of historically low-nutrient ecosystems. With increased nutrient availability, plant biodiversity is expected to decline, and the abundance of copiotrophic taxa is anticipated to increase in bacterial communities. Here, we address how bacterial communities associated with different plant functional types (forb, grass) shift due to long-term nutrient enrichment. Unlike other studies, results revealed an increase in bacterial diversity, particularly of oligotrophic bacteria in fertilized plots. We observed that nutrient addition strongly determines forb and grass rhizosphere composition, which could indicate different metabolic preferences in the bacterial communities. This study highlights how long-term fertilization of oligotroph-dominated wetlands could alter diversity and metabolism of rhizosphere bacterial communities in unexpected ways.",
"introduction": "INTRODUCTION The soil microbiome is critical for plant health, fitness, and diversity, especially in nutrient-limited environments ( 1 – 4 ). In particular, within the rhizosphere, plants provide carbon (C) resources to soil microorganisms in exchange for nutrients such as nitrogen (N) and phosphorus (P). However, nutrient enrichment has been documented to disrupt plant-microbe mutualisms ( 2 ). Over the last century, agricultural fertilization and the burning of fossil fuels and biomass have indirectly led to nutrient deposition onto historically low-nutrient ecosystems ( 5 – 8 ). Nutrient enrichment generally causes reduced plant species diversity ( 9 , 10 ) sometimes as a shift in plant functional types with an increase in grass biomass and loss of forb diversity ( 11 – 13 ). Fertilization has also been shown to decrease soil microbial diversity across cropland, grassland, forest, and tundra ecosystems ( 14 – 16 ). Despite patterns that have emerged from these bulk soil studies, it is less clear how changes in soil microbial diversity due to nutrient additions influence rhizosphere microbial community assembly and diversity. We address this knowledge gap by comparing changes in rhizosphere bacterial community composition of a grass and forb within a long-term fertilization experiment. Both bulk soil matrix (i.e., not in contact with plant roots) properties and plant identity influence rhizosphere microbial communities. The bulk soil matrix is the reservoir of microbial diversity from which rhizosphere-associated microbial communities are selected; therefore, shifts in bulk soil microbial communities affect rhizosphere assemblages ( 17 – 19 ). In many cases, N, N and P, and N-P-K (nitrogen-phosphorus-potassium) fertilization decreases soil bacterial diversity ( 14 – 16 ). Additionally, nutrient enrichment selects for more copiotrophic (i.e., fast-growing, r-strategists) microbial heterotrophs that preferentially metabolize labile C sources versus oligotrophic (i.e., slow-growing, K-strategist) microbial species, which can metabolize complex C sources ( 20 – 23 ). A molecular marker to identify life history strategy (i.e., copiotroph or oligotroph) is rRNA ( rrn ) gene copy number ( 23 – 26 ). Bacterial taxa are estimated to contain 1 to 15 rRNA gene copies, with faster-growing taxa containing higher numbers of gene copies than slower-growing taxa ( 20 , 23 – 27 ). Specifically, bacterial growth rate is limited by the transcription rate of rRNA, such that growth rate is estimated to double with doubling of rRNA gene copy number ( 23 ). Further, several studies indicate that fertilization increases the abundance of copiotrophic bacterial groups within Actinobacteria , Alphaproteobacteria , and Gammaproteobacteria and decreases abundance in oligotrophic bacterial groups within Acidobacteria , Nitrospirae , Planctomycetes , and Deltaproteobacteria of bulk soils ( 15 , 21 , 28 , 29 ). Additionally, copiotrophic taxa within Alpha -, Beta -, and Gammaproteobacteria , Actinobacteria , Firmicutes , and Bacteroidetes are dominant members of some rhizosphere communities ( 17 , 30 , 31 ). While the bulk soil environment is the primary source of rhizosphere diversity, plant species also influence rhizosphere bacterial community assembly due to variation in rhizodeposition ( 30 – 33 ). Rhizodeposits include nutrients, exudates, root cells, and mucilage released by plant roots ( 34 ). Plants allocate 5 to 20% of photosynthetically fixed C belowground ( 35 – 37 ). Some estimates suggest that up to 40% of fixed C is translocated belowground ( 38 ), and grasses are suggested to be near that upper limit with ∼30% of fixed C allocated belowground ( 39 ). These rhizodeposits also include root exudates which are composed of sugars, organic acids, phenolic compounds, and amino acids ( 1 , 17 , 40 , 41 ). Differences in plant physiology influencing the quantity and composition of root exudates can affect rhizosphere bacterial community composition. For example, C 4 grasses have higher photosynthetic rates (i.e., fix more C) and greater root biomass allocation compared to C 3 plants, resulting in a greater quantity of root exudates ( 42 , 43 ). C 3 plant root exudates can contain a greater variety of organic acids and amino acids along with the sugars mannose, maltose, and ribose compared to C 4 plant root exudates, which can contain several sugar alcohols (i.e., inositol, erythritol, and ribitol) ( 44 ). However, N fertilization has been shown to increase C assimilation in plants but decrease belowground allocation of assimilated C while increasing total C into soils as rhizodeposits ( 39 , 45 ). Prior studies revealed that root exudation of organic C can be higher in both low-nutrient scenarios ( 46 , 47 ) and high-nutrient scenarios ( 48 , 49 ). Further, differences in soil nutrient status can change the composition (i.e., carbohydrates, organic acids, and amino acid concentrations) of root exudates ( 46 , 50 ). Thus, fertilization and plant-specific rhizodeposition patterns of C 3 forbs and C 4 grasses are predicted to differentially affect rhizosphere bacterial community structure. In this study, we address the following question: to what extent does long-term fertilization (N-P-K) of bulk soil shift rhizosphere bacterial communities of two plant species representing distinct functional types (i.e., a C 3 forb and a C 4 grass)? First, we hypothesize that nutrient addition will decrease bacterial species diversity and increase the abundance of copiotrophic taxa in all soils, especially rhizosphere soils due to increased availability of labile C from root exudates. We expect that fertilization will stimulate microbial activity of faster-growing copiotrophic species, which would outcompete slower-growing oligotrophic species and result in decreased bacterial diversity. This effect is predicted to be amplified within plant rhizospheres due to the availability of labile C substrates in root exudates, which should preferentially select for copiotrophic bacteria. Second, we hypothesize that fertilization will be the primary factor determining differences in rhizosphere communities and plant identity will secondarily influence the rhizosphere community. If bulk soil is the reservoir for the rhizosphere community, then fertilization will more strongly determine rhizosphere bacterial diversity and community composition. In addition, plant type can also affect rhizosphere communities due to differences in root exudate composition; however, fertilization effects will constrain rhizosphere effects. As a result, plant species are expected to associate with unique core microbiomes that differ between fertilization treatments. To test these hypotheses, bulk and rhizosphere soils were sampled from two plant species (a grass and a forb) from fertilized and unfertilized plots at a long-term disturbance and fertilization experiment (established in 2003). Bacterial communities were identified using 16S rRNA amplicon sequencing which allowed binning of bacterial taxa as copiotrophic or oligotrophic by estimating the average rRNA ( rrn ) gene copy number. By evaluating differences in taxonomic information and 16S rRNA gene copy numbers of bulk and rhizosphere soils of two plant species with associated soil properties (i.e., ammonium, nitrate, soil pH, carbon, and moisture), we provide insight into biotic and abiotic processes that are contributing to rhizosphere bacterial community assembly.",
"discussion": "DISCUSSION In this study, nutrient addition increased bacterial species diversity (H′) and richness in bulk and rhizosphere soils. These results were similar to the results of O’Brien et al. ( 51 ) but contrary to our prediction and the results of other studies ( 14 – 16 ). Overall, bulk soils had the greatest bacterial diversity and highest pH values compared to rhizosphere soils. Since pH is known to be a strong driver of bacterial diversity, which can have a positive relationship with pH ( 52 , 53 ), this increase in diversity may be due, in part, to the greater bulk soil pH compared to rhizosphere soil pH. The difference in pH between soil types is possibly due to organic acids in plant root exudates released into the rhizosphere ( 41 ); however, we did not analyze the composition of root exudates. Additionally, pH tended to be lower in unfertilized treatments, and diversity was more strongly related to pH in unfertilized soils than in fertilized soils. This may be due to the sensitivity of bacteria to acidic soils ( 53 ). The increase in bacterial diversity is likely the result of soil pH and niche differentiation due to fertilization increasing nutrient availability and rhizodeposition by plants, which introduces organic C resources for heterotrophs ( 17 , 32 ). In dilution to extinction experiments, decreases in microbial diversity can result in loss of microbial functional diversity ( 54 , 55 ). Therefore, increases in microbial diversity could result in increased microbial functional diversity, which could increase C cycling and promote N mining particularly in plant rhizospheres ( 56 ). Bacterial taxa identified in rhizosphere samples are putatively involved in nutrient cycling and disease-suppressive functions. For example, fertilized forb rhizospheres were enriched in taxa from the family Streptomycetaceae , of which many produce antibiotics ( 57 ) and Sphingomonadaceae , which include taxa with disease suppression potential against fungal pathogens ( 58 ) ( Fig. 5 ). This increase in disease-suppressive bacterial taxa suggests a potential increase in plant-pathogenic taxa within fertilized rhizospheres; however, this study did not specifically address disease suppression in soils. In contrast, fertilized grass rhizospheres were enriched with taxa putatively involved in N 2 fixation ( Acetobacteraceae ) ( 59 ) and also Chitiniphagaceae and Conexibacteraceae , which have been implicated in decomposition of recalcitrant C sources ( 60 , 61 ) ( Fig. 4 ). Bacterial taxa in the Xanthomonadaceae family, which have previously been found in environments containing glyphosate ( 62 ), and Caulobacteraceae , which grows optimally on pesticides ( 63 ), are also more abundant in fertilized grass rhizospheres ( Fig. 4 ). Since fertilization increased bacterial diversity and shifted composition, it is possible that fertilization has stimulated root exudation. The relative increase in complex C-degrading bacterial taxa in the grass rhizosphere could also be due to greater inputs of phenolics and terpenoids used as allelochemicals by the plant as revealed in past studies ( 64 , 65 ). These differences in bacterial composition between the two plant species could be due to differences in composition of root exudates released into the rhizosphere ( 37 ); however, we did not analyze the composition of root exudates in the present study. Together, results suggest that nutrient addition enriches forb rhizospheres with putatively disease-suppressive bacteria and grass rhizospheres with taxa capable of decomposing complex C sources. Within bulk soil bacterial members, putative nitrogen cycling taxa in the order Rhizobiales were enriched across all fertilization treatments ( 66 , 67 ). This is not surprising, considering the limited amount of nitrogen in both unfertilized and fertilized soils at the study site. Despite the increase in taxa capable of N 2 fixation in fertilized rhizospheres, these bacteria will acquire soil N if it is available ( 68 ). Therefore, these taxa may be less cooperative with plant associates than the same taxa from unfertilized soils, thereby reducing plant benefit ( 2 , 69 ). This was not specifically tested in this study but could be an important future research topic. Contrary to our prediction, bulk soils had a higher copiotroph-to-oligotroph ratio (based on rrn gene copy number) than rhizospheres. Characteristic of the copiotrophic life history strategy is the ability to rapidly decompose labile C sources; therefore, we expected that C-rich root exudates in the rhizosphere would support higher proportions of copiotrophic species ( 17 ). Additionally, fertilization did not increase the relative abundance of copiotrophic taxa. Rather, the observed copiotroph-to-oligotroph ratios were low in all samples with unfertilized bulk soils having the greatest proportion (0.22) and unfertilized grass rhizospheres having the lowest (0.13) copiotroph-to-oligotroph ratios. We suggest that the dominance of oligotrophs reflects the low-nutrient history of this wetland ( 29 , 70 ), which is in contrast to agricultural systems that undergo regular fertilization at target rates intended to support high nutrient requirements for enhanced crop production (e.g., corn). These results are in contrast to our first hypothesis and in agreement with our second hypothesis. Analyses of bacterial diversity and copiotroph-to-oligotroph ratios revealed an increase in bacterial diversity in response to fertilization and dominance of oligotrophs across all treatments within the study wetland. The low-nutrient history of the study site is likely the primary factor shaping bacterial community composition within the wetland. In agreement with our second hypothesis, comparisons of bulk and rhizosphere bacterial communities revealed that rhizospheres were more similar to each other than to bulk soil bacterial communities within fertilization treatments. Core plant microbiomes were predominantly composed of broadly distributed taxa; therefore, changes in bulk soil bacterial composition due to nutrient enrichment can directly alter plant microbiome composition and indirectly diminish benefits to plants if nutrient enrichment selects for more competitive bacterial taxa. These results highlight the importance of bulk soils as reservoirs of diversity for plant rhizospheres, which could have further implications for agricultural plant species in maintaining beneficial microbial communities. Overall, this study revealed that long-term fertilization of oligotroph-dominated soils in low-nutrient wetlands increases bacterial species diversity. This increase in bacterial diversity has the potential to result in increased C and nutrient cycling that could lead to declines of wetland C storage potential. Nutrient enrichment also differentially alters plant rhizosphere composition in a way that suggests metabolic changes within soil bacterial communities. These metabolic changes could indirectly impact plant species diversity by providing an advantage to one species versus another through disease suppression or by increasing plant-available N through promotion of soil organic matter decomposition. If indirect fertilization supports rhizosphere bacterial communities that can enhance recalcitrant or labile C decomposition, wetland C storage potential could decline. Based on this study, bacterial taxonomic characterization sheds light on fertilization effects on plant-bacterial relationships. As such, nutrient enrichment effects on the metabolic diversity of bacterial communities could be even more pronounced in naturally low-nutrient ecosystems and warrants further investigation."
} | 4,085 |
36179033 | PMC9524829 | pmc | 8,617 | {
"abstract": "Nonlinear phenomena in physical systems can be used for brain-inspired computing with low energy consumption. Response from the dynamics of a topological spin structure called skyrmion is one of the candidates for such a neuromorphic computing. However, its ability has not been well explored experimentally. Here, we experimentally demonstrate neuromorphic computing using nonlinear response originating from magnetic field–induced dynamics of skyrmions. We designed a simple-structured skyrmion-based neuromorphic device and succeeded in handwritten digit recognition with the accuracy as large as 94.7% and waveform recognition. Notably, there exists a positive correlation between the recognition accuracy and the number of skyrmions in the devices. The large degrees of freedom of skyrmion systems, such as the position and the size, originate from the more complex nonlinear mapping, the larger output dimension, and, thus, high accuracy. Our results provide a guideline for developing energy-saving and high-performance skyrmion neuromorphic computing devices.",
"introduction": "INTRODUCTION Artificial neural networks, mimicking human brains, exhibit extraordinary abilities in several tasks, such as image recognition ( 1 ), machine translation ( 2 ), and a board game ( 3 ). Nowadays, most artificial neural networks rely on silicon-based general-purpose electronic circuits, such as a central processing unit and a graphics processing unit. However, these circuits consume a large amount of energy and are approaching the physical limits of downscaling ( 4 ). Therefore, developing devices specialized for brain-inspired computing, namely, neuromorphic devices, is highly required ( 4 , 5 ). In particular, nonlinearity and short-term memory effects are essential functions for neuromorphic devices that various spintronic devices can offer ( 6 – 21 ). Among them, we focus on a topological spin structure called magnetic skyrmion ( 22 – 31 ). So far, skyrmion-based neuromorphic devices, such as reservoir computing devices ( 9 – 14 ), synapse devices ( 15 , 16 ), and probabilistic computing devices ( 17 , 18 ), have been studied to bring about high performance. However, a fully experimental evaluation of its ability for neuromorphic tasks such as pattern recognition is still lacking. We design the skyrmion neuromorphic computer on the basis of a reservoir computing model ( 7 – 13 , 32 – 39 ). The conventional reservoir computing model consists of two parts ( Fig. 1A ). The first part, called the “reservoir part,” performs a complex nonlinear transformation of input data into high-dimensional output data. Here, the dimension is the number of linearly independent outputs. In this process, the reservoir part temporally stores the information of past input to make the output depend on both present and past inputs (short-term memory effect). The second part conducts a linear transformation of the outputs from the reservoir part. The coefficient parameters of this linear transformation are optimized by using a training dataset so that the final output becomes a desirable one. Incidentally, the nonlinear transformation of input into high-dimensional outputs is the essence of reservoir computing; the linearly inseparable data can become linearly separable in the high-dimensional space, enabling complex data classification as in the kernel method ( 40 ). Optimizing parameters (i.e., training) in reservoir computing is unnecessary for the reservoir part. In other words, the reservoir part performs the complex nonlinear transformation with fixed parameters. Hence, we can implement the reservoir part using a physical system with the complex nonlinearity and memory effect (or equally hysteresis) with short-term properties ( 7 – 13 , 33 – 37 ). As shown below, skyrmion systems also exhibit nonlinearity and short-term memory effects. Moreover, the skyrmion system has large degrees of freedom because each can take various states with different positions and sizes. This feature theoretically brings about a complex transformation of input data and high performance ( 9 – 13 ). However, it has not been experimentally explored well. We experimentally found that the skyrmion-based physical reservoir device exhibits good abilities in recognition tasks. Notably, although the structure of the present device is quite simple, the recognition accuracy as high as 94.7% is obtained in a handwritten digit recognition task, indicating an advantage of the skyrmion system in neuromorphic computing. Fig. 1. Concept of skyrmion-based neuromorphic computing. ( A ) Schematic for the conventional reservoir computing model. ( B ) Schematic illustration of a Hall bar device and a magnetic skyrmion. ( C ) Conceptual diagram for the data conversion in a subsection. ( D ) Schematic illustration of a skyrmion-based neuromorphic computer. Polar Kerr images of the subsection with various constant magnetic fields ( H const ) in the absence of a time-dependent magnetic field [ H AC ( t )] are also presented.",
"discussion": "DISCUSSION Last, we discuss the origin of the better recognition accuracy obtained using the skyrmion-based neuromorphic device than the ferromagnetic domain–based one. First, the creep motion of ferromagnetic domains decreases the recognition accuracy. In fig. S3, we present the 41 output signals ( H const = −1.6 to 1.6 Oe) in the waveform recognition task for skyrmions (device A) and ferromagnetic domains (device C). In the case of ferromagnetic domains, the center of the oscillation (the red lines in fig. S3F) gradually changes with time at low H const . This tendency originates from a slow change in the total magnetization in the Hall bar due to the thermally induced creep motion of the ferromagnetic domains. Such a gradual change in the background must reduce the recognition accuracy because even if we input the same signal, the outputs might be different depending on time, causing false recognition. In contrast, in the case of skyrmion (device A), the output signals oscillate around the time-independent values. This is because thermal agitation has a lower impact on the magnetization in the skyrmion-based device (i.e., the total number of skyrmions) compared with the magnetization in the ferromagnetic domain–based device due to the topological stability of skyrmions (i.e., a finite energy barrier between skyrmions and ferromagnetic state). Hence, the profiles of the output signals are reproducible and determined by the form of the input signal. As shown in fig. S4 and Supplementary Text, the output signals and the number of skyrmions are reproducible. Although some skyrmions are created/annihilated stochastically because of the thermal effect, the stochastic fluctuations are averaged out since many skyrmions exist in the device. Second, the larger number of output data dimensions, which originates from the large degree of freedoms of the skyrmion system, also contributes to better recognition accuracy. As mentioned above, the complex nonlinear mapping into high-dimensional space is a crucial factor for the present neuromorphic computing. Because of the particle nature of skyrmions, skyrmion systems have many degrees of freedom, such as position and skyrmion size, causing different spin structural responses to the input signals H AC ( t ). This results in high-dimensional mapping. However, the ferromagnetic domain state consists of only two internal states (up and down domains). Hence, the transformation should be less complex than the skyrmion system. To further discuss the dimensionality, we evaluate the dimensionality of the experimentally obtained output signals. The dimensionality is defined by the linearly independent outputs from the subsections. Thus, we plot an output signal of the i th subsection ( V i ) obtained in the waveform recognition task as a function of an output signal of the j th subsection with a different H const value ( V j ) ( i ≠ j ) (fig. S5). If V i and V j are linearly dependent (i.e., V i = CV j , where C is a coefficient), the profile becomes a straight line. However, if V i and V j are linearly independent, the curve shape becomes nonmonotonous. As shown in fig. S5A, the profiles for the skyrmion-based device tend to be nonmonotonous smooth curves. In contrast, the ferromagnetic domain–based device profiles are relatively straight and squarish (fig. S5C). These results indicate that the number of linearly independent outputs in the skyrmion-based device is more than that in the ferromagnetic domain device. In other words, the skyrmion-based device has a larger dimensionality than the ferromagnetic domain–based device. This fact contributes to the better recognition accuracy in the skyrmion-based device. We experimentally conclude that the skyrmion system is a promising candidate for neuromorphic computing. The high degree of freedom and topological stability of the skyrmions lead to reproducible, complex, and high-dimensional mapping and, consequently, better recognition accuracy. The present skyrmion-based neuromorphic system consists of less than 10 simple-shaped and microscale Hall bars. Nevertheless, the recognition accuracy in the handwritten digit recognition task is better than other neuromorphic devices ( 15 , 33 , 37 ), which require the fabrication of a large number of nanoscale objects. Moreover, using nanometric skyrmions ( 45 ), current-induced dynamics of skyrmions ( 12 , 46 ), and magnetic tunnel junctions ( 47 ) can further improve the performance. In addition, other spin textures with high degrees of freedom and the stability against thermal agitation, such as anti-skyrmions ( 48 ) and skyrmion strings ( 49 ), might also be used for the neuromorphic system. Our findings provide a previously unknown pathway for designing a high-performance neuromorphic computer."
} | 2,459 |
27601031 | PMC5011921 | pmc | 8,618 | {
"abstract": "Background Rhizobia are soil bacteria that establish symbiotic relationships with legumes and fix nitrogen in root nodules. We recently reported that several nitrogen-fixing rhizobial strains, belonging to Rhizobium phaseoli, R. trifolii, R. grahamii and Sinorhizobium americanum, were able to colonize Phaseolus vulgaris (common bean) seeds. To gain further insight into the traits that support this ability, we analyzed the genomic sequences and proteomes of R. phaseoli (CCGM1) and S. americanum (CCGM7) strains from seeds and compared them with those of the closely related strains CIAT652 and CFNEI73, respectively, isolated only from nodules. Results In a fine structural study of the S. americanum genomes, the chromosomes, megaplasmids and symbiotic plasmids were highly conserved and syntenic, with the exception of the smaller plasmid, which appeared unrelated. The symbiotic tract of CCGM7 appeared more disperse, possibly due to the action of transposases. The chromosomes of seed strains had less transposases and strain-specific genes. The seed strains CCGM1 and CCGM7 shared about half of their genomes with their closest strains (3353 and 3472 orthologs respectively), but a large fraction of the rest also had homology with other rhizobia. They contained 315 and 204 strain-specific genes, respectively, particularly abundant in the functions of transcription, motility, energy generation and cofactor biosynthesis. The proteomes of seed and nodule strains were obtained and showed a particular profile for each of the strains. About 82 % of the proteins in the comparisons appeared similar. Forty of the most abundant proteins in each strain were identified; these proteins in seed strains were involved in stress responses and coenzyme and cofactor biosynthesis and in the nodule strains mainly in central processes. Only 3 % of the abundant proteins had hypothetical functions. Conclusions Functions that were enriched in the genomes and proteomes of seed strains possibly participate in the successful occupancy of the new niche. The genome of the strains had features possibly related to their presence in the seeds. This study helps to understand traits of rhizobia involved in seed adaptation. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3053-z) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusion The seed-borne, nitrogen-fixing rhizobia strains represent an extended symbiotic model of the interaction with legume plants. Genomic differences such as rearrangement and reduction of transposases in the chromosomes possibly resulted from the adaptation to the seeds. Some functions such as stress response and biosynthesis of coenzymes, cofactors, carbohydrates and fatty acids appeared enriched in the seed strains. Comprehensive genomic studies, such as those presented here help to reveal global differences between the rhizobial seed strains and those isolated only from nodules.",
"discussion": "Discussion The seed-borne strains R. phaseoli CCGM1 and S. americanum CCGM7 described in this work were obtained through assays with noninoculated bean plants that nodulated and fixed nitrogen [ 12 ]. As described previously, the first non efficient strain tested was an Agrobacterium tumefaciens devoid of pTi and carrying instead a pSym derived from R. etli strain CFN42 [ 12 ]. Using these procedures, ten strains were isolated that showed plasmid profiles not observed previously. We reported that strain CCGM1 encoded several prophages (the firsts reported in Rhizobium ), toxin/antitoxin pairs, queuosine, cellulosome anchoring system and other genes possibly related to the interaction with the plants [ 12 ]. The strain was a biotin auxotroph that showed a growth decline in serial subcultures, accumulated poly-beta-hydroxybutyrate (PHB) and had low pyruvate dehydrogenase (PDH) activity (as typical of some strains of its species), yet had optimal nodulation and nitrogen fixation ability [ 12 ]. Here, we compared the genomes and proteomes of rhizobial strains isolated only from nodules with isolates from bean seeds. The R. phaseoli strains were CIAT652 and CCGM1, respectively, and the S. americanum strains were CCGM7 and CFNEI73 [ 15 ]. This last strain was sequenced twice and, together with CCGM7 resequencing, allowed us to make a fine structural genome comparison (Fig. 1 ). The S. americanum strains each have three plasmids: a megaplasmid of about 2 Mb, the symbiotic plasmid ranging from 450 to 550 Kb, and a smaller plasmid between 200 and 400 kb. The main observation of the structural study was the high synteny of the chromosomes and the megaplasmids. However, their symbiotic plasmids showed important differences in the region surrounding the symbiotic gene clusters. Furthermore, the smallest plasmid of CCGM7 apparently derived from a segment of the megaplasmid, but the smallest plasmid of CFNEI73 was almost completely unrelated. The plasmids in these S. americanum strains were difficult to observe. However, it was easy to observe the plasmids of other strains of S. americanum, CCBAU051121 and CCBAU051127 [ 30 ]. In our previous report, we did not observe plasmids in strain CCGM7 [ 12 ]. However, given the genome assembly and the report that CFNEI73 contained three plasmids [ 15 ], additional efforts were made to detect its plasmids. To avoid the action of nucleases that possibly degrade the nucleic acids when the cells are lysed, the protocols were modified as described in Methods . CFNEI73 also had some features that we previously found only in strain CCGM7: a nifV gene for homocitrate synthesis, hydrogenase uptake genes ( hup ) and two RubisCO clusters. The strains shared the three nodA and the five nodD reiterations (see the phylogeny in Fig. 4a ), with some of them being identical and others having slight differences. The expansion of genetic families appears as an adaptative trait, as observed in Leishmania [ 31 ]. Also, we observed that chromosomes of seed-borne strains had less transposases and strain-specific genes in comparison to the typical strains, indicating reduced potential for rearrangement and possibly gene loss as a requisite for seed prevalence. This could be analogous to the genome reduction observed in obligate intracellular bacteria [ 32 , 33 ]. The S. americanum strains had interesting metabolic abilities. CCGM7 had high PDH activity, grew without decline in serial cultures of minimal medium and, like CFNEI73, had the complete gene set for biotin synthesis, thus making them biotin prototrophs [ 12 ]. In the proteome analysis of abundant proteins, CCGM7 showed a protein set enriched for energy generation, response to stress, metal detoxification, translation and carbohydrate and ion metabolisms (Table 3 ). CFNEI73 proteins appeared enriched for amino acid transport and metabolism. The abundant proteins of seed strains participated in the metabolic pathways of biosynthesis of carbohydrates, fatty acids and cofactors. On the other hand, the nodule strains had better coverage of amino acyl tRNA charging, and biosyntheses of amino acids and cell structures (Additional file 5 : Figure S3). It is important to mention that only five hypothetical proteins were abundant in the proteins analyzed (from total 173). Apparently the main difference between the strains is related to the form in which the metabolism is performed, using the same main pathways. Also, 136 proteins had names, with specific function, and the rest 36 only generic functions. Only two proteins were abundant in both seed strains, namely PurH (Bifunctional phosphoribosyl amino imidazole carboxamide formyl transferase/IMP cyclo hydrolase) and SucB (Dihydrolipoamide succinyl transferase). This pair of proteins may be considered specific markers of the seed strains in minimal medium. The differential metabolic functions of the identified abundant proteins were found even when the strains were growing at the same rate. Although the physiological meaning of these particular proteins in each strain can be matter of speculation, the data contribute to the characterization of the peculiarities of the strains. For example, the majority of abundant proteins (93 out 173) had no signal in the other strain, thus appearing as specific traits for each one. In E. coli it has been found that the core proteome is significantly enriched in nondiferentially expressed genes and depleted in differentially expressed genes [ 34 ]. The nodule strain CIAT652 had abundant proteins for energy generation, translation, and more dehydrogenases (related to redox and energy processes). We previously performed symbiotic and physiological characterization of this strain, qualifying it as a highly efficient strain [ 14 , 26 ]. The abundant proteins of seed strain CCGM1 were enriched for synthesis of coenzymes and cofactors (Tables 3 and 4 ). The seed bacteria must develop great adaptative traits because the spermosphere is a new niche with high competition between seed borne and soil microorganisms occurring at the time of seed emergence [ 35 ]. It will be of interest to determine the host range of the S. americanum strains because they are relatively newly described species and their closest relatives belong to the very broad host range S. fredii strains NGR234 and USDA257, which can nodulate up to 112 and 79 legume species, respectively [ 36 ]. We have so far determined that CCGM7 can nodulate and fix nitrogen with P. vulgaris and Medicago truncatula ; CFNEI73 can nodulate Acacia farnesiana [ 15 ] and P. vulgaris . As mentioned, the strains present five nodD reiterations and possibly these have a role in the host range. In a relevant recent paper, Del Cerro et al. determined that the five nodD genes of R. tropici CIAT899 were necessary to engage the microsymbiont in nodulation with different legume plants [ 37 ]. A factor that might be crucial for our ability to isolate rhizobia from seed was that the seeds were cropped from plants irrigated previously with nitrogen. Apparently, the seed strain is more adapted to the presence of nitrogen. On the other hand, nodulation is the main process by which Rhizobium colonizes the plants, but the seed niche is a less constrained environment that relaxes the selective pressure on the symbiotic genes. Although the strain persistence in seeds can represent an advantage for the plants given their potential metabolic capabilities, the seed rhizobia can also lose the symbiotic capability in the seed environment, without apparent consequences for the plant. Thus, originally the nodulation ability was a necessary feature for entry into the plants, but in the seed isolates it is not an essential feature."
} | 2,695 |
33884875 | PMC8153535 | pmc | 8,623 | {
"abstract": "Biological cilia\noften perform metachronal motion, that is, neighboring\ncilia move out of phase creating a travelling wave, which enables\nhighly efficient fluid pumping and body locomotion. Current methods\nfor creating metachronal artificial cilia suffer from the complex\ndesign and sophisticated actuation schemes. This paper demonstrates\na simple method to realize metachronal microscopic magnetic artificial\ncilia (μMAC) through control over the paramagnetic particle\ndistribution within the μMAC based on their tendency to align\nwith an applied magnetic field. Actuated by a 2D rotating uniform\nmagnetic field, the metachronal μMAC enable strong microfluidic\npumping and soft robot locomotion. The metachronal μMAC induce\ntwice the pumping efficiency and 3 times the locomotion speed of synchronously\nmoving μMAC. The ciliated soft robots show an unprecedented\nslope climbing ability (0 to 180°), and they display strong cargo-carrying\ncapacity (>10 times their own weight) in both dry and wet conditions.\nThese findings advance the design of on-chip integrated pumps and\nversatile soft robots, among others.",
"conclusion": "3 Conclusions and Perspective Metachronal μMAC arrays,\nactuated with a uniform magnetic\nfield, have been successfully created using a facile and repeatable\nfabrication method that induces programmable magnetic anisotropy.\nSpecifically, the paramagnetic particle distribution in the μMAC\narray is well controlled by placing a rod-shaped magnet array arranged\nwith an alternating dipole orientation between neighboring magnets\nunderneath the mold during the sample curing step. The working principle\nis based on the fact that the paramagnetic particles form structures\nthat tend to align with the applied magnetic field. This mechanism\nalso enables the design of a variety of smart stimulus-responsive\nstructures and micromachines. 45 Owing to\nthe difference in the magnetic particle arrangement between adjacent\nμMAC, neighboring μMAC in the array exhibit a phase-shifted\ndeformation when actuated in a uniform magnetic field, while the amplitude\nof the μMAC bending varies with varying magnetic field magnitudes,\nsee Figure 2 and Movie S2 . This behavior of the μMAC arrays\nin a uniform magnetic field offers an alternative solution for creating\nreversible adhesion, 46 switchable wettability, 47 particle and droplet manipulation, 48 controlled liquid spreading, 49 cell/tissue stimulation by applying stretching and/or squeezing\nforces on cells, as well as microgrippers. 17 Note that the concentration of the magnetic particles has an important\nimpact on the μMAC behavior and that the bending angle of the\nμMAC scales nonlinearly with the applied magnetic field. When the μMAC are placed in a 2D rotating magnetic field,\na metachronal wave is observed to travel along the μMAC array.\nThis metachronal wave enables superior fluid pumping and locomotion\ncapability compared with the synchronous motion. Specifically, in\nterms of pumping efficiency, the metachronal μMAC exhibit twice\nthe pumping efficiency than the synchronously moving control μMAC\nin water. In glycerol, with a viscosity 1000 times larger than that\nof water, the control μMAC cannot induce a flow, while the metachronal\nμMAC are still able to generate significant flow. The individual\nμMAC exhibit a 2D whip-like motion consisting of two strokes\n(a magnetic stroke and an elastic stroke), with no noticeable shape\nasymmetry but with different timescales. Hence, depending on the precise\nconditions (magnetic field rotation frequency, fluid viscosity), the\ngenerated flow is caused by the combination of inertial effects (in\nwater) and metachrony (in water and in glycerol). For the previously\nreported artificial cilia with controlled anisotropic magnetic properties,\nthe individual cilia motion is different: these exhibit 3D asymmetric\nmotion, 19 curved film oscillation, 27 2D rotational buckling, 28 or rotation, 29 and therefore, the mechanism\nof the fluid flow generation of our cilia is different. A quantitative\ncomparison with the flows reported by two studies of cilia with controlled\nanisotropic magnetization 19 , 28 shows that our metachronal\nμMAC generate similar or larger global flow in a microchannel,\nand the normalized flow speed has the same order of magnitude for\nall studies (see Table S1 ). Concerning\nthe locomotion capability of the ciliated robots based\non inverted μMAC arrays, the metachronal robots move at a speed\nof approximately 0.7 cilia length per beating cycle, which is 3 times\nfaster than the control robots. Due to the continuous contact between\nthe metachronal μMAC and the surface, the metachronal robots\nshow an unprecedented slope climbing ability (0 to 180°). The\nmetachronal robots also exhibit heavy cargo carrying capacity (over\n10 times its own weight) in both dry and wet conditions. We quantitatively\ncompare the performance of our ciliated soft microrobot to that of\na similar robot presented previously. 19 Apart from our ciliated robot being substantially smaller, the locomotion\nspeed is much larger (up to 2 mm/s vs 0.1 mm/s) due to the higher\nactuation frequency used, and the maximal normalized walking speed\nis larger as well, namely, at least 1 versus 0.5 (see Figure 5 E). This is due to the differences\nin individual cilia motion as mentioned above and differences in adhesive\nand frictional interactions between the cilia and the surface. The demonstrated abilities of the metachronal μMAC facilitate\nthe design of on-chip integrated pumps as well as advanced versatile\nsoft robots. Compared to previously reported metachronal cilia with\nspatially controlled magnetic anisotropy, our metachronal μMAC\nprovide a number of key advantages, including (i) the ease of fabrication,\n(ii) the need for only a simple actuation scheme, (iii) the relatively\nsmall size that is compatible with common microfluidic chips, (iv)\nexcellent fluid pumping capacity, and (iv) the capability to create\nversatile climbing soft robots. While scaling of the programmable\nμMAC array is feasible\nin theory according to the predicted magnetic particle distribution\nby the COMSOL simulation, the use of the rod-shaped magnets imposes\nlimits to the design of more complex and sophisticated structures\nbecause of their specific periodical magnetic field. More systematic\nstudies need to be done in order to find the best magnetic particle\narrangement in terms of pumping and locomotion efficiency. Besides\nthe magnetic properties of the μMAC array, the cilia surface\nadhesion also has an impact on the locomotion efficiency of the metachronal\nsoft robots. Therefore, a theoretical-numerical model is needed to\ncalculate the bending performance of the metachronal μMAC in\na given magnetic field both in a liquid and in contact with a solid\nsurface and to predict the corresponding fluid pumping and locomotion\nefficiency. The pumping efficiency of our metachronal μMAC outperforms\nmost reported artificial cilia ( Table S1 and Figure S7 ), 19 , 21 − 23 , 26 , 38 , 40 and is competitive with that of most of the existing microfluidic\npumping methods including electrohydrodynamic, piezoelectric, electroosmotic,\nand electrostatic micropumps ( Figure S8 ), 50 , 51 while the proposed concept requires no physical\nconnection to peripheral equipment, reduces the usage of reagents\nby minimizing “dead volumes”, avoids undesirable electrical\neffects, and accommodates a wide range of different fluids. However,\nto create fully integrated on-chip pumps, more work needs to be done\nincluding standardizing the geometry of the μMAC and the microfluidic\nchips, building a benchmark database for the corresponding pumping\ncapability, and creating a portable magnetic actuation setup. To move\ntoward the real applications of the metachronal soft robots, more\nstudies need to be performed, for example, on the effect of surface\nroughness on the robot locomotion and on biocompatibility for biomedical\napplications. Furthermore, we have shown that the metachronal robots\ncan move both in air and in liquid, however, to obtain fully amphibious\nrobots requires them to be able to transit through the liquid–air\ninterface, which we have not been able to demonstrate. This may require\nsurface modification or multimodal locomotion capacities including\nstrong global body deformations. 2 , 3 In some cases, a biodegradable\nrobot body may be preferred.",
"introduction": "1 Introduction Stimuli-responsive materials have drawn extensive attention in\nvarious applications including industrial soft robots, 1 small-scale biomedical robots, 2 − 4 implantable\nand wearable devices, 5 sensors, 6 droplet and particle manipulators, 7 self-cleaning and anti-fouling surfaces, 8 , 9 and on-chip integrated liquid mixers and pumps. 10 Magnetically responsive materials, among other materials\nsuch as light-driven, 11 pH-driven, 12 pressure-driven, 1 and electric field-driven materials, 13 enable the integration of such merits as a fast and reversible response\nand remote activation without the need for physical connections to\nan external actuation setup. Moreover, magnetic fields can easily\nand harmlessly penetrate most biological and synthetic materials. 14 These advantages boost the research on magnetic\nartificial cilia (MAC), a type of magnetic-responsive structures inspired\nby nature, for applications including fluid pumping, 10 , 15 liquid mixing, 16 particle manipulation, 17 as well as soft robots. 18 , 19 Fluid pumping is one of the paramount functions in lab-on-a-chip\ndevices where biological analyses and chemical syntheses are performed\nin small volumes (typically from 10 –4 to 10 –8 L). 20 Nevertheless, current\nmethods for fluid pumping suffer from the need for large peripherals\nsuch as syringe pumps and tubing connections, which makes the platform\ncumbersome and nonportable. Recent advances in the study of using\nartificial cilia as a means of fluid pumping pave the way for the\ndesign and fabrication of on-chip integrated pumps. 21 − 24 In terms of pumping capability,\nmetachronal cilia, where neighboring cilia move slightly out-of-phase\ncreating a travelling wave, can lead to a 3-fold increase in propulsion\nvelocity and a 10-fold increase in efficiency compared to synchronous\ncilia. 25 This has shifted the focus of\nresearchers from the design and fabrication of synchronous cilia to\nmetachronal cilia. The mechanism of creating metachronal cilia can\nbe summarized into two categories: (i) applying different forces to\neach cilium within an array of cilia 23 , 26 , 27 and (ii) designing an array of cilia with different\nresponses to a uniformly applied stimulus such as a uniform magnetic\nfield. 19 , 27 − 30 The latter is beneficial due\nto its much simpler actuation method. The fabrication approaches presented\nin previous papers, however, are either time-consuming because complex\nassembly steps are needed 28 , 29 or they are based on\nexpensive raw materials, costly processes, or complex actuation devices. 19 , 27 , 28 , 30 Moreover, the relatively large size of the reported metachronal\ncilia, typically several millimeters or larger, 19 , 28 − 30 render them difficult to be integrated in common\nmicrofluidic devices. Two of these previous publications demonstrated\nfluid flow induced by the metachronal artificial cilia. 19 , 28 In nature, metachronal wave motion of biological cilia enables\npropulsion and locomotion of many creatures, 19 , 31 in addition to egg cell transportation, 32 mucus clearance in the human respiration system, 33 feeding assistance, 34 and self-cleaning\nand antifouling. 35 Locomotion is a fundamental\nfunction desired for future biomedical robots that has been shown\nto potentially enable capabilities such as minimally invasive surgery,\ntargeted therapeutics and diagnostics, tissue engineering, and single-cell\nanalysis. 36 , 37 Due to the diverse, dynamic, and complex\nnature of tissues, biomedical robots should possess a high degree\nof mobility and be able to overcome different obstacles with different\nslope angles in order to navigate in hard-to-reach regions of the\nhuman body. A few studies have demonstrated the obstacle-crossing\ncapability of soft robots, such as crossing a small hill 18 and a small tunnel, 2 as well as transiting at the air–liquid interface. 2 However, in these demonstrations either the reported\nactuation method is sophisticated or the design of the robots is complex,\nlimiting their common application. Here, we demonstrate a facile\nway to create metachronal microscopic\nMAC (μMAC) by controlling the paramagnetic particle distribution\nwithin the cilia array. Specifically, the metachronal μMAC are\nfabricated using a micromolding process, during which the distribution\nof the paramagnetic particles in the μMAC is precisely controlled\nby placing a rod-shaped magnet array, arranged to have an alternating\ndipole orientation between consecutive magnets, underneath the mold.\nBecause the paramagnetic particles tend to align with the applied\nmagnetic field, neighboring cilia will assume different paramagnetic\nparticle distributions, and they will, therefore, have different magnetic\nproperties. Consequently, the geometrically identical μMAC exhibit\nnonidentical bending behaviors in a static uniform magnetic field\nand perform a metachronal motion in a 2D rotating uniform magnetic\nfield. The metachronal μMAC show excellent microfluidic pumping\ncapacity and a promising capability to realize versatile climbing\nrobots. Our metachronal μMAC outperform most previously reported\nμMAC in terms of the fluid pumping efficiency characterized\nby a normalized global flow speed. We also show that the metachronal\nμMAC enable superior climbing soft robots that can carry cargos\n10 times their own weight, functioning in both dry and wet conditions.\nMoreover, the ciliated robots are able to climb slopes with angles\nranging from 0 to 180°. Compared to the previously reported metachronal\ncilia with spatially controlled magnetic anisotropy, our metachronal\nμMAC provide a number of key advantages: (i) the ease of fabrication\nthat allows for the replication of artificial cilia in large numbers,\n(ii) the need for only a simple actuation scheme, (iii) the excellent\nfluid pumping capacity, and (iv) the creation of versatile climbing\nsoft robots. In addition, our metachronal μMAC are smaller in\nsize than the previously reported metachronal magnetic cilia which\nfacilitates their real applications in diverse fields including on-chip\nintegrated micropumps and particle manipulation. The capability to\ncreate versatile climbing soft robots creates new options to realize\nbiomedical robots.",
"discussion": "2 Results and Discussion 2.1 Microscopic MAC (μMAC) The\npolydimethylsiloxane (PDMS)- and paramagnetic carbonyl iron powder\n(CIP)-based μMAC are fabricated using a highly reproducible\nmicromolding process ( Figure 1 A, details are available in the Experimental\nSection ). Based on the fact that the paramagnetic particles\ntend to form chains that are aligned with the direction of the applied\nmagnetic field, we placed an array of rod-shaped magnets, which are\norganized to have an alternating dipole orientation between consecutive\nmagnets, underneath the mold ( Figure 1 A(v)). In this way, a nonuniform but periodic magnetic\nfield is generated ( Figure 1 B) such that the paramagnetic particle chains are expected\nto have different orientations in neighboring cilia ( Figure 1 A(vi)). Movie S1 shows the alignment process of the paramagnetic particles\nwhen the mold is approached by the rod-shaped magnet array. In all\nexperiments reported in this paper, the fabricated μMAC are\narranged in a rectangular grid ( Figure 1 C), and each cilium has a cylindrical shape with a\ndiameter of 50 μm and a height of 350 μm ( Figure 1 D), standing on a transparent\nnonmagnetic PDMS substrate. As the rod-shaped magnets have a diameter\nof 4 mm, the period of the generated magnetic field is also 4 mm ( Figure 1 B). The length of\nthe μMAC array was chosen approximately equal to the magnetic\nfield period, that is, 4 mm, and the width was chosen to always have\n10 cilia. Thus, for the μMAC array with a pitches of 350, 450,\nand 550 μm between adjacent cilia, there are 12 × 10 =\n120 cilia, 9 × 10 = 90 cilia, and 8 × 10 = 80 cilia, respectively.\nNote that the period of the μMAC array can be tuned by using\nmagnets with different diameters, and the width can be reduced to\nminimally 2 cilia (∼400 μm). Figure 1 Magnetic field-assisted\nfabrication of metachronal μMAC.\n(A) Micromolding process of the metachronal μMAC, during which\nthe paramagnetic particle distribution can be programmed by placing\na magnet-array underneath the mold in the sample curing step. See Movie S1 . (B) Snapshot of a COMSOL simulation\nof the magnetic flux density B induced by the rod-shaped\nmagnet-array that is arranged with an alternating dipole orientation\nbetween adjacent magnets. The horizontal white line indicates the\ncentral position of the μMAC array, that is, 0.7 mm above the\ntop surface of the magnet array. The red arrows indicate both the\ndirection and the magnitude of B at the locations of\neach cilium, with a pitch of 350 μm. See Figure S1 for more details about the generated magnetic field.\n(C,D) Top-view and side-view SEM images of the fabricated μMAC\narray showing the arrangement in a rectangular grid and their cylindrical\nshape. The cylindrical μMAC have a diameter, a height, and a\npitch of 50, 350, and 350 μm, respectively. (E) Schematic drawing\nof the alignment direction of the paramagnetic particles (black line\nsegments) in one row of μMAC (pitch = 350 μm) according\nto the COMSOL simulation in panel (B), by assuming that the particle\nchain direction is in the same the direction as the magnetic field.\nThe middle of the μMAC array is assumed to be placed at x = 0 in panel (B). The theoretically predicted acute angle\n(θ) between the magnetic particle chain and the vertical direction\nis shown above the corresponding cilium. See Figure S1 for results of other configurations of the magnet array.\nIllustration is not to scale. (F) Side-view optical microscopy image\nof one row of μMAC (pitch = 350 μm) showing the experimentally\nobtained acute angle (θ) between the magnetic particle chain\nand the vertical direction. The red arrows shown in Figure 1 B indicate the magnetic flux density B applied on the central part of the μMAC array, which is approximately\n700 μm (half of the cilia height, ∼175 μm, plus\nthe thickness of the silicon wafer, ∼525 μm) above the\nsurface of the magnet array. The details of the magnetic field are\navailable in Figure S1 , which also shows\na comparison between different magnet array configurations. According\nto the simulated direction of B , the expected alignment\nof the paramagnetic particles in one row of the μMAC array with\na pitch of 350 μm is depicted in Figure 1 E, where the acute angle θ between\nthe expected direction of the paramagnetic particle chain and the\nnormal direction to the PDMS substrate is shown above the corresponding\ncilium. The experimental results of the μMAC array made of the\nPDMS/CIP composite with a weight ratio of PDMS/CIP = 2:1 are shown\nin Figure 1 F, showing\ngood agreement with the simulation results. Thanks to the well-controlled\nconcentration of paramagnetic particles, the difference in the CIP\nchain orientation is expected to result in a difference in magnetic\nproperties of neighboring cilia and is the basis for the metachronal\nbehavior of the μMAC array. Note the difference in the distribution\nof paramagnetic particles between the cilia at symmetric positions,\nas shown in Figure 1 F, which is probably the result of a slight misalignment of the magnet\narray. 2.2 Magnetic Particle Distribution and μMAC\nBending Behavior Figure 2 A shows the magnetic particle distribution at different\nconcentrations and the corresponding μMAC bending behavior in\na static vertical uniform magnetic field of 280 mT generated by the\nelectromagnetic setup reported by Wang et al. 38 The control μMAC, which have a unique paramagnetic particle\ndistribution which is along the long axis of the cilia, are fabricated\nusing the micromolding method reported in our earlier work. 39 In contrast to the control μMAC that do\nnot bend at all, the μMAC fabricated using the process, as shown\nin Figure 1 A, display\nbending angle differences between neighboring cilia for all three\nmagnetic particle concentrations. This is due to the fact that the\nmagnetization direction of each cilium is close to the magnetic particle\nalignment direction: the magnetic anisotropy of the μMAC is\ncaused by both the shape anisotropy of the cilium itself and the anisotropy\nof the magnetic particle alignment, with the latter anisotropy dominating\nin our case. 40 Consequently, due to the\ninduced magnetic torque in a uniform magnetic field, a cilium tends\nto bend so as to align its magnetic particle chain direction with\nthe applied magnetic field ( Figure 2 B); the extent of the bending depends on the direction\nand magnitude of the magnetization as well as the cilia stiffness.\nBecause of the difference in the magnetic particle alignment between\nadjacent cilia, the μMAC in an array exhibit different bending\nbehaviors, which is needed to generate metachronal behavior in a uniform\nmagnetic field. The bending is smallest for the highest particle concentration\n(PDMS/CIP = 1:1) because the level of particle alignment anisotropy\nis the smallest, and the stiffness is the highest. Figure 2 Magnetic particle distribution\nand μMAC (pitch = 350 μm)\nbending behavior in a static uniform vertical magnetic field. (A)\nOptical microscopy images of μMAC arrays made with different\nmagnetic particle concentrations, showing the particle distribution\nand cilia bending behavior in a static uniform magnetic field of 280\nmT. (B) Schematic drawing of the mechanism behind the cilia bending,\nwhere B is the applied magnetic field, m is the magnetization of the cilia, and T is the resulting\nmagnetic torque acting on the cilia. Illustration is not to scale.\n(C,D) Quantitative results of the magnetic particle distribution and\nthe bending angle [α in panel (A)] of the μMAC arrays.\nThe particle chain direction in panel (C) represents θ in Figure 1 F. The bending angle\nis denoted by the angle between the substrate normal and the straight\nline connecting the tip and the bottom of each μMAC. (E) Phase\ndifference deducted from the bending angle differences between the\nneighboring cilia calculated from the results shown in panel (D).\n(F) Bending angle of the cilia at the two ends of the μMAC array\n(2 PDMS/1 CIP) as a function of the magnitude of the static vertical\nmagnetic field (see Movie S2 ). Each experimental\ndata point was obtained by averaging the results of at least five\nidentical but independent experiments. The quantitative results of the particle alignment are shown in Figure 2 C. The particle chain\ndirection is characterized by the acute angle θ to the substrate\nnormal, as shown in Figure 1 F. Clearly, the particle alignment agrees well with the theoretical\nprediction, as shown in Figure 1 E, for the two arrays with PDMS to CIP weight ratios of 4:1\nand 2:1. For the cilia array with a PDMS to CIP weight ratio of 1:1,\nthe particle alignment shows quite some variation for cilia in the\nsame column as is evident from the large error bars. This is probably\nbecause the high particle concentration results in an unwanted particle\nchain connection along the long axis of the cilia. For the control\nμMAC, on the contrary, the particle chain direction is almost\nperfectly oriented along the long axis of the cilia as expected. The\nbending angle (α in Figure 2 A) of these μMAC arrays in an uniform vertical\nmagnetic field of 280 mT is depicted in Figure 2 D. The results show a similar trend to that\nin Figure 2 C, while\nμMAC made using our new method with a PDMS to CIP weight ratio\nof 2:1 show the smoothest transition in the bending angle, which is\nthe most promising for generating metachrony. Note that a relatively\nlow magnetic particle concentration renders not only a smaller magnetization\nbut also a lower stiffness, which is why the μMAC made of PDMS\nto CIP weight ratios of 2:1 and 4:1 show a similar bending behavior.\nIn order to confirm that a weight ratio of 2:1 between PDMS and CIP\nresults in the most promising μMAC for generating a controlled\nmetachronal motion, we calculated the bending angle difference (i.e.,\nphase difference) between neighboring cilia based on the results shown\nin Figure 2 D and plotted\nthe results in Figure 2 E. Indeed, the phase difference of the μMAC made of a PDMS\nto CIP weight ratio of 2:1 is the smoothest, although there is some\nperturbation. The large variation for the μMAC made of a PDMS\nto CIP weight ratio of 4:1 is mostly due to the inhomogeneity of the\nmagnetic mixture, which results in a relatively large variation in\nthe magnetic properties of the μMAC in the same column. Note\nthat the angle differences between the neighboring cilia is largely\nconsistent during continuous actuation using a rotating magnetic field\n(see the following section), resulting in a stable phase difference\nfor the metachronal movement. Based on the above results, we\nchoose μMAC made with a PDMS\nto CIP weight ratio of 2:1 for the following experiments. Figure 2 F shows the bending\nbehavior of the cilia at the two ultimate sides of one row of μMAC\nas a function of the magnitude of the applied vertical magnetic field.\nAs expected, the two cilia exhibit a symmetrical response. Their bending\nangle initially increases slowly up to 80 mT, then increases steeper\nbetween 80 and 120 mT, and moderately increases along with the applied\nmagnetic field above 120 mT. This behavior is the result of the competition\nbetween the elastic stiffness of the cilium and the magnetic torque\nacting on the cilium. Note that the magnetization of the paramagnetic\nparticles has a nonlinear relationship with an applied magnetic field. 40 The bending behavior of the whole row of μMAC\nat different magnetic fields can be found in Movie S2 . 2.3 Metachronal μMAC\nMotion in a 2D Rotating\nMagnetic Field and Fluid Pumping Capability As shown above,\nthe deformation of adjacent μMAC in an array exhibits an angle\ndifference in a static uniform magnetic field. Here, we investigate\nwhether this leads to metachronal motion in a uniform rotating field.\nTherefore, we built a setup consisting of two permanent magnets (50\n× 50 × 12.5 mm 3 ) with their opposing magnetic\npoles facing each other at a distance of 50 mm ( Figure 3 A). The magnets are mounted in a frame driven\nby an electric motor. In this way, a 2D rotating quasi-uniform magnetic\nfield of approximately 150 mT is generated at the central space between\nthe two magnets (see Figure S2 for details),\nwhere the μMAC array (PDMS/CIP = 2:1) is located. The μMAC\narray is covered with a microfluidic chip ( Figure 3 B) and is positioned within a square circulatory\nchannel with a rectangular cross section (height 2 mm and width of\n6 mm). A camera mounted on a microscope is used to observe the motion\nof the μMAC array in the 2D rotating uniform magnetic field\nand the generated liquid flow from the side ( Figure 3 A). Unless otherwise specified, the rotating\ndirection of the motor is counterclockwise as seen from the microscope\nfor the experiments reported in this section. Figure 3 Metachronal μMAC\nmotion in a 2D rotating uniform magnetic\nfield and the fluid pumping capability. (A) Schematic diagram of the\nmagnetic actuation setup for creating a 2D rotating magnetic field.\nThe details of the generated magnetic field are available in Figure S2 . Illustration is not to scale. (B)\nSchematic drawing of the square microfluidic chip including the μMAC,\nindicating the observation area of the generated flow. Illustration\nis not to scale. (C) Snapshots of the metachronal motion of one row\nof μMAC with a pitch of 550 μm during one beating cycle\nat 1 Hz in water (see Movie S3 ). The electric\nmotor rotates counterclockwise. The white arrow indicates the traveling\ndirection of the metachronal wave. The red arrows indicate the direction\nof the applied magnetic field. The encircled numbers indicate the\nsequence of the μMAC for reference in later analyses. (D) Side-view\ntime-lapse images of cilium motion at 1 Hz in both water and glycerol,\nshowing a 2D symmetric motion. β represents the opening angle\nof the cilium motion. The blue dashed line and the red dashed line\nindicate the tip trajectory during the magnetic stroke and elastic\nstroke, respectively. The motion of cilium 4 during the elastic stroke\nin both water and glycerol is available in Movie S6 . The motion of the whole μMAC array in both water\nand glycerol can be found in Figure S3 .\nNote the difference in the recording speed for the metachronal μMAC\nand the control μMAC, as well as for in water and in glycerol.\nSee Experimental Section for details. (E,F)\nGenerated flow speed of water (E) and glycerol (F) as a function of\nthe beating frequency of the metachronal μMAC array with pitches\nof 350, 450, and 550 μm, respectively, as well as the control\nμMAC array with a pitch of 550 μm. A positive result represents\na flow above the ciliated area in the same direction as the metachronal\nwave traveling direction. The trajectories of the tracer particles\nin both water and glycerol can be found in Figure S4 . The corresponding volumetric flow rate and pressure drop\nare available in Figure S5 . Each data point\nwas obtained by averaging the results of at least ten measurements. Figure 3 C shows\nthe motion of one row of μMAC (pitch = 550 μm) in the\n2D rotating uniform magnetic field at a beating frequency of 1 Hz\nin water (see also Movie S3 ). Note that\nthe μMAC beating frequency is 2 times the rotating frequency\nof the electric motor due to the symmetry of the generated magnetic\nfield in the first and the second halves of one rotating cycle of\nthe motor. Even though the directions of the magnetic fields are opposite\nto each other in the two different cycle halves, this has no effect\non the bending behavior of the μMAC. It is clearly shown in Figure 3 C that the μMAC\narray performs a wave-like motion, and thus indeed exhibits metachrony.\nThe metachronal wave traveling direction is to the right as seen from\nthe microscope. In contrast, Movie S4 shows\nthat the control μMAC perform a 2D synchronous motion. We chose\nthe μMAC array with a pitch of 550 μm instead of 350 μm\nbecause the neighboring cilia are too close to move freely for 350\nμm pitch, touching each other (see Movie S5 ). Figure 3 D shows that each cilium performs a 2D symmetric whip-like motion\nin the vertical plane consisting of two strokes: (i) a magnetic stroke\nwhen the cilium mostly follows the applied magnetic field and bends\nto the left, thereby accumulating elastic energy (indicated by the\nblue dashed line), and (ii) an elastic stroke when the cilium tip\nstarts to move upward and whips back to its original position by releasing\nthe accumulated elastic energy (indicated by the red dashed line).\nA high-speed video of the elastic stroke part of the 2D symmetric\nmotion of cilium 4 in both water and glycerol is available in Movie S6 . This movie shows that the cilium vibrates\nfor a certain amount of time at the end of the elastic stroke before\nit reaches its equilibrium state in water but not in glycerol. This\nis a result of the competition between the elastic forces, magnetic\nforces, and the fluidic viscous drag, the latter being around 1000\ntimes larger in glycerol based on Stokes’ law. The motion of\nthe whole μMAC array can be seen in Figure S3 . It shows that neighboring cilia exhibit motions with different\nopening angles. Specially, the motion of cilia 4, 5, and 6 has a larger\nopening angle than that of the others; this is caused by the fact\nthat cilia at the central part of the μMAC array (cilia 4, 5,\nand 6) contain longer magnetic particle chains and thus stronger magnetization\nthan the cilia at both ends (cilia 1, 2, 3, 7, and 8). Also, the left\npart of the μMAC array performs almost exactly the same motion\nas the right part of the μMAC array, that is, the behavior is\nsymmetric with respect to the center of the array. This is because\ncilia at opposing positions contain similar magnetic particle distribution.\nIt is important to stress that the 2D symmetric whip-like motion shown\nin this article is similar to that we recently reported, 26 but not exactly the same because the motion\nshown in the current work does not contain the so-called sliding stroke,\nand thus, it is a symmetric motion (see Figure 4 for details). Metachrony is termed symplectic,\nantiplectic, and laeoplectic when the metachronal wave travels in\nthe same, opposite, and perpendicular directions as the effective\nstroke, respectively. 41 The effective stroke\nis conventionally defined as the stroke during which one cilium moves\nmore straight and thus sweeps a larger area than during the backward/recovery\nstroke, when the cilium performs an asymmetric motion. This definition,\nhowever, cannot be applied to our current results as our μMAC\nperform a symmetric motion, as seen in Figure 3 D, and therefore, the common definition of\nsymplectic or antiplectic metachrony cannot be applied here as well.\nIn our experiments, the metachronal wave always travels in the same\ndirection as the fast elastic stroke, which is to the right in Figure 3 . Figure 4 Quantitative analyses\nof the μMAC motion in both water and\nglycerol. (A) Tip speed of the μMAC array with a pitch of 550\nμm during one beating cycle at 1 Hz. T = 0\ncorresponds to t = 0 s as shown in Figure 3 A. The peak of the tip speed\noccurs during the elastic stroke of each cilium, and the time difference\nbetween these peaks represents the phase difference of the μMAC\nmotion. (B) Calculated local maximum Reynolds number at 1 and 10 Hz\nbased on the maximum tip speed in panel A and Figure S6A . See S7 for details\nabout the calculation. (C) Swept area by the μMAC during the\nmagnetic stroke and the elastic stroke at 1 Hz, respectively, showing\nthat there is no difference in the swept area during the two strokes,\nindicating that the μMAC motion is symmetric. (D) Opening angle\n(β in Figure 3 D) of the μMAC motion at 1 and 10 Hz. The tip speed and swept\narea of the μMAC motion at 10 Hz in both water and glycerol\ncan be found in Figure S6 . Each data point\nin panel (C,D) was obtained by averaging the results of at least three\nmeasurements. Despite the symmetry of the metachronal\nμMAC motion, they\ncan generate substantial water flow ( Figure 3 E) and glycerol flow ( Figure 3 F) in the aforementioned microfluidic chip\n( Figure 3 B). Here,\nthe positive results indicate that the flow direction above the ciliated\narea is in the same direction as the metachronal wave traveling direction,\nand the negative results indicate that the flow direction above the\nciliated area is in the opposite direction to the metachronal wave\ntraveling direction. In other words, the metachronal μMAC induce\nwater flow and glycerol flow with opposite directions even though\nthey perform the same metachronal motion. Figure S4 shows the time-lapse images of the used tracer particles\nover a specific period in both water and glycerol, indicating the\nflow speed and direction. Note that due to the cyclic nature of the\ncilia motions, cyclically pulsating flow may be expected to occur.\nHowever, we did not observe the pulsating flow of either water or\nglycerol at the recording frame rates of 10 and 0.1 fps, respectively.\nThis is likely due to the fact that our recording frame rate is not\nhigh enough to detect the pulsating flow. The maximum water flow velocity\nis 220 μm s –1 , which is generated by the metachronal\nμMAC with a pitch of 350 μm at 10 Hz (the limit of our\nactuation setup). This corresponds to a volumetric flow rate of 85\nμL min –1 and a local pressure drop of 0.027\nPa in our microfluidic channel (see S5 for\ndetails). The maximal glycerol flow velocity is 5.5 μm s –1 generated by the same μMAC array with a pitch\nof 350 μm at 10 Hz, which corresponds to a volumetric flow rate\nof 2.1 μL min –1 and a local pressure drop\nof 1 Pa in our channel (see S5 for details).\nDue to the ease of control over the rotation of the electric motor,\nversatile flows can be generated in principle, for example, with time-varying\nflow rates, just as we reported before. 26 , 40 Several\nobservations can be made from Figure 3 E,F: (1) the generated water flow is in the\nsame direction as the metachronal wave traveling direction and the\nelastic stroke direction as well, while the glycerol flow is in the\ndirection opposite to the metachronal wave traveling direction; (2)\nthe control μMAC cannot induce any significant flow in glycerol;\n(3) the μMAC array with a smaller pitch, thus containing more\ncilia, generates a higher water flow, but does not always generate\na higher glycerol flow; (4) the water speed increases linearly with\nthe beating frequency of the μMAC, and the glycerol speed has\na slightly less than linear relationship with the cilia beating frequency;\nand (5) the metachronal μMAC generates a water flow of approximately\ntwice that generated by the control μMAC. The mechanisms underlying\nthese observed phenomena are similar to what we recently reported, 26 namely, inertial effects, asymmetric motions,\nand metachrony. As shown in Figure 4 A, the maximum cilia tip speed during the elastic stroke\nin water is in the order of m s –1 , which leads to\na maximum local Reynolds number ( Re max ) in the order of hundreds, independent of the beating frequency\n( Figure 4 B, see S7 for details about the calculation). Note that\nthe average Re during the magnetic stroke in water\nis around 0.1 and 1 at 1 and 10 Hz, respectively. This means that\ninertial effects dominate over viscous effects in water during the\nelastic stroke but not during the magnetic stroke. In glycerol on\nthe other hand, the cilia move much slower, and Re max is much smaller than 1 during the whole cycle, and\nthus viscous effects prevail. Note that in Figure 4 A, the beginning of the x axis corresponds to t = 0 s, as shown in Figure 3 C, when the magnetic\nfield is perpendicular to the cilia substrate and that the phase difference\nbetween the peaks of the tip speed also represents the phase difference\nof the μMAC motion. Figure 4 C shows that each cilium sweeps the same area during\nthe magnetic stroke as that during the elastic stroke, which means\nthat the μMAC motion is a symmetric motion, both in water and\nin glycerol. Based on these analyses, the explanations of the\ndirection of the\nwater flow and the glycerol flow [observation (1)] are as follows.\nIn glycerol, only metachrony works, and the out-of-phase motion of\nthe μMAC array creates a net pressure gradient, which results\nin a unidirectional glycerol flow whose direction is opposite to the\ndirection of the metachronal wave. Detailed discussion of the relationship\nbetween the directions of the glycerol flow and metachrony can be\nfound in ref ( 42 ).\nTherefore, we conclude that the metachrony of our μMAC array\ngenerates a flow in the opposite direction to the metachronal wave\ntraveling direction in glycerol. Next, the explanation for the water\nflow direction is the following. In water, in addition to the metachrony,\ninertial effects operate which induce a flow in the elastic stroke\ndirection (i.e., metachronal wave travelling direction) as observed.\nThe contribution of metachrony remains unclear, however. Based on\nthe glycerol results, inertia and metachrony could potentially counteract\neach other, and this means that the resulting water flow could be\na competition between these two mechanisms with the inertial effects\ndominant over metachrony in water. This suggests that it might be\nbeneficial to design a μMAC array whose elastic stroke direction\nis opposite to the metachrony traveling direction to further improve\npumping efficiency in low viscosity liquids. However, the metachronal\nmotion may well enhance the inertially driven flow, as can be deduced\nfrom earlier numerical studies; 42 this\nis supported by observation (5) but needs to be studied in more detail\nusing advanced numerical modeling in the future. Observation (2) confirms\nfrom another angle that the metachronal wave does contribute to the\npumping efficiency in the low Re regime in glycerol.\nAs for the relationship between the flow speed and the number of cilia\n[observation (3)], we believe it is a result of the competition between\nthe pumping efficiency of each cilium and the dependency of the pumping\nefficiency of the metachrony on the cilia pitch. Detailed discussions\ncan be found in refs, 25 , 43 and. 44 Note that inertia\nplays an important role in water but not in glycerol. This indicates\nthat the optimal μMAC configuration for pumping efficiency can\nbe different for liquids with different viscosities. As for observation\n(4), the relationship between the flow speed and the beating frequency\nof the μMAC is due to the fact that the net water flow induced\nby the μMAC array per beating cycle is almost constant as the\nμMAC motion is almost independent of the beating frequency,\nwhile the net glycerol flow induced by the μMAC array per beating\ncycle decreases as the μMAC motion diminishes at a higher beating\nfrequency in glycerol ( Figure 4 D). The change in the μMAC motion results from the competition\nbetween the elasticity of the μMAC and the viscous drag, with\nviscous drag dominating in glycerol at high beating frequencies. 26 Observation (5) does not contradict observation\n(1) because the enhanced water flow is not necessarily because of\nthe metachrony but could also result from the difference in the magnetic\nproperties of the metachronal μMAC and the control μMAC\ndue to their difference in the magnetic particle arrangement, which\ncan be seen from the tip speed ( Figure 4 A) and the motion opening angle ( Figure 4 D). This asks for more in-depth numerical\nanalysis as mentioned. In any case, observation (5) does underline\nthat the pumping efficiency can be substantially enhanced by tuning\nthe magnetic particle distribution within the μMAC. 2.4 Ciliated Soft Robots Soft robots\nhave shown remarkable potential for applications ranging from bioengineering\nto minimally invasive surgery. 36 , 45 Magnetic programmable\nrobots offer potential to become the next generation of microsystems\nwith advanced locomotion and manipulation capabilities. 14 Here, we demonstrate the superior mobility of\nciliated soft robots enabled by the metachronal motion of the μMAC\non flat surfaces and slopes with angles ranging from 0 to 180°\nin air. We also show their remarkable cargo carrying capability in\nboth air and liquid. The ciliated soft robots reported here are inverted\nμMAC arrays with a pitch of 350 μm consisting of 12 ×\n10 = 120 cilia; thus, the robots are approximately 4 mm long and 3.5\nmm wide. For easy referencing, we name the ciliated robots made from\nthe metachronal μMAC “metachronal robots”, and\nthe ciliated robots made from the synchronously moving μMAC\n“control robots”. The high-speed locomotion of\none metachronal robot in air during one beating cycle at 1 Hz is shown\nin Figure 5 A (see also Movie S7 for the high-speed free μMAC motion\nin air and the locomotion of the metachronal robot). Note that all\nsurfaces mentioned here are made of glass coated with a thin layer\nof lubricant oil in order to tune the adhesion between the μMAC\nand the surface (see Experimental Section ).\nFor too low adhesion (resulting from too much lubricant oil), the\nciliated robot will remain stuck to the surface showing no forward\nmovement; for too high adhesion (resulting from no or too little lubricant\noil), the robot will not be able to move at all. Figure 5 B quantifies the displacement\nof the central point of the robot body in both horizontal and vertical\ndirections. Initially ( t = 0 s), the applied magnetic\nfield is perpendicular to the glass surface and the μMAC bend\nin a direction that depends on their location in the array with the\ncenter of the body as the mirror plane, to the left on the left side,\nand to the right on the right side. When the magnetic field starts\nto rotate clockwise due to the rotation of the electric motor (0–0.2\ns), the front μMAC (on the right) tend to bend to the left (corresponding\nto the magnetic stroke, as shown in Figure 3 D, see Movie S7 ). Due to the adhesion between the cilia and the surface, this motion\ncauses the body of the metachronal soft robots (150 μm thick)\nto move forward. Meanwhile, the back μMAC tend to move to the\nright (corresponding to the elastic stroke in Figure 3 D), but due to the cilia surface friction,\nthey remain to be bent more toward the left and move forward together\nwith the robot body. When the magnetic field rotates further (0.2–0.6\ns), the central part of the robot body moves downward along with the\nbending of the μMAC at the middle part of the array (corresponding\nto the magnetic stroke in Figure 3 D). This pushes the robot forward further. Later on\n(0.6–0.8 s), the μMAC array bends to the right (corresponding\nto the elastic stroke in Figure 3 D) in a metachronal fashion, which lifts the robot\nupward and pushes the robot slightly backward as a result of the competition\nbetween the frictional and adhesive forces acting on the front μMAC\nand the back μMAC, respectively. Lastly (0.8–1 s), the\nfront μMAC return to their original orientation and drive the\nrobot forward further. As a result, the metachronal robot has moved\nahead by a distance of approximately one cilia length, that is, 0.35\nmm. Due to the symmetry of the μMAC array in terms of the particle\ndistribution, the ciliated robot can walk in the opposite direction\nwhen the rotating direction of the magnetic field is reversed ( Figure 5 C, see also Movie S8 ), demonstrating bi-directional locomotion\ncapability. Figure 5 Locomotion of ciliated soft robots in air. (A) Snapshots of the\nlocomotion of the metachronal robot in air during one beating cycle\nat 1 Hz when the magnetic field rotates clockwise. See Movie S7 . (B) Central body displacement along\nboth the horizontal (blue square line) and vertical (red circle line)\ndirections during one beating cycle at 1 Hz. The data points are connected\nby the B-Spline function in Origin. The trajectory of the central\nbody over five beating cycles can be found in Movie S7 . (C) Demonstration of bi-directional walking capability\nof the metachronal robot by reversing the rotating direction of the\nmagnetic field. See Movie S8 . (D) Walking\nspeed of the metachronal robot in air as a function of the beating\nfrequency, including the walking speed of the control robot at 1 Hz\nas a reference. Note the two y axes, with the left y axis indicating the absolute value of the walking speed\nand the right y axis indicating the speed relative\nto the cilia length. (E) Normalized walking speed by dimensionless\nnumber V fl , representing the locomotion\ndistance per beating cycle relative to the cilia length. Each data\npoint was obtained by averaging the results of at least five identical\nbut independent experiments. Figure 5 D shows\nthat the metachronal robot moves 3 times faster than the control robot.\nThe locomotion of the control robot can be found in Movie S9 , which shows that the control robot vibrates as a\nwhole in the rotating magnetic field due to the synchronous motion\nof the μMAC array. Also, its body remains straight in contrast\nto that of the metachronal robots of which the body performs a wave-like\ndeformation, as can be seen in Figure 5 A,B, as well as in Movie S7 . This suggests that the more efficient locomotion of the metachronal\nrobot may be partly explained by this effect, in addition to the metachronal\nmotion of the cilia. In fact, previous studies have demonstrated magnetic\nsoft robots shaped as rectangular thin films (without cilia or legs)\nthat exhibit net motion precisely due to the undulating wavy deformation\nof the soft body. 2 , 30 However, the locomotion speed\nof our ciliated metachronal robot is substantially larger than that\nof the film-shaped robot demonstrated by Shinoda et al., 30 as detailed in Section S9 . This suggests that the contribution of the wavy deformation to\nthe total locomotion is minor relative to the effect of the metachronal\nmotion of the cilia, acting as the robot’s legs. Nevertheless,\nthe efficient locomotion of the metachronal robots might benefit from\nthe soft nature of the robot body, which can be investigated in the\nfuture by varying the body’s stiffness. The walking speed\nof our metachronal robot scales slightly less\nthan linear with the beating frequency of the μMAC, as can be\nseen in Figure 5 D.\nThe maximum walking speed is approximately 2.3 mm/s at 10 Hz, corresponding\nto almost 7 times the cilia length per second, a proportion similar\nto humans at running speed. 18 Note that\nthe maximum walking speed is limited by our actuation setup and can\npotentially be increased using an actuation setup that can generate\na faster magnetic field rotation. We also quantified the walking speed\nof our metachronal robots using a dimensionless number V fl , that is, the walking distance per beating cycle relative\nto the cilia length ( Figure 5 E). It shows clearly that the walking efficiency of the metachronal\nrobot slightly drops with the increasing beating frequency. On average,\nthe metachronal robot moves at a speed of approximately 0.75 cilia\nlength per beating cycle, which is 3 times better than the control\nrobot. The decrease in the walking efficiency at a higher beating\nfrequency is probably due to an increased slip of the μMAC on\nthe surface especially during the magnetic stroke, which renders the\nμMAC not being able to fully push the robot body forward sufficiently\nduring a single beating cycle. Obstacle-crossing and cargo-carrying\ncapabilities are essential\nfor biomedical robots. Our metachronal robots possess such capabilities\n( Figure 6 ). Note that\nall surfaces used here are made of polymethyl methacrylate (PMMA)\ncoated with a thin lubricant layer (see Experimental\nSection ). Note also that the beating frequency of the μMAC\narray is 2 Hz. Figure 6 A shows that the metachronal robot is able to climb across a small\nhill with a slope of 45° in air (see Movie S10 ). The metachronal robot struggles at the corners of the\nhill (40–90 and 100–130 s, respectively). This is mainly\nbecause only part of the μMAC array can touch the surface at\nthose locations, resulting in reduced work from the μMAC array\nper beating cycle. Consequently, the moving speed is slower than when\nthe whole μMAC array can touch the surface. This suggests an\ninteresting topic of study on the contribution of different parts\nof the μMAC array to the moving speed of the ciliated robots.\nThis is, however, out of the scope of the current proof-of-principle\nwork. Figure 6 Demonstration of the versatility of the ciliated metachronal robots.\n(A) Snapshots of the metachronal robot climbing across a PMMA hill\nat 2 Hz in air. See Movie S10 . (B) Normalized\nclimbing speed of the metachronal robots in air by V fl on PMMA slopes with an angle ranging from 0 to 180°.\nSee Movie S11 . Each data point was obtained\nby averaging the results of at least five identical but independent\nexperiments. (C) Demonstration of cargo (>10 own weight) carrying\ncapability of the metachronal robot in both air and liquid. The cargo\nis a cubic glass grain of 3 × 3 × 1 mm 3 . The\nliquid is pure ethanol. See Movie S12 . To demonstrate the versatility of our metachronal\nrobots further,\nwe performed experiments using slopes at different angles and experiments\nwith an extra weight placed on top of the metachronal robot in both\ndry and wet conditions. The results shown in Figure 6 B demonstrate that our metachronal robots\nare capable of climbing slopes with angles ranging from 0 to 180°\nin air, which is unprecedented (see Movie S11 ). The remarkable climbing capability, especially on 90° (i.e.,\nvertical) and 180° (upside down) slopes, is possible due to the\ncilia-surface adhesion overcoming gravity, while preserving continuous\ncontact between the metachronal μMAC and the surface, which\nis unachievable by the control robots. The climbing speed initially\ndecreases with the sloping angle, from a maximum speed of 0.7 cilia\nlengths per cycle on a horizontal surface to a minimum speed of 0.25\ncilia lengths per cycle on a 45° slope. Increasing the slope\nfurther results in an increase in the speed to 0.55 cilia lengths\nper cycle on a 90° slope. This behavior is probably caused by\nthe combined effects of adhesion, gravity, and friction. The adhesive\nforces that drive the locomotion remain similar for all slopes. As\nthe slope increases from 0 to 90°, the gravity component along\nthe surface that counteracts the robot motion increases, slowing down\nthe motion. However, at the same time, the gravity component normal\nto the surface decreases, which leads to a decrease in the frictional\nforce that also counteracts the locomotion of the robot, speeding\nup the motion. These two effects cross over at a slope of 45°,\nresulting in the observed behavior. Figure 6 C shows that a metachronal robot of 2 mg\ncan carry a glass grain of 25 mg (3 × 3 × 1 mm 3 ), which is more than 10 times heavier than its own weight, in both\nair and ethanol at speeds of 0.2 and 0.3 cilia lengths per beating\ncycle, respectively (see Movie S12 ). This\nis substantially slower than the speed without cargo (which is 0.9\ncilia lengths per beating cycle in air at 2 Hz, see Figure 5 E), showing that the cargo\nweight reduces the locomotion speed. The slightly faster speed in\nethanol is probably attributed to the reduced forces on the metachronal\nrobots due to the buoyance of the glass grain. Ethanol instead of\nwater was chosen in order to maintain the lubricant layer intact,\nas the lubricant layer would float in water."
} | 13,508 |
34448326 | PMC9293036 | pmc | 8,624 | {
"abstract": "Abstract The algal cell wall is an important cellular component that functions in defense, nutrient utilization, signaling, adhesion, and cell–cell recognition—processes important in the cnidarian–dinoflagellate symbiosis. The cell wall of symbiodiniacean dinoflagellates is not well characterized. Here, we present a method to isolate cell walls of Symbiodiniaceae and prepare cell‐wall‐enriched samples for proteomic analysis. Label‐free liquid chromatography–electrospray ionization tandem mass spectrometry was used to explore the surface proteome of two Symbiodiniaceae species from the Great Barrier Reef: Breviolum minutum and Cladocopium goreaui . Transporters, hydrolases, translocases, and proteins involved in cell‐adhesion and protein–protein interactions were identified, but the majority of cell wall proteins had no homologues in public databases. We propose roles for some of these proteins in the cnidarian–dinoflagellate symbiosis. This work provides the first proteomics investigation of cell wall proteins in the Symbiodiniaceae and represents a basis for future explorations of the roles of cell wall proteins in Symbiodiniaceae and other dinoflagellates.",
"conclusion": "CONCLUSIONS This study explored the cell wall protein composition of two cultured Symbiodiniaceae species: B . minutum and C . goreaui . We developed a method to isolate Symbiodiniaceae cell walls and performed a proteomic analysis of cell‐wall‐enriched samples. Transporters, translocases, hydrolases, and polypeptides involved in protein–protein interactions and cell signaling were identified in this analysis of the algal cell wall proteome. The majority of proteins identified have yet to be functionally characterized in Symbiodiniaceae, so this study flags numerous interesting candidates for further investigation to better understand this iconic symbiosis. We found little overlap between the cell wall proteomes of B . minutum and C . goreaui . The proteins identified here refer to cultured algae. Various traits of Symbiodiniaceae biology are likely to change between the two lifestyles of the organism (free‐living and in hospite ) as a result of symbiosis (Maruyama & Weis, 2020 ). Furthermore, previous studies showed that the Symbiodiniaceae cell wall adjusts its thickness when switching from cultured to being in hospite (Palincsar et al., 1988 ; Wakefield et al., 2000 ). Thus, it is feasible that the suite of proteins populating the cell wall might change as the algae switch between their two lifestyles. Our development of protocols to isolate and characterize CWPs in free‐living algae paves the way for future work to compare the cell wall proteome of free‐living vs. symbiotic Symbiodiniaceae. This would provide precious insights into the molecules that are critical to the mutualistic state and the effect that symbiosis has on the mutualistic partners.",
"discussion": "RESULTS AND DISCUSSION In this study, we prepared CWPs of two Symbiodiniaceae species ( B . minutum and C . goreaui ) by adapting a method developed in the model plant Arabidopsis (Feiz et al., 2006 ). Complete separation of cell wall from internal membranes and organelles of the algal cell was difficult to achieve. Our cell‐wall‐enriched fraction likely includes algal membranes, particularly the plasma membrane and the alveolar sac membranes (also known as amphiesmal vesicles). A series of centrifugations, aimed to separate cell walls from membranes and organelles of the Symbiodiniaceae cell, was performed (Figure 1A ). The efficacy of the cell wall isolation was then confirmed microscopically (Figure 1B and Figure S1 ). Intact Symbiodiniaceae cells showed algal chlorophyll autofluorescence (in red) surrounded by a continuous layer of cellulose (stained in blue by calcofluor white). In comparison, the cell wall isolates appeared as calcofluor white‐stained cellulosic fractions, and the chlorophyll autofluorescence was not detected, suggesting that the algal cell lost pigments and inner cell structures during the cell wall isolation procedure. This permitted the proteomic analysis of Symbiodiniaceae cell‐wall‐enriched samples. We identified a total of 786 proteins for B . minutum and 1487 proteins for C . goreaui . Filtering of these proteins according to their potential relevance to the cell wall resulted in 70 high confidence CWPs, of which 25 were identified for B . minutum and 54 for C . goreaui (Table 1 and Table S1 ). It is likely that a few scarce or labile proteins were lost during preparation of the samples. The identified CWPs clustered accordingly to the Symbiodiniaceae species. Surprisingly, only a few proteins were shared between cultured B . minutum and C . goreaui (Figure 2 ), suggesting that the phylogenetic differences between the two Symbiodiniaceae may be reflected by their surface proteome. The proteins were annotated as integral components of the membrane and grouped into five main functional categories: transporters (12), hydrolases (4), translocases (9), uncharacterized proteins (32), and other functions (12; Figure 3 ). TABLE 1 List of cell wall proteins identified in the Symbiodiniaceae species Breviolum minutum and Cladocopium goreaui \n GOterm Protein ID Protein name Gene name Functional category Peptide counts \n B. minutum \n \n C. goreaui \n A0A1Q9E3Y3 TRINITY_DN81331_c0_g1_i1.p1 Choline transporter‐like protein 5‐A slc44a5a AK812_SmicGene15055 Transporters 4 x A0A1Q9DXM6 TRINITY_DN12400_c0_g1_i1.p1 Divalent metal cation transporter MntH mntH AK812_SmicGene17473 Transporters 4;3;3;1 x A0A1Q9CX31 TRINITY_DN81400_c0_g1_i1.p1 GTP‐binding protein yptV4 YPTV4 AK812_SmicGene31283 Transporters 3 x A0A1Q9EVT1 TRINITY_DN8565_c0_g2_i3.p1 High‐affinity nitrate transporter 2.5 NRT2.5 AK812_SmicGene4635 AK812_SmicGene4659 Transporters 2;2;2;2;2;2;2;2;1;1 x A0A1Q9F6C9 TRINITY_DN75323_c0_g1_i1.p1 Protein ZINC INDUCED FACILITATOR‐LIKE 1 ZIFL1 AK812_SmicGene469 Transporters 17 x A0A1Q9D599 TRINITY_DN589_c0_g1_i1.p1 Putative ABC transporter ATP‐binding protein AK812_SmicGene28158 Transporters 8;3 x A0A1Q9EZY9 TRINITY_DN1165_c0_g1_i4.p1 Putative E3 ubiquitin‐protein ligase HERC1 HERC1 AK812_SmicGene3039 Transporters 3;3;3;3;2 x A0A1Q9E7B9 TRINITY_DN4547_c0_g4_i1.p1 Putative transporter YrhG yrhG AK812_SmicGene13733 Transporters 2 x A0A1Q9E7B9 TRINITY_DN2129_c0_g1_i3.p1 Putative transporter YrhG yrhG AK812_SmicGene13733 Transporters 2;2;2;2;2;2 x A0A1Q9CTV0 TRINITY_DN331_c0_g1_i9.p1 Sodium‐dependent phosphate transport protein 2A SLC34A1 AK812_SmicGene32588 Transporters 2;2;2;2;2;1;1;1;1 x A0A1Q9D6M3 TRINITY_DN22423_c0_g2_i1.p1 Solute carrier family 12 member 7 SLC12A7 AK812_SmicGene27515 Transporters 2 x A0A1Q9CNB5 TRINITY_DN66597_c0_g1_i1.p1 Synaptic vesicle 2‐related protein SVOP AK812_SmicGene34731 Transporters 2 x A0A1Q9EBY9 TRINITY_DN61236_c0_g1_i1.p1 Vignain CYSEP AK812_SmicGene11951 Transporters 4;1 x A0A1Q9E5T8 TRINITY_DN68576_c0_g1_i1.p1 (3S)‐malyl‐CoA thioesterase mcl2 AK812_SmicGene14316 Hydrolases 15 x A0A1Q9D1Y5 TRINITY_DN1384_c0_g1_i12.p1 AAA domain‐containing protein AK812_SmicGene29413 Hydrolases 2;2;2;2;2;1;1 x A0A1Q9F599 TRINITY_DN2468_c0_g1_i1.p1 Metalloendopeptidase (EC 3.4.24.‐) AK812_SmicGene955 Hydrolases 4 x A0A1Q9CVW0 TRINITY_DN12416_c0_g1_i1.p1 Putative isochorismatase family protein YddQ yddQ AK812_SmicGene31815 Hydrolases 3 x A0A1Q9CTN2 TRINITY_DN6337_c0_g1_i1.p1 Acyl_transf_3 domain‐containing protein AK812_SmicGene32637 Translocases 4 x A0A1Q9C4Z0 TRINITY_DN8204_c0_g1_i1.p1 Canalicular multispecific organic anion transporter 2 (Fragment) Abcc3 AK812_SmicGene41887 Translocases 2;1 x A0A1Q9BYL0 TRINITY_DN294_c0_g1_i6.p1 dTDP‐4‐amino‐4,6‐dideoxygalactose transaminase rffA AK812_SmicGene44383 Translocases 12;12;3;3;3;3;3;1;1 x A0A1Q9EF77 TRINITY_DN3047_c0_g1_i4.p1 H(+)‐exporting diphosphatase (EC 7.1.3.1) AK812_SmicGene10685 Translocases 6;1 x A0A1Q9EF77 TRINITY_DN1694_c0_g1_i1.p1 H(+)‐exporting diphosphatase (EC 7.1.3.1) AK812_SmicGene10684; Translocases 7 x A0A1Q9EUX6 TRINITY_DN1401_c0_g1_i3.p1 Hematopoietic prostaglandin D synthase HPGDS AK812_SmicGene4996 Translocases 5;5 x A0A1Q9CDQ7 TRINITY_DN3840_c0_g1_i1.p1 Proton‐translocating NAD(P)(+) transhydrogenase (EC 7.1.1.1) NNT AK812_SmicGene38460; Translocases 6;6 x A0A1Q9CDQ7 TRINITY_DN1231_c0_g1_i1.p1 Proton‐translocating NAD(P)(+) transhydrogenase (EC 7.1.1.1) NNT AK812_SmicGene38460; Translocases 13 x A0A1Q9EXD2 TRINITY_DN11568_c0_g1_i2.p1 Proton‐translocating NAD(P)(+) transhydrogenase (EC 7.1.1.1) pntA AK812_SmicGene3948 Translocases 13;13;13 x A0A1Q9EGY9 TRINITY_DN55363_c0_g1_i1.p1 Protein translocase subunit SecA secA AK812_SmicGene9992 Translocases 5;1 x A0A1Q9F1J3 TRINITY_DN14529_c0_g1_i1.p1 Putative serine/threonine‐protein kinase‐like protein CCR3 CCR3 AK812_SmicGene2484 Translocases 2 x A0A1Q9CEJ3 TRINITY_DN47369_c0_g1_i1.p1 Serine/threonine‐protein kinase Nek5 NEK5 AK812_SmicGene38102 Translocases 14 x A0A1Q9EGG1 TRINITY_DN2237_c0_g1_i2.p1 14 kDa zinc‐binding protein ZBP14 AK812_SmicGene10174 Other 5;5 x A0A1Q9D706 TRINITY_DN775_c0_g1_i1.p1 ANK_REP_REGION domain‐containing protein AK812_SmicGene27441 Other 8 x A0A1Q9DP71 TRINITY_DN19289_c0_g1_i3.p1 ANK_REP_REGION domain‐containing protein AK812_SmicGene20729 Other 4;4;3;3;2;2 x A0A1Q9F7U4 TRINITY_DN2962_c0_g1_i1.p1 Ankyrin repeat domain‐containing protein 50 ANKRD50 AK812_SmicGene83 Other 3;3;3;2;1;1;1 x A0A1Q9CVK2 TRINITY_DN47063_c0_g1_i1.p1 Ankyrin‐1 ANK1 AK812_SmicGene31912 Other 10 x A0A1Q9CVK2 TRINITY_DN49615_c0_g1_i1.p1 Ankyrin‐1 ANK1 AK812_SmicGene31912 Other 10 x A0A1Q9CRV7 TRINITY_DN9643_c0_g1_i2.p1 Long‐chain fatty acid‐‐CoA ligase 4 ACSL4 AK812_SmicGene33346 Other 2;2;2;2;2;2;2 x A0A1Q9EJE9 TRINITY_DN55895_c0_g1_i1.p1 Long‐chain fatty acid‐‐CoA ligase ACSBG2 ACSBG2 AK812_SmicGene9054 Other 4 x A0A1Q9D7L1 TRINITY_DN1621_c0_g1_i1.p1 Oxygen‐independent coproporphyrinogen‐III oxidase‐like protein AK812_SmicGene27203 Other 2 x A0A1Q9DK01 TRINITY_DN757_c0_g4_i1.p1 Potassium voltage‐gated channel subfamily H member 5 Kcnh5 AK812_SmicGene22354 Other 2 x A0A1Q9CCS4 TRINITY_DN55369_c0_g1_i1.p1 Reticulocyte‐binding protein 2‐like a AK812_SmicGene38827 Other 3 x A0A1Q9ERN9 TRINITY_DN16676_c0_g1_i1.p1 Reticulocyte‐binding protein 2‐like a AK812_SmicGene6218 Other 3 x A0A1Q9D5J5 TRINITY_DN67878_c0_g1_i1.p1 Transmembrane protein 87A tmem87a AK812_SmicGene27971 Other 2 x Note GOterm, protein ID, protein name, gene name, functional category, and number of peptides identified are reported for each protein. Uncharacterized proteins have been omitted and are included in Table S1 . John Wiley & Sons, Ltd FIGURE 2 Venn diagram of cell wall proteins identified in Breviolum minutum (purple) and Cladocopium goreaui (blue). B . minutum = 16 proteins; C . goreaui = 45 proteins; B . minutum and C . goreaui = 9 proteins FIGURE 3 Relative proportions of proteins identified on the cell wall of Breviolum minutum and Cladocopium goreaui . The proteins are grouped in five main functional categories: hydrolases, translocases, transporters, uncharacterized proteins, and other (oxidases, protein–protein interactions, cell adhesion) Transporters Membrane transport proteins regulate the selective passage of molecules across membranes, and so their presence in the algal cell wall is unsurprising (Table 1 , Figure 3 , Table S1 ). We identified zinc (Zn) and nitrate ( NO 3 ‐ ) transporters as potential CWPs. Both Zn and NO 3 ‐ are used by the dinoflagellate cell in several biological functions, ranging from cell growth to metabolism (Karim et al., 2011 ) and must be acquired from the surrounding environment. The presence of Zn induced facilitator‐like‐1a and high affinity NO 3 ‐ transporters has been previously shown in plants (Lezhneva et al., 2014 ; Ricachenevsky et al., 2011 ), so their presence is not surprising in the cell wall of Symbiodiniaceae. The high affinity NO 3 ‐ transporter 2.5, in particular, is well studied in higher plants, where it is localized to the plasma membrane and involved in NO 3 ‐ acquisition and remobilization (Lezhneva et al., 2014 ). Nitrate—together with ammonium ( NH 4 + )—are two common forms of available nitrogen (N) found in natural ecosystems and are key nutrients for cellular growth and development. In the cnidarian–dinoflagellate symbiosis, there is a highly efficient system of N cycling and assimilation (Pernice et al., 2012 ). Indeed, Symbiodiniaceae are specialized in assimilating dissolved inorganic nitrogen (DIN) from the environment and, although both host and symbiont have the enzymes to incorporate DIN, it is the symbiont that accounts for the majority of NO 3 ‐ and NH 4 + acquired from the surrounding water (Pernice et al., 2012 ). N transporters are, therefore, important players in the regulation of the mutualism between cnidarians and their symbiotic dinoflagellates. A choline‐like transporter (CTL) involved in the movement of choline for phospholipid synthesis (Michel et al., 2006 ) was detected among the possible CWPs of C . goreaui . CTLs are enriched in the Symbiodiniaceae genome compared to other eukaryotes (Aranda et al., 2016 ), and genes for this class of transporters were found to be differentially expressed in the algae when in culture compared to in hospite (Maor‐Landaw et al., 2020 ). When symbiotic with cnidarians, the dinoflagellate produces lipids that are transferred to the host (Peng et al., 2011 ). CTL proteins may, thus, have a role in regulating lipid biosynthesis and metabolism of the cnidarian–dinoflagellate mutualism (Maor‐Landaw et al., 2020 ). ABC transporters belonging to the ATP‐binding cassette (ABC) superfamily are characterized by two regions: a highly conserved ABC and a transmembrane domain. These transporters hydrolyze ATP to promote the import/export of various substrates, including small ions, metabolic products, lipids, and sterols (Higgins, 2001 ; Wang et al., 2011 ). Here, a putative ABC transporter was found in the CWPs of B . minutum . This transporter has previously been identified in the cnidarian–Symbiodiniaceae symbiosis in both the symbiont (Aranda et al., 2016 ) and the host (Meyer & Weis, 2012 ), and on the symbiosome membrane (Peng et al., 2010 ). The cell walls of the green alga Haematococcus pluvialis (Wang et al., 2004 ) and the dinoflagellate Alexandrium catenella (Wang et al., 2011 ) also possess several ABC transporters, suggesting a role for these proteins in nutrient transport. Although these transporters remain uncharacterized in the cnidarian–dinoflagellate symbiosis, it is possible that they are involved in inter‐partner nutrient flux. Hydrolases Hydrolases catalyze hydrolysis reactions, the cleavage of a covalent bond by the addition of a water molecule. This class of enzymes is commonly found on the walls of bacteria (Gumucio & Ostrow, 1991 ) and higher plants (Minic, 2008 ). In this study, we identified four hydrolases: (3S)‐malyl‐CoA thioesterase, AAA domain‐containing protein, metalloendopeptidase, and putative isochorismatase family protein YddQ (Table 1 , Figure 3 , Table S1 ). \n Breviolum minutum CWPs showed an AAA domain‐containing protein belonging to a large family of proteins that are characterized by a conserved 230 amino acid residues. The protein identified here contains an AAA ATPase domain in residues 87–180, which has 71% similarity to the canonical conserved AAA domain. AAA domain protein activity combines the chemical energy provided by the hydrolysis of ATP and alterations in their structural conformation to induce conformational changes in a wide range of macromolecules (Erzberger & Berger, 2006 ). They are involved in many cellular processes and their functions range from membrane fusion to signal transduction (Hanson & Whiteheart, 2005 ). \n Cladocopium goreaui CWPs contained a metalloendopeptidase, a protein belonging to a diverse group of enzymes that catalyzes the hydrolysis of internal, α‐peptide bonds of a polypeptide chain. Previously found in Symbiodiniaceae (Baumgarten, 2013 ), the integral membrane metalloendopeptidases play a role in physiological and pathological processes and are involved in cell adhesion (Bond & Beynon, 1995 ; Sauer et al., 2004 ). Translocases Translocases are proteins that use enzyme activity to move molecules across membranes. Nine translocases were found in the cell wall fractions of B . minutum and C . goreaui (Table 1 , Figure 3 , Table S1 ). Two translocases were common to the cell wall of both Symbiodiniaceae species: H(+)‐exporting diphosphatase and proton‐translocating NAD(P)(+) transhydrogenase. The first enzyme uses the energy from diphosphate hydrolysis to move protons across the membrane, while the second one catalyzes the transfer of hydride equivalents from NADH to NADP+, hence regenerating NADPH in the cell (Sauer et al., 2004 ). We identified the hematopoietic prostaglandin D synthase (HPGDS) among the CWPs of C . goreaui . HPGDS is a key enzyme in the synthesis of prostaglandins (PGs; Kanaoka & Urade, 2003 ), as the name suggests. PGs are derivatives of polyunsaturated fatty acids that commonly act as mediators in a range of physiological and pathological processes (Di Dato et al., 2020 ). First described in higher vertebrates, these molecules have also been discovered in marine invertebrates (Cnidaria, Mollusca and Crustacea), macroalgae, and microalgae (Di Costanzo et al., 2019 ). Animal‐like PGs in unicellular photosynthetic eukaryotes have been proposed to play a role in mediating intracellular and extracellular (cell–cell) signaling (Di Dato et al., 2017 ; Rosset et al., 2020 ). Although the function of these proteins has not yet being described in Symbiodiniaceae, they might play a similar role in dinoflagellates. A subunit of the Sec pathway, the SecA protein, was also found. The SecA translocase protein is a cell membrane‐associated subunit of the Sec pathway and has been described in diatoms (Chan et al., 2011 ). It has the functional properties of an ATPase and translocates macromolecules involved in the biogenesis of cell walls and signaling (Vrontou & Economou, 2004 ). Other Proteins with several other functions (oxidase, protein–protein interaction, and cell adhesion) were also identified on the cell wall of Symbiodiniaceae and are considered here (Table 1 , Figure 3 , Table S1 ). The ANK repeat (ANKr) is a conserved domain of approximately 33 amino acids, characteristic of the components of the Ankyrin family, one of the most common protein families across all kingdoms of life (Brüwer et al., 2017 ). We found proteins containing this domain in both B . minutum and C . goreaui CWPs. In general, ANKr functions as a protein–protein interaction domain and mediates cross‐talk between host and symbiont in various endosymbioses (Liu et al., 2018 ; Thomas et al., 2010 ). For instance, four ANKr proteins were discovered in γ‐proteobacteria when symbiotic with sponges and proposed to prevent phagocytosis, thus allowing the symbiont to escape digestion by the sponge host (Nguyen et al., 2014 ). In some cases, this protein motif is part of the signaling network of viral infections and works against host innate immunity by preventing apoptosis of infected cells (Al‐Khodor et al., 2010 ; Brüwer et al., 2017 ). In the Symbiodiniaceae genome, ANKrs represent the second largest gene family (after the EF‐hand family; Shinzato et al., 2014 ). Although the function of ANKr is not well described in the Symbiodiniaceae, these polypeptides may play a role in suppressing phagocytosis, as they appear to do in the bacterial‐sponge symbiosis. One CWP that caught our attention is a putative homologue of the malaria parasite reticulocyte‐binding‐like (RBL) protein 2a. RBL proteins are sialic acid receptors crucial to host cell recognition and invasion by Plasmodium , the malaria‐causing pathogen (Rayner et al., 2001 ). Dinoflagellates and Plasmodium are closely related, both being members of the Infra‐Kingdom Alveolata, and it is widely held that Apicomplexa, the group of parasites to which Plasmodium belongs, arose from photosynthetic, endosymbiotic mutualists akin to modern day Symbiodiniaceae (Berney & Pawlowski, 2006 ). Thus, at face value, RBL homologues in the cell walls of Symbiodiniaceae might point to an ancient, conserved mechanism of host cell recognition/invasion. However, the sequence similarity between A0A1Q9CCS4, A0A1Q9ERN9 in the S . microadriaticum genome (Aranda et al., 2016 ) and RBL homologues of Plasmodium is confined to low complexity repeats. Whether or not CWPs A0A1Q9CCS4 and A0A1Q9ERN9 have a role in dinoflagellate–cnidarian symbiosis remains to be determined."
} | 5,184 |
28810712 | PMC5553392 | pmc | 8,625 | {
"abstract": "Abstract A characteristic feature of the order Rhodobacterales is the presence of a large number of photoautotrophic and photoheterotrophic species containing bacteriochlorophyll. Interestingly, these phototrophic species are phylogenetically mixed with chemotrophs. To better understand the origin of such variability, we sequenced the genomes of three closely related haloalkaliphilic species, differing in their phototrophic capacity and oxygen preference: the photoheterotrophic and facultatively anaerobic bacterium Rhodobaca barguzinensis , aerobic photoheterotroph Roseinatronobacter thiooxidans , and aerobic heterotrophic bacterium Natronohydrobacter thiooxidans . These three haloalcaliphilic species are phylogenetically related and share many common characteristics with the Rhodobacter species, forming together the Rhodobacter-Rhodobaca ( RR ) group. A comparative genomic analysis showed close homology of photosynthetic proteins and similarity in photosynthesis gene organization among the investigated phototrophic RR species. On the other hand, Rhodobaca barguzinensis and Roseinatronobacter thiooxidans lack an inorganic carbon fixation pathway and outer light-harvesting genes. This documents the reduction of their photosynthetic machinery towards a mostly photoheterotrophic lifestyle. Moreover, both phototrophic species contain 5-aminolevulinate synthase (encoded by the hemA gene) incorporated into their photosynthesis gene clusters, which seems to be a common feature of all aerobic anoxygenic phototrophic Alphaproteobacteria . Interestingly, the chrR-rpoE (sigma24) operon, which is part of singlet oxygen defense in phototrophic species, was found in the heterotrophic strain Natronohydrobacter thiooxidans . This suggests that this organism evolved from a photoheterotrophic ancestor through the loss of its photosynthesis genes. The overall evolution of phototrophy among the haloalkaliphilic members of the RR group is discussed.",
"introduction": "Introduction The order Rhodobacterales ( Alphaproteobacteria ) encompasses a highly diverse ensemble of species ( Simon et al. 2017 ). They largely differ in their phenotype, metabolic traits, and ecological niches they inhabit. Its members conduct many fundamental metabolic processes such as aerobic respiration, anaerobic fermentation, sulfur oxidation, autotrophic carbon fixation, nitrogen fixation, or hydrogen production in various combinations ( Garrity et al. 2005 ; Androga et al. 2012 ). One of the most interesting features of Rhodobacterales is the presence of many species that perform anoxygenic photosynthesis. Members of the Rhodobacter ( Rba .) and Rhodovulum ( Rdv. ) genera represent classical examples of purple nonsulfur bacteria—organisms capable of photoautotrophic growth under anaerobic conditions utilizing the Calvin cycle, or heterotrophic growth in the presence of oxygen ( Androga et al. 2012 ). On the other hand, there exist many marine photoheterotrophic species belonging to the so called Roseobacter group, which grow and conduct photosynthesis in the presence of oxygen ( Wagner-Döbler and Biebl 2006 ; Moran et al. 2007 ; Brinkhoff et al. 2008 ), do not contain RuBisCO and use light only as an additional energy source ( Koblížek et al. 2013 ). In the last 20 years, seven haloalkaliphilic photoheterotrophic species were isolated from several saline and soda lakes in Africa, America, Asia, and Antarctica. Remarkably, these organisms are phylogenetically closer to freshwater Rhodobacter species than to marine photoheterotrophs. Rhodobaca (Rca.) bogoriensis was isolated from soda lakes in the East African Rift Valley ( Milford et al. 2000 ), and is capable of both aerobic and anaerobic growth on organic substrates, however it does not contain Calvin cycle enzymes and thus cannot grow photoautotrophically. A closely related organism with the same physiology, Rca. barguzinensis , was later isolated from a small soda lake in the Barguzin valley in south-eastern Siberia ( Boldareva et al. 2008 ). In contrast, the closely related aerobic anoxygenic phototrophic (AAP) bacterium Roseinatronobacter (Rna.) thiooxidans , isolated from a soda lake in the Kunkurskaya steppe (Chita region, south-easter Siberia) grows solely under aerobic conditions ( Sorokin et al. 2000b ; Stadnichuk et al. 2009 ). Later, a very similar species, Rna. monicus was isolated from the hypersaline Soda Mono lake in California, USA ( Boldareva et al. 2006 ). Closely related to the above mentioned species are two Antarctic AAP isolates Roseibaca ekhonensis and Roseicitreum antarcticum , which were collected from Lake Ekho ( Labrenz et al. 2009 ), and sandy intertidal sediments ( Yu et al. 2011 ), respectively. The last described AAP bacterium in this group is Roseibacula alcaliphilum , which was isolated from Lake Doroninskoe, East Transbaikal region, Russia ( Boldareva and Gorlenko 2014 ). This photoheterotrophic group is complemented by some nonphototrophic species such as the haloalkaliphilic heterotrophic bacterium Natronohydrobacter ( Nhb. ) thiooxidans isolated from a soda lake in Kenya. Based on the close phylogenetic proximity of photoautrophic, photoheterotrophic, and lithoautotrophic species, Keppen et al. (2013) suggested that the photoheterotrophic species such as Rhodobaca and Roseinatronobacter represent the intermediate species on the regressive evolutionary pathway leading from photoautotrophic purple nonsulfur bacteria to heterotrophic species. A similar scenario has also been proposed for photoheterotrophic and heterotrophic species in the marine Roseobacter group ( Koblížek et al. 2013 ). However, the existence of photoheterotrophic species can be explained using an alternative scenario where the originally heterotrophic species acquired their photosynthesis genes via horizontal gene transfer (HGT). A typical feature of purple phototrophic bacteria is that most of their genes involved in the light-phase of photosynthesis are organized in photosynthesis gene clusters (PGCs) ( Zsebo and Hearst 1984 ; Zheng et al. 2011 ). It has been speculated that clustering of photosynthesis genes in PGCs can facilitate HGT. In line with this hypothesis is the report that the whole PGC was likely transferred between Proteobacteria and Gemmatimonadetes ( Zeng et al. 2014 ). Rhodobacterales species often carry various extra chromosomal elements ( Petersen et al. 2013 ), transposable elements ( Vollmers et al. 2013 ), or gene transfer agents ( Luo and Moran 2014 ), which can facilitate gene transfer. Moreover, PGC-containing plasmids have been identified in some Roseobacter -group representatives ( Petersen et al. 2013 ). HGT has also been proposed for puf genes encoding the protein subunits of the bacterial reaction centers within the Roseobacter group ( Koblížek et al. 2015 ). To elucidate the most likely evolutionary pathway, we decided to sequence genomes of three closely related (on average 98.7% 16S rRNA pairwise similarity) haloalkaliphilic organisms differing in their metabolic capacities and oxygen preference. Photoheterotrophic Rca. barguzinensis strain alga05 exhibits both aerobic and anaerobic growth. It lacks RuBisCO and nitrogenase, but it has the capacity to utilize thiosulfate and sulfide as electron donors ( Boldareva et al. 2008 ). The second organism, Rna. thiooxidans strain ALG1, is a photoheterotrophic bacterium combining aerobic respiration and photophosphorylation, but it can also oxidize thiosulfate and sulfide during aerobic lithoheterotrophic growth ( Sorokin et al . 2000b ). The last organism, Nhb. thiooxidans strain AH01, is a strictly aerobic, nonphototrophic bacterium. It is capable of growing lithoheterotrophically with acetate and thiosulfate, sulfide, polysulfide, and elemental sulfur which are oxidized to sulfate, and in addition it can oxidize H 2 ( Sorokin et al. 2000a ). We mostly focused on the presence of photosynthesis genes, their organization and phylogeny in order to test the two proposed evolutionary scenarios.",
"discussion": "Discussion Two evolutionary scenarios explaining the presence of phototrophic and heterotrophic strains among haloalkaliphilic species in the RR -group were considered in this study (1) the “regressive evolution” scenario, where the ancestors of the haloalkaliphilic RR -group were photoautotrophic organisms, which later lost part (or all) of their photosynthesis gene, or (2) the ancestors of the RR -group were heterotrophs, which adopted their photosynthesis genes via HGT of photosynthesis genes. Our data indicates that phototrophy is ancestral in the RR -group. There are two main lines of evidence. First, all the studied phototrophic organisms belonging to the RR -group shared an almost identical organization of their PGCs ( fig. 2 ). A unique feature for all phototrophic RR -species is the divergent orientation of superoperons bchFNBHLM - IhaA - puh and crt - bchCXYZ - puf . This orientation has not been found in any other Rhodobacterales species ( Zheng et al. 2011 ), which represents the main evidence against the hypothesis that the RR -species adopted the PGC through HTG from other Rhodobacterales genera. Another distinctive feature present in all studied phototrophic members of the RR -group is the presence of the pufX gene, and the absence of the pufC gene ( table 2 ). The gene pufX is also present in several marine AAP species, but they have likely received it via the HGT as discussed before ( Koblížek et al. 2015 ). Second, the performed phylogenetic analyses on the photosynthesis genes bchIDHLNB , acsF and pufM ( fig. 4 , supplementary figs. 2 and 3 , Supplementary Material online) document that the RR -group always cluster together, with an exception of the pufM tree where the RR -group is mixed with several marine AAP species ( supplementary fig. 3 , Supplementary Material online). An important finding is the presence of genes connected with the singlet oxygen defense system in chemotrophic strain AH01. This strain contains the same singlet-oxygen-stress-response mechanism (encoded in rpoE-chrR gene operon) as Rba. sphaeroides ( Anthony et al. 2004 ), as well as in ALG1 and alga05 strains. When bound to its anti-sigma factor ChrR, RpoE is inactive in the cell. The presence of singlet oxygen leads to a dissociation of the heterodimer, thus the activation of RpoE and subsequently its more than 180 target genes ( Anthony et al. 2004 ). A similar response has been shown for the AAP species Rsb. litoralis ( Berghoff et al. 2011 ) and Drb. shibae ( Tomasch et al. 2011 ). As the main source of singlet oxygen in bacteria is BChl a ( Borland et al. 1989 ), all the phototrophic Rhodobacterales contain the RpoE–ChrR system to protect their cellular machinery against damage. However, chemotrophic organisms (which lack BChl a ) should not be prone to the formation of singlet oxygen, and the presence of RpoE–ChrR system is unnecessary. This suggests that the ancestor of AH01 was originally a phototrophic bacterium, which later lost its photosynthetic genes, but yet retained the RpoE–ChrR system. From the presented pieces of evidence, we conclude that the ancestors of the haloalkaliphilic members of the RR -group were phototrophic organisms. A more difficult question to answer is whether these ancestral species were photoautotrophic and contained RubisCO. There are two possible scenarios ( fig. 6 ). The first [compatible with Keppen et al. (2013) ] assumes that the ancestors of the RR -group were photoautotrophs similar to most of the modern Rhodobacter species. Then, during evolution, some RR -lineages lost a part or all of their photosynthesis genes producing photoheterotrophic, lithoheterotrophic, or chemoheterotrophic species. In the second scenario, the ancestors of the RR -group were photoheterotrophs. Here, some lineages (e.g., Rhodobacter species) gained RubisCO genes and became photoautotrophs, whereas some lineages lost all their photosynthesis genes and became heterotrophs ( fig. 6 ). These two scenarios are difficult to reconcile based on the available data, but based on the performed phylogenetic analyses we prefer the photoautotrophic ancestor scenario. Indeed, the photoautotrophic Rhodobacter species (especially Rba. veldkampii ) are located closer to the root of the RR -group in our 16S rRNA tree, whereas the photoheterotrophic species branch off later ( fig. 1 ). The weakness of this argument is the very low statistical support of the 16S phylogenetic tree. However, a similar conclusion can also be made based on the ALAS phylogeny. The ALAS ( hemA/hemT gene) tree shows a major split, documenting the existence of two different forms of the gene in Rhodobacterales (hemA -like and hemT -like form). These two forms probably originate from a gene duplication event. Since the hemA and hemT genes of Rba. sphaeroides lay close to the split, it seems that the gene duplication occurred in the ancestors of Rhodobacter -like species and all the subsequent photoheterotrophic ancestors retained the two forms of the gene, which gradually diverged. Photoheterotrophic species always contain both forms, which indicate that they only evolved after Rba. sphaeroides . Later, the AAP species incorporated the hemT -like form of ALAS into the PGC. The presence of ALAS in the PGC seems to be a common characteristic of all the AAP species belonging to Alphaproteobacteria ( Zheng et al. 2011 ). This arrangement is probably advantageous for convenient regulation of BChl a synthesis in AAP species, which need to tightly control the initial step of the tetrapyrrol pathway with the final part of the BChl a pathway. Indeed, this has been documented in Drb. shibae , where the inducible ALAS gene located in the PGC ( hemT -like form) was under strong light regulation together with all bch genes ( Tomasch et al. 2011 ). Interestingly, all these ALAS genes present in the PGC cluster together form a distinct subclade of the entire hemT -like group ( fig. 3 ). This indicates that the hemA split occurred first in photoautotrophic Rhodobacter -like species, which probably needed to differentially regulate the tetrapyrrol biosynthesis pathway for heme ( hemA form) and BChl a synthesis ( hemT form). Later, after the evolution of the AAP species, the hemT form of the gene was moved under the common regulation of the PGC cluster. Moreover, in some species ( Drb. shibae and Roseobacter species), the hemT form of ALAS underwent a second duplication, indicating the need for even more delicate regulation of the gene.\n Fig. 6.— Proposed schemes of the evolution of phototrophy among the members of the RR -group: (A) Regressive evolution model compatible with Keppen et al. (2013) . (B) Mixed model assuming a photoheterotrophic ancestor of all modern RR -species—heterotrophs, photoheterotrophs, and photoautotrophs. In conclusion, the presented data indicate that the ancestors of the haloalkaliphilic members of the RR -group were phototrophic species. The heterotrophic species have evolved through the regressive loss of their photosynthetic apparatus. In addition, we have demonstrated that the important step enabling the evolution of photoheterotrophic species was the duplication of the hemA gene, and its later incorporation into the PGC of the AAP species. Many interesting questions were left to be answered such as if there is an alternative pathway of descent from photoautotrophic species through the facultatively autotrophic aerobic or facultatively anaerobic lithotrophs, such as Paracoccus (loss of the PGC), to aerobic chemolithoheterotrophs (loss of RubisCO) and finally organoheterotrophs? What was the role of the hydrogenase in this alternative scenario? Did Rba. capsulatus evolve regressively from a Rba. sphaeroides- like species through the loss of genes ( acsF, hemF, hemT , and anti-sigma factor), or does it represent a former (more ancient) photoautotrophic species? We believe, that the constantly expanding genomic data, will allow us to address these questions in the near future."
} | 4,048 |
39333559 | PMC11436852 | pmc | 8,627 | {
"abstract": "Climate change is causing widespread impacts on seawater pH through ocean acidification (OA). Kelp forests, in some locations can buffer the effects of OA through photosynthesis. However, the factors influencing this variation remain poorly understood. To address this gap, we conducted a literature review and field deployments of pH and dissolved oxygen (DO) loggers within four habitats: intact kelp forest, moderate kelp cover, sparse kelp cover and barrens at one site in Port Phillip Bay, a wind-wave dominated coastal embayment in Victoria, Australia. Additionally, a wave logger was placed directly in front of the intact kelp forest and barrens habitats. Most studies reported that kelp increased seawater pH and DO during the day, compared to controls without kelp. This effect was more pronounced in densely populated forests, particularly in shallow, sheltered conditions. Our field study was broadly consistent with these observations, with intact kelp habitat having higher seawater pH than habitats with less kelp or barrens and higher seawater DO compared to barrens, particularly in the afternoon and during calmer wave conditions. Although kelp forests can provide local refuges to biota from OA, the benefits are variable through time and may be reduced by declines in kelp density and increased wave exposure. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-024-72801-5.",
"introduction": "Introduction Climate change is having widespread impacts on coastal ecosystems through rising seawater temperatures, altered weather patterns and changes in seawater chemistry 1 . Since the beginning of the industrial revolution the sustained absorption of atmospheric CO 2 by oceans has led to increased concentrations of dissolved inorganic carbon (DIC) and lowered sea surface pH by approximately 0.1 unit, termed ‘ocean acidification’ (OA) 2 . OA is predicted to have widespread impacts on key taxa, commercial fisheries 3 , and other critical ecosystems services 4 , 5 . There is an increasing interest in understanding the role of marine macrophytes (such as macroalgae) in mitigating the effects of OA 6 , 7 . Large brown seaweeds or kelps (orders Laminariales and Fucales) form extensive forests in temperate coastal reef systems worldwide 8 . Kelp forests can alleviate the impacts of OA by locally increasing pH and dissolved oxygen (DO) in seawater through the process of photosynthesis 9 – 11 . However, the effects of kelp forests on seawater chemistry vary diurnally 9 – 11 . During the day, kelps actively draw in DIC from the surrounding seawater through photosynthesis which increases seawater DO and pH while at night kelps respire resulting in declines in seawater DO and pH (Fig. 1 ). The ability of kelp forests to buffer OA, therefore, depends on whether net photosynthesis outweighs net respiration 9 , 12 , 13 . Fig. 1 Schematic showing the effects of kelp on seawater chemistry during ( a ) day and ( b ) at night. There are, however many other abiotic and biotic factors that could influence a kelp forest’s ability to influence seawater chemistry 9 , 13 . Dense kelp forests can reduce seawater flow, internal motion and mixing within the centre of the forest and acceleration on the forest edge 14 , 15 . Longer seawater residence times within the centre of intact Macrocystis forests allows for increased uptake of DIC by kelp during the day, increasing local seawater DO and pH levels 14 . Studies have further suggested that the seawater pH is higher in the surface water of dense Macrocystis forests in sheltered waters compared to that of deeper or more exposed waters 12 , 14 , 16 . The ability of Macrocystis and other kelp forests to influence hydrodynamics and/or seawater chemistry could therefore be dampened by stressors which reduce densities of kelp or where the entire forest is removed through overgrazing by sea urchins (i.e. barrens habitat) 12 , 17 , or during periods of high wave exposure 12 , 14 , 16 . Previous studies on the effects of kelp forests on seawater chemistry have shown mixed evidence that kelp forests can buffer the effects of OA. Some studies comparing seawater chemistry inside vs. outside of kelp forests demonstrate consistently higher pH and DO inside than outside kelp forests 18 , while others show temporally variable effects 19 or no differences 10 , 14 . Much of the research on the effects of kelp forests on seawater chemistry has focused on Macrocystis forests (but see 9 , 19 , 20 ). There is a clear need for further study to understand the key driver(s) of this variability, across a range kelp species. Here we conducted a literature review as well as field measurements at one site in Port Phillip Bay Victoria, Australia. Our aim was to assess how the varying abiotic and biotic characteristics of the dominate kelp forests in this region, primarily formed by Ecklonia radiata , influence seawater chemistry. Our prediction for the field measurements was that the benthic seawater pH and DO would be higher in E. radiata forests compared to barrens habitats, primarily due to photosynthetic draw down by the kelps. However, we also hypothesized that habitats with lower densities of kelp or during periods of increased wave height would have lower seawater pH and DO due to the reduced influence of E. radiata on these parameters.",
"discussion": "Discussion This study combines findings from a literature review with field measurements to assess the effects of kelp forests on seawater chemistry. Our results offer critical insights into some of the abiotic and biotic drivers of temporal variation in kelp forest influence on seawater chemistry. The literature search revealed that kelp forests increase seawater pH and DO levels during the day, relative to areas without kelp, but the effects differ among kelp species and increase with density of kelp and are more pronounced in shallower waters and sheltered locations. Our field measurements were broadly consistent with these observations, with kelp forests significantly altering seawater chemistry, resulting in higher pH and DO levels inside the forests compared to outside. Specifically, the intact E. radiata forest showed higher values for daily maximum (0.11 units and 1.00 mg/L), mean (0.01 units) and range (0.11 units and 2.09 mg/L) but lower minimum pH and DO (− 0.07 units and − 0.97 mg/L) compared to the barrens habitat. Diurnal patterns in seawater pH and DO suggest that the photosynthetic activity of kelp and other seaweeds 26 in the intact E. radiata forest and turf algae 27 in the barrens habitat play a pivotal role in driving these patterns. These results emphasize the significant role of kelp forests in regulating seawater chemistry and the need for continued research in this area to better understand the impacts of climate change and other stressors on their ecological functions and services (Fig. 5 ). Fig. 5 Schematic showing the (1) effects of intact kelp forests on seawater chemistry (2) threats to kelp forests and (3) effects of reducing kelp forest density and increased wave exposure on seawater chemistry. Our study findings align with prior research conducted in Tasmania 9 , 19 which also demonstrated positive impact of intact E. radiata forests on local seawater chemistry. Consistent with previous studies 9 , 19 we observed the highest peaks in seawater pH and DO in the late afternoon when the higher light availability (12:00–15:00), warmer seawater temperatures (15:00–20:00) and calm conditions (T. Graham unpublished data) potentially stimulated greater photosynthetic activity and water residence time. The lowest values of seawater pH and DO were recorded in the early morning during the period of highest respiration 9 , 19 . However, the daily range in pH and DO for the intact E. radiata forest were consistently positive, suggesting that net photosynthesis outweighs respiration. Furthermore, our research indicates the effects of kelp forests on seawater chemistry varied among species, which may be linked to their morphology. For instance, studies on Macrocystis pyrifera , a species of kelp that reaches the sea surface, have revealed minimal differences in benthic seawater chemistry inside and outside the kelp forest. However, more pronounced differences in seawater pH and DO within Macrocystis forests have been observed in surface waters where kelp biomass and light availability are greatest 10 , 28 . By contrast, shorter taxa such as Ecklonia , Laminaria and Sargassum kelp forests have exhibited significant increases in seawater DO and pH in benthic waters relative to barren areas 19 , 29 . These finding highlight that different kelp species may exhibit varying effects on seawater chemistry, emphasizing the important of considering species-specific characteristics and their ecological role in understanding these dynamics. The impacts of kelp forests on seawater chemistry varied, depending on both kelp density 30 , 31 and local hydrodynamic conditions 10 , 14 , 16 , 29 . We observed that the intact E. radiata forest had elevated seawater daily maximum seawater pH and DO (0.1 unit and1.00 mg/L) levels compared to habitats with lower densities of kelp and the barrens. Notably, there in the intact E. radiata forest, we identified negative relationships between seawater pH and DO levels and wave heights—the predominant driver of water motion in our study site. Conversely, the same relationships were not observed in the barrens habitat. These findings suggest that fragmented E. radiata forests and barrens characterised by lower density of kelp due to overgrazing by sea urchins 26 have a weaker biogeochemical signature owing to the reduced photosynthetic activity 32 and decreased water retention time 14 , 33 . Hence, the ability of E. radiata forests to buffer OA could be compromised by increasing wave exposure and storminess associated with climate change. As the impacts of climate change intensify, understanding the role of kelp forests in creating local refuges for associated organisms from OA is increasingly important. Previous research has demonstrated that benthic organisms are particularly sensitive to lower pH conditions 34 . Negative effects of sustained lower seawater pH have been demonstrated on various taxa including reduced survival of amphipods (pH 7.6 35 ), decreased growth rates in molluscs such as juvenile blacklip abalone (pH 6.79 36 ), declined growth rate and gene expression of echinoderms including purple sea urchins (pH 7.6 37 ) and diminished calcification rates of calcareous algal taxa like corallines (pH 7.6 38 ). All these organisms inhabit the benthos of E. radiata forests 32 . Our results, therefore, support arguments that intact kelp forests in sheltered locations such as Port Phillip Bay could provide local refuges from OA for certain associated taxa 39 , 40 . Nonetheless, the higher variability in seawater pH in intact E. radiata forests might also have adverse implications for the calcification rates of other taxa 38 . There is increasing interest in understanding how marine macrophytes such as kelp forests can provide key ecosystems services 41 . Kelp forests can buffer the impact of OA on key marine taxa including fished species 36 . At the global scale there is a paucity of data about the conditions in which kelp forests can increase seawater pH and DO. Here we conclude that E. radiata and other kelps can buffer the effects of OA, but the effects are confined to daylight periods 42 – 44 , calm conditions 14 , 16 , and intact forests 17 , 31 . Kelp forests include a diversity of species and morphologies 45 , thus there is a need to understand which species can consistently increase seawater pH and DO, at different spatial and temporal scales, heights in the water column and in what environmental conditions. This will involve collection of more field data across different species, forest characteristics (density, area etc.), water depths and wave exposures. Kelp is one of the dominant habitat-forming organisms of temperate reefs. In many locations, kelp beds are, however, in decline 46 , 47 , due to a combination of local and global threats 48 . A greater understanding of the suite of ecosystem services being lost will underpin more effective management of kelp forests both in Port Phillip Bay and elsewhere."
} | 3,099 |
35046027 | PMC8794849 | pmc | 8,628 | {
"abstract": "Significance Current high-throughput cell screening tools do not select cells based on their behavior in production or commercial environments, making it difficult to translate selected cells from the laboratory to commercialization. With PicoShells, we are able to perform high-throughput sorting of cells based on their phenotypic behavior in production-relevant environments, like stirred flasks, and in the presence of background cells, potentially speeding up the development of new biotechnology products by several months to years. In particular, PicoShells enable the selection of clonal colonies based on their overall accumulated growth or production of, for example, chlorophyll over a set period of time, potentially creating a cell selection tool that will improve yields of desired bioproducts.",
"discussion": "Discussion Advantages of PicoShells. Several key aspects of the PicoShell workflow suggest that it can aid in the selection/evolution of cells and cell-based products, including the following. 1) Cell behavior and growth are significantly enhanced in PicoShells compared with water-in-oil droplet emulsions. 2) PicoShells containing desired cells/colonies can be selected using commercial fluorescence-activated cell sorters. 3) Selected cells/colonies can be successfully released from the PicoShells and recultured. 4) Selected populations maintained a high-growth phenotype postprocess at least for several generations. Importantly, PicoShells can be placed and remain stable in more production-relevant environments (e.g., a shaking culture flask, bioreactor, outdoor cultivation farms) that are not feasible with other high-throughput selection technologies (e.g., droplet technology, microwells, etc.) ( SI Appendix , Fig. S11 ). The porous outer shell enables solution exchange with the external environment, likely allowing replenishment of nutrients; diffusive transport and dilution of cytotoxic cellular waste; access to quorum-sensing factors from external cells/colonies; and exposure to natural concentration, temperature, light, or physical gradients in the culture environment. As a result, PicoShell technology may provide a high-throughput screening tool that enables cell-line developers and researchers to select cells based on their behavior in production-relevant environments, making it much more likely that selected populations will exhibit the desired phenotypic properties when scaled up for real-world applications. Transport across PicoShells is likely responsible for distinct growth phenotypes for encapsulated cells compared with droplets in oil. Previous studies demonstrated that droplet size affects the division of cells in microfluidic droplets surrounded by oil ( 31 , 32 ). Another recent study has demonstrated that yeast cells cultured in large flasks, large droplets, and small droplets differed morphologically ( 14 ). There may be several reasons for this phenomenon. For example, smaller droplets have less nutrients, which could be depleted more rapidly when cells are placed within droplets and begin to grow and divide. Similar effects are expected with sealed nanowell arrays. With PicoShells, we have demonstrated that nutrients, such as the amino acid leucine, present in solution outside of the shell affect the growth and division of cells within ( SI Appendix , Fig. S6 ). Based on these data, we conclude that leucine is able to transport across into the internal environment of the PicoShell, and we postulate that other factors and nutrients will be able to transport as well. In addition, it is well understood that cells release elements to the external environment that change its pH. For example, CHO cells release lactate that lowers the pH of the external media ( 33 ), and yeasts release alcohol that increases the pH of the external media ( 34 ). Charged species that modulate pH are expected to not easily transport out of droplets in oil or through solid barriers of nanowell arrays. Lastly, it is probable that proteins or other small molecules that are used for cell communication cannot pass through the walls of nanowell arrays or easily partition and transport through the oil phase of water-in-oil droplet emulsions. Since the outer shell of PicoShells is porous and has a molecular mass cutoff above 70 kDa, it is likely that these factors can pass through the outer matrix. This is supported by our data demonstrating that the presence of cells in the external solution can affect the growth behavior of cells within the cavity of the PicoShells ( SI Appendix , Fig. S4 ). In addition, inclusion of a solid surface for adherent cell lines to attach to in PicoShells likely enhances the growth properties of adherent CHO cells in PicoShells compared with in droplets. It is important to note that the lack of adherent CHO cell growth in droplets that we observed may have resulted from reduced gas exchange through the mineral oil cap we placed on top of the droplets, which we use to reduce evaporation. However, we have demonstrated that S. cerevisiae do not have different growth properties within droplets capped or not capped with mineral oil ( SI Appendix , Fig. S8 ). Growth of other algae species in droplets has been previously shown ( 11 , 12 , 35 , 36 ), making it intriguing why Chlorella in particular does not survive when encapsulated into water-in-oil droplet emulsions. While it is unclear exactly why this particular phenomenon occurs, we believe that the lack of cell survival is related to the restricted gas exchange across the oil barrier. This particular species is grown in autotrophic media and is very sensitive to gaseous CO 2 concentrations. We have observed that bulk cultures of this particular species cannot grow when not cultured in an incubator that regulates CO 2 or not cultured with media that are not supplemented with sodium bicarbonate ( 37 ). While previous studies have shown that gases can generally pass through fluorinated oil ( 38 , 39 ), this diffusion may be limited or altered to an extent that sensitive species are greatly affected unlike more robust cell types ( Chlamydomonas reinhardti , Euglena gracilis , etc.). Regardless of the root cause for the lack of growth in droplets, the results demonstrate that the environments in PicoShells and droplets are different enough that we can observe a noticeable effect on cell behavior, a result that is substantiated by the improved growth properties of S. cerevisiae in PicoShells. Potential for Chemically Degradable PicoShells. We have explored multiple mechanisms to chemically release cells from PicoShells by including chemically degradable motifs in the outer PEG shell. Currently, we can consistently fabricate PicoShells cross-linked with PEG-MAL and DTT. These are compatible with multiple cell types, including Chlorella , S. cerevisiae , and E. gracilis ( SI Appendix , Fig. S15 ). PicoShells cross-linked with PEG-MAL and DTT can be broken down with the addition of sodium periodate (NaIO 4 ) due to the presence of a diol in DTT. Unfortunately, NaIO 4 can be toxic ( 40 ) and likely kills or has large negative impacts on many cell types. Previously, we have made hydrogel particles with degradable peptide cross-linkers ( 20 ), and similar incorporation of degradable cross-linkers could enable enzymatic or chemical degradation of particles to release selected cells/colonies. As an initial proof of concept of this approach, we developed PicoShells that contain disulfide linkages that can be degraded via the addition of DTT or Tris(2-carboxyethyl)phosphine (TCEP). S. cerevisiae encapsulated in these particles remain viable, grow, and can be chemically released ( SI Appendix , Fig. S16 and Movie S5 ). Unfortunately, a chemical precursor we use to form these particles (four-arm polyethylene glycol ortho-pyridyldisulfide [PEG-OPSS]) is toxic to Chlorella ( SI Appendix , Fig. S17 ), suggesting that the chemical formulation of the PicoShell should be matched to the cell type. We have also encapsulated and grown C. reinhardtii in PicoShells cross-linked with matrix metalloproteinase (MMP)–degradable peptides ( SI Appendix , Fig. S18 ) that can be degraded with the addition of trypsin ( Movie S6 ). Unfortunately, C. reinhardtii (and likely other cell types) naturally secrete MMPs that often prematurely break down the particles ( 41 ). While the mechanical mechanism of release that we demonstrate works well for releasing bulk populations of selected particles, it is likely difficult to adapt the process to separately release individual colonies (e.g., a single particle in a single well). Such single-particle isolation is important if a researcher wishes to explore the different strategies for hyperperformance and the various underlying genetic mechanisms that result in such phenotypes. While it may be possible to engineer tools to mechanically break down a single particle, release of cells using these tools may be complicated and inefficient. In addition, there are studies that indicate that shear stress can negatively impact cells. For example, while shear stress may not induce cell death, such stress on microalgae or yeast may cause a decreased growth rate ( 42 , 43 ). Shear stress can even reduce the expression of a recombinant protein in CHO cells ( 44 ). Hence, it may be necessary to fully develop PicoShells that are chemically degradable and compatible with several cell types. Although we have engineered disulfide cross-linked PicoShells that are compatible for yeast applications, we have also shown that it is difficult to discover chemistries that enable chemical degradation and maintain cell viability for more sensitive cell types. Limitations on Throughput. We have also found that there is a tendency for cross-linked material to stick to the walls near the droplet generation junction, causing a disruption in the flow ( SI Appendix , Fig. S19 ). Since we use pH-induced gelation and the gellable materials (PEG-MAL and DTT) come into close proximity briefly before droplet generation, gelled material often forms at the junction, inducing jetting and disruption of particle formation ∼15 min after initial particle formation. As a result, the device needs to be replaced each time particle formation is halted, reducing the overall number of PicoShells that can be manufactured to 370,000 particles per device. The jetting of reagents due to premature formation of gelled material that sticks to the walls of the droplet generator limits the overall throughput of PicoShell generation. One potential way to address this is to use a coaxial device geometry to reduce the amount of gelled material that sticks to the walls of the device ( 45 ). Use of UV-induced cross-linking mechanisms can also address this problem since gelation would occur downstream of droplet generation ( 29 , 46 , 47 ) unlike pH-induced mechanisms, where mixing of reagents immediately prior to droplet generation often results in gelled material forming in the droplet generation junction over time that disrupts the overall flow. UV cross-linking could also enable PicoShell manufacturing approaches with higher throughput ( 48 ). However, use of UV-induced cross-linking likely creates issues for particular cellular applications, as previously discussed. At the same time, UV-induced cross-linking may be used for workflows involving resilient cell types (e.g., bacteria) or workflows where cells are mutagenized prior to selection, and UV-induced mutations would be potentially beneficial. A summary of the different types of PicoShells that we can currently fabricate and their advantages and disadvantages is shown in Table 1 . Table 1. Summary of current PicoShell variations Fabrication throughput (PicoShells/h) Chemical release mechanism Primary advantage Primary disadvantage DTT cross-linked with UV 2.5 million NaIO 4 High fabrication throughput Unclear how UV affects cells DTT cross-linked via pH 1.3 million NaIO 4 Compatible with most cell types Limited to mechanical degradation to viably release cells Peptide cross-linked via pH 1.3 million MMPs or trypsin Cells can be chemically released Cells may prematurely release themselves via enzyme secretions Disulfide cross-linked via pH 1.3 million DTT or TCEP Cells can be chemically released Only compatible with robust cell types such as bacteria and yeast Information is based on cells and chemistries explored in this study and previous studies. Potential Future Applications. Despite these solvable limitations, the experimental evidence we have presented shows that PicoShell technology has significant advantages. The workflow can be potentially used for directed evolution of cell populations ( 49 ) where mutagenized cells are placed under selection pressures to generate strains based on unique selection criteria that are time dependent (e.g., growth and production of pigments), at the colony level (multicellular construct formation), or require solution exchange steps (lipid staining, enzyme-linked immunosorbent assays [ELISAs]). For example, the technology may be used to produce microalgae strains that overperform in lipid accumulation rates without significantly reducing their rate of growth for biofuel applications. The technology may also be used to generate yeast strains that maintain a high growth rate at higher ethanol concentrations, potentially enhancing the overall production of ethanol biofuels ( 50 , 51 ), plastics ( 52 ), materials ( 53 ), and alcoholic beverages ( 54 ). Additionally, PicoShells have now enabled the ability to select single cells and/or clonal colonies based on their behavior in environments that have not been previously possible. For example, we have demonstrated that S. cerevisiae can grow in a bioreactor that has cells external to the PicoShells, with constant stirring, and temperature controls ( SI Appendix , Fig. S20 ). Such a culture environment is not possible to achieve with other nanoliter-scale screening technologies, indicating the PicoShells may enable unique assays and applications that have previously been impossible. Given that PicoShells remain stable within our custom bioreactor and can be reisolated, it is probable that PicoShells can be placed into other commercial bioreactors, outdoor cultivation farms, and other unique environments and later isolated for screening and/or selection. The outer shell’s PEG material is also able to be modified, enabling the technology to be potentially used for relevant mammalian cell applications. For example, affinity motifs such as antibodies and peptides can be added to the solid matrix that can capture cellular secretions ( 29 ). Antibody-conjugated PicoShells may be used to produce hypersecreting and hypergrowing CHO cell populations based on their behavior in bioreactors for scaled production of protein therapeutics. The pore size of the particles may also be modulated by changing the molecular weight (MW) of PEG used to cross-link the solid phase ( 55 ) or by including nonfunctionalized PEG ( 56 ), gelatin ( 57 ), or hyaluronic acid ( 58 ) in the PEG phase. Adherence motifs, such as arginine-glycine-aspartate (RGD) peptides, fibronectin, or poly- l -lysine, may be also added to the outer PEG matrix so that stem cells, adherent CHO cells, or other adherent cell types have a solid surface to adhere to, further expanding the potential applications of the PicoShell workflow. In summary, we have shown that PicoShells may enable cell-line developers to develop cell populations based on their behavior in production environments. Unlike previously developed high-throughput screening tools, individual cells may be compartmentalized, placed into relevant environments such as bioreactors, exposed to natural stimuli, and selected based on their time-dependent behavior and growth in such environments via widely used flow cytometers. As a result, the technology has the exciting potential to rapidly accelerate the development of cell-derived bioproducts, such as biodiesel, materials, cell-derived agriculture, nutrient supplements, and protein therapeutics."
} | 4,032 |
37110452 | PMC10141921 | pmc | 8,629 | {
"abstract": "Bioleaching processes or microbially mediated iron/sulfur redox processes in acid mine drainage (AMD) result in mineral dissolution and transformation, the release of mercury and other heavy metal ions, and changes in the occurrence forms and concentration of mercury. However, pertinent studies on these processes are scarce. Therefore, in this work, the Fe/S redox-coupled mercury transformation mediated by Acidithiobacillus ferrooxidans ATCC 23270 under aerobic and/or anaerobic conditions was studied by combining analyses of solution behavior (pH, redox potential, and Fe/S/Hg ion concentrations), the surface morphology and elemental composition of the solid substrate residue, the Fe/S/Hg speciation transformation, and bacterial transcriptomics. It was found that: (1) the presence of Hg 2+ significantly inhibited the apparent iron/sulfur redox process; (2) the addition of Hg 2+ caused a significant change in the composition of bacterial surface compounds and elements such as C, N, S, and Fe; (3) Hg mainly occurred in the form of Hg 0 , HgS, and HgSO 4 in the solid substrate residues; and (4) the expression of mercury-resistant genes was higher in earlier stages of growth than in the later stages of growth. The results indicate that the addition of Hg 2+ significantly affected the iron/sulfur redox process mediated by A. ferrooxidans ATCC 23270 under aerobic, anaerobic, and coupled aerobic–anaerobic conditions, which further promoted Hg transformation. This work is of great significance for the treatment and remediation of mercury pollution in heavy metal-polluted areas.",
"conclusion": "4. Conclusions This work studied the mercury transformation behavior coupled with iron/sulfur redox mediated by A. ferrooxidans ATCC 23270 under aerobic, anaerobic, and coupled aerobic–anaerobic conditions. The presence of Hg 2+ significantly inhibited microbial growth by decreasing the iron/sulfur redox activities of A. ferrooxidans ATCC 23270, but the cells grown under aerobic–anaerobic coupling conditions could more quickly release this inhibition than other groups due to the strong adsorption of pyrite on Hg 2+ . The addition of Hg 2+ caused a significant change in the composition of bacterial surface compounds and elements such as C, N, S, and Fe, and thus affected the speciation transformation of Hg and S, where Hg was mainly present in the form of Hg 0 , HgS, and HgSO 4 in the solid substrate residues for all groups. The presence of Hg 2+ seriously inhibited the expression of functional genes relevant to the iron and sulfur metabolism of A. ferrooxidans ATCC 23270, and the expression of mercury-resistant genes was higher in earlier stages of growth than in the later stages of growth for the groups with aerobic iron and sulfur oxidation and the anaerobic Fe 3+ reduction coupled with S 0 oxidation. For the aerobic–anaerobic coupling group, when comparing with gene expression at the aerobic stage, we can see that the expression level of mercury reduction genes increased, and the stress effect was weakened after A. ferrooxidans ATCC 23270 entered the anaerobic stage.",
"introduction": "1. Introduction Heavy metal pollution is a major environmental problem in the world today, and continued heavy metal pollution poses a potentially significant threat to almost all forms of life in the environment [ 1 , 2 ]. Mercury is a pollutant with high toxicity, persistence, enrichment, and mobility that can exist in inorganic forms, such as divalent mercury ions (Hg 2+ ) and elemental mercury (Hg 0 ), and can be transformed in situ to methylated forms. The toxicity of mercury is determined by its speciation and its occurrence forms [ 3 , 4 , 5 ]. The microbially mediated dissolution of sulfur-containing minerals is an important source of mercury pollution in mining areas [ 6 ]. Mining areas are an important site of mercury release and migration and pose a major threat to the surrounding soil and water environment. During the occurrence and development of mercury pollution, extremophilic microorganisms in the mining areas (such as extremely acidophilic, thermophilic, salt-tolerant, and heavy metal ion-tolerant microorganisms) occur in the main niche [ 7 , 8 ], which promotes the interaction between minerals, microorganisms, and metals; leads to the further dissolution and transformation of minerals; and significantly changes the occurrence forms and toxicity of mercury [ 9 , 10 ]. In the acid mine drainage environment, chemoautotrophic acidophilic iron- and sulfur-oxidizing microorganisms can oxidize and leach sulfide ores, and chemoautotrophic microorganisms metabolize organic carbon to produce organic acids and promote the dissolution of secondary oxidized minerals, which can lead to the release of mercury into water and soil, resulting in mercury pollution [ 11 ]. Acidithiobacillus ferrooxidans , a typical chemoautotrophic Gram-negative bacterium, can survive and reproduce in the acid mine drainage (AMD) of sulfide mineral mining environments. Under aerobic conditions, A. ferrooxidans can oxidize Fe 2+ and S 0 and reduce sulfur compounds to obtain energy [ 12 , 13 ]. At the same time, A. ferrooxidans can also obtain energy under anaerobic conditions with S 0 , H 2 , and Fe 2+ as electron donors and Fe 3+ and S 0 as electron acceptors [ 14 , 15 ]. Through a genomic study of A. ferrooxidans , it was found that it has resistance to mercury and other heavy metals [ 16 ]. As early as 1982, it was found that the resistance mechanism of A. ferrooxidans to mercury mainly depends on mercury reduction by merA encoded by the mer operon [ 17 , 18 ]. In addition to the mercury reduction mechanism involved in the mer operon, cytochrome c oxidase also plays an important role in mercury reduction [ 18 ]. However, there are few studies on the combination of iron and sulfur metabolism in A. ferrooxidans and its mercury transformation mechanism. Additionally, the relationship between iron/sulfur speciation transformation and mercury fate has rarely been studied. Thus, in the present study, by changing the oxygen state in the environment, the coupling relationship between iron and sulfur redox and mercury transformation behavior under aerobic, anaerobic, and aerobic–anaerobic coupling conditions was revealed, and the mercury transformation mechanism mediated by iron and sulfur redox of A. ferrooxidans was expounded. This study lays a foundation for further understanding the role and regulatory mechanism of extreme acidophilic microorganisms in the geochemical cycle of mercury.",
"discussion": "3. Results and Discussion 3.1. Bacterial Growth and Fe/S Oxidation Figure 1 shows the curves of cell density and Fe/S oxidation over time for all experimental groups with 0 and 1 mg/L [Hg 2+ ]. Under the condition of aerobic Fe 2+ oxidation, the group with Hg 2+ showed an obvious lag phase at 0–88 h, in which the bacterial density did not increase significantly, then it entered the logarithmic phase at 88–158 h, and the bacterial growth reached the stationary phase at 158 h. In contrast, the group without Hg 2+ had no obvious lag phase, and the bacteria entered the logarithmic phase within 10 h and reached the stationary phase after 88 h ( Figure 1 a). [Total Fe] and [Fe 2+ ] showed a decreasing trend in all systems ( Figure 1 b and Figure S2a ). [Total Fe] and [Fe 2+ ] for the group without Hg 2+ decreased rapidly without a significant delay and finally reached the lowest concentration of 0.68 mg/L. However, there were no significant changes in [Total Fe] or [Fe 2+ ] within 0–88 h in the group of bacteria with Hg 2+ , and they then rapidly decreased after 88 h. Compared with the group without Hg 2+ , the decreases in [Total Fe] and [Fe 2+ ] in the solution of the group with Hg 2+ were slower. These results indicate that the bacterial growth was inhibited in the presence of 1 mg/L Hg 2+ due to the toxic effect of Hg 2+ , with the inhibition of microbial Fe 2+ oxidation at the early phase. Furthermore, in addition to the oxidation of A. ferrooxidans ATCC 23270, oxygen in the air also contributes to a small degree to the oxidation of Fe 2+ in the solution. When Fe 2+ is oxidized to produce a large amount of Fe 3+ , jarosite will be formed under the appropriate pH value, resulting in a decrease in the total Fe concentration in the solution. Under aerobic S 0 oxidation, the bacterial density of all systems showed a trend of rising at the initial stage and then changing a little ( Figure 1 c). There was no significant change in 0–62 h, indicating that the bacterial growth was in the lag phase. Compared with the group without Hg 2+ , the group with Hg 2+ showed slower bacterial growth and a longer time to reach the stationary phase. The presence of Hg 2+ has a strong stress effect on the early stage of microbial growth, leading to the extension of the lag phase. With the adaptation of the mercury-resistant mechanism and the gradual release of mercury stress, A. ferrooxidans ATCC 23270 gradually enters the logarithmic phase. There was no significant change in [SO 4 2− ] for the sterile control group, while [SO 4 2− ] for all bio groups (with A. ferrooxidans ATCC 23270) showed an increasing trend and increased with bacterial growth ( Figure 1 d). [SO 4 2− ] had no significant change before 88 h in the bio group with Hg 2+ . After 88 h, it began to increase rapidly, and the inhibition was relieved to a certain extent. However, compared with the control group without Hg 2+ , the increase in [SO 4 2− ] was slower over time. This result shows that A. ferrooxidans ATCC 23270 significantly inhibits the oxidation process of elemental sulfur to produce acid, and the presence of Hg 2+ has a certain negative effect on A. ferrooxidans ATCC 23270 sulfur metabolism. Under the conditions of anaerobic Fe 3+ reduction coupled with S 0 oxidation, the bacterial density in the solution showed an overall increasing trend ( Figure 1 e). There was no obvious lag phase for the group without Hg 2+ , and the bacterial concentration began to enter the logarithmic phase at day 2. On the other hand, bacteria in the experimental group supplemented with 1 mg/L [Hg 2+ ] were in the lag phase at 0–5 days, and their growth was significantly inhibited, which may have been due to the stress effect of Hg 2+ leading to the low activity of bacteria. The concentration of bacteria increased significantly after 5 days. [Fe 2+ ] in the solution of the bacterial system showed an increasing trend ( Figure 1 f), while [Total Fe] had no significant change ( Figure S2b ). In the control group without Hg 2+ , [Fe 2+ ] increased rapidly immediately after inoculation, indicating that the bacteria grew well with the high activity of the anaerobic Fe 3+ reduction coupled with S 0 oxidation ( Figure 1 f). However, the bacteria under the influence of Hg 2+ showed a weaker Fe 2+ reduction ability and slower [Fe 2+ ] increase compared with the group without Hg 2+ . There was no significant change in the Fe 2+ concentration in the aseptic system. The concentration changes of different forms of Fe in solution indicate that A. ferrooxidans ATCC 23270 in the system reduces Fe 3+ . The reason why there was no significant change in [Total Fe] may be that most Fe existed in the solution during bacterial action and did not migrate to the substrate residue and cell surface. Meanwhile, [SO 4 2− ] for the bio group showed an increasing trend, while [SO 4 2− ] for the sterile group showed no significant change ( Figure 1 f). In the bacterial system without Hg 2+ , [SO 4 2− ] increased rapidly due to the oxidation of bacteria and reached the highest level at day 20. However, [SO 4 2− ] for the bio group in the presence of Hg 2+ showed an increasing trend from day 10, and the increase was slower than that for the bio group without Hg 2+ . Under the conditions of the aerobic–anaerobic coupling experiment ( Figure 1 g), the bacterial density showed an increasing trend under aerobic conditions and reached a stable concentration at day 8. After entering the anaerobic environment, the concentration of bacteria increased again and then decreased and reached stability at day 21. This was because, during the aerobic culture stage, the addition of Hg 2+ inhibited the initial growth of bacteria. With the progress in the mercury conversion process, the stress effect of Hg 2+ was almost lifted at day 11. Therefore, after entering the anaerobic culture stage, the growth process of bacteria was hardly inhibited by Hg 2+ . Under aerobic conditions, the oxidation of pyrite by bacteria resulted in an increase in [Fe 3+ ] in solution ( Figure 1 h), and the [Total Fe] also showed an increasing trend ( Figure S2c ). After entering the anaerobic stage, the iron reduction of bacteria resulted in a decrease in [Fe 3+ ] in the solution, but [Total Fe] did not change significantly. Since most Fe in the solution exists in the form of Fe 3+ , when Fe 3+ is reduced by bacteria under anaerobic conditions, it exists in the form of Fe 2+ in the solution. As a result, [Total Fe] shows no obvious change when the bacterial growth environment is anaerobic. There was no significant change in [Fe 3+ ] in the sterile system, but [Total Fe] increased slightly, which may have been caused by the dissolution of pyrite in the medium. In addition, compared with the control group without Hg 2+ , the increase in [Fe 3+ ] in the solution of the bacterial system with Hg 2+ was slower with time, but [Fe 3+ ] was higher after day 11. This result indicates that the presence of Hg 2+ inhibited the activity of bacterial growth at the initial stage, and the metabolic activity of bacterial growth was improved after the removal of the inhibition effect. [SO 4 2− ] in the solution of all experimental systems showed an increasing trend due to the leaching effect of pyrite by bacteria and the dissolution effect of pyrite itself at low pH values ( Figure 1 h). The addition of Hg 2+ has an inhibitory effect on the growth and metabolism of bacteria, resulting in the slow elevation of solution [SO 4 2− ]. 3.2. Changes in pH and [Hg 2+ ] in Aqueous Solution For the group with Fe 2+ oxidation by A. ferrooxidans ATCC 23270 under aerobic conditions ( Figure 2 a and Figure S2d ), the overall pH value increased initially and then decreased. Compared with the bio group without Hg 2+ , the pH value of the bacterial experimental group with 1 mg/L [Hg 2+ ] increased and decreased slowly, and the lowest pH value (1.76) was relatively high. [Hg 2+ ] in the solution for the bio group with Hg 2+ showed an overall downward trend, and [Hg 2+ ] fluctuated with time during 0–88 h and rapidly decreased after 88 h ( Figure 2 b). This may have been because A. ferrooxidans ATCC 23270 can reduce the total Hg concentration by reducing Hg 2+ to Hg 0 . In addition, the formation of ferrosite substances also has a certain adsorption effect on Hg [ 24 ]. In contrast, there was no significant change in [Hg 2+ ] for the sterile control group. For the groups with S 0 oxidation by A. ferrooxidans ATCC 23270 under aerobic conditions ( Figure 2 c and Figure S2e ), the pH value of the solution showed a decreasing trend with time within 0–244 h. There was no obvious change in pH value for the bacterial group within 0–88 h. The overall pH value decreased slowly and finally reached the lowest pH value (244 h). A. ferrooxidans uses S 0 as the energy substrate for oxidation, which is an acid-producing process, resulting in a decrease in solution pH value. However, Hg 2+ can obviously inhibit the process, resulting in a slower pH decline in the group with Hg 2+ . There was no significant change in the sterile control group [Hg 2+ ] ( Figure 2 d). For the bacterial group with [Hg 2+ ], the effect of mercury stress was the weakest when the concentration of Hg 2+ reached 0.83 mg/L. Hg 2+ in the solution reacts with SO 4 2− to form HgSO 4 , which can also be complexed with S 0 and adsorbed to the surface of S 0 particles [ 25 ]. It can also bind Hg 2+ through sulfhydryl groups on the surface of bacteria, resulting in a decrease in [Hg 2+ ] in the solution. For the group with the anaerobic Fe 3+ reduction coupled with S 0 oxidation by A. ferrooxidans ATCC 23270 ( Figure 2 e and Figure S2f ), the overall pH value showed a downward trend. The pH value of the group without Hg 2+ decreased rapidly and reached the lowest value (1.28) at day 12. Compared with the bacterial group with 1 mg/L [Hg 2+ ], the pH value decreased more slowly and reached the lowest level at day 20 (1.33). There was no significant change in pH value over time for the sterile group with Hg 2+ . As seen from the pH change trend for each system, anaerobic Fe 3+ coupled with S 0 oxidation is an acid-producing process, and the presence of Hg 2+ has a certain inhibitory effect on its growth and metabolism. The bacterial group with Hg 2+ showed a decreasing trend ( Figure 2 f). When [Hg 2+ ] was 0.8 mg/L, the stress effect on A. ferrooxidans ATCC 23270 was the weakest. In contrast, there was no significant change in [Hg 2+ ] for the sterile group. For the aerobic–anaerobic coupling group ( Figure 2 g and Figure S2g ), the overall pH value decreased and showed a gentle trend over time during the aerobic stage (0–13 d) due to bacterial iron and sulfur oxidation. After entering the anaerobic stage (13–30 d), the pH value of the solution further dropped to the lowest value and then reached stability. At the same time, the pH value of the sterile system decreased slightly due to abiotic oxidation, but there was no significant change trend. In the aerobic culture stage, the pH value of the bacterial group with 1 mg/L [Hg 2+ ] decreased more slowly than that of the bacterial group without Hg 2+ and reached the lowest pH value (1.58) at day 12. There was no significant difference in pH value in the anaerobic culture stage. In addition, the overall pH of the sterile control group with Hg 2+ was higher than that without Hg 2+ . For the group with Hg 2+ , [Hg 2+ ] showed a rapid decline and then a stable trend ( Figure 2 h). Compared with the sterile group, [Hg 2+ ] decreased slower and was higher for the bio group, indicating that pyrite enables the strong adsorption of Hg 2+ . Due to the Hg transformation of bacteria, Hg exists in the solution for a long time [ 26 ]. With the utilization of pyrite by A. ferrooxidans ATCC 23270, a small amount of Hg 2+ returns to the solution through the mercury transformation of bacteria [ 27 ], resulting in the fluctuation of [Hg 2+ ] in the solution for the bio groups over time. When [Hg 2+ ] is 1 mg/L, the stress effect produced by A. ferrooxidans ATCC 23270 is the strongest. In the process of bacterial growth, [Hg 2+ ] floats within the range of 0.15 ± 0.03 mg/L and finally reaches [Hg 2+ ] min of 0.14 mg/L in the anaerobic stage ( Figure 1 g). 3.3. Changes in Cell Morphology and Element Distribution A. ferrooxidans ATCC 23270 cells of different sections for the Fe 2+ oxidation group showed clear cell boundary outlines and cell content shadows ( Figure 3 a,b). Black spots appeared in the cell sections for the group with Fe 2+ oxidation with Hg 2+ , and the black spots were distributed outside the cells. The main components inside the cells were N, O, S, and Fe. The bacterial group with Hg 2+ had a small amount of Hg inside the cells ( Figure 3 c). In addition, the proportion of Fe in the group with Hg 2+ was higher than in that without Hg 2+ , but the proportion of S was lower than in that without Hg 2+ ( Figure S3a ). Through the analysis of the distribution of elements in the cells of the Fe 2+ oxidation group, it was found that the main element composition of the black particle region in the cells of the group with Hg 2+ was probably Fe. Since no additional S 0 was added in the Fe 2+ oxidation group, the bacteria had a weak ability to enrich Hg 2+ , resulting in low Hg content in bacterial cells, which were difficult to detect. For the group with S 0 oxidation with Hg 2+ , the cross-section of cells shows numerous small black spots on the outside and inside of the cell wall ( Figure 3 d,e). In addition to N, O, S, P, and other elements, Hg was also detected at a higher proportion in the cells for the bio group with Hg 2+ ( Figure 3 f). In addition, the proportion of S was significantly higher here than in the group without Hg 2+ ( Figure S3b ). Comprehensive analysis of the composition of elements inside cells in the S 0 oxidation group showed that a large amount of Hg elements were distributed in the black particle area inside the cells of the group with Hg 2+ , which further proved that Hg 2+ was captured by bacteria through the extracellular polymer and transported to the cell for transformation. For the anaerobic Fe 3+ reduction group, the cell outline was clear, and the contents were obviously shaded ( Figure 3 g,h). Here, there were a few black particles in the middle of the cell walls of the cells without Hg 2+ ( Figure 3 g). Under the influence of Hg 2+ , a small amount of black granular material was adsorbed on the cell surface, and a shadow of flocculent material was evident around the cell ( Figure 3 h). The cell interior was mainly composed of N, O, S, P, Fe, and other elements ( Figure 3 i). The proportion of N and O elements in cells supplemented with Hg 2+ was significantly higher than that in cells without Hg 2+ , indicating that cells would secrete more organic substances under the influence of Hg 2+ ( Figure S3c ). In addition, a very small amount of Hg was detected inside the cells of the system supplemented with Hg 2+ , and it is possible that most Hg was mainly distributed on the cell surface. Through comprehensive analysis of the above results, it was found that the black particles in the cells without Hg 2+ addition may have been Fe, while the black particles on the cell wall surface in the system with Hg 2+ addition may have been related to Hg, which further proves that A. ferrooxidans ATCC 23270 can capture Hg 2+ through the extracellular polymer on the cell surface. In addition, under the influence of Hg 2+ , A. ferrooxidans ATCC 23270 may secrete more carbohydrates, lipids, proteins, and other substances for the mercury conversion process. For the aerobic–anaerobic coupling group, in the aerobic phase, there were large black particles inside the bacterial cells and outside the cell wall with the addition of Hg 2+ ( Figure 3 j,k). The main components of the cells were N, P, O, S, and Fe ( Figure 3 l). The proportion of N and P elements in bacteria without Hg 2+ was significantly higher than that in bacteria with Hg 2+ under aerobic conditions ( Figure S3d ). In addition, the bacterial group with Hg 2+ had a higher proportion of Fe. Combined with TEM analysis, it was found that the black particles in the cells were likely to be Fe complexes. This result was consistent with the change in [Fe 3+ ] in the bacterial leaching process ( Figure 1 h), indicating that the metabolic activity of bacteria was significantly improved after the removal of Hg stress under the addition of Hg 2+ . There was more flocculent material around the cells growing in the anaerobic stage ( Figure 3 m,n). After entering the anaerobic stage, the proportion of N and O elements in bacteria in the bacterial group with Hg 2+ increased significantly, which was almost the same as the result for the bacteria without Hg 2+ ( Figure 3 o and Figure S3e ). This result indicates that the Hg 2+ stress effect on bacteria was greatly reduced at this stage. In the bacterial system with added Hg 2+ , only a very small amount of Hg was detected inside the cells under aerobic and anaerobic conditions ( Figure 3 o), possibly because most of the added Hg 2+ was adsorbed on the surface of pyrite, so the concentration of Hg on the surface and inside the cells was very low. 3.4. Changes in Solid Residue Morphology and Element Distribution The SEM-EDS results for the substrate residue at 244 h in the A. ferrooxidans ATCC 23270 Fe 2+ oxidation process are shown in Figure 4 a,b. There were no significant differences in the morphologies of the substrate residues in the two systems, and the residues were agglomerated into spheroids with sharp edges and good crystals ( Figure 4 a,b). The main components of the substrate residue were O, Fe, S, and K. The results show that there was no significant change in the proportion of the main components of the substrate residue in the Hg 2+ system compared with that in the system with addition, and no Hg was detected. In summary, the above results indicate that the main component of the substrate residue formed in the Fe 2+ oxidation group may have been jarosite. In addition, the reason why Hg could not be detected in the substrate residue of the group with Hg 2+ may have been that the EDS could not be detect it due to the low content of Hg adsorbed on the surface of jarosite. Further detection approaches are needed to analyze the Hg element on the jarosite surface. In the sulfur oxidation group, the substrate residue surface in the system without Hg 2+ was obviously eroded by bacteria and the surface was rough, but the elemental sulfur particles were intact ( Figure 4 c). In addition, compared with the bacterial group, the substrate residues of the Hg 2+ system showed smaller particle sizes and less aggregation, with fewer surface pits but a greater depth ( Figure 4 d). The EDS results show that the substrate residue surface was still dominated by S, and trace Hg was detected on the surface of the Hg 2+ system. In conclusion, bacteria in the group without Hg 2+ were mainly attached to the surface of sulfur for erosion, thus leaving dense indentations, while the group with Hg 2+ may have eroded into the interior of the sulfur, resulting in fewer but deeper indentations on the surface of the substrate residue. In addition, Hg 2+ in solution can be transformed and adsorbed to the surface of sulfur under the action of bacteria and can also combine with the surface of sulfur through chemical action. For the anaerobic Fe 3+ reduction coupled with S 0 oxidation group, the substrate residue of the system without Hg 2+ was rough, uneven, and covered with pits, and more granular debris was attached ( Figure 4 e). This may have been caused by bacterial erosion. There were fine cracks and more pits on the surface of the substrate residue in the group with Hg 2+ added, as well as smaller fragments attached to the surface, and the whole surface was looser ( Figure 4 f). Compared with the substrate residue without Hg 2+ , the degree of bacterial erosion was slightly less, indicating that the presence of Hg 2+ had an effect on the growth and metabolism of bacteria. Since only S 0 was added to the solid substrate, EDS analysis showed that the main components of the substrate residue were S followed by C, O, and a small amount of Fe. A large proportion of Hg was detected on the substrate residue surface of the Hg 2+ system ( Figure 4 f), indicating that the Hg transformation mechanism of A. ferrooxidans ATCC 23270 helps to transfer Hg 2+ in solution to the substrate residue surface and fix it by combining it with S. In the aerobic and anaerobic coupled system, pits caused by bacterial erosion appeared on the mineral surface at the aerobic stage, and a small amount of flocculent substance was present around the pits ( Figure 4 g,h). After a period of anaerobic culture, patches of regularly shaped erosion pits appeared on the mineral surface, accompanied by a large amount of flocculent material ( Figure 4 i,j). The EDS results show that the proportions of C, N, O, P, and other elements in substrate residues were slightly higher, indicating that organic substances such as proteins, lipids, and sugars may be secreted on the surface of minerals during the interaction between bacteria and minerals. The proportion of C element (22.96%, 45.64%) on the substrate residue surface of the system without Hg 2+ addition in the aerobic stage and anaerobic stage was significantly higher than that in the system with Hg 2+ addition (0.64%, 32.07%), indicating that the flocculent material around the bacterial erosion pit was organic material composed of C. The addition of Hg 2+ had a strong inhibitory effect on the growth and metabolism of bacteria. In addition, the proportion of Hg on the surface decreased gradually from the aerobic stage to the anaerobic stage until no Hg was detected at the end of the anaerobic stage, indicating that the interaction between bacteria and minerals accompanied the mercury transformation process. 3.5. Changes in Morphology, Composition, and Phase of Solid Residue XRD was used to characterize substrate residues in different culture systems, and the results are shown in Figure 5 . The XRD results for the substrate residue for the group with Fe 2+ oxidation ( Figure 5 a) show that the main component of the solid substance was jarosite when cultured to 244 h, indicating that the Fe 2+ in the bacterial oxidation solution produced a large amount of Fe 3+ , which reacted with SO 4 2− and K + in the solution to produce jarosite precipitation [ 28 ]. A small amount of HgS was found for the group with the addition of Hg 2+ . The results for the group with S 0 oxidation are shown in Figure 5 b. The main component of the substrate residue was still mainly S 0 , indicating that, because the products of bacterial oxidation of S 0 are mainly water-soluble, there was little residue on the surface, and it could not be detected. In addition, the XRD results for the substrate residues in each system showed no significant differences in peak shape and intensity, and it was found that diffraction peaks related to HgS and HgSO 4 basically coincided with S 0 diffraction peaks, so it was impossible to accurately judge whether Hg was present on the surfaces of substrate residues in the Hg 2+ system. For the group with anaerobic Fe 3+ reduction coupled with S 0 oxidation, the main component of the substrate residue in different systems was still S 0 at day 16 ( Figure 5 c). Due to the low concentration of Hg 2+ applied and the fact that most of the XRD peaks of mercury compounds coincide with the characteristic peaks of S 0 , the phase changes on the substrate residue surface could not be accurately characterized ( Figure 5 c). For the aerobic–anaerobic coupling group, the substrate residue was mainly composed of pyrite at day 13 of cultivation under aerobic conditions, and a small jarosite diffraction peak appeared in the bio group ( Figure 5 d). After entering the anaerobic environment for a period of time, the main components of the substrate residue for the bio groups were still pyrite and a small amount of jarosite, but the intensity of the jarosite diffraction peak decreased significantly, indicating that the anaerobic conditions under the action of bacteria may lead to partial jarosite dissolution ( Figure 5 e). In addition, the HgS diffraction peak appeared in the same position in the bacterial system with Hg 2+ at day 13 and the sterile system with Hg 2+ at day 30, which was consistent with the SEM-EDS results, confirming that the contents of Hg on the substrate residue surfaces of the bacterial system and the sterile system were different. This indicates that A. ferrooxidans ATCC 23270 mercury transformation can lead to the migration of mercury elements between the surface of the substrate residue and the solution. To explore the compositions of and changes in substrate residues on the surface of A. ferrooxidans ATCC 23270 cells, FT-IR spectroscopy was undertaken according to a methodology from the literature [ 29 , 30 ], and the results are shown in Figure 6 . In the group with Fe 2+ oxidation by A. ferrooxidans ATCC 23270, sharp absorption peaks were found at 624, 985, 1080–1200, 1424, and 1642 cm −1 , and wide and strong absorption peaks were found at 2021, 3217, and 3561 cm −1 ( Figure 6 a). Among these, the peaks at 624 and 1080–1200 cm −1 represent the absorption of PO 2 − on the surface of phosphate groups or phospholipids [ 31 ] or were generated by SO 4 2− stretching vibration [ 32 ]. The absorption peak at 985 cm −1 represents the deformation vibration of OH, and the absorption peak at 1642 cm −1 represents the deformation vibration of HOH [ 33 , 34 ]. The range from 3200 to 3600 cm −1 is the vibration absorption of -OH, and the range from 1424 cm −1 is the absorption of -CH 3 , indicating the presence of lipid or carbohydrate adsorption. The A. ferrooxidans ATCC 23270 Fe 2+ oxidation system formed in the substrate residue was the main component of jarosite. In the S 0 oxidation system, the substrate residue surface of the bacterial group with Hg 2+ showed higher absorption peaks at 1052, 1203, 1442, 1535, 1665, and 1720 cm −1 than in that without Hg 2+ ( Figure 6 b). The peak at 1052 cm −1 represents the vibration absorption of S=O, the peaks at 1203 and 1442 cm −1 represent the absorption generated by the bending vibration of -CH 2 and -CH 3 in lipids, and the peaks at 1535 and 1650–1850 cm −1 represent the absorption peak generated by the vibration of -NH 2 and -C=O. These results indicate that the substrate residues in the bacterial group with Hg 2+ formed a large number of organic components, such as proteins and lipids, after bacterial action at 244 h. It was found that the bacteria in the group without Hg 2+ began to adsorb and grow on the substrate sulfur at the early stage (14 h), reached the highest metabolic intensity at the middle stage (110 h), and entered the declining stage later (244 h), and the amount of organic matter adsorbed on the surface of the substrate residue decreased. However, the bacteria in the group with Hg 2+ grew slowly in the early and middle stages (14 h, 110 h) due to the inhibiting effect of Hg 2+ . After the bacteria gradually came to tolerate Hg 2+ through the mechanism of mercury conversion, Hg 2+ stress was finally relieved, and the growth and metabolism of bacteria reached the most vigorous stage ( Figure 6 b and Figure S4a,b ). A large number of absorption peaks with different strengths were present on the substrate residue surface at 500–2000 cm −1 , among which 843 cm −1 is the characteristic peak of S 0 [ 35 ], 1060 cm −1 is the absorption peak generated by S=O vibration, and 1219 cm −1 is the absorption peak of the C-O bond ( Figure 6 c). The absorption peak of carbohydrate or lipid -CH 3 groups is 1427 cm −1 ; 1480–1800 cm −1 is the absorption peak generated by the vibration of protein amide bonds; 2700–3700 cm −1 is a strong and wide peak and is considered to be related to -OH and -NH. It was found that the peak intensity of the bacterial system in the carbohydrate-, lipid-, and protein-related absorption peak region was significantly higher than that of the sterile group, indicating that the bacterial growth and metabolism intensity in the system was high, and there was a greater amount of adsorbed bacteria on the substrate residue surface. All the substrate residues showed different intensity absorption peaks at 662, 1056, 1428, 1513, 1676, 2359, and 2700–3370 cm −1 ( Figure 6 d,e). Here, 662 and 1056 cm −1 represent the absorption peaks generated by the bending vibration and asymmetric stretching vibration of SO 4 2− , 1428 cm −1 represents the absorption peak of -CH 3 , and 1513 and 1676 cm −1 represent the absorption peaks related to amide bonds in proteins; 2359 cm −1 is generally considered to be the absorption peak produced by CO 2 , and the strong and wide peak at 2700–3370 cm −1 represents the stretching vibration of the -OH group. It was found that the SO 4 2− (662 and 1056 cm −1 ), -CH 3 (1428 cm −1 ), and -OH (2700–3370 cm −1 ) absorption peaks on the substrate residue surface of the sterile control group were stronger, and the SO 4 2− absorption peaks decreased with the increase in time. After bacterial action, the -CH 3 - and -OH-related absorption peaks gradually decreased with the increase in time, while the protein-related absorption peaks at 1513 and 1676 cm −1 were significantly enhanced, indicating that bacterial action resulted in changes in the crystal structure of the mineral surface, increased the adsorption of organic matter on the substrate residue surface, and made the bacterial metabolism more vigorous. The Raman spectra show that the main component of the substrate residue for the group with Fe 2+ oxidation by A. ferrooxidans ATCC 23270 was jarosite ( Figure 7 a). The substrate residue of the bacterial system with Hg 2+ had an obvious and sharp absorption peak at 378 cm −1 , which was FeS 2 , indicating that the addition of Hg 2+ leads to the formation of Fe-S bonds on the surface of the jarosite. The substrate residues for the groups with aerobic S 0 oxidation and anaerobic Fe 3+ reduction coupled with S 0 oxidation had obvious S 0 peaks at 151, 217, 245, 436, and 472 cm −1 ( Figure 7 b,c). For the bio group with Hg 2+ , the substrate residue showed obvious signal noise, indicating the presence of Hg 0 on the surface of the substrate residue. This then continued to transform into other forms of Hg. In the Raman spectra for the aerobic–anaerobic coupling group, the pyrite characteristic peaks were found at 344, 380, and 429 cm −1 ( Figure 7 d,e). In addition, the characteristic peaks of jarosite appeared at 138, 220, 1005, and 1100 cm −1 , and the characteristic peaks of jarosite ( Figure 7 d) in the aerobic stage were stronger than those in the anaerobic stage ( Figure 7 e). At the same time, the characteristic peaks of S n 2− and other polysulfides produced during the oxidation of pyrite appeared at 476 cm −1 , which indicates that the surface of the mineral was oxidized by bacteria and formed jarosite and polysulfides [ 36 ]. In addition, the sterile control group with Hg 2+ showed the characteristic peaks of Fe-O in jarosite and Hg-S in HgS at 218 and 280 cm −1 , indicating that Hg 2+ in the solution mainly combined with S on the surface of pyrite to form Hg-S bonds and affected the structure of surface minerals. In the S K-edge XANES analysis, elemental sulfur (S 0 ), sodium thiosulfate (Na 2 S 2 O 3 ), pyrite (FeS 2 ), sodium sulfate (Na 2 SO 4 ), copper sulfide (CuS), and potassium persulfate (K 2 S 2 O 8 ) were selected as S-containing reference samples ( Figure 8 a). In the S 0 oxidation group, the characteristic peak of the substrate residue surface S 0 was at 2.4724 keV, and the characteristic peak of SO 4 2− was at 2.4825 keV ( Figure 8 b). With the oxidation of S 0 in the bio group, the surface of substrate S 0 was oxidized to SO 4 2− . In addition, the SO 4 2− characteristic peak signal on the substrate residue surface was the strongest in the bacterial group at 244 h, which was consistent with the FT-IR result relating to the substrate residue ( Figure 6 b), confirming that Hg 2+ may be present in the substrate residue in the form of HgSO 4 . With the interaction between A. ferrooxidans ATCC 23270 and substrate S 0 , the substrate residue surface showed a strong absorption peak at 2.4825 keV, which gradually increased with time ( Figure 8 c). It was found that the sulfur speciation transformed from S 0 to SO 3 2− and SO 4 2− , indicating that the substrate S 0 in each group was gradually oxidized. Compared with the bacterial group without Hg 2+ , the SO 4 2− absorption peak intensity was lower at day 5 and day 10, while the peak intensity was significantly increased at day 16. The results indicate that the bacteria were strongly inhibited by Hg 2+ in the early and middle stages of growth, and the growth and metabolic intensity were significantly improved after the inhibition of Hg 2+ was lifted in the later stage. The characteristic peaks of FeS 2 and SO 4 2− were located at 2.4721 eV and 2.4826 eV, respectively ( Figure 8 d). With time, in the bacterial system, the absorption peak at SO 4 2− shifted to the right to 2.4828 eV, and the peak intensity continued to increase, indicating that the S species on the substrate residue surface were continuously oxidized, with the formation of jarosite and other intermediates. In addition, the SO 4 2− absorption peak intensity on the substrate residue surface for the bio group with Hg 2+ was much higher than that of other systems at day 30, which was the same result as in Figure 8 b,c, indicating that the metabolic activity of bacteria was greatly improved after the removal of the inhibition of Hg 2+ . Via the comprehensive analysis of the XPS spectral fitting results for the substrate residue surface Hg 4 f (see Figure S4 and the relevant description in the Supplementary Materials ) and the S K-edge XANES spectra for all experimental groups, it was found that the morphological transformations of S, Fe, and Hg are closely related to the Fe/S oxidation of A. ferrooxidans ATCC 23270. A. ferrooxidans ATCC 23270 oxidizes the substrate, then the S/Fe morphology changes, and SO 4 2− /Fe 3+ is generated. 3.6. Transcriptome Analysis In the group with Fe 2+ oxidation ( Figure 9 a, Table S1 ), the expressions of cytochrome c oxidase and other functional genes of bacteria in the presence of Hg 2+ were increased, indicating that the iron oxidation function and antioxidant function were enhanced. The expressions of functional genes, such as outer membrane protein, periplasmic solute binding protein, ATP-dependent protease La, and chaperone protein, were increased. The results indicate that the presence of Hg 2+ improves the protein transcription and translation efficiency of A. ferrooxidans ATCC 23270. In the presence of Hg 2+ , A. ferrooxidans ATCC 23270 showed enhanced iron and sulfur oxidation metabolism in the system simply using Fe 2+ /S 0 as a substrate. For the S 0 oxidation group, the expression levels of some functional genes were significantly different ( Figure 9 a, Table S1 ). The expression levels of functional genes such as peripheral solute binding protein and ncRNA decreased, indicating that A. ferrooxidans ATCC 23270 stress in the presence of Hg 2+ leads to a reduction in protein transcription and translation efficiency. The expressions of cytochrome c oxidase, pyridine nucleotide disulfide reductase, and other functional genes increased, while the expression of isodisulfide reductase was not significantly different. Thioneone reductase is involved in the reaction between hydrogen sulfide and panquinone, catalyzing the oxidation of hydrogen sulfide into sulfide [ 37 ]. Pyridine nucleotide disulfide reductase is involved in the catalytic oxidation of elemental sulfur [ 38 ]. The isodisulfide reductase complex plays a leading role in the oxidation of elemental sulfur [ 39 ]. The results indicate that Hg 2+ improves the partial sulfur catalytic oxidation function of A. ferrooxidans ATCC 23270. The expression of cytochrome c oxidase was significantly increased under the influence of Hg 2+ , and this enzyme is involved in the reduction of Hg 2+ , indicating that the mercury reduction function of A. ferrooxidans ATCC 23270 in the presence of Hg 2+ may depend on this pathway. The expression of the Hg 2+ transporter and Hg 2+ reductase related to the cellular stress detoxication mechanism was upregulated in the Fe 2+ oxidation group ( Figure 9 b). The results indicate that the presence of Hg 2+ inhibited the overall growth and metabolism function of A. ferrooxidans ATCC 23270, but the expression of mercury reduction-related functional genes was active. In the S 0 oxidation system ( Figure 9 b), the effects of Hg 2+ on the expression levels of various functional genes of A. ferrooxidans ATCC 23270 were mainly regulated, and only the expression levels of cell-related functional genes were downregulated. Studies have shown that A. ferrooxidans ATCC 23270 can increase the activities of superoxide dismutase, glutathione reductase, and thioredoxin reductase due to stress behavior when leaching sulfide ore containing heavy metal ions [ 40 ]. Therefore, the upregulated expression of glutathione-related functional genes indicates that A. ferrooxidans ATCC 23270 has an active sulfur oxidation function in the system solely metabolized with S 0 as the substrate, and the detoxification mechanism of Hg 2+ is jointly produced by glutathione-related mercapto coupling and the reduction of Hg 2+ reductase. Compared with the control group with Hg 2+ (SF0b), Hg 2+ stress inhibited the synthesis of logarithmic preglycosyltransferase I and a variety of unknown proteins and promoted the expressions of cytochrome c (Cyc2), cytochrome c 552 (Cyc1), thioredoxin-dependent adenylate sulfate reductase (CysH), and other genes ( Figure 9 c, Table S2 ). Cyc2 and Cyc1 participate in the electronic chain transfer pathway for the A. ferrooxidans ATCC 23270 ferrous oxidation group, and studies have shown that the two cytochrome c genes are related to anaerobic iron reduction coupled with sulfur oxidation [ 41 ]. Cytochrome c oxidase downstream of the electron transport chain can participate in the reduction of Hg 2+ [ 18 ]. CysH is involved in the sulfur reduction process in sulfur metabolism. The above results indicate that, under the influence of Hg 2+ , A. ferrooxidans ATCC 23270 has a low expression of glycosyltransferase, indicating that Hg 2+ has a negative effect on its biomolecular function. The upregulated expression of cytochrome c oxidase also promoted the expressions of functional genes of iron and sulfur metabolism, which had positive effects on the detoxification mechanism of Hg 2+ , the activity of iron and sulfur metabolism, and the motor function of bacteria. When the bacteria in the experimental group grew to the middle and late logarithmic stages, the total amount of differential genes was very small compared with that in the control group, indicating that, with the growth of bacteria and the progress of mercury conversion, the stress effect of external Hg 2+ on bacteria was reduced such that they could return to normal growth and metabolism levels ( Figure 9 d). The expression levels of a few genes related to the localization function of biological processes, cell surface protein complexes, and molecular transport activities were downregulated, suggesting that the metabolic function of bacterial cells was still affected to some extent after most of the Hg 2+ stress was relieved. A. ferrooxidans ATCC 23270 gene expressions in the aerobic stage and anaerobic stage were compared ( Figure 9 e, Tables S3 and S4 ). A. ferrooxidans ATCC 23270 was greatly influenced by Hg 2+ in the aerobic stage, which mainly manifested in the enhanced expression of functional genes related to mercury conversion. The expression of functional genes related to physiological processes such as transcription and translation was reduced. After entering the anaerobic stage, due to the change in oxygen concentration, the electron transfer pathway of the A. ferrooxidans ATCC 23270 metabolic process changed greatly, resulting in a great difference between the expressions of each gene and the samples in the aerobic stage. However, there was little difference in gene expression between different cases in the anaerobic stage, indicating that the influence of Hg 2+ was light in this stage, which is consistent with previous experimental results. In addition, the expression of Hg 2+ reductase gradually increased and then decreased over time, indicating that the stress effect of Hg 2+ on A. ferrooxidans ATCC 23270 was enhanced in the late aerobic stage and then weakened in the anaerobic stage. A. ferrooxidans ATCC 23270 showed an increase in differentially expressed genes in the middle and late stages of the aerobic stage, and the overall level was downregulated, while the expressions of merC and merA were upregulated ( Figure 9 f). The results indicate that the middle and late stages of the aerobic stage are greatly influenced by Hg 2+ . A. ferrooxidans ATCC 23270 cells greatly improved the fixation, transfer and reduction function of Hg 2+ . The overall differential gene expression of A. ferrooxidans ATCC 23270 affected by Hg 2+ showed a downward trend compared with the control group ( Figure 9 f). However, in the late anaerobic stage, the total amount of differentially expressed genes was significantly reduced, indicating that A. ferrooxidans ATCC 23270 essentially completed the process of detoxification and developed tolerance to Hg 2+ under anaerobic conditions. However, because there was still a small amount of Hg 2+ in the solution, the expressions of merC and merA showed upregulated trends compared with the control group. 3.7. Correlation between the Iron/Sulfur Redox Mediated by Acidithiobacillus ferrooxidans ATCC 23270 and Mercury Transformation Therein The present study shows that the iron/sulfur redox of Acidithiobacillus ferrooxidans ATCC was obviously affected by the addition of Hg 2+ , and mercury transformation was thus closely related to the energy substrates and the growth conditions; the proposed correlation mechanism is shown in Figure 10 . It was found that, for all the groups of A. ferrooxidans ATCC 23270 grown on different energy substrates under different aerobic and anaerobic conditions, Hg 2+ significantly inhibited cell growth and reproduction. For the groups with S 0 , Fe 2+ , and Fe 3+ /S 0 as energy substrates, the inhibition effects were gradually reduced as the culture time increased, and they were eliminated at the late stage of the experiment. Compared with the simple aerobic/anaerobic environment with S 0 , Fe 2+ , and Fe 3+ /S 0 as the energy substrates, the aerobic–anaerobic coupling environment simulating the AMD environment was more complex. However, due to the adsorption effect of pyrite on Hg 2+ , the aerobic–anaerobic coupling group had little influence on the growth and metabolism and other physiological processes of A. ferrooxidans ATCC 23270 at the beginning. Later, with the utilization of the substrate pyrite by A. ferrooxidans ATCC 23270, the Hg 2+ adsorbed on the surface of pyrite was gradually released, gradually inhibiting the growth and reproduction of bacteria. At the same time, the contents of extracellular polymers increased and changed. The bonding mode and distribution on the bacterial surface also changed. At the same time, A. ferrooxidans ATCC 23270 enhanced the functions of Hg 2+ stress, mercury transformation, and intercellular signal transduction. It was also found that Hg 2+ significantly changed the composition of polymer on the surface of A. ferrooxidans ATCC 23270 and the distribution of C, N, Fe, S, and other elements inside the cells. Particularly in the group containing substrate S, A. ferrooxidans ATCC 23270 was more likely to enrich Hg outside and inside the cell walls due to the affinity of S to Hg. Furthermore, A. ferrooxidans ATCC 23270 can change the form of Hg, transfer it in the solution and solid phases through iron sulfur redox coupled with mercury conversion, and participate in the mercury cycle together with other mercury-tolerant bacteria. In mercury conversion, the bacteria attached Hg elements to the surface of the substrate sulfur residue or the iron vitriol dregs in the form of Hg 0 , HgS, and HgSO 4 in all groups to reduce the concentration of Hg 2+ in solution. Hg 2+ combined with the SO 4 2− generated during the metabolism of iron and sulfur to form HgSO 4 . In addition, secondary products, such as jarosite, generated by the oxidation of Fe 2+ can also adsorb Hg 2+ in the solution. At the same time, Hg 2+ can also be transported into the cell under the action of A. ferrooxidans ATCC 23270 and converted into Hg 0 by the mercury reduction of A. ferrooxidans ATCC 23270. Hg 0 will combine with S 0 to produce HgS. However, A. ferrooxidans ATCC 23270 underwent slightly different mercury reduction pathways in the three groups. In the aerobic system, the sulfur oxidation group mainly adsorbed and transformed the Hg 2+ in solution through the glutathione-associated sulfhydryl coupling pathway, mer operon-related mercury reduction, and mercury reduction associated with cytochrome c oxidase. The mercury reduction pathways in the other three groups were related to mer operons. Notably, the responses of the bacteria to the three different conditions were different. Under single aerobic or anaerobic conditions, the expressions of functional genes related to mercury transformation and the iron/sulfur redox of A. ferrooxidans ATCC 23270 increased. Under the aerobic–anaerobic coupling conditions, the strong adsorption of pyrite on Hg 2+ enhanced the continuous stress of the Hg element, which significantly inhibited the expression of iron and sulfur metabolism function genes of A. ferrooxidans ATCC 23270 in the middle and late aerobic stages. However, this gradually recovered during the anaerobic phase."
} | 13,302 |
24860565 | PMC4030187 | pmc | 8,630 | {
"abstract": "The presence and association of fungi with sessile marine animals such as coral and sponges has been well established, yet information on the extent of diversity of the associated fungi is still in its infancy. Culture – as well as metagenomic – and transcriptomic-based analyses have shown that fungal presence in association with these animals can be dynamic and can include “core” residents as well as shifts in fungal communities. Evidence for detrimental and beneficial interactions between fungi and their marine hosts is accumulating and current challenges include the elucidation of the chemical and cellular crosstalk between fungi and their associates within the holobionts. The ecological function of fungi in association with sessile marine animals is complex and is founded on a combination of factors such as fungal origin, host health, environmental conditions and the presence of other resident or invasive microorganisms in the host. Based on evidence from the much more studied terrestrial systems, the evaluation of marine animal–fungal symbioses under varying environmental conditions may well prove to be critical in predicting ecosystem response to global change, including effects on the health of sessile marine animals.",
"introduction": "INTRODUCTION There is no consensus on the definition of marine fungi, even though it is clear that the grouping of marine fungi is primarily based on an ecological rather than a taxonomical basis ( Kohlmeyer and Kohlmeyer, 1979 ; Hyde et al., 2000 ). Commonly used descriptions include “marine-derived” or “marine-associated” fungi, yet “facultative-marine” and “obligate-marine” would, most likely, best distinguish between fungi isolated from marine niches versus those requiring the marine environment. Much of the research on these organisms has focused on fungi from marine environments such as obtained or found associated with mangroves, wood substrates, sediments ( Jones, 2011b and references within), as well as on fungal infections of marine mammals ( Higgins, 2000 ). The occurrence of fungal associations with other organisms within the marine environment has been reported and discussed for over a century, including the concerns whether fungi can, at all, grow in sea water ( Murray, 1893 ). One of the earliest reports on actual parasitism by a marine fungus (albeit on an algae) was documented that same year by Church (1893) . A significant landmark in the study of fungi from the marine environment was the report by Barghoorn and Linder (1944) who in addition to their descriptions of marine-derived fungi stated that “The fact that a score or more of species have been described as occurring in the sea is of importance since it shows that fungi not only tolerate salt water, but indeed that marine conditions furnish a normal habitat for the relatively small number of fungi that have become adapted to it.” Since then, the number of fungal species (over 800) described from marine environments and the rate at which they are currently being described indicates that the marine fungal community is larger than originally considered ( Jones et al., 2009 ; Jones, 2011a , b ). Nonetheless, whereas the Symbiodinium and marine prokaryotic and viral communities and their possible contributions to the niches they reside in received increased attention during the last decades (e.g., Breitbart, 2012 ; Garren and Azam, 2012 ; Weber and Medina, 2012 ; Moitinho-Silva et al., 2014 ), our current understanding of the fungal communities in marine environments is still extremely limited ( Amend et al., 2012 ). The fungal kingdom is estimated to be comprised of approximately 1.5 million species, with less than 10% of them described to date ( Hawksworth, 2001 ). As the identification of fungi associated with sessile marine animals and the realization of the ecological significance they may have is fairly recent, it is highly likely that many new species (some with unique attributes/ecological roles) within these niches have yet to be isolated/identified. In this short review, evidence accumulated for the presence and association of fungi with corals, sponges, and ascidians will be provided as well as discussion of the potential impact fungi may prove to have in these niches."
} | 1,064 |
36067300 | PMC9477243 | pmc | 8,631 | {
"abstract": "Significance Enzymatic proteins are the engines of life, generating energy, fixing CO 2 into organic matter, and building biomass. At scale, these biological catalysts have the power to drive global elemental cycles. These microbial machines were responsible for the oxygenation of the atmosphere, can mediate the formation and destruction of greenhouse gases, and potentially influence cloud formation. Here, we used metaproteomics across the central Pacific Ocean to investigate how microbial protein distributions change across vertical and horizontal scales, identify interactions among microbial consortia, and characterize metabolic hot spots in line with major geochemical features. These observations provide insights into microbial community dynamics and can act as empirical constraints for biogeochemical ecosystem models to improve understanding of microbial interactions on a changing planet.",
"conclusion": "Conclusions The distribution of microbial proteins across large transects spanning major ecosystems enables comparisons of microbial processes in various biogeochemical provinces, providing a holistic view to investigate microbial function across large geospatial scales. We identified how protein diversity of the microbial community, from both taxonomic and functional capacities, was dominated by a few major protein groups and a multiplicity of lower-abundance groups, while highlighting the interconnectedness among microbial consortia and the biogeochemical cycles of nitrogen, carbon, oxygen, and sulfur. Notably, many of the critical oxidoreductase enzymes are metalloenzymes or utilize metals as substrates (like MnxG), thus also impacting global trace metal cycling. Community and enzyme shifts along vertical scales attest to the rapid growth and reproduction in the euphotic zone and the need to meet energetic demands at depth. Community shifts were also observed along regional horizontal scales associated with varying oxygen concentrations and nutrient availability. The array of detected and quantified enzymes reflects carbon utilization and nitrification pathways including the production and consumption of volatiles like CO, CO 2 , NO, methanethiol, and methylamines. Chemical interactions between members of the microbial consortia were also observed in the neural net, for example, between the nitrite-oxidizing Nitrospina and ammonia-oxidizing Thermoproteota, connections between photosynthetic production by Cyanobacteria and methyltrophy, and the catabolism of organics by heterotrophs supporting chemoautotrophic ammonia oxidation by Thermoproteota. Direct measurements of these critical microbial enzymes as the engines of biochemical transformations can provide high-resolution empirical data to refine complex global biogeochemical models ( 12 , 58 ) and improve our understanding of the ocean in response to a changing climate.",
"discussion": "Results and Discussion To interrogate this large dataset, we utilized multiple statistical and modeling approaches to identify the major patterns in protein distributions ( Materials and Methods ). Machine learning clustering analyses of hydrographic and geochemical data resulted in partitioning of microbial communities along the transect into cohesive depth bins along a broad regional scale ( Fig. 1 G ). Four major depth groups resulted from k -means clustering: surface, cline, twilight, and deep depth groups ( SI Appendix , Fig. S2 ). Hierarchical clustering also divided stations into two regions: North Pacific (stations 4 to 10) and South Pacific regions (stations 11 to 14; SI Appendix , Fig. S3 ). These two clusters corresponded to multiple biogeochemical provinces: North Pacific stations captured the low-nutrient surface waters of the North Pacific Subtropical Gyre (NPSG; stations 4 and 5) and the western flank of the Eastern Tropical North Pacific (ETNP) oxygen deficient zone (ODZ) within the cline and twilight depths. In contrast, South Pacific stations captured the highly productive and relatively nutrient-replete area of equatorial upwelling, with corresponding enhanced particulate organic carbon (POC), NO 2 − , and NH 4 + in the surface and cline ( Fig. 1 E , F , and I ). To better understand vertical stratification of proteins, we performed a community attenuation analysis by fitting a power law model to protein abundance through the water column. Microbial protein attenuation ( c ) is an indicator of the importance of specific proteins to microbial communities with increasing depth through the water column ( Figs. 1 E and H and 2 A and SI Appendix , Fig. S4 ). For example, the average c for all proteins is −1.25. Very negative values of c indicate proteins that are predominantly found in the surface relative to communities at depth. Less negative values of c indicate proteins that are more important to microbial communities in deeper depths as they have a less pronounced decline in abundance with depth through the water column, with positive values indicating the few proteins that increase in abundance with depth even though total biomass dramatically decreases through the water column. Finally, an artificial neural network analysis provided insight across these biogeochemical provinces by highlighting protein distributions characteristic to specific samples, generating sample-specific “fingerprints,” while simultaneously providing an exploration of microbial consortia interactions among proteins of varying functions and taxonomic origin. Fig. 2. Taxonomic and functional diversity within the ProteOMZ metaproteome. ( A ) The large concentric circles represent the relative abundance of peptides attributed to taxonomic groups according to an LCA analysis of peptides per depth group. The legend for each depth group also displays the percentage contribution of each major domain to the taxonomic profile with LUCA assigned to peptides which are highly conserved and thus found in multiple domains. Unknown indicates peptides which have no taxonomic homology to known organisms. The size of blue circles under the depth group names represents the relative contribution of peptides from that depth group to the overall metaproteome. ( Inset ) The swamp plot displays the abundance of peptides in sc corr /L sw per depth group, colored by region. ( B ) A cumulative summation plot categorically displaying KO groups in rank order of abundance along the x axis and the relative contribution of each KO group to the total peptides in the KO-identifiable metaproteome along the y axis. ( C ) The relative abundance of peptides assigned to the major Enzyme Commission categories according to depth group. The contribution of different taxa to the microbial community proteome varied along both vertical and regional dimensions. A least common ancestor (LCA) analysis of individual peptide constituents was conducted, with the relative contributions from each taxonomic group reported along depth groups ( Fig. 2 A ) ( 22 ). The overall microbial proteome was dominated by marine Bacteria, with 85.1% of peptides assigned to bacterial groups in the surface, twilight, and deep depth groups. Bacteria had the lowest relative contribution in the cline at 81.7%. Cyanobacteria were the primary source of peptides in the surface (36.1%) with Prochlorococcus as the dominant taxon (25.8%). Pelagibacter was the primary contributor among the Alphaproteobacteria, the second-largest source of peptides in the surface (20.8%). Both the Cyanobacteria and Alphaproteobacteria peaked in abundance in the South Pacific near the region of equatorial upwelling. This coincided with the peak in POC (Pearson correlation coefficient r ≥ 0.94; Fig. 1 E and H ). The dominance of these two groups inferred from metaproteomics is consistent with taxonomic distributions from metagenomic data collected from similar pelagic regions ( 23 – 27 ). Integrating over all depth groups, Proteobacteria were the single largest source of peptides, contributing 32.6% overall. Eukaryotes, and Nitrospinae bacteria were the next largest groups, contributing 3.8 and 2.1% of the total peptides, respectively. Eukaryotic peptides were primarily found within the euphotic zone and were associated with the picoeukaryotic phytoplankton Pelagamonas and diatoms because the size fraction analyzed here (0.2 to 3.0 µm) precluded most eukaryotes and large particle-associated organisms ( 9 ). Archaeal peptides were most abundant below the surface depth group, ranging from 3.0 to 3.2%. About 2/3 of archaeal peptides were from Euryarchaeota in which the dominant peptides were associated with L-amino acid transport and metabolism, and 1/3 from Thermoproteota (Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota), in which the bulk of the peptides were associated with the uptake and metabolism of nitrogenous compounds, in line with prior findings from studies of Archaea ( 28 ). Interestingly, Archaea are known to be highly abundant by cell number in the mesopelagic ( 29 , 30 ), but their small cell size and slow metabolism appeared to result in a small contribution to overall microbial protein abundance. The same pattern was also recently observed in global marine metatranscriptomes ( 31 ). A relatively small contribution to the overall proteome was made by peptides that lacked association with any known taxonomic group (0.6%) demonstrating the capacity for dark environmental DNA to be translated into “dark protein.” The functional capabilities of the metaproteome were investigated by assigning traits using KEGG Orthology (KO) and Enzyme Commission (E.C.) identifiers. Seventy percent of the normalized spectral counts were assigned functional traits with KOs. Notably, out of 2,037 functional KO groups identified ( Movie S1 ), over 51.6% of the peptide abundance was associated with only 25 KO groups ( Fig. 2 B and SI Appendix , Fig. S5 ). The chaperone protein GroEL was the single most abundant functionally characterized protein across the dataset ( Fig. 3 and SI Appendix , Fig. S6 ), accounting for 6.4% of the KO-identifiable normalized spectral counts. The sheer abundance of the GroEL protein has not been previously observed using metagenomic or metatranscriptomic approaches highlighting the differences between transcription and protein abundance and the universal importance of protein folding in marine microbes. Fig. 3. Summary table of protein abundance and attenuation across the transect. Data in the table include the gene name, Kegg Ontology identifier (KO), specific taxonomic group determined by LCA analysis of peptides (blank taxa indicate all taxa for that protein presented), total abundance (sc corr /L sw : spectral counts per liter of seawater), community attenuation ( c ) through the water column (dash indicates lack of data for calculating; Materials and Methods ). The heat map represents an aggregate of the individual depths (surface, cline, twilight, and deep) across the regions (north and south). The colors in the heat map represent the log2 fold change of the average abundance for each depth/region combination compared to the overall average abundance for all samples. Lines around groups represent where a protein in a particular location is significantly more abundant than other locations, with a dashed line indicating P ≤ 0.05 and a thick solid line indicating P ≤ 0.01. Lines around an entire region indicate where a protein is significantly more abundant than the other region as assessed by a Mann–Whitney u test. Lines around individual depths indicate where a protein is significantly more abundant in one or more depths when compared to the other depths as assessed by a Kruskal–Wallis H test with post hoc Dunn’s tests. Asterisk indicates KO groups that contain multiple different functional proteins, such as K00370, which contains both NarG and NxrA proteins ( Materials and Methods and SI Appendix , Figs. S10–S15 ). Caret indicates peptides identified through parsimony analysis from protein group inference in the software package Scaffold as opposed to LCA analysis which was utilized elsewhere due to the high conservation of the peptides among taxonomic groups ( Materials and Methods ). Section profiles of the distribution of these proteins can be found in Fig. 4 or SI Appendix , Figs. S6–S9 . A photosynthesis-centered taxonomic community characterized the surface depth group, while a more diverse community was present at depth responding to more variable and diffuse sources of energy ( Fig. 2 A ). The functional focus of the microbial community also changed in the cline, twilight, and deep depth groups. E.C. numbers classify the chemical reactions carried out by enzymes into seven broad categories ( Fig. 2 C ). Evaluation of these broad reaction categories revealed that the microbial catalytic focus shifted from that associated with rapid growth and reproduction in the surface, characterized by abundant transferases (E.C. class 2), to a focus on energy harvesting dominated by oxidoreductases (E.C. class 1) at depth ( Fig. 2 C ). The transferase DNA-directed RNA polymerase (2.7.7.6) was the most abundant enzyme in the surface and throughout the entire dataset, consistent with microbial growth and associated transcription ( 32 ). In accordance with the biomass production and growth in the surface, peptides from lyase class (E.C. class 4) including ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO, RbcLS), the major CO 2 fixation enzyme, and the HCO 3 − consuming phosphoenolpyruvate carboxylase (PEPC), which participates in the anaplerotic synthesis of oxaloacetate in Cyanobacteria ( 33 , 34 ), were most abundant in the surface ( Fig. 3 ). The transferases were the most abundant enzyme group in the photosynthesis-dominated surface layer where the majority of proteinaceous biomass occurred. The relative contribution of oxidoreductase enzymes increased with depth through the mesopelagic where the microbial focus shifted to maintaining life-sustaining energetic demands and were the most abundant enzyme class throughout the entire water column, composing 30% of total enzyme abundance. Transport proteins were abundant, contributing at least 28% of the total proteome, similar to the findings of microbial metaproteomes from the pelagic Atlantic Ocean ( 2 , 5 ). Transport protein distributions in the surface ocean corroborated prior findings of nitrogen stress in Cyanobacteria from the oligotrophic NPSG ( 7 ) while expanding upon the biomarker catalog ( Fig. 3 ). Proteobacterial iron transporters were vertically stratified, with the iron(III) transport system substrate-binding protein (AfuA) significantly more abundant in the surface and the TonB-dependent siderophore receptor protein (TC.FEV.OM) deeper in the euphotic zone. The community attenuation coefficients ( c ) of −4.13 and −1.28 for AfuA and the TonB-dependent transporter, respectively, indicated the greater importance of the TonB-dependent iron transporter at depth because it attenuated more slowly through the water column than AfuA at a level more similar to the attenuation for all proteins ( c = −1.25; Fig. 3 ). This stratification hints at the differing microbial strategies for uptake of limiting nutrients, suggesting ligand-bound mechanisms are preferentially utilized by free-living microbes deeper in the water column. Some organic carbon transport proteins also displayed vertical and regional-scale variations. The simple sugar transport substrate-binding protein (ABC.SS.S: c = − 4.36) attenuated more quickly than the multiple sugar transport substrate-binding protein (ABC.MS.S: c = −1.17), suggesting a shift in the DOC pool with depth ( Fig. 3 ). Notably, the inositol-phosphate transport substrate-binding protein of Proteobacteria increased in abundance in the deep, indicative of the importance of this protein to community success in the mesopelagic (InoE: c = +0.26; Figs. 3 and 4 A ). Some transport proteins displayed variation across regional scales, suggestive of variation in DOC utilization along horizontal gradients; for example, the fructose transport substrate-binding protein (FcrB), primarily produced by Alphaproteobacteria, was significantly more abundant in the surface of the North Pacific ( Fig. 3 ). Variations in transport protein abundance can act as biological indicators (or biomarkers) of scarce nutrient distributions and stress in microbial communities ( 5 , 7 , 35 ). Fig. 4. Attenuation profiles and distributions of the most abundant oxidoreductases in the mesopelagic. ( A ) Attenuation lines calculated by fitting a power law through abundance data of total extracted protein ( c = −1.15), POC ( c = −0.7), PON ( c = −1.05), transport, and oxidoreductase enzymes that dominate the mesopelagic. The attenuation for all proteins combined was c = −1.25; more negative attenuations mean that proteins are more abundant in the surface and are reduced more quickly from communities at depth. Proteins with relatively slow attenuation rates, like the low-oxygen formate dehydrogenase (FdoG; c = −0.46), which oxidizes the C1 compound formic acid to CO 2 , and carbon monoxide dehydrogenase (CoxL; c = −0.31), which oxidizes carbon monoxide to CO 2 , are shown. Also presented is the aerobic formate dehydrogenase (FDH; c = −19.56), which is associated with methylotrophy and attenuates rapidly as it is far more abundant in the surface than in the mesopelagic. The inositol-phosphate transport system substrate-binding protein (InoE; c = +0.26), which increases in abundance with depth, is also shown. ( B ) Abundance of the most abundant oxidoreductase enzyme, nitrite oxidoreductase (NxrA), across the transect. This enzyme is abundant in the mesopelagic as well as in the surface waters near the equatorial upwelling region in the south. ( C ) Ammonium monooxygenase alpha subunit (AmoA) of Thermoproteota is most abundant at the interface of the surface and cline and is also more abundant in the south. ( D ) Bacterial nitrite reductase (NirK) peaks in abundance in the low-oxygen waters above the ETNP ODZ. ( E ) Archaeal nitrite reductase (NirK) is present in the waters above the ETNP ODZ but peaks in abundance in the surface waters associated with equatorial upwelling. ( F ) The oxidizing form of dissimilatory sulfite reductase (DsrA) is found within the mesopelagic waters. ( G ) The copper monooxygenase from Nitrospinae, putatively a Mn oxidase (MnxG), is most abundant at the top of the ETNP ODZ and is also found at similar depths in more oxygenated waters in the South Pacific. ( H ) MnxG shows a similar distribution pattern in the South Pacific to the bacterial catalase (KatG), which is indicative of a region characterized by oxidative stress. ( I ) The third most abundant oxidoreductase protein in the mesopelagic, formate dehydrogenase (FdoG), is most abundant along oxic transitional regions. ( J ) The abundant protein carbon monoxide oxidoreductase (CoxL) is also found throughout the mesopelagic, peaking in abundance in the South Pacific. ( K ) The formate dehydrogenase (FDH), however, is most abundant in the South Pacific surface near high POC. ( L ) The distribution of multiple ammonifying catabolic enzymes like alanine dehydrogenase (Ald) is tightly correlated with ammonia monooxygenase. The patterns in the distribution and abundances of oxidoreductase enzymes indicated carbon stress, oxidative stress, and use of alternative respiratory pathways. The oxidoreductase class of enzymes catalyze redox reactions and often utilize transition metals at their catalytic sites, most often using Fe cofactors, as well as Mo, Cu, and W, among others ( 36 ). Oxidoreductases are critical components of respiration and include the CO 2 -evolving enzymes pyruvate dehydrogenase (PdhA) and isocitrate dehydrogenase (IDH), both enriched in the surface ( Fig. 3 ) in conjunction with POC and O 2 ( Fig. 1 C and E ). Many biogeochemically relevant oxidases support chemolithoautotrophy by mediating the oxidation of reduced substrates. Distributions of oxidoreductase enzymes associated with nitrification were linked with major biogeochemical features along this transect, specifically within the ETNP ODZ and equatorial upwelling. The Mo- and Fe-containing enzyme nitrite oxidoreductase (NxrAB) is responsible for the final step in nitrification through the oxidation of NO 2 − to NO 3 − ( SI Appendix , Fig. 10 ). NxrAB was the most abundant oxidoreductase protein along this transect ( Figs. 3 and 4 B ) and in the mesopelagic central Pacific Ocean, in general, at over 60 billion molecules per liter ( 8 ). While NxrAB was extraordinarily abundant in the mesopelagic with low attenuation coefficients ( c = −0.38 and −0.55 for NxrA and NxrB, respectively), it peaked in abundance in the oxygenated surface region of equatorial upwelling at station 12 ( Fig. 4 B ). The abundance of NxrAB here may be supported through elevated production of NO 2 − via remineralization of photosynthetically derived organic matter and aerobic ammonia oxidation. The Cu-containing archaeal ammonia monooxygenase (including subunits AmoABC) is responsible for the first step of nitrification through the oxidation of NH 4 + ( SI Appendix , Figs. S11–S13 ). AmoABC peptides were associated with Thermoproteota and peaked in abundance at the interface of the surface and cline depth groups ( Figs. 3 and 4 C ). The Cu-utilizing nitrite reductase (NirK), which reduces NO 2 − to NO, displayed variable distribution patterns related to taxonomic origin. Bacterial NirK was primarily found in low-oxygen waters ( Figs. 1 B , 4 D , and 5 ) with the explicit functional role of NirK in these free-living communities still uncertain. Similarly, the function of archaeal NirK is also uncertain but has been suggested as a source of NO, an essential intermediate of ammonia oxidation ( 37 , 38 ). Archaeal NirK occurred at the top of the ETNP ODZ at an abundance roughly similar to that of bacterial NirK; however, archaeal NirK was significantly more abundant in the South Pacific near the equatorial upwelling region of station 12 ( Fig. 4 E ). Archaeal NirK is located near NxrAB from Nitrospina in the neural net ( 39 ) ( Fig. 5 C ) and is closely correlated ( r = 0.78 for NxrA and archaeal NirK) showing that these enzymes have similar distribution patterns indicative of related geochemical niches linked to NO 2 − . The high abundance of NO 2 − utilizing enzymes in conjunction with the typically lower standing NO 2 − concentrations in the surface ocean above the primary nitrite maximum suggests active chemical cycling where the available NO 2 − was consumed as quickly as it was produced in these regions ( Figs. 1 E and 4 B and E ). Notably, higher concentrations of N 2 O were observed at the top of the ETNP ODZ at station 7 ( Fig. 1 B ), where archaeal NirK and AmoABC peaked in the North Pacific. This cooccurrence of N 2 O and archaeal ammonia oxidation proteins in the ETNP ODZ may be due to the activity of archaeal ammonia oxidizers, whose N 2 O yield from ammonia oxidation increases at low oxygen concentrations ( 40 ). Purified archaeal NirK has been shown to catalyze both the formation of N 2 O from hydroxylamine under aerobic conditions, and the reduction of NO 2 − with hydroxylamine to form N 2 O under anaerobic conditions ( 41 ). Further, production of labeled N 2 O from NH 4 + has been demonstrated over a range of oceanic oxygen concentrations where archaea are the only ammonia oxidizers ( 42 , 43 ). However, we cannot conclusively rule out that this N 2 O was instead advected from a remote zone of production further east in the ETNP and not locally produced. Fig. 5. Distribution of select oxidoreductases across the transect, according to oxygen concentrations, and all proteins as analyzed by an artificial neural network. ( A ) Distributions of proteins binned by oxygen concentrations (10 µmol/kg) and normalized by the number of samples per bin. Gray outlines of protein distributions show the shape of the distribution for each individual enzyme. The colored interiors of AmoA, Archaeal and Bacterial NirK, DsrA, SoxA, MnxG, and KatG are distributions scaled to the relative abundance of Archaeal NirK, the most abundant of these proteins. NxrA is presented separately as it is significantly more abundant than the other proteins (20× more than Archaeal NirK). NarG and IdrA are presented together as they both have the same distribution pattern and only occur in the lowest oxygen bin. The samples column displays the discrete number of samples in each oxygen bin by depth illustrating the variability in sampling across oxygen concentrations. ( B ) Neural network feature maps of individual samples showing the unique fingerprints of each sample, highlighting the major underlying protein distributions associated with each sample. Note how the fingerprints change with depth as well as along regional scales. The neural net is composed of 900 individual nodes (a 30 × 30 matrix) using periodic boundary conditions. ( C ) The integrated differences of weights across all samples in the neural net are displayed in the background. Overlaid on top are points which represent the location of individual proteins according to nodes in the neural net (best matching unit). Individual points within a single node are offset slightly to show density of points. Nodes closer to each other with lower weight differences between them are more similar to each other. The three nitrification enzymes (NxrAB, Archaeal NirK, and AmoABC) also cooccurred within the low-oxygen transition into the ETNP ODZ in the North Pacific ( Fig. 1 C ), where anaerobic respiratory pathways can occur in free-living communities within the pelagic water column ( 44 ). The high oxygen affinities of archaeal ammonia oxidation and Nitrospina- mediated nitrite oxidation support these metabolic processes in the ETNP ODZ ( 45 ) and the Eastern Tropical South Pacific (ETSP) ODZ ( 46 ). The first step of heterotrophic denitrification is the dissimilatory reduction of nitrate. While particles can be hot spots of denitrification ( 44 ), the size fraction analyzed here precludes most particle-associated microorganisms. However, some peptides from nitrate reductase were identified (NarG and NapA) within the ODZs but only in samples from locations with oxygen concentrations less than 5 µmol kg −1 ( Figs. 3 and 5 A ). Aside from bacterial NirK, other signatures of bacterial denitrification such as nitrite reductase (NirS), nitrous oxide reductase (NosZ) which reduces N 2 O to N 2 , or nitric oxide reductase (NorBC) which reduces NO to N 2 O were not found in the free-living community using our two-dimensional (2D) metaproteomic analysis ( 16 ), although representative sequences were present in the metagenome, indicating the proteins either were not present or were rare and below detection. Additionally, the marker protein for anammox, hydrazine dehydrogenase (Hdh/Hzo), was not identified in the metaproteome or the metagenome. These proteins may have been primarily associated with particles and thus were not identified in the free-living pelagic community, or an exact representative may not have been available in the corresponding environmental metagenome used for peptide identification ( 47 ). Additional anaerobic respiratory enzymes were identified in association with the ETNP ODZ. The distribution of heterotrophic dissimilatory iodate reductase (IdrA, formerly called AioA-Like; SI Appendix , Fig. S14 ) ( 48 ) followed the pattern of oxygen depletion ( Figs. 1 C , 3 , and 5 A ). IdrA was identified in similar abundance to the oxidizing form of dissimilatory sulfite reductase, DsrA ( SI Appendix , Fig. S15 ), similar to prior observations of transcripts in Pacific ODZs ( 49 ), implying a reliance on iodate metabolism comparable to that of sulfur oxidation in ODZs. Within the ETNP ODZ samples, these two proteins had similar abundances (sum from stations 6 to 8, 29.7 and 37.0 sc corr /L sw for IdrA and DsrA, respectively). Proteins associated with sulfur oxidation (SoxA and the oxidizing form of DsrA) were significantly more abundant below the surface group but did not display a clear relationship with the ODZ regions ( Figs. 3 and 4 F ), although they were negatively correlated with O 2 concentrations (r = −0.32 and −0.53 for SoxA and DsrA, respectively; Fig. 5 A ). Some nonrespiratory enzymes also displayed noteworthy relationships with oxygen concentrations across regional scales. The second most abundant oxidoreductase from Nitrospinae, manganese oxidase (MnxG), peaked in abundance in the cline of the ETNP ODZ ( Figs. 4 G and 5 A ) and was negatively correlated with O 2 concentrations (r = −0.47). While the Mn-oxidizing function of this specific multicopper oxidase in Nitrospinae has not been experimentally confirmed, the homology of this sequence with known Mn-oxidizing proteins suggests this as a likely function ( 50 ), warranting further investigation of this environmentally important enzyme. In the oxygen-depleted north region, Nitrospinae MnxG cooccurred with Nitrospina NxrA ( r = 0.64); however, in the oxygenated south, MnxG peptides did not correlate with NxrA but instead correlated with catalase (KatG; r = 0.64). The abundance of KatG in these samples ( Fig. 4 H ) was indicative of a region of high oxidative stress, likely driven by the generation of reactive oxygen species as by-products of ammonia oxidation ( 51 ). In keeping with this, archaeal Fe-Mn superoxide dismutase (SOD2) that converts superoxide radicals into H 2 O 2 , AmoAB, and archaeal NirK all peaked in abundance at station 12, 100 m. While no catalase or peroxiredoxin associated with Thermoproteota were identified, KatG displayed a similar distribution to other ammonia oxidation proteins in the south ( r = 0.73 with AmoA; Fig. 5 A ). Aerobic ammonium oxidation may be enhancing oxidative stress in the South Pacific which is then moderated by catalases produced by Bacteria, a commensal process previously demonstrated in laboratory cocultures of Thermoproteota and heterotrophic bacteria ( 51 ). Additionally, the abundance of MnxG suggests a coupling of N and Mn cycles catalyzed by Nitrospinae. This coupling may manage oxidative stress ( 50 ) by oxidizing reduced Mn and removing it from the water column through Mn-oxide particle formation ( 52 ). Across the entire transect, the most abundant oxidoreductases in the dark mesopelagic—in the twilight and deep groups—were the Fe- and Mo-containing formate dehydrogenase, FdoGH ( Figs. 3 A and 4 I ), and Cu- and Mo-containing carbon monoxide oxidoreductase CoxLM ( Figs. 3 and 4 J ). Both FdoGH and CoxLM help sustain microbial energy demands during periods of nutritional or oxidative stress ( 53 , 54 ). The large Mo-containing subunits FdoG and CoxL were the third and fourth most abundant oxidoreductase proteins in the mesopelagic. The importance of these enzymes to microbial communities at depth was evidenced by their slow attenuations through the water column ( c = −0.46 and −0.31, for FdoG and CoxL, respectively; Fig. 4 A ). CoxLM likely supports mixotrophic growth under organic carbon stress at depth: the vast majority of CoxLM-harboring environmental microorganisms are carboxydovores capable of scavenging CO at subatmospheric levels for electrons to support aerobic respiration producing CO 2 when organic carbon is limiting ( 53 ). CoxLM proteins, primarily from Bacteria—including from Bacteroidetes, Actinobacteria, Chloroflexi, Alpha- and Gammaproteobacteria, among others—were found throughout the dark mesopelagic and were significantly more abundant in deeper depths compared to the surface ( Fig. 3 ). The importance of CoxLM to mesopelagic communities is consistent with a high genomic capacity for CO oxidation in free-living microbial communities in the bathypelagic ( 55 ). Regional variations in the abundance of CoxLM was observed, where CoxLM was significantly more abundant in the South Pacific ( Figs. 3 and 4 G ). Formate dehydrogenases oxidize another C1 compound, formic acid, to CO 2 . Formate dehydrogenases are classified into two families, both of which were observed in the proteome: one uses FeS catalytic subunits, such as FdoGH, and the other, FDH, utilizes NAD(P)+ as electron acceptors ( 56 ). FdoGH, primarily associated with Bacteria—including from Alpha- and Gammaproteobacteria, candidate division NC10, Candidatus Tectomicrobia, and Actinobacteira among others—peaks in abundance in the cline; however, it does not show a significant regional bias overall. Laboratory studies have shown that FdoGH assists in oxic/anoxic transitions, supporting substrate-level bioenergetic conservation in anaerobic chemoorganotrophic microbial respiration with NO 3 − or NO 2 − ( 54 ). In culture, the nitrifier Nitrospira was also shown to increase FdoGH protein in response to the onset of oxygen limitation to support cellular energetic requirements ( 57 ). In contrast, the NAD+ dependent formate dehydrogenase, FDH, functions optimally in aerobic conditions and has a very different distribution than FdoGH across this dataset as this protein is primarily found in the more productive South Pacific surface group, correlating with total extracted protein ( r = 0.78; Figs. 1 E and H , 3 , and 4 I ). FDH supports methylotrophy as the final catabolic step in conversion of C1 compounds to CO 2 and can account for 10 to 15% of cellular protein content in methylotrophs ( 56 ). Given that the methylotrophy-related FDH is significantly more abundant in the surface upwelling region ( Figs. 3 and 4 K ), the variability in the distributions of FDH and CoxLM demonstrates the use of metaproteomics for constraining microbial production and consumption of gases such as CO that are generally sparsely sampled at depth ( 58 ). Other oxidoreductase enzymes that can support methylotrophic growth also showed a similar regional distribution to FDH. Peptides from the Cu-containing methanethiol oxidase (MtoX), primarily produced by Proteobacteria, were significantly more abundant in the surface and cline of the South Pacific ( Fig. 3 ). The gas methanethiol can be generated through the degradation of sulfur-containing amino acids ( 59 ) and is also an intermediate in the biotic degradation of the phytoplankton metabolite dimethylsulfoniopropionate (DMSP) and the volatile dimethylsulfide (DMS), a source of sulfur to the atmosphere and hypothesized contributor to cloud formation ( Fig. 3 ) ( 60 – 62 ). Notably, MtoX peptides were found to positively correlate with eukaryotic RbcL ( r = 0.75), suggesting a tight relationship between MtoX, which can be used by methylotrophs, and the likely source of the enzyme’s substrate: picoeukaryotic phytoplankton. Trimethylamine monooxygenase (Tmm), which catalyzes the oxidation of trimethylamine (TMA) to trimethylamine N -oxide (TMAO), can also support methylotrophy ( Fig. 3 ) ( 63 , 64 ). The Tmm peptides predominantly originated from Pelagibacter and were also significantly more abundant within the surface of the South Pacific. Peptides of the methylamine–glutamate N -methyltransferase enzyme associated with methylamine oxidation (MgsBC) also peaked in abundance in the South Pacific; however, these were found deeper in the water column ( c = −0.23 and −0.53 for MgsB and MgsC, respectively) and were also abundant above the ETNP ODZ. Methylamines make up a significant portion of both the volatile and dissolved C and N pools with oxidation of these compounds able to provide an exogenous source of ammonia ( 65 ) that can cross feed to other organisms ( 64 ). Nitrification and C1 metabolisms described above are dependent on the activities of other enzymes and members of the microbial consortium, including enzymes involved in the catabolism of organic matter. Numerous catabolic hydrolases (E.C. class 3) that participate in ammonification had distribution patterns linked to major biogeochemical features and showed distribution patterns where these degradative enzymes incidentally support other members of the microbial consortia, namely, Thermoproteota. The hydrolase formamidase (FmdA), which produces formate and ammonia as by-products, displayed a similar distribution pattern to the formate dehydrogenase FdoGH ( r = 0.71 with FdoG). Other bacterial ammonia-producing hydrolases like beta-ureidopropionase (PydC) primarily from Actinobacteria and Proteobacteria, amidase (AmiE) primarily from Alphaproteobacteria, and N,N -dimethylformamidase (DmfA) from Alpha- and Gammaproteobacteria were significantly more abundant in the South Pacific near equatorial upwelling. These enzymes were positively correlated with archaeal AmoA ( r = 0.78, 0.60, and 0.62) and were located close to AmoABC in the neural net ( Fig. 5 C ). Additionally, the ammonifying catabolic enzyme alanine dehydrogenase (Ald; Fig. 4 L ) from Proteobacteria was one of the most tightly correlated with AmoA ( r = 0.87). The cooccurrence of these ammonifying enzymes with AmoABC suggests a consortial syntrophic relationship between exogenous ammonia production and archaeal ammonia oxidation ( Fig. 5 C ). Ammonifying hydrogenases also can support ammonia oxidation within the organism that produces them. For example, the hydrolytic Ni-containing urease enzyme (UreC) that releases ammonia from urea produced by Thermoproteota was significantly more abundant in the South Pacific and peaked in abundance in the twilight depth here ( Fig. 3 ). This enzyme supports ammonia oxidation in Thermoproteota and displayed noteworthy taxonomic variability as the UreC of Cyanobacteria, used when Cyanobacteria are nutrient stressed, was significantly more abundant in the surface of the North Pacific associated with the oligotrophic waters of the NPSG. Notably, the Thermoproteotal UreC, which was colocated with the urea symporter (DUR3) in the neural net ( Fig. 5 C ), did not have as strong of a correlation with AmoA ( r = 0.28 and 0.20 for UreC and DUR3, respectively) as the ammonium transporter AMT ( r = 0.66), suggesting archaeal use of urea when free ammonia is scarce. Ammonifying hydrolases, in addition to methylamine oxidation, likely contribute to the significant abundance of nitrification enzymes in the surface upwelling region of the South Pacific."
} | 9,614 |
23552964 | PMC3615570 | pmc | 8,632 | {
"abstract": "Fluorescent proteins undergoing green to red (G/R) photoconversion have proved to be potential tools for investigating dynamic processes in living cells and for photo-localization nanoscopy. However, the photochemical reaction during light induced G/R photoconversion of fluorescent proteins remains unclear. Here we report the direct observation of ultrafast time-resolved electron transfer (ET) during the photoexcitation of the fluorescent proteins EGFP and mEos2 in presence of electron acceptor, p-benzoquinone (BQ). Our results show that in the excited state, the neutral EGFP chromophore accepts electrons from an anionic electron donor, Glu222, and G/R photoconversion is facilitated by ET to nearby electron acceptors. By contrast, mEos2 fails to produce the red emitting state in the presence of BQ; ET depletes the excited state configuration en route to the red-emitting fluorophore. These results show that ultrafast ET plays a pivotal role in multiple photoconversion mechanisms and provide a method to modulate the G/R photoconversion process.",
"discussion": "Discussion This ability of electron acceptor (here BQ) to control the G/R photoconversion in EGFP and EGFP like photoconvertible fluorescent proteins (mEos2, Kaede 7 , DendFP 24 , mcavRFP and rfloRFP 25 ) suggests the central role of ET in red chromophore formation in PCFP. In summary, the present work shows an active role of light triggered ET in G/R photoconversion. This strengthens the legitimate use of the photoconversion of EGFP as detectors of electron acceptors as suggested earlier 11 . However, light-induced electron donors can affect the redox balance in the cell by providing reducing equivalents upon photo-excitation, and this may account for some of the photo-toxicity of the fluorescent proteins 26 . For the mEos2 protein, although it has a similar ground state chromophore ( Fig. 6 ), our experiments suggest that ET causes a depletion of the chromophore itself by channeling the reaction in the excited state towards a different fate, effectively competing with the photoconversion process. This raises a cautionary note about using mEos2 proteins to quantitatively detect intracellular protein distribution and dynamics. The presence of high concentration of electron acceptors in areas such as the mitochondrion, will cause a rapid photochemical conversion of the excited states of these proteins, rendering them of limited applicability. Finally, the femtosecond/picosecond-resolved fluorescent transients presented here provide evidence for the ultrafast time scales associated with ET during the photoconversion process, that unequivocally implicate ET in sculpting spectral changes in emission wavelengths and photochemical changes upon irradiation."
} | 683 |
37579173 | PMC10450442 | pmc | 8,633 | {
"abstract": "Significance While water is essential to life, it is detrimental to semiconductors and electronics. However, the development of encapsulation methods to prevent the penetration of water has fallen behind the evolution of semiconductors and devices. Here, we report a bioinspired liquid encapsulation platform that not only protects water-sensitive materials from various water forms but also provides additional attractive features, including flexibility, transparency, self-cleaning, and self-healing. This technology is based on a polymer scaffold infused with a hydrophobic oil, whose exceptionally low water transmission is due to the small pore size of the scaffold and the low diffusion of water in the oil. The encapsulation layer is easily replenishable, resulting in long-term, renewable protection of the enclosed devices.",
"discussion": "Discussion The liquid encapsulation platform we have presented displays an ultralow water transport rate to prevent water infiltration while providing customizable wetting, mechanical, and optical properties. Using halide perovskite as a model system because of its ability to change color upon dissolution, we demonstrated that water damage is drastically reduced when the sensitive layer is protected by a hydrophobic polymeric matrix filled with a matching hydrophobic oil. While this work mainly focused on understanding the fundamental water transport mechanisms, our further research aims to apply this platform to a detailed study of the device performance characteristics and degradation of function in situ. We note that the choice of a fluoropolymer gel and fluorinated lubricants in this work was dictated by the following reasons: as we show, the PFOEA-PFPE copolymer can be synthesized in different volume ratios of the components, and thus, its physicochemical properties can be easily tunable. The resulting fluorogels were, therefore, excellent candidates for our fundamental study, since we could comprehensively describe the water diffusion characteristics as a function of 1) the mechanical properties of the polymer (from a soft elastomer to a rigid plastic); 2) its crystallinity; 3) the pore sizes of the network available for lubricant incorporation; 4) polymer hydrophobicity; and 5) lubricant molecular weight and viscosity. It is important to emphasize, however, that the concept of a liquid encapsulation platform is not limited to fluorogels and fluorinated lubricant oils, whose potential may be influenced by the regulations of per- or polyfluorinated alkyl substances (PFAS) due to environmental effects ( 38 , 39 ). The presented principle could be extended to various hydrophobic polymer matrices, such as acrylic, poly(vinyl chloride) polymers, or silicone gels, as long as they work as conventional conformal coatings, combined with an infused hydrophobic oil, such as silicone, mineral, or vegetable oil, that is compatible with the supporting polymer network and does not react with the protected, water-sensitive materials. This makes the proposed platform amenable for environmentally friendly real-world applications, including the protection of various complex three-dimensional devices as the fluidic property of the lubrigel makes it possible to coat and infuse the entire shape via dip coating ( 40 ). From a fundamental mechanistic perspective, we established general design guidelines for creating multifunctional water-impermeable coatings for emerging semiconducting materials and devices. Through tuning the dynamics of lubricant constraint, vapor diffusion, and replenishment, we can optimize this liquid encapsulation platform for different applications. In particular, using both experiments and simulation, we systematically investigated the effects of polymer scaffolding and lubricant oil on the stability of perovskite films, elucidating the basic transport mechanisms of water diffusion in the complex polymer matrix/infused oil environment. We found that water tends to aggregate into water clusters, which are extremely sensitive to the polymer network pore size and the lubricant viscosity, leading to an ultralow water transmission rate. (We note that the elevation of temperature will increase the diffusion coefficient of water molecules based on Stokes–Einstein equation, which will cause a slight decrease in water resistance and the associated reduced longevity.) Furthermore, similar to the frog’s ability to secrete and replenish the wax on its surface, a key advantage of the developed encapsulation device is the potential to refresh the oil overlayer, effectively removing all the water from this overlayer. We argue that this important property is due to the unique regeneration mechanism involving the reversal of the diffusion direction of water clusters. Such a reversed concentration gradient leads to water diffusion away from the water-sensitive components, extending the longevity of the protected materials. We believe that the routine replenishment of hydrophobic oils in solar cell farm scenarios, for example, could be possible if integrated with regular maintenance of solar cells, and the replenishment frequency could be accurately predicted based on the diffusion model demonstrated here. In summary, the liquid encapsulation platform provides a new strategy to address the long-standing challenge of water protection, suggesting opportunities in a variety of applications, such as perovskite solar cells and bioelectronics. In addition to the exceptional water protection performance, this approach also offers other attractive properties, including optical transparency, high flexibility, and self-healing—critical characteristics, which are impossible to achieve simultaneously for traditional hermetic encapsulation methods. Importantly, this design principle can be readily applied to other fluid–polymer hybrids, and, more broadly, can open up an exciting path to controlling the molecular transport properties of soft materials."
} | 1,488 |
36438421 | PMC9682352 | pmc | 8,634 | {
"abstract": "Iron is an essential micronutrient for most living organisms, including cyanobacteria. These microorganisms have been found in Earth's driest polar and non-polar deserts, including the Atacama Desert, Chile. Iron-containing minerals were identified in colonized rock substrates from the Atacama Desert, however, the interactions between microorganisms and iron minerals remain unclear. In the current study, we determined that colonized gypsum rocks collected from the Atacama Desert contained both magnetite and hematite phases. A cyanobacteria isolate was cultured on substrates consisting of gypsum with embedded magnetite nanoparticles. Transmission electron microscopy imaging revealed a significant reduction in the size of magnetite nanoparticles due to their dissolution, which occurred around the microbial biofilms. Concurrently, hematite was detected, likely from the oxidation of the magnetite nanoparticles. Higher cell counts and production of siderophores were observed in cultures with magnetite nanoparticles suggesting that cyanobacteria were actively acquiring iron from the magnetite nanoparticles. Magnetite dissolution and iron acquisition by the cyanobacteria was further confirmed using large bulk magnetite crystals, uncovering a survival strategy of cyanobacteria in these extreme environments.",
"conclusion": "4 Conclusions and outlook We identified magnetite and hematite phases in the gypsum rocks collected from the Atacama Desert. Microorganisms were clustered on or near the iron oxide minerals occluded within these rocks, indicating these inorganic minerals may be used as an iron source in this extreme environment. A previously isolated Chroococcidiopsis strain was cultured with synthesized magnetite nanoparticles. Shrinkage of nanoparticle size and the concurrent emergence of amorphous domains surrounding the nanocrystals indicated a process of magnetite dissolution in presence of the cyanobacterial biofilm. The production of EPSs in embedded magnetite nanoparticles and siderophores in a liquid medium supplemented with magnetite nanoparticles further suggest that cyanobacteria were able to extract iron from the magnetite solid phase. The dissolution of solid magnetite phase was further verified in large bulk magnetite crystals, revealing phase transformation from magnetite to hematite during culture experiments, most likely as the result of oxygen production by photosynthesis. These experiments demonstrated that magnetite minerals can be used as an iron source by cyanobacteria in culture experiments and may similarly enable the survival of microorganisms living in extreme environments. To understand this process thoroughly, future work will involve additional in-situ analyses to monitor and understand the interactions between living cells and inorganic minerals in real-time. The oxidation of magnetite to hematite at room temperature by living organisms could provide potential design strategies for engineered living materials. Thus, smart and intelligent materials that react to environmental stimuli can be made by incorporating living microorganisms in fabrication processes such as additive manufacturing.",
"introduction": "1 Introduction Microbial communities inhabit rock substrates (endoliths) as a survival strategy in arid deserts around the world such as the Atacama Desert, Chile [ [1] , [2] , [3] ]. Cyanobacteria, a major component of endolithic communities in hyper-arid environments, utilize crystalline water from gypsum rocks as a source of moisture for metabolic activity [ 4 ]. In fact, it is these rocks that provide refuge for a variety of microbial taxa that include not only Cyanobacteria , but also Actinobacteria , Chloroflexis , and Proteobacteria [ 5 , 6 ]. Beyond water, cyanobacteria also acquire trace elements from their rocky habitat, such as iron, an essential micronutrient for many enzymatic processes and for photosynthesis [ 7 , 8 ]. Although iron is in relatively high abundance in the environment, its accessibility to microorganisms is low because it is found primarily as minimally soluble ferric (Fe 3+ ) ions within oxide minerals such as hematite, magnetite, siderite, etc. [ 9 ]. As a result, microorganisms have evolved several iron acquisition pathways including siderophores, heme and transferrin/lactoferrin uptake (i.e., hemophores), and reduction of Fe(III) to Fe(II) with subsequent transport of Fe(II) [ 8 ]. Siderophores are of particular interest in extreme environments where heme-producing hosts are lacking; Siderophores are low molecular-weight organic ligands with high affinity and specificity for Fe. Siderophores mobilize Fe by competitive complexation or dissolution of iron-bearing minerals for delivery to cells via specific receptors [ [10] , [11] , [12] , [13] ]. In the past few years, the uptake and transport pathways of iron by siderophores have been thoroughly investigated in bacteria [ 14 ]. However, the pathways by which ferric iron is extracted from minerals at neutral pH in the presence of bacteria and the interactions between bacteria and iron-based mineral surfaces remain unclear [ 15 , 16 ]. Significant progress had been made in understanding how dissimilar iron reducing bacteria (DIRB) such as Shewanella putrefaciens can transform magnetite to siderite or vivianite and thus utilized in biogeochemical reactions including redox balance and oxidation of organic carbon [ [17] , [18] , [19] , [20] ]. In contrast, it was reported that chemolithoautotrophic bacteria form organic carbon compounds by reducing carbon dioxide while oxidizing manganese minerals. In this case, Mn(II) in MnCO 3 was oxidized to Mn(IV)O 2 in presence of the microorganisms, in which the inorganic manganese phase serves as an electron donor [ 15 ]. In this work, gypsum was collected from the Atacama Desert to investigate interactions between microbial cells and minerals within the context of iron acquisition. In addition to revealing survival strategies of cyanobacteria in an extreme desert, the modifications of geological minerals and synthetic ceramics by microorganisms could lead to new insights that are important in biomineralization [ [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] ] as well as biomedical applications such as bioimaging or targeted drug delivery [ [30] , [31] , [32] , [33] , [34] , [35] , [36] , [37] ] and could also provide inspiration for the design and fabrication of functional living materials [ [38] , [39] , [40] ]. An engineered living material is a category of smart materials that incorporate living cells within matrices (scaffolds), thus providing active responses to environmental stimuli [ 41 ]. Applications of living materials in the biomedical field include wound healing and biosensing, while environmentally friendly building composites have been developed by researchers in civil engineering [ 42 , 43 ]. Beyond this, understanding these organic-mineral interactions could also yield new strategies for the synthesis of new functional materials with novel properties for a broad range of applications [ [44] , [45] , [46] , [47] , [48] , [49] , [50] ]. The interactions between living cells with the materials matrices (polymers or ceramics) occur in extreme environments, providing crucial information for the extreme biomimetic designs, leading to novel material synthesis and fabrication strategies under extreme biological conditions [ 51 , 52 ].",
"discussion": "3 Results and discussions 3.1 Magnetite and hematite minerals in gypsum from the Atacama Desert Gypsum rocks collected from the Atacama Desert, Chile ( Fig. 1 a), harbored black and orange-colored particles most likely due to iron minerals inclusions (inset, Fig. 1 a). The presence of iron in the black particles and its distribution inside the rock were confirmed by optical microscopy and SEM/EDX mapping ( Fig. 1 c). A closer examination of the sample using scanning electron microscopy (SEM) revealed the presence of filamentous microorganisms within the rocky substrate ( Fig. 1 d). Based on previous studies of gypsum rocks from several locations of the Atacama Desert [ [57] , [58] , [59] ], filamentous microorganisms likely belong to actinobacteria as well as cyanobacteria from the Nostocales and Oscillatoriales orders. Chroococcidiopsis , although non-filamentous, were also a well-represented taxon in the gypsum communities. These cyanobacteria have been found in aggregates around sepiolite minerals where water was available for metabolism [ 57 ]. EDX mapping ( Fig. 1 e of the same area from Fig. 1 d) provided the elemental distribution of the rock surface, in which iron (Fe) and the organic content, suggestive of bacterial occurrence, were observed from C, S and N abundances. Based on the color and presence of iron, the observed black particles within the gypsum rocks were likely magnetite. To further confirm this, we obtained elemental distributions and determined mineral groups present at the micron scale using polished flat surfaces of the black particles and synchrotron micro X-ray fluorescence (μXRF) microscopy and X-ray absorption near edge structure (μXANES) spectroscopy. Micro-XRF mapping revealed that iron, calcium, potassium, and sulfur were the major elements detected in the rock, and that iron was found in both ferric and ferrous forms ( Fig. 1 f and S 1 , Table S1 ). Least-square linear combination fitting of Fe K-edge XANES data ( Fig. S2 ) provided mineral phase information and further indicated the presence of magnetite within the gypsum rocks. Additional analyses of these minerals by powder and wide-angle X-ray diffraction ( Figs. S3a and b , respectively) highlighted both magnetite and hematite phases. Fig. 1 Iron minerals in gypsum rocks collected from the Atacama Desert. (a) Field photo of the Atacama Desert. Inset photo shows the rocks collected from the desert. (b) Optical microscopy image of black and yellow particles found in gypsum rock, inset of (a). (c) SEM micrograph of the polished surface in (b) and EDS mapping of iron (inset). (d, e) SEM micrograph of inset of (a) showing particles embedded within rock and microbe colonies. The EDS mapping showing distribution of iron (purple) and microorganisms (red and lavender). (f) Synchrotron micro X-ray fluorescence microscopy of the gypsum rock sample in (b). The distribution of iron, calcium and sulfur are shown in red, green and blue, respectively. Pixel size is 1.5 μm × 1.5 μm. (g, h) Raman spectroscopy and mapping of the mineral sample of the inset (a), indicating the existence of magnetite and hematite phases. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 1 Raman spectroscopy and mapping were performed on the rock samples to provide the spatial distribution of magnetite and hematite phases. Both magnetite and hematite phases were confirmed by Raman spectroscopy ( Fig. 1 g). Raman mapping ( Fig. 1 h) depicted regions of magnetite within the rock (as selected by highlighting the peak at 666 cm −1 , which is the most intense peak of magnetite phase) [ 60 ]. Furthermore, the red Raman spectrum in Fig. 1 g, which was based on the red spot in Fig. 1 h, showed peaks at 1615, 1382, and 1271 cm −1 that were assigned to pigments such as xanthophylls, carotenoids, and amide in cyanobacteria [ 57 , 61 ]. The presence of iron-based minerals and biological pigments at the same location in the gypsum samples suggested potential cyanobacteria-mineral interactions for iron acquisition. 3.2 Interactions between cyanobacteria and magnetite nanoparticles To understand the interactions between cyanobacteria and iron minerals, Chroococcidiopsis sp. G-MTQ-3P2 were cultured with gypsum substrates either with or without embedded synthesized magnetite nanoparticles ( Fig. S5 ). Small changes (even at nanoscale) to the size or phase of these magnetite nanoparticles can be monitored via transmission electron microscopy (TEM). Fig. 2 a shows the three groups utilized for the experiments: I) experimental group: cyanobacteria with coupons of gypsum and synthesized magnetite nanoparticles; II) control group: cyanobacteria with coupons of gypsum without synthesized magnetite nanoparticles; III) control group: coupons of gypsum and synthesized magnetite nanoparticles without cyanobacteria. After 21 days of culturing, the surface of sample I demonstrated the presence of green cyanobacteria colonies within a biofilm attached to the substrate ( Fig. 2 b). To compare the effect of the growing colonies on any phase changes in magnetite, Raman spectroscopy and mapping were performed on samples I and III after the culture experiments. In sample III (control with no cyanobacteria), only magnetite was observed ( Fig. 2 c), whereas in sample I, both hematite and magnetite phases were present ( Fig. 2 d). Fig. 2 Cyanobacteria cultures on gypsum substrates with or without magnetite nanoparticles. (a) Three different groups of the culture experiments were conducted: I) Gypsum substrate with embedded magnetite nanoparticles and cultured with cyanobacteria. II) Gypsum substrate without magnetite nanoparticles and cultured with cyanobacteria. III) Gypsum substrate with embedded magnetite nanoparticles without cyanobacteria (negative control). (b) Optical micrograph showing the cyanobacteria and magnetite nanoparticles in the substrate. Inset SEM image shows the cyanobacteria and surrounding biofilm attached to the gypsum substrate. (c) Raman spectroscopy of sample (III). The inset image shows the distribution (red is highest, blue is lowest) of magnetite. (d) Raman spectroscopy of sample (I) from region circled in black (inset). Hematite and magnetite phases, as well as organics are observed. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 2 We further investigated potential size and phase changes of the magnetite nanoparticles during the culture experiments with high resolution transmission electron microscopy (HRTEM) imaging of the nanoparticles in samples I and III. Aggregates of nanoparticles shown within control sample III (i.e., no cyanobacteria, Fig. 3 a), with selected area electron diffraction (SAED, inset 3a) and Fast Fourier Transformation (FFT, Fig. 3 b), revealed a randomly oriented magnetite phase. The average size of these highly crystalline nanoparticles ( Figs. 3 c and d) was ∼15.8 ± 3.1 nm, which was comparable to the original synthesized nanoparticles that were introduced before the culturing experiments ( Fig. S4 ). Conversely, nanoparticles within aggregates from sample I (containing cyanobacteria) had a significantly smaller size (∼7.1 ± 1.1 nm, Fig. 3 e) than the initial magnetite nanoparticles introduced before culturing. Phase analysis of these particles using FFT verified the presence of hematite after the culturing with cyanobacteria ( Fig. 3 f). HRTEM images of the hematite nanoparticles showed both crystalline and amorphous domains ( Figs. 3 g and h). A close comparison of the magnetite nanoparticle aggregates in Fig. 3 a and e revealed the presence of what appeared to be organic biofilms surrounding the hematite nanoparticle aggregates in sample I ( Fig. 3 e). The organic biofilm was detected in SEM and validated with EDX before TEM sample preparation ( Fig. S6 ). This observation corroborated our Raman spectrum acquired from the same specimen (i.e., Sample I, Fig. 2 d). HRTEM images and the FFT pattern suggested the presence of amorphous films ( Figs. 3 i and j) surrounding hematite-containing nanoparticles; these were likely organic biofilms leading to the dissolution of iron-based mineral nanoparticles ( Figs. 3 k and l). Indeed, abundant extracellular polymeric substances (EPS) have been reported in endolithic communities from arid deserts [ 3 ]. EPSs are synthesized by cyanobacteria mainly from sugars, forming a protective shield around cells [ 62 ]. EPSs are the principal component of microbial biofilms, they are essential for retaining moisture and nutrients and, with associated organic acids, contribute to mineral weathering [ 3 , 63 , 64 ]. For example, cyanobacteria, specifically Chroococcidiopsis , were shown to trigger the weathering of sandstone by abrupt pH changes as the result of photosynthetic activity [ 65 ]. Furthermore, decreases in pH by microbial metabolites were shown to promote iron solubilization under low iron bioavailability. Conversely to the reactions carried out by Chroococcidiopsis in this study that lead to dissolution and oxidation to hematite, DIRB organisms such as Shewanella putrefaciens have demonstrated reduction reactions that transform iron oxides. A seeming commonality between these systems is the presence of the organic biofilms that surround the mineral and likely play a significant role in solubilization. In fact, multiple studies on bacteria-mineral interactions [ 18 , [66] , [67] , [68] , [69] ] revealed direct contact of cells and further hypothesized that extracellular polymers (i.e., exopolysaccharides, EPS) are responsible for the changes to mineral surfaces. The composition of these extracellular polymer substances are currently unknown, and merits a significant future effort in their investigation. Fig. 3 HRTEM of magnetite nanoparticles after culturing with cyanobacteria. (a) Magnetite nanoparticle aggregates in the substrates without cyanobacteria cultures. Inset: SAED pattern indicates magnetite phase. (b) FFT of sample from the blue box in (a). (c) HRTEM image of a single magnetite nanoparticle in (a). (d) FFT of the nanoparticle is shown in (c). (e) Magnetite nanoparticle aggregates in the substrates with cyanobacteria cultures. Inset: Higher resolution image from (e). (f) FFT of sample from the yellow box in (e), indicating the existence of hematite phase. (g, h) HRTEM of the hematite nanoparticles in (e). (i, j) HRTEM of the regions surrounding the nanoparticle aggregates in (e, red box). Amorphous domains are observed. (k, l) Hematite nanocrystals are noticed in the surrounding amorphous domains. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 3 In addition to studying the changes of iron-based mineral nanoparticles, the activities of cyanobacteria cultures were monitored. Because cells and related biological products were difficult to collect on gypsum coupon surfaces, we used liquid culture experiments, in which magnetite nanoparticles were directly added to the culture media. Different sets of experiments were performed, and are shown in Table 1 . Chl a concentration in the culture, as a proxy for cell abundance, revealed that the samples cultured in low-iron medium with added magnetite nanoparticles had a larger number of cyanobacteria than samples without magnetite ( Table 1 ). The production of siderophores was also higher in the iron-depleted medium compared to the iron-rich medium, suggesting the use siderophores by cyanobacteria to acquire iron from magnetite nanoparticles. However, it is important to note that electron transfer between iron-based mineral and cyanobacteria leading to the phase transformation cannot be ruled out. Table 1 Chlorophyll a content and siderophore production in G-MTQ-3P2 cyanobacteria cultures in different media after 21 days incubation indicating increased cell numbers in cultures with nanoparticles and no significant differences in iron-depleted medium with or without nanoparticles. Table 1 Experiment group Chlorophyll a content (μg/ml) Siderophore content (μM DFOM equivalents) Cyanobacteria + iron depleted medium 1.62 ± 0.13 9.4 ± 1.1 Cyanobacteria + iron depleted medium + nanoparticles 3.65 ± 0.20 10.4 ± 1.3 Iron depleted medium N/A N/A Cyanobacteria + iron containing medium 2.01 ± 0.33 4.8 ± 1.3 3.3 Iron acquisition by cyanobacteria from bulk magnetite crystals To understand the mineral-microorganisms relation at larger scales, we used cyanobacteria cultured in the presence of bulk magnetite crystals (ca. Those at the millimeter scale). Bulk magnetite crystals were embedded in epoxy and polished to expose the mineral surfaces ( Fig. 4 a, top). Raman mapping was performed to confirm the original magnetite mineral phase (with almost no hematite) on sample surfaces before the culture experiments ( Fig. 4 a, bottom). Culture experiments were conducted in both low and high concentrations of soluble iron ( Fig. 4 b): the upper row consists of 3 samples cultured in iron-depleted media with a limited soluble iron (i.e., samples 1A, 1B, 1C), while the bottom row were samples cultured in regular BG11 media containing iron ions (i.e., samples 2A, 2B, 2C). By conducting this experiment, we aim to determine whether cyanobacteria under iron-limiting conditions could actively acquire iron from a magnetite substrate, causing the dissolution and oxidation of magnetite crystals. Fig. 4 Experiments of cyanobacteria cultured on large geologic magnetite crystals embedded in epoxy resin. (a) Epoxy coupons with and without large magnetite crystals. Raman mapping verifies the magnetite phase on the substrate (i.e., from large crystals). (b) Two series of different experimental conditions are applied: the upper row are samples (1A, 1B and 1C) cultured in iron-depleted media, while the bottom row are samples (2A, 2B and 2C) cultured in normal media with sufficient concentrations of iron ions. Controls without magnetite and without cyanobacteria were also performed. Fig. 4 Raman spectroscopy and mapping was used to evaluate the different cultured samples ( Fig. 5 ). Note, samples 1A and 2A are not shown as no changes to the epoxy surface were observed. Analysis of the samples cultured in iron-depleted media, reveals that sample 1C (with magnetite and cyanobacteria in iron-depleted media) had a large area of hematite phase (large green area in Figs. 5 f and S7 ) that developed during the culture experiments. SEM images of the same sample ( Figs. 5 g and h) showed cyanobacteria colonies attached to the surfaces of magnetite crystals. Grooves and cracks were observed on the surface, indicating physical changes likely caused by the dissolution of magnetite when cultured with cyanobacteria ( Fig. 5 g). Higher magnification imaging in Fig. 5 h showed the biofilm on the nanograins of magnetite substrate. However, almost no hematite phase or surface changes were observed in the control sample 1B (magnetite but no cyanobacteria in iron-depleted media, Figs. 5 a–d). In contrast, when magnetite substrates were cultured in media with sufficient iron ions (samples 2A-C described from Fig. 4 ), almost no hematite phase was observed. SEM images from sample 2C (cyanobacteria plus magnetite in normal media), showed that cyanobacteria were attached to the surface of the magnetite substrates. However, the underlying substrate was flat, with no obvious grooves and physical changes observable ( Figs. 5 o and p). These experimental results indicated that cyanobacteria could acquire iron ions from the bulk magnetite crystals when experiencing iron deficiency (i.e., liquid culture medium in our experiments). Dissolution of nanoparticles and modification of large magnetite crystal surfaces were observed in the presence of biofilms ( Fig. 3 , Fig. 5 ). This is likely due to the local low pH of these biofilms, which consist of organic acids that have been shown to etch gypsum rocks [ 4 ], and the higher solubility of magnetite in acidic environments [ 18 , 70 ]. This process can be described by the following reaction [ 71 ]: [ F e 2 3 + F e 2 + ] O 4 ( M a g n e t i t e ) + 8 H + → F e 2 + + 2 F e 3 + + 4 H 2 O Fig. 5 Interactions between cyanobacteria and magnetite bulk crystals. (a–h) Raman mapping and SEM images of samples cultured in iron-depleted media. The first row (a–d) is the control sample without cyanobacteria culture, corresponding to sample 1B in Fig. 4 . The second row (e–h) is the experimental sample of 1C. (a, e) Optical microscopy images of sample surfaces after culturing. (b, f) Raman mapping of control and experimental samples, respectively. (c, d, g and h) SEM images of sample surfaces after cyanobacteria culturing. (i–p) Raman mapping and SEM images of samples cultured in media with sufficient iron ions. The first row (i–l) and second row (m–p) correspond to samples 2B and 2C, respectively, from Fig. 4 . In the Raman maps, the red color indicates magnetite phase (Raman shift 666 cm −1 ), while the green color indicates hematite phase (Raman shift 217 cm −1 ). A large area of hematite phase is observed in sample 1C. SEM of sample 1C (g, h) and 2C (o, p) show cyanobacteria attaching to the substrate surfaces. Grooves and dissolution of the large magnetite substrate particles are noticed in the SEM images from sample 1C (g, h). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 5 Furthermore, the use of ferric iron (Fe 3+ ) by the cyanobacteria during photosynthesis and metabolism could drive the reaction forward, leading to additional dissolution. This may also explain observations in sample 2C ( Fig. 5 ), where almost no dissolution of magnetite was observed since there was sufficient iron ions in the culture media, which could impede the dissolution reactions. In addition, the presence of hematite was observed concurrently with the dissolution of magnetite. Because photosynthetic releases oxygen, components of the reaction can be modified such that: [ 71 ] 3 [ F e 2 3 + F e 2 + ] O 4 ( M a g n e t i t e ) + 2 H + + 1 / 20 2 → F e 2 + + 4 [ F e 2 3 + ] O 3 ( h e m a t i t e ) + H 2 O Hematite phase is barely observed in sample 2C, further indicating that the magnetite to hematite phase transformation only happened when magnetite dissolution occurred. The oxidation of magnetite to hematite phases was only observed at either high temperatures (>200 °C) or under hydrothermal (>120 °C) conditions [ 72 , 73 ]. We showed here that in presence of metabolically active cyanobacteria, this process could occur under atmospheric pressure and at room temperature. In our experiments, the magnetite to hematite transformation was facilitated by Chroococcidiopsis . The higher amount of chlorophyll – a proxy for cell density - we observed in the magnetite nanoparticles containing liquid medium, the changes in nanoparticles size, and the production of siderophores indicated a biological origin for the magnetite to hematite phase transformation."
} | 6,673 |
35910633 | PMC9329127 | pmc | 8,635 | {
"abstract": "The breaking silence between the plant roots and microorganisms in the rhizosphere affects plant growth and physiology by impacting biochemical, molecular, nutritional, and edaphic factors. The components of the root exudates are associated with the microbial population, notably, plant growth-promoting rhizobacteria (PGPR). The information accessible to date demonstrates that PGPR is specific to the plant's roots. However, inadequate information is accessible for developing bio-inoculation/bio-fertilizers for the crop in concern, with satisfactory results at the field level. There is a need to explore the perfect candidate PGPR to meet the need for plant growth and yield. The functions of PGPR and their chemotaxis mobility toward the plant root are triggered by the cluster of genes induced by the components of root exudates. Some reports have indicated the benefit of root exudates in plant growth and productivity, yet a methodical examination of rhizosecretion and its consequences in phytoremediation have not been made. In the light of the afore-mentioned facts, in the present review, the mechanistic insight and recent updates on the specific PGPR recruitment to improve crop production at the field level are methodically addressed.",
"conclusion": "Concluding Remarks Since the 1980s, plant growth-promoting rhizobacterial inoculants have been developed, but few of them revealed irregular performance at the field level. Although several researchers have developed the consortia of plant growth-promoting rhizobacteria, but with more or less similar outcomes in the farmer's field, the solution to these problems is somehow hidden in the root exudates and root microenvironment. Thus, the present review has concentrated on the remarkable views for future research to manage the challenges at the field level with PGPR inoculants. Several components of root exudates have functional interplay with PGPR either directly or through their gene expression. The recruitment of plant growth-promoting rhizobacteria through root exudates can enhance plant growth-promoting rhizobacteria root colonization, specifically, and induce close sustainable relationships between them for a long time. The hypothesis of specific recruitment would address the key gap for warranting the perfect plant growth-promoting rhizobacteria candidate and opening a new horizon of research in biofertilizer technology. It would be a promising technique for reducing the asymmetrical performance of plant growth-promoting rhizobacteria in the farmer's field.",
"introduction": "Introduction According to the World Health Organization (WHO), the food shortage for sustaining the human population is on a steep upward trajectory, mainly owing to the quickly booming human population that is expected to cross the 10 billion mark by 2050 (DESA UN, 2015 ). Both WHO and the United Nations have proposed to intensify global food production by 50% in the near future. The agriculturally important microorganisms (AIMs) can play a pivotal role in realizing this colossal target considering the fact that fertile lands are sharply shrinking owing to urbanization and industrialization. AIMs not only improve plant growth and yield but provide sustained protection against a variety of phytopathogens (Bhattacharyya and Jha, 2012 ; Glick, 2012 ; Compant et al., 2019 ). The beneficial microbes of the rhizosphere zone interact positively with mutually guided components of root exudates, i.e., rhizodeposits (Hassan et al., 2019 ). During the rhizodeposition process, the plant roots secrete carbohydrates, fatty acids, essential amino acids, organic acids, hydrolytic enzymes, growth-regulating hormones, vitamins, nucleotides, flavonoids, polyphenols, sterols, and volatile organic compounds (Hartmann et al., 2009 ; Hu et al., 2018 ; Ankati and Podile, 2019 ). In the last century, the word “rhizosphere” was introduced as a microbial hot spot in the area of the rootsystem (Hartmann et al., 2008 ). The rhizospheric region, a specific zone around the root and harbors various kinds of microorganisms, primarily bacteria, fungi, nematodes, insect larvae, mites, amoebas, and protozoa (Bonkowski et al., 2009 ). The bacterial colonies residing in the rhizospheric zone are called rhizobacteria (Hartmann et al., 2009 ). The rhizospheric zone supports the plant root system (Ahemad and Kibret, 2014 ) and modulates the physico-chemical and biological properties of the soil (Ahemad and Kibret, 2014 ; Zhalnina et al., 2018 ). The rhizosphere zone provides a shelter for the exchange of biochemical components that establish inter-species relationships between the roots and microorganisms (Gupta et al., 2020 ). Plant roots release various types of enzymes/compounds in the soil that mediate the interaction between microorganisms and plants (Ankati and Podile, 2019 ). Factors influencing soil microbial population include soil quality, soil moisture, soil pH, and rhizospheric secretion (Bagyalakshmi et al., 2012 ; Upadhyay and Singh, 2015 ; Hu et al., 2018 ). There are various physical and chemical parameters of the rhizospheres that impact the function of microorganisms, which ultimately affect several mechanisms, such as the respiratory process, the secretion of organic acids by the roots, the breakdown of soil organic matter, nutrient uptake, symbiotic nitrogen fixation, etc. (Reinhold-Hurek et al., 2015 ; Mahmud et al., 2021 ). The rhizosphere plays an important role in root excretion, microbial activity, genetic exchange, improving nutrient use efficiency, and gradient diffusion, which are jointly referred to as the rhizosphere effect (Badri and Vivanco, 2009 ; Ladygina and Hedlund, 2010 ; Mendes et al., 2013 ). Rhizobacteria associated with the plant root are often referred to as plant growth-promoting rhizobacteria (PGPR). The functions of plant growth-promoting rhizobacteria, such as direct and indirect mechanism, metabolism, chemotaxis, secretion, antibiotic production, etc., are mediated by its gene cluster that triggers host–PGPR interactions (Mark et al., 2005 ; Matilla et al., 2007 ; Ramachandran et al., 2011 ; Zhang et al., 2015 ; Bashir et al., 2021 ; South et al., 2021 ). Ultrastructure of the root cell wall mediated PGPR interaction, which was induced by the gene expression of the plant. Ryu et al. ( 2003 ) demonstrated that out of 38 genes, 30 genes of Bacillus subtilis -GB03 were associated with a change in the Arabidopsis root-ultrastructure and promote plant growth. Azospirillum irakense vitalized polygalacturonase gene (PG genes) in the roots of rice plant (Sekar et al., 2000 ). Among PG genes, PbrPG6 is responsible for fruit-soothe (Zhang et al., 2019 ). The root exudation and root exudates are relevant for the survivability of plants against various environmental conditions. The root exudates aid in the selection of microbial populations around the rhizosphere (Mendes et al., 2013 ; Zhang et al., 2015 ). In the purview to tackle this aspect, the review discusses the mechanisms of root exudation, the current updates on the selective plant growth-promoting rhizobacteria aggregation and their role in plant–microbe interface, and most importantly, the future developments in plant–PGPR interactions for sustainable agriculture."
} | 1,818 |
36364530 | PMC9657928 | pmc | 8,636 | {
"abstract": "Photothermally assisted superhydrophobic materials play an important role in a variety of applications, such as oil purification, waste oil collection, and solar desalination, due to their facile fabrication, low-cost, flexibility, and tunable thermal conversion. However, the current widely used superhydrophobic sponges with photothermal properties are usually impaired by a high loading content of photothermal agents (e.g., gold or silver nanoparticles, carbon nanotubes), low photothermal efficiency, and require harmful processes for modification. Here, a one-pot, simple composite consisting of two-dimensional (2D) selenium (Se) nanosheets (NSs) and commercially used melamine sponge (MS) is rationally designed and successfully fabricated by a facile dip-coating method via physical adsorption between 2D Se NSs and MS. The loading content of 2D Se NSs on the skeleton of the MS can be well controlled by dipping cycle. The results demonstrate that after the modification of 2D Se NSs on the MS, the wettability transition from hydrophilicity to hydrophobicity can be easily achieved, even at a very low loading of 2D Se NSs, and the highly stable photothermal conversion of the as-fabricated composites can be realized with a maximum temperature of 111 ± 3.2 °C due to the excellent photothermal effect of 2D Se NSs. It is anticipated that this composite will afford new design strategies for multifunctional porous structures for versatile applications, such as high-performance solar desalination and photothermal sterilization.",
"conclusion": "4. Conclusions In this study, 2D Se NSs with a lateral size of 180–380 nm were successfully fabricated by a facile LPE method, and were directly employed to fabricate Se@MS by a simple dip-coating method via physical absorption between 2D Se NSs and MS. Through the absorption of 2D Se NSs by the MS in sequence, Se@MSs with different loadings of 2D Se NSs are obtained, as evidenced by SEM result that the loadings of 2D Se NSs on the MS are 2.8 ± 0.5 wt%, 4.4 ± 0.5 wt% and 5.1 ± 0.8 wt%, respectively, and the elemental Se is uniformly distributed on the MS skeleton. The apparent wettability transition from hydrophilicity (0°) to hydrophobicity (137 ± 0.8°~151 ± 0.3°) has been achieved after the modification of 2D Se NSs on the MS, even at an extreme loading, indicating that the Se@MSs have great potential in self-cleaning applications. In addition, the high photothermal conversion of all Se@MSs is obtained due to the high photothermal effect of the Se NSs on the MS skeleton, but the Se@MS-3 exhibits relatively poor photothermal performance due to the severe aggregation at a high loading of 2D Se NSs. The highest temperature can reach up to 111 ± 3.2 °C at 1.0 W cm −2 with good stability. Because of the facile fabrication of 2D Se NSs and Se@MS, rapid wettability transition from hydrophilicity to hydrophobicity, quick photothermal response, and high stability, it is anticipated that this Se@MS holds great promises in various applications, including solar desalination, photothermal sterilization, etc.",
"introduction": "1. Introduction The Group VI element selenium (Se), one of Xenes (phosphorus [ 1 , 2 ], tellurene [ 3 , 4 ], bismuthene [ 5 , 6 ], antimonene [ 7 , 8 ], etc.), is an important semiconductor that offers intriguing properties, including anisotropic thermal conductivity, excellent photoconductivity, and superior piezoelectric and thermoelectric response [ 9 , 10 ]. Se has been reported to have excellent photothermal efficiency, which has been widely applied in biomedical applications, such as photothermal radiotherapy [ 11 ] and imaging-guided synergistic chemo-photothermal therapy [ 12 , 13 ]. The large photothermal effect, cost-effective fabrication, and relatively low cytotoxicity of Se nanostructures make them competitive candidates in many applications, such as waste oil collection, oil purification, solar desalination, and photothermal-assisted antibacterial application. The rapid development of the 5G era and the multifunctionality of miniaturized equipment have yielded many impressive benefits for humans [ 5 , 14 , 15 , 16 ]. Among multifunctional devices, photothermally assisted superhydrophobic materials play an important role in the fields of waste oil collection, oil purification, and solar desalination due to their facile fabrication, low-cost, flexibility, and tunable thermal conversion [ 17 , 18 , 19 ]. For example, in 2021, Han et al. [ 18 ] reported high-efficiency photothermal conversion material MXene nanosheets (NSs) and low thermal conductivity silica (SiO 2 ) coated on a hydrophilic poly(tetrafluoroethylene) (HPTFE) membrane by a commercial continuous spraying system to fabricate a SiO 2 /MXene/HPTFE Janus membrane, and demonstrated that the film had high stability, light absorption, salt resistance, and self-cleaning ability. Moreover, in 2020, Li et al. [ 20 ] passivated 2D black phosphorus (BP) NSs with hydrophobic SiO 2 by hydrolytic co-condensation of 3-aminopropyl-triethoxysilane and tetraethoxysilane, which exhibited high efficiency and stability in solar evaporation without sacrificing the intrinsic properties of BP NSs. In the past decade, many researchers have focused on the superhydrophobic materials with high thermal efficiency and broad-spectrum light absorption to maximum the light-to-heat conversion [ 21 , 22 , 23 , 24 ]. As a typical kind of 3D materials, melamine sponge (MS) was widely used as a universal substrate in the fields of oil/water separation and solar desalination due to its large adsorption capacity, high stability, and low cost [ 25 , 26 ]. However, the intrinsic hydrophilicity and extremely low light-to-heat transition efficiency greatly restrict the work efficiency of MS in related industries [ 27 , 28 ]. In this scenario, hydrophobization by low surface energy materials and doping of photothermal agents are common strategies for the improved performance of the MS [ 29 ]. Although photothermally assisted hydrophobic materials have undergone great progress, the reported fabrication of the multifunctional materials is usually time-consuming, complicated, environmentally unfriendly, and difficult to be realized for industrial production. For example, the reported modifiers for the hydrophobization of MXene NSs and BP NSs, such as polydimethylsiloxane and 1H,1H,2H,2H-perfluorooctyltriethoxysilane, are usually harmful to the environment and organisms, which goes against the principles of green chemistry. Therefore, the development of a one-pot and simple fabrication of superhydrophobic materials with both excellent hydrophobicity and high thermal efficiency in an environmentally friendly manner is crucial. In this study, two-dimensional (2D) Se nanosheets (NSs) were successfully fabricated by a facile LPE method, and then directly employed for the production of composites, abbreviated as Se@MS, composed of the 2D Se NSs and melamine sponge (MS) via physical absorption by a dip-coating method. The loading content of 2D Se NSs on the skeleton of the MS is easily controlled by the dipping cycle. Three kinds of the Se@MSs are obtained with the loading content of the 2D Se NSs of 2.8 ± 0.5 wt%, 4.4 ± 0.5 wt%, and 5.1 ± 0.8 wt%. After the modification of the 2D Se NSs, the wettability of the pristine MS rapidly changes from hydrophilicity to hydrophobicity, even at a low loading of the 2D Se NSs (2.8 ± 0.5 wt%). The photothermal result demonstrates that the as-fabricated Se@MS has an excellent photothermal efficiency with a maximum photothermal temperature of 111 ± 3.2 °C, significantly higher than that of pristine MS (55 ± 2.1 °C) under the same conditions. In addition, at a high loading of 2D Se NSs (5.1 ± 0.8 wt%), the photothermal conversion declines mainly due to the relatively serious aggregation of the 2D Se NSs on the skeleton of the MS, which largely reduces the efficient specific surface area. Due to the facile and environmentally friendly fabrication of Se@MS with low cost, rapid wettability transition from hydrophilicity to hydrophobicity, and excellent photothermal efficiency, it is anticipated that this Se@MS can pave the way for new designs of multifunctional porous structures for versatile applications, such as high-performance solar desalination and photothermal sterilization.",
"discussion": "3. Results and Discussion Figure 1 presents the structural characterization of the 2D Se NSs as fabricated by a facile LPE method. The XRD pattern ( Figure 1 a) shows that the as-fabricated nanostructures have the characteristic peaks of bulk Se, in good agreement with the standard HA (JCPDS No. 86-2246) diffraction peaks, and no impurity peaks (such as SeO 2 ) were observed. The TEM image ( Figure 1 b) shows that the as-fabricated Se nanostructures present 2D structural morphology with a lateral size of 180–380 nm. Size distribution of the Se NSs was evaluated by the dynamic light scattering (DLS). Figure S2 shows an average diameter of Se NSs measured by DLS is 480 ± 2.5 nm, which is in good accordance with the TEM results. The HRTEM image ( Figure 1 b inset) shows a lattice fringe of 0.30 nm, which can be indexed to the (101) plane of the Se crystal [ 31 ]. The selected area electron diffraction (SAED) pattern ( Figure 1 c) also confirms the successful fabrication of the 2D Se NSs by a LPE method. The fabrication of the 2D Se NS-based MS was performed by a simple dip-coating method via physical adsorption. As the number of dipping cycles increased, the loading of the 2D Se NSs on the MS gradually increased ( Figure 2 a), i.e., the weight percentages of the 2D Se NSs on the MS are 2.8 ± 0.5 wt%, 4.4 ± 0.5 wt% and 5.1 ± 0.8 wt%, for Se@MS-1, Se@MS-2, and Se@MS-3, respectively. The weight percentage of the selected Se@MS-2 was also confirmed by EDS (N: 41.8 ± 0.6 wt%, C: 38.5 ± 0.5 wt%, O: 15.1 ± 0.4 wt%, Se: 4.0 ± 0.1 wt%, Figure 2 b). The difference in weight percentage was due to the different characterization techniques. The change in morphology of Se MS on the skeleton with the increasing weight percentage of Se can be seen in Figure 3 a–f. The 2D Se NSs are well distributed on the surface of MS skeleton, and with an increase in dipping cycle, the density of the 2D Se NSs on the MS remarkably increases. It is noted that the aggregation of 2D Se NSs in Figure 3 e,f can be mainly attributed to the high loading of the 2D Se NSs on the MS (5.1 ± 0.8 wt%) for the Se@MS-3 sample. Due to the high porosity of the Se@MS, the surface areas are measured to be 55.45 ± 0.9 m 2 g −1 ; 44.69 ± 1.2 m 2 g −1 ; 40.05 ± 0.8 m 2 g −1 , and 37.40 ± 1.1 m 2 g −1 for the pristine MS, Se@MS-1, Se@MS-2, and Se@MS-3, respectively ( Figure S3 ), in good agreement with the SEM result. In addition, EDS analysis ( Figure 3 g) of the Se@MS-2 illustrates that the elemental C, N, O, and Se are uniformly distributed, and the location of the elemental Se is in good accordance with the loading position in the SEM image. The wettability of a water droplet on the surface of the pristine MS and Se@MS was studied, as shown in Figure 4 . It can be seen in Figure 4 a,b and Figure S4 that the water droplet can quickly spread and penetrated into the pristine MS when it was dropped onto the surface of the sample. On the contrary, for the Se@MS samples, the water droplet clearly beads up, demonstrating the strong in-air hydrophobicity after the modification of 2D Se NSs. Besides, it can be also observed that when the pristine MS and as-fabricated Se@MS-2 were immersed into a glass of water, the pristine MS quickly sinks into the bottom while the as-fabricated Se@MS-2 keep floating on the surface due to its excellent hydrophobicity ( Figure S5 ). The optical images of the MS and Se@MS confirm that the wettability of the commonly used MS can be distinctly switched by surface modification of the 2D Se NSs at a relatively low loading ( Figure 4 a). The wetting transition of MS from hydrophilicity to hydrophobicity after Se NSs treatment is induced by the hydrogen bonding effect between the exposed Se atoms on the Se NSs and the N-H groups in the skeleton of MS. The formation of -N-H∙∙∙Se changed the surface chemical states of MS and greatly lowered its surface energy, and thus the wetting property of MS was reversed [ 32 ]. The similar wettability reversion of MS was also reported in the formation of metal-ion-induced cross-linkage [ 27 , 33 ] and -N-H∙∙∙F hydrogen bonds [ 17 ]. Notably, for the three studied Se@MS samples, the water CA increases with the dipping cycle, i.e., the water CAs of the Se@MS-1, the Se@MS-2, and the Se@MS-3 are 137 ± 0.8°, 151 ± 0.3°, and 144 ± 0.7°, respectively ( Figure 4 b). In addition, the excellent stability of water contact angle for the as-fabricated Se@MS-2 ( Figure 4 c) over one month indicates that the Se@MS holds great promise for practical applications. The rapid wettability transition from hydrophilicity to hydrophobicity by the modification of the 2D Se NSs on the MS is expected to have great potential in anti-fouling and self-cleaning smart equipment in the biomedical field. Given the superior photothermal effect of Xenes, such as phosphorene [ 1 , 34 , 35 ], bismuthene [ 6 , 36 ], and MXenes [ 37 , 38 , 39 ], the photothermal conversion of the as-fabricated Se@MS with excellent hydrophobicity was also studied, as shown in Figure 5 . It can be observed in Figure 5 a–e that at a fixed light power density, the temperatures of Se@MS-2 and Se@MS-3 remarkably increase under illumination for 300 s compared with that of the pristine MS, e.g., under light illumination with a power density of 0.8 W cm −2 for 300 s, the temperatures of the Se@MS-2 and Se@MS-3 quickly increase to 102 ± 4.8 °C and 86 ± 3.7 °C, respectively, significantly higher than that (57 ± 2.3 °C) of the pristine MS ( Figure 5 d), verifying that the Se NSs employed in the Se@MS indeed have an excellent photothermal efficiency. Note that the Se@MS-2 achieved the best photothermal effect compared with Se@MS-1 and Se@MS-3, which can be mainly ascribed to the suitable loading of Se NSs and uniform distribution on the surface of MS skeleton. Here, the loading of 2D Se NS in the Se@MS-1 is very low (2.8 ± 0.5 wt%), leading to an inconspicuous temperature change in comparison with the pristine MS at the studied power density range (0.2–1.0 W cm −2 ), while the loading of 2D Se NS in the Se@MS-3 is fair yet the relatively serious aggregation of 2D Se NSs make a remarkable reduction in the efficient photothermal conversion due to the apparent reduction in specific surface area. Moreover, it can be observed that the temperature of the Se@MS distinctly increases with the power density, i.e., the temperatures of the Se@MS-3 are 57 ± 1.4 °C (0.2 W cm −2 , Figure 5 a), 61 ± 3.9 °C (0.4 W cm −2 , Figure 5 b), 80 ± 2.1 °C (0.6 W cm −2 , Figure 5 c), 86 ± 3.7 °C (0.8 W cm −2 , Figure 5 d), and 93 ± 2.8 °C (0.8 W cm −2 , Figure 5 e), respectively, which demonstrates that the photothermal effect can be rationally controlled by the external power density and loading of 2D Se NSs. In addition, the temperature cycle of the Se@MS-2 at a high light power density of 1.0 W cm −2 ( Figure 5 f) shows that the as-fabricated Se@MS has a very stable photothermal conversion, even at a high photothermal temperature of 111 ± 3.2 °C. The high thermal conversion efficiency of Se NSs can be attributed to the narrow band gap energy ( E g ) due to nonradiative relaxation. The 2D Se NSs capture the solar light, and stimulate the fall of electrons back to the low-energy states and thus the energy is released through radiating photons or nonradiative phonons. In the nonradiative mode, the heat is produced when the phonon interacts with the lattice, establishing a temperature gradient based on the optical absorption and electron–hole recombination feature [ 11 , 12 ]. The infrared thermal photos of the as-fabricated Se@MS in one on/off temperature cycle can be seen in Figure 6 . Upon illumination at 1.0 W cm −2 , the surface temperature of the as-fabricated Se@MS-2 rapidly rises up from room temperature (29.8 ± 1.6 °C) to a maximum (111 ± 3.2 °C) within 300 s, and quickly recovers when the light illumination is off, suggesting the rapid photoresponse behavior and excellent reproducibility under strong light illumination. Table 1 briefly summarizes the photothermal performance of representative reported photothermal agents, such as multiwalled carbon nanotubes (MWCNTs) [ 19 , 40 ] and BP NSs [ 20 ]. Notably, the hydrophobic Se@MS in this work is superior/comparable to these photothermal agents for photothermal efficiency. Apart from that, it is noted that there is no obvious morphology change for the as-fabricated Se@MS before and after photothermal investigation at such a higher light power density ( Figure S6 ). This, combined with excellent hydrophobicity of the Se@MS, indicates that the sample Se@MS-2 is an ideal biomedical candidate for both high-performance self-cleaning and photothermal conversion, which has great potential for practical biomaterial scaffold and biological packaging materials."
} | 4,274 |
39315602 | PMC11420662 | pmc | 8,638 | {
"abstract": "Abstract Wastewater treatment plants are one of the major pathways for microplastics to enter the environment. In general, microplastics are contaminants of global concern that pose risks to ecosystems and human health. Here, we present a proof‐of‐concept for reduction of microplastic pollution emitted from wastewater treatment plants: delivery of recombinant DNA to bacteria in wastewater to enable degradation of polyethylene terephthalate (PET). Using a broad‐host‐range conjugative plasmid, we enabled various bacterial species from a municipal wastewater sample to express FAST‐PETase, which was released into the extracellular environment. We found that FAST‐PETase purified from some transconjugant isolates could degrade about 40% of a 0.25 mm thick commercial PET film within 4 days at 50°C. We then demonstrated partial degradation of a post‐consumer PET product over 5–7 days by exposure to conditioned media from isolates. These results have broad implications for addressing the global plastic pollution problem by enabling environmental bacteria to degrade PET.",
"introduction": "INTRODUCTION Plastics are versatile materials that are lightweight, strong and chemically resistant. These desirable properties have made them an integral part of daily activities, infrastructure and economies since their invention in the 20th century (Andrady & Neal, 2009 ). Although plastics have been an economic boon, particularly in packaging and construction applications, their use has produced unsustainable levels of waste. From 1950 to 2015, about 4900 megatons, accounting for 59% of all plastics ever produced, have been discarded into landfills and the environment (Geyer et al., 2017 ). Ninety percent of plastics produced—consisting of polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polyethylene terephthalate (PET), polyurethane (PU) and polystyrene (PS) (Geyer et al., 2017 )—are projected to persist for hundreds of years; they are not biodegradable on timescales relative to their end of use (Chamas et al., 2020 ). Consequently, their bioaccumulation poses threats to ecosystems and, potentially, to human health (Wang et al., 2019 ; Xu et al., 2020 ; Yuan et al., 2022 ). Annual plastic waste generation is projected to almost triple by 2060 compared to 2019 levels if trends in current plastic usage continue (OECD, 2022 ). Even with immediate implementation of ambitious strategies for waste management, environmental recovery and reduction of plastic production, conservative estimates suggest that plastic waste entering terrestrial and aquatic ecosystems will exceed or remain close to 2016 levels between 2030 and 2040 (Borrelle et al., 2020 ; Lau et al., 2020 ). Although PE, PP, PVC, PET, PU and PS are referred to as non‐biodegradable, some microbes and insects have ways to metabolise these plastics into their constituent molecules, which can then be used as carbon and energy sources (Brandon et al., 2018 ; Chattopadhyay, 2022 ; Jeon & Kim, 2016 ; Maheswaran et al., 2023 ; Yoshida et al., 2016 ). These natural processes are relatively slow, taking weeks to months for significant biodegradation to occur (Gao & Sun, 2021 ; Kumar et al., 2023 ; Maheswaran et al., 2023 ; Sekhar et al., 2016 ; Shah et al., 2008 ; Yoshida et al., 2016 ). Among the most produced plastics, PET is a hydrolysable polymer that is widely used in the packaging and textile industries. Compared to other common plastics, PET is more amenable to biodegradation due to the presence of hydrolysable ester bonds. Substantial progress has been made in enzymatic PET depolymerisation for resource recovery (Liu et al., 2023 ). For example, PET hydrolase (PETase) from Ideonella sakaiensis has been subject to numerous protein engineering efforts that have resulted in PETase variants with depolymerisation rates orders of magnitude higher than the wild‐type enzyme, and with greater thermo‐ and chemostability (Bell et al., 2022 ; Cui et al., 2021 ; Lu et al., 2022 ; Son et al., 2019 ; Zurier & Goddard, 2023 ). Efforts have also been made to optimise expression and secretion of PET‐degrading enzymes in some bacterial species other than Escherichia coli (Li et al., 2023 ; Wang et al., 2020 ; Yan et al., 2021 ), and developments are ongoing for employing naturally occurring and designer microbial communities to degrade PET (Gao & Sun, 2021 ; Maheswaran et al., 2023 ; Qi et al., 2021 ; Roberts et al., 2020 ). Effluent and sludge from wastewater treatment plants are major pathways by which microplastics enter the environment (Liu et al., 2021 ; Sun et al., 2019 ; Zurier & Goddard, 2021 ). Notably, a recent study reported that 6% of plastic processed at a recycling facility was found as microplastics in the facility's wash water post‐filtration (Brown et al., 2023 ). Secondary treatment processes and digestion of sludge offer opportunities for the removal of microplastics through bioaugmentation. As reviewed by Zurier and Goddard ( 2021 ), microbial communities could be supplemented with engineered bacteria that secrete plastic‐degrading enzymes (i.e., cell bioaugmentation); this can be more cost‐effective than continuously supplementing a cocktail of purified enzymes into a secondary treatment unit. A major challenge with the cell bioaugmentation approach is persistence of the introduced microorganisms, which are often not adapted to the environment at hand and are therefore likely to be quickly eliminated (Albright et al., 2022 ; Ma et al., 2022 ). As an alternative strategy that would bypass these barriers to establishment, we propose genetically engineering microbes native to the environment to express metabolic pathways for biodegradation of plastic waste. This approach, known as genetic bioaugmentation, can be achieved through in situ delivery of broad‐host‐range conjugative plasmids. Genetic bioaugmentation has been shown to improve persistence of the introduced functionality when using donor strains native to their environment (Ronda et al., 2019 ). Genetic bioaugmentation has not yet been applied for bioremediation of plastic waste, but this technique has been used to introduce catabolic genes into microbial communities for bioremediation of non‐plastic‐based pollutants in laboratory and pilot scale settings (Bathe et al., 2004 ; Chettri et al., 2023 ; French et al., 2020 ; Garbisu et al., 2017 ; Ke et al., 2022 ; Ren et al., 2018 ; Top et al., 2002 ; Venkata Mohan et al., 2009 ). In this proof‐of‐concept study, we lay a foundation for the use of genetic bioaugmentation for bioremediation of PET. We genetically engineered bacteria in a municipal wastewater sample to degrade PET plastics by delivering a broad‐host‐range, conjugating plasmid carrying the FAST‐PETase gene (Lu et al., 2022 ), which codes for an engineered PET hydrolase that is more robust to pH and temperature ranges and is orders of magnitude more efficient than the wild‐type enzyme (Lu et al., 2022 ). We assessed degradation of commercial PET film using purified FAST‐PETase from several engineered isolates. We then measured degradation of post‐consumer PET using conditioned media from some of the engineered isolates. This report provides a foundation to support the next steps in assessing the performance and efficacy of such a genetic bioaugmentation approach.",
"discussion": "DISCUSSION Engineering microbiomes to enable in situ degradation of plastics in diverse ecological niches remains a key milestone for deploying biological solutions for plastic waste at scale (Engineering Biology Research Consortium, 2022 ). Biotechnological plastic degradation and upcycling can offer a means to achieve a circular life cycle for plastic waste in a sustainable and cost‐effective manner, facilitating a broader move towards a circular economy. Developments in biotechnological approaches to plastic waste management will ideally complement related strategies for reduction of single‐use plastics and innovations in biodegradable plastics. In this work, we demonstrated a proof of concept for genetically engineering bacteria from municipal wastewater in situ to degrade PET. We achieved this by using a broad‐host‐range conjugative plasmid carrying the FAST‐PETase gene. Compared to cell bioaugmentation efforts, genetic bioaugmentation bypasses the adaptation barrier of introducing exogenous microbes into a new environment and thus could be used to achieve long‐term bioremediation solutions. In particular, an approach focused on degradation of specific plastic compounds could be useful for removing microplastics from the waste streams of plastic manufacturing and recycling facilities, which are a notable source of microplastic output into wastewater (Brown et al., 2023 ). The approach demonstrated here may generalise to a variety of ecological niches: several studies have utilised the RK2/RP4 conjugation machinery coupled with a broad‐host‐range origin of replication (or a library of such origins) to engineer microbes from diverse environments: soil (Klümper et al., 2015 ), mammalian guts (Ronda et al., 2019 ) and marine ecosystems (Goodman et al., 1993 ). A broad‐host‐range genetic engineering platform could facilitate the construction of microbial consortia for degradation of individual types of plastic polymers that have known biodegradation pathways (e.g. PET). Such libraries of consortia could eventually be further expanded for degradation of other common plastic polymers, potentially resulting in modular tools for degradation of mixed‐waste plastics. Our results demonstrate that active FAST‐PETase released by the engineered isolates can degrade PET without the need for enzymatic purification (Figure 4 ). The assays using purified enzyme (Figure 3 ) illustrate the plastic‐degrading potential of the engineered isolates, which can guide future optimisation efforts. However, we found that the solution conditions had a substantial effect on FAST‐PETase activity. Our preliminary attempts to degrade commercial PET film in LB medium using a purified enzyme solution showed no activity (purified from WW5; data not shown). Furthermore, our attempts at degrading PET using untreated and pH‐adjusted supernatant also proved unsuccessful (Figure S4 ), highlighting that further developments are necessary for effective in situ degradation. It was necessary to dilute the supernatant to achieve significant PET degradation, consistent with previous observations that FAST‐PETase activity is inhibited at high enzyme concentrations (Avilan et al., 2023 ) and acidic conditions (Lu et al., 2022 ). We found that the secreted enzyme fraction performed better than the intracellular enzyme fraction at similar dosages (e.g. WW5 and WW7 in Figure 3B,D ), with a lower abundance of contaminating proteins in the purified secreted fraction compared to the intracellular fraction (Figure 3A,C ), suggesting that the presence of other proteins in the medium may hinder FAST‐PETase activity. Improved activity could be achieved by a design in which expressed FAST‐PETase is anchored to the cell surface; immobilising PETase enzymes has been shown to improve the stability and activity of the enzyme (Chen et al., 2020 ), including under simulated wastewater conditions (Zhu et al., 2022 ; Zurier & Goddard, 2022 ). Further optimisation of enzyme activity could be achieved by addressing environmental factors such as pH, temperature and biotic factors such as other secreted enzymes and metabolic waste products. Alongside improving operating conditions, PET degradation efficiency could be enhanced by improving the genetic vector design. In pFAST‐PETase‐cis, FAST‐PETase expression was regulated by the inducible P BAD promoter for testing purposes. Regulating expression using a constitutive promoter could enable autonomous plastic degradation. Alternatively, expression induced by specific environmental conditions could be employed to minimise metabolic load and avoid unintended plastic degradation in, for example, products in use and infrastructure. Optimisation of ribosome‐binding sites, transcriptional terminators and screening of signal peptides in members of the microbiome of interest could likewise prove beneficial (Low et al., 2013 ; Xu et al., 2019 ). To achieve complete conversion of PET to its constituents, TPA and ethylene glycol (EG), the PETase activity investigated here could be complemented by a MHETase enzyme, which would enable conversion of the depolymerisation product, MHET, into TPA and EG; alternatively, MHETase might be present in the environment (produced by, e.g. I. sakaiensis (Yoshida et al., 2016 )). The turnover rate of wild‐type MHETase from I. sakaiensis ( k \n cat ~ 25 s −1 at 30°C) (Knott et al., 2020 ) is much higher than that of FAST‐PETase ( k \n cat ~ 0.016 s −1 at 50°C), 1 so depolymerisation by PETase will likely be the rate‐limiting step for the conversion of PET into TPA and EG. Moreover, MHETase has been observed to improve PETase activity by relieving product inhibition caused by MHET monomers (Erickson et al., 2022 ; Knott et al., 2020 ). Recently, a MHETase that is thermostable at the optimum operating temperature for FAST‐PETase has been designed (Zhang et al., 2023 ), offering an attractive dual‐enzyme system for applications in PET biodegradation and upcycling. If production of large quantities of TPA and EG poses ecological or environmental concerns, bioaugmentation of additional consortia could be considered, using, for example the consortium designed by Bao et al. ( 2023 ) for efficient conversion of TPA and EG into biomass. Using E. coli as a donor strain, we found that pFAST‐PETase‐cis could conjugate on a filter‐mating setup and in native wastewater conditions at efficiencies high enough to allow isolation of transconjugants by plating. Improvements in conjugation efficiency could be achieved by exploring alternative donor strains, conjugation systems and plasmid incompatibility classes; working towards applications, such efforts would need to account for environmental conditions (e.g. pH, temperature, seasonal variability). Moreover, the concentration of suspended solids (not measured) could affect conjugation efficiency by increasing the total surface area available for attachment, promoting spatial proximity between donor and recipient bacteria (Hamilton et al., 2019 ). We observed low taxonomic diversity among the transconjugant isolates, likely in part due to our reliance on mCherry expression, driven by the P bs promoter whose activity has only been previously reported in Escherichia coli , Bacillus subtilis and Saccharomyces cerevisiae (Yang et al., 2018 ). Moreover, it is possible that conjugation efficiency from E. coli is higher towards closely related species (Stewart & Levin, 1977 ). Finally, the laboratory growth conditions likely had an impact on the results; reduced diversity of transconjugants has been reported in mating conditions that are less conducive to growth of diverse species (Ronda et al., 2019 ). Additional improvements for selection of the plasmid could be made by using a promoter library for selection markers and origins of replication (e.g. Ronda et al., 2019 ). Such efforts could target specific hosts that might be best suited to application (e.g. non‐pathogenic strains known to be abundant in the conditions of interest). We observed that pFAST‐PETase‐cis was lost in the absence of selection to varying extents based on the host species (Figure 2 ). This could be attributed to copy number variance arising from differences in host‐encoded replication and regulatory proteins, plasmid oligomer formation in recombination‐proficient hosts, and differences in metabolic load (Summers, 1991 ). Nonetheless, our observations highlight that long‐term bioremediation applications will require efforts towards improving genetic stability. Improved maintenance of the function of interest could be achieved by incorporating plasmid maintenance systems such as toxin‐antitoxin systems (Kroll et al., 2010 ; Lin et al., 2023 ), gene entanglement (Blazejewski et al., 2019 ; Chlebek et al., 2023 ) and plasmid partitioning systems (Danino et al., 2015 ) into the plasmid design, or by integrating the gene of interest into the chromosome of the host (Ronda et al., 2019 ). Chromosomal integration of the genetic cargo could be further constrained to non‐pathogenic species by using species‐ and site‐specific gene‐editing tools (Rubin et al., 2022 ). Complementary selection efforts may be required to ensure long‐term in situ maintenance of engineered strains (Raper et al., 2018 ). Deployment of genetically engineered microorganisms for degrading plastics will require careful consideration of environmental and public safety and adhere to regional regulatory policies. To date, there have been few pilot‐scale or field studies utilising genetic bioaugmentation for bioremediation purposes (Ripp et al., 2000 ; Sayler & Ripp, 2000 ; Venkata Mohan et al., 2009 ). One notable case was a field release in Estonia in 1989 of Pseudomonas putida cultures carrying a gene for metabolising phenol into a watershed contaminated from a phenol release caused by a subterranean oil shale mine fire (Peters et al., 1997 ). Six years after the release, the introduced operon was still found to be present in native microbiota (Peters et al., 1997 ). This study suggests that an introduced functionality could persist long‐term in the environment if under selection for metabolism of an abundant pollutant. It will be prudent to test the use of engineered microbes for plastic degradation in contained systems while simultaneously assessing ecological risk, including: characterising the modified microorganisms and nature of the genetic modification, tracking the fate of these microorganisms and the introduced genes of interest, assessing the environmental impact of the release, and monitoring the effects of the release on non‐target microorganisms (Gustafsson & Jansson, 1993 ). Genetic biocontainment of the genetic vector using conditional lethality, conditional genetic regulation, and conditional fitness control could help prevent unintended escape of the modified microorganisms (Lee et al., 2018 ). In wastewater treatment plants, disinfection through tertiary treatment operations could also offer an additional means of biocontainment. Moreover, the controlled environments in wastewater treatment units offer opportunities to assess ecological risks and explore the performance of in situ microplastic degradation at scale."
} | 4,655 |
30760820 | PMC6374434 | pmc | 8,640 | {
"abstract": "Marine sponges are early-branching, filter-feeding metazoans that usually host complex microbiomes comprised of several, currently uncultivatable symbiotic lineages. Here, we use a low-carbon based strategy to cultivate low-abundance bacteria from Spongia officinalis . This approach favoured the growth of Alphaproteobacteria strains in the genera Anderseniella , Erythrobacter , Labrenzia , Loktanella , Ruegeria , Sphingorhabdus , Tateyamaria and Pseudovibrio , besides two likely new genera in the Rhodobacteraceae family. Mapping of complete genomes against the metagenomes of S . officinalis , seawater, and sediments confirmed the rare status of all the above-mentioned lineages in the marine realm. Remarkably, this community of low-abundance Alphaproteobacteria possesses several genomic attributes common to dominant, presently uncultivatable sponge symbionts, potentially contributing to host fitness through detoxification mechanisms (e.g. heavy metal and metabolic waste removal, degradation of aromatic compounds), provision of essential vitamins (e.g. B6 and B12 biosynthesis), nutritional exchange (especially regarding the processing of organic sulphur and nitrogen) and chemical defence (e.g. polyketide and terpenoid biosynthesis). None of the studied taxa displayed signs of genome reduction, indicative of obligate mutualism. Instead, versatile nutrient metabolisms along with motility, chemotaxis, and tight-adherence capacities - also known to confer environmental hardiness – were inferred, underlying dual host-associated and free-living life strategies adopted by these diverse sponge-associated Alphaproteobacteria .",
"introduction": "Introduction Determining the ecological and evolutionary forces that shape the structure of marine sponge microbiomes is fundamental to current marine microbiology research due to the relevance of these symbiotic communities to ecosystem functioning 1 – 4 and biotechnology 5 – 8 . Fifty-two bacterial phyla have been reported to inhabit sponges 3 , with Proteobacteria (mostly Alpha- and Gammaproteobacteria ) being by far the most abundant, followed by Acidobacteria , Actinobacteria , Chloroflexi , Nitrospirae , Cyanobacteria and the candidate phylum Poribacteria 3 , 9 . Sponge-associated bacteria engage in nutritional exchange with their hosts and as such are considered to play an important role in benthic biogeochemical cycling 4 , 10 , 11 . Moreover, they are believed to produce most of the secondary metabolite repertoire of sponges 6 , 12 – 17 , and thus hold potential value for applications in medicine and pharmacy 7 , 16 . Alphaproteobacteria display great versatility in their association with multicellular organisms, with interactions ranging from mutualistic over commensal to parasitic and pathogenic 18 . Microbial diversity surveys performed on different sponge species from various geographic locations have noted Alphaproteobacteria as regular sponge associates 9 , 19 – 21 . Particularly, a variety of currently uncultivatable lineages in the families Rhodobacteraceae and Rhodospirillaceae have been found as dominant members of the marine sponge microbiome 22 – 24 . Recent metagenomic “binning” studies, that sort metagenomic sequences into genomes that are assumed to constitute separate taxa, uncovered versatile metabolisms among diverse uncultivated Alphaproteobacteria symbionts of sponges, in which import and utilization of organic nitrogen and sulphur emerged as conspicuous features 2 , 25 , 26 . Among cultivated sponge-associated Alphaproteobacteria , the genera Pseudovibrio and Ruegeria likely rank as the best-described groups. They have been consistently isolated from various host species across the globe 19 , 20 , 27 – 29 , and based on recent genomic surveys, are considered to be well equipped for a symbiotic life-style 30 – 33 . In contrast, our understanding of the potential contribution of most cultivatable sponge-associated bacteria to holobiont functioning remains hindered by scarce knowledge of their genome content and architecture. The known taxonomic diversity of sponge-derived culture collections is still limited, with 1% to 14% of the total sponge bacterial community estimated to be cultivatable using different methods 19 , 27 , 34 , 35 . Indeed, the most abundant bacterial symbionts of sponges, in particular, remain uncultivated 36 , 37 . Complicating factors for the cultivability of these bacteria are the initial sample processing method, the nature of the growth medium, and the incubation conditions 38 . The in-situ implantation of nutrient medium-containing diffusion growth chambers (DGCs) 39 into sponge specimens and their subsequent incubation in the field 40 , or the concomitant use of several solid or liquid media (with and without antibiotics) 27 , 41 , for example, have been attempted to enlarge the phylogenetic breadth of marine sponge symbionts captured in the laboratory, and have shown promising results. However, continuous effort to cultivate hitherto “uncultivatable” symbionts or novel representative lineages within taxa less prone to cultivation is needed if we are to harness the metabolism of the marine sponge microbiome in a comprehensive fashion. In this study, to attempt the isolation of “difficult-to-culture” bacterial symbionts of sponges - defined here as any organism detected in association with the sponge host regardless of whether the interaction is beneficial or obligatory 36 - we used simple modifications to growth medium preparation and incubation conditions. First, we replaced the solidifying agent agar, which may inhibit the growth of certain bacterial taxa 42 , 43 , with the nontoxic agent gellan gum. In addition, to favour the cultivation of putatively slow-growing bacteria 44 , we prepared a low-carbon culture medium and utilized a lower incubation temperature (19 °C) with a prolonged incubation period (8 weeks). Our conditions favoured the cultivation of taxonomically diverse Alphaproteobacteria strains, especially of the genus Ruegeria , prompting us to (1) investigate the functional features of ten distinct genera spanning three Alphaproteobacteria orders ( Rhodobacterales , Sphingomonadales and Rhizobiales ) in detail, and (2) define the core functional attributes of Alphaproteobacteria species cultivated from the model sponge host Spongia officinalis . Cultivation-independent methods were employed to infer the relative abundance of the studied lineages in the S . officinalis microbiome, enabling us to critically contextualize the implications of genomic blueprints of symbiosis, identified across all these lineages, as possible factors enhancing host fitness.",
"discussion": "Discussion To date most bacteria isolated from sponges have been affiliated with the phyla Actinobacteria , Bacteroidetes , Firmicutes , and Proteobacteria 29 , 35 , 60 , 61 . Our cultivation method (see File S1 for a detailed discussion) promoted the controlled growth of diverse Alphaproteobacteria species from S . officinalis , in line with the observations of Sipkema et al . (2011) who retrieved a majority of Alphaproteobacteria strains from Haliclona sp. with diverse oligotrophic media. Our bacterial isolation procedure placed sharp focus on distinct colony morphologies, enabling us to cultivate 14 bacterial genera within 48 cultures and to foster unprecedented, deep genome mining of putatively novel genera (Alg231-04 and Alg231-35, File S1 ), of the first-described genome sequence in the Anderseniella genus - found to possess many adaptive signatures for a symbiotic life-style (File S1 ), and of several other understudied lineages within the Alphaproteobacteria class. Indeed, only few genome assemblies are currently available on public databases for Labrenzia (22 assemblies), Sphingorhabdus (9), Loktanella (17), Tateyamaria (3), Pseudovibrio (24), Ruegeria (33), and Erythrobacter (89) species (our own assemblies included), in comparison with the number of genome assemblies available for intensively studied marine bacteria such as Vibrio spp. (2,798 genome assemblies). Moreover, although the abovementioned genera have already been cultivated before, most of the strains sequenced in this study (7 in 10) share less than 99 or 98% 16S rRNA gene similarity with the type strain of their closest, described species (Table S1 ). Finally, the genome-centred strategy employed in this study can be useful in solidifying the phylogeny of unresolved groups, which is likely the case of Pseudovibrio and Labrenzia strains and their placement within the Alphaproteobacteria 58 , 59 . It can also aid in the proposal of novel taxa (the case of strains Alg231-04 and Alg231-30) as supported, for instance, by genome-wide ANI/AAI estimates 62 , 63 . It is possible that technical limitations such as insufficient metagenome sequencing depth and/or the usage of only short read lengths which may not align properly with reference genomes 64 , 65 contribute to an underestimation of relative abundances calculated with the metagenome-genome mapping approach used in this study. Nevertheless, for all strains the percentages of aligned metagenome-genome reads were highest in seawater, followed by sediments and only then by sponge microbial metagenomes, a pattern corroborated by genus-level assessment of relative abundances using MG-RAST functional annotation (see File S1 ). Moreover, we verified that metagenome-genome mapping estimates delivered higher relative abundances for uncultivatable alphaproteobacteria representing dominant sponge symbionts. Altogether, these results indicate that (1) marine sponges are not the primary habitat of the here cultivated Alphaproteobacteria species, (2) bacterial culturing methods tend to sample rare members of the marine sponge microbiome (as suggested by Montalvo, et al . 66 and extensively discussed by Hardoim, et al . 37 , (3) low-abundant sponge symbionts usually captured in culture evolve adaptive features that support a biphasic particle- (“free-living”)/host-associated life-style. Indeed, numerous “symbiosis factors” have been identified in the genomes of Pseudovibrio and Ruegeria spp., prompting extensive discussion on their potential roles in promoting host fitness 30 , 31 , 67 . The debate has, however, often disregarded the in-situ densities of the studied organisms, raising concerns about the net effect of the presumed adaptive features on holobiont functioning 68 . Evidence exists for the presence of these symbiosis factors in the genomes of both free-living and host-associated representatives of cultivatable sponge symbionts 68 , reinforcing the biphasic mode of living hypothesis, and such factors have been proposed to underlie the evolution of canonical commensal bacteria such as Escherichia coli 69 . Here, we delineate the core functional traits of sponge-associated alphaproteobacterial cultures and address their relevance as genomic hallmarks of symbiosis and bimodal life strategies. In nutritional terms, the presence of arylsulfatase-encoding genes in all genomes, an attribute enriched in the marine sponge microbiome 23 and revealed to be common among several uncultivated lineages of sponge symbionts 25 , 26 , underlies one possible role of this pool of Alphaproteobacteria species in consuming sulphated polysaccharides. The potential ability to break-down taurine, identified earlier as one adaptive feature of a currently uncultivatable and sponge-enriched Rhodospirillales clade (“SERC” 26 ), was identified, in this study, among many low-abundance and cultivatable Alphaproteobacteria spp. In addition, several genes encoding for vitamin B biosynthesis were shared among our strains. Biotin (B7), thiamine (B1) and cobalamin (B12) biosynthesis capacities are common, for instance, among members of the Roseobacter clade 70 and evidence exists for the participation of symbiotic Alphaproteobacteria in nourishing vitamins B1 and B12 required by a marine dinoflagellate ( Lingulodinium polyedrum ) for growth 71 . In line with this view, Alpphaproteobacteria spp. could likewise play an important role in providing essential nutrients for sponge growth and functioning. Each of the studied isolates possessed hundreds of genes conferring resistance to antibiotics and toxic compounds. Particularly intriguing in this regard was the ubiquitous presence of genes encoding arsenate reductase that mediates the reduction of arsenate (As(V)) to arsenite (As(III)) in arsenic detoxification processes 72 , 73 . This feature has been recently assigned for the sponge symbiont Entotheonella sp. which mineralizes arsenic and barium in intracellular vesicles 74 . Furthermore, genes involved in ABC-type multidrug efflux systems, hydrolases of the metallo-beta-lactamase superfamily, and remediation of ROS stress (e.g. gluthathione metabolism genes) underline how versatile the mechanisms of cell detoxification employed by these organisms can be 75 , 76 . Such capabilities may substantially increase bacterial fitness within dense and chemically-rich microbial communities, and have been generally reported as distinguishing features of the marine sponge microbiome in cultivation-independent studies 12 , 23 , 26 , 54 . All of the analysed alphaproteobacterial genomes have ELPs which are known sponge symbiosis factors because of the role they play in the modulation of cellular protein-protein interactions and in the prevention of symbiont phagocytosis by host cells 77 , 78 . Functional genome Groups II and III had altogether higher proportions of ELPs than Group I, suggesting higher affinity of members of the former groups in establishing favourable or more stable interactions with marine sponges. This seems to be particularly true for the Anderseniella and Labrenzia strains, which possessed the higher ELP counts among the surveyed genomes and showed the highest relative abundance values, respectively, in the S . officinalis microbiome. It remains to be determined whether ELPs could likewise be involved in bacterial adaptation to other marine hosts, supporting an emerging, generalist pattern of occurrence of cultivatable Alphaproteobacteria across multiple sessile invertebrates such as ascidians 79 , corals 80 , and bryozoans 81 . Particularly intriguing was also the presence of genes required for the tight-adherence (Tad) pilus secretion machinery in all strains. The Tad locus underlies the assembly of Flp (fimbrial low-molecular-weight protein) pili fundamental for cell aggregation, biofilm formation, surface attachment, host colonization and pathogenesis 82 – 84 . Along with protein domains known to mediate biofilm formation (e.g. EAL and CGDEF domains involved c-di-GMP metabolism) and a multitude of other cell motility and chemotaxis factors, the Tad locus equips their host cells not only with host-colonization aptitude but also environmental hardiness. All these genomic features were de-selected in the S . officinalis endosymbiotic consortium while being more pronounced, for instance, in sediment metagenomes 23 , suggesting that they might be more required for persistence in other microniches. We therefore posit that such traits have been subjected to purifying selection to favour the maintenance of a dual life-style among the studied organisms. Using antiSMASH, we could detect several antibiotic biosynthetic gene clusters across the studied genomes, in line with accumulating in vitro evidence for mild to high antimicrobial activities by sponge-associated Alphaproteobacteria such as Ruegeria , Pseudovibrio , and Labrenzia 28 , 29 , 31 , 85 – 87 . Particularly, both terpene-synthase and polyketide-synthase (PKS) biosynthetic gene clusters were common among the studied strains, each being present in eight out of ten genomes, while COG annotations predicted PKS-encoding genes for all genomes. The roles and activities of polyketides from sponge symbiotic bacteria have been largely explored in the last fifteen years 6 , 13 – 15 , however much less is known about the potential contribution of bacterial symbionts as producers of terpenoids in marine sponges 23 . Intriguingly, terpenoid biosynthesis has been regularly documented in keratose marine sponges 61 , 88 , 89 , including Spongia officinalis 90 . Yet the origin of the biosynthesis (host or symbionts) has, to our knowledge, not been specifically addressed by regular chemical screening studies. Sponge-derived diterpenoids have shown antimicrobial activity against pathogenic bacteria such as Pseudomonas aeruginosa 88 . Dihydrogracilin A, a terpene extracted from Dendrilla membranosa , has been shown to possess immune modulatory and anti-inflammatory action 89 . In addition, except for Anderseniella , all other Alphaproteobacteria strains possessed the potential to produce bacteriocins commonly regarded to inhibit growth of closely related strains and, as such, considered to be major molecules shaping the structure of microbial communities in situ 91 . Our results reveal that polyketide, terpene and bacteriocin biosynthesis capacities, recently documented in several Pseudovibrio genomes 31 , 32 , are widespread across diverse sponge-associated Alphaproteobacteria , suggesting a pivotal contribution of this clade to the chemical complexity, natural product biosynthesis repertoire and taxonomic composition of the marine sponge microbiome. In conclusion, the use of simple modifications to regular culture conditions coupled to dedicated genome-wide analysis of marine sponge symbionts enabled unprecedented access to highly versatile metabolisms across diverse understudied Alphaproteobacteria . To improve our capacity to domesticate the so-far uncultivatable portion of the marine sponge microbiome, the design of future culture media should consider our improved understanding of the nutritional requirements of these symbionts acquired via recent metagenomic binning studies 25 , 26 , which allow strain-level, deep insights into the physiology of uncultivated bacteria. Here, we disclose manifold genomic blueprints of the marine sponge microbiome 12 , 23 , 54 across the genomes of several low-abundance, cultivatable symbionts of Spongia officinalis , providing support for the convergent evolution of symbiosis traits above the genus level within a class known for its widespread occurrence in association with sponge hosts, encompassing hundreds of cultivatable and so far uncultivable sponge-associated lineages 9 , 22 , 26 . Certainly, the genomic attributes revealed here are to be found among closely-related, cultivatable Alphaproteobacteria - as emphasized above for Pseudovibrio and Ruegeria strains - retrieved not only from sponges but also from other particle- and host-associated microniches, suggesting that such traits are widespread across diverse lineages of generalist marine bacteria. Taken together, the outcomes compiled here contribute to novel insights into the potential roles of alphaproteobacterial communities in mediating molecular interactions and shaping the structure of the marine sponge microbiome. They further open new opportunities for study regarding the roles of low-abundace microorganisms as consistent reservoirs of functional redundancy within nature’s microbiomes, likely promoting the resilience of host-associated microbial assemblages in the marine realm."
} | 4,867 |
24031728 | PMC3768758 | pmc | 8,644 | {
"abstract": "Fourteen strains of Grifola frondosa (Dicks.) S. F. Gray, originating from different regions (Asia, Europe and North America) were tested for lignin degradation, ligninolytic enzyme activities, protein accumulation and exopolysaccharide production during 55 days of cultivation on oak sawdust. Lignin degradation varied from 2.6 to7.1 % of dry weight of the oak sawdust substrate among tested strains. The loss of dry matter in all screened fungi varied between 11.7 and 33.0%, and the amount of crude protein in the dry substrate varied between 0.94 to 2.55%. The strain, MBFBL 596, had the highest laccase activity (703.3 U/l), and the maximum peroxidase activity of 22.6 U/l was shown by the strain MBFBL 684. Several tested strains (MBFBL 21, 638 and 662) appeared to be good producers of exopolysaccharides (3.5, 3.5 and 3.2 mg/ml respectively).",
"introduction": "INTRODUCTION Grifola frondosa is a white-rot basidiomycete thatproduces a highly nutritious fruit body used as food in differentparts of the world. It has also been reported to contain bioactivemetabolites, which exhibit various medicinal properties such asantitumor, antiviral, antioxidant, antidiabetic, immunomodulation ( 11 , 14 , 23 , 12 , 13 ). Different plant waste material has been used for the cultivation of G. frondosa ( 15 ). In commercial cultivation, sterilized hardwood sawdust of alder and poplar is often used ( 16 ). Chung ( 7 ) used sawdust and cotton seed composts, while Xing et al. ( 22 ) reported cultivation of this fungus on a substrate consisting of beech sawdust, wheat bran and corn meal. G. frondosa secretes ligninolytic enzymes to degrade the lignocellulose substrate from which it obtains needed nutrients for its growth and development. Extracellular laccase activity was detectable in liquid cultures of G. frondosa during the early/middle stages of primary growth ( 22 ). Total peroxidase and manganese independent peroxidase were found in brewery waste substrates used in solid-state fermentation involving G. frondosa ( 18 ). Polysaccharides are secreted during G. frondosa cultivation in both liquid and solid substrates used for its cultivation ( 3 , 24 ). G. frondosa is of huge economic importance as a result of its nutritional and medicinal properties. Favorable conditions for growing it exist in the southeastern United States, where oak sawdust is abundant. However, poor yields persist, despite huge supplementation. This situation calls for basic research into substrate degradation and utilization, as well as into how the strains originating from different regions may affect enzyme production and substrate utilization. Therefore, a total of 14 isolates of G. frondosa, originating from North America, Europe and Asia were studied for ligninolytic enzymatic activities, lignin degradation rates, and exopolysaccharide production during cultivation on un-supplemented oak sawdust.",
"discussion": "RESULTS AND DISCUSSION Results of the substrate utilization as measured by dry matter (biomass) loss revealed that most of the G. frondosa strains have weak abilities to utilize oak sawdust ( Fig. 1 ). For most of the strains, dry matter decreased steadily between days 25 through day 55 of cultivation. However, the Asian strains (MBFBL 660, 684 and 662) and European strains (MBFBL 637, 638 and 649) showed a significant decrease in substrate weight much earlier (at day 15) during the cultivation period. MBFBL 662, 21 and 34 were found to be the best performing strains in terms of substrate utilization, and were associated with 1.3 – 1.5 times dry matter loss, compared to the control ( Table 1 ). The Asian strains (MBFBL 660 and 684) and Northeast USA strains (MBFBL 598 and 605) produced the lowest rate of substrate utilization. Statistical analysis of dry matter loss results revealed significant differences ( P < 0.05) among strains tested (not shown in Fig.1). Our findings are consistent with the report of Chen et al. ( 6 ), who observed differences among G. frondosa strains in biological and physiological characteristics, with different strains showing different mycelia colonization rates on agar plates and in solid-state fermentation in flasks. Figure 1 Changes in substrate dry matter during cultivation of G. frondosa strains The lignin content in the uninoculated substrate was 16.4%. At the end of 55 days, the lignin loss ranged from 2.6 to 7.1% ( Table 1 ). The lignin degradation during the first 25 days was low compared to the values obtained after 35 days of cultivation ( Fig. 2 ). The Asian strain, MBFBL 662, showed the highest lignin degradation rate (7.1%), followed by the North East USA strains (MBFBL 598, 605). Arora and Sandhu ( 2 ) reported an angiospermic wood sawdust total weight loss of 6% accompanied by a 14% lignin loss during 60 days of incubation with Pleurotus ostreatus. Figure 2 Changes in residual lignin level in dry matter during cultivation of G. frondosa strains Total protein content varied between strains and length of cultivation ( Table 2 ). After 55 days, crude protein content in the substrates increased from 1.48% in the uninoculated substrate to values ranging from 1.85 to 2.55%. Asian strains showed the highest protein accumulation, ranging from 1.98 – 2.55%. Northwest USA strains also showed comparatively high protein content (2.21 – 2.37%). Tabata et al . ( 19 ) reported a 1.66% substrate protein accumulation during fructification of G. frondosa in rice bran supplemented sawdust. Table 2 Crude protein accumulation in dry matter during cultivation of G. frondosa species Strains 10 days 25 days 35 days 45 days 55 days MBFBL 660 1.58 2.08 1.97 1.97 2.11 MBFBL 684 1.27 0.94 1.12 1.50 1.98 MBFBL 662 1.72 1.89 2.30 2.42 2.55 MBFBL 637 1.66 1.70 1.75 1.81 1.94 MBFBL 638 1.63 2.03 1.96 1.94 2.00 MBFBL 649 1.96 1.98 2.19 2.18 2.48 MBFBL 596 1.35 1.37 1.75 2.01 2.21 MBFBL 621 1.70 1.77 2.15 2.06 2.25 MBFBL 611 1.74 1.80 2.21 2.03 2.37 MBFBL 598 1.51 1.92 1.69 1.99 1.91 MBFBL 605 1.49 1.61 2.05 2.09 1.85 MBFBL 21 1.72 1.69 2.17 1.97 2.08 MBFBL 26 1.35 1.74 2.11 2.21 1.92 MBFBL 34 1.82 2.25 2.09 2.20 2.40 Results are presented as % of substrate dry weight Uninoculated substrate consists of 1.48 % crude protein The data on ligninolytic enzyme activities ( Table 3 and 4 ) shows that all strains produced the highest amount of laccase enzyme on day 15. The strong correlation (0.817), between substrate utilization and laccase activity was obtained only on strain MBFBL 21. The highest laccase activity (703.3 U/l) was recorded for the G. frondosa strain MBFBL 596, and the lowest activities were in MBFBL 598 and 605 strains, 13.2 and 10.7 U/l, respectively. Xing et al. ( 22 ) showed that during liquid cultivation of G. frondosa, laccase activity reached a maximum value of 70 U/l after 52 days. Vikineswary et al. ( 21 ) observed degradation of rubberwood sawdust by Pycnoporus sanguineus, where maximal laccase productivity reached 5.7 U/g on day 11. Table 3 Laccase activities among G. frondosa strains during 55 days of cultivation Strains 15 days 25 days 35 days 45 days 55 days MBFBL 660 50.06 ±1.98d 29.81 ±1.27cde 5.92 ±0.46abc 5.46 ±0.79bcd 3.64 ±0.91cd MBFBL 684 38.46 ±1.78c 34.36 ±2.17de 36.41 ±2.41d 15.02 ±1.58e 10.92 ±0.79e MBFBL 662 44.60 ±1.38cd 54.15 ±2.41f 6.37 ±0.46abc 17.29 ±0.91e 5.01 ±0.45d MBFBL 637 6.37 ±0.60a 30.94 ±1.20cde 3.19 ±0.91ab 2.28 ±0.91 ab 1.82 ±0.91abc MBFBL 638 14.56 ±0.99ab 19.79 ±1.04abc 6.37 ±0.46abc 8.19 ±0.79d 3.64 ±0.46cd MBFBL 649 10.47 ±0.60ab 38.23 ±1.81e 6.83 ±0.79abc 3.64 ±0.46abc 2.73 ±0.79bc MBFBL 596 703.30 ±12.43e 688.52 ±12.32g 145.17 ±4.04e 52.33 ±3.28f 0.91 ±0.46ab MBFBL 621 11.60 ±0.79ab 31.85 ±2.77de 4.55 ±1.98abc 4.55 ±0.45 abd 2.73 ±0.79bc MBFBL 611 21.39 ±1.21b 8.19 ±0.39a 2.28 ±0.45a 1.82 ±0.45ab 0 MBFBL 598 11.38 ±0.82ab 13.20 ±1.21ab 8.42 ±0.60c 6.83 ±1.37cd 0.46 ±0.46a MBFBL 605 5.01 ±0.23a 10.69 ±0.60a 1.82 ±0.45a 0.91 ±0.46a 0 MBFBL 21 16.38 ±0.79ab 10.24 ±0.79a 9.10 ±0.46c 7.28 ±0.45cd 2.73 ±0.79bc MBFBL 26 12.52 ±1.38ab 23.89 ±1.18bcd 7.74 ±1.98bc 2.28 ±0.45ab 0 MBFBL 34 18.20 ±0.60b 9.56 ±0.79a 3.19 ±0.46ab 7.74 ±0.45d 0 Results of laccase activities are presented in U/l Each value is expressed as mean ± SD (n = 3) Different letters in each column indicate significant differences at P < 0.05 Table 4 Peroxidase activities in G. frondosa strains during 55 days of cultivation Strains 15 days 25 days 35 days 45 days 55 days MBFBL 660 5.45±0.55d 6.67±0.28g 3.03±0.21a 4.00±0.73a 6.55±0.36bc MBFBL 684 7.00±0.72e 8.67±0.76h 22.55±0.55h 9.82±0.63e 8.36±0.36de MBFBL 662 5.39±0.56d 6.61±0.46g 7.45±0.18f 15.18±0.91e 10.06±0.42f MBFBL 637 3.76±0.37bc 5.58±0.38de 5.09±0.48cde 8.97±0.56de 7.52±0.76cd MBFBL 638 3.27±0.33b 4.18±0.18ab 3.33±0.10a 4.97±0.21 a 6.79±1.87bc MBFBL 649 4.21±0.41c 5.21±0.lOfg 5.88±0.28de 7.03±0.56e 13.33±0.56g MBFBL 596 4.15±0.19c 6.36±0.18cde 3.88±0.46ab 9.70±0.56bc 5.70±0.56b MBFBL 621 2.36±0.09a 4.79±0.28bc 5.45±0.48cde 6.79±0.56b 7.15±0.56cd MBFBL 611 3.64±0.24bc 5.27±0.09i 3.58±0.46a 7.39±1.llbc 4.36±0.63a MBFBL 598 2.39±0.14a 3.76±0.38a 6.00±0.48e 4.12±0.56a 6.42±0.56bc MBFBL 605 2.61±0.19a 6.24±0.28 fg 5.03±0.28cd 7.39±0.21bc 9.33±0.42ef MBFBL 21 4.85±0.46d 4.97±0.21cd 5.82±0.58g 10.06±0.76e 7.03±0.92bcd MBFBL 26 3.36±0.18b 5.88±0.46ef 4.67±0.21bc 6.42±0.56b 9.45±0.73ef MBFBL 34 2.33±0.10a 5.45±0.48cde 5.45±0.48cde 8.00±0.96cd 5.70±0.42b Results of peroxidase activities are presented in U/1 Each value is expressed as mean ± SD (n = 3) Different letters in each column indicate significant differences at P < 0.05 Among the strains, MBFBL 684 appeared to be the best producer of peroxidase (22.6 U/l) at 35 days. Mn-Peroxisade activity, though measured, showed insignificant activity in strains tested (data not shown). The correlation between substrate utilization and peroxidase activities was poor, and the relationship between substrate utilization and enzyme activities is not linear. Kadimaliev et al . ( 10 ) observed considerably lower laccase activity during 14 days of growing Lentinus tigrinus on pine sawdust (2.3 U/g) compared to birch sawdust (20 U/g), while peroxidase activity measured by o -dianisidine ranged from 0.6 and 0.65 U/g on birch and pine sawdust, respectively. G. frondosa MBFBL 21 and 662 produced the highest yields exopolysaccharides (3.5 and 3.2 mg/ml) on day 45 of cultivation ( Table 5 ). A positive correlation between dry matter loss and polysaccharide secretion was obtained only in MBFBL 26. Zhou et al . ( 24 ) showed a 3.81 mg/ml exopolysaccharide accumulation by G. frondosa mycelium in a sucrose-brain medium. Bae et al . ( 3 ), obtained 7.2 mg/ml exopolysaccharide on day 4 during cultivation of G. frondosa in a fermenter. Table 5 Polysaccharides secretion in G. frondosa strains during cultivation on oak wood sawdust Strains 15 days 25 days 35 days 45 days 55 days MBFBL 660 1.00±0.10c 1.23±0.06e 0.87±0.06bcd 1.77±0.15ab 1.13±0.06ab MBFBL 684 0.93±0.06c 1.27±0.06e 1.00±0.10cde 0.87±0.06ab 1.70±0.10e MBFBL 662 1.30±0.00de 1.10±0.10de 1.47±0.06g 3.23±0.15 3.00±0.60f MBFBL 637 1.23±0.06de 0.53±0.23a 0.93±0.06bcd 1.77±0.15ab 0.83±0.06a MBFBL 638 0.97±0.06c 0.80±0. lObc 1.23±0.15f 2.17±0.06ab 1.47±0.12bcde MBFBL 649 1.50±0.10e 0.93±0.15bcd 0.40±0.10a 2.37±0.06ab 1.20±0.26bc MBFBL 596 0.23±0.06a 2.83±0.12f 1.43±0.15g 2.67±0.15ab 1.67±0.06de MBFBL 621 2.23±0.64f 0.53±0.15a 1.17±0.06ef 1.67±0.15ab 1.30±0.10bcd MBFBL 611 0.70±0. lObc 2.83±0.15f 1.73±0.15h 2.90±0. lOab 1.33±0.06bcde MBFBL 598 2.67±0.12g 0.83±0.06bc 1.03±0.06de 1.53±0.06ab 1.33±0.06bcde MBFBL 605 1.07±0.06cd 1.00±0.10cd 0.83±0.12bc 2.93±0.15b 1.50±0.10bcde MBFBL 21 0.43±0.06ab 3.10±0.10g 1.90±0.1 Oh 3.50±3.90b 1.57±0.15cde MBFBL 26 0.97±0.06c 0.77±0.06b 1.13±0.12ef 1.97±0.21 ab 1.43±0.06bcde MBFBL 34 1.53±0.25e 0.90±0. lObcd 0.80±0.10b 1.50±0.10ab 1.67±0.15de Results of polisaccharides presented in mg/ml Each value is expressed as mean ± SD (n = 3) Different letters in each column indicate significant differences at P < 0.05 It appears that G. frondosa is not as hardy a lignin degrader as Pleurotus spp, Lentinula edodes, Phanerochaete chrysosporium and Ganoderma colossum , which have been reported to have lignin degradation of 14% on angiospermic wood sawdust ( 2 ), 39–60% on Eucalyptus sawdust ( 5 ), 12% on red oak and 16.7% on white fir ( 15 ), respectively. It fruits off living roots of trees as a weak parasite, which does not kill its host quickly ( http://botit.botany.wisc.edu/toms_fungi/nov2006.html ). From a particular tree in Greensboro NC where G. frondosa has been collected continuously for 5 years, each time the fruit body is picked up, latex was seen oozing from the point of collection of the G. frondosa fruit body from the oak tree root. It is possible that the photosynthetic system of their host (live oak trees) is exploited, in addition to minimal substrate degradation, to acquire the nutrients that the fungus needs to make fruit bodies in nature; that might explain the relative difficulty in cultivation of this fungus for fruit body production. The results showed that ligninolytic enzymes production, sawdust substrate degradation and exopolysaccharide production appears to be strain specific and not affected by the origin of strains tested. In general, G. frondosa seems to be a weak degrader of sawdust, although it is found associated with oak trees in the wild. Our results have helped us to detect strains that seem to be the best oak substrate degraders, which we are now applying in mass production studies, polysaccharide secretion and breeding to obtain improved strains needed for other biotechnological applications."
} | 3,414 |
24348470 | PMC3845345 | pmc | 8,645 | {
"abstract": "Geological CO 2 sequestration in unmineable subsurface oil/gas fields and coal formations has been proposed as a means of reducing anthropogenic greenhouse gasses in the atmosphere. However, the feasibility of injecting CO 2 into subsurface depends upon a variety of geological and economic conditions, and the ecological consequences are largely unpredictable. In this study, we developed a new flow-through-type reactor system to examine potential geophysical, geochemical and microbiological impacts associated with CO 2 injection by simulating in-situ pressure (0–100 MPa) and temperature (0–70°C) conditions. Using the reactor system, anaerobic artificial fluid and CO 2 (flow rate: 0.002 and 0.00001 ml/min, respectively) were continuously supplemented into a column comprised of bituminous coal and sand under a pore pressure of 40 MPa (confined pressure: 41 MPa) at 40°C for 56 days. 16S rRNA gene analysis of the bacterial components showed distinct spatial separation of the predominant taxa in the coal and sand over the course of the experiment. Cultivation experiments using sub-sampled fluids revealed that some microbes survived, or were metabolically active, under CO 2-rich conditions. However, no methanogens were activated during the experiment, even though hydrogenotrophic and methylotrophic methanogens were obtained from conventional batch-type cultivation at 20°C. During the reactor experiment, the acetate and methanol concentration in the fluids increased while the δ 13 C acetate , H 2 and CO 2 concentrations decreased, indicating the occurrence of homo-acetogenesis. 16S rRNA genes of homo-acetogenic spore-forming bacteria related to the genus Sporomusa were consistently detected from the sandstone after the reactor experiment. Our results suggest that the injection of CO 2 into a natural coal-sand formation preferentially stimulates homo-acetogenesis rather than methanogenesis, and that this process is accompanied by biogenic CO 2 conversion to acetate.",
"introduction": "Introduction In addition to causing dramatic increases in the surface temperature of the Earth, the release of anthropogenic greenhouse gasses into the atmosphere is considered to have had a major effect on changes to the oceans and climate (e.g., sea-level changes, ocean acidification) (Crowley, 2000 ; Alley et al., 2003 ; Karl and Trenberth, 2003 ). A variety of potential methods have been proposed to reduce anthropogenic CO 2 emissions. Of the methods proposed to date, CO 2 capture and storage followed by geologic CO 2 sequestration (GCS) have been proposed as effective means to prevent the potentially dangerous consequences of climate change (White et al., 2003 ; Anderson and Newell, 2004 ; IPCC Special Reports, 2005; Davison, 2007 ; Zakkour and Haines, 2007 ; Benson and Cole, 2008 ; Kirk, 2011 ). As a result, a number of potential CO 2 storage sites have been identified in a wide variety of geological settings, and feasibility assessments have been undertaken by some corporations and national governments (e.g., European Union, United States and Australia), which leads to draft a variety of legal instruments to accommodate commercial interest in GCS projects (White et al., 2004 ; Zakkour and Haines, 2007 ). One such example is the Utsira sandstone formation, an aquifer 800 m beneath the North Sea, into which 1 Mt of CO 2 has been injected per year by Statoil since 1996 (Eiken et al., 2000 ). Preventing CO 2 leaking from geologic formations is an essential component of minimizing maintenance costs and avoiding the extensive environmental damage that would result from a large-scale leak. Some studies have examined admissible quantities of CO 2 leakage and operational costs, as well as the efficiencies of impermeable cap-rock layers and sealing mechanisms (Anderson and Newell, 2004 ; Davison, 2007 ; Van der Zwaan and Gerlagh, 2009 ; Song and Zhang, 2013 ). To prevent CO 2 leakage and reduce costs, the offshore subsurface has been considered as potential realm of GCS. In the deep subseafloor environment, CO 2 can exist as a liquid/supercritical phase or it can dissolve into ambient seawater under conditions of high pressure and low temperature, facilitating migration into subsurface geologic formations. Since CO 2 dissolved in seawater and supercritical CO 2 are less dense than ambient seawater, they are buoyant after sequestration, whereas liquid CO 2 has a higher density than ambient seawater, causing it to sink (House et al., 2006 ; Benson and Cole, 2008 ). However, at water depths of <3000 m and a few hundred meters of sediment, the generation of CO 2 hydrate disturbs the seepage of high-density liquid CO 2 through deep-sea sediments (House et al., 2006 ), suggesting superiority of deep-sea subsurface environment as a GCS site. At present, the major techniques employed for trapping CO 2 are capillary trapping, solubility trapping and mineral trapping (Mitchell et al., 2010 ; Jun et al., 2013 ). All of these mechanisms require empty space inside geological formations for storing gaseous/liquid CO 2 or precipitated carbonates. Sandstones are considered to be well suited for GCS because of their high porosity and their ubiquity (Bachu, 2003 ). Hydrocarbon reservoirs (e.g., unmineable subsurface oil/gas fields and coal beds) have also been considered for use as potential geologic CO 2 repositories. Recovery of coal-bed CH 4 (CBM) associated with hydrocarbon reservoirs can be facilitated by CO 2 injection, which potentially contributes to decreasing the energy costs associated with such a venture (Gunter et al., 1997 ; White et al., 2005 ). The geophysical, geochemical and ecological impacts of CO 2 sequestration in the natural subsurface environment have largely remained unknown. The injection of CO 2 can promote the precipitation of carbonates through a reaction between the CO 2 and surrounding rocks (Oelkers et al., 2008 ; Rosenbauer et al., 2012 ), probably decreasing rock porosity/permeability and resulting in secondary cap-rock generation (Kharaka et al., 2006 ), however, compared to capillary trapping or solubility trapping, carbonate precipitation occurs too slowly for it to be considered as an effective means of capturing CO 2 (Gilfillan et al., 2009 ). On the other hand, the changes in the chemical environment associated with GCS by CO 2 injection can have a marked impact on the microbial consortia within sediments. Supercritical CO 2 or water containing large amounts of dissolved CO 2 kills microorganisms by disrupting their cell membranes (Bertoloni et al., 2006 ; Wu et al., 2010 ), whereas the maximum limits of microbial CO 2 tolerance are still poorly understood. Numerous studies have confirmed that methanogens are capable of growth in aqueous media containing dissolved CO 2 concentrations that are considerably higher than those of natural conditions (Yakimov et al., 2002 ; Videmsek et al., 2009 ; Oppermann et al., 2010 ). The microbiological and geochemical characteristics of the deep-sea CO 2 seep site at the Yonaguni Knoll IV hydrothermal system, which is characterized by CO 2 seepage, suggest that habitat segregation of anaerobic bacteria and methanogens occurs in response to differences in CO 2 concentrations and associated chemical conditions in marine sediments (Inagaki et al., 2006 ; Konno et al., 2006 ; Yanagawa et al., 2013 ). It is also expected that some microbes would be activated by CO 2 injection under subsurface conditions, because CO 2 is an important carbon source for autotrophic and mixotrophic microorganisms Previous studies on the activation of microbes associated with Fe 3+ , SO 2− 4 reduction and methanogenesis by CO 2 injection suggest that microbial CO 2 conversion to available carbon species lead to novel sustainable CO 2 recycling system (Kirk, 2011 ; Mayumi et al., 2013 ). Although laboratory-based GCS experiments could potentially clarify the impacts of CO 2 injection on geologic formations, such studies have not yet been attempted. On the other hand, high-pressure incubators have been developed to culture microbes under in-situ conditions (Zobell and Oppenheimer, 1950 ; Yayanos et al., 1979 ; Orcutt et al., 2008 ; Sauer et al., 2012 ) and to simulate subsurface hydrothermal alteration of basaltic rocks (Seyfried and Janecky, 1985 ). In an attempt to simulate GCS conditions, a high-pressure flow-through reactor system was developed at the Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science and Technology (JAMSTEC) in Kochi, Japan, by referring to previous studies on high-pressure instruments. In this study, we investigated changes in the geophysical features, constituent minerals and microbial community structures in a column comprised of bituminous coal and sand before and after CO 2 injection under simulating in-situ subsurface conditions and discussed various impacts of CO 2 injection to geologic formations. CO 2 was supplemented with anaerobic artificial fluids into a coal-sand column, which were sub-sampled after passing through the column. Concentrations and carbon isotope compositions of dissolved gases and volatile organic carbons in the sub-sampled fluids were determined to monitor CO 2 impact during experiment. Sub-sampled fluids were also incubated to determine microbes survived through a CO 2 injection experiment. Conventional batch-type cultivation was performed using same bituminous coal sample to investigate the potential for biological carbon conversion in sample pre-coal. Fresh bituminous coal and associated sandstone were obtained from a subterranean coal mine and used as an analog of a subsurface coal-sand formation. As mentioned above, coal and sandstone are both considered to be well suited for use as CO 2 repositories. In addition to the recovery of CBM by CO 2 injection, immature hydrocarbon reservoirs (e.g., oil, bituminous coal and lignite) contain a variety of organic molecules and gases (e.g., H 2 and CO 2 ) generated during maturation of carbonaceous compounds in the hydrocarbon reservoirs accompanied by sedimentation. These compounds, in turn, are utilized by a variety of microorganisms in the nutrient-limited subsurface sediments. Consequently, development of microbial communities that consist of various bacteria, fungi and methanogenic archaea has been reported in these hydrocarbon reservoirs (Fakoussa, 1988 , 1990 ; Edwards and Grbic-Galic, 1994 ; Nazina et al., 1995 ; Krüger et al., 2008 ; Strapoć et al., 2008 ), suggesting the possibility of biological CO 2 conversion system (Bio-CCS) that responds to the GCS.",
"discussion": "Discussion Migration and pressure tolerance of bacteria Cloning analysis of samples pre-coal, pre-sand, post-coal and post-sand revealed that bacteria migrated from coal to surrounding sand. Furthermore, some of these bacteria were moved to the sub-sampling cell by CO 2 -containing fluid due to the high porosity of the sand. The mechanisms of microbial migration have been investigated in a variety of natural environments, either for purposes of bioremediation or to prevent microbial contamination (e.g., drinking water pumped from an aquifer) (Ferguson et al., 2003 ; Smith and Perdek, 2004 ). Numerous laboratory experiments and field observations of microbial migration have been examined using various mathematical models (Harvey et al., 1995 ; Johnson et al., 2001 ; Harvey and Harms, 2002 ; Tufenkji, 2007 ). However, the change of microbial communities associated with migration is still poorly understood. Cloning analyses of sub-sampled fluids and the cultivated samples showed that, with the exception of a few genera, the microbial communities differed markedly from those in the coal-sand column. These results can be explained by two sorting events of bacterial communities as follows: microbial effluence to CO 2 -injected fluid from the coal-sand column and decompression after sample recovery. The coal-sand column would retain some bacteria and prevent them from being transported in the flow of CO 2 -injected fluids. Cultivation experiments using sub-sampled fluids revealed that Bacillus, Cupriavidus , and Microbacterium were capable of surviving pressurization/decompression, suggesting that certain terrestrial bacteria in coal-sand formations have the ability to resist marked changes in pressure. Furthermore, it is conceivable that spore formation might be a possible function to survive drastic environmental changes associated with the GCS. Although anaerobic conditions were maintained in all lines and vessels, aerobic Lysinibacillus bacteria were dominant in the microbial community of coal-sand column. It is possible that Lysinibacillus was able to survive the anaerobic conditions in coal-sand column by producing spores. Indeed, Lysinibacillus was not observed in any of the fluid samples collected, suggesting that Lysinibacillus does not produce spores under ambient pressure conditions or they could not migrate with fluid flow and remained in the column. Microbial migration associated with fluid flow is expected to occur in natural subsurface formations if the formation has enough pore-throat connectivity. In addition, it is considered that artificial CO 2 injected will positively enhance microbial migration. Thus, considering that the microbial communities in coal and sand became similar after experiment, it is possible that bacterial communities may be very homogeneous in porous subsurface coal-sand formations used as geological CO 2 repositories. Origin of dissolved CH 4 The relationship between δ 13 C CH4 and δ 13 C DIC is good indicator of the biogenic production of CH 4 during experiment (e.g., Whiticar, 1999 ). When the CH 4 is produced by hydrogenotrophic methanogenesis or acetoclastic methanogenesis during experiment, the δ 13 C CH4 value should be affected by the δ 13 C values of precursors of CH 4 such as δ 13 C DIC and δ 13 C acetate . However, the δ 13 C CH4 remained constant through the experiment and was similar to δ 13 C CBM , even though the δ 13 C DIC and δ 13 C acetate were depleted during the experiment. Those carbon isotopic compositions suggest that the CH 4 dissolved in fluids not originated from biogenic but from CBM in pre-coal. Biogenic CH 4 was not detected likely because of the slow metabolic rate or inactivation of methanogens in the experimental condition. It is most likely that CH 4 discharge was enhanced by the CO 2 -injected fluid that was supplemented into the coal-sand column as previously reported (Gunter et al., 1997 ; White et al., 2005 ). Indeed, the results of the PCR and cloning analysis showed that known methanogen was not observed in any examined samples, which is consistent with the lack of any biogenic changes in δ 13 C CH4 values. H 2 , CO 2 , acetate, formate and methanol concentrations and carbon isotope compositions The CO 2 concentration in sub-sampled fluids was consistently lower than the total amount of CO 2 that was added to the system. This decrease in CO 2 can be explained by adsorption on the coal, mineral trapping, or consumption by bacteria. Previous reports have suggested that several gases are adsorbed by immature coal (White et al., 2005 ; Bustin and Clarkson, 1998 ; Pan and Connell, 2007 ). The FE-SEM-EDS results of this study suggested that mineral trapping of CO 2 by carbonate precipitation could have accounted for the slightly lower than expected CO 2 concentrations. On the other hand, the increase of acetate concentration in the sub-sampled fluids was observed during the experiment, which, in conjunction with decreases in H 2 and CO 2 concentrations, implies that homo-acetogenesis occurred during the experiment. The lower δ 13 C value of increased acetate during the experiment than that in normal total organic carbon (−20 to −30‰) also suggests that acetate was produced by homo-acetogenesis. This is because, acetate synthesized via the acetyl-CoA pathway during homo-acetogenesis by acetogen is depleted in 13 C compared with its precursor (House et al., 2003 ). Indeed, the presence of Sporomusa -related 16S rRNA genes, a homo-acetogenic bacterium, detected in the cloning analysis of post-sand also supports this interpretation. δ 13 C CO2 simply reflects the value of supplied CO 2 and bicarbonate in pore water, which ranged from −24.4 to −19.2‰. A decrease of formate concentrations and enrichment of 13 C in δ 13 C formate also likely indicate bacterial consumption of formate in which 12 C would be preferentially consumed. Interestingly, we observed increase of methanol concentration during the CO 2 injection experiment (Figure 9 ; Table 5 ). The carbon isotopic compositions of methanol were relatively constant at around −40‰ although the δ 13 C values were notably lower than those of DIC. Given the available data set, it is still difficult to identify the source and/or production mechanisms of methanol at this point; however, we infer that continuous injection of the CO 2 and fluid might abiotically release absorbed methanol from the coal-formation sample. It might also be conceivable that microbial activity stimulated by the CO 2 and fluid supply might contribute to methanol production (e.g., as a secondary product via degradation of organic matter). Homo-acetogenesis vs. methanogenesis Batch-type cultivation of methanogens at ambient temperature and pressure followed by cloning analysis revealed that Methanobacterium and Methanosarcina were indigenous to the Kushiro bituminous coal examined in this study. This finding is consistent with previous reports describing the presence of methanogens in coal (Krüger et al., 2008 ; Strapoć et al., 2008 ). However, no methanogens were activated during CO 2 injection experiment under in-situ subsurface conditions. To constrain the habitability of bacteria and methanogens, we calculated ΔG of acetoclastic methanogenesis, hydrogenotrophic methanogenesis and homo-acetogenesis using in-situ geochemical data obtained in the present study (Tables 5 , 6 ; Figure 10 ). We could not calculate ΔG of acetoclastic methanogenesis on day 34, hydrogenotrophic methanogenesis on day 56, and homo-acetogenesis on days 34 and 56 because the concentrations of acetate in sample 34d and the dissolved H 2 in sample 56d were below detection limit. On days 14 and 34, the ΔG of hydrogenotrophic methanogenesis was the lowest of all reactions, indicating that hydrogenotrophic methanogenesis is most favorable under the conditions examined (Figure 10 ). It is thus enigmatic that hydrogenotrophic methanogenesis did not occur, even though methanogens are indigenous to the examined coal and sufficient H 2 and CO 2 were supplemented into the coal-sand column during experiment. Slow metabolic rates of methanogens might be responsible for the result. Under conditions of supplemented H 2 and CO 2 , homo-acetogens and hydrogenotrophic methanogens would compete against each other because both utilize H 2 and CO 2 . Sporomusa , the homo-acetogenic bacterium observed in post-sand, has been reported to be capable of demethylating aromatic compounds and of breaking the bonds of aromatic rings (Mechichi et al., 1999 ), many of the aromatic organic compounds supplied by pyrolysed/pressurized bituminous coal could be used by Sporomusa as nutrients, resulting in the predominance of homo-acetogenesis in the system. Alternatively, despite the sub-sampling and reactor experiment were carried out under the anaerobic condition, we cannot deny the possibility that small oxygen contamination during the initial sampling might negatively affect methanogenesis and if so, it would be outcompeted by homo-acetogenesis under trace oxygen conditions (Leadbetter and Breznak, 1996 ). Table 6 Gibbs free energies and enthalpies of possible microbial reactions in CO 2 injection experiment . Reaction ΔH◦(KJ) ΔG◦(KJ) ΔG◦ 40, 41 (KJ) * ΔG◦ 40, 41 (KJ) * 14d 34d 56d Acetoclastic methanogenesis CH 3 COO + H 2 O→CH 4 + HCO − 3 7.1 −31.2 −34.4 −61.2 NA −56.6 Hydrogenotrophic methanogenesis HCO − 3 + 4H 2 + H + →CH 4 + 2H 2 O −225.7 −246.2 −250.2 −131.8 −114.8 NA Homo-acetogenesis 2HCO − 3 + 4H 2 + H + →CH 3 COO − + 4H 2 O −232.8 −215.1 −215.8 −65.4 NA NA ΔH◦ and ΔG◦ represent enthalpy and standard Gibbs free energy (25◦C, 1 atm) . * ΔG◦ 40, 41 and * ΔG 40, 41 represents Gibbs free energies at equilibrium and calculated using in-situ geochemical data . Figure 10 Effects of HCO − 3 activity (a HCO − 3 ) on ΔG of acetoclastic methanogenesis, hydrogenotrophic methanogenesis and homo-acetogenesis . All lines in figure were calculated using geochemical data of samples 14d, 34d, and 56d, except Dissolved inorganic carbon (DIC) concentrations. Solid circles indicate ΔG of acetoclastic methanogenesis, hydrogenotrophic methanogenesis and homo-acetogenesis at the DIC concentrations examined in this study. A previous study suggested that an increase in the partial pressure of CO 2 could promote acetoclastic methanogenesis in crude oil reservoirs (Mayumi et al., 2013 ). However, in this study, an increase in DIC concentration and a decrease in the acetate concentration associated with acetoclastic methanogenesis were not observed, indicating that acetoclastic methanogenesis did not occur even though the fluid supplemented into coal-sand column contained sufficient acetate for growth. Higher ΔG values of the acetoclastic methanogenesis on days 14and 56 than those obtained for hydrogenotrophic methanogenesis and homo-acetogenesis is also consistent with our results, which can be explained by differences H 2 utilization in each system. The influence of dissolved H 2 concentration on the ΔG of hydrogenotrophic methanogenesis was also observed in this study (blue lines in 14d and 34d; Figure 10 ). The steep gradient of the curves in Figure 10 suggest that an increase in HCO − 3 activity is more effective for promoting homo-acetogenesis than hydrogenotrophic methanogenesis. In summary, our results suggest that homo-acetogenesis is possible reaction in GCS settings involving unmineable subsurface coal-sand formations. Aromatic organic compounds supplied by bituminous coal can activate homo-acetogenic bacteria. These findings indicate that microbial conversion of CO 2 to acetate under subsurface conditions is feasible. However, to gain our knowledge of the potential response of subsurface microbial ecosystem to CO 2 sequestration, more detailed comparative geochemical and microbiological studies using tracer incubation experiments and high-throughput sequencing will be necessary for in-situ and ex-situ conditions. In addition, to accelerate the biological CO 2 conversion to reduced compounds (i.e., Bio-CCS), supply of electron and/or molecular hydrogen would be essential. In this regard, we need to investigate the place where natural H 2 concentration is remarkably high due to the thermal degradation of organic matter (Head et al., 2003 ) or other H 2 -producing geologic systems (e.g., serpentinization), or to consider the utilization of natural electric resources for electromethanogenesis (Cheng et al., 2009 ; Kuramochi et al., 2013 ). These are our on-going foci. 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."
} | 5,877 |
35423415 | PMC8695389 | pmc | 8,646 | {
"abstract": "With rapid advancement in water filtration materials, several efforts have been made to fabricate electrospun nanofiber membranes (ENMs). ENMs play a crucial role in different areas of water treatment due to their several advantageous properties such as high specific surface area, high interconnected porosity, controllable thickness, mechanical robustness, and wettability. In the broad field of water purification, ENMs have shown tremendous potential in terms of permeability, rejection, energy efficiency, resistance to fouling, reusability and mechanical robustness as compared to the traditional phase inversion membranes. Upon various chemical and physical modifications of ENMs, they have exhibited great potential for emerging applications in environment, energy and health sectors. This review firstly presents an overview of the limiting factors influencing the morphology of electrospun nanofibers. Secondly, it presents recent advancements in electrospinning processes, which helps to not only overcome drawbacks associated with the conventional electrospinning but also to produce nanofibers of different morphology and orientation with an increased rate of production. Thirdly, it presents a brief discussion about the recent progress of the ENMs for removal of various pollutants from aqueous system through major areas of membrane separation. Finally, this review concludes with the challenges and future directions in this vast and fast growing area.",
"conclusion": "Conclusion and outlook Electrospinning is a prevailing technology to produce advanced nanofibrous materials for various applications. The water purification performance of ENMs depends on the properties such as high surface area, high porosity, high surface roughness, and surface chemistry (hydrophobicity/hydrophilicity). These properties are often altered by the influence of both limiting factors of electrospinning and the chosen functional materials. This provides a promising platform to tune the properties such as surface roughness, porosity, and thickness of ENMs. Hence, firstly in this review, we explored the influence of limiting factors on morphology of polymeric nanofibers. Secondly, we discussed about the recent developments in electrospinning, enabling faster production of nanofibers with different morphology, including their alignment in a required direction. Finally, we briefly discussed the recent and last progresses in performance and potential of ENMs to expel pollutants from aqueous system. The ENMs have been successfully applied for different membrane separation areas, such as MF, RO, FO, MD, heavy metal removal, and oil/water separation. Unquestionably, ENMs have established a promising platform for water treatment, where (i) fabricating ENMs by doping a polymer solution with nanomaterials from ceramics, metals/metal oxide and carbon, and (ii) treating the surface of ENMs by functionalization, (iii) developing a top selective layer by interfacial polymerization for TFNC, and (iv) grafting and chemical etching strategies have well extended the potential of ENMs. However, more research progress is needed in the areas of (i) using biodegradable polymers with negative surface charge to enhance fouling resistance of microfiltration ENMs, (ii) developing a superhydrophobic microporous ENMs with high porosity for MD application, (iii) developing a reusable and fouling resistant high specific surface area ENMs with ability to adsorb more than one type of heavy metal ions, and (iv) surface coating of ENMs by various sputtering/coating techniques for tuning their surface wettability for high flux gravity driven oil/water separation application. These are some of the emerging promising strategies which will further extend the potential of ENMs in different areas of water purification. The design, development and implementation of cost-effective and high performance ENMs in membrane technology via innovative electrospinning technologies is anticipated to contribute a lot in addressing the world's fresh water scarcity and pollution problems in the near future.",
"introduction": "Introduction The rapid growth in global population, urbanization, and industrialization has led to several complications such as depletion of natural energy resources, environmental pollution causing climate change, and potential health risks including scarcity of food and potable water. Efficient treatment of wastewater and sustainable utilization of renewable energy sources to harvest clean water and energy could be the most promising approach to tackle these issues. 1 In this regard, the ENMs are fascinating materials due to their tuneable versatile properties making them promising materials for water treatment applications. Several techniques have been proposed to produce nanofibers such as template synthesis, 2 self-assembly, 3 solvothermal synthesis, electro hydrodynamic direct writing, 4 centrifugal jet spinning, 5 plasma induced synthesis, 6 solution blow spinning, 7 CO 2 laser supersonic drawing, 8 and sonochemical synthesis. 9 Electrospinning is an effective and key technique for enabling continuous production of nanofibers for fabrication of porous materials. The use of electrospun nanofibers has been proved to be a promising choice not only for filtration materials but also for various other applications such as, environmental remediation, protection sensors in defence and security, optical application, conducting and insulating nanofibers in electrical applications, scaffolds, films and composites in health care, and packaging applications. 10,11 The production of nanofibers by electrospinning was first patented in the year 1934. 12 However, significant progress has been made by Reneker's group after 1990. This invention greatly influenced researchers to study polymer nanofibers for various applications in the broad areas like energy, healthcare and environment. A typical electrospinning instrument has different parts, a high voltage power supply, a metallic spinneret and an electrically earthed metallic collector as shown in Fig. 1 . When a high voltage direct current is applied, a drop of a polymer solution suspended at the tip of the spinneret will turn into a conical shaped droplet from its semi sphere shape known as the Taylor cone. 13 When high voltage is applied, the electrostatic repulsive force acting on the surface of the droplet of a polymer solution counteracts the surface tension and forms the Taylor cone followed by the formation of liquid jet that is deposited onto the collector placed at a specified distance to form nanofibers. The recent tremendous progress in experimental design and upscaling, and vast opportunity for materials choice (polymers, ceramics, functional molecules, carbon and metal/metal oxides) have enabled the production of a wide variety of morphology of nanofibers, such as hollow fibers, core–sheath nanofibers, nanoribbons and nanofibers with various surface topographies (porous, rough surface). 14 Fig. 1 Schematic diagram showing the different components of a typical electrospinning. Since fresh water sources are gradually depleting and getting contaminated by verities of pollutants, it is necessary to fabricate membrane materials of high rejection efficiency, flux performance and reusability, to treat wastewater in a simple and cost effective way. These kind of complex issues require the use of electrospun fabricated purification materials such as membranes, absorbents, adsorbents, and scaffolds. From the past few years, many efforts have been made across the world to develop suitable energy efficient ENMs to expel various types of pollutants from aqueous system. The membranes obtained from conventional phase inversion techniques have many disadvantages such as low permeance, fouling propensity, uneven pore size and poor mechanical strength. Nanofiber membranes fabricated via electrospinning have been often used as the best substitutes that can outperform conventional membranes due to their high interconnected porosity, tuneable thickness and pore size distribution from few nanometres to several microns, and high surface area for tuning the surface chemistry. 15 Moreover, solvent induced fusion of inter fiber junctions and enhanced crystallinity of nanofibers, impart mechanical robustness to ENMs, and due to high surface area, the surface chemistry of ENMs can be easily and effectively modified. In addition, the recyclable and reusable ENMs of eco-friendly polymers have significantly controlled the negative impact of non-degradable commodity polymer based phase inversion membranes on environment. The present review firstly aims at understanding the limiting factors of electrospinning influencing the properties of nanofibers on the basis of important morphological investigations reported earlier for various different polymeric nanofibers. Secondly, it introduces an overview of the recent advancements in electrospinning technique and its advantages to produce variety of nanofibers. Thirdly, it provides an overview of use of variety of polymeric ENMs and their efficiency of the expulsion of various pollutants from aqueous system via different membrane separation processes, such as microfiltration, desalination ( via membrane distillation (MD), reverse osmosis (RO), and forward osmosis (FO)), heavy metal removal and oil/water separation. Finally, the review provides a summary and outlook for the future in these vast fields."
} | 2,367 |
28955887 | PMC5613248 | pmc | 8,649 | {
"abstract": "Magnetosomes are membrane-enveloped bacterial organelles containing nano-sized magnetic particles, and function as a cellular magnetic sensor, which assist the cells to navigate and swim along the geomagnetic field. Localized with each magnetosome is a suite of proteins involved in the synthesis, maintenance and functionalization of the organelle, however the detailed molecular organization of the proteins in magnetosomes is unresolved. MamA is one of the most abundant magnetosome-associated proteins and is anchored to the magnetosome vesicles through protein-protein interactions, but the identity of the protein that interacts with MamA is undetermined. In this study, we found that MamA binds to a magnetosome membrane protein Mms6. Two different molecular masses of Mms6, 14.5-kDa and 6.0-kDa, were associated with the magnetosomes. Using affinity chromatography, we identified that the 14.5-kDa Mms6 interacts with MamA, and the interaction was further confirmed by pull-down, immunoprecipitation and size-exclusion chromatography assays. Prior to this, Mms6 was assumed to be strictly involved with biomineralizing magnetite; however, these results suggest that Mms6 has an additional responsibility, binding to MamA.",
"introduction": "1 Introduction In 1975, magnetotactic bacteria (MTB) were first discovered by Blakemore [1] , [2] . These bacteria contain unique organelles called magnetosomes that biomineralize magnetic minerals using specific proteins that are only associated with the organelle [3] . In 1996, the first magnetosome associated protein was isolated, sequenced and found to consist of tetratricopeptide repeat (TPR) motifs which are known to mediate protein-protein interactions [4] . Since 1996, this protein has been designated MamA (Mam22), and researchers in the field of biochemistry and genetics have studied it, and recently the structures of four different MamA proteins from four different MTB have been resolved [5] , [6] , [7] . However, even using cutting edge techniques, researchers have merely confirmed the idea that MamA interacts with other magnetosome-associated protein(s), but the function of MamA still remains enigmatic. We have made a major discovery towards identifying the binding partner of MamA, which gives a significant clue to its function. MamA is conserved in all known MTB [8] , and even though it is a soluble cytoplasmic protein [9] , it localizes in the magnetosome matrix, a proteinaceous layer surrounding magnetosome vesicles of Magnetospirillum species [10] , [11] . The entire primary structure of MamA consists of five TPR motifs and one putative TPR motif [9] . These motifs consist of a helix-turn-helix fold, which has been known to promote protein-protein interactions [12] . Proteins with TPR motifs are important to cells which use them in a wide variety of ways such as protein transport, protein folding, transcription and splicing, and cell cycle control [13] . Two different functions for MamA have been proposed. Based on studies of a mamA deletion mutant, MamA appears to activate or prime preformed magnetosomes for biomineralization [14] . A different study used the atomic force microscopy (AFM) to observe chains of magnetosomes with and without MamA and proposed that MamA is anchored to the magnetosome membrane and may stabilize the magnetosome chain [11] . According to the MamA crystal structures, the five TPR motifs form a superhelix structure which has at least three putative protein binding sites, and one of the sites specifically binds to one of the magnetosome-associated proteins [5] , [6] , [7] , However, the question remains as to which magnetosome-associated protein(s) interacts with MamA. In this study, we used MamA affinity chromatography to screen the proteins from the magnetosomes of Magnetospirillum magneticum AMB-1 which bind to MamA. We found that Mms6, a magnetosome membrane-bound protein, binds to MamA. We further confirmed this binding using immuno-precipitation, pull-down and size-exclusion chromatography experiments. In addition to this, we established that two different types of Mms6 exist in the magnetosome membrane, a 14.5-kDa and 6.0-kDa version. Until now, Mms6 was thought to be exclusively involved in biomineralization, however these new results imply an additional function of Mms6 within magnetosomes and provide a clue to answer the question of how MamA binds to magnetosomes in M. magneticum AMB-1.",
"discussion": "3 3. Results and discussion 3.1 Screening MamA binding proteins in magnetosomes Previous studies demonstrated that when MamA was removed from magnetosomes, purified MamA could be added back to the MamA-eliminated magnetosomes and bind to them [10] , [11] ; our objective was to identify which magnetosome-associated proteins were binding to MamA. We began by extracting proteins from MamA-eliminated magnetosomes ( Fig. S1 ), running them through a MamA-affinity chromatography column and loading the eluted fractions onto an SDS-PAGE gel ( Fig. S3 ). Thirteen protein bands were detected and analyzed by tandem mass spectrometry ( Fig. 1 ). Six different MamA bands were detected (bands 7–11 and 13) which represent recombinant MamA and truncated recombinant MamA that detached from the column. Bands 2, 4, and 6 were identified as proteins derived from E. coli . These proteins were contaminated in the purified His-MamA sample, which was used to make the column, and probably detached from the affinity column during the elution process. Five of the bands were identified as proteins from M. magneticum AMB-1; band 1 is a methyl-accepting chemotaxis protein (amb1418) , band 3 is a hypothetical protein (amb3421) , band 5 is a porin (amb0025) , and band 12 is two proteins, the ATP synthase epsilon chain (amb4138) and Mms6 (amb0956). Of these five proteins, Mms6 is the only magnetosome-associated protein. The other four proteins are inner or outer membrane-bound proteins, which are contaminants, derived during the magnetosome purification process [21] . A control was performed using BSA to screen for MamA binding proteins. The SDS-PAGE gel profile showed no proteins specifically bound to the BSA column. The 63.7 and 54.0-kDa protein bands that were found in all lanes in both columns represent contaminated proteins from the electrophoresis procedure ( Fig. S3 ). Our goal was to understand which magnetosome associated protein binds to MamA, therefore we focused on Mms6. However, the other four membrane proteins could also be MamA binding candidates specifically, the cytoplasmic membrane proteins, amb1418, amb3421, and amb4138, which come in direct contact with MamA in the cell. There is also the possibility that other MamA binding magnetosome-associated proteins still remain, because some magnetosome membrane proteins may not have been solubilized by sucrose monocaprate prior to the affinity chromatography step. Fig. 1 SDS-PAGE gel profile of proteins eluted from the His-MamA column and their apparent molecular masses; lane M, protein markers (Precision Plus protein standards; Bio-Rad); lane 1, eluted proteins. The eluted fractions were concentrated approximately 200 times for SDS-PAGE. These 13 bands were analyzed using tandem mass spectrometry and identified. Bands 2, 4, and 6 were proteins belonging to E. coli ; bands 1, 3, 5, and 12 were proteins belonging to M. magneticum AMB-1; and bands 7–11, and 13 were recombinant MamA proteins. Only two of the bands were identified as magnetosome associated proteins, Mms6 and MamA. The gel was stained with Coomassie Brilliant Blue G-250. Fig. 1. 3.2 Presence of a 14.5-kDa Mms6 in magnetosomes After we screened MamA binding proteins and analyzed them by SDS-PAGE, we determined that the 14.5-kDa Mms6 is the primary binding candidate ( Fig. 1 ). Previous to this result, Mms6 is generally known as a 6.0-kDa peptide that is tightly bound to magnetite crystals and is involved in the biomineralization of cubo-octahedral magnetite crystals both in vitro \n [22] , [23] and in vivo \n [24] , [25] , [26] . Arakaki et al. [22] identified Mms6 as a 6.0-kDa mature protein consisting of 59 amino acids (from a. a. 75–133), but the mms6 gene sequence shows that the full-length Mms6 protein is 133 amino acids (deduced from the 14.5-kDa peptide) ( Fig. S4 ). The 6.0-kDa Mms6 could have been present during the elution of MamA bound proteins but was not detected because the gel that was used could not separate low molecular mass proteins. In order to resolve the conflict of why we found an abundance of 14.5-kDa Mms6 instead of 6.0-kDa Mms6, we first wanted to confirm the presence of the 14.5-kDa version of Mms6 in magnetosomes. To do this, we generated anti-Mms6 1–133 polyclonal antibodies which were used for the immunoblotting analysis of cellular fractions. We found two bands that were specifically localized in the magnetosome fraction, one at 14.5-kDa and another at 6.0-kDa ( Fig. 2 (A)). As a control experiment, we performed the immunoblotting with an excess amount of Mms6 1–133 (antigen), confirming that the cross-reactions of these two bands, 6.0-kDa and 14.5-kDa, were specific ( Fig. 2 (A)). Also, we confirmed that the anti-Mms6 1–133 antibodies could recognize both recombinant protein bands of Mms6 1–133 and Mms6 75–133 ( Fig. S2 ). Using immunoblotting, we quantified the ratio of 14.5-kDa and 6.0-kDa Mms6 bands in the magnetosome extracts using two different preparation methods. Method 1: incubating in 2% SDS at 37 °C for 1 h, method 2: incubating in boiling 1% SDS for 1.5 h and taking an aliquot every 30 min (the same method used by Arakaki et al. [22] ) ( Fig. 2 (B)). In each method, both types of Mms6 were detected, but are present in different amounts. We calculated the ratio of 14.5-kDa and 6.0 -kDa Mms6 amounts from the intensities of the protein bands in the immunoblots. The signal intensity for the Mms6 1–133 band was 23 times stronger than that for the Mms6 75–133 band for an equal weight of proteins ( Fig. S2 ). The ratios were 63% and 37% for 14.5-kDa Mms6 and 6.0-kDa Mms6, respectively for method 1; and 38% and 62% for 14.5-kDa Mms6 and 6.0-kDa Mms6, respectively for method 2. This result showed, for the first time, that two different sizes of peptides of Mms6 exist in the magnetosome, and they are present in roughly equal amounts depending on the method of preparation. Previous studies demonstrated that the 6.0-kDa Mms6 (Mms6 75–133 ) binds magnetite crystals and controls the crystal morphology [23] , [24] , [25] , [26] . Furthermore, peptides mimicking the C-terminal region of Mms6 formed cubo-octahedral shaped crystals [27] . These insights indicate that the purpose of the 6.0-kDa version of Mms6 is for magnetite biomineralization, but there may be a separate function for the 14.5-kDa version of Mms6. Fig. 2 (A) Immunoblotting of M. magneticum AMB-1 extracts labeled with anti-Mms6 1–133 polyclonal antibodies [left]. Two different Mms6 bands are evident, one at 14.5-kDa (arrow) and the other at 6.0-kDa (arrowhead). The 14.5-kDa Mms6 has a higher intensity than the 6.0-kDa Mms6. In the control experiment, the immunoblotting was carried out with an excess amount of Mms6 1–133 antigen. In the control, the 14.5-kDa and 6.0-kDa bands were not detected [right]. S: soluble fraction; M: membrane fraction; MA: magnetosome fraction. (B) Two methods were used to extract Mms6 from the magnetosomes and then analyzed using immunoblotting. Method I used 2% SDS at 37 °C for 1 h to extract Mms6 which produced two Mms6 bands, one at 14.5-kDa and another at 6.0-kDa, but the 14.5-kDa band has higher intensity. Method II was performed by Arakaki et al. [22] which extracted Mms6 by boiling magnetosomes in 1% SDS for 1.5 h with three aliquots taken every 30 min lane 1, 2, and 3. This resulted in two distinct Mms6 bands (14.5-kDa [arrow] and 6.0-kDa [arrowhead]) in the first aliquot but only one band (14.5-kDa) in the second two aliquots. Fig. 2. 3.3 Confirmation of the interaction between MamA and 14.5-kDa Mms6 We confirmed the protein-protein interaction between MamA and 14.5-kDa Mms6 (Mms6 1–133 ) by immunoprecipitation and pull-down assay ( Fig. 3 ). Immunoprecipitation was performed using His-tagged Mms6 1–133 and His-tagged MamA, and two different antibodies, anti -MamA and anti-Mms6 1–133 , in different combinations to prove the binding between the two peptides ( Fig. 3 (A)). This demonstrated that Mms6 1–133 co-precipitated with MamA ( Fig. 3 (A)). In the control experiment, there was no interaction ( Fig. 3 (A)). Additionally, the Ni-NTA pull-down assay designed to test the specific interaction between MamA and Mms6 1–133 demonstrated that they did co-precipitate ( Fig. 3 (B)). We also confirmed the interaction between MamA and Mms6 1–133 using size-exclusion chromatography (SEC) ( Fig. S5 ). According to SEC, MamA ( Fig. S5A ) and Mms6 1–133 ( Fig. S5B ) were individually eluted in different fractions from the column. For example, MamA and Mms6 1–133 formed large oligomers with different molecular mass, ~500-kDa and >1000-kDa, respectively, which is consistent with previous studies [11] , [28] . Whereas, when we applied the mixture of MamA and Mms6 1–133 to the column they were eluted in the same fractions at near the void volume of the column ( Fig. S5C ). Even though these results show the interaction between MamA and Mms6 1–133 , the question remains as to whether the interaction is due to the nonspecific binding between the hydrophobic regions of the putative TPR motif in MamA [9] and the transmembrane region in Mms6 1–133 \n [22] . To reconcile this, we examined the interaction between MamA and a hydrophobic transmembrane protein, cytochrome a 1 -like hemoprotein [29] , by SEC. The MamA elution profile was not affected by adding the cytochrome a 1 -like hemoprotein ( Fig. S6 ), indicating that the MamA-Mms6 1–133 interaction is specific. Fig. 3 (A) SDS-PAGE analyses of the immunoprecipitation assays. A mixture containing His-MamA and Mms6 1–133 -His was precipitated with anti -MamA (left) or anti-Mms6 1–133 (right) antibodies and clearly show that Mms6 (arrow) co-precipitates with MamA (arrowhead). When normal serum was used, there was no band for either MamA or Mms6 (right lanes). (B) SDS-PAGE analyses of the Ni-NTA agarose pull-down assay. The arrows indicated the His-MamA and the His-tag removed MamA protein bands; the arrowheads indicated the Mms6 1–133 -His and His-tag removed Mms6 1–133 protein bands. Both the immunoprecipitation and pull-down assays confirm the interaction between MamA and Mms6 1–133 . The molecular mass standards (Precision Plus protein standards; Bio-Rad) are indicated on the left side of the gels. The gels were stained with Coomassie Brilliant Blue G-250. Fig. 3. We determined the interaction between MamA and 14.5-kDa Mms6 by affinity chromatography, immunoprecipitation, pull-down, and size-exclusion chromatography. The 14.5-kDa Mms6 (Mms6 1–133 ) has a larger N-terminal soluble domain (a. a. 1–88) ( Fig. S4 ), therefore it could extend into the cytosolic space and might anchor the MamA at the magnetosome surface. It is possible that MamA can bind to 6.0-kDa Mms6, however due to the sample limitation, we are unable to confirm this by the affinity chromatograph experiment. MamA was shown to cover the outside of the magnetosome and to play a role in maintenance processes such as protein sorting or activating magnetosome vesicles [11] , [14] . Our results suggest a direct interaction between MamA and Mms6. We propose that Mms6 localizes in the magnetosome membrane and is a factor controlling MamA localization. Because MamA homogenously surrounds the magnetosomes and are attached to Mms6, these proteins must also be homogenously spaced around the magnetosome as well. This homogeneous localization of Mms6, which controls the magnetite crystal shape, may affect the growth of the magnetite crystals. Therefore, in cells with the mamA gene deleted, the magnetite crystals may be altered. This may account for the results shown by Komeili et al. [14] who demonstrated that Δ mamA AMB-1 cells contained fewer crystals in the magnetosomes vesicles. There are at least 30 proteins associated with the magnetosome, one of which is MamA, a key protein for the process of constructing the organelle. By identifying that Mms6 is the binding partner of MamA, we found a major piece of the puzzle, which allows other researchers to continue the work on MamA and other magnetosome-associated proteins. Over the 40 year history of research on magnetotactic bacteria, a great deal of progress has been made, however many questions remain. For example, does MamA bind to the 6.0-kDa Mms6 and is it the same binding site as the 14.5-kDa Mms6? An important next step involves performing in vivo studies to examine the function of the MamA-Mms6 interaction. On the other hand, even though MamA is conserved in all known MTB, Mms6 exits only in MTB belonging to Alphaproteobacteria . Therefore, MamA in MTB belonging to non- Alphaproteobacteria should bind to a different magnetosome-associated protein in order to be anchored to the magnetosome. This hypothesis gives a new view that should inspire further studies into the protein-protein interactions in magnetosome bacterial organelles."
} | 4,335 |
35539930 | PMC9080891 | pmc | 8,651 | {
"abstract": "Thin and flexible elastomeric membranes are frequently used in many microfluidic applications including microfluidic valves and organs-on-a-chip. The elastic properties of these membranes play an important role in the design of such microfluidic devices. Bulge testing, which is a common method to characterize the elastic behavior of these membranes, involves direct observation of the changes in the bulge height in response to a range of applied pressures. Here, we report a microfluidic approach to measure the bulging height of elastic membranes to replace direct observation of the bulge height under a microscope. Bulging height is measured by tracking the displacement of a fluid inside a microfluidic channel, where the fluid in the channel was designed to be directly in contact with the elastomeric membrane. Polydimethylsiloxane (PDMS) and polyurethane (PU) membranes with thickness 12–35 μm were fabricated by spin coating for bulge testing using both direct optical observation and the microfluidic method. Bulging height determined from the optical method was subject to interpretation by the user, whereas the microfluidic approach provided a simple but sensitive method for determining the bulging height of membranes down to a few micrometers. This work validates the proof of principle that uses microfluidics to accurately measure bulging height in conventional bulge testing for polydimethylsiloxane (PDMS) and polyurethane (PU)eElastomeric membranes.",
"conclusion": "Conclusion We have demonstrated the ability to characterize elastic modulus of polymeric membranes using a simple microfluidic platform. In spite of its simplicity, our platform showed higher precision compared to conventional bulging tests. This method will be suitable to perform precise and sensitive measurements with a wide range of polymeric membranes applications including micro-valves and organs-on-a-chip. The ability to incorporate various types of elastic membranes in our platform and rapidly obtain the elastic properties can also be an important quality control step to verify fabrication variation, batch to batch consistency, and membrane stability.",
"introduction": "Introduction Elastic thin membranes integrated into microfluidic devices have been widely used for cell biology, tissue engineering, and drug discovery. 1 Some of these thin membranes are elastically deformable and have been used in different platforms such as micro-valves, 2,3 micro-pumps, 4–6 pressure sensors, 7,8 and optofluidic devices. 9,10 For biological applications, elastic membranes have also been used for tissue engineering, 11 lung-on-a-chip, 12 and gut-on-a-chip. 13 The elastic properties of these membranes are important parameters to determine their functionality and modes of operation in these particular micro-environments. Typically, the elastic properties of polymeric membranes are characterized by pressure loading such as bulge test or blister test 14–18 and point loading techniques such as indentation and microtensile tests. 19–22 These membrane characterization processes involve multiple steps, which include: (1) handling and mounting the membranes to the measuring device; (2) controlling the applied force to obtain deformation; (3) observation and data processing to determine the deformation characteristic; (4) using developed mathematics to calculate the elastic modulus. However, these techniques currently require specialized tools such as indenters, high amplification microscopes, and interferometers to carry out the characterization. For example, use of interferometers is common in the semiconductor industry to accurately determine the deformation characteristic of the membrane (bulging height). However, the dynamic range of these instruments is limited to account for large deflections, especially for highly elastic membranes. Moreover, these dedicated tools are very sensitive to vibrations. Alternately, high amplification microscopes integrated with high-resolution cameras have also been used to observe the deformation of the elastic membrane. 16,20 Nevertheless, translucent or reflective samples may not be compatible with these imaging techniques. These instruments may also not be readily accessible due to cost concerns. A recent study has shown the use of a liquid displacement approach in a microfluidic device to determine the peak deflection of the membrane without using expensive equipment. 23 The peak deflection can be altered by modifying the dimension of the embedded membrane in the device. However, the results are still preliminary and were not validated against a common elastic modulus characterization method. Furthermore, the fabrication of microfluidic device and membrane still involves the photolithography process, limiting the types of the membrane. As indicated above, the handling of the elastic membranes during testing is also an important consideration so that they can be mounted on a measuring device. Thin (<35 μm) elastic membranes tend to become wrinkled when removed from the substrate. Typically, they are clamped using a special screw-based clamping tool, which sometimes can cause air leaks between the mounting gaskets. 17 The objective of this work is to present a simple, microfluidic method that can rapidly and accurately measure the bulging height of elastomeric membranes and validate the method by comparing results with a typical microscopic observation based bulging test. The platform utilizes displacement of fluids inside a microfluidic channel caused by the deformation of the elastic membrane. By measuring the displacement of the fluid, the elastic modulus of the membrane can be estimated. Proof of concept was demonstrated by measuring the stress–strain relationship of thin PDMS and PU membranes. Theory \n Fig. 1(a) shows the principle of measuring the elastic modulus of thin polymeric membranes using the current approach. The platform is composed of two compartments. The first compartment (liquid compartment) is connected to a microfluidic channel that is partially filled with an indicator liquid. The microfluidic channel is designed such that movement of the indicator liquid is visible with the naked eye or via an easily accessible digital microscope. The second compartment (pneumatic compartment) is connected to a pressure source that can allow the users to determine the applied force on the membrane. The membrane is installed between the two compartments. As the membrane is deformed by a known pressure ( P ), the deformation of the membrane is indicated by the displacement of the indicator inside the microfluidic channel. The indicator displacement length due to the membrane deformation is noted as Δ l and the bulging height of membrane at the center is noted as w 0 . The bulging volume is equal to the displaced indicator volume (Δ l × h × w , where h is the height of the microchannel and w is the width). If the bulging volume ( V sp ) was assumed as a spherical cap, it will be equal to 1/3 × π × w 0 2 × (3 r − w 0 ), where r is the radius of the opening for bulging membrane. The bulging height ( w 0 ) can then be calculated using the length of the displaced fluid: 1 Fig. 1 (a) The principle of elastic modulus measurement in a microfluidic device. (b) Schematic showing the individual components of the device. (c) Image of membrane elastic modulus measurement device. Scale bar = 1 cm. Now, the material elastic properties can be then obtained from eqn (2) , which is governed by the equi-biaxial expression of Hooke's Law: 2 where σ is the stress applied to the membrane, E is the elastic modulus of the membrane, ν is Poisson ratio of membrane and ε is strain on the membrane. For the thin-wall sphere, the stress on the thin membrane can be expressed as: 14 3 where t is the membrane thickness. The strain on the membrane derived from circular membrane can be expressed in terms of bulging height: 14 4 Therefore, the elastic modulus of the membrane can be obtained by plotting the stress–strain curve with the known Poisson ratio. In this case, w 0 is determined by measuring the liquid displacement (Δ l ) in our microfluidic channel. Even the smallest bulging in the membrane can cause sufficiently large displacement in the microchannel. For example, if we assume that a displacement of 1 mm can be seen with the naked eye in a channel having the width of 1 mm, a 50 μm deep channel can allow the detection of a bulging height about 3 μm (Table. S1 † ). Design and fabrication One challenge of integrating membranes into microfluidic devices is the requirement of placing and fixing flexible membranes into planar systems. Although thin polydimethylsiloxane (PDMS) membranes can be fabricated in situ and assembled in the measurement device, 15 it is not trivial to integrate flexible membranes using common fabrication techniques available for microfluidics. To integrate the membrane into the microfluidic devices, we use layer-by-layer stacking technique to sandwich membrane layer between the liquid and pneumatic compartments. Fig. 1(b) shows the different layers of the measurement device. The patterns for each layer were designed using Solid Edge 2D Drafting ST4 software (Siemens PLM Software). The membrane holder and microfluidic channel portion were cut with a CO 2 laser cutter (M-360, Universal Laser Systems) on PET film (0.25 mm; McMaster-Carr) laminated with adhesive tapes (9122, 3M Company) on both sides. PDMS thin membranes were fabricated by mixing of degassed pre-polymer and curing agent (10 : 1 ratio; Sylgard 184; Dow Corning) and spin coating on a polycarbonate (PC) film (0.125 mm; McMaster-Carr) temporarily bonded to a rigid substrate such as acrylic. Typically, thin PDMS membranes (<25 μm) are molded or spin coated on rigid substrates such as glass or silicon. To remove a thin membrane from the substrate and to place it onto the measurement device may require precise handling. The bonding between the substrate and PDMS elastic membrane can be sufficiently strong that it makes peeling the thin material challenging without tearing. In addition, once the membrane is peeled off from the substrate, we found that the thin membranes less than 50 μm also tend to crumple together [Fig. S1(a) † ] and it is difficult to stretch the membranes back to their original state. These handling and fixation issues may cause instability, require more samples for the measuring process, and eventually affect the results of the measurement. To address these challenges, we used a rapid prototyping method based on laser-based micro-patterning and lamination techniques as described previously. 11,24 This technique can allow us: (1) to fix the radius of the window ( r ) for membrane elasticity measurement [Fig. S1(b) † ]; (2) peel off the membrane from the PC substrate rapidly and easily using an adhesive layer due to the adhesion of the PDMS film on the PC is weaker than on silicon or glass substrates [Fig. S1(c) † ]; (3) transport the membrane with the rigid holder [Fig. S1(d) † ]; and (4) integrate the membrane into the microfluidic device to perform the measurement. Currently, the free-standing ultrathin PDMS membrane (less than 1 μm in thickness) is possible to be fabricated and transferred to a ring support for the ease of handling. 25 It is also applicable to use the lamination approach mentioned here to transfer the ultrathin membrane into the microfluidic-based measurement device. Polyurethane (PU) membranes were fabricated by mixing two components (1 : 1 ratio; 1552-2; GS Polymers, Inc.) and spin coating on the silicon wafer. Here silicon wafers were used as the substrate because the adhesion of PU to PC films was too strong for follow-on processing. On the other hand, it was simple to peel the PU membrane from the silicon substrate after soaking in water for 24 hours. Different thicknesses of PDMS membranes and PU membranes were fabricated and tested in this work. The thickness of each membrane was confirmed using a scanning electron microscope (Fig. S2 † ). Once the measurement device was assembled by using layer-by-layer stacking fabrication technique, PEEK (polyether ether ketone) tubing was placed and glued to the air injection port [ Fig. 1(b) ] to complete the device [ Fig. 1(c) ]. The device was designed with the specific dimensions so that the displacement of the indicator can be easily observed under a stereomicroscope ( h = 0.35 mm, w = 1 mm and r = 2 mm). The liquid compartment was partially filled with a fluorinated oil (Novec 7500, 3M) solution as a liquid indicator to reduce the friction in the microchannel. The fluorinated oil is a lubricant with excellent thermal stability and chemical resistance. Thus, it can reduce the interaction between the membrane and liquid and avoid the evaporation during the operation. However, if bubbles are trapped during the filling of the channels with the oil – it can have a significant impact on the experimental result. Therefore, care needs to be taken while filling the device with the fluorinated oil. The design of the platform integrates this requirement by including a liquid injection port and a vent port [ Fig. 1 (b)] in the liquid channel such that no air bubble is trapped during filling. To ensure reproducibility, the device is held vertically with the injection port connected with a syringe during the filling operation. The oil is injected using the syringe against the gravity, which can simply push any air bubble out through the vent port. With this arrangement, it was simple to fill the channel with the desired amount of oil in a reproducible and rapid fashion. The membrane was deformed by applying a known pressure using a pneumatic pump (PneuWave Pump; CorSolutions, LLC). The deformation of the membrane causes the indicator to be displaced along the microchannel. The displacement of the indicator can be recorded from the difference of length based on a scale imprinted on the device [ Fig. 2(a) ]. The scale with millimeter range was engraved using the laser cutter on the surface of the device [ Fig. 2(b) ]. When a digital microscope is used, the displacement of the indicator can be recorded more accurately (down to 0.01 cm). Therefore, the sensitivity of this platform can be tailored to measure the elastic modulus of membranes with very small deflection. In order to validate the measurements with the microfluidic method, the membranes were also subjected to a conventional bulging test under a stereo microscope. 16 After the images were captured, the bulging height was determined using ImageJ software (National Institutes of Health). Fig. 2 (a) The procedure of measurement. (b) Photograph of microfluidics with liquid under a light microscope. The liquid volume can be precisely measured according to the engraved scale (unit in centimeter). In this example, the distance would be measured as 0.79 cm. (c) Increased length of liquid displacement corresponds to applied pressure. N = 3.",
"discussion": "Results and discussion Conventionally, bulging height of membrane can be directly measured using high amplification microscopes. 15,16 The bulging shape is in a form of spherical cap and the bulging height is determined by measuring the length between the peak point and base point (Fig. S3 † ). Although the measurement technique can be viewed as a straightforward method, this imaging technique is sometimes not suitable for reflective or transparent samples. For example, planar PDMS still has about 5% reflectance of light. 26 Light reflection can affect the observation as well. Fig. S3 † shows an example of our membrane under the inflated condition where it is difficult to pinpoint the exact peak and base points of the bulged membrane. In addition, the determination of these points can also vary depending on the observation angle. Due to these reasons, the bulging height might vary with experimental setup or the observer's judgment that can cause measurement errors between samples. On the other hand, using our microfluidic-based technique enabled interpretation of the bulging magnitude to obtain a stable and quantitative measurement. Fig. 2(c) shows the applied pressure-displacement curve for thin PDMS membrane (35 μm of thickness) integrated into the measurement device. The result demonstrates that the displacement length in the liquid compartment is proportional to the applied pressure in the pneumatic compartment. The main task of a bulging test is to obtain the bulging height of the membrane. In our study, we can simply translate the length of displacement into the bulging height of membrane using eqn (1) . In order to validate the bulging height obtained from the microfluidic-based device, we used the conventional bulging test to observe the change of bulging height under a microscope as well. Initially, the colorless membrane/substrate made it difficult to determine the peak and base points of the bulging membrane under the microscope. We have used one fixed peak and two base points (as indicated by the orange line in Fig. 3 and Fig. S3 † ) to determine the observed bulging height. Fig. 3(a) and (b) shows the comparison between the microfluidic-based measurement device and the conventional bulging test. We were able to confidently observe the movement of the indicator fluid and obtain the length of displacement [ Fig. 3(c) and (d) ], whereas one could obtain different bulging height depending on their selection of base/peak point in the conventional bulging test [indicated by orange and red lines in Fig. 3(a) ]. A range of pressure was then applied to obtain load-deflection curves for the membranes. The result shows that although the same setup of conventional bulging height was observed, the standard deviation of each measurement varied by 50–80 μm. The microfluidic-based measurement, on the other hand, the standard deviation was only within 2–10 μm. Furthermore, the load-deflection curve obtained from the conventional approach varied significantly based on where we chose the location of the base/peak points [ Fig. 3(e) ]. Therefore, we found that our microfluidic-based approach revealed results that are more reliable and repeatable eliminating human errors and light reflection effects. For the materials with a higher elastic modulus ( e.g. PU), the microfluidic-based technique might be a useful tool to measure very small bulging of the membranes. The microfluidic-based approach can be made more sensitive by reducing the dimension of the channel in a microfluidic chip. Fig. 3 Slightly changes of pressure were applied between 1.2 (left column) psi and 1.3 psi (right column) using (a), (b) microscope-based observation and (c), (d) microfluidic-based bulging tests to obtain bulging height and liquid displacement of PDMS membrane, respectively. Unit of length is in centimeter. (e) Bulging height plots with applied pressure for the conventional bulging test with base point 1 (red dots), conventional bulging test with base point 2 (orange dots), and microfluidic-based bulging tests (blue dots). N = 3. In this work, the elastic modulus of PDMS membrane was measured by analyzing the bulging height of membrane integrated into the microfluidic-based measurement device. First, the stress–strain curve of each membrane was plotted by calculating the applied stress and strain of the membrane from eqn (3) and (4) ( Fig. 4 ). The elastic modulus of each test membrane was then determined by fitting the slope of the stress–strain curve to eqn (2) (assuming ν = 0.5). 27 Table 1 shows the elastic modulus results for the PDMS membrane with 12 and 35 μm of thicknesses. We have obtained two different elastic moduli of the PDMS membrane for the two different thicknesses. It is not uncommon to obtain different elastic modulus for PDMS depending on the processing conditions and fabrications method. 28 Thickness-dependent arises from shear stress during fabrication, which can be dependent on the processing conditions and surface energy of the substrate. Unfortunately, it is difficult to compare literature data with our findings as the processing conditions and substrates used in our work is unique. Fig. 4 Stress–strain curves for PDMS membrane. All data were obtained using the measurement devices ( N = 3) with known dimensions ( h = 0.25 mm, w = 1 mm, r = 2 mm). The PDMS membranes used for this experiment have a thickness of 35 μm. The calculated elastic modulus for various samples \n \n a Elastic modulus obtained based on the optical measurement with base point 1 in Fig. 3 . b Elastic modulus obtained based on the optical measurement with base point 2 in Fig. 3 . The same method was also used to measure the elastic modulus of PU membrane (assuming ν = 0.49) 29 with 15 μm of thickness ( Table 1 and Fig. S4 † ). The high elastic modulus of PU meant lower bulging height for the same applied pressure compared to PDMS. We were able to detect PU bulging height differences that were as a little as 10 μm, which were not detectable using the conventional bulging test performed under the same condition. The elastic moduli obtained by using microscope-based observation were also calculated to demonstrate the range of inaccuracy if different base points were chosen. The microfluidic method to measure the bulging height follows a simple principle. The platforms in this work were fabricated using a rapid prototyping method that uses laser cutters and lamination method. However, the basic working principle is independent of the fabrication technique used in this work and should be amenable for integration with other common fabrication methods. One unique attribute of our method is the bulging test is carried out in a microfluidic environment. Therefore, the current method is capable of producing testing conditions which are similar to the ultimate applications of the membranes ( e.g. microfluidics valves, organ on a chip etc. ). This provides a great advantage over other bulging tests since mechanical properties of polymeric membranes. However, the microfluidic method in its current form may not be suitable for testing porous membranes or highly air permeable membranes. Similarly, care should be taken to utilize this technique with materials that can interact with the indicator fluid. Since the objective of this work was to validate the proof of principle that uses microfluidics to accurately measure bulging height in conventional bulge testing, we have used fluorinated oil as the indicator fluid. Nevertheless, we believe that the current method will provide new opportunities for mechanical characterization of a wide range of materials."
} | 5,671 |
28630897 | PMC5473674 | pmc | 8,652 | {
"abstract": "Compliant battery design strategy for wearable power sources with high degree of flexibility and stretchability.",
"introduction": "INTRODUCTION There is currently a great deal of interest in incorporating electronic functions into clothing and wearable devices for applications such as sensing and health care ( 1 ). Flexible and stretchable batteries play an important role in achieving the vision of wearable and conforming electronics. In recent years, several approaches have been developed to achieve compliant batteries. The initial demonstrations were flexible batteries based on conventional planar structures, assembled through stacking of the battery components ( 2 , 3 ). These designs evolved into more advanced form factors that enabled omnidirectional flexibility. Batteries in the shape of a fiber or wire ( 4 – 17 ), for example, can be twisted, tied, and woven into fabrics, allowing integration with wearable garments. In addition, several approaches to design stretchable batteries have been proposed, particularly using concepts of kirigami ( 18 ), origami ( 19 ), bridge-island battery design ( 20 ), arched electrode architecture ( 21 ), winding fibers around elastic support ( 14 ), embedding battery active materials within stretchable fabrics ( 22 ), and embedded nanowire elastic conductors ( 23 ). Despite innovative design strategies, there are no reports of wire batteries that exhibit fatigue resistance sufficient for applications in wearable systems that are likely to undergo thousands of flex cycles throughout their lifetime. In the case of stretchable batteries, none of the systems offer safety, compliance along multiple axes, and flexibility of electrode components simultaneously. The compliant battery design concept introduced here addresses the aforementioned limitations of existing stretchable and wire-shaped battery systems. This strategy could be applied to a number of material composites and is demonstrated here on the silver-zinc (Ag-Zn) system, which has the advantage of high energy density combined with intrinsic safety. The core of the approach is in the utilization of metal current collectors with enhanced mechanical design, such as helical springs, serpentines, and spirals, as a structural support and backbone for the rest of the battery components. These architectures effectively accommodate stress imposed by mechanical deformation, thus minimizing strain experienced by the electrodes without compromising their surface area. Depending on the choice of current collector geometry, batteries can be fabricated with flexible or stretchable form factors to match the mechanical properties of wearable electronic systems while using the same battery chemistry, cell components, and fabrication steps. We demonstrate the concept through fabrication and electrochemical-mechanical characterization of batteries with two form factors—flexible wire and stretchable serpentine. We achieve flexible wire batteries by shaping the current collector–electrode as a helical band spring. The wire batteries show linear capacity of 1.2 mA·hours cm −1 , are resilient to repetitive dynamic mechanical load, and can withstand >17,000 bending cycles to the bending radius of 0.5 cm under continuous operation mode without a decrease in electrochemical performance. Although using a current collector in the form of a helical band spring enabled omnidirectional flexibility of the battery, its elongation remained limited. Therefore, to achieve batteries that can be readily stretched, we used a current collector with serpentine ribbon geometry. In this structure, stretchability is facilitated by the out-of-plane rotations of serpentine ribbons, and batteries can operate under 100% stretch. The degree and direction of stretching can be modified by changing the serpentine geometry. In addition to stretching, the battery based on the serpentine ribbon can accommodate flexible motions in one plane. Thus, the omnidirectional, fatigue-resistant flexibility of the wire-shaped battery based on the helical band spring makes it the preferred geometry for flexible applications. On the other hand, the serpentine-shaped battery that can be readily stretched along two axes is preferred for integration with stretchable electronics. Integrating compliant batteries with energy-harvesting devices is crucial for widespread realization of autonomous wearable power sources. Therefore, it is important not only to design batteries with compliant form factors but also to study their performance as a part of practical wearable systems. We explore a jewelry-integrated power source design in the form of a bracelet that includes a compliant battery and a photovoltaic module. The battery comprises part of the wristband that is expected to undergo flexing motions throughout the lifetime of the accessory. The battery with wire geometry is chosen because of its omnidirectional flexibility and resilience to flexing motions. However, if stretchability is one of the design considerations, then an accessory with the same energy-harvesting and storage capabilities can be achieved using a battery with serpentine architecture. The photovoltaic module charges the battery under multiple lighting conditions, including time-varying illumination that mimics the light conditions a wearable device may be exposed to during a typical daily routine. Such an accessory can harvest and store energy and provide power ranging from microwatts to milliwatts, depending on the illumination.",
"discussion": "DISCUSSION We demonstrate a new design concept to fabricate stretchable and flexible batteries. This strategy relies on using mechanically robust current collector geometries such as serpentines or helical springs to serve as a structural support for the rest of the battery components. The choice of current collector geometry determines the mechanical properties of the battery. To demonstrate the concept, we explored two different current collector geometries—helical band spring and serpentine ribbon. The battery fabricated around the helical band spring current collector has the form factor of a flexible wire. Such geometry ensured omnidirectional flexibility and fatigue resistance to flexing motions. Electrochemical-mechanical characterization of flexible wire–shaped batteries based on the helical band spring current collector showed that they can withstand flexing >17,000 times to the bending radius of 0.5 cm. The elongation of the wire battery is limited by the mechanical properties of the polymer electrolyte, cellophane layer, and silver electrode. To achieve devices that can be readily stretched, we fabricated batteries around serpentine ribbon current collectors. Serpentine-shaped batteries retained their electrochemical performance while being stretched to 100% and can accommodate flexible motions in one plane. Furthermore, we have shown that biaxial stretching can be realized by the utilization of a self-similar serpentine current collector. Therefore, using current collectors with spring and serpentine geometries as a backbone for the battery components represents a promising fabrication approach to compliant batteries with a range of mechanical properties. Integration with the organic solar module into a wearable energy bracelet and study of the performance of such an accessory under conditions simulating a day of use demonstrate the suitability of these batteries for real-life applications."
} | 1,858 |
27631692 | PMC5025190 | pmc | 8,653 | {
"abstract": "Basic research on biodiversity has concentrated on individual species—naming new species, studying distribution patterns, and analyzing their evolutionary relationships. Yet biodiversity is more than a collection of individual species; it is the combination of biological entities and processes that support life on Earth. To understand biodiversity we must catalog it, but we must also assess the ways species interact with other species to provide functional support for the Tree of Life. Ecological interactions may be lost well before the species involved in those interactions go extinct; their ecological functions disappear even though they remain. Here, I address the challenges in studying the functional aspects of species interactions and how basic research is helping us address the fast-paced extinction of species due to human activities."
} | 213 |
33602869 | PMC7919410 | pmc | 8,655 | {
"abstract": "Here, Opachaloemphan et al. investigated the temporal dynamics of the social behavior and molecular mechanisms underlining the caste transition and social dominance in the ant society of Harpegnathos saltator . They show that molecular changes in the brain serve as earliest caste predictors compared with other tissues, and behavioral and molecular data indicate that despite the prolonged social upheaval, the gamergate fate is rapidly established, suggesting a robust re-establishment of social structure.",
"discussion": "Discussion Organized social structure and cooperation are fundamental for survival in a social setting. Social insects, and in particular the ant H. saltator , have fascinating superorganism resilience, allowing them to tolerate the loss of germ cells in its colony (gamergates and queens) ( Hölldobler and Wilson 2008 ; Straub et al. 2015 ). We took advantage of the caste plasticity in H. saltator that possesses both rigid and flexible adult caste systems ( Opachaloemphan et al. 2018 ) to study the molecular features that underlie the re-establishment of the social hierarchy structure. In response to the lack of queen pheromones, multiple H. saltator workers initiate aggressive antennal dueling to produce new reproductives (gamergates). Unlike other hostile competitions, the aggressiveness of antennal dueling has no apparent negative effects among the contestants ( Sasaki et al. 2016 ). Instead, after a rapid exchange in which most workers return to work (winner–loser), dueling among gamergates appears to promote reproduction and social dominance hierarchy in a winner–winner configuration ( Liebig et al. 1999 ; Sasaki et al. 2016 ). Other types of social interactions, such as biting, infrequently occur during the caste transition and strongly inhibit the targeted individuals from continuing to duel. However, bitten and policed workers were not included in this study. By tracking the dueling behavior during the months-long caste transition, we were able to show that future gamergates are determined expediently within the first few days of the dueling tournament. Future gamergates show significantly increased dueling activity compared with the workers, allowing us to predict the gamergate fate with 86% accuracy at 3 d postinitiation of dueling ( Fig. 2 C). Activation of the ovary of the future gamergates is rapid, with egg laying being apparent at day 10 ( Fig. 1 E). The ovary consists of eight ovarioles in total. At day 3 postdueling initiation, the dueler's ovary is partially activated and occasionally develops a yolky oocyte in one of the ovarioles. By day 10, prospective gamergates have developed yolky oocytes in approximately three of the eight ovaries ( Supplemental Fig. S2B ). Dueling activity peaks during days 6–10 ( Fig. 1 D), followed by a decrease that corresponds to an increase in egg-laying events after day 10 ( Fig. 1 E), implying that the cost of social dominance is swiftly reinvested in reproduction after the re-establishment of social structure. Nevertheless, dueling has not yet ended, and the prospective gamergates constantly duel to show and maintain their social dominance, suggesting a well-balanced energy investment between reproduction and dominance hierarchy. In contrast, workers with low or no dueling invest their energy in colony maintenance tasks, including foraging, brood caring, nest defending, and waste collecting, rather than in social competition and reproduction. Altogether, H. saltator rapidly senses and responds to the absence of a reproductive female: New gamergates are rapidly determined, emphasizing a remarkable resiliency in the wake of disturbance to the social hierarchy in H. saltator . Recent modeling of H. saltator during the worker-to-gamergate transition has underscored the importance of dueling in maintaining shared dominance amongst gamergates ( Sasaki et al. 2016 ). The prolonged dueling among gamergates after their fate is established is a “winner–winner” scenario in which individuals that duel mutually benefit, resulting in the production of several gamergates in a colony, likely necessary for colony maintenance given that the gamergates retain the smaller worker body and cannot produce as many eggs as actual queens ( Peeters et al. 2000 ). Our observations during the early stages of the dueling tournament indicate that most, but not all, individuals that duel were destined to become actual gamergates. Some early duelers gave up on the dueling tournament and eventually remained as workers, suggesting that the antennal dueling is not always a winner–winner scenario, at least during the unstable social structure in the early transition. Once duelers (immature gamergates) reach a mature stage and fully develop their production of queenlike pheromones, dueling activity considerably decreases ( Fig. 1 D). The other workers (subordinates) in the colony recognize the mature gamergates as the reproductives and do not challenge them ( Liebig et al. 1999 , 2000 ), and their reproductive capacity is repressed by the pheromones of the mature gamergates ( Ghaninia et al. 2017 ). Other social interactions (for example, dominant biting) influence the gamergate determination as bitten individuals are excluded from the dueling tournament, although not all of the transient dueling behavior ceases because of biting ( Liebig et al. 1999 ; Sasaki et al. 2016 ). Therefore, the transient dueling behavior in some workers during the early period ensures that no cheaters threaten the upcoming social hierarchy. Once the gamergate fate is established, the sustained dueling interactions reinforce the developmental plasticity as a positive-feedback loop that positively influences the shared dominance among duelers. Because we could predict future gamergates as early as at 3 d, we could identify multiple molecular markers associated with the dueling behavior and the transition to gamergate, as evidenced by the tissue-specific gene expression profiles from dueling versus nondueling workers. We observed both differential behavioral dynamics and differential molecular gene expression in distinct tissues, indicating physiological changes during the early worker-to-gamergate transition. Each tested tissue showed different responsiveness to the absence of the queen pheromones. The brain of nonduelers and duelers starts diverging as early as day 3 postdueling initiation, and these differences become more apparent at day 10. On the other hand, the abdominal fat body has an initial rapid response at day 3, but most of the early response gene differences between duelers and nonduelers disappear by day 10. Several of the late response genes, involved in pheromone production and vitellogenesis processes, may be subsequently induced by changes in the brain. Vitellogenin is transported to the ovary and supports oogenesis. Therefore, the ovary appears to act as a downstream tissue receiving signals from the other tissues and responding to them extensively after day 3 and until day 10 ( Supplemental Fig. S7 ). We propose a model whereby the brain serves as a primary tissue driving the activation of other peripheral tissues during the antennal dueling tournament. Expression levels of several genes in the brain show a rapid divergence between prospective gamergates and workers and serve as early molecular markers of gamergate fate. Gp-9-like pheromone-binding protein (PBP) is down-regulated in the brain, fat bodies, and ovaries of prospective gamergates ( Fig. 4 A; Supplemental Fig. S3B ′′ , S4B ) . In Lepidoptera, expression of PBPs is very high in antennae ( Mao et al. 2016 ) in which the proteins facilitate pheromone perception by olfactory receptors ( Chang et al. 2015 ). However, in the red fire ants ( Solenopsis invictia ), expression of many PBPs, including Gp-9, is less abundant in the antenna compared with the rest of the head and the thorax ( Zhang et al. 2016 ), and Gp-9 was proposed to function as a transporter of chemical compounds that acts beyond olfaction. It could also serve as a protein circulating in the hemolymph that functions as a carrier of specific hormones or signaling molecules ( Wang et al. 2013a ) that has an inhibitory effect on the queenlike phenotypes. Vitellogenin expression is increased in the brain of prospective gamergates. The expression of vitellogenin in the fat body is known to be crucial for oogenesis and is repressed by corazonin, neuroparsin, and JH ( Corona et al. 2007 ; Libbrecht et al. 2013 ; Yang et al. 2014 ; Gospocic et al. 2017 ), but its expression in the brain is not well understood. Vitellogenin expression is found in the red fire ant workers, which have no ovaries, suggesting additional roles of vitellogenin besides being an egg yolk protein in ants ( Wurm et al. 2011 ). Several studies in ants and honeybees propose that the vitellogenin precursor may mediate responsiveness to social cues such as broods and food and consequently affect social behaviors by increasing parental care ( Roy-Zokan et al. 2015 ; Kohlmeier et al. 2018 ). Membrane metallo-endopeptidase-like 1 ( MMEL1 ) and kunitz-type protease inhibitor ( HCRG1 ) are up-regulated in the early gamergate-destined brain; however, their roles in the insect brain are unknown. On the other hand, proteases and serine protease inhibitors are widely reported in the vertebrate brain to influence inflammation, synaptic plasticity, and behavior ( Almonte and Sweatt 2011 ) by processing extracellular matrix molecules that contribute to neuronal outgrowth ( Rivera et al. 2010 ). Their up-regulation in the dueling brain may facilitate structural changes in the brain that allow neuronal growth or migration during caste establishment. Following the initial response of the gamergate brain, we observed a cascade of downstream events in the brain, fat body, and ovary that appear to promote queenlike and suppress worker behaviors in the prospective gamergates ( Fig. 6 ). Down-regulation of corazonin in the brain suppresses foraging behavior and derepresses the expression of vitellogenin in the fat body and, hence, oocyte maturation ( Gospocic et al. 2017 ). The reduction of IGF in the brain results from the lowering of JH levels, likely mediated by the reduction of Kr-h1 ( Fig. 5 B). This may subsequently contribute to the increase of vitellogenin expression. Global reduction of Kr-h1 leads to up-regulation of Ec biosynthesis in the ovary ( Zhang et al. 2018 ). Similar to other social insects ( Robinson et al. 1992 ; Pamminger et al. 2016 ; Norman et al. 2019 ), JHA treatment shows negative effects on ovary growth in H. saltator workers, whereas 20E treatment promotes ovary maturation in workers. As the JHA or 20E treatments do not affect Vg expression in the brain and fat body of workers ( Supplemental Fig. S5A, A′ ), this suggests that vitellogenesis in H. saltator is controlled by other signaling ( Gospocic et al. 2017 ), as observed in the black garden ant ( Pamminger et al. 2016 ). Finally, we observed an increase in the expression levels of brain Ins and ovarian IGF in prospective gamergates. However, although brain IGF in workers is induced by JH treatment, both gamergate-biased genes (brain Ins and ovarian IGF ) are not affected by JH or by 20E. The function of worker-brain and gamergate-ovarian IGFs is unclear, but brain injection of Ins in clonal raider ants stimulates oocyte maturation ( Chandra et al. 2018 ). In summary, we showed here that high JH in nonduelers induces brain IGF expression and promotes the worker fate while high ecdysone in duelers promotes ovary maturation ( Fig. 6 ). Figure 6. A hypothetical pathway during the early caste transition. The absence of inhibitory queen pheromones unleashes the antennal dueling in workers and subsequent molecular changes in the different tissues. We propose that initial gene expression changes in the brain, such as down-regulation of Gp-9 - like pheromone-binding protein and up-regulation of vitellogenin in the destined gamergates, trigger a cascade of gene expression changes in the brain, fat body, and ovary that promote queenlike phenotypes, such as queen pheromone biosynthesis and oocyte development, and suppress worker-like phenotypes, such as foraging. High juvenile hormone (JH) enables the worker fate, while increased ecdysone (Ec) promotes reproduction in duelers or prospective gamergates. Ins is the homolog of human insulin , whereas IGF is the homolog of human IGF1 . Ins was previously referred to as llp1 and IGF to Ilp2 in H. saltator ( Bonasio et al. 2010 ; Gospocic et al. 2017 ), while the Ins homolog in honeybees and clonal raider ants was designated as llp2 ( Corona et al. 2007 ; Wang et al. 2013b ; Chandra et al. 2018 ). Genes down-regulated in the dueling ants are indicated by red arrows , whereas up-regulated genes are indicated by green arrows. Solid lines indicate a known functional role of a gene. Broken lines represent hypothetical relationships. In conclusion, we monitored the behavior and gene expression profiles at the very beginning of caste transition. The absence of queen pheromones rapidly initiates behavioral changes (antennal dueling) in workers with changes in gene expression in the brain of prospective gamergates. We discovered that the gamergate fate is rapidly determined and can be efficiently predicted at 3 d after dueling initiation, suggesting a rapid restoration of social structure in H. saltator . Furthermore, altered gene expression in the brain appears to be the primary genetic factor for caste switching. These early molecular alterations in the brain are likely due to a response to the absence of inhibitory queen pheromones and to the antennal dueling behavior, which subsequently lead to other systemic effects in peripheral tissues that might be mediated by JH signaling that appears to play a crucial role in the maintenance of the worker fate while ecdysone (Ec) promotes reproduction in gamergates ( Fig. 6 )."
} | 3,513 |
38033806 | PMC10683474 | pmc | 8,657 | {
"abstract": "Microorganisms can be genetically engineered to transform\nabundant\nwaste feedstocks into value-added small molecules that would otherwise\nbe manufactured from diminishing fossil resources. Herein, we report\nthe first one-pot bio-upcycling of PET plastic waste into the prolific\nplatform petrochemical and nylon precursor adipic acid in the bacterium Escherichia coli . Optimizing heterologous gene expression\nand enzyme activity enabled increased flux through the de\nnovo pathway, and immobilization of whole cells in alginate\nhydrogels increased the stability of the rate-limiting enoate reductase\nBcER. The pathway enzymes were also interfaced with hydrogen gas generated\nby engineered E. coli DD-2 in combination with a\nbiocompatible Pd catalyst to enable adipic acid synthesis from metabolic cis , cis -muconic acid. Together, these optimizations\nresulted in a one-pot conversion to adipic acid from terephthalic\nacid, including terephthalate samples isolated from industrial PET\nwaste and a post-consumer plastic bottle.",
"conclusion": "Conclusions In summary, the development of new sustainable\nbio-based methods\nto valorize waste carbon into industrial small molecules is an elegant\napproach to creating a circular chemicals economy. Through a series\nof chemical and genetic optimizations, this study reports the first\nbioproduction of the prolific platform chemical adipic acid directly\nfrom terephthalic acid generated in situ from industrial\nPET waste and a post-consumer plastic bottle. The reaction occurs\nin engineered E. coli cells through an eight-gene,\nsix-enzyme de novo biosynthetic pathway within calcified\nalginate beads. Product conversion is high (79%, 115 mg/L) and occurs\nin aqueous media under ambient conditions (room temperature, pH 7.4)\nin 24 h. We believe this is the first report of the bioproduction\nof adipic acid from a plastic waste source, substantiating the use\nof microbial biotechnology as a solution to the valorization of this\nabundant “waste” feedstock while also diverting chemical\nmanufacturing routes away from the sole use of raw petrochemicals.\nFuture work from our lab will include process intensification focused\non cofactor recycling and parameters such as terephthalate import,\nBcER engineering, scale-up, and extension of this pathway to encompass\nthe microbial synthesis of other chemical targets of industrial significance.",
"introduction": "Introduction Synthetic pathways to industrial chemicals\ncan be designed and\nassembled in living cells using modern synthetic biology. This enables\nthe bioproduction of target compounds from renewable resources via\nfermentation and is emerging as an elegant and viable alternative\nto multistep synthesis from diminishing fossil fuels. 1 , 2 Many of these pathways proceed via the fermentation of carbohydrate\nfeedstocks via primary metabolic reactions in vivo . However, this approach also enables the upcycling of waste carbon\nfrom existing industrial processes to create circular economies, avoiding\nthe environmental consequences of landfill and/or incineration processes.\nThis includes the upcycling of plastic-waste-derived small molecules\nfrom post-consumer polyethylene terephthalate (PET)—a thermoplastic\nmaterial used throughout the modern chemical industry to create a\nwealth of everyday products. The global demand for this material exceeds\n30 M ton/year, of which >80% is designed to be single use, leading\nto ca. 25 M ton/year of post-consumer PET waste and contributing to\nthe global plastic waste crisis. 3 , 4 Although chemical\nand biological approaches to the depolymerization\nand recycling of PET waste are being investigated, bio-upcycling technologies\nto convert plastic waste into higher value small molecules are less\nestablished. 5 − 12 This approach is attractive as the PET depolymerization products\nethylene glycol and terephthalic acid (TA) are microbial metabolites\nand therefore viable substrates for de novo metabolic\npathway design. To this end, Kim et al. previously reported the bioconversion\nof PET-derived ethylene glycol into glycolic acid in Gluconobacter\noxydans and TA into vanillic acid, muconic acid, gallic acid,\nand pyrogallol in engineered E. coli MG1655 in 33–93%\nyield. 13 More recently, Werner et al. reported\nthe high-level bioproduction of β-ketoadipate from bis (2-hydroxyethyl)terephthalate (BHET) in 76% yield in engineered Pseudomonas putida KT2440. 14 This\nwas also achieved by Sullivan et al. in 73% yield using P.\nputida AW307 and benzoate isolated from chemically modified\nmixed plastic waste. 15 In 2021, our lab\nreported the conversion of post-consumer PET from a waste plastic\nbottle into the vanilla flavor compound vanillin in 79% yield in engineered E. coli MG1655 RARE. 16 Following on from this work, we sought to expand the range of small\nmolecules that can be accessed via microbial synthesis from terephthalic\nacid. Adipic acid (AA) is an aliphatic 1,6-dicarboxylic acid and prolific\nplatform chemical that is used throughout the materials, pharmaceuticals,\nfragrances, and cosmetics industries. It is currently manufactured\non a 2.6 M ton/year scale from petrochemically derived benzene via\nthe nitric acid-catalyzed oxidation of cyclohexanol and cyclohexanone.\nThe process is highly energy intensive and releases a mol/mol equivalent\nof nitrous oxide into the atmosphere. These emissions have been shown\nto significantly contribute to global greenhouse gas levels; 17 1 kg of N 2 O equates to 298 kg CO 2 equivalents. As a result, the bioproduction of adipic acid\nfrom renewable feedstocks has been an active area of research. Recent work has included the high-level production of the adipate\nanalogue β-ketoadipate from d -glucose by Rorrer et\nal. in engineered P. putida KT2440 and the one-pot\nbioconversion of lignin-derived guaiacol to adipic acid by our laboratory\nin engineered Escherichia coli ( Figure 1 A). 18 , 19 However, the microbial synthesis of adipic acid directly from waste\nPET remains an outstanding challenge in the field of chemical biotechnology.\nHerein we report the first one-pot bioproduction of adipic acid from\nterephthalic acid and terephthalate waste in engineered Escherichia\ncoli . The reaction proceeds in aqueous media at room temperature\nand produces adipic acid in 79% conversion (115 mg/L) in 24 h when\ncells are immobilized in alginate hydrogels ( Figure 1 B). Together, this study validates the use\nof microbial cells as a viable biotechnology for the upcycling of\nplastic-derived small molecules and PET plastic waste. Figure 1 Microbial biotransformation\nand fermentation approaches to adipic\nacid and adipate analogues. (A) Carbohydrate fermentation in P. putida and valorization of lignin aromatics in E. coli . (B) Proposed bio-upcycling of terephthalic acid\nto adipic acid.",
"discussion": "Results and Discussion We began by assembling the eight\ngenes required for adipic acid\nsynthesis from terephthalate in E. coli ( Figure 2 A, Table S2 ). The heterologous pathway begins with two enzymes\nfrom Comamonas sp. : TPADO, a heterotrimeric O 2 -dependent dioxygenase consisting of TphA1–2 and TphB2\nsubunits, and DCDDH, a NAD + -dependent dehydrogenase. Together,\nthese enzymes catalyze the oxidative decarboxylation of terephthalate\nto protocatechuate (PCA). Protocatechuate is then transformed into\nadipic acid by four additional enzymes: AroY, KpdB, CatA, and BcER.\nAroY is a protocatechuate decarboxylase from Klebsiella pneumoniae ; KpdB is the B-subunit of 4-hydroxybenzoate decarboxylase from K. pneumoniae that activates AroY by generating prenylated\nFMN (prFMN); CatA is a non-heme Fe(III)-dependent dioxygenase from Pseudomonas putida ; and BcER is a [4Fe–4S]-dependent\noxidoreductase from Bacillus coagulans . 20 Figure 2 Initial pathway construction and whole-cell activity.\n(A) The de novo biosynthesis pathway to adipic acid\nfrom terephthalic\nacid. (B) Whole-cell mixing experiment. Microbial biocatalysis reactions\nwere performed at OD 600 120 at 21 °C in sealed Hungate\ntubes for 24 h. Product concentrations were determined by reverse-phase\nHPLC relative to an internal standard of caffeine. All data shown\nare an average of three replicate experiments to one standard deviation.\nSt1, E. coli BL21(DE3)_pQlinkN- aroY - kpdB ; St2b, E. coli BL21(DE3)_pAA2(pQlinkN- catA - bcER ); St3, E. coli BL21(DE3)_pVan1( tpado-dcddh ). The aroY and kpdB genes were\ninserted into an empty pQLinkN backbone ( Figure S1 ), and the plasmid was transformed into E. coli BL21(DE3). Similarly, our previously reported pAA and pAA2 plasmids\n(pETDuet-1 and pQLinkN encoding catA and bcER , respectively 19 ) and pVan1\nplasmid (encoding TPADO and DCDDH 16 ) were\nalso transformed into E. coli so as to conduct an\ninitial whole-cell mixing experiment. This was to determine whether\nAA could be detected when PCA or TA was added to suspended whole-cell\nmixtures of E. coli BL21(DE3)_pQLinkN- aroY - kpdB (termed St1), E. coli BL21(DE3)_pAA\n(termed St2), E. coli BL21(DE3)_pAA2 (termed St2b),\nand E. coli BL21(DE3)_pVan1 (termed St3; Figure 2 B and Figure S9 ). To this end, cells were grown\nto mid-log phase and protein expression\nwas induced for 24 h. Cells were isolated, resuspended in equal amounts\nto OD 600 120 in M9 media containing 5 mM PCA/TA, and incubated\nat 21 °C for a further 24 h. Gratifyingly, St1 was able to convert\nPCA to catechol in 76% conversion. St2 was able to convert catechol\nto adipic acid in 79% conversion. A co-culture of St1 and St2 collectively\nharboring AroY, KpdB, CatA, and BcER was able to convert PCA to cis , cis -muconic acid (ccMA) in 48% conversion\nby HPLC, with no AA detected. In comparison, a co-culture of St1 and\nSt2b (harboring the pAA2 plasmid) transformed PCA to adipic acid in\n49% yield, presumably due to mild T5 as opposed to strong T7-induced\nexpression of BcER from pAA2 in vivo ( Figure S1 and Table S1 ). However, when St1, St2b,\nand St3 were combined, ccMA was produced as the major product in 19%\nyield, with the remaining material being unreacted TA—indicating\nboth TPADO and BcER activity as pathway bottlenecks. Having\nconfirmed that AA and ccMA could be produced from PCA and\nTA, respectively, in a multicell biotransformation, we hypothesized\nthat localization of all the pathway enzymes to a single cell would\nincrease product conversions. We therefore moved on to design genetic\nconstructs that could be used to balance the expression and activity\nof TPADO and BcER with the aim of producing a single E. coli strain to produce AA from TA. To this end, the expression cassette\nencoding the tphA1–2 / B2 and dcddh genes was transferred from our reported pVan1 plasmid\nand ligated into a pACYC-derived backbone, creating pPCA1 ( Figure 3 and Figure S2 ). The pACYC vector is a medium/low\ncopy-number plasmid with a p15A origin-of-replication and Cam R selection marker that are compatible with the pQLinkN vector\nharboring the remaining pathway genes. The aroY and kpdB genes were inserted into pAA2 by ligation-independent\ncloning to generate pAA3 ( Figure S1 ). Plasmids\npPCA1 and pAA3 were then transformed into E. coli BL21(DE3), and a whole-cell biotransformation was conducted to determine\nwhether adipic acid could be detected when TA was added to suspended\nwhole cells. Unfortunately, no AA was observed from these reactions\nby HPLC. The major product was ccMA in 92% yield, reconfirming that\nthe expression and/or activity of BcER was a limiting step in the\npathway. The increased yield of ccMA, however, validated that single\ncell co-expression of the pathway enzymes was sufficient to increase\nproduct flux. Figure 3 Maps of the pPCA1 and pAA4 plasmids and chromosomal loci\nfor tpado\nand dcddh integration in E. coli . We therefore designed a revised pAA3 plasmid (pAA4, Figure 3 and Figure S2 ) to express the aroY , kpdB , catA , and bcER genes as part\nof a polycistronic mRNA. Here, bcER was assembled\nat the 5′ end of the operon and contained a strong ribosomal\nbinding site (RBS), followed by aroY (medium RBS), kpdB (medium RBS), and catA (weak RBS)\n( Figure 3 ). The latter\nis the most active enzyme in the pathway and therefore required minimum\nexpression for maximum product flux. We also hypothesized that this\narrangement would maximize expression of bcER and\ntherefore enable increased conversion of ccMA to AA from TA. We also\nintegrated the PCA1 cassette into the genome of E. coli BL21(DE3) via λ-Red recombineering using CRISPR/Cas9 as a\nnegative selection tool, 20 with the aim\nof decreasing the overall metabolic burden to the host cell ( Figure 3 ). IS6, SS3, and\nSS9 loci were selected in the E. coli BL21(DE3) genome\n(positions 2580897, 1308935, and 3979535, respectively) as equivalent\nsites in E. coli BW25113 were originally reported\nto be suitable for genomic integration, 21 generating the strains E. coli IS6::PCA1, E. coli SS3::PCA1, and E. coli SS9::PCA1.\nSuccessful knock-ins were confirmed by colony PCR using primers that\nbind to genomic regions flanking the corresponding insertion loci.\nThe pAA4 plasmid was transformed into these strains, or co-transformed\ninto E. coli BL21(DE3) with pPCA1 and used in a whole-cell\nbiotransformation experiment. Singly transformed PCA1 integrants were\nalso co-transformed with pACYC plasmids encoding for the molecular\nchaperones DnaK-DnaJ-GrpE (pKJE7), GroEL-GroES (pGro7), and Trigger\nFactor (pTf16) to facilitate soluble folding of the heterologous pathway\nenzymes. Unfortunately, no adipic acid was produced from E. coli BL21(DE3)_pPCA1_pAA4 cells after 24 h at 21 °C.\nAdipic acid\nwas detected in 19% yield from this strain when cells were fed ccMA\n( Figure 4 B, Figures S11 and S12 ). The genome integration\nof PCA1 to IS6, SS3 or SS9 loci did not increase adipic acid production\nand reduced the overall levels of ccMA ( Figure 4 A,B). Comparison of PCA production from TA\nin E. coli IS6::PCA1, E. coli SS3::PCA1,\nand E. coli _pPCA1 confirmed that PCA formation was\nsignificantly increased in strains containing pPCA1 ( Figure 4 A and Figure S9 ). Chaperone co-expression reduced and/or abolished ccMA\nlevels in all cells expressing pAA4 and PCA1 and did not result in\nany detectable AA from any PCA1 integrated strains. The PCA and AA\nexpression cassettes from pPCA1 and pAA4 were also swapped (generating\nplasmids pAA5 and pPCA2; Figure S3 ) to\nincrease the copy number of tpado and dcddh genes and increase flux to PCA from TA. However, kinetic analysis\nof PCA and ccMA production indeed confirmed that PCA was produced\nmore rapidly in E. coli _pPCA2_pAA5 from 2 to 4 h\nbut that ccMA titers were ultimately lower in this strain (31% yield)\nwhen compared to E. coli _pPCA1_pAA4 (93% yield; Figure S14 ). Due to low TPADO activity and increased\nccMA titers using pPCA1 and pAA4, we decided to proceed with this\ntwo-plasmid system and to focus our studies on optimization of the\nwhole-cell biotransformation. Figure 4 Comparing the reactivity of PCA1 plasmid and\ngenome integration\nstrains. (A) Whole-cell reactions to protocatechuate. (B) Whole-cell\nreactions to muconic acid and adipic acid. Product concentrations\nwere determined by reverse-phase HPLC relative to an internal standard\nof caffeine. All data shown are an average of three replicate experiments\nto one standard deviation. [a] ccMA was added instead of TA. We began by assessing AA production from TA by\nour engineered strains\nunder fermentation conditions, anticipating that addition of TA immediately\nafter synthesis of the pathway enzymes would mitigate the instability\nof TPADO and/or BcER. As such, TA was added to cultures at OD 600 0.5 and reactions were sampled periodically over 7 days.\nInterestingly, no AA could be detected from cells transformed with\npPCA1 and pAA3 plasmids. Cells transformed with pPCA1 and pAA4 produced\nadipic acid in 6% yield, and E. coli IS6::PCA1_pAA4\nand E. coli IS6::PCA1_pAA4_pGro7 strains produced\nAA in 6% and 5% yield, respectively. Altering the fermentation growth\nmedia or carbon source eliminated AA production from all strains and\nproduced PCA as the primary product. BcER activity therefore\ncontinued to be the rate-limiting step\nin the pathway, so we progressed to investigating methods to overcome\nthis using chemical approaches ( Figures S17–S20 ). Chemical methods included the use of biocompatible chemistry to\nreplace the activity of BcER by converting ccMA to AA using a H 2 -generating strain of E. coli (DD-2) and\na membrane-bound Pd catalyst. Microbial H 2 (g) has been\nshown to reduce ccMA in vitro using the Royer Pd\ncatalyst, 22 but this has not been combined\nwith metabolic ccMA generation. To this end, ccMA was produced from E. coli _pPCA1_pAA4 before cells were removed by centrifugation\nand the supernatant containing ccMA introduced to a culture of E. coli DD-2. This engineered strain contains an insulated\npathway consisting of a pyruvate ferredoxin oxidoreductase (PFOR)\nfrom Desulfovibrio africanus , hydrogenase maturation\nfactors from Chlamydomonas reinhardtii , and a ferredoxin\nand [Fe–Fe] hydrogenase from Clostridium acetobutylicum , which together enable the anaerobic production of H 2 (g) from d -glucose. 23 Indeed,\nthis enabled the bio-hydrogenation of metabolic ccMA to AA in 80%\nyield using the biocompatible Royer Pd catalyst ( Figure 5 ). Intriguingly, both Pd/CaCO 3 and Pd/C were biocompatible but inactive bio-hydrogenation\ncatalysts, affording 14% and 64% of the monoreduced product 2-hexenedioic\nacid (2-HDA), respectively. The increased reactivity of Royer Pd is\ntherefore likely due to an attractive electrostatic interaction between\nthe cells, ccMA, and the positively charged polyethyleneimine catalyst\nsupport. Hydrogen-gas-producing E. coli and biocompatible\nchemistry can therefore be used to overcome the lack of activity of\nBcER within larger heterologous biosynthetic pathways. Figure 5 Bio-hydrogenation of\nmetabolic cis , cis -muconic acid from E. coli BL21(DE3)_pPCA1_pAA4,\nfollowed by E. coli DD-2 and biocompatible Pd catalysts.\nProduct concentrations were determined by reverse-phase HPLC relative\nto an internal standard of caffeine. Data shown are an average of\nthree replicate experiments. 2HDA = 2-hexenedioic acid. Finally, we examined the use of cells supported\nin alginate hydrogel\nas a method to increase the activities of TPADO and BcER and thus\nthe yield of AA. The use of calcified alginate hydrogels is known\nto increase the stability of enzymes in vitro ( 24 , 25 ) and to improve the downstream purification of whole-cell biotransformations;\nhowever, few studies report the use of alginate immobilization to\nincrease the stability of heterologous enzymes in de novo pathways in E. coli . More specifically, this has\nnot been applied to stabilizing BcER within a microbial adipic acid\npathway despite reported instability in vivo . 20 , 26 To this end, we were delighted to observe that, when TA was added\nto cells of E. coli _pPCA1_pAA4 supported in alginate\nhydrogels (termed alg- E. coli ), AA conversion was\nincreased from 0% to 79% ( Figure 6 B). Adipic acid was not detected in control samples\nlacking cells, alginate, and/or pathway enzymes or in the presence\nof supported dead cells, confirming that this was a microbe-mediated\nchemical transformation. A time-course experiment confirmed that alginate\nimmobilization increased the stability of BcER, showing rapid formation\nof 2-hexenedioic acid from ccMA after 6 h and then gradual conversion\nto AA over 24 h ( Figure 6 C). In comparison, no reduction of ccMA to AA was observed after\n24 h in the absence of the alginate support. Finally, adipate-rich\nproduct streams could be readily isolated from alg- E. coli reactions by filtration of the cell-containing alginate beads from\nthe reaction. Muconic acid reduction in reactions with non-immobilized\ncells or in alg- E. coli with smaller bead sizes also\nproduced less adipic acid, indicating that the alginate support likely\nimproves the oxygen tolerance and/or stability of the [4Fe–4S]-containing\nBcER enzyme. This was confirmed by observing loss of BcER activity\nin <6 h in the absence of the alginate support ( Figure S20 )—a time point which precludes the accumulation\nof ccMA by upstream pathway enzymes in E. coli _pPCA1_pAA4\nstrains. Figure 6 E. coli supported in calcium alginate beads enables\nbio-adipic acid production from a post-consumer plastic bottle. (A)\nPET depolymerization and upcycling in whole cells and in encapsulated\nwhole cells. Reactions were carried out at OD 600 60 in\nM9 media with 166 mg/L TA at 21 °C with shaking at 220 rpm for\n24 h. (B) Photograph of alg- E. coli cells. (C) Metabolite\nformation during biotransformation reactions. Product concentrations\nwere determined by reverse-phase HPLC relative to an internal standard\nof caffeine. Data shown are an average of three replicate experiments\nto one standard deviation. Following this finding, three factors were explored\nto improve\nthe activity of the de novo pathway: (i) increased\ncofactor availability and recycling, (ii) pH-dependent TA diffusion\ninto the cell, and (iii) BcER inhibition by upstream intermediates.\nFirst, NADH availability was identified as a potential key limitation\nas the de novo pathway to adipic acid from TA generates\n3 mol equivalents of NAD + and only regenerates 1 mol equivalent\nof NADH. We therefore co-expressed the NAD + -dependent formate\ndehydrogenase Fdh from the methylotrophic bacterium Pseudomonas sp. 101 (EC 1.17.1.9) downstream of medium or strong constitutive\npromoters in modified pPCA1 plasmids (pPCAX1–3, Table S1 ) in immobilized alg- E. coli _pPCA X _pAA4 strains, generating 1 mol of NADH from\n1 mol each of NAD + and formate. 27 However, fdh co-expression resulted in no change\nin adipic acid formation at increased TA concentrations in either\nthe presence or absence of exogenous formate. Such strategies have\nbeen successfully applied to counteract nicotinamide redox imbalance\nin other metabolically engineered strains. 28 Reactions containing Fdh and run in the presence of formate also\nbecame alkaline over time—conditions that are known to inhibit\nTA diffusion into the cell by increasing repulsive ionic interactions\nwith the negatively charged outer membrane (p K a 1 3.5 and p K a 2 4.5 in H 2 O). This was confirmed by observing increased\nconversion of TA into PCA in E. coli _pPCA1_pAA4 cells\nat pH 5. However, this was accompanied by decreased downstream pathway\nactivity ( Figure S22 ). We therefore moved\non to examine the use of increased concentrations of glucose and the\nuse of alternative carbohydrate feedstocks as a source of NADH in vivo . 29 , 30 Switching the carbon source from d -glucose to d -mannitol or d -sorbitol—two\nhexose sugar alcohols that generate more NADH equivalents than glucose\nduring glycolysis—had no effect on adipic acid levels, nor\ndid the co-addition of glucose and sorbitol at 1:1 mol equivalent\nor increasing the concentration of glucose 2-fold ( Figure S24 ). Together, these data combined with the observed\nincrease in adipic acid production from alginate reactions run in\ndiluted media with reduced glucose concentration ( Figure 6 A,C) make cofactor availability\nand redox balance an unlikely limiting factor at this scale but nevertheless\none that should be a primary consideration in subsequent strain and\nprocess designs. Finally, we examined the inhibition of BcER by TA.\nInterestingly, TA was found to inhibit BcER activity at high substrate\nconcentrations, presumably due to similarities in three-dimensional\nstructure between the cis -oid diacid in ccMA and\nthe 1,4-disubstituted aromatic diacid in TA ( Figure S22 ). Inhibition of BcER by TA in combination with pH-dependent\nTA diffusion and flux at physiological pH are therefore principal\nconsiderations that will be the focus of our future work. Having\nconfirmed that we can convert TA into AA using engineered E. coli , we set out to examine whether alg- E. coli _pPCA1_pAA4 could be used to valorize post-consumer plastic waste.\nTo this end, a discarded PET bottle was depolymerized using aq. NaOH\nand ethanol (90 °C, 1 h), yielding white flakes of pure TA by 1 H NMR analysis. To our delight, addition of crude TA samples\nto alg- E. coli _pPCA1_pAA4 cells resulted in 65 mg/L\nAA by HPLC. To further demonstrate the applicability of this system\nand avoid the compositional variability of PET bottles, 31 we also examined the use of pure industrial\nPET waste. Hot stamping foils (HSFs) are used across multiple industries\nfor the rapid depositing of ultrathin release single-use lacquer and\nadhesive labels. In 2022, the global demand for HSFs was 2.5 billion\nm 2 , and this is estimated to generate 40,000 tons of PET\nwaste per annum. Pleasingly, depolymerization of HSF samples under\nidentical alkaline hydrolysis conditions (aq. NaOH, EtOH, 90 °C,\n1 h) yielded pure TA by 1 H NMR, which could be converted\nto AA under our optimized biotransformation conditions in 66% yield\n(96 mg/L) using alg- E. coli _pPCA1_pAA4 cells ( Figure 7 ). This increased\nconversion establishes HSFs as a source of PET waste that is highly\namenable to microbial upcycling processes. Figure 7 Microbial upcycling of\nindustrial PET stamping foil waste. (A)\nImage of PET stamping foils. (B) 1 H NMR spectrum of foil-TA.\n(C) Bio-upcycling of PET/TA samples into adipic acid. Data shown are\nan average of three replicate experiments to one standard deviation."
} | 6,375 |
26839587 | PMC4736557 | pmc | 8,658 | {
"abstract": "Background The acyl carrier protein (ACP) is an essential and ubiquitous component of microbial synthesis of fatty acids, the natural precursor to biofuels. Natural fatty acids usually contain long chains of 16 or more carbon atoms. Shorter carbon chains, with increased fuel volatility, are desired for internal combustion engines. Engineering the length specificity of key proteins in fatty acid metabolism, such as ACP, may enable microbial synthesis of these shorter chain fatty acids. Results We constructed a homology model of the Synechococcus elongatus ACP, showing a hydrophobic pocket harboring the growing acyl chain. Amino acids within the pocket were mutated to increase steric hindrance to the acyl chain. Certain mutant ACPs, when over-expressed in Escherichia coli , increased the proportion of shorter chain lipids; I75 W and I75Y showed the strongest effects. Expression of I75 W and I75Y mutant ACPs also increased production of lauric acid in E. coli that expressed the C12-specific acyl-ACP thioesterase from Cuphea palustris . Conclusions We engineered the specificity of the ACP, an essential protein of fatty acid metabolism, to alter the E. coli lipid pool and enhance production of medium-chain fatty acids as biofuel precursors. These results indicate that modification of ACP itself could be combined with enzymes affecting length specificity in fatty acid synthesis to enhance production of commodity chemicals based on fatty acids. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0430-4) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions In sum, we have shown that ACP, an essential protein in fatty acid metabolism, can be modified by site-directed mutagenesis to skew cellular lipid pools toward smaller acyl chain lengths. Specifically, expressing certain mutant ACPs enhanced the level of C14 fatty acids in membrane lipids, and by co-expressing mutant ACPs with a chain-length specific thioesterase production of a medium-chain free fatty acid (lauric acid) was enhanced. These results are consistent with a hypothesis that bacterial ACPs influence lipid chain-length during fatty acid synthesis. Other enzymes involved in fatty acid synthesis also likely affect chain-length, and engineering modified acyl chain specificity has been similarly achieved. For example, FabB and FabF catalyze elongation of fatty acid chains (Fig. 1 ), and have a clearly defined pocket that should accommodate carbon chains up to about 18 [ 19 ]. Val et al. engineered the FabF pocket to accommodate a maximum of six carbons [ 20 ]. Similarly, the cyanobacterial aldehyde decarbonylase solved structure [ 21 , 22 ] contains electron density corresponding to a C18 fatty acid or aldehyde; Khara et al. modified this enzyme to have specificity for medium-chain substrates [ 22 ]. The C8-, C12-, and C14-specific plant-derived acyl-ACP thioesterases apparently also control length of fatty acid products, although the underlying structural mechanisms have not been identified. Since FFAs contain the hydrophilic carboxylic acid functional group, they are not ideal fuel molecules. Instead, FFAs can act as precursors to further enzymatic modification for transformation into highly desired fuel molecules such as fatty alcohols and alkanes. Engineering such enzymes (e.g., aldehyde decarbonylases, acyl-ACP reductases, and carboxylic acid reductases) toward shorter carbon chain substrate recognition will likely be key to tailoring biofuel formulations. To achieve the ultimate goal of efficient biofuel synthesis, it may be necessary to engineer the length specificity of several enzymes—most such enzymes have evolved to handle chains of 16–18 carbons, but shorter chains are desired in fuels. This technology could help to optimize biofuel yield and molecular makeup, which would benefit the goal of developing energy sources alternative to fossil fuels.",
"discussion": "Results and discussion To enhance production of medium-chain fatty acids, we constructed mutants of ACP designed to decrease the acyl chain pocket size (Fig. 2 ). Variants of the cyanobacterial ( S. elongatus ) ACP were expressed in an E. coli host. We chose S. elongatus ACP due to its potential compatibility with recently discovered enzymes of the cyanobacterial alkane biosynthesis pathway [ 15 ], which could enable microbial synthesis of fatty alcohol or alkanes. The native E. coli ACP gene was left intact, as we found that its knockout could not be rescued by complementation from expression of wild-type E.coli ACP encoded on a plasmid (data not shown). To determine which hydrophobic residues of S. elongatus ACP lined the inner, acyl chain pocket, we constructed a structural homology model using the published crystal structure of E. coli ACP bound to a C10 fatty acyl chain (2FAE) as a template (Fig. 2 ). We constructed a number of single amino acid mutants by exchanging small hydrophobic side-chain residues, such as isoleucine or leucine, with bulkier hydrophobic side-chains such as phenylalanine, methionine, tyrosine, or tryptophan. ACPs initially fold into an inactive apo state. Conversion to the active holo state is achieved through post-translational modification whereby 4′-phosphopantetheine is transferred from co-enzyme A (CoA) to a specific serine residue on the apo-ACP (Ser39 on S. elongatus ACP) [ 8 , 16 ]. Acyl carrier protein over-expression may reduce the CoA pool and lead to toxic accumulation of apo-ACP, which inhibits sn-glycerol-3-phosphate acyltransferase [ 16 , 17 ], so as a quick check for functional expression of recombinant ACPs, we measured culture growth kinetics over 15 h. Compared to controls, cells over-expressing wild-type (‘WT’) E. coli ACP (Ec-ACP), WT S. elongatus ACP (Se-ACP), or mutant Se-ACPs all showed suppressed growth at low levels of induction and worsened at higher induction levels (Additional file 1 : Figure S1; Additional file 2 : Figure S2), suggesting that these recombinant cyanobacterial ACPs were expressed and properly folded. To analyze the effect of mutant Se-ACPs on lipid pools, we used gas chromatography–mass spec (GC–MS) to characterize fatty acid methyl esters (FAMEs) derived from lipid pools in Se-ACP over-expressing cells. We compared ratios of FAME peak areas for each sample to minimize effects of differences in growth and sample extraction. We detected peaks for FAMEs derived from the naturally most abundant palmitic acid (C16) and the shorter, less abundant myristic acid (C14) and quantified these peaks in all sample spectra and calibrated to molar concentrations based on a standard curve (Additional file 3 : Figure S3). Together, C14 and C16 accounted for >90 % of total fatty acids extracted in all samples (Additional file 4 : Figure S4; Additional file 5 : Figure S5). The concentration ratios of C14–C16 were calculated and compared across controls and cells expressing Se-ACP point mutants. For all uninduced samples, the C14:C16 ratio was around 0.1 (Fig. 3 a). After induction, only the I75 W and I75Y Se-ACP mutants demonstrated a statistically significant increase in the C14:C16 ratio relative to cells expressing WT Se-ACP: the mutants, respectively, caused 3- and 2.7-fold increases ( p < 0.05, two-tailed student-t test; Fig. 3 b), indicating that their lipid pools had shifted toward shorter acyl chains. Mutants that replaced Leu49 or Ile57 did not increase the proportions of shorter fatty acids compared to over-expressing WT ACPs. The side chain of isoleucine 75 is positioned in the hydrophobic pocket close to the terminus of the acyl chain, more so than residues 49 and 57, which contact the side of the acyl chain (Fig. 2 a) [ 12 ]. Mutating Ile75 to phenylalanine or methionine may cause slight shifts in lipid pool chain-length composition (Fig. 3 ). Homology modeling indicated that the Tyr75 and Trp75 side-chains protrude roughly two carbon–carbon bond distances further into the hydrophobic acyl chain pocket than an isoleucine at this position (Fig. 2 b, c; only I75 W shown). Therefore, I75 W and I75Y Se-ACP mutants may directly hinder elongation from C14 to C16 in fatty acid synthesis and skew the fatty acid pool toward shorter chain lengths. Fig. 3 GC–MS analysis of cellular lipids in single ACP mutants. a Ratios of C14–C16 molar concentrations for uninduced ( black ) and induced ( red ) strains: no vector (NO), empty vector (MT), WT E. coli ACP (EC), WT S. elongatus ACP (SE). b Fold changes of induced vs. uninduced C14:C16 ratios. The I75 W and I75Y mutants have significantly increased C14:C16 ratios as compared to expressing WT Se-ACP (* p < 0.05, two-tailed student- t test). Data represent triplicate biological measurements. Error bars are standard error of the mean (S.E.M) To explore the potential to further skew cellular lipids toward short-chain lengths, particularly those shorter than 14 carbons long, we introduced secondary point-mutations in addition to the Se-ACP I75 W or I75Y mutations. Amino acids with small hydrophobic side-chains such as isoleucine, valine, or alanine were exchanged for a bulkier methionine, a polar glutamine, or a hydrophilic arginine. Double mutant Se-ACPs did not significantly increase the C14:C16 ratio beyond either single I75 W or I75Y mutation alone (Additional file 3 : Figure S3), and did not cause observable production of chains shorter than C14. As an additional control, the Se-ACP serine 39 residue, which is post-translationally modified with 4-phosphopantetheine, was mutated to alanine (S39A), thereby generating an inactive, obligate apo-ACP. Over-expressing this inactive ACP resulted in similarly low C14:C16 ratio compared to WT (Fig. 3 ). Growth was suppressed by over-expressing this mutant protein, suggesting that the protein was correctly folded [ 16 , 17 ]. These results indicated that expression of mutant ACPs could be used to enhance production of a medium-chain fatty acid. To explore conditions for optimal production, we characterized C14:C16 ratios over a 24-h time course. The lipid pool composition shows that the highest C14:C16 ratio occurs around 5-h post-induction (Fig. 4 ). Longer induction times resulted in a decreased C14:C16 ratio for all strains, particularly for Se-ACP I75 W and I75Y mutants, which fell and became indistinguishable from controls by 24 h. This highlights the importance of growth phase on lipid composition. During exponential growth, when cells are actively dividing and building new membranes, fatty acid metabolism is highly active, and an abundance of mutated ACPs with reduced pocket sizes likely biases the fatty acid pool toward shorter acyl chains [ 18 ]. It may be that membrane synthesis proceeds with greater fidelity as cell growth slows. Alternatively, short-chain fatty acids may be actively replaced with fatty acids of the correct length, which would be more apparent in stationary phase when new C14 fatty acids are not being added to membrane lipids. Fig. 4 Time Course of C14:C16 Ratios Se-ACP I75 W and I75Y demonstrating the highest C14:C16 cellular lipid ratio at 5 h after induction during the growth phase. As the cell cultures saturate past 14 h, the ratios decrease to the baseline of around 0.05–0.1. Data represent triplicate biological measurements. Error bars are S.E.M We next tested the effect of mutant ACPs on production of lauric acid (C12). A thioesterase that specifically produces 12-carbon chains ( UcFatB2 from Cuphea palustris ) [ 6 ] was co-expressed with wild-type and mutant Se -ACPs, and FFA production was measured by GC–MS analysis of fatty acid ethyl esters (FAEE) derived from the produced FFAs (Fig. 5 ). We hypothesized that increased levels of shorter chain acyl-ACPs would serve as substrates to the medium chain-specific thioesterase and further increase the yield of medium chain FFAs. In conjunction with expressing the C12 thioesterase, strains over-expressing I75 W or I75Y mutant ACPs significantly increased medium chain FFA yields (Fig. 5 ); all controls produced less FFA than the I75 W or I75Y mutants. (There were significant differences between the various controls, presumably reflecting the fact that overproducing various forms of ACP can affect fatty acid metabolism by, for example, depleting CoA or non-productively interacting with other enzymes [ 16 , 17 ]). Combining mutations did not further enhance FFA production (Additional file 6 : Figure S6). In addition, FFA yields were uncorrelated to differences in growth rates among all the strains (Additional file 7 : Figure S7) and were not affected by beta oxidation knock out (Additional file 8 : Figure S8). Fig. 5 Free fatty acid production by C12 thioesterase. a Representative GC-MS trace of FAEEs derived from cell cultures shows thioesterase specificity toward 12-carbon acyl chains. b FFA concentrations measured from cell cultures at 6 h ( blue ) and 24 h ( black ) post-induction of both the C12 thioesterase and the indicated ACP. The Se-ACP I75 W and I75Y mutants and their derivatives yield more FFA than controls. Data represent triplicate biological measurements. Error bars are S.E.M"
} | 3,302 |
27119630 | PMC4850321 | pmc | 8,659 | {
"abstract": "Over the last decade, functionally designed DNA nanostructures applied to lipid membranes prompted important achievements in the fields of biophysics and synthetic biology. Taking advantage of the universal rules for self-assembly of complementary oligonucleotides, DNA has proven to be an extremely versatile biocompatible building material on the nanoscale. The possibility to chemically integrate functional groups into oligonucleotides, most notably with lipophilic anchors, enabled a widespread usage of DNA as a viable alternative to proteins with respect to functional activity on membranes. As described throughout this review, hybrid DNA-lipid nanostructures can mediate events such as vesicle docking and fusion, or selective partitioning of molecules into phase-separated membranes. Moreover, the major benefit of DNA structural constructs, such as DNA tiles and DNA origami, is the reproducibility and simplicity of their design. DNA nanotechnology can produce functional structures with subnanometer precision and allow for a tight control over their biochemical functionality, e.g., interaction partners. DNA-based membrane nanopores and origami structures able to assemble into two-dimensional networks on top of lipid bilayers are recent examples of the manifold of complex devices that can be achieved. In this review, we will shortly present some of the potentially most relevant avenues and accomplishments of membrane-anchored DNA nanostructures for investigating, engineering, and mimicking lipid membrane-related biophysical processes.",
"conclusion": "Conclusions DNA nanotechnology is a rapidly evolving field promising unique advantages for controllable nanoscale engineering of biomolecules and biologically relevant processes. The compelling self-assembly properties of DNA have opened up a wide spectrum of biological applications besides its original role as information storage. Of particular recent interest is the engineering of DNA-based tools for lipid membrane biophysics. The possibility to functionalize oligonucleotides with cholesteryl or other lipophilic moieties is of major relevance to enable the implementation of DNA nanostructures into lipidic environments. Considering the partitioning properties of membrane-anchoring tags and the specificity of DNA interactions, it is thus possible to build all kinds of dynamic and functional membrane-active modules. In addition, since the development of the DNA origami technique by Rothemund in 2006 ( 5 ), the complexity of DNA-based nanodevices has dramatically increased. Biomimetic devices performing advanced biofunctional tasks can now be conceived and created. As an illustrative example, we can particularly highlight origami structures mimicking membrane-sculpting proteins, e.g., involved in endocytosis. The capability of those artificial nanostructures to undergo isotropic-nematic transitions on lipid bilayers ( 57 ), to show membrane-assisted oligomerization ( 60 ) and most notably, to macroscospically scaffold and deform giant vesicles ( 61 ), is of great biological interest. Although these phenomena have been all demonstrated with planar origami structures, a next key step would be to develop curved DNA origami amphipathic objects that mimic the properties of coat (e.g., clathrin) or BAR domain proteins and, in this sense, investigate in a controllable manner the influence of structure, in particular, curvature on membrane bending. Another future hallmark for such nanostructures would be the implementation of externally controllable conformational switches that, in a minimalistic way, allow modeling the structural dynamics involved in biological membrane transformation and curvature recognition events. In summary, DNA nanotechnology appears as an extremely valuable and unrivaled bioengineering tool. The biocompatibility of DNA, its sequence specificity, and controllable self-assembly makes this molecule particularly exciting for nanofabrication of biomimetic components. When decorated with lipid moieties, DNA nanodevices can be excellent artificial systems for tackling and recreating minimal tasks of membrane-embedded proteins. These intrinsic properties make DNA nanotechnology an optimal toolkit for reconstitution of biophysical processes in bottom-up synthetic biology."
} | 1,070 |
39817101 | PMC11730487 | pmc | 8,662 | {
"abstract": "Cyanobacteria are widespread, photosynthetic, gram-negative bacteria that generate numerous bioactive secondary metabolites via complex biosynthetic enzymatic machinery. The model cyanobacterium Picosynechococcus sp. strain PCC 7002, hereafter referred to as PCC 7002, contains a type I polyketide synthase (PKS), termed olefin synthase (OlsWT), that synthesizes 1-nonadecene and 1,14-nonadecadiene: α-olefins that are important for growth at low temperatures. The putative biochemistry encoded by the PKS domains suggests that OlsWT will create an olefin with one additional carbon relative to the original substrate (+1 mechanism). The first domain in the multi-module OlsWT protein has homology to fatty acyl-AMP ligases (FAALs) that typically activate free fatty acids prior to creating novel thioester linkages. Paradoxically, unmodified wildtype PCC 7002 is not known to maintain a substantial pool of free fatty acids, and prior work demonstrated conversion of exogenous pentadecanoic acid to 1-octadecene instead of the expected 1-hexadecene. In this study, we developed PCC 7002 as a heterologous host to facilitate the expression and study of Ols proteins in effort to discover their true substrates. Here, we report the successful expression of two Ols homologs from Geminocystis sp. NIES-3709 and Xenococcus sp. PCC 7305 in PCC 7002 that generated 1-heptadecene and 1-pentadecene, respectively. Through the additional deletion of a gene encoding an acyl–acyl carrier protein (ACP) synthetase (Aas) responsible for activation of exogenous free fatty acids, we demonstrated the expected conversion of exogenously provided odd-chain fatty acids to α-olefins containing one additional carbon. These data suggest that short-lived fatty acids liberated from lipid membranes are the Ols substrate. We subsequently confirmed OlsWT activity on octadecanoic acid via in vitro chrome azurol S assay using a purified FAAL module. Collectively, this work clarifies the in vivo substrate of Ols FAAL domains and identifies the FAAL module as a target for future bioengineering to allow access to desired α-olefins.",
"conclusion": "Conclusions It was previously hypothesized that the Ols substrates were the abundant acyl-ACPs similar to the Aar/Ado pathway. However, the initiating FAAL domain of Ols has homology (29.9% similarity) to characterized FAAL domains that activate free fatty acids. 38,39 Therefore, we sought to confirm free fatty acids as the Ols substrate while also investigating the heterologous expression of Ols homologs capable of synthesizing shorter chain α-olefins. Through deletion of olsWT in PCC 7002 and subsequent genomic integration of ols04 and ols08 , we were able to access 1-pentadecene and 1-heptadecene, respectively. To further confirm free fatty acids as the Ols substrates, feeding studies utilizing odd-chain fatty acids were performed. This led to the production of α-olefins with a net addition of three carbons, indicating that fed free fatty acids are quickly incorporated into fatty acid biosynthesis via Aas, which is consistent with Aas being involved in fatty acid intake and activation to acyl-ACPs. 24,40 The deletion of aas was performed and the direct incorporation of odd-chain free fatty as n + 1 α-olefins was observed. Additionally, in vitro activation of free fatty acids by the OlsWT FAAL-ACP shows that these FAAL domains may also have a substrate selectivity mechanism to preferentially select specific chain lengths. Similar results were observed in the production of secondary metabolite nocuolin A where a FAAL domain fused to an ACP domain preferentially loads hexanoic or octanoic acid to initiate biosynthesis. 14 In this case, the authors were able to observe incorporation of exogenously provided free fatty acids in producing strain Nodularia sp. LEGE 06071, which may be due to lower incorporation of these short-chain fatty acids into the fatty acyl-ACP pool by Aas. Overall, this work indicates that free fatty acids are the substrate of Ols enzymes. Given that free fatty acids do not accumulate in cyanobacteria, these substrates are likely a result of free fatty acids being liberated from the lipid and thylakoid membranes by lipases prior to their reactivation by Aas ( Fig. 2C ). Unfortunately, only a few putative lipases have been characterized in cyanobacteria. One example is LipA in Synechocystis sp. PCC 6803, which hydrolyzes glycolipids at the sn -1 position to increase fatty acyl recycling and photosystem repair under high light conditions. 41,42 However, the LipA homolog in PCC 7002 (SYNPCC7002_A1441) has not been investigated for its involvement in these processes nor as the source of the free fatty acid substrate for OlsWT. Another ORF from Synechocystis sp. PCC 6803, sll0482, is suspected to be responsible for diacylation at the sn -2 position of glycolipids, but there is no homolog of this lipase in PCC 7002 to perform an analogous function. 43 During this work, we identified a putative hydrolase encoded directly upstream of olsWT in PCC 7002 (SYNPCC7002_A1174), which contained conserved domains typically associated with alpha/beta hydrolases. We hypothesized that this uncharacterized enzyme could be at least partially responsible for liberation of these fatty acids for Ols incorporation. Deletion of this hydrolase produced no changes in α-olefin production (Fig. S5, ESI † ). Although, this may be a result of a redundant lipase capable of functional complementation. A more complete analysis of the lipase environment in PCC 7002 will facilitate future engineering efforts to increase α-olefin production.",
"introduction": "Introduction Cyanobacteria synthesize and maintain cell and thylakoid membranes that differ significantly from other bacteria. Cyanobacterial membranes consist mostly of glycolipids with the major phospholipid found in the thylakoid membrane being phosphatidylglycerol. 1 In addition to these lipid components, cyanobacterial membranes contain hydrocarbons in either alkane or alkene forms. Cyanobacteria hydrocarbons – including alkanes, internal olefins, and terminal olefins – are implicated in maintaining cell size, growth, and membrane properties. 2 These alkanes/alkenes also play an important role in cyanobacterial halotolerance and temperature adaptation, through modulating cyclic electron flow. 3–5 The more widely distributed pathway for alkane biosynthesis is the fatty acyl-ACP reductase/aldehyde deformylating oxygenase (Aar/Ado) pathway. 6 The Aar reductively cleaves acyl-ACPs to produce a fatty aldehyde that undergoes deformylation catalyzed by Ado to produce an alkane with the net loss of one carbon. 7 Cyanobacterial terminal alkenes (α-olefins) are produced by a type I polyketide synthase (PKS) termed olefin synthase (OlsWT). OlsWT was first identified in Picosynechococcus sp. strain PCC 7002, hereafter referred to as PCC 7002, and has been previously evaluated by our group and others. 5,8–10 The primary hydrocarbons produced by OlsWT in PCC 7002 are 1-nonadecene and 1,14-nonadecadiene. OlsWT is a multidomain enzyme consisting of a fatty acyl-AMP ligase (FAAL) domain hypothesized to initiate transfer of a fatty acyl chain to the first acyl carrier protein (ACP o ) domain. The acyl chain is subsequently extended by a two-carbon unit ( via malonyl-CoA) by the ketosynthase (KS) and acyltransferase (AT) domains. The resulting ACP-domain-linked β-keto intermediate is reduced by the ketoreductase (KR) domain. Finally, the β-hydroxyl group of the acyl chain is activated via sulfation by the sulfotransferase (ST) domain. The sulfate provides a leaving group for simultaneous decarboxylation and sulfate elimination catalyzed by the thioesterase (TE) domain. Overall, the combined enzymology releases an odd-numbered n + 1 α-olefin relative to the n -carbon substrate ( Fig. 1A ). The exact substrate for the initial fatty acyl chain activated by the FAAL domain of OlsWT remains unclear. Canonically, FAALs activate free fatty acids via a two-step adenylation mechanism utilizing adenosine triphosphate (ATP) to first form the acyl adenylate (acyl-AMP) and eject pyrophosphate. 11 The activated acyl chain is then transferred to the 4′-phosphopantetheinyl arm of the downstream ACP domain. This second step distinguishes FAALs from fatty acyl-CoA ligases (FACLs) that transfer the acyl-AMP to coenzyme A. 12,13 FAAL domains in cyanobacterial secondary metabolite biosynthetic gene clusters have been described and evaluated for substrate usage previously. For example, the incorporation of free fatty acids into the scaffolds of nocuolin A, the hapalosins, and the chlorosphaerolactylates has been demonstrated using in vivo labeling and in vitro assays. 14–16 However, cyanobacteria have not been found to actively produce or accumulate a large pool of free fatty acids directly from either acyl-CoAs or acyl-ACPs. 17,18 Likewise, the presence of triacylglycerols (TAGs) has been observed in only a small minority of cyanobacteria, namely the Nostacales order. 19,20 Like other bacteria, cyanobacteria generate fatty acids and lipids through fatty acid biosynthesis with pathway intermediates covalently linked to ACPs as acyl-ACP thioesters. 21 To date, no native acyl-ACP thioesterases (TEs) have been identified in cyanobacteria that would hydrolyze free fatty acids directly from ACPs involved in fatty acid biosynthesis. In contrast, native plant acyl-ACP TEs, such as FATA or FATB, directly hydrolyze acyl-ACPS from fatty acid biosynthesis to produce free fatty acids. 22,23 Fig. 1 (A) The OlsWT FAAL domain selects fatty acyl chains from primary metabolism for conversion to α-olefin hydrocarbons. (B) Optimized genetic manipulations in Picosynechococcus sp. PCC 7002 using homologous recombination with a selective marker followed by removal via a CRE recombinase to provide a markerless deletion. (C) α-Olefin production in PCC 7002 strains expressing Ols homologs. All data represent the mean ± s.d. of biological triplicates. In general, cyanobacteria also lack complete, active β-oxidation pathways, 24,25 as they do not contain the genes encoding for key β-oxidation enzymes such as 3-hydroxyacyl-CoA dehydrogenase (FadB), acyl-CoA dehydrogenase (FadE), and 3-ketoacyl-CoA thiolase (FadI). 16 FadD acyl-CoA ligase homologs that would activate long chain fatty acids to the CoA species have been annotated for several cyanobacterial species within the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. 26 However, these are proposed to be mis-annotations of acyl-ACP synthetases (Aas), which are also adenylate-forming enzymes that cyanobacteria use to incorporate exogenous fatty acids into lipid and fatty acid biosynthesis via the formation of a thioester to an ACP. 21 These acyl-ACP Aas products are then elongated via fatty acid biosynthesis and ultimately condensed into lipids. In prior work, we detected elevated production of 1-octadecene in PCC 7002 strains fed pentadecanoic acid, but not the expected n + 1 product 1-hexadecene. 8 Therefore, in this study, we developed PCC 7002 as a heterologous host to facilitate the expression and study of Ols proteins in effort to discover the source of their true substrates. Our findings indicate that free fatty acids liberated from lipid membranes are the native substrates of Ols, raising the possibility of engineering Ols variants that could access shorter chain α-olefins for use as fuel additives.",
"discussion": "Results and discussion Heterologous host PCC 7002 synthesizes variable chain length α-olefins encoded by Ols homologs This work was initially focused on the production of shorter chain α-olefins via expression of Ols homologs known to generate products of varying length. The genes encoding Ols homologs are present in only a small subset of cyanobacterial species (∼10%), none of which have been evaluated in a heterologous host. 27,28 In order to introduce Ols homologs into the PCC 7002 genome, the endogenous olsWT gene was first deleted by introducing a gentamicin resistance cassette via homologous recombination directed by long homology arms. The CRE-lox recombinase was then employed to remove the antibiotic resistance cassette, allowing the generation of a markerless deletion and recycling the selective marker for future genetic manipulations ( Fig. 1B ). 29 Subsequently, genes coding for individual Ols homologs were integrated downstream to the glpK gene in the unmarked Δ olsWT PCC 7002 mutant strain under an inducible synthetic promoter cLac143. 30 Homologs Ols04 from Xenococcus sp. PCC 7305 and Ols08 from Geminocystis sp. NIES-3709 were chosen since they should not produce the endogenous 1-nonadecene. Hydrocarbon products of Ols04 were previously found to be 1-pentadecene and 2-pentadecene, as extracted directly from Xenococcus sp. PCC 7305. While the hydrocarbon profile for Ols08 was not directly quantified, it was hypothesized that it would produce 1-heptadecene based on the previous phylogenetic analysis. 27 The cultures containing Ols homologs were induced with 1 mM IPTG and subsequently extracted and analyzed via gas chromatography/mass spectrometry (GC/MS). Strain AYG042 encoded for the homolog Ols04 from Xenococcus sp. PCC 7305; initial results showed only trace amounts of the expected 1-pentadecene ( Fig. 1C ). PCC 7002 does not accumulate high levels of the 1-pentadecene precursor, tetradecanoic acid, which we hypothesized led to lower titers of this α-olefin product. This was confirmed by a total lipid extraction and fatty acid methyl ester (FAME) analysis of the culture, which showed only small amounts of tetradecanoic species (Fig. S1, ESI † ). For strain AYG043, integration of homolog Ols08 from Geminocystis sp. NIES-3709 resulted in the production of 1-heptadecene and a small amount of 1-nonadecene ( Fig. 1C ). Ols08 was expected to only produce the 1-heptadecene due to its homology to previously characterized olefin synthases. 27 The ability for Ols08 to accept both hexadecanoic and octadecanoic acyl chains was not completely unexpected as OlsWT has been shown to accept both the octadecanoic and heptadecanoic substrates, indicating that these enzymes are moderately flexible in which acyl chain they will load to the initial ACP domain. There was also a small amount of these α-olefins present in the non-induced cultures indicating leaky gene expression from our genetic cassette. The precursor strain to both of these Ols homolog strains, AYG032 (Δ olsWT :: Gm R ), lacked any olefin production confirming the olsWT deletion. These results established that Ols homologs can be successfully expressed in PCC 7002 to generate shorter chain olefins. These results, however, did not answer the fundamental question of the in vivo substrate of the Ols FAAL domain. Even though FAAL domains canonically utilize free fatty acids as substrates, cyanobacteria do not accumulate free fatty acids, and PCC 7002 has no homologs for FATA/B acyl-ACP TE enzymes responsible for the direct hydrolysis of fatty acyl-ACPs (Table S1, ESI † ). 31 Ols homologs utilize free fatty acid to generate α-olefins In order to further investigate the FAAL domain substrate in Ols homologs, cultures were supplemented with non-native odd-chain free fatty acids to track the incorporation of these species into the lipid profile. If free fatty acids are the substrate of the FAAL domain, we hypothesized that the Ols homologs would be able to directly utilize supplemented odd-chain fatty acids. Therefore, strain AYG042 encoding Ols04 was supplied with 0.5 mM tridecanoic acid. Analysis of these lipid products did not contain the expected n + 1 α-olefin product, 1-tetradecene. Interestingly, a new 1-hexadecene product was observed, consistent with the addition of n + 3 carbons, albeit in similarly low titers ( Fig. 2A ). Strain AYG043 encoding homolog Ols08 was also supplemented with 0.1 mM pentadecanoic acid, which was expected to yield 1-hexadecene. Again, upon extraction of olefins following cell growth, we did not observe the n + 1 α-olefin but instead substantial amounts of the n + 3 product, 1-octadecene ( Fig. 2B ). Based on these results, we hypothesized that the fed odd-chain fatty acids were internalized and activated by the acyl-ACP synthetase (Aas) to produce a bound acyl-ACP species that could then enter the fatty acid synthesis (FAS) cycle for an elongation by two carbons before being intercepted by the Ols homologs. This hypothesis was bolstered by analyses of the entire lipid environment to demonstrate that tridecanoic acid was being activated and elongated to pentadecanoic and heptadecanoic acids in AYG042 (Fig. S2A, ESI † ). A corroborating result was observed for strains AYG043 when fed pentadecanoic acid; the levels of heptadecanoic acid increased in the fed cultures as compared to control (Fig. S3A, ESI † ). Therefore, the odd-chain free fatty acids are likely first activated by Aas to produce the ACP-bound acyl chain followed by FAS elongation prior to being intercepted by Ols. While these data indicate that ACP-bound acyl species may be the Ols substrate, there is no precedent for the FAAL domain to transfer an ACP-bound acyl chain to a trans ACP. Rather, FAAL domains activate free fatty acids to the acyl-AMP for subsequent transfer to the 4′-phosphopantetheinyl arm of a holo -ACP. 11,32 Therefore, we sought to identify a pathway that allows liberation of the elongated ACP-bound acyl species to free fatty acids. Fig. 2 Ols homologs utilize free fatty acids as in vivo substrates to generate α-olefins (A) evaluation of α-olefins extracted from heterologous expression of Ols04 in PCC 7002. Deletion of aas results in the loss of the n + 3 product 1-hexadecene from fed tridecanoic acid. (B) Evaluation of α-olefins extracted from heterologous expression of Ols08 in PCC 7002. Deletion of aas results in a decrease of the n + 3 product 1-octadecene from fed pentadecanoic acid. (C) Clarified α-olefins biosynthesis in cyanobacteria. Free fatty acids are quickly activated by Aas to acyl-ACPs, which are extended in fatty acid biosynthesis and/or integrated into membrane lipids. Lipases liberate free fatty acids in lipid remodelling to provide short-lived substrates for Ols. All data represent the mean ± s.d. of biological triplicates. Since no acyl-ACP thioesterases have been identified in cyanobacteria to date, we hypothesized that the Ols free fatty acid substrate may be a result of lipases/hydrolases liberating free fatty acids from the lipid or thylakoid membranes. 1 These free fatty acids would eventually be re-activated to ACP-bound acyl chains by Aas, but may also be briefly available to Ols for α-olefin synthesis ( Fig. 2C ). To test this, the gene encoding for Aas was deleted and replaced with a gene encoding kanamycin resistance in strains AYG042 and AYG043 to generate strains AYG046 and AYG047, respectively. The same odd-chain feeding strategy was employed, and olefins were extracted for analysis. Supplementation of AYG046 with tridecanoic acid resulted in only a small amount of direct conversion to the n + 1 olefin 1-tetradecene. Conversely, the production of 1-hexadecene was completely abolished in AYG046, indicating that the deletion of aas prevented extension to the pentadecanoate product. ( Fig. 2A ). In strain AYG047, the exogenously provided pentadecanoic acid was converted to the n + 1 olefin 1-hexadecene, with only a small amount of the n + 3 olefin 1-octadecene being observed ( Fig. 2B ). A total lipid extraction of AYG047 showed an increase in the amount of accumulated pentadecanoate species as compared to the fed AYG043 strain, while simultaneously showing a decrease in the amount of conversion to the extended n + 2 heptadecanoate acyl chains (Fig. S3A and B, ESI † ). The lack of significant production of 1-tetradecene in AYG046 supplemented with tridecanoic acid may be a result of poor internalization of the tridecanoic acid following deletion of the gene encoding Aas. The total lipid extraction of AYG042 shows a dramatic increase in the n + 2 pentadecanoate acyl chains when tridecanoic acid is fed (Fig. S2A, ESI † ). However, upon deletion of aas , strain AYG046 does not accumulate either tridecanoate nor pentadecanoate species in its lipid environment, indicating that very little of the tridecanoic acid is entering the cell to act as substrate for Ols04 to produce 1-tetradecene (Fig. S2B, ESI † ). Together, however, these results indicate that deletion of aas significantly diminished the sequestration of the fed free fatty acids to the ACP-bound forms to allow the Ols homologs to directly access these substrates for α-olefin production. Deciphering OlsWT acyl-chain loading in vitro To provide further support that the free fatty acid was the substrate of the FAAL domain of OlsWT, an in vitro analysis was completed. The region of olsWT that encodes for the di-domain containing the FAAL domain and the initial ACP 0 was cloned into a protein expression vector that introduced a C-terminal histidine tag and overproduced in E. coli BAP1 containing the promiscuous 4′-phosphopantetheinyl transferase Sfp. This truncated OlsWT enzyme was evaluated for fatty acyl activation in vitro using the colorimetric assay chrome azurol S (CAS) assay ( Fig. 3A ). 33 In order to make the forward adenylation reaction to form the acyl-AMP by the FAAL domain irreversible, hydroxylamine was added to the in vitro assay. The pyrophosphate released from this reaction then sequesters iron from the CAS-Fe 3+ complex (blue in colour) to produce free CAS (yellow in colour) resulting in a decrease in the absorbance measured at 630 nm. Fig. 3 The FAAL domain of OlsWT demonstrates significant preference for stearic acid in vitro . (A) Overview of CAS colorimetric assay in vitro . The hydroxylamine reagent drives the FAAL adenylation reaction forward by capturing the acyl-AMP intermediate as a hydroxamic acid. Accumulated pyrophosphate (PP i ) chelates Fe 3+ from the CAS reagent to decrease absorbance at 630 nm. (B) In vitro FAAL activation for Ols LD-ACP di-domain demonstrating a significant preference for octadecanoic acid over other fatty acids. All data represent the mean ± s.d. of biological triplicates. P values were analysed based on student two-tailed t test assuming equal variances. * P = 0.0126, ** P < 0.0001. The CAS assay additionally afforded an assessment of selectivity for specific fatty acyl chain lengths by the FAAL domain of Ols. From these results, we observed that the FAAL-ACP didomain of OlsWT shows a significant preference in fatty acyl chain loading based on chain length. Octadecanoic acid was demonstrated to be the preferred substrate for OlsWT ( Fig. 3B ). To confirm these results, we also analysed related fatty acyl compounds including fatty alcohols and fatty methyl esters, which should not be activated by the FAAL domain of OlsWT. As expected, these fatty acyl compounds did not result in a significant change in the absorbance of the CAS reagent (Fig. S4, ESI † ). We next aimed to assess free fatty acid activation by the full-length OlsWT. Unfortunately, we were not successful in purifying a soluble preparation of this large (300 kDa) enzyme (data not shown). This may be a result of improperly folded protein lacking key secondary and tertiary structures required for function. Together, these data further establish free fatty acids as the substrate for the FAAL domain of Ols and demonstrate chain length selectivity with octadecanoic acid as the preferred substrate. These results are also consistent with our data that deletion of the gene encoding aas prevented the sequestration of the fed free fatty acids to the ACP bound forms to allow Ols to directly intercept the free fatty acid substrate. Olefin synthases are widely distributed in cyanobacteria Previous groups have shown that olefin synthases are found across different morphotypes of cyanobacteria, including unicellular, colonial, and filamentous. 27,28 We updated the list of predicted Ols homologs by confirming previous sequences and utilizing them for blastp searches against cyanobacterial genomes from GenBank (Tables S3 and S4, ESI † ). New homologs were confirmed to contain the sulfotransferase (ST) domain responsible for sulfonation of the β-hydroxy acyl intermediate. Any homologs that lacked the FAAL domain responsible for loading were excluded. Homologs that do not contain the FAAL domain were often found to be encoded upstream or downstream of genes coding for other PKS or NRPS modules, indicating that they belonged to non-olefin biosynthetic gene clusters such as the marine cyanobacterial natural product curacin A. 34,35 Finally, homologs were confirmed to contain a terminal TE domain responsible for the termination and release of the product to allow for subsequent decarboxylation and desulfonation. Protein homologs containing the ST domain but lacking a TE domain are likely to result in sulfated metabolites instead of α-olefins. 36,37 Publicly available cyanobacteria genomes were also reviewed for both major types of Ols architectures. The open reading frame (ORF) for OlsWT from PCC 7002 encodes for all domains of the enzyme (FAAL-ACP-KS-AT-KR-ACP-ST-TE) in a single protein. Other Ols homologs are arranged in two contiguous ORFs with the first encoding for the first two domains (FAAL-ACP) in trans and the second encoding for the remaining domains (KS-AT-KR-ACP-ST-TE). In total, we identified 48 new Ols homologs that contain all domains in a single ORF and 22 new Ols homologs with the FAAL-ACP domains on separate ORFs. A previously identified Ols homolog with the FAAL encoded by the first ORF and the subsequent domains (ACP-KS-AT-KR-ACP-ST-TE) on a second ORF from Cyanobacterium stanieri PCC 7202 was also evaluated. No homologs containing this third, rare architecture were identified. Two phylogenetic trees of both types of Ols architectures were constructed using the CurM domain from the curacin A pathway as the outgroup ( Fig. 4 ). Previously reported hydrocarbon profiles along with our observed heterologous hydrocarbon production demonstrate that related Ols pathways produce similar α-olefins. However, these hydrocarbon profiles are not exclusive to either Ols architecture. For example, the production of 1-heptadecene is widespread in both types of Ols organization. The production of shorter chain 1-pentadecene appears to be the rarest with only two confirmed strains capable of its biosynthesis. While this may be due to substrate selection via the FAAL domain, it could also be attributed to substrate availability. Chroococcidiopsis sp. PCC 6712 and Xenococcus sp. PCC 7305, which contain Ols homologs capable of synthesizing 1-pentadecene, both produce tetradecanoyl lipid chains. Conversely, strains that contain Ols homologs capable of producing 1-nonadecene tend to accumulate longer hexadecanoyl and octadecanoyl lipids. Fig. 4 (A) Phylogeny of 71 Ols homologs with fused, cis modules identified from available cyanobacterial genomes. (B) Phylogeny of 29 Ols homologs with decoupled, trans modules with FAAL-ACP on a separate ORF. Ols homologs with confirmed α-olefins production are highlighted as follows: red, 1-nonadecene; green, 1-heptadecene; blue, 1-pentadecene. Homologs directly studied in this work in bold. Trees was generated by the maximum likelihood method with 500 bootstrap replicates. Bootstrap values >0.5 are shown. CurM from the curacin pathway was used to root both trees."
} | 6,910 |
25393313 | PMC4230839 | pmc | 8,663 | {
"abstract": "Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.",
"conclusion": "Conclusions We assimilated our results into a model of C. phytofermentans polysaccharide catabolism that shows degradation by active CAZymes and uses mRNA expression profiles to predict how these substrates are transported and metabolized ( Fig. 6 ). Unlike other clostridia that transport sugars with numerous phosphotransferase systems (PTS) [35] \n [36] , C. phytofermentans encodes a single, lowly expressed PTS and also lacks the symporters to transport xylose and arabinose [37] . Instead, C. phytofermentans responds to carbon sources by up-regulating between two (galacturonic acid) and twenty-two (arabinan) ABC transporters ( Fig. 6 ). Expression changes support that oligosaccharides and monosaccharides are uptaken by distinct transporters. For example, different ABC transporters are up-regulated on xylose and xylan. Similarly, different transporters respond to glucose, cellobiose, and cellulose. Intracellular cellodextrins are cleaved by at least one cellodextin phosphorylase (GH94); hexoses are phosphorylated, likely by a ROK hexokinase (Cphy0329) and a putative galactokinase (Cphy2237), and fed into glycolysis. While hexokinases may have wide substrate activity [38] , poor growth on mannose could be due to inefficient mannose phosphorylation. The pentoses xylose and arabinose are isomerized and metabolized by the pentose phosphate pathway (PPP). Weak growth on arabinose could be due to inefficient transport or the lack of the phosphoketolase in the PPP enabling rapid L-arabinose metabolism by C. acetobutylicum \n [39] . 10.1371/journal.pgen.1004773.g006 Figure 6 Model of polysaccharide degradation and metabolism by C. phytofermentans . CAZymes (shown as the number of enzymes in CAZy families) are based on purified activities and are intra- or extracellular based on putative secretion signals. Metabolic enzymes are shown as NCBI numbers and are proposed based on mRNA expression. Rhamnose transport and assimilation is based on pathway from [55] . Abbreviations are D-galacturonic acid (GA), L-rhamnose (R), D-mannose (M), D-glucose (Gc), D-galactose (G), D-xylose (X), L-arabinose (A), fructose (F), phosphate (P), pentose phosphate pathway (PPP), dihydroxyacetone-phosphate (DHAP), glyceraldehyde-3-phosphate (G3P). For each substrate, the number of significantly up-regulated extracellular solute binding proteins (ESB) and ABC transporters (ABC) are shown. Shaded regions show metabolism of glucose (green), mannose (blue), xylose and arabinose (yellow), rhamnose (orange), and galacturonic acid (red). Plant degrading microbes differ widely in their abilities to depolymerize and metabolize polysaccharides, likely reflecting niche differentiation to alleviate resource competition. Among soil clostridia, C. thermocellum ferments cellulose, but not xylan [40] . C. cellulolyticum grows faster on xylose than xylan and faster on cellobiose than glucose [41] , both of which differ from C. phytofermentans . Similar specialization exists in the human gut microbiome where microbes catabolize different glycans in dietary fiber [20] . The strategy presented here of high-resolution anaerobic growth measurements, RNA sequencing, and CAZyme assays complements other methods such as proteomics [42] and metagenomics. Elucidating how microbes metabolize polysaccharides is key to understanding the function of plant-degrading microbial communities and to develop improved enzyme mixtures and recombinant microbes for industrial processing of plant biomass.",
"introduction": "Introduction Plants annually produce 200 billion tons of lignocellulosic biomass [1] , which is metabolized by specialized microbes in diverse environments. For instance, recycling of plant biomass by soil [2] and marine [3] microbes is a key part of the global carbon cycle and intestinal bacteria ferment indigestible plant fiber to short chain fatty acids that constitute 60–85% of calories in ruminants and 5–10% in humans [4] . Further, as only 2% of cellulosic biomass is currently used by humans [5] , it is a vast potential feedstock that industrial microbes could convert into energy and commodities. Elucidating how microbes depolymerize and metabolize plant biomass is thus important to understand carbon flow in the environment, to promote healthy human nutrition and prevent disease [6] , and to develop industrial processes based on cellulosic bioconversion. Most of plant biomass is in the cell wall, a macromolecular network of phenolic lignin and three types of polysaccharides (cellulose, hemicelluloses, and pectins) whose relative abundances vary widely among species and tissues ( Table S1 ). The load bearing structure of the cell wall consists of cellulose fibrils tethered by various types of hemicellulose. Hemicellulose is enriched in xylan [7] and xyloglucan [8] in dicots, arabinoxylan in monocots [9] , and galacto- and glucomannans in gymnosperms [10] . Outside the cell wall, mannans also act as storage polysaccharides in seeds [11] , similar to starch. Pectins are cross-linked galacturonic acid-based polysaccharides that act in cellular adhesion and primary wall extension. More than 60% of pectin is often homogalacturonan (HG) [12] , which is esterified with methanol to various degrees. Rhamnogalacturonan I (RGI) [13] , the second most abundant pectin, can have galactan and arabinan side chains on the rhamnose residues [14] . Because plant tissues are composed of such heterogeneous polysaccharides, plant-degrading microbes express a myriad of carbohydrate-active enzymes (CAZymes) [15] , each of which modifies or cleaves a specific type of sugar linkage. Here we demonstrate a strategy for systematic analysis of the enzymatic machinery used by microbes to degrade and metabolize plant polysaccharides. Among these microbes, the plant-fermenting clostridia are of particular interest for being a dominant group in the human gut microbiome [6] and top candidates to transform cellulosic biomass into fuels and commodities [16] , [17] . We studied Clostridium phytofermentans \n [18] , a soil bacterium with 171 CAZyme-encoding genes ( Table S2 ) including 116 glycoside hydrolases in 44 different families. We first quantified growth on comprehensive panel of plant polysaccharides and sugars ( Table S3 ). Strand-specific RNA sequencing revealed all genes whose expression changed on the various substrates. In particular, we focused on up-regulated CAZyme genes and determined how they are organized into regulons that respond to specific polysaccharides. A set of 56 up-regulated CAZymes were cloned, purified, and an “each enzyme versus each substrate” screen quantified their abilities to bind and cleave plant polysaccharides. These enzymes were then tested in combination to identify synergies for polysaccharide degradation. We discuss how the results can be integrated to further our knowledge of how microbes metabolize plant biomass.",
"discussion": "Results/Discussion Growth on polysaccharides and sugars We developed a high resolution, microtiter anaerobic growth assay that shows C. phytofermentans ferments diverse plant polysaccharides ( Fig. 1 ) and their constituent monosaccharides ( Fig. S1 ), but with widely varying cell yields and growth rates ( Table S4 ). It also forms colonies on solid medium containing each polysaccharide except arabinogalactan II (AGII) ( Fig. S2 ). Growth was fastest on HG ( Fig. 1A , generation time 0.70h), similar to rumen microbes that digest pectin more rapidly than cellulose and hemicellulose [19] . Although C. phytofermentans ferments both galacturonic acid ( Fig. S1F ) and rhamnose ( Fig. S1H ), cell yield was low on RGI ( Fig. 1B ). C. phytofermentans grows well on galactan ( Fig. 1C ), xylans ( Fig. 1F–G ), mannans ( Fig. 1H–I ), xyloglucan ( Fig. 1J ), and starch ( Fig. 1L ). Limited growth on AGII ( Fig. 1E ) relative to galactan supports that C. phytofermentans cleaves β-1,4 galactan, but not the β-1,3 and β-1,6-galactose bonds in AGII. Poor growth on arabinan ( Fig. 1D ) is similar to arabinose ( Fig. S1G ), suggesting this sugar is transported or metabolized inefficiently. C. phytofermentans grows well on cellulose plates ( Fig. S2 ) and solubilizes cellulosic substrates such as filter paper and raw corn stover ( Fig. S3 ), but weak growth on carboxymethylcellulose (CMC) might result from either lack of a suitable endoglucanase or carboxymethyl side groups inhibiting its metabolism. 10.1371/journal.pgen.1004773.g001 Figure 1 \n C. phytofermentans growth on pectic A–E, hemicellulosic F–J, and glucan K–L. Polysaccharides: homogalacturonan A , rhamnogalacturonan I B , galactan C , arabinan D , arabinogalactan II E , xylan F , arabinoxylan G , glucomannan H , galactomannan I , xyloglucan J , carboxymethylcellulose K , starch L . Growth was measured as OD 600 every 15 minutes. Each point is the mean of six cultures; red lines show one standard deviation. \n C. phytofermentans shows diauxic growth on the mixed sugar polysaccharides galactomannan ( Fig. 1I ) and xyloglucan ( Fig. 1J ). For each of these substrates, one of the component sugars (galactose or glucose) supports faster growth than the other (mannose or xylose) ( Fig. S1 , Table S4 ). Growth on various mixtures of galactose/mannose ( Fig. S4 ) and of glucose/xylose ( Fig. S5 ) shows rapid metabolism of the preferred sugar followed by slower growth on the other one. However, in both cases when the favored sugar reached 75% of the total, the other sugar does not appear to be metabolized. Similar to some ruminal [19] and human gut microbes [20] , C. phytofermentans often grows faster on polysaccharides than the constituent sugars ( Table S4 ). When presented with mixtures of xylan and xylose, this bacterium shows diauxic growth with preferential metabolism of xylan ( Fig. S6 ), which is surprising because xylan must be cleaved to xylose to be metabolized. Growth on polysaccharides could be energetically favorable if significant ATP is saved by simultaneous transport of multiple sugar units in a single oligosaccharide [21] or by intracellular phosphorolysis of oligosaccharides [22] . C. phytofermentans encodes at least a dozen phosphorylases [23] \n [24] , which cleave oligosaccharides without using ATP. Although the mechanisms regulating sugar metabolism in C. phytofermentans are unknown, diauxic growth supports carbon catabolite repression prioritizes growth on preferred sugars and polysaccharides. Gene expression We quantified mRNA expression by strand-specific RNA sequencing during log-phase growth on 8 polysaccharides, 3 monosaccharides, and raw corn stover as a complex biomass substrate. An average of 17.3 million mRNA reads were mapped per sample ( Table S5 ), yielding expression (RPKM) values ( Table S6 ) that were highly correlated (r 2 = 0.96–0.99) between duplicate cultures for all conditions ( Fig. S7 ). The reads were also highly strand-specific ( Fig. S8 ), which will facilitate their future use for de novo transcriptome assembly, gene annotation and detection of antisense transcription. The fraction of reads mapping to CAZymes during growth on glucose was 2.0%, but this increased greatly on polysaccharides, especially cellulose (11.9%) and stover (31.0%). We assessed which genes were significantly differentially expressed on each polysaccharide relative to glucose using DESeq [25] ( Table S7 ). Expression of CAZyme genes on polysaccharides relative to glucose ( Fig. 2 ) shows that between 15 (cellobiose) and 40 (stover) CAZymes were significantly up-regulated per treatment ( Table S8 ) with a total of 92 CAZymes up-regulated on at least one polysaccharide. 10.1371/journal.pgen.1004773.g002 Figure 2 mRNA expression of all 171 CAZymes during growth on pectins A–C, hemicelluloses D–E, glucans F–H, and raw corn stover I relative to expression on glucose. Expression was quantified as log 2 (RPKM) with significantly differentially expressed genes on a given polysaccharide shown as triangles and unchanged genes as circles. The 56 purified CAZymes are red and others are blue. The differentially expressed CAZymes are putatively classified by the CAZy database as 67 glycoside hydrolases, 6 carbohydrate esterases, 4 polysaccharide lyases, 14 glycosyl transferases, and 2 CBM proteins. We analyzed the specificity of the CAZyme transcriptional response by K-means clustering the expression profiles of these genes ( Fig. 3 , Table S9 ). Cluster A consists six genes that were highly up-regulated on multiple substrates: the GH26 cphy1071 , the GH11 cphy2105 , two GH18 chitinases cphy1799 and cphy1800 \n [26] , the GH9 cellulase cphy3367 \n [27] \n [28] and the GH48 cellulase cphy3368 \n [29] . Clusters B–F respond to specific polysaccharides such as homogalacturonan (clusters B,C), starch (cluster D), xylan (cluster E), and cellulose/arabinan (cluster F). C. phytofermentans thus perceives signals from individual polysaccharides and responds by up-regulating specific transcriptional regulons that enable it to tailor its complement of CAZymes to the polysaccharide substrate. 10.1371/journal.pgen.1004773.g003 Figure 3 CAZymes clustered based on gene expression patterns (clusters A–I) show that some genes respond to multiple carbon sources while others are substrate-specific. mRNA expression changes (log 2 expression ratios relative to glucose) for all 92 CAZyme genes differentially expressed on at least 1 polysaccharide relative to glucose were separated into nine clusters using K-means. Plot centers are expression on glucose and concentric rings show log 2 up-regulation on the following carbon sources: cellobiose (Cb), filter paper cellulose (Cl), starch (Sa), xylose (Xo), xylan (Xy), arabinan (Ar), galacturonic acid (Ga), homogalacturonan (Hg), galactan (Gl), galactomannan (Gm), raw corn stover (Co). Gene membership of clusters is shown in Table S9 . CAZyme activities A set of 56 CAZymes up-regulated on polysaccharides were His-tagged, overexpressed, purified, and their abilities to bind and cleave polysaccharides were quantified. The CAZy database classifies these enzymes putatively as 47 glycoside hydrolases, 4 polysaccharide lyases, and 4 carbohydrate esterases ( Table S2 ); putative glycosyltransferases were not examined as they are not involved in polysaccharide catabolism [6] . Thirty-two enzymes have significant cleavage or binding activities ( Fig. 4 , Table S11 ). Some substrates such as β-1,4-galactan appear to be cut by a single, highly active enzyme, while multiple CAZymes from the same family degrade other substrates such as xylan (GH10), mannan (GH26), starch (GH13), and HG (PL9). CAZymes from multiple families together depolymerize substrates such as xyloglucan (GH2,5,12,31), glucomannan (GH5,GH9,GH26) and galactomannan (GH5,GH26). 10.1371/journal.pgen.1004773.g004 Figure 4 Cleavage A, binding B, and CAZy database classification C of purified enzymes. \n A Polysaccharide cleavage was quantified as nmol reducing sugar released per milligram enzyme per minute: >160 red, 80–160 orange, 40–80 yellow, 20–40 green, <20 gray. B Binding to insoluble polysaccharides was quantified as percentage enzyme bound to substrate: >30% red, 20–30% orange, 15–20% yellow, 10–15% green, <10% gray. C CAZy database classifications: glycoside hydrolases (GH), carbohydrate esterases (CE), polysaccharide lyases (PL), and carbohydrate binding domains (CBM). Among 56 purified CAZymes, only the 32 enzymes for which activities were found are shown. We found 15 CAZymes that bind insoluble polysaccharides, most commonly cellulose and mannan ( Fig. 4B ). Unexpectedly, no CAZyme bound corn stover, suggesting that partial digestion of raw biomass is needed to facilitate enzyme binding. Nine enzymes that bound substrates have carbohydrate binding modules (CBM), but some enzymes such as the cellulase Cphy1163 can bind their substrate without one. While CBM are known to discriminate between polysaccharides such as cellulose and mannan [30] , we observed overlap with cellulase CBMs binding mannan and vice versa. Further, CBM from xylanases can bind cellulose and mannose, but with lower affinity, showing that CBM often bind a range of polysaccharides. Consistent with their cleavage activities, GH13 were the only enzymes to bind starch. Enzymes with CBM usually also have catalytic modules, but Cphy1713, a CAZyme with a CBM32 and no catalytic module, binds galactomannan. CBM32 are known to bind galactose and this protein may function similar to one in Yersinia that is proposed to bind oligosaccharides to prevent them from leaking out of the cell [31] . Thirty-two CAZy families have multiple members, which often have divergent cleavage activities and cellular localizations. Cphy1510 has the highest activity among the four GH10 active on xylan ( Fig. 5A ). Cphy3010, the GH10 with lowest activity, is the only one lacking a secretion signal, supporting it acts intracellularly on xylo-oligosaccharides while the other GH10 are extracellular. Members of the GH5 family act on a wide range of polysaccharides [32] . C. phytofermentans encodes 3 GH5 enzymes, among which one is active on galactomannan and two on xyloglucan ( Fig. 5B ). The GH5 Cphy1163 has no activity on either of these substrates, but is the most active on cellulose and glucomannan. The 3 GH26 also vary in substrate specificities ( Fig. 5C ); all the GH26 are similarly active on β-mannan, but only Cphy1071 has cellulase activity and it has lower activity on gluco- and galactomannan. Sequenced-based families are thus useful to make general substrate predictions for CAZymes, but experiments are needed to determine substrate range and catalytic efficiency. 10.1371/journal.pgen.1004773.g005 Figure 5 Members of the same CAZy family vary in polysaccharide cleavage activities and CAZymes can by potentiated by other enzymes. \n A Variation in cleavage activities of GH10 enzymes on xylan. B GH5 and C GH26 family members differ in their activities and substrate specificities on amorphous cellulose (red), glucomannan (green), xyloglucan (violet), galactomannan (yellow), mannan (gray). Enzyme activities in A – C are nmol reducing sugar released per milligram enzyme per minute. D – G CAZyme mixtures have higher activities than the individual enzymes. D Cphy1163 and Cphy3367 alone and together on amorphous cellulose. E Cphy2105, Cphy3009, and Cphy3207 alone and the latter two enzymes plus Cphy2105 on xylan. F Cphy1719 and Cphy1071 alone and together on glucomannan. G Cphy1687, Cphy2567, and Cphy3310 alone and the latter two enzymes plus Cphy1687 on homogalacturonan. In D – G , enzyme activities are shown as reducing sugar (nmol) produced by individual and combined enzymes. The fraction of the reducing sugar produced by the mixed enzymes that exceeds the sum of the individual enzymes is shown in green. CAZymes mixtures can degrade polysaccharides more efficiently than individual enzymes. We assessed pairwise interactions between each CAZyme and a second enzyme on cellulose (Cphy3367), xylan (Cphy2105), glucomannan (Cphy1071), and homogalacturonan (Cphy1687) ( Fig. 5D–G ). Similar to results showing synergy between the GH9 Cphy3367 and a B. subtilis GH5 [33] , we found that a mix of Cphy3367 and the GH5 Cphy1163 has higher activity on cellulose than either enzyme alone ( Fig. 5D ), supporting they have complementary roles in cellulolysis. CAZymes can also potentiate other enzymes that have no activity by themselves. For example, the xylanase Cphy2105 activates the putative xylosidases Cphy3009 and Cphy3207 on xylan ( Fig. 5E ). Similarly, Cphy1071 activates the putative mannosidase Cphy1719 on glucomannan ( Fig. 5F ). Activities of the GH28 Cphy2567 and Cphy3310 are enhanced by the carbohydrate esterase Cphy1687 ( Fig. 5G ), supporting this enzyme demethylesterifies homogalacturonan to facilitate its degradation. This carbohydrate esterase did not, however, increase cleavage by the PL9 enzymes that were the most active on homogalacturonan. Global correlations between CAZyme mRNA expression and cleavage activities were weak for all polysaccharides ( Fig. S12 ), mostly because many CAZyme genes are up-regulated on substrates upon which they have no activity. CAZymes up-regulated on multiple substrates ( Fig. 3 , cluster A) may act as ‘carbon scouts’ [34] that degrade complex substrates into inducing molecules used to fine-tune the expression of hydrolytic enzymes. As described above, some CAZymes such as xylosidases ( Fig. 5D ) are inactive on intact xylan, but are potentiated by other xylanases. The GH18 Cphy1799 and Cphy1800 are the most highly-upregulated CAZymes on cellulose ( Fig. S12 ), but are chitinases with no activity on cellulose or other plant substrates [26] . As such, the set of up-regulated CAZymes is useful to identify active enzymes, but strong up-regulation does not necessarily indicate activity on a given substrate. Conclusions We assimilated our results into a model of C. phytofermentans polysaccharide catabolism that shows degradation by active CAZymes and uses mRNA expression profiles to predict how these substrates are transported and metabolized ( Fig. 6 ). Unlike other clostridia that transport sugars with numerous phosphotransferase systems (PTS) [35] \n [36] , C. phytofermentans encodes a single, lowly expressed PTS and also lacks the symporters to transport xylose and arabinose [37] . Instead, C. phytofermentans responds to carbon sources by up-regulating between two (galacturonic acid) and twenty-two (arabinan) ABC transporters ( Fig. 6 ). Expression changes support that oligosaccharides and monosaccharides are uptaken by distinct transporters. For example, different ABC transporters are up-regulated on xylose and xylan. Similarly, different transporters respond to glucose, cellobiose, and cellulose. Intracellular cellodextrins are cleaved by at least one cellodextin phosphorylase (GH94); hexoses are phosphorylated, likely by a ROK hexokinase (Cphy0329) and a putative galactokinase (Cphy2237), and fed into glycolysis. While hexokinases may have wide substrate activity [38] , poor growth on mannose could be due to inefficient mannose phosphorylation. The pentoses xylose and arabinose are isomerized and metabolized by the pentose phosphate pathway (PPP). Weak growth on arabinose could be due to inefficient transport or the lack of the phosphoketolase in the PPP enabling rapid L-arabinose metabolism by C. acetobutylicum \n [39] . 10.1371/journal.pgen.1004773.g006 Figure 6 Model of polysaccharide degradation and metabolism by C. phytofermentans . CAZymes (shown as the number of enzymes in CAZy families) are based on purified activities and are intra- or extracellular based on putative secretion signals. Metabolic enzymes are shown as NCBI numbers and are proposed based on mRNA expression. Rhamnose transport and assimilation is based on pathway from [55] . Abbreviations are D-galacturonic acid (GA), L-rhamnose (R), D-mannose (M), D-glucose (Gc), D-galactose (G), D-xylose (X), L-arabinose (A), fructose (F), phosphate (P), pentose phosphate pathway (PPP), dihydroxyacetone-phosphate (DHAP), glyceraldehyde-3-phosphate (G3P). For each substrate, the number of significantly up-regulated extracellular solute binding proteins (ESB) and ABC transporters (ABC) are shown. Shaded regions show metabolism of glucose (green), mannose (blue), xylose and arabinose (yellow), rhamnose (orange), and galacturonic acid (red). Plant degrading microbes differ widely in their abilities to depolymerize and metabolize polysaccharides, likely reflecting niche differentiation to alleviate resource competition. Among soil clostridia, C. thermocellum ferments cellulose, but not xylan [40] . C. cellulolyticum grows faster on xylose than xylan and faster on cellobiose than glucose [41] , both of which differ from C. phytofermentans . Similar specialization exists in the human gut microbiome where microbes catabolize different glycans in dietary fiber [20] . The strategy presented here of high-resolution anaerobic growth measurements, RNA sequencing, and CAZyme assays complements other methods such as proteomics [42] and metagenomics. Elucidating how microbes metabolize polysaccharides is key to understanding the function of plant-degrading microbial communities and to develop improved enzyme mixtures and recombinant microbes for industrial processing of plant biomass."
} | 6,461 |
39120150 | PMC11409717 | pmc | 8,664 | {
"abstract": "ABSTRACT Phosphatidylcholine (PC) is critical for the nitrogen-fixing symbiosis between rhizobia and legumes. We characterized three PC biosynthesis pathways in Rhizobium leguminosarum and evaluated their impact on nitrogen fixation in clover nodules. In the presence of choline, a PC synthase catalyzes the condensation of cytidine diphosphate-diacylglycerol with choline to produce PC. In the presence of lyso-PC, acyltransferases acylate this mono-acylated phospholipid to PC. The third pathway relies on phospholipid N -methyltransferases (Pmts), which sequentially methylate phosphatidylethanolamine (PE) through three rounds of methylation, yielding PC via the intermediates monomethyl-PE and dimethyl-PE. In R. leguminosarum , at least three Pmts participate in this methylation cascade. To elucidate the functions of these enzymes, we recombinantly produced and biochemically characterized them. We moved on to determine the phospholipid profiles of R. leguminosarum mutant strains harboring single and combinatorial deletions of PC biosynthesis genes. The cumulative results show that PC production occurs through the combined action of multiple enzymes, each with distinct substrate and product specificities. The methylation pathway emerges as the dominant PC biosynthesis route, and we pinpoint PmtS2, which catalyzes all three methylation steps, as the enzyme responsible for providing adequate PC amounts for a functional nitrogen-fixing symbiosis with clover. IMPORTANCE Understanding the molecular mechanisms of symbiotic nitrogen fixation has important implications for sustainable agriculture. The presence of the phospholipid phosphatidylcholine (PC) in the membrane of rhizobia is critical for the establishment of productive nitrogen-fixing root nodules on legume plants. The reasons for the PC requirement are unknown. Here, we employed Rhizobium leguminosarum and clover as model system for a beneficial plant-microbe interaction. We found that R. leguminosarum produces PC by three distinct pathways. The relative contribution of these pathways to PC formation was determined in an array of single, double, and triple mutant strains. Several of the PC biosynthesis enzymes were purified and biochemically characterized. Most importantly, we demonstrated the essential role of PC formation by R. leguminosarum in nitrogen fixation and pinpointed a specific enzyme indispensable for plant-microbe interaction. Our study offers profound insights into bacterial PC biosynthesis and its pivotal role in biological nitrogen fixation.",
"introduction": "INTRODUCTION The gram-negative soil bacterium Rhizobium leguminosarum has been thoroughly studied for its ability to form symbiotic relationships with legumes. Upon infection of the host plant, nodules are formed in which bacteria differentiate into bacteroids. These bacteroids can convert atmospheric nitrogen into ammonia through nitrogen fixation, thereby providing a crucial nitrogen source for the host plant. Based on the preferred symbiotic interaction partner of R. leguminosarum , its species can be further divided into three biovars ( 1 , 2 ). R. leguminosarum bv. viciae forms nitrogen-fixing nodules with Pisum (pea) or Vicia (vetch), bv. phaseoli interacts with Phaseolus spp. (bean), and bv. trifolii with Trifolium spp. (clover) ( 3 – 7 ). The host range is primarily determined by the secretion of specific flavonoids by the plant, which upon perception regulate nodulation ( nod ) genes in the bacterium. Conversely, Nod factors produced by the rhizobia are also crucial determinants of host range ( 8 – 11 ). Additional factors affecting the symbiosis of R. leguminosarum include the production and composition of exopolysaccharides and lipopolysaccharides as well as the presence of unusually long and multi-unsaturated fatty acyl chains, indicating the significant role of cell surface properties in host interaction ( 12 – 16 ). In various rhizobia species, the phospholipid composition, particularly the presence of phosphatidylcholine (PC), plays a pivotal role in bacterium-host interactions [for reviews see references ( 17 ) and ( 18 )]. For instance , Bradyrhizobium diazoefficiens, formerly known as Bradyrhizobium japonicum ( 19 ) , is a nodule-forming and nitrogen-fixing symbiont of soybeans. Mutants of B. diazoefficiens exhibiting only 10%–15% PC levels compared to the wild type produced nodules inefficient in nitrogen fixation ( 20 – 22 ). Interestingly, Bradyrhizobium sp. SEMIA 6144 showed functional nodules with a 50% reduction in PC levels, suggesting the existence of a threshold of PC necessary for effective nitrogen fixation ( 23 ). A puzzling case is a Bradyrhizobium toxin-antitoxin mutant that produces incomplete lipopolysaccharides and lacks PC, but nonetheless forms about two-thirds of the nodules that the wild type forms ( 22 ). How effective these nodules are in nitrogen fixation activity has not been analyzed. In the absence of PC, Sinorhizobium meliloti, the symbiont of alfalfa, fails to form nitrogen-fixing nodules ( 24 , 25 ). Interestingly, PC does not only play a role in rhizobia-legume interactions, but it is also crucial for tumor formation by the plant pathogen Agrobacterium tumefaciens ( 26 ), underscoring the significance of PC in both mutualistic and pathogenic plant-microbe interactions. Notably, the zwitterionic phospholipid PC is not commonly found in bacterial membranes. It has been estimated that approximately 15% of bacterial species have the capability to synthesize PC ( 17 ). Remarkably, a significant proportion of these PC-synthesizing species engages in interactions with eukaryotic hosts. In addition to the plant-interacting species introduced above, also the human pathogens Brucella abortus and Legionella pneumophila produce PC, and mutants deficient in PC production exhibit various degrees of attenuated virulence ( 27 – 29 ). Bacteria utilize three pathways for PC production, with the methylation and the PC synthase pathway being the primary ones, while the acylation pathway has been described in only a few bacteria ( 30 ). In the latter, glycerophosphocholine (GPC) is first acylated to lyso-PC (LPC) and then further to PC using acyl-CoA as acyl donor. In the PC synthase pathway, the ubiquitous phospholipid precursor cytidine diphosphate diacylglycerol (CDP-DAG) is condensed with choline, a reaction catalyzed by the PC synthase (Pcs). On the other hand, the methylation pathway involves a three-step N -methylation of phosphatidylethanolamine (PE) utilizing S -adenosylmethionine (SAM) as methyl donor. This pathway yields the intermediates monomethyl-PE (MMPE) and dimethyl-PE (DMPE), ultimately leading to the production of PC. The methylation reactions are catalyzed by phospholipid N -methyltransferases (Pmts). Bacterial Pmts can be categorized into two main groups based on their amino acid sequence homology. Pmts resembling the enzyme found in S. meliloti are classified as S - type, while those similar to Rhodobacter sphaeroides PmtA belong to the R - type. Certain Pmts, such as those from A. tumefaciens, R. sphaeroides, and Rubellimicrobium thermophilum , catalyze the complete methylation process from PE to PC ( 31 – 34 ). In contrast, other Pmts only catalyze specific methylation steps. For instance, Pmts from Xanthomonas campestris and various thermophilic actinobacteria produce MMPE, and some also produce DMPE but not PC ( 30 , 34 ). The underlying structural and functional determinants of these differences in substrate and product preferences among Pmt enzymes remain elusive. The situation becomes even more complicated when multiple Pmt paralogs exist within a single bacterium. In B. diazoefficiens , for instance, at least four distinct Pmts with varying substrate specificities have been identified ( 20 ). Their biochemical characterization was not possible due to the poor solubility of the purified recombinant enzymes. R. leguminosarum is another plant-beneficial Rhizobium species with a complex membrane lipid profile. The membrane of R. leguminosarum bv. trifolii ANU843 in late exponential growth phase is characterized by substantial quantities of methylated PE derivatives. While PE constitutes 15% of the entire membrane composition, MMPE (47%) is the dominant phospholipid in this bacterium ( 35 ). DMPE and PC are present in lesser amounts at 9% and 2%, respectively. The biosynthetic enzymes accountable for this unusual membrane composition are largely unexplored. The presence of MMPE and DMPE strongly suggests an active methylation pathway. Indeed, studies using radiolabeled SAM have detected methylated PE derivatives in cell-free R. leguminosarum extracts ( 36 ), and sequence homology searches have uncovered one R-type and two S-type Pmt candidates ( 37 ), along with a Pcs candidate ( 17 , 36 ). The principle aim of this study was to understand the mechanisms underlying the complex PC biosynthesis in R. leguminosarum . Through a comprehensive combination of in silico , in vivo, and in vitro methodologies, we identified and characterized three distinct pathways responsible for the formation of methylated PE lipids in R. leguminosarum bv. t rifolii ATCC14479. Finally, we demonstrated the indispensability of PC for symbiotic nitrogen fixation in clover plants and narrowed down the Pmt enzyme primarily responsible for effective symbiosis.",
"discussion": "DISCUSSION Nitrogen-fixing plant symbionts, such as R. leguminosarum, are of significant interest in agriculture due to the frequent limitation of crop yields by nitrogen availability ( 48 ). Therefore, a comprehensive understanding of the underlying mechanisms governing this symbiosis is important. One crucial aspect influencing the interaction between a bacterium and its eukaryotic host often is the presence of PC in the bacterial membrane ( 17 , 18 ). Previous research has demonstrated the severe impact of PC deficiency on the nitrogen fixation activity of B. diazoefficiens and S. meliloti ( 20 , 21 , 25 ). Hence, it was reasonable to assume that the symbiosis of R. leguminosarum might also be influenced by the presence of PC. It had been suggested that the clover symbiont possesses the capability to produce PC through the PC synthase and methylation pathways ( 36 ). However, the functionality of specific PC biosynthesis enzymes and their contribution to the outcome of plant-microbe interaction remained elusive. In this study, we elucidated how R. leguminosarum synthesizes PC and demonstrated the importance of this phospholipid for the nitrogen-fixing symbiosis with clover. One out of four phospholipid N -methyltransferases is crucial for nitrogen fixation PC biosynthesis in R. leguminosarum is remarkably complex and flexible ( Fig. 10 ). Among the array of pathways available to produce PC from both external and internal substrates, the methylation pathway emerged as the predominant pathway necessary for the establishment of productive root nodules. Out of the four Pmt candidates, PmtS2 turned out to be the key enzyme responsible for carrying out all three methylation reactions from PE to PC, thus rendering it indispensable for symbiotic nitrogen fixation. Now the question arises why R. leguminosarum possesses multiple Pmt enzymes, given that PmtS2 alone is capable of synthesizing PC, at least under the conditions tested. One explanation might be that multiple Pmt enzymes offer redundancy and robustness to the biosynthetic pathway. In situations where one enzyme lacks substrate or is inactivated due the mutations or inhibitors, redundancy guarantees that the organism can still generate PC or other methylated PE derivatives through alternative Pmts. MMPE, the most abundant phospholipid with largely unexplored functions in R. leguminosarum , must also be considered. Some Pmts, especially PmtS1, and probably PmtS3 under certain conditions, might play a pivotal role in maintaining the exceedingly high MMPE levels. Fig 10 Overview of PC biosynthesis pathways in R. leguminosarum . The bacterium can synthesize PC via the methylation pathway, for which three out of four Pmts (PmtS2, PmtS1, and PmtR1) were shown to be active in this study. PmtS3, which was not detected under laboratory conditions in R. leguminosarum but was active in E. coli, is grayed out. In addition, the Pcs enzyme produces PC by condensation of CDP-DAG with choline, which can be acquired from the environment. Moreover, R. leguminosarum forms PC in the presence of LPC by the action of one or more yet unidentified acyltransferase(s) (AT). CMP, cytidine monophosphate; CoA, coenzyme A; SAM, S -adenosyl methionine; SAH, S -adenosyl homocysteine. In comparison, PC biosynthesis in S. meliloti appears straightforward. The organism encodes one PmtA enzyme catalyzing all three methylation steps and one Pcs enzyme. The double-knockout strain exhibits reduced growth ( 49 ), loss of motility, succinoglycan overproduction, and an inability to form nitrogen-fixing root nodules on alfalfa ( 25 ). In contrast, PC biosynthesis in R. leguminosarum resembles an equally complex situation in B. diazoefficiens . In B. diazoefficiens, the methylation pathway operates in a two-step process: the S-type PmtA methylates PE to MMPE and DMPE, followed by the R-type PmtX1 utilizing MMPE and DMPE as substrates for subsequent methylation to PC ( 20 ). The pmtA mutant was strongly impaired in nitrogen fixation, while a pmtX1 mutant could not be constructed, underscoring the critical role of PC in bacterial physiology ( 21 , 50 ). Two additional Pmt candidates from B. diazoefficiens were found to be active in E. coli with different substrate specificities. While the corresponding genes were not expressed in B. diazoefficiens under standard laboratory conditions, one of them was upregulated in the pmtA mutant suggesting potential compensation for the absence of PmtA ( 50 ). Similarly, R. leguminosarum harbors four Pmts with distinct substrate and product specificities ( Fig. 10 ). Through successful purification and biochemical characterization of three of these enzymes, we obtained a coherent picture of the PC biosynthesis pathways corroborated by multiple lines of evidence both in vitro and in vivo (expression in E. coli ; deletion mutants and FLAG-tag strains in R. leguminosarum ). PmtS3 is a notable exception for several reasons. The protein was undetectable in both the R. leguminosarum wild type and Δ pmtS2 Δ pmtS1 mutant. Unlike all other genes involved in PC biosynthesis, pmtS3 is located on one of the four plasmids. The genome of R. leguminosarum strains typically consists of a chromosome and a variable number of plasmids (Table S5). The core chromosome is highly conserved, containing most essential genes, while plasmid-encoded genes are often regarded as accessory and adaptive ( 51 , 52 ). The extrachromosomal DNA exhibits high variability between strains, e.g., to facilitate adaptation to specific symbiotic partners. Consequently, certain genes are only present in a subset of R. leguminosarum strains. This is evident with pmtS3, which is absent from 6 out of 21 fully sequenced genomes available in the NCBI database (Table S5). Remarkably, all other chromosomally encoded pmt genes, as well as pcs , are conserved across all genomes. The absence of pmtS3 in one-third of the strains investigated underscores its minor importance for R. leguminosarum . It is possible that an evolutionary ancestor originally encoded only three Pmts and that the fourth gene was later acquired by certain strains. Alternatively, it could be hypothesized that an evolutionary ancestor encoded four Pmts and that pmtS3 was lost from the plasmid in some strains through recombination events. Little selection pressure due to the functional redundancy of PmtS3 with PmtS1, which is encoded on the chromosome, likely contributed to this loss. Multiple ways to produce PC The methylation pathway in R. leguminosarum is complemented by additional PC biosynthesis options ( Fig. 10 ). Beyond the canonical choline condensation process, the Pcs enzyme was able to accept MMEA and DMAE as substrates, yielding MMPE and DMPE, respectively. This broad substrate spectrum seems to be a conserved feature of Pcs enzymes ( 34 , 42 – 44 ). Presumably, this substrate promiscuity equips the bacterium with significant adaptability to synthesize all three methylated PE derivatives via Pcs and/or Pmt activity, depending on the availability of precursors from the environment. Bacteria generally cannot produce choline de novo and thus rely on the uptake of exogenous choline from the environment ( 26 , 27 , 36 , 53 ). To attract symbiotic bacteria, plants are believed to exude choline into the environment ( 54 ). When choline was available, Pcs effectively replenished PC in the R. leguminsarum pmtS2 mutant to levels comparable to the wild type (increasing from 0% without choline to 31% with choline; Table S3). However, it appears that the choline levels in the plant infection assay were inadequate for PC production, as evidenced by the severely compromised nitrogen fixation activity in the pmtS2 mutant but not in the pcs mutant. R. leguminosarum produced PC when CDP-choline was supplemented to the growth medium ( Fig. 10 ). Notably, previous in vitro assays with A. tumefaciens Pcs revealed that the enzyme did not utilize CDP-choline and DAG as substrates for PC production (data now shown). This suggests that CDP-choline in R. leguminosarum was metabolized within the cell to choline, which was then condensed with CDP-DAG to form PC through the conventional Pcs activity ( Fig. 10 ). Pcs-dependent PC production was also observed in the presence of GPC (Fig. S2B). While certain bacteria, such as X. campestris, can acylate GPC to LPC, which is then utilized for PC production, R. leguminosarum exclusively generated PC from LPC in a Pcs-dependent manner. Analogous to CDP-choline, it is likely that the bacterium derives choline from GPC. When LPC was provided, R. leguminosarum was able to acylate it into PC ( Fig. 10 ). In contrast to the CDP-choline and GPC-dependent PC formation, this activity was found to be independent of Pcs, which strongly suggests the existence of one or more acyltransferase(s) capable of adding an acyl chain to the lysolipid. While R. leguminosarum was able to produce large amounts of PC from LPC under laboratory conditions, it remains uncertain whether and how much LPC the bacterium encounters in its natural habitats. A recent study on lipids in soil organic matter revealed that LPC constitutes approximately 4% of all lipids in soil and around 8% of all lipids in roots ( 55 ). However, it is unclear how much of this LPC exists in a free state, available for incorporation and acylation by R. leguminosarum, vs being part of the membranes of other organisms present in the soil. Nevertheless, it is conceivable that R. leguminosarum may encounter trace amounts of LPC in its environment, leading to the production of minor quantities of PC via the acylation pathway. PC is crucial for symbiotic nitrogen fixation To the best of our knowledge, R. leguminosarum is now the third rhizobia member demonstrated to depend on PC for effective symbiosis with host plants ( 21 , 25 ). Similarly, deficiency in PC within the phytopathogen A. tumefaciens abrogated tumor formation on plants ( 26 ). Interestingly, the severity of these defects in plant-microbe interactions varies. In the case of the S. meliloti pmtA/pcs mutant, the absence of PC completely abolished nodule formation at an early developmental stage ( 25 ). Conversely, both the B. diazoefficiens pmtA mutant and the R. leguminosarum pmtS2 mutant, which retained some capacity to produce residual PC, progressed to nodule formation ( 21 ). However, these nodules appeared pale and lacked the ability to fix nitrogen. Despite these striking phenotypes, the molecular mechanisms underlying the significance of PC in plant-microbe interactions remain poorly understood. It has been suggested that signal transduction processes, such as the functionality of the sensor kinase VirA in A. tumefaciens , may be disrupted in PC-depleted membranes ( 56 ), but this hypothesis requires further investigation. Additional support for the connection between PC and membrane-associated signal transduction processes comes from a recent suppressor screen conducted with the S. meliloti PC-deficient mutant ( 25 ). This strain exhibits a loss of swimming ability and an overproduction of succinoglycan due to reduced transcripts necessary for flagellum formation and increased transcripts for succinoglycan biosynthesis. Several suppressor mutants from this screen, which regained normal swimming ability and succinoglycan levels, showed alterations in the ExoS sensor kinase. The authors proposed that the physicochemical properties of PC-deficient membranes induce conformational changes in the membrane-embedded sensor protein, leading to premature activation of the ExoS-dependent signaling cascade. Other suppressor mutants from the same screen exhibited various amino acid substitutions in ExoS, the ChvI response regulator, the sigma factor RpoH1, or FabA ( 57 ). Intriguingly, the mutations restored the ability to form nodules on alfalfa roots, albeit to varying degrees. However, nodules formed by the suppressor strains appeared white instead of pink and were unable to fix nitrogen, resulting in yellow, stunted host plants. The precise point at which the blockage between nodule formation and successful nitrogen fixation occurs in PC-deficient rhizobia is currently unknown. It appears that some step after infection thread formation is compromised. Despite adjustments in membrane composition by PC-deficient S. meliloti fabA supressor mutants, characterized by lower levels of unsaturated fatty acids and higher levels of saturated and shorter chain fatty acids to be compensated for the lack of PC, the tri-methylated phospholipid seems indispensable for later stages of nodule development and efficient nitrogen fixation activity ( 57 ). Lipidomic and transcriptomic profiling of developing soybean nodules has revealed that active fatty acid and lipid metabolism are essential for nodulation and productive symbiosis. Notably, PC ranks among the most abundant lipids in developing nodules ( 58 ). Our study further underscores the importance of PC in symbiotic plant-microbe interactions. Future research is needed to uncover both common and divergent principles in the requirement for PC in the symbioses between S. meliloti and alfalfa, Bradyrhizobium and soybean, and R. leguminosarum and clover."
} | 5,700 |
28880150 | PMC5589413 | pmc | 8,665 | {
"abstract": "Methyl-coenzyme M reductase (MCR), found in strictly anaerobic methanogenic and methanotrophic archaea, catalyzes the reversible production and consumption of the potent greenhouse gas methane. The α subunit of MCR (McrA) contains several unusual post-translational modifications, including a rare thioamidation of glycine. Based on the presumed function of homologous genes involved in the biosynthesis of thioviridamide, a thioamide-containing natural product, we hypothesized that the archaeal tfuA and ycaO genes would be responsible for post-translational installation of thioglycine into McrA. Mass spectrometric characterization of McrA from the methanogenic archaeon Methanosarcina acetivorans lacking tfuA and / or ycaO revealed the presence of glycine, rather than thioglycine, supporting this hypothesis. Phenotypic characterization of the ∆ ycaO-tfuA mutant revealed a severe growth rate defect on substrates with low free energy yields and at elevated temperatures (39°C - 45°C). Our analyses support a role for thioglycine in stabilizing the protein secondary structure near the active site.",
"introduction": "Introduction Methyl-coenzyme M reductase (MCR) is a unique enzyme found exclusively in anaerobic archaea, where it catalyzes the reversible conversion of methyl-coenzyme M (CoM, 2-methylmercaptoethanesulfonate) and coenzyme B (CoB, 7-thioheptanoylthreoninephosphate) to methane and a CoB-CoM heterodisulfide ( Ermler et al., 1997 ; Scheller et al., 2010 ): C H 3 − S − C o M + H S − C o B ⇄ C H 4 + C o M − S − S − C o B This reaction, which has been proposed to proceed via an unprecedented methyl radical intermediate ( Wongnate et al., 2016 ), plays a critical role in the global carbon cycle ( Thauer et al., 2008 ). In the forward direction, MCR catalyzes the formation of methane in methane-producing archaea (methanogens). In the reverse direction, MCR catalyzes the consumption of methane in methanotrophic archaea (known as ANMEs, for an aerobic oxidation of me thane). Together, these processes produce and consume gigatons of methane each year, helping to establish the steady-state atmospheric levels of an important greenhouse gas. MCR displays an α 2 β 2 γ 2 protein domain architecture and contains two molecules of F 430 , a nickel porphinoid cofactor ( Ermler et al., 1997 ; Zheng et al., 2016 ; Moore et al., 2017 ). The reduced Ni(I) form of F 430 is essential for catalysis ( Goubeaud et al., 1997 ), but is highly sensitive to oxidative inactivation, a feature that renders biochemical characterization of MCR especially challenging. As a result, many attributes of this important enzyme remain uncharacterized. An unusual feature of MCR is the presence of several modified amino acids within the active site of the α-subunit. Among these are a group of methylated amino acids, including 3-methylhistidine (or N 1 -methylhistidine), S -methylcysteine, 5(S)-methylarginine, and 2(S)-methylglutamine, which are presumed to be installed post-translationally by S -adenosylmethionine-dependent methyltransferases ( Selmer et al., 2000 ; Kahnt et al., 2007 ). 3-methylhistidine is found in all MCRs examined to date, whereas the presence of the other methylated amino acids is variable among methane-metabolizing archaea ( Kahnt et al., 2007 ). A didehydroaspartate modification is also observed in some, but not all, methanogens ( Wagner et al., 2016 ). Lastly, a highly unusual thioglycine modification, in which the peptide amide bond is converted to a thioamide, is present in all methanogens that have been analyzed to date ( Figure 1—figure supplement 1 ) ( Ermler et al., 1997 ; Kahnt et al., 2007 ). The function(s) of the modified amino acids in MCR has/have not yet been experimentally addressed; however, several theories have been postulated. The 3-methylhistidine may serve to position the imidazole ring that coordinates CoB. This methylation also alters the p K a of histidine in a manner that would promote tighter CoB-binding ( Grabarse et al., 2000 ). The variable occurrence of the other methylated amino acids suggests that they are unlikely to be directly involved in catalysis. Rather, it has been hypothesized that they tune enzyme activity by altering the hydrophobicity and flexibility of the active site pocket ( Kahnt et al., 2007 ; Grabarse et al., 2000 ). A similar argument has been made for the didehydroaspartate residue ( Wagner et al., 2016 ). In contrast, three distinct mechanistic hypotheses have implicated the thioglycine residue in catalysis. One proposal is that the thioglycine facilitates deprotonation of CoB by reducing the p K a of the sulfhydryl group ( Grabarse et al., 2001 ). A second proposal suggests that thioglycine could serve as an intermediate electron carrier for oxidation of a proposed heterodisulfide anion radical intermediate ( Horng et al., 2001 ). Lastly, cis-trans isomerization of the thioamide during catalysis could potentially play an important role in coupling the two active sites via a previously proposed two-stroke mechanism for the enzyme ( Goenrich et al., 2005 ). Thioamides are rare in biology. While cycasthioamide is plant-derived ( Pan et al., 1997 ), most other naturally occurring thioamides are of bacterial origin. Among these are the ribosomally synthesized and post-translationally modified (RiPP) peptide natural products thioviridamide ( Hayakawa et al., 2006 ) and methanobactin ( Kim et al., 2004 ; Kenney and Rosenzweig, 2012 ; Kenney and Rosenzweig, 2013 ), as well as closthioamide, which appears to be an unusual non-ribosomal peptide ( Lincke et al., 2010 ; Chiriac et al., 2015 ). The nucleotide derivatives thiouridine and thioguanine ( Coyne et al., 2014 ), and two additional natural product antibiotics, thiopeptin and Sch 18640 ( Puar et al., 1981 ; Hensens and Albers-Schönberg, 1983 ), also contain thioamide moieties. Although the mechanism of thioamide installation in peptides has yet to be established, the identification of the thioviridamide biosynthetic gene cluster provides clues to their origin ( Izawa et al., 2013 ). Two of the proteins encoded by this gene cluster have plausible roles in thioamide synthesis. The first, TvaH, is a member of the YcaO superfamily, while the second, TvaI, is annotated as a ‘TfuA-like’ protein ( Izawa et al., 2013 ). Biochemical analyses of YcaO-family proteins indicate that they catalyze the ATP-dependent cyclodehydration of Cys, Ser, and Thr residues to the corresponding thiazoline, oxazoline, and methyloxazoline ( Figure 1 ). Many ‘azoline’ heterocycles undergo dehydrogenation to the corresponding azole, which are prominent moieties in various RiPP natural products classes, such as linear azol(in)e-containing peptides, thiopeptides, and cyanobactins ( Dunbar et al., 2012 ; Dunbar et al., 2014 ; Arnison et al., 2013 ; Burkhart et al., 2017 ). The YcaO-dependent synthesis of peptidic azol(in)e heterocycles often requires a partner protein, which typically is the neighboring gene in the biosynthetic gene cluster ( Burkhart et al., 2015 ; Dunbar et al., 2015 ; Wright et al., 2006 ). Based on enzymatic similarity, the TfuA protein encoded adjacent to the YcaO in the thioviridamide pathway may also enhance the thioamidation reaction, perhaps by recruiting a sulfurtransferase protein. Although not RiPPs, thiouridine and thioguanine biosynthesis requires the use of sulfurtransferases ( Coyne et al., 2013 ; Palenchar et al., 2000 ). 10.7554/eLife.29218.002 Figure 1. Comparison of reactions catalyzed by YcaO proteins. Top , Biochemically characterized YcaO proteins involved in the synthesis of azol(in)e-containing ribosomal natural products catalyze the ATP-dependent cyclodehydration of cysteine, serine, and threonine residues. Shown is the conversion of peptidic cysteine to thiazoline. This reaction proceeds via an O -phosphorylated hemiorthoamide intermediate. Bottom , An analogous reaction is believed to occur in the biosynthesis of the thioamide bond in thioviridamide. Rather than an adjacent cysteine acting as the nucleophile, an exogenous source of sulfide (S 2- ) is required for this reaction. 10.7554/eLife.29218.003 Figure 1—figure supplement 1. A view of the MCR active site using the crystal structure of M. barkeri (Protein DataBank entry 1E6Y). The peptide backbone of McrA in the vicinity of the thioglycine (Gly 462 -Leu 468 ) is shown in gray sticks with the thioamide sulfur of Gly 465 shown as a yellow sphere. Coenzymes B, M, and F 430 are shown with several interatomic distances to McrA-Asn 501 . The biosynthesis of the thioglycine in MCR was proposed to occur by a mechanism similar to that used to produce thioamide-containing natural products ( Kahnt et al., 2007 ), a prediction made six years prior to the discovery of the thioviridamide biosynthetic gene cluster ( Izawa et al., 2013 ). Given their putative role in thioamidation reactions, it is notable that YcaO homologs were found to be universally present in an early analysis of methanogen genomes, resulting in their designation as ‘methanogenesis marker protein 1’ ( Basu et al., 2011 ). Genes encoding TfuA homologs are also ubiquitous in methanogens, usually encoded in the same locus as ycaO , similar to their arrangement in the thioviridamide biosynthetic gene cluster. Therefore, both biochemical and bioinformatic evidence are consistent with these genes being involved in thioglycine formation. In this report, we use the genetically tractable methanogen Methanosarcina acetivorans to show that installation of the thioamide bond at Gly 465 in the α-subunit of MCR requires the ycaO-tfuA locus ( Figure 1—figure supplement 1 ). As MCR is essential for growth and survival ( Rother et al., 2005 ), the viability of ycaO-tfuA mutants precludes the hypothesis that the thioamide residue is essential for MCR activity. Instead, our phenotypic analyses support a role for thioglycine in maintaining a proper structural conformation of residues near the active site, such that absence of this modification in MCR might be particularly detrimental on growth substrates that have low free energy yields as well as under additionally destabilizing conditions such as elevated temperatures.",
"discussion": "Discussion The loss of the thioglycine modification in the ∆ ycaO, ∆tfuA, and Δ ycaO-tfuA mutants shows that both of these genes are required for the thioamidation of McrA. Although this could be an indirect requirement, we believe it is more likely that the YcaO/TfuA proteins directly catalyze modification of McrA. This conclusion is mechanistically compatible with biochemical analyses of YcaO homologs. YcaO enzymes that carry out the ATP-dependent cyclodehydration of beta-nucleophile-containing amino acids have been extensively investigated ( Burkhart et al., 2017 ). Such cyclodehydratases coordinate the nucleophilic attack of the Cys, Ser, and Thr side chain on the preceding amide carbonyl carbon in a fashion reminiscent of intein splicing ( Perler et al., 1997 ) ( Figure 1 ). The enzyme then O -phosphorylates the resulting oxyanion and subsequently N -deprotonates the hemiorthoamide, yielding an azoline heterocycle. An analogous reaction can be drawn for the YcaO-dependent formation of peptidic thioamides. The only difference is that an exogenous equivalent of sulfide is required for the thioamidation reaction, rather than an adjacent beta-nucleophile-containing amino acid for azoline formation ( Figure 1 ). Most YcaO cyclodehydratases require a partner protein for catalytic activity. The earliest investigated YcaO partner proteins are homologs of the ThiF/MoeB family, which are related to E1 ubiquitin-activating enzymes ( Dunbar et al., 2014 ; Schulman et al., 2009 ). These YcaO partner proteins, as well as the more recently characterized ‘ocin-ThiF’ variety ( Dunbar et al., 2015 ), contain a ~ 90 residue domain referred to as the RiPP precursor peptide Recognition Element (RRE), which facilitates substrate recognition by interacting with the leader peptide. Considering these traits of azoline-forming YcaOs, it is possible that thioamide-forming YcaOs require the TfuA partner to facilitate binding to the peptidic substrate. Alternatively, TfuA may recruit and deliver sulfide equivalents by a direct or indirect mechanism. In this regard, it is noteworthy that the ycaO-tfuA locus can be found adjacent to genes involved in sulfur and molybdoterin metabolism ( Figure 2 ). Many of these genes encode proteins with rhodanese-like homology domains, which are well-established sulfurtransferases. These enzymes typically carry sulfur in the form of a cysteine persulfide, a non-toxic but reactive equivalent of H 2 S ( Palenchar et al., 2000 ; Matthies et al., 2005 ). Akin to rare cases of azoline-forming, partner-independent YcaOs, certain methanogens lack a bioinformatically identifiable TfuA (e.g. Methanopyrus kandleri and Methanocaldococcus sp., Figure 2 ). Whether these YcaOs act independently or use an as yet-unidentified partner protein remains to be seen. Clearly, further in vitro experimentation will be required to delineate the precise role of TfuA in the thioamidation reaction. The viability of the Δ ycaO-tfuA mutant raises significant questions as to the role of thioglycine in the native MCR enzyme, especially considering its universal presence in all MCRs examined to date. We considered three hypotheses to explain this result. First, we examined the possibility that thioglycine modification is involved in enhancing the reaction rate ( Kahnt et al., 2007 ; Horng et al., 2001 ). Although it has not been explicitly determined, MCR is thought to catalyze the rate-limiting step of methanogenesis ( Scheller et al., 2010 ; Wongnate et al., 2016 ). Therefore, the absence of thioglycine might lead to a corresponding decrease in the growth rate, with more pronounced defects on substrates that lead to the fastest growth. However, we observed the opposite: the most pronounced defects were observed with growth substrates that support the slowest WT growth rates ( Table 1 ). Next, we considered the possibility that the thioglycine influences substrate affinity. C 1 units enter methanogenesis at the level of N 5 -methyl-tetrahydrosarcinapterin (CH 3 -H 4 SPt) during growth on acetate ( Galagan et al., 2002 ; Deppenmeier et al., 1999 ), but at the level of methyl-CoM during growth on DMS, methanol, and TMA ( Figure 6 ) ( Bose et al., 2008 ; Fu and Metcalf, 2015 ). Significantly, these entry points are separated by an energy-dependent step that is coupled to production or consumption of the trans-membrane Na + gradient. As a result, intracellular levels of methyl-CoM, CoM, and CoB could possibly be significantly different depending on the entry point into the methanogenic pathway. Because we observed growth defects on substrates that enter at both points (i.e. DMS and acetate), we suspect that the growth deficiency phenotype is unlikely to be related to changes in substrate affinity. A third explanation we considered was that thioglycine increases the stability of MCR. In this model, unstable MCR protein would need to be replaced more often, creating a significant metabolic burden for the mutant. Consistent with our results, this additional burden would be exacerbated on lower energy substrates like DMS and acetate, especially given that MCR comprises ~10% of the total protein ( Rospert et al., 1990 ). Further, one would expect that a protein stability phenotype would be exaggerated at higher temperatures, which we observed during growth on methanol ( Figure 5 ). Thus, multiple lines of evidence support the idea that the growth-associated phenotypes stemming from the deletion of TfuA and YcaO are caused by decreased MCR stability. However, the melting temperature of MCR from the ∆ ycao-tfuA mutant was modestly higher than that purified from the WT. As the thioglycine is buried deep within the active site of MCR ( Ermler et al., 1997 ), these data are consistent with the notion that thioamidation does not affect the global stability of the protein. Instead, it suggests that the thioglycine modification impacts the local stability in the vicinity of the buried active site of MCR ( Figure 1—figure supplement 1 ), which in turn might have a detrimental effect under catalytic turnover conditions. 10.7554/eLife.29218.018 Figure 6. An overview of methanogenic metabolism in M. acetivorans . Methylotrophic substrates such as methanol (CH 3 OH) or dimethylsulfide (DMS, CH 3 -S-CH 3 ) enter the methanogenic pathway via S -methylation of coenzyme M (CoM) and subsequently disproportionate to methane (CH 4 ) and carbon dioxide (CO 2 ; metabolic flux is shown as blue arrows). Notably, the first step in oxidation of CH 3 -CoM to CO 2 is the energy-requiring transfer of the methyl moiety to generate methyl-tetrahydrosarcinapterin (CH 3 -H 4 SPt). In contrast, acetic acid (CH 3 COOH) enters the pathway at the CH 3 -H 4 SPt level, followed by reduction to CH 4 (green arrows). Thus, the second step of the pathway is exergonic. The chemical properties of amides relative to thioamides are consistent with our hypothesis. Although amides tend to be planar, their rotational barriers are lower than for the corresponding thioamide and thus they are more conformationally flexible. This is especially true for glycine. Considering the negligible electronegativity difference between sulfur and carbon, the thioamide carbonyl is not polarized like an amide carbonyl ( Wiberg and Rablen, 1995 ). Further, sulfur has a larger van der Waals radius than oxygen resulting in a thioamide bond length that is ~40% longer than the amide bond (1.71 Å versus 1.23 Å) ( Petersson et al., 2014 ), which presents additional steric hindrances to backbone flexibility. Finally, the p K a of thioamides is lower than amides, making thioglycine a stronger hydrogen bond donor than glycine ( Lee et al., 2002 ), which again could reduce conformational flexibility. Taken together, it is chemically reasonable to state that the increased flexibility of the unmodified glycine in the Δ ycaO-tfuA mutant would render the contorted conformation of the peptide backbone in the Gly 462 -Leu 469 region of McrA considerably less stable ( Figure 1—figure supplement 1 ). Indeed, this region adopts a rather strange configuration, with the side chain of Phe 463 bending back towards the thioamide of Gly 465 . The molecular alignment and distance (3.5 Å) suggests a potential π-cation interaction between these residues( Figure 1—figure supplement 1 ) The thioamide sulfur is also optimally distanced (3.6 Å) and geometrically positioned to form an H-bond with the side chain of Asn 501 and the backbone N-H of Leu 469 . There also appears to be a hydrophobic interaction between the thioamide and the beta carbon of Leu 469 , which would be energetically unfavorable with a more polarized amide. These observations suggest that the local stability in the vicinity of the active site imparted by the thioamide is required for protein stability and increased half-life of the MCR protein. A conclusive test of this hypothesis will require comprehensive structural and biochemical characterization of the active MCR variant lacking the thioglycine modification, which is beyond the scope of this work. Finally, the temperature-sensitive phenotype of the Δ ycaO-tfuA mutant has potential implications regarding the evolution and ecology of methanogenic archaea. Based on this result, it seems reasonable to speculate that the thioglycine modification would be indispensable for thermophilic methanogens. It is often posited that the ancestor of modern methanogens was a thermophilic organism ( Gribaldo and Brochier-Armanet, 2006 ; Forterre, 2015 ; Weiss et al., 2016 ; López-García et al., 2015 ). If so, one would expect the thioglycine modification to be present in most methanogenic lineages, being stochastically lost due to genetic drift only in lineages that grow at low temperatures where the modification is not required. In contrast, if methanogenesis evolved in a cooler environment, one might expect the distribution of the modification to be restricted to thermophilic lineages. Thus, the universal presence of the thioglycine modification supports the thermophilic ancestry of methanogenesis. Indeed, the ycaO-tfuA locus is conserved even in Methanococcoides burtonii , a psychrophilic methanogen isolated from Ace Lake in Antarctica, where the ambient temperature is always below 2°C ( Franzmann et al., 1992 ). It will be interesting to see whether this modification is maintained by other methanogenic and methanotrophic archaea growing in low temperature environments."
} | 5,179 |
33155797 | null | s2 | 8,666 | {
"abstract": "Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified, but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5'-phosphate (PLP)-dependent enzyme (Fub7), and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic α,β-unsaturated aldehyde. By harnessing the stereoselective C-C bond-forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl-, 5,5-dialkyl-, and 5,5,6-trialkyl-l-pipecolic acids of high diastereomeric ratio."
} | 235 |
34874024 | null | s2 | 8,667 | {
"abstract": "Microbes, such as bacteria, can be described, at one level, as small, self-sustaining chemical factories. Based on the species, strain, and even the environment, bacteria can be useful, neutral or pathogenic to human life, so it is increasingly important that we be able to characterize them at the molecular level with chemical specificity and spatial and temporal resolution in order to understand their behavior. Bacterial metabolism involves a large number of internal and external electron transfer processes, so it is logical that electrochemical techniques have been employed to investigate these bacterial metabolites. In this mini-review, we focus on electrochemical and spectroelectrochemical methods that have been developed and used specifically to chemically characterize bacteria and their behavior. First, we discuss the latest mechanistic insights and current understanding of microbial electron transfer, including both direct and mediated electron transfer. Second, we summarize progress on approaches to spatiotemporal characterization of secreted factors, including both metabolites and signaling molecules, which can be used to discern how natural or external factors can alter metabolic states of bacterial cells and change either their individual or collective behavior. Finally, we address "
} | 328 |
24834901 | null | s2 | 8,671 | {
"abstract": "As swollen polymer networks in water, hydrogels are usually brittle. However, hydrogels with high toughness play critical roles in many plant and animal tissues as well as in diverse engineering applications. Here we review the intrinsic mechanisms of a wide variety of tough hydrogels developed over the past few decades. We show that tough hydrogels generally possess mechanisms to dissipate substantial mechanical energy but still maintain high elasticity under deformation. The integrations and interactions of different mechanisms for dissipating energy and maintaining elasticity are essential to the design of tough hydrogels. A matrix that combines various mechanisms is constructed for the first time to guide the design of next-generation tough hydrogels. We further highlight that a particularly promising strategy for the design is to implement multiple mechanisms across multiple length scales into nano-, micro-, meso-, and macro-structures of hydrogels."
} | 242 |
38959044 | PMC11252946 | pmc | 8,672 | {
"abstract": "Significance Directional liquid transport within closed spaces is crucial for both fundamental studies and industrial applications. Despite extensive progress in engineering diverse open surfaces to guide liquid transport, the implementation of diode-like liquid transport in closed spaces is still challenging. In this study, we present a flexible liquid-diode microtube designed specifically for directional liquid transport in closed spaces, achieved through microfluidics. Additionally, a flow lithography modality is introduced to produce short microtube segments to construct a fluidic–electronic circuit with the ability to execute dual logic control.",
"discussion": "Discussion In summary, we have presented a type of flexible liquid-diode microtube created through a piezoelectric microfluidic platform. These microtubes are formed by pulsating a core sacrificial jet and curing a surrounding sheath stream. This results in an asymmetric inner wall of the microtube with overlapped truncated cone-shaped cavities, each presenting a sharp edge and corner along the axial direction. Precise control over the structural features is achieved by adjusting the microfluidic and piezoelectric operation parameters. The unique structure makes the microtubes liquid diodes, allowing for directional liquid transport within confined spaces due to Laplace gradient and asymmetric pinning effect. The continuous fabrication of the microtubes, provided by microfluidics, enables long-distance liquid transport; the flexibility of the microtubes, owing to the rational choice of the component material, facilitates liquid transport along arbitrary pathways. Apart from long-length tubes, short-length tubes are prepared through flow lithography. Such liquid diode segments can serve as fundamental components in a fluidic–electronic circuit capable of executing logic operations, potentially paving the way for advancements in wearable technology, especially in health monitoring or sweat sensing. The above results indicate the multifunction of the liquid diodes. Future efforts can focus on incorporating stimuli-responsive nanoparticles or utilizing stimuli-responsive materials in the outer phase, potentially allowing these liquid diodes to possess active switching or pumping capabilities. For the fabrication platform, the dynamic flow control system can be integrated with advanced lithography technique or multichannel design of the microfluidic device to achieve multicomponent tubes with multifunctional properties. Additionally, combination of the piezoelectric microfluidic spinning platform with 3D printing may lead to the creation of macroscale entangled tube networks in various 3D shapes, thereby expanding their applications in real-world scenarios."
} | 686 |
36844546 | PMC9948187 | pmc | 8,673 | {
"abstract": "Silkworm silk proteins\nare of great importance in several fields\nof science owing to their outstanding properties. India generates\nwaste silk fibers, also known as waste filature silk, in abundance.\nUtilizing waste filature silk as reinforcement in biopolymers enhances\nits physiochemical properties. However, the hydrophilic sericin layer\non the surface of the fibers makes it very difficult to have proper\nfiber–matrix adhesion. Thus, degumming the fiber surface allows\nbetter control of the fiber properties. The present study uses filature\nsilk ( Bombyx mori ) as fiber reinforcement\nto prepare wheat gluten-based natural composites for low-strength\ngreen applications. The fibers were degummed in sodium hydroxide (NaOH)\nsolution from a 0 to 12 h duration, and composites were prepared from\nthem. The analysis exhibited optimized fiber treatment duration and\nits effect on the composite properties. The traces of the sericin\nlayer were found before 6 h of fiber treatment, which interrupted\nhomogeneous fiber–matrix adhesion in the composite. The X-ray\ndiffraction study showed enhanced crystallinity of the degummed fibers.\nThe FTIR study of the prepared composites with degummed fibers showed\nthat shifted peaks toward lower wavenumbers supported better bonding\namong the constituents. Similarly, the tensile and impact strength\nof the composite made of 6 h of degummed fibers showed better mechanical\nproperties than others. The same can be validated with the SEM analysis\nand TGA as well. This study also showed that prolonged exposure to\nalkali solution reduces the fiber properties, thus reducing composite\nproperties too. As a green alternative, the prepared composite sheets\ncan potentially be applied in manufacturing seedling trays and one-time\nnursery pots.",
"conclusion": "5 Conclusions In this study, the tensile, impact, thermal\nand morphological behavior,\nand molecular spectroscopy were systematically investigated for WG-WFS\ncomposites for different durations of fiber treatment in NaOH media.\nThe main findings are as follows, (i) The XRD study showed increased crystallinity\nfor the degummed fibers as the degumming duration increased. More\ncrystallinity means straighter and lustrous fibers, which helped the\ncomposites perform better under tensile and impact loading. (ii) The FTIR analysis shows\nenhanced\nβ-sheet formation as the fiber treatment time increases. It\nsupports the enhanced mechanical properties of the TFs for extended\nhours. On the other hand, shifting peaks to the lower wavenumbers\nat CTF-6 justifies better interaction among the composite constituents. (iii) The surface morphology\nshowed enhanced\nfiber–matrix interaction as the surface of the TFs gradually\ngot degummed. CTF-6 showed the best surface morphology among other\ncomposites. (iv) CUF-0\nshowed remarkable properties\nbecause of the support of the glue-like sericin layer. As the degumming\nprogressed, the composites from CTF-0.5 to CTF-4 showed gradual improvement\nin their mechanical properties. The overall mechanical properties\nof CTF 6 excel all other composites in terms of ultimate strength,\nelongation, and impact energy. However, Young’s modulus of\nsCTF-8 stands highest as more alkali treatment tends to increase the\nβ-sheet formation. The mechanical strength reduces further because\nof thinning of the fiber due to excessive NaOH exposure. (v) The TGA showed an almost similar trend\nfor all the composites, but CTF-6 performed marginally better than\nother composites in terms of thermal stability. Based on all the thermal and physiochemical observations,\nit can\nbe concluded that the composite prepared by NaOH-treated WFS for 6\nh delivers the best combination of strength, elongation, and thermal\nproperties. This work also provides future possibilities to conduct\nsimilar studies using other alkali solutions. The prepared sheets\nare used to develop 100% degradable nursery seedling pots and trays,\nwhich can be directly planted into the soil without discarding the\npot.",
"introduction": "1 Introduction Wheat gluten is a natural polymer consisting of a complex protein\nstructure. Because of its high biodegradability, low cost, non-toxicity,\nand renewability, it is often considered a potential substitute for\nsynthetic resins in natural composites. 1 , 2 Regardless\nof its impeccable properties, wheat gluten is very brittle in nature\nand absorbs ample amounts of moisture after processing. However, these\nshortcomings can be tailored by introducing synthetic or natural fibers\nas fiber reinforcements into it. 3 − 5 Natural fibers are fundamentally\nsustainable considering their fast biodegradability, non-toxicity,\nfast renewability, minimum carbon emission, and significantly fewer\nhealth hazards. 6 Natural silk ( Bombyx mori ) is one of the most popular fiber reinforcements\nto produce natural protein-based green composites. 7 , 8 The\nwaste natural silk, also known as waste filature silk (WFS), produced\nin textile industries during silk processing, possesses excellent\nmechanical and thermal properties. 9 − 12 India is the second-highest silk\nmanufacturer after China on the global map. 13 Along with the production of high-end clothing, the Indian silk\nindustry generates huge silk waste yearly, making it an economical\noption to use as fiber reinforcement. 14 , 15 The well-aligned\ninternal structure of the filature silks makes them susceptible to\nmore tensile load, high elongation, and good resilience. 16 Due to long chains of amino acids, these silks\npossess higher heat resistance capacity. 17 The WFS consists of two parts, fibroins (75%) in the inner section\nand sericin (25%) on the outer layer. The silk fibroins have a semicrystalline\nstructure that provides inner strength to the silk fibers, and sericin\nacts like amorphous glue, holding the fibroins and structure together. 18 Among the 18 amino acids that sericin possesses,\nserine aspartic acid and glycine are the three most essential amino\nacids responsible for its extreme hydrophilicity. 19 , 20 Research showed that hydrophobic surfaces bond with proteins more\ndefinitely than hydrophilic surfaces. Hydrophobic surfaces attach\nto another hydrophobic surface more viciously because of a good adsorption\nprocess. 21 Because of the hydrophilicity\nof the sericin layer, it is often challenging to have proper fiber–matrix\nadhesion with natural polymers like wheat gluten. 19 Although removing the sericin layer reduces the toughness\nof the silk fiber, the process may benefit the fiber–matrix\nbonding, thus improving the overall composite properties. The sericin\nlayers can be removed by various degumming methods like washing the\nfibers off with different solutions like sodium hydroxide (NaOH) 22 and sodium carbonate, 23 soap washing, 24 succinic acid, urea, 25 citric acid, 26 hot\nwater, 27 and hydrogen peroxide. 28 The degummed structure of WFS consists of a\nrandom coil amorphous structure and a periodic β-sheet structure. However,\nthe amorphous coil structure may change into a regular β-sheet\nstructure upon mechanical stretching and moisture absorption during\nthe degumming process. 29 Conversion of\nrandom and α-helix structures to more β-sheet structures\nmakes the fibers straight, lustrous, and stiffer. 30 The main strength of any natural fiber is acquired from\nthe hydrogen bond present in the core protein structure. 31 Thus, preserving those hydrogen bonds while\ndegumming should be of utmost priority. The degumming process can\nreadily remove sericin layers, but sometimes, too much exposure to\nthe alkali solution may cause detrimental changes to the fiber’s\nmechanical, morphological, and chemical properties. Alkali water solution\nhas been used from ancient times for simple and effective degumming\nof raw silk cocoons. Alkali degumming removes the sericin binding\nagent without damaging the nature of the fiber. 32 NaOH and water solution are widely used for deep cleaning,\ndegumming, and enhancing the properties of the raw silk fibers. 22 − 34 The primary purpose of degumming the raw fiber is to hydrolyze the\nsericin layer and dissolve it into the degumming solution or media,\nmaking it detangled and open. 16 , 25 When used in developing\ncomposites, these detangled fibers help in distributing the stress\nevenly in the structure, thus improving the overall properties of\nthe composite. Although NaOH treatment improves many properties of\nthe fiber, exposure to the alkali solution for a longer duration disturbs\nthe WFS core structure. Only a handful of studies reported the effect\nof degumming duration on the texture and core structure of different\nkinds of silk fibers. 25 , 32 , 35 , 36 The main objective of this study is to optimize\nthe fiber treatment time or degumming time without affecting the core\nstructure of the WFS fibers so that the prepared composites can adhere\nbest to physiochemical properties. In this study, degummed silk fibers\nwere used to prepare a wheat gluten-based biocomposite where the lemon\nextract was used as a crosslinker and castor oil was used as a plasticizer.\nThe methodology and process optimization to prepare these composite\nsheets were thoroughly studied in our previous work where the samples\nwere prepared with raw WFS (non-degummed). 11 , 37 The fibers were treated in 5% NaOH–water solution for 0,\n0.5, 1, 2, 4, 6, 8, 10, and 12 h, and the mechanical thermal, morphological,\nand molecular analysis were done on them. 38 , 39 For consistency, the NaOH solutions were kept at 5% for all the\ndegumming periods. However, variation of the NaOH concentration for\ndegumming of the fibers is currently out of this paper’s scope\nand is considered a future study. The composite prepared by degummed\nfibers showed improved physiochemical and thermal properties. It was\nalso observed that extended exposure to alkali solution reduces the\nfiber and composite properties drastically.",
"discussion": "3 Results and Discussion 3.1 XRD Analysis of the Fibers XRD analysis\nwas performed on all the silk samples before and after degumming to\nstudy the change in crystalline and amorphous structure in the WFS\nfor different conditions. It is shown in Figure 3 that the major diffraction peaks got generated\nat 21° (2θ), which generally depicts the secondary β-structure\nor crystallinity of the fibers. It is observed that the raw silk fiber\nUF-0 showed a broad peak around 21.3°, which indicated the natural\npattern of amorphous silk. As degumming begins, the peak intensity\nincreases gradually from CF-0.5 to CF-10. The increase in peak intensity\nand height conveys the increase in the crystallinity of the fibers\nupon degumming. 41 However, the peak intensity\ndrops slightly for CF-12, imparting that over-exposure to NaOH solution\nmay have started to affect the core structure of the silks. Table 3 quantifies the percentage\nof amorphous and crystalline structure for all the fibers as per the\nXRD-generated data. Figure 3 X-ray diffraction curves of the waste filature silk before\nand\nafter degumming in NaOH solution for different duration of time. Table 3 Change in Percentage of Crystallinity\nand Amorphous Region of Waste Filature Silk before and after Degumming\nin NaOH Solution for Different Durations sample name % crystallinity % amorphous UF-0 38.5 61.5 CF-0.5 39.4 60.6 CF-1 40.4 59.6 CF-2 40.9 59.1 CF-4 43.7 56.3 CF-6 44.8 55.2 CF-8 48.4 51.6 CF-10 48.9 51.1 CF-12 47.0 53 3.2 Fourier Transform Infrared (FTIR) Spectroscopy Building strong interfacial cohesion between silk fibroins and\nthe adjacent polymer is challenging. Removing the sericin layer of\nsilk by NaOH treatment improves the composite’s overall properties.\nFTIR shows the underlying factors of structural changes to understand\nthe variation in mechanical properties of the prepared composites.\nHence, FTIR spectra are used here to identify the structure of untreated\nfiber (UF), simple WG, treated fiber (TF), and the composites prepared\nby them. The primary structure of the silk consists of amino acids\nof glycine, alanine, and serine in a repetitive order. 16 They take up to 90% of the protein in silk,\nand the last 10% consists of glutamic, valine, and aspartic acids. 35 They act like side chains and are primarily\nresponsible for the elasticity and strength of the silk. The secondary\nstructure of the silk is mainly dominated by β-pleated sheets\nconnected to each other with hydrogen bonds. 42 Apart from this, α-helix, turn, and random coils also play\na dominating part in the secondary structure of silk. FTIR spectra\nare widely sensitive to the secondary structure of silk fibroins,\nproviding molecular validation of the change in silk structure. 43 , 44 The signature absorption peaks for WFS are found around 1620 cm –1 (Amide I), 1515 cm –1 (Amide II),\nand 1260 cm –1 (Amide III). These peaks are conformation\npeaks of the crystalline β sheet structure, and the silk shows\nsimilar peaks in all degumming conditions until the core structure\nstarts to break. 36 The Amide I band of\nthe silk fibroin (between 1600 and 1700 cm –1 ) is\nmainly associated with C=O stretching vibration (70 to 85%) and is\ndirectly related to the backbone conformation. The Amide II band results\nfrom the N–H bending vibration (40–60%) and the C–N\nstretching vibration (18–40%). The Amide III band is conformationally\nsensitive and very rarely traced with sharp peaks. The Amide II peaks\nare found where microsphere or aqueous formation takes place. The\nformation of microspheres confirms a more stable state of the fibroins.\nThe stretched vibration peaks around 1600 cm –1 confirm\nthe presence of stronger C=O stretching. The definite peaks for the\nAmide I and Amide II regions at 1630 and 1520 cm –1 indicate crystalline β-sheet conformation. 45 As the degumming occurs, some subtle change occurs around\n1000 cm –1 , as shown in Figure 4 . The peak intensity increases for the TFs,\nindicating enhanced crystalline β structure formation. 36 Degumming generally increases the formation\nof crystalline β-sheets of WFS. Although lesser exposure to\nNaOH treatment does not affect the amorphous peptide formation of\nthe silk, prolonged exposure of silk to alkali solution may affect\nthe silk surface morphology. 32 , 46 The alkali treatment\nremoves the amorphous region and makes the silk more crystalline,\nthus reducing the silk elongation rate. Increasing the duration of\nfiber treatment reduces the elongation and creates more brittleness\nin the WFS and the composites. The gelatinization of WG occurs in\nthe presence of lemon extract, NaOH, and castor oil. These components\nhelp to have better crosslinking in the WG and WG with WFS. Better\ncrosslinking leads to better interaction between functional and proton\ndonor groups. Enhanced interaction forces the proton donor group to\nmake them give up their electron and reduce electron density. Reduced\nelectron density also reduces the vibrational energy of the different\nfunctional groups, thus generating characteristic peaks at lower wavenumbers. 47 The signature peaks of lower wavenumber in FTIR\nstudies indicate better interaction among the constituents of the\nmatrix. 11 The reduced intensity of WG to\nCUF-0 at 3200 peaks represents reduced hydroxyl groups after crosslinking.\nThe peaks between 1613 and 1632 cm –1 are assigned\nfor β-sheets of the WG protein structure. The β structure\nchanges with the increase in hydration. The spectra generated around\n1000 cm –1 are the combined peaks for WG and WSF\nboth. The peaks and their absorption range are represented in Table 4 . The tensile and\nthermal properties of the composites increase because of the presence\nof opened-up fibroins due to their ability to interact with WG protein\nto form complex protein structures. The reduced wavenumbers on and\nafter CTF-4 indicated enhanced interaction among the constituents\nof the composite after the complete removal of the sericin layer.\nThis data also supports the composites’ enhanced tensile and\nthermal properties, as described in the upcoming sections. Figure 4 Comparison\ngraph of FTIR analysis between treated and untreated\nwaste filature silk, wheat gluten, and the composites prepared by\nthem. Table 4 FTIR Absorption Band\nof Different\nCompound Classes Present in Filature Silk and Wheat Gluten Composites 48 absorption (cm –1 ) appearance group compound class 3300–2500 strong broad O–H stretching carboxylic acid 3000–2800 strong\nbroad N–H stretching amine 1750–1725 strong C=O stretching esters 1650–1580 medium N–H bending amine 1550–1500 strong N–O stretching nitro compound 1250–1020 medium C–N stretching amine 1085–1050 strong C–O stretching primary alcohol 3.3 Scanning Electron Micrographs\n(SEMs) The SEMs showed the microlevel changes in the fibers\nand composites\nfor all nine sets of samples. The micrographs shown in Figure 5 elaborate on the gradual removal\nof the sericin layer from the fibers, composite cross section, and\nadhesion between the major constituents of the composites. Samples\nwere collected from the fractured surface of the composites to study\nthe behavior of fiber–matrix bonding under applied load. Figure 5 a shows CUF-0, which\nclearly shows the thick sericin layer and the heavily tangled fibroins\nin it. Fiber clustering renders poor mechanical properties. As the\nuntreated WFS shows a hydrophilic nature, the fiber–matrix\nadhesion was very poor for the prepared composite. The micrograph\nof the WFS collected from the fractured surface showed lack of adhesion\nof WG, in the fiber periphery. This slip of fiber from WG results\nin average mechanical properties. The TFs for the first three samples,\nCTF-0.5, CTF-1, and CTF-2, had medium to low traces of sericin present\nin them. The semi-open fibers and the presence of sericin created\nuneven adhesins of fiber–matrix and interlayer voids, as shown\nin Figure 5 b–d.\nThe samples prepared by TFs from 4 h onward had low to no traces of\nthe sericin layer. The complete removal of sericin made the fibers\nstraighter and more lustrous. This helped to develop a better composite\nstructure that was almost free from voids and poor adhesion, as shown\nin Figure 5 e,f. CTF-4\nand CTF-6 showed the most promising properties among all their counterparts.\nThe interface is strongest for CTF-6 as it showed the least number\nof voids and a better interface among all the samples. A better fiber–matrix\ninterface leads to better stress distribution, enhancing the prepared\ncomposites’ load bearing capacity under tensile and impact\nloading. Although the fiber–matrix adhesion in CTF-8, CTF-10,\nand CTF-12 was also good, the fibroins’ thinning due to extensive\nalkali exposure weakened the fibers. As a result, they cannot carry\nthe tensile and impact loads ensuing in easy failure, as seen in Sections 3.4 and 3.5 . Figure 5 SEM micrograph of (a)\nCUT-0, (b) CTF-0.5, (c) CTF-1, (d) CTF-2,\n(e) CTF-4, (f) CTF-6, (g) CTF-8, (h) CTF-10, and (i) CTF-12, analyzing\nthe microstructure of the composite cross section, fibers at different\nstages, and fiber–matrix adhesion of waste filature silk and\nwheat gluten. Figure 6 Comparison graph of the single-fiber tensile\nproperties of the\nwaste filature silk before and after treatment. (a) Young’s\nmodulus, (b) ultimate tensile strength, and (c) % elongation. 3.4 Tensile Properties The tensile properties\nof the fibers and composites were compared among the nine sets of\nsamples. The first sample, CUF-0, was prepared from UFs where the\nsericin layer glued the fibroins randomly. The fibroins were badly\ntangled and unable to stretch themselves upon tensile loading, resulting\nin the brittle behavior of the composite. 22 The tensile strength is comparatively higher for CUF-0, but the\nelongation is critically low, supporting the brittle nature of the\ncomposite. The first four sets of TF-based composites CTF-0.5, CTF-1,\nCTF-2, and CTF-4 showed a gradual increase in their tensile properties,\nas shown in Figure 7 . The micrograph in Section 3.3 clearly showed the partially present hydrophilic sericin\nlayer among the semi-opened fibroins, which delivers uneven sipping\nof WG into fibers. This leads to the formation of internal voids in\nthe composite, resulting in early failure upon tensile loading. As\nthe sericin layer was completely removed after 6 h of NaOH treatment,\nthe fiber–matrix adhesion improved drastically, resulting in\nimproved tensile properties. The fibers of 6 h of treatment showed\nimproved elastic properties as they were entirely free from gluey\nsericin binding. It increased the tenacity of the fibers, which increased\nthe interfacial adhesion of the fiber and matrix. Hence, CTF-6 showed\nthe best elongation among all the composites. CTF-8, CTF-10, and CTF-12\nshowed excellent Young’s modulus but drastically reduced maximum\ntensile strength and elongation. Exposure of the fibers in NaOH solution\nfor a prolonged period resulted in the formation of more β-sheets,\nmaking them stiffer. 35 It also started\nto peel the microfibrils, making the fibers thinner and weaker. It\nreduced the fibers’ strength, which resulted in the samples’\nearly failure. The same characteristic pattern can be seen in the\ntensile properties of the fibers, as shown in Figure 6 . Although the degumming process increased\nthe crystallinity of the fibers, thus making them stiffer, gradual\nremoval of the sericin layer increased the fibers’ elongation\nas they can move freely. It further verified the tensile behavior\nof the composite influenced by the tensile properties of the fiber\nas the degumming time increased. Figure 7 Comparison graph of tensile properties\nfor the composite manufactured\nby wheat gluten and untreated and NaOH treated waste filature silk.\n(a) Young’s modulus, (b) ultimate tensile strength, and (c)\n% elongation. Because of all these reasons,\nthe Young’s modulus for CUF-8\nis maximum showing enhanced adhesion among the fiber and wheat gluten,\nbut it lacks elongation. As fiber failure occurred drastically upon\nincreased load, wheat gluten overpowered the composite’s mechanical\nproperties, thus making it more brittle. The same trend can be seen\nfor CTF-10 and CTF-12. The tensile comparison proved optimized properties\nfor CTF-6 as it showed good Young’s modulus, best tensile strength,\nand elongation among its peers. 3.5 Impact\nStrength During the impact\ntest, impact energy gets dissipated in the form of matrix breakage,\nfiber breakage, matrix–fiber interface failure, delamination\nof layers, and crushing of the core structure of the composite elements.\nThe impact test finds the damage tolerance of the composite as impact\nload reduces the design strength of the composite. The low-velocity\nIzod test primarily finds the damage resistance in terms of absorbed\nenergy or impact energy, which is directly proportional to impact\nstrength. Figure 8 shows\nthat the impact energy for CUF-0 is 0.644, indicating higher impact\nstrength than CTF-0.5 and CTF-1 composites. The sericin layer of the\nUFs works like external support, increasing the composite’s\nstrength. However, at the same time, sericin does not offer good fiber–matrix\nadhesion with WG, being hydrophilic. The tangled fibers tightly packed\nunder the sericin layer also fail to distribute stress under impact\nload. This results in the early failure of the composites. On the\nother hand, CTF-0.5 and CTF-1 show early failure due to randomly removed\nsericin layers. The semi-opened and semi-tangled fibers fail under\nimpact load due to uneven stress distribution and non-homogeneous\norientation of the fibroin. The impact strength increases CTF-2 onward\nas sericin starts to peel off entirely from the fibers and the fibroins\nstart to untangle. NaOH-TF removes the sericin layer, which activates\nalanine and glycine compounds. As both these compounds are hydrophobic\nin nature, it induces a higher affinity toward hydrophobic WG and\ndevelops a stronger bond. 22 , 35 It increases the fiber–matrix\ninterface, thus increasing impact strength. The interface is most\nsubstantial for CTF-6 as it showed the best fiber and matrix adhesion\nand very few voids, as shown in Section 3.3 . The impact strength further reduces for\nCTF-8, CTF-10, and CTF-12 as the fiber diameter reduces, making them\nweak and breaking faster under impact. Figure 8 Comparison graph of impact\nenergy for the composite manufactured\nby wheat gluten and untreated and NaOH treated waste filature silk. 3.6 TGA TGA shows\nthe thermal stability\nof the composites at elevated temperatures. The first stage of 0 to\n5% weight reduction occurs due to the evaporation of trapped moisture\nand air in the voids of the sample. The integral moisture absorption\nproperty of WG attracts a lot of residual moisture. This moisture\nand air evaporate upon heating, which occurs in the first stage of\nweight reduction. The subsequent 5 to 10% weight reduction occurs\ndue to the evaporation of plasticizer in the composites, which is\ncastor oil for this case. The next stage of drastic weight reduction\noccurs at the temperature range of 200 to 250 °C, as shown in Figure 9 , due to the breakage\nof amino acid and hydroxyl groups present in the composite. As most\nof the graphs are overlapping, an enlarged view of the TGA graph at\n220 °C is as shown in Figure 9 a. The graph shows CTF-6 showing maximum stability\nof 90.5 wt % at the said temperature. Better thermal stability represents\nbetter structural integrity and molecular interaction among the constituents\nof the composite. At 220 °C, the samples that showed good thermal\nstability after CTF-6 are CTF-8 with 90.2 wt % and CTF-10 with 89.7\nwt %. It again proved to have good structural integrity, as mentioned\nin previous sections. The following composites, CTF-1, CUF-0, and\nCTF-0.5, were protected by the amino acid bonds of the sericin layer\nfrom the extreme burning of the samples. However, due to structural\ndefects, it cannot excel among its other counterparts. CTF-4 showed\ncomparatively better thermal stability (87.2 wt % at 220 °C)\nbecause of better interaction among the constituents, as mentioned\nin previous sections. CTF-2 and CTF-12 showed poor property, probably\nbecause of many voids in the CTF-2 structure and thinning of fiber,\nfollowed by poor strength for the CTF-12 composite. In the third stage\nof TGA, the drastic weight reduction occurs above 450 °C, where\nthe remaining N–H, N–O, C–O, and C–O links\ncollapse and the sample completely decompose. 37 Figure 9 b shows the\nenlarged view at 480 °C, where the CTF-6 shows dominance with\n37 wt % over other samples. It is followed by CTF-0.5 at 36.8 wt %,\nCTF-2 at 35.5 wt %, CTF-8 at 34 wt %, CTF-1 at 33 wt %, CTF-4 at 33\nwt %, CTF-12 at 32.3 wt %, CTF-10 at 31.9 wt %, and CTF-0 at 28. 5\nwt %. They are all segregated with a very narrow margin showing good\nthermal stability for all the samples. At the last stage, all the\ncomposite samples completely decompose at the temperature range of\n550 to 600 °C. Although CTF-6 showed elevated properties for\nthe first three stages, it completely decomposes at 550 °C, which\nis lesser than CUF-0, CTF-8, CTF-10, and CTF-12. They survived more\nin the later stage probably because of the presence of the sericin\nlayer, and CTF-8, CTF-10, and CTF-12 survived because of better adhesion\namong fiber and matrix, which required more energy to burn out completely. Figure 9 Comparison\ngraph of TGA for the composite manufactured by wheat\ngluten and untreated and NaOH-treated waste filature silk. (a) Enlarged\nview at 220 °C and (b) enlarged view at 480 °C."
} | 6,845 |
22319314 | PMC3274235 | pmc | 8,676 | {
"abstract": "This paper presents a novel method to fabricate temperature sensor arrays by dispensing a graphite-polydimethylsiloxane composite on flexible polyimide films. The fabricated temperature sensor array has 64 sensing cells in a 4 × 4 cm 2 area. The sensor array can be used as humanoid artificial skin for sensation system of robots. Interdigitated copper electrodes were patterned on the flexible polyimide substrate for determining the resistivity change of the composites subjected to ambient temperature variations. Polydimethylsiloxane was used as the matrix. Composites of different graphite volume fractions for large dynamic range from 30 °C to 110 °C have been investigated. Our experiments showed that graphite powder provided the composite high temperature sensitivity. The fabricated temperature sensor array has been tested. The detected temperature contours are in good agreement with the shapes and magnitudes of different heat sources.",
"conclusion": "5. Conclusions A flexible temperature sensor array consists of a graphite-PDMS composite, metal sensing electrodes, and flexible polyimide film has been successfully fabricated. There are 8 × 8 sensor cells in a 4 × 4 cm 2 area with good flexibility and robustness. Our experiment shows that polymer mixed with conductive fillers exhibits resistance variation when it is subjected to ambient temperature change. Graphite powders mixed with PDMS is found to have the highest temperature sensitivity and higher stability compared with the composites using other carbon fillers. As the volume fraction of graphite powders reaches the percolation threshold, the resistivity of the composites drops substantially. Different volume fractions of graphite powders have been investigated. The composite with 15% graphite powder are suitable for on/off devices while the one with 20% graphite powder provides sufficient dynamic range for continuously sensing temperature changes. The function of the fabricated temperature array has been verified through a resistance scanning system. The detected temperature contours agree well with the shape and magnitude of the applied heat source. An automatic dispensing method has been developed to fabricate this temperature sensor array. Larger sensor array can be easily achieved using the same process for robotic applications.",
"introduction": "1. Introduction Recently, intensive efforts have been taken to develop sensing systems for intelligent robots. One of the focuses on this subject is the design and fabrication of humanoid artificial skin. The successful implementation of artificial skin relies on the improvement of its sensitivity and mechanical flexibility. Most investigations of artificial skin emphasize the touch or tactile sensing to imitate the somatic sense of real human skin. Tajima et al. [ 1 ] developed a multi-layer touch sensor integrated with soft materials by microelectromechanical systems (MEMS) technology. Engel et al. [ 2 ] and Someya et al. [ 3 ] presented soft and flexible tactile sensor arrays for robotic applications. As a matter of fact, the sensory functions of human skin include not only the touch sensation but also pressure, temperature, pain, or other compound reactions from receptors and nerve endings. In the development of more sophisticated skin-like sensing systems, Siegel et al. [ 4 ] mounted 8 × 8 force sensing cells and 4 × 4 temperature sensors in a stacked rigid substrate, which was then implemented on a robot finger. Their temperature sensors are based on detecting heat loss due to ambient temperature variations. To develop flexible sensor array, Someya et al. [ 5 ] fabricated networks of pressure and thermal sensors using active matrices of organic transistors. Han et al. [ 6 ] employed platinum microresistors to form a one-dimensional flexible temperature sensor array. Temperature sensing systems have yet to become more commonplace in the field of robotic sensation. Besides Someya et al. ’s work [ 5 ], reports about large arrays of temperature sensors on flexible substrate are still very rare. The development of a flexible sensor array relies on an appropriate sensing mechanism and a suitable large-area fabrication technique. Our preliminary study showed that a polymer dielectric filled with conductive particles could be a good candidate to provide both mechanical flexibility and temperature sensitivity to the sensor array [ 7 ]. When the volume fraction of conductive particles inside a polymer dielectric approaches a percolation threshold, the electrical resistivity of the composite dramatically drops. This is because the number of the conductive particles reaches a critical value at which some “electrical pathways” are formed in the polymer matrix [ 8 ]. The associated material model is based on general effective media theory and has been intensively studied [ 9 – 12 ]. The electrical resistivity of the conductor-polymer composite could change significantly in response to mechanical stress or temperature variations. In theory, conductive fillers with suitable conductivity and aspect ratio can make the electrical resistivity of the composite sensitive to temperature changes [ 11 ]. Although the piezoresistivity of similar composites subjected to external stresses has been extensively studied [ 11 , 13 – 18 ], the temperature response and associated sensor development have not been reported. In this paper, we investigate the resistivity-temperature characteristics of a graphite-polydimethylsiloxane (PDMS) composite. A bending test is also conducted to verify the resistivity response of the composite. To fabricate a sensor array based on the investigated carbon-polymer composites, we demonstrate a mass production process which employs an automatic dispensing system that is used to apply the composites onto a flexible circuit board. The function of the fabricated flexible sensor array will be shown.",
"discussion": "4. Experiments and Discussion PDMS with three different volume fractions of graphite powder (15% and 25%) were chosen to determine the resistance variations by altering temperature on a hotplate in an air atmosphere. These volume fractions were chosen for higher temperature sensitivity as they are in the range of the percolation threshold. After degassing and curing, the sheets of the graphite-PDMS composites were cut into 1 × 1 cm 2 pieces and then sandwiched by copper plates. The thickness of these PDMS sheets was fixed at 0.5 mm by using a scraper before polymerization. The copper plates were connected to a multimeter for resistance measurement while the temperature on the composite sheets was increased from 25 °C to 110 °C on the hotplate. Before each measurement, the temperature was increased by 5 °C and then maintained as a constant for 10 minutes to assure thermal equilibrium between testing sample and hotplate. The relative humidity of the testing environment was maintained at 60%. Figure 7 shows the measured resistance variation of the graphite-PDMS composites subjected to different temperature. Platinum thin film as a temperature sensor was also characterized for comparison. The temperature coefficient of resistance (TCR) can be calculated by:\n (1) α = Δ R / R 0 T − T 0 where T 0 is the ambient temperature, and R 0 is the initial resistance of tested sample. Equation (1) is used to extract the TCRs in our experiment. The fitting curves are shown as the dashed lines in Figure 7 . The experimental results agree with the linear model. According to the data, the platinum TCR is determined to be 0.0055 K −1 . The TCRs of the composites are 0.042 K −1 and 0.286 K −1 for the graphite volume fractions of 25% and 15%, respectively. The testing results indicate that the sensitivity of the graphite-PDMS composites is higher than that of typical platinum temperature sensors [ 29 ]. Although the uniformity of the graphite-PDMS composites still has to be improved, these composites have been demonstrated to have potential applications in automatic or robotic systems. Prominent increase of resistance in these composites is also observed as the volume fraction of graphite powder decreases. However, the sensation range also drops with decreasing volume fraction. For the 15% volume fraction, the temperature sensing polymer becomes insulating when the temperature is greater than 40 °C. The thermal expansion of the PDMS could break contact between graphite particles in the composite [ 30 ]. Because the sample of 15% volume fraction is close to the lower bound of the percolation region as shown in Figure 3(a) , all the conductive paths in the composite are broken at over 40 °C. Therefore, this temperature sensing polymer is suitable to be a switch apparatus for protecting objects from high temperature sources because of its sharp increase in resistance. The experimental result in Figure 7 implies that the 25% volume fraction composite has lower TCR than the 15% volume fraction composite. More rigorous experiment is required for fully understanding the TCRs of this composite in the future. The flexibility of the fabricated sensing composite with 20% volume fraction of graphite powder has been characterized. In this test, the sensing composite in 1 cm 2 area was sandwiched by a conductive copper tape and a conductive carbon tape on the flexible printed circuit board, as shown in Figure 8 . The configuration in Figure 8(b) is to shift the material away from the neutral axis so that the material can be completely under tensile stress. The sensing composite was then wrapped around steel rods, and its resistance change was measured. The steel rods have different curvature radii from 2 mm to 10 mm with 1 mm increment. The testing results in Figure 9 indicate that the resistance decreases with the radius of curvature. This is because the composite was put at the convex side in the test and hence was subjected to tensile stress. The tensile stress stretches the composite laterally, causing the thickness to decrease. Thinner composite has smaller resistance. This bending test implies that the sensing signal of the composite could be affected by external stress. To remove the bending effect, one can simply put the composite close to the neutral axis by change the thickness ratio of the material to the substrate. The fabricated temperature sensor array of 20% volume fraction of graphite powder was connected to the scanning circuits for full-function evaluation. At room temperature, the resistance varies randomly between 1.43 MΩ and 1.76 MΩ across the sensor array. For easy observation, a 4 × 4 resistance map of the array is shown in Figure 10 . The resistance variation could be due to the thickness and shape non-uniformity since they were controlled by applying the thin plate for flattening all sensor cells. Nevertheless, measurement uniformity could be achieved by conducting independent calibration of each sensor cell. We also improved the performance uniformity of the sensor array by adjusting scanning circuit program. Then heat sources with different shapes were applied 5 mm above the temperature sensor array. The heated ring is made of iron wire. The heat rod is soldering iron. The heated plate is made of bulk aluminum. The temperature increase is successfully detected by each sensor cell, and the overall temperature is displayed by the contour map shown in Figure 11 . These contours are in well agreement with the shapes and temperature scales of the applied heat sources. The non-uniform temperature distribution could be caused by the heat source, background noise, or ambient fluctuations."
} | 2,895 |
34316509 | null | s2 | 8,677 | {
"abstract": "Although mechanical signals presented by the extracellular matrix are known to regulate many essential cell functions, the specific effects of these interactions, particularly in response to dynamic and heterogeneous cues, remain largely unknown. Here, we introduce a modular semisynthetic approach to create protein-polymer hydrogel biomaterials that undergo reversible stiffening in response to user-specified inputs. Employing a novel dual-chemoenzymatic modification strategy, we create fusion protein-based gel crosslinkers that exhibit stimuli-dependent intramolecular association. Linkers based on calmodulin yield calcium-sensitive materials, while those containing the photosensitive LOV2 (light, oxygen, and voltage sensing domain 2) protein give phototunable constructs whose moduli can be cycled on demand with spatiotemporal control about living cells. We exploit these unique materials to demonstrate the significant role that cyclic mechanical loading plays on fibroblast-to-myofibroblast transdifferentiation in three-dimensional (3D) space. Our moduli-switchable materials should prove useful for studies in mechanobiology, providing new avenues to probe and direct matrix-driven changes in 4D cell physiology."
} | 306 |
33857258 | PMC8049308 | pmc | 8,678 | {
"abstract": "Wastewater treatment plants (WWTPs) are important for pollutant removal from wastewater, elimination of point discharges of nutrients into the environment and water resource protection. The anaerobic/anoxic/oxic (A2/O) process is widely used in WWTPs for nitrogen removal, but the requirement for additional organics to ensure a suitable nitrogen removal efficiency makes this process costly and energy consuming. In this study, we report mixotrophic denitrification at a low COD (chemical oxygen demand)/TN (total nitrogen) ratio in a full-scale A2/O WWTP with relatively high sulfate in the inlet. Nitrogen and sulfur species analysis in different units of this A2/O WWTP showed that the internal sulfur cycle of sulfate reduction and reoxidation occurred and that the reduced sulfur species might contribute to denitrification. Microbial community analysis revealed that Thiobacillus , an autotrophic sulfur-oxidizing denitrifier, dominated the activated sludge bacterial community. Metagenomics data also supported the potential of sulfur-based denitrification when high levels of denitrification occurred, and sulfur oxidation and sulfate reduction genes coexisted in the activated sludge. Although most of the denitrification genes were affiliated with heterotrophic denitrifiers with high abundance, the narG and napA genes were mainly associated with autotrophic sulfur-oxidizing denitrifiers. The functional genes related to nitrogen removal were actively expressed even in the unit containing relatively highly reduced sulfur species, indicating that the mixotrophic denitrification process in A2/O could overcome not only a shortage of carbon sources but also the inhibition by reduced sulfur of nitrification and denitrification. Our results indicate that a mixotrophic denitrification process could be developed in full-scale WWTPs and reduce the requirement for additional carbon sources, which could endow WWTPs with more flexible and adaptable nitrogen removal.",
"conclusion": "Conclusion Mixotrophic denitrification was reported in the A2/O process with low COD/TN and high sulfate in the influent. Metagenomic analyses revealed the mixotrophic denitrification potential based on sulfur reduction and oxidation cycles at the level of microbial communities and functional genes. High abundances of sulfur oxidation, sulfate reduction and denitrification genes were found in our study. Thiobacillus dominated the microbial communities, and the narG gene was mainly harbored by Thiobacillus and Thauera , however, denitrification genes were enriched in heterotrophic denitrifiers with high abundance. Both autotrophic and heterotrophic denitrifiers contributed to nitrogen removal. Reduced sulfur showed no inhibition of the expression of nitrifying and denitrifying genes. Further investigation aimed at manipulating the denitrification process will help to further identify the key autotrophic denitrification metabolism pathways involved in nitrogen removal and the important factors related to denitrification under different conditions.",
"introduction": "Introduction With the increasing realization of the impacts of excess nitrogen (N) discharge on the environment and human health, N effluent regulations have become increasingly stringent worldwide. Until now, the biological process of nitrification/denitrification has been the most prevalent wastewater treatment plant (WWTP) used to remove N from wastewater. Normally, the ratios of COD (chemical oxygen demand)/TN (total nitrogen) and BOD (biological oxygen demand)/TN are required to be higher than 15 and 8, respectively, to supply a sufficient carbon source for the traditional nitrification/denitrification process [ 1 , 2 ]. To ensure N removal, external organics are often required, and the additional consumption of carbon and energy has become a challenge in some WWTPs. As sustainable wastewater treatment becomes a priority, autotrophic denitrification processes based on sulfur oxidation have become increasingly popular [ 3 – 5 ]. Sulfur-based autotrophic denitrification, such as the sulfate reduction autotrophic denitrification nitrification integrated (SANI ® ) process and sulfur-limestone autotrophic denitrification (SLAD), has been comprehensively studied [ 3 , 5 – 8 ]. In these processes, chemolithotrophic sulfide-oxidizing denitrifying bacteria (SONB) ( Thiobacillus sp., Sulfurimonas denitrificans , Beggiatoa sp., and Thiothrix sp.) and heterotrophic sulfide-oxidizing denitrifiers ( Thauera -like taxa, Azoarcus , Pseudomonas , and Dechloromonas ) cooperate in the processes of N removal [ 3 , 5 , 9 – 14 ]. Moreover, sulfate reducing bacteria (SRB), such as Desulfobacteraceae , Desulfonema , and Thermotogaceae , in activated sludge (AS), which convert sulfate to sulfur, sulfide, and poly-S, could cooperate with SONB and enhance N removal by providing electron donors [ 3 , 8 , 15 ]. Normally, studies of sulfur-based denitrification have focused on wastewater containing high concentration of sulfate, such as saline sewage [ 3 , 16 ]. In fact, some inland WWTPs, especially WWTPs located in industrial parks, also treat influent containing high sulfate concentrations due to the high concentration of sulfate in industrial wastewater [ 17 , 18 ], which might boost the development of sulfur-based denitrification. In our previous study, we analyzed the performance of a full-scale WWTP (referred to hereafter as the YXM WWTP) based on an anaerobic/anoxic/oxic (A2/O) process in an industrial park in Yixing, China, and found that the WWTP could achieve efficient N removal under a low ratio of COD/TN [ 19 ]. In the YXM WWTP, approximately 40% of the influent was collected from an industrial park, and the average concentration of SO 4 2- -S in the inlet was over 100 mg L -1 , which could provide the sulfate needed in mixotrophic denitrification. Moreover, organic carbon, N and sulfate coexist in actual wastewater. Mixotrophic denitrification, which integrates the advantages of heterotrophic and autotrophic denitrification, is promising in removing N, and it can match the actual conditions of wastewater and reduce the requirement for carbon sources [ 6 , 11 , 20 ]. Furthermore, the A2/O process has temporal changes in redox conditions that create an environment favorable for sustaining sulfide for extended time periods, and previous researchers have reported sulfide-related corrosion in the A2/O process [ 14 ]. Accordingly, we hypothesized that the N removal under a low COD/TN ratio in the YXM WWTP was due to mixotrophic denitrification facilitated by the sulfur cycle. In this study, we sought to reveal i) whether mixotrophic denitrification facilitated by sulfur-based autotrophic denitrification could be developed in full-scale WWTPs using A2/O through acclimation to low COD/TN and high sulfate concentrations and ii) the underlying microbial and genetic mechanisms of mixotrophic denitrification in full-scale A2/O WWTPs. Until now, A2/O process, i.e., anoxic denitrification followed by aerobic oxidation of organic and N, and then recycling of nitrate back to the anoxic reactor for denitrification to N 2 is still the most common mainstream biological N and phosphorus removal process used in full-scale WWTPs [ 2 ]. Despite innovations leading to energy-efficient N management, the improvement of existing WWTPs to fulfil stringent N regulation will be increasingly valuable [ 14 ]. Understanding the underlying mechanisms of mixotrophic denitrification in full-scale A2/O WWTPs will help to design and reform existing full-scale A2/O WWTPs to improve the N removal efficiency, cost savings and sludge minimization.",
"discussion": "Results and discussion Sulfur-cycling facilitated nitrogen removal The influent of the YXM WWTP was characterized by a low ratio of COD/TN (< 6) [ 19 ] and a high sulfate concentration (> 100 mg SO 4 2- -S L -1 ). Additionally, the ratio of BOD/COD in the YXM WWTP was only approximately 0.35. However, the effluent quality could meet the first class A criteria of effluent discharge, and the average removal efficiency of COD, TN and NH 4 + -N could reach 83%, 72.4%, and 98.6%, respectively [ 19 ]. Both COD/TN and ΔCOD/ΔTN showed no significant relationship with TN removal efficiency ( p > 0.05, Fig 1 ), which indicated that the ratio of COD/TN did not significantly influence the N removal processes in the YXM WWTP. 10.1371/journal.pone.0250283.g001 Fig 1 The relationship of COD/TN (A) and ΔCOD/ΔTN (B) with TN removal efficiency. In the PRAN unit, NO 3 - -N decreased from 7.41 ± 0.44 mg L -1 in the influent to 1.03 ± 0.25 mg L -1 in the effluent, and the concentration of NO 3 - -N continued to decrease to 0.41 ± 0.08 mg L -1 in the ANA unit effluent ( Table 1 ). Meanwhile, approximately 12 mg L -1 SO 4 2- -S was reduced, and 10 mg L -1 TDS was produced in the ANA unit. In AN, the main denitrification unit in the A2/O process received 400% nitrate-rich internal influx from the AO unit ( S1 Fig ), and approximately 17 mg L -1 COD was removed, which at most corresponded to approximately 7 mg L -1 TN removal. Previous studies reported that 2.86 mg L -1 BOD was needed for complete denitrification of 1 mg L -1 nitrate [ 29 ]. However, approximately 8 mg L -1 TN was removed in AN, which indicated that heterotrophic denitrification alone could not meet the need for N removal even though all of the consumed COD was used for denitrification. In addition to COD reduction, we also found that approximately 9 mg L -1 TDS was oxidized to SO 4 2- -S in AN, which stoichiometrically corresponded to approximately 6 mg L -1 TN removal following the equation of 5HS - + 8NO 3 - + 3H + → 5SO 4 2- + 4N 2 + 4H 2 O [ 30 ]. Thus, the combined data showed that the internal sulfur cycle of sulfate reduction and re-oxidation did occur in the A2/O system, and sulfur-based autotrophic denitrification could be a supplement to the integrated denitrification capacity. 10.1371/journal.pone.0250283.t001 Table 1 Performance of the PRAN, ANA, AN and POAN chambers. Parameter PRAN influent PRAN effluent ANA effluent AN effluent POAN effluent COD (mg L -1 ) 74.23 ± 5.01 65.27 ± 3.02 77.06 ± 2.34 60.63 ± 3.03 38.65 ± 3.56 TN (mg L -1 ) 21.2 ± 1.55 18.85 ± 0.98 18.2 ± 0.39 10.38 ± 1.61 9.76 ± 0.24 NH 4 + -N (mg L -1 ) 9.38 ± 0.37 13.22 ± 0.56 15.09 ± 0.48 7.95 ± 0.21 0.67 ± 0.08 NO 3 - -N (mg L -1 ) 7.41 ± 0.44 1.03 ± 0.25 0.41 ± 0.08 1.48 ± 0.15 8.56 ± 0.31 SO 4 2- -S (mg L -1 ) 110.02 ± 10.08 109.35 ± 11.13 98.65 ± 10.31 107.22 ± 15.34 109.26 ± 13.06 TDS (mg L -1 ) 11.13 ± 1.51 2.33 ± 0.32 < 0.1 DO 0.3–0.5 0.01–0.1 0.1–0.5 -0.4–0.7 PRAN: Pre-anoxic unit, ANA: Anaerobic unit, AN: Anoxic unit, POAN: Post-anoxic unit. Simultaneous sulfur-based autotrophic and heterotrophic denitrification has been reported to be achieved in pilot and full-scale bioreactors [ 6 , 31 , 32 ]. Wu et al. [ 16 ] reported that a high sulfate-to-COD ratio (> 1.25 mg SO 4 2- /mg COD) facilitated the development of sulfur-based autotrophic denitrification. Accordingly, the average concentration of SO 4 2- -S was over 100 mg L -1 in the inlet of the YXM WWTP, which was high enough to provide sufficient sulfur through sulfate reduction. In addition, the A2/O process has varying redox environments, including anaerobic and anoxic zones, which are favorable for sulfate reduction and sulfur oxidation. Normally, the redox potential needed for sulfate reduction is lower than that needed for denitrification, and NO 3 - -N can interfere with sulfate reduction. In the YXM WWTP, NO 3 - -N and DO were consumed in PRAN before entering the ANA unit ( Table 1 ), which facilitated the establishment of an anaerobic environment suitable for sulfate reduction in ANA [ 33 ]. Furthermore, the floc micro-environment might also play an important role in sulfur-based denitrification in the A2/O process. AS flocs are partially penetrated by oxygen, and the outer portion of the flocs is aerobic, while the inner portion of the flocs will be anoxic and/or anaerobic [ 34 ], which could provide a critical micro-environment for the harmonious coexistence of SRB, heterotrophic denitrifiers and sulfur-based autotrophic denitrifiers. Overview of the microbial community and key bacteria involved in sulfur cycling facilitated nitrogen removal To reveal the underlying microbial and genomic mechanisms of the observed denitrification under low COD/TN, the metagenome of the AS in the YXM WWTP was analyzed. The AS microbial communities were composed of 86 phyla and over 1,700 genera. Bacteria (5,612,582–6,168,910 reads, 98.22%) dominated the AS microbial communities, followed by Archaea (36,272–45,988 reads, 0.72%), Eukarya (13,566–17,280 reads, 0.27%), and viruses (25,632–42,440 reads, 0.80%). For bacteria dominant in AS microbial communities, we focused our analysis on the domain of bacteria. Similar to other full-scale N removal WWTPs [ 19 , 35 ], Proteobacteria (35.2%-38.8%), Bacteroidetes (20.8%-23.0%), Ignavibacteriae (8.4%-9.6%), Nitrospirae (4.1%-6.3%), Chloroflexi (5.6%-7.4%), Acidobacteria (2.9%-3.2%), and Firmicutes (2.5%-2.9%) were the dominant bacterial phyla ( Fig 2 ). At the class level, β-proteobacteria was the most dominant class, accounting for approximately 20.64%-23.25% of the total bacterial reads, much more than other Proteobacteria. The dominance of β-proteobacteria in sulfur-based N removal AS bacterial communities was also reported in other metagenomics studies [ 15 , 35 ]. 10.1371/journal.pone.0250283.g002 Fig 2 Bacterial community composition at the phylum level, determined using metagenomic sequencing, in the four denitrification units. At the genus level, the top 20 abundant genera collectively accounted for 42.1–44.1% of the total bacterial reads ( Fig 3 ). Many members among the top 20 genera were associated with nitrification and sulfur-based denitrification. Nitrospira (4.0%-6.3%) and Nitrosomonas (0.8%-1.1%), typical nitrifiers, also dominated in other AS communities [ 19 , 35 ]. Ignavibacterium (7.3%-8.4%) was the most dominant genus in the AS community, which was associated with sulfur-based autotrophic denitrifying processes, converting NO 2 - and NO to N 2 [ 36 , 37 ]. Thiobacillus (4.9%-5.6%) was the third most abundant taxon, which was previously reported to be the dominant taxon in sulfur-based autotrophic denitrification bioreactors and could also cooperate with heterotrophic denitrifiers in heterotrophic environments [ 8 , 11 , 15 , 32 ]. Sulfuritalea (0.80%-0.91%) and Thauera (0.65%-0.76%) were also found to act as sulfur-driven autotrophic denitrifiers [ 13 ]. Dechloromonas (1.40%-2.30%), a heterotrophic denitrifier, was also found to contain sulfur oxidation genes [ 38 ]. 10.1371/journal.pone.0250283.g003 Fig 3 Bacterial community composition at the genus level in the four denitrification units. Sulfate reduction by SRB provides the reduced S species for sulfur-assisted denitrification. The relative abundance of SRB (e.g., Geobacter , Desulfobulbaceae , Pelobacteraceae , and Desulfobacteraceae ) was 1.74%-1.88% of the total bacterial reads. Unlike the mainstream sulfur-driven denitrification process [ 3 , 8 , 15 ], SRB were not the dominant taxa in the A2/O process. However, the guild of SRB showed high diversity in our study (40 genera) ( S3 Table ). The high diversity of SRB could facilitate the adaption and activity of SRB guilds when they went through units with different environments in A2/O facilities. In addition, Ignavibacterium might also act as the potential SRB because it was reported to have polysulfide, thiosulfate reductases, and tetrathionate reductases [ 36 ]. Meanwhile, Ignavibacterium , together with other fermenters, could provide organic acids (butyrate, lactate, fumarate) or alcohols (ethanol), which could be used by SRBs and heterotrophic denitrifiers [ 33 , 36 , 39 ]. Generally, the metagenomics data showed the co-existence of nitrifiers, anaerobic SRB, autotrophic SOB denitrifiers and heterotrophic denitrifiers in AS, which could provide a substantial microbial basis for sulfur-based mixotrophic N removal. Functional genes related to sulfur reduction and oxidation Sulfate reduction mediated by SRB was anticipated to provide reduced S species for electron donors in sulfur-based autotrophic denitrification, and the genes involved in dissimilatory sulfate reduction, including sulfate adenylyltransferase ( sat ), adenylylsulfate reductase ( aprAB ) and dissimilatory sulfite reductase ( dsrAB ), were found in high abundance in the AS communities of the YXM WWTP ( Fig 4 and Table 2 ). In addition, a high abundance of the poly-S formation gene coding sulfide quinone oxidoreductase ( sqr ) was found in the AS community ( Fig 4 and Table 2 ). Poly-S could also serve as electron and energy storage material for denitrification [ 8 , 16 , 40 ]. 10.1371/journal.pone.0250283.g004 Fig 4 Internal cycling of sulfur through the cooperation of enzymes encoded by sulfate reduction and sulfur oxidation genes. 10.1371/journal.pone.0250283.t002 Table 2 Sulfur oxidation and sulfate reduction genes in the sludge samples. Gene(s) PRAN ANA AN POAN Sulfur oxidation sqr 2188 2378 2286 2474 soxZ 330 320 336 348 soxA 700 704 682 676 soxY 702 586 676 718 soxB 1022 1030 1024 1130 soxX 428 426 424 452 hdrC 284 316 360 358 hdrB 760 822 852 800 hdrA 750 838 956 912 Sulfate reduction dsrB 432 502 462 504 dsrA 588 626 646 694 sat 4044 4560 4746 4908 aprB 262 268 254 264 aprA 864 802 948 944 PRAN: Pre-anoxic, ANA: Anaerobic, AN: Anoxic, POAN: Post-anoxic. Sulfur oxidation genes, including those for sulfur oxidation multienzyme complex ( sox ) and heterodisulfide reductase ( hdr ), were detected in the AS communities ( Fig 4 and Table 2 ). The Sox enzyme system oxidizes thiosulfate, which is produced by SRB or chemical oxidation of H 2 S [ 33 , 41 , 42 ]. A previous study showed that the sox and hdr genes were simultaneously expressed with denitrification genes in Thiobacillus and Thauera [ 13 ], indicating the important role of sox and hdr in sulfur-based denitrification. Therefore, the metagenomic analysis of internal sulfur cycling-related genes showed that the AS of the YXM WWTP could proceed with the internal cycling of sulfur through the cooperation of enzymes encoded by sulfate reduction and sulfur oxidation genes, which consolidated the possibility of sulfur-based autotrophic denitrification without additional TDS supplementation ( Fig 4 ). Functional genes related to nitrogen transformation and taxonomic classification of denitrification genes Of the detected functional genes related to N cycling, those related to denitrification had the highest number of hits, followed by ammonification and nitrification genes. The functional gene related to the anammox process ( hzo , encoding hydrazine oxidoreductase) was not detected in this study, which suggested that denitrification was the main pathway of N removal in the YXM WWTP. Among the denitrification genes, the abundance of narG was the highest (4,320–4,660 reads), followed by napA (2,366–2,618 reads), nosZ (2,122–2,296 reads), and norB (1,130–1,330 reads), whereas the abundances of nirK (748–798 reads) and nirS (1,044–1,120 reads) were much lower ( Table 3 ). 10.1371/journal.pone.0250283.t003 Table 3 The abundances of genes related to nitrogen removal. Gene(s) PRAN ANA AN POAN Ammonification gdhA 984 1016 1004 1110 ureE 84 68 60 64 ureD , ureH 6 12 16 8 ureC 236 238 210 196 Nitrification pmoA-amoA 88 106 86 74 pmoB-amoB 108 98 72 134 hao 536 602 550 636 Denitrification narG 4320 4660 4440 4562 napA 2366 2578 2486 2618 nosZ 2122 2296 2138 2146 norB 1130 1214 1192 1330 nirS 1044 1082 1062 1120 nirK 778 748 772 798 norC 340 416 324 342 Dissimilatory nitrate reduction nirB 1012 1094 1074 1024 nrfA 606 818 718 810 Assimilatory nitrate reduction narB 10 30 22 24 nasA 612 624 604 628 nirA 238 170 158 162 PRAN: Pre-anoxic, ANA: Anaerobic, AN: Anoxic, POAN: Post-anoxic. The taxonomic origins of the denitrification genes were also analyzed. The results showed that denitrification genes were unevenly distributed in eighty genera, whereas most of them were affiliated with β-proteobacteria ( Fig 5 ), which was consistent with the dominance of β-proteobacteria in the AS communities. The narG gene was mostly associated with Thauera , Pseudomonas , Thermus , Azoarcus , Stenotrophomonas , Acidovorax , Rhodanobacter , and Thiobacillus . The napA gene was mainly associated with Thauera , Thioalkalivibrio , Sulfuritalea , Dechloromonas , Cupriavidus , and Leptothrix ( Fig 5 ). Among the genes required for the transformation of NO 2 - -N to NO, nirS was mainly associated with Labrenzia , Pseudomonas , Alicycliphilus , Thauera , Sulfuritalea , and Dechloromonas , while nirK was mainly affiliated with Azospirillum , Rhodanobacter , and Polaromonas . The norB gene was mostly associated with Thauera , Rubrivivax , Hyphomicrobium , Hydrogenophaga , and Variovorax . In our metagenomics data, 23 genera were found to harbor nosZ , predominately Dechloromonas , Sulfuritalea , and Candidatus Accumulibacter phosphatis . The diversity of denitrifying gene associated taxa showed that every step of denitrification was metabolized by a combination of gene products that originated from different bacterial species. Complete denitrification is achieved through the combined activity of taxonomically diverse co-occurring bacteria performing successive metabolic steps. The diverse denitrifiers catalyze N transformation at very different rates, however, the diverse denitrification consortia were suitable to adapt to varying environments in different units in the A2/O process. 10.1371/journal.pone.0250283.g005 Fig 5 Taxonomic distribution of denitrification genes (a) and relative abundance of taxa (b). The genes encoding enzymes initiating nitrate reduction, narG and napA , were mainly associated with autotrophic Thiobacillus , Thioalkalivibrio , and Sulfuritalea ( Fig 5 ). Consistent with the dominance of Thiobacillus in the microbial communities, the results showed the important roles of autotrophic sulfur-based denitrifiers at the functional gene level. Thauera was associated with a high relative abundance of the narG , napA , nirS , norB , and nosZ genes ( Fig 5 ). Both Thauera and Thiobacillus can use reduced sulfur as the electron donor and contribute to sulfur-driven autotrophic denitrification [ 12 , 13 ]. The analysis provided insight into microbial and functional gene foundations for the occurrence of sulfur-based mixotrophic denitrification in the A2/O process. It must be noted that most of the denitrifier taxa and high abundance of the denitrification genes narG , napA , nirS , norB , and nosZ were associated with heterotrophic denitrifiers, such as Dechloromonas , Pseudomonas , and Rhodanobacter . Dechloromonas , which carries the napA , nirS , norB , and nosZ genes, accounted for a relatively high percentage of the denitrifiers ( Fig 5 ). Dechloromonas , which was found to act as a heterotrophic sulfur oxidizing denitrifier [ 43 ], might also contain sulfur oxidation genes [ 38 ]. The results were not surprising because heterotrophic denitrification was still the main N removal pathway in the A2/O process. Notwithstanding, the coexistence of autotrophic and heterotrophic denitrifiers provided evidence that denitrification in the YXM WWTP could be a mixotrophic process. Mixotrophic denitrification involves the use of fewer carbon sources and less sulfate production than heterotrophic or autotrophic denitrification and thus allows for a relatively good rate of nitrate removal over a long period of time while achieving efficient COD and N removal [ 44 ]. Notably, the mixotrophic denitrification process could achieve high efficiency of NH 4 + -N removal, as indicated in our study and others [ 43 ]. We should keep in mind that electron donors significantly impact the microbial community structure and composition [ 32 ]. Our previous study also showed that the addition of external carbon source could improve the efficiency of TN removal, however, the external carbon source elevated the proportion of heterotrophic denitrifiers in the AS communities and incurred feedback for more carbon sources [ 45 ]. Additional exploration of the communities in sulfur-based mixotrophic denitrification, with clearer variations in performance parameters, as well as the interactions between “ Thiobacillus ” and heterotrophic denitrifiers in A2/O, will facilitate the development of low-cost and more flexible denitrification processes. Activities of nitrification and denitrification genes Although SONB can tolerate a certain concentration of sulfide, a high concentration of sulfide can inhibit their denitrification activity [ 12 ]. A previous study also showed that the activities of nitrification were inhibited by sulfide because ammonia oxidizers were sensitive to sulfide [ 14 ]. To evaluate the influence of TDS on mixotrophic denitrification in the YXM WWTP, the expression activities of functional genes related to N removal were analyzed using RT-qPCR. In our study, a high amoA mRNA/DNA ratio ( S4 Table ) was found in all four units (from 0.43 in POAN to 2.88 in AN), and the expression of amoA had no significant relationship with the content of reduced sulfur, which indicated that amoA expression was not inhibited by sulfide, as reported in a previous study [ 14 ]. The high-level expression of amoA ensured a high rate of NH 4 + -N transformation and subsequent denitrification in the A2/O process despite the low abundance of nitrifiers ( Fig 6 ) [ 46 ]. 10.1371/journal.pone.0250283.g006 Fig 6 Quantitative PCR analysis of the activity of nitrifying and denitrifying bacteria based on the abundance (DNA) and expression (RNA) of nitrogen-associated genes in the four denitrification units, a: napA ; b: narG ; c: nirK ; d: nirS ; e: norB ; f: nosZ ; g: amoA ; h: the expression levels of nitrogen-related genes in the four denitrification units. In agreement with the metagenomics data, the abundance and expression of the narG gene were higher than those of napA ( Fig 6 ). Moreover, the expression of narG and napA in ANA and AN was significantly higher than that in PRAN and POAN. The high expression of narG confirmed the important role of Thiobacillus and Thauera in denitrification, as Thiobacillus and Thauera were the main taxa carrying narG ( Fig 5 ). We also found a higher mRNA/DNA ratio of narG and napA in the ANA chamber than in other units ( S4 Table ), indicating that the anaerobic environment was favorable to the expression of narG and napA , while narG and napA were not sensitive to TDS ( Fig 6 ). However, the low content of nitrate and short hydraulic retention time (HRT) implied no significant denitrification in the ANA chamber. The abundance of nirS was higher than that of nirK , and the mRNA abundance of nirS was 3.1- to 93.3-fold higher than that of nirK , indicating that nirS was a key player in NO 2 - -N reduction ( Fig 6 ). Although among the denitrification genes, the abundance of norB was relatively high, its expression was low, as evidenced by its mRNA/DNA ratio, which only varied from 0.05 to 0.21. The nosZ gene is often used as a marker of complete denitrification [ 47 ]. The activity of nosZ is critical for the elimination of N 2 O in the denitrification process. Some recent studies have shown that sulfur-based autotrophic denitrification could promote the production and accumulation of N 2 O as a significant intermediate product [ 48 ]. In this study, the abundances of the nosZ gene and mRNA were the lowest among the denitrification genes ( Fig 6 ). Therefore, the bioaugmentation of nosZ -type denitrifiers, including Thauera and Candidatus Accumulibacter phosphatis , might facilitate the enhancement of the TN removal performance of WWTPs. The correlation of the expression of amoA and denitrification genes and the content of TDS was analyzed, and no significant relationship was found between them ( p > 0.05). Our results suggested that reduced sulfur did not inhibit or stimulate the potential of N removal in the AS of the YXM WWTP. The expression pattern of functional genes might be attributed to mixotrophic denitrification using both reduced sulfur and organic compounds as electron donors, which compensate for the influence of high reduced sulfur levels on functional gene activity. In the cooperation of SRB with SONB and other heterotrophic bacteria, metabolic diversity helps to relieve the inhibition of sulfide. As discussed above, HS - produced by SRB might be transformed to poly-S and thiosulfide, and these reduced S species could also be oxidized by Thiobacillus in denitrification."
} | 7,222 |
28701946 | PMC5487483 | pmc | 8,682 | {
"abstract": "NEST is a simulator for spiking neuronal networks that commits to a general purpose approach: It allows for high flexibility in the design of network models, and its applications range from small-scale simulations on laptops to brain-scale simulations on supercomputers. Hence, developers need to test their code for various use cases and ensure that changes to code do not impair scalability. However, running a full set of benchmarks on a supercomputer takes up precious compute-time resources and can entail long queuing times. Here, we present the NEST dry-run mode, which enables comprehensive dynamic code analysis without requiring access to high-performance computing facilities. A dry-run simulation is carried out by a single process, which performs all simulation steps except communication as if it was part of a parallel environment with many processes. We show that measurements of memory usage and runtime of neuronal network simulations closely match the corresponding dry-run data. Furthermore, we demonstrate the successful application of the dry-run mode in the areas of profiling and performance modeling.",
"introduction": "1. Introduction The neuronal network simulator NEST (Gewaltig and Diesmann, 2007 ) has an active and growing community of developers and users. Advances in computer hardware on the one hand and the requirements of novel computational models on the other hand push the development of new simulation technology and have made NEST a tool for a broad spectrum of applications. It enables simulations of spiking neuronal networks that differ greatly in size and complexity, and depending on the requirements of the models researchers can run the simulations on their laptops or they can make use of high-performance computing (HPC) facilities. A simulation with NEST consists of two main phases. During the build phase (or setup phase ) NEST creates neuron and synapse objects and the data structures to access these objects. Afterwards the actual simulation phase starts, in which the main simulation loop is repeated iteratively. Each iteration comprises the update of synapses and neurons, the exchange of recent spikes between MPI ranks and the delivery of these spikes to their local targets. A description of the fundamental data structures and the main simulation loop of NEST follows in Section 2.1. The software-development framework around NEST is becoming ever more comprehensive. A testsuite (Eppler et al., 2009b ) and continuous integration technology (Zaytsev and Morrison, 2012 ) help to ensure code quality; models of memory usage (Kunkel et al., 2012 ) and runtime (Adinets et al., 2015 ; Schenck et al., 2014 ) enable the structured analysis of existing code and the design of new, more scalable data structures and algorithms. However, in order to confirm the model predictions and to check for errors that occur only in the supercomputing regime, NEST developers still need to carry out actual simulations on supercomputers that may employ hundred thousands of MPI processes. Running such tests and benchmarks takes up compute-time resources and slows down the code-development process due to long queuing times. In this manuscript, we present a method, where only one MPI process carries out its part of a distributed simulation with NEST. In the build phase, the process sets up all local data structures as if it took part in a parallel simulation with many processes, and in the simulation phase, it creates fake spike s to represent input from other MPI ranks. We refer to this method as the dry-run mode of NEST and to the actual distributed neuronal network simulation that corresponds to a specific dry run as real run . Furthermore, we distinguish between static and dynamic dry-run mode. In the static mode, the MPI process creates fake spikes according to a predefined firing rate while in the dynamic mode the process uses the spikes of its local neurons to invent the fake remote spikes. In Section 2.2, we provide details on the implementation of the dry-run mode and also an example script that shows how the mode can be enabled. In computer science, the term “dry run” often refers to hardware tests under controlled conditions, but it is also used in the context of software development to refer to the static analysis of an algorithm by mentally evaluating each step. The NEST dry-run mode enables dynamic code analysis and hence differs from the latter definition. A NEST dry run corresponds more to a hardware dry run: Simulation code is executed in the controlled environment of a single MPI process such that failure cannot cause any severe damage as the simulation does not use any allocated HPC resources. The definition of the term “dry run” is rather vague as the concept is rather uncommon. Examples of programs that provide a dry-run option are among others the rsync utility (Tridgell, 1999 ) and the GPAW project (Enkovaara et al., 2010 ). The command-line tool rsync enables file synchronization and transfer; a dry run produces an output of the potential changes, which the user can check before performing a real run. GPAW enables electronic structure calculations; a dry run allows the user to estimate the memory usage and to inspect the parallelization settings for a given number of MPI processes. In this way, the dry-run mode of GPAW is conceptually similar to the dry-run mode of NEST concerning the build phase. However, we are not aware of any simulation software which is able to perform a dry run of the simulation dynamics. The dry-run mode of NEST allows developers to investigate the performance of different parts of the code without consuming precious computing time. This requires, however, that dry run and real run exhibit similar memory-access patterns, which may seem questionable given that in dry-run mode most spikes do not originate from real network dynamics. Hence, in Section 3.1 we compare spiking activity, memory usage, and runtime of dry-run and real-run simulations for different network sizes and number of processes. Within the software-development framework of NEST, the dry-run mode complements the previously developed models of memory usage and runtime as it is an economical way to obtain a realistic performance estimate of a distributed simulation. Kunkel et al. ( 2012 ) used a preliminary version of the dry-run mode to verify the predictions of the memory-usage model and in Helias et al. ( 2012 ) and Kunkel et al. ( 2014 ) the network sizes for the maximum-filling benchmarks were determined using the preliminary dry-run mode, which saved a lot of time and HPC resources. Dry-run simulations are also compatible with established profiling tools (see e.g., Schenck et al., 2014 ) because it is one and the same NEST binary which is used for conventional NEST operation and for dry runs. Besides, because the dry-run mode only requires a single compute node, developers can debug their NEST code for different regimes of number of processes without requiring access to HPC facilities—a single workstation is sufficient. Dry-run simulations can also help NEST users to estimate the HPC resources that they need to request for planned simulations. In order to demonstrate the usefulness of the dry-run mode we give several sample use cases in Section 3.2. The conceptual and algorithmic work described here is a module in our long-term collaborative project to provide the technology for neural systems simulations ( http://www.nest-initiative.org ). We devised the dry-run mode as a software-development method for NEST. However, the concepts that we present here are transferable to other simulators.",
"discussion": "4. Discussion 4.1. General remarks We presented the dry-run mode for NEST, by which it is possible to mimic the behavior of large-scale simulations on a single compute node. The results show that the generated data, such as spike patterns, memory consumption, and runtimes, is similar to corresponding real runs, at least for the dynamic version of the dry-run mode. Furthermore, two use cases for developers were explained in detail: With the help of the dry-run mode, large amounts of data for profiling and performance modeling can be collected without the need to employ more than one compute node for each data point. This saves huge amounts of core hours on large clusters or supercomputers and shortens development cycles. Furthermore, new algorithms can be tried out without wasting precious supercomputer resources, and the suite of automated software tests for NEST (Eppler et al., 2009b ) can be extended to cover also large-scale simulations. In a similar vein, users of NEST can use the dry-run mode to estimate in advance the required memory consumption and runtime of large-scale simulations. This helps to make better use of the available computing resources by saving core hours on test runs, by running full simulations with optimal parameter settings, and by enabling users to generate scaling data for compute-time proposals in an inexpensive way. It is important to note that the same cannot be achieved by just running small simulations instead of large simulations. The connection infrastructure generated during the build phase of NEST differs considerably depending on simulation size, partly qualitatively, and of course quantitatively. The subsequent simulation phase uses these data structures for processing and shows therefore also different runtime behavior depending on overall simulation size. 4.2. Restrictions of the dry-run mode The dry-run mode in its current form has two restrictions. The first restriction belongs inherently to the concept: the non-consideration of MPI communication and synchronization, i.e., in NEST terms the non-consideration of the gather step during the simulation phase. This restriction is not severe as we have shown in the results section. Generally, the gather step is very short in real runs. In the data from the supercomputer JUQUEEN reported in Section 3.1.2, it consumes between 1 and 8% of the overall simulation runtime. In case it is desired to estimate the time required for the gather step as well in advance, the dry-run mode helps at least insofar as it also predicts the size of the send buffers used for MPI operations. In combination with benchmarks of the MPI_Allgather operation on the respective cluster, the time required for MPI communication can be calculated. The second restriction concerns the current focus on balanced random networks where the inhibitory and the excitatory subpopulation show the same firing rates and spike characteristics (especially the same synchronization patterns). Such network models are good-natured because their overall spike patterns are invariant with regard to network size if the neurons are sparsely connected (Brunel, 2000 ). These are important reasons why dynamic dry runs exhibit very similar spike patterns and frequencies as the corresponding real runs. For NEST developers and their typical use cases the confinement to balanced random networks is unproblematic because these networks can be parameterized in various ways to cover most base scenarios relevant for profiling and performance modeling (e.g., low vs. high connectivity, low vs. high spike rate, or with vs. without plastic synapses). However, NEST users may want to simulate networks with rich internal structure, consisting of many different subpopulations with varying characteristics, resulting in complex spike patterns. For such networks it is less clear whether a dry run will yield a simulation runtime similar to a real run. In any case, operations during the build phase are exactly the same between dry and real runs, and since nearly all of the required memory is allocated during network wiring, at least the estimation of the memory usage and of the runtime of the build phase will be quite accurate even for complex networks. To get reliable estimates of simulation runtimes even for complex structured networks via the dynamic dry-run mode, it will be necessary to identify each subpopulation in the network and to extrapolate the spiking activity of each subpopulation on the single existing rank to the whole network. This is a topic of ongoing research. Furthermore, it is planned to investigate how the dry-run mode could be extended to cover very recent and advanced NEST features like structural plasticity (Diaz-Pier et al., 2016 ) and gap junctions (Hahne et al., 2015 ), which require MPI communication outside the standard spike-communication framework. 4.3. Applicability of the dry-run concept In conclusion, the dry-run mode is an important contribution to the software-development framework of NEST. It facilitates the maintenance and further development of NEST as an open source project with a general-purpose orientation and extreme scalability. Furthermore, the basic idea of the (dynamic) dry-run mode is applicable to other parallel applications in a straightforward way if the following preconditions are fulfilled: The buildup of basic data structures on each rank does not depend on actions on other ranks. Statistical properties of the data that is generated during the simulation can be inferred from the single existing rank in dry-run mode. Especially, this needs to be true for those properties which are relevant for the simulation runtime, and which are employed to generate fake data in place of real data. The replacement of real data by fake data does not change the overall simulation dynamics in a way that considerably affects simulation runtimes or memory consumption. These preconditions hold in principle for parallel implementations of Monte Carlo methods in which each compute node carries out an equally sized set of random experiments which is large enough to be representative for the whole simulated sample, or in which the impact of random events is computed in parallel (e.g., Carvalho et al., 2000 ). Furthermore, simulation algorithms based on spatial decomposition are candidates for a dry-run mode, at least if work is distributed equally over all compute nodes and stays (nearly) constant throughout the simulation (e.g., during the simulation of the dynamics of homogeneously distributed molecules with software like “ls1 mardyn”; Niethammer et al., 2014 ). And last but not least, simulators used in neuroscience like NEURON (Carnevale and Hines, 2006 ; Migliore et al., 2006 ) or NCS (“NeoCortical Simulator”) (Tanna, 2014 ) could profit from a dry-run mode similar to the one in NEST because the basic challenges are very similar (i.e., neurons distributed over MPI ranks, spike exchange via MPI communication)."
} | 3,658 |
33452484 | PMC8163825 | pmc | 8,686 | {
"abstract": "Asgard archaea are widely distributed in anaerobic environments. Previous studies revealed the potential capability of Asgard archaea to utilize various organic substrates including proteins, carbohydrates, fatty acids, amino acids and hydrocarbons, suggesting that Asgard archaea play an important role in sediment carbon cycling. Here, we describe a previously unrecognized archaeal phylum, Hermodarchaeota, affiliated with the Asgard superphylum. The genomes of these archaea were recovered from metagenomes generated from mangrove sediments, and were found to encode alkyl/benzyl-succinate synthases and their activating enzymes that are similar to those identified in alkane-degrading sulfate-reducing bacteria. Hermodarchaeota also encode enzymes potentially involved in alkyl-coenzyme A and benzoyl-coenzyme A oxidation, the Wood–Ljungdahl pathway and nitrate reduction. These results indicate that members of this phylum have the potential to strictly anaerobically degrade alkanes and aromatic compounds, coupling the reduction of nitrate. By screening Sequence Read Archive, additional genes encoding 16S rRNA and alkyl/benzyl-succinate synthases analogous to those in Hermodarchaeota were identified in metagenomic datasets from a wide range of marine and freshwater sediments. These findings suggest that Asgard archaea capable of degrading alkanes and aromatics via formation of alkyl/benzyl-substituted succinates are ubiquitous in sediments.",
"introduction": "Introduction Alkanes and aromatic hydrocarbons are abundant and prevalent in the environment. Although they are major components of petroleum, living organisms are also their important sources. These compounds are inactive in molecular structure, which makes them relatively inert substrates. Under aerobic conditions, alkanes can be oxidized by monooxygenase or dioxygenase in aerobic microorganisms, which use oxygen to supply a reactive oxygen species. The resulting alcohols are further oxidized to aldehydes by dehydrogenases, which are then converted to fatty acids [ 1 ]. For the anaerobic oxidation of alkanes, several mechanisms have been found in anaerobic microorganisms. Sulfate-reducing bacteria (SRB) are capable of activating n-hexadecane [ 2 ], propane, and n-butane [ 3 ] through the addition to fumarate, producing alkyl-substituted succinates. This is analogous to the anaerobic activation of aromatic hydrocarbons, yielding benzylsuccinate as the first intermediate, in the denitrifier Thauera aromatica [ 4 ]. For Archaea, only a few anaerobic species are currently known to have the ability to grow on hydrocarbons, with the exception of methane. Ferroglobus placidus can degrade benzene at 85 °C, coupling reduction of Fe(III) [ 5 ]. A thermophilic sulfate-reducing archaeon, Archaeoglubus fulgidus , is found to be able to oxidize long-chain n-alkanes anaerobically. It is inferred that Archaeoglubus fulgidus may activate alkane through binding to fumarate, which is catalyzed by alkylsuccinate synthase [ 6 ]. Recently, a thermophilic archaea ( Candidatus Syntrophoarchaeum ) is shown to activate butane via alkyl-coenzyme M formation under anaerobic conditions, which is similar to anaerobic activation of methane [ 7 ]. Genes encoding a similar methyl-coenzyme M reductase (MCR) complex have also been identified in genomes of uncultivated Bathyarchaeota [ 8 ], Hadesarchaeota [ 9 ] and Helarchaeota [ 10 ]. The recently discovered Asgard superphylum is a group of archaea with many eukaryotic features including six distinct phyla: Loki-, Thor-, Odin-, Heimda-, Hel-, and Gerd-archaeota [ 10 – 12 ]. These archaea possess so-called eukaryotic signature proteins (ESP), which, in eukaryotes, are involved in membrane-trafficking processes, vesicle biogenesis and trafficking, cytoskeleton formation and remodeling, endosomal sorting complexes required for transport-mediated protein degradation, and endosomal sorting; therefore, they are deemed representative of the closest archaeal relatives of eukaryotes [ 11 ]. Diversity investigations have revealed that Asgard archaea are widely distributed in various anoxic environments, including mangrove sediments, estuarine sediments, freshwater sediments, hydrothermal habitats, marine sediments, cold seeps, hot springs, mud volcanos, and soils [ 10 , 11 , 13 , 14 ]. Genomic analysis suggests that Asgard archaea may primarily be organoheterotrophs but some of them, such as Lokiarchaeota, Thorarchaeota and Gerdarchaeota, may also be mixotrophs, which can perform carbon fixation via the Wood–Ljungdahl pathway (WLP) [ 11 , 12 , 14 ]. The versatile lifestyles of Lokiarchaeota, Thorarchaeota and Gerdarchaeota have been supported by metatranscriptomics [ 12 , 14 ]. More recently, Helarchaeota from hydrothermal deep-sea sediments is suggested to have the potential to oxidize short-chain hydrocarbon using MCR-like enzymes [ 10 ], similar to the butane-degrading archaea C. Syntrophoarchaeum ; Lokiarchaeota from deep Costa Rica sediments is found to contain genes encoding glycyl-radical enzyme and benzoyl-CoA reductase, suggesting their potential ability to degrade alkanes or aromatic hydrocarbons [ 15 ]. These results underscore the roles of Asgard archaea in global carbon cycling. Here, we present the discovery of metagenome-assembled genomes (MAGs) recovered from anoxic mangrove sediment belonging to a new Asgard phylum that has the potential to carry out anaerobic oxidation of alkanes and aromatic compounds through binding to fumarate producing alkyl/benzyl-substituted succinates, which further extends our knowledge on carbon metabolism of Asgard archaea.",
"discussion": "Results and discussion Identification of Hermodarchaeota genomes from mangrove swamps A total of 360 gigabases of raw sequence data were obtained from above-mentioned six sediment samples (Supplementary Fig. 1 and Supplementary Table 1 ). Metagenomic de novo assembly and binning generated the reconstruction of over 112 archaeal genomes (>50% complete). Among them, 22 genomes belonged to the Asgard superphylum. Partial high-quality Asgard genomes are shown in Supplementary Table 2 . Maximum likelihood phylogenic trees were reconstructed using 56 concatenated ribosomal proteins or 122 concatenated archaeal-specific marker proteins. The phylogenetic analyses revealed seven MAGs representing a novel group of the Asgard archaea (Fig. 1a and Supplementary Fig. 3 ). These MAGs are located in distinct lineages and form a distantly related cluster with the Odinarchaeota in a phylogenetic tree with high bootstrap support (Fig. 1a ). They had a bin size ranging from 1.86 to 5.10 Mbp and a 43.1–48.7% mean GC content (Table 1 ) . Genome completeness of the MAGs ranged from 74.7% to 92.7% and there was almost no contamination of other genome fragments detected. These MAGs were recovered from top-layer (0.15–0.2 m) and mid-layer (0.4–0.45 m) samples of mangrove swamp sediment. Fig. 1 Phylogenetic placement of Hermodarchaeota within the Asgard archaea superphylum. a Phylogenomic tree of 56 concatenated ribosomal proteins reconstructed using IQtree with the LG + C60 + F + G + PMSF model. Nodes with ultrafast bootstrap values ≥80 are indicated by black circles. The distribution of key genes involved in degradation of hydrocarbons in Asgard archaeal genomes is also presented. Ass/Bss alkyl/benzyl-succinate synthase gene, Ass/Bss AE alkyl/benzyl-succinate synthase activating enzyme gene, bcrABCD benzoyl-CoA reductase ABCD subunit genes. b Maximum-likelihood phylogenetic tree of 16S rRNA gene sequences belonging to Hermodarchaeota were inferred using IQtree with GTR + F + I + G4 model. Black circles indicate bootstrap values ≥80. Table 1 Genomic features of Hermodarchaeota bins. Bin ID h02s_80 h02m_131 h02s_68 h02m_52 h02m_117 h03m_104 h02s_26 Completeness (%) 92.67 89.5 86.22 77.46 74.69 78.04 76.42 Contamination (%) 0 1.87 0.47 0 0 0 1.94 Strain heterogeneity (%) 0 0 0 0 0 0 0 Number of coding genes 3636 3895 4833 2582 1785 2561 2508 GC content (%) 44.54 43.21 43.91 44.52 44.71 43.05 48.74 Contig/scaffold number 267 863 903 357 269 469 677 Estimated genome size (Mbp) 3.76 4.22 5.10 2.68 1.86 2.66 2.53 N50 (bp) 20636 6335 6054 11896 8731 6995 4119 Longest contig/scaffold (bp) 99342 54136 46141 68536 36381 32924 28150 Genome completeness, contamination, and heterogeneity were estimated using CheckM 43 . Two partial 16S rRNA genes (1066 bp and 506 bp in length) were identified from MAGs of h02s_26 and h02m_131, respectively (Supplementary Table 3 ). By blasting against all 16S rRNA gene sequences from the six samples, two additional 16S rRNA genes were found to have more than 95% identity with that of h02s_26 (Supplementary Tables 3 and 7 ). Phylogenetic analyses revealed that the four 16S rRNA gene sequences formed a phylogenetically distinct group from the Odinarchaeota, and their position was similar to that of the above-mentioned seven MAGs in the genomic trees inferred by concatenated marker proteins (Fig. 1b ). The 16S rRNA gene sequences of h02s_26 and h02m_131 showed a phylum level divergence with a DNA identity of 72.9–83.7% [ 65 ] when compared with other Asgard archaeal 16S rRNA gene sequences (83.7% and 79.9% sequence similarity to that of Odinarchaeota, respectively) (Supplementary Table 7 ). Here, we propose Candidatus “Hermodarchaeota” as the name of this new group, after Hermod, the son of the god Odin in Norse mythology. An analysis of the average amino acid identity (AAI) revealed that these genomes have an AAI of 41.12–47.48% to other Asgard archaea (Supplementary Fig. 4 ) and fall within the range (40–52%) recommended for the phylum-level classification [ 66 ]. This further supports classification of the novel group as a separate phylum in Asgard superphylum. The genomic phylogenetic analysis and AAI suggest that these MAGs represent three different genera within the Hermodarchaeota (Fig. 1a and Supplementary Fig. 3 ). The MAGs h02m_117, h02s_80, h02m_52, h02s_68 and h03m_104 represent one genus, h02m_131 represents the second, and h02_124 represents the third. In addition, Hermodarchaeota MAGs possessed a suit of eukaryotic signature proteins (ESPs) that have been identified in other Asgard archaea (Supplementary Table 8 ). Metabolic reconstruction of Hermodarchaeota Similar to Lokiarchaeota and Thorarchaeota [ 62 ], metabolic analysis of Hermodarchaeota uncovered the presence of genes involved in the complete WLP (Fig. 2 , Supplementary Table 9 ). The WLP is traditionally connected with methanogenesis in Archaea. However, all Hermodarchaeota genomes lacked genes that encode MCR and genes of key subunits that encode Na + -translocating methyl-THMPT:coenzyme M methyltransferase (MTR). Therefore, Hermodarchaeota are unable to perform hydrogenotrophic CO 2 -reducing methanogenesis. Each Hermodarchaeota genome contained three to five copies of gene encoding trimethylamine methyltransferases ( mtt ) and corresponding corrinoid proteins ( mttc ), as well as one to two copies of gene for methylcobamide: CoM methyltransferase ( mtbA ), which is required for methyl-coenzyme M production from trimethylamine [ 67 ]. All of the Hermodarchaeota harbored two to four copies of mtrH subunit genes, and one to two of them were found to be collocated with a gene encoding corrinoid protein homolog (Supplementary Table 9 ). The gene operon has been suggested to be involved in transfer of methyl group directly to tetrahydromethanopterin (H 4 MPT) in some methylotrophic methanogens [ 8 , 68 , 69 ]. This may allow the Hermodarchaeota to use methyl compound by establishing a link between methyl-H 4 MPT and methyl-coenzyme M or methyl compound (Fig. 2 ), as suggested for recently reported Thorarchaeota [ 62 ]. Fig. 2 Key metabolic pathways in Hermodarchaeota genomes. The presence or absence of enzymes in these pathways is indicated with dots with different colors. If the dot corresponding to a genome is colorless, it indicates that the enzyme is absent in the genome. Red dotted lines represent electron flow. Genes related to the pathways presented in this figure are given in Supplementary Table 9 . WLP Wood–Ljungdahl pathway, TCA tricarboxylic acid, DMA dimethylamine, TMA trimethylamine, MP methanophenazine, Fd ferredoxin. In h02m_131, h02s_68, h02m_117 and h02m_52 genomes, five homologs of alkylsuccinate synthase (Ass) or benzylsuccinate synthase (Bss) were identified (Fig. 2 , Supplementary Table 9 ). Their amino acid sequences exhibited 29–33% identity with AssA1 (ABH11460) from D. alkenivorans strain AK-01 (bit score: 262–320; e-values: e−98–7e−79) and 29–32% identity with BssA (YP158060) from A. aromaticum EbN1 (bit score: 234–294; e-values: 3e−90–e−68), but only had 24–27% identity with the pyruvate formate lyase Pfl (NP415423) from Escherichia coli (bit score: 113–165; e-values: 4e−46–3e−29) (Supplementary Table 10 ). Furthermore, higher similarity was observed between Ass/Bss of Hermodarchaeota and PflD (AAB89800) of Archaeoglobus fulgidus (identity: 33–38%; bit score: 336–463). The PflD has been suggested to possess an Ass activity in A. fulgidus [ 6 ]. The multiple sequence alignment revealed that, similar to other AssA and BssA, the five homologs in Hermodarchaeota harbor only one conserved cysteine which is used to receive the radical from the glycyl residue and initiate the reaction, whereas pyruvate formate lyases, such as Pfl from E. coli , possess two neighboring conserved cysteines in the region [ 6 ] (Fig. 3a ). Based on the 12 known archaeal sequences (Fig. 3a ), AssA/BssA in archaea tends to substitute the first cysteine with glycine at the conserved region for PFLs. We analyzed conformation of h02s_68 Ass/Bss using I-TASSER [ 47 ]. The displayed structural model was superimposed on PflD crystal structure (PDB ID: 2f3oA) of A. fulgidus with high alignment confidence (TM-score: 95.6%) (Fig. 3c ). However, among eight active sites reported in the PflD, amino acids of four sites were changed (Fig. 3d ). It is unclear if these amino acid changes affect the type of substrates. A phylogenetic analysis was performed using Hermodarchaeota Ass or Bss sequences and their closest homologs (Fig. 4 ). This revealed that Ass/Bss encoded by Hermodarchaeota are not monophyletic, but intermixed with bacterial sequences, indicating that these Ass/Bss sequences were likely obtained through horizontal gene transfers from bacteria, similar to the pflD of A. fulgidus [ 6 ]. Specifically, it is inferred that at least two separate horizontal gene transfers occurred between different bacterial donors and members of Hermodarchaeota (Fig. 4 ). Fig. 3 Partial sequence alignment of Hermodarchaeota alkylsuccinate synthase (Ass)/benzylsuccinate synthase (Bss) with known Ass/Bss, and Hermodarchaeota Ass or Bss-activating enzyme (Ass/Bss AE) with known Ass/Bss AE, and structural modeling and active sites of the h02s_68 Ass/Bss. a Sequence comparison of Ass/Bss. The regions containing the conserved cysteine residue (**, shaded in red) and the conserved glycine residue (*, shaded in red) are presented. Vertical black line in the middle of sequences represents truncated section. D. A_ AK-01, D. alkenivorans strain AK-01. b Sequence comparison of Ass/Bss AE. Boxes 1 and 2 correspond to the CxxxCxxC sequence motif and two cysteine-rich regions, respectively, and they are involved in FeS cluster binding. c Model of the h02s_68 Ass (red) superimposed on PflD crystal structure (green) (PDB ID: 2f3oA) of A. fulgidus . A high alignment confidence (TM-score: 95.6%) was observed between the h02s_68 and A. fulgidus crystal structure. d Model of the active sites of the h02s_68 Ass (red) overlaid onto PflD (green) of A. fulgidus . Fig. 4 Maximum-likelihood tree of alkyl/benzyl-succinate synthases (Ass/Bss) identified in Hermodarchaeota genomes and homologs from nr database reconstructed using IQtree with LG + I + G4 substitution model. Hermodarchaeota and Thorarchaeota alkyl/benzyl-succinate synthases were red-coded. The verified alkyl-succinate synthases were shaded in yellow. Archaeal homologs were shaded in blue. The Nodes with ultrafast bootstrap values ≥ 80 are indicated by black circles. In addition to Ass/Bss genes, among seven Hermodarchaeota genomes, five contained one gene encoding the Ass or Bss-activating enzyme (Ass/Bss AE) (Supplementary Table 9 ), which is needed for Ass/Bss. A Blastp [ 37 ] search found that they had greater similarity with AssD2 (YP_002431363) and AssD2’ (YP_002429341) from D. alkenivorans strain AK-01, PflC (KUJ94427) of A. fulgidus , and BssD (CAA05050) from T. aromatica K172, compared to the PflA-activating enzyme (NP_415422) of E. coli (Supplementary Table 11 ). The N-terminal regions of Hermodarchaeota Ass/Bss AEs contained a CxxxCxxC sequence motif (box 1) and two cysteine-rich regions (box 2) (Fig. 3b ). Box1 is necessary for the Fe-S cluster of SAM-radical enzymes [ 70 ] while box 2 is involved in Fe-S cluster binding, which is unique to Ass/Bss AE and not found in pyruvate formate lyase-activating enzymes [ 6 ]. Generally, these data indicate that Hermodarchaeota possess Ass or Bss and activating enzymes. Furthermore, a publicly available metatranscriptome (SRR11241197) from mangrove sediment was assembled. Among the generated gene fragments, two were found to be highly homologous to Hermodarchaeota Ass/Bss genes (>60% amino acid identity, >93% alignment length) (Supplementary Table 12 ), likely suggesting that Hermodarchaeota Ass/Bss -like genes may be expressed in mangrove sediment. Once alkanes are activated, the alkyl-substituted succinates formed will be subjected to thioesterification, carbon-skeleton rearrangement, and decarboxylation [ 71 ]. At present, the genes involved in these reactions remain unclear. In D. alkenivorans strain AK-01, it is postulated that these steps were catalyzed by acyl-CoA synthetase (ligase) (AMP-forming), methylmalonyl-CoA mutase, and methylmalony-CoA carboxyltransferase [ 71 ]. The genes for all of these were present in each Hermodarchaeota genome (Supplementary Table 9 ). Subsequently, acyl-CoA produced from alkane oxidation can be oxidized to acetyl-CoA by related enzymes of the beta-oxidation including acyl-CoA dehydrogenase (Acd), enoyl-CoA hydratase (Ech), 3-hydroxyacyl-CoA dehydrogenase (Hadh), and acetyl-CoA acyltransferase (Fad), and the genes encoding these enzymes have been identified in Hermodarchaeota genomes (Supplementary Table 9 ). In addition, each Hermodarchaeota genome contained genes for 10–19 Acds, two to three electron transfer flavoprotein complexes (ETF), and one to four FeS oxidoreductases (Supplementary Table 9 ). This could produce reduced ferredoxin or NADH by electron bifurcation in the ACD/ETF complex for anabolism (Fig. 2 ) [ 72 , 73 ]. The genes for acetyl-CoA decarbonylase/synthase:CO dehydrogenase complex (ACDS/CODH) were identified in Hermodarchaeota (Supplementary Table 9 ); these are key enzymes in the metabolism of acetyl-CoA from beta-oxidation. This suggests that acetyl-CoA can be further oxidized into CO 2 and yield reduced ferredoxin via the oxidative WLP as previously shown for butane oxidation in Ca. Syntrophoarchaeum [ 7 ] (Fig. 2 ). For anaerobic oxidation of aromatic hydrocarbons, the first intermediate formed by the addition of fumarate, benzylsuccinate, is further oxidized to benzoyl-CoA, which is regarded as a primary aromatic intermediate in the anaerobic oxidation of plentiful aromatic hydrocarbons [ 74 ]. Hermodarchaeota genomes contained almost all genes found in the benzoyl-CoA pathway (Fig. 2 ; Supplementary Table 9 ). Next in this pathway, the conversion from benzoyl-CoA to 3-hydroxypimelyl-CoA, is catalyzed by four key enzymes in T. aromatica including benzoyl-CoA reductase (Bcr), cyclohexa-1,5-dienecarbonyl-CoA hydratase (Dch), 6-hydroxycylohex-1-ene-1-carboxyl-CoA dehydrogenase (Had), and 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase (Oah) [ 75 ], of which all genes were identified in h02s_80, h02s_68, h03m_104 and h02s_26 genomes (Supplementary Table 9 ). In addition, the genes encoding BcrABCD subunits in these genomes were found collocated in a contig (Fig. 5 ), forming a gene cluster; in h02s_80, the contig also contained genes for an BzdV protein, a ferredoxin, a Dch, a Had, a NADP-dependent oxidoreductase, and two anaerobic benzoate catabolism transcriptional regulators (Fig. 5 ). The arrangement of the gene cluster for benzoate catabolism was analogous to that in T. aromatica [ 48 ]. This indicates that ferredoxin probably acts as electron donor for the reduction of benzoyl-CoA in Hermodarchaeota while NADP-dependent oxidoreductase regenerates reduced ferredoxin as previously shown for Azoarcus evansii [ 76 ]. Subsequently, 3-hydroxypimelyl-CoA will be further oxidized to acetyl-CoA by the related enzymes via beta-oxidation including Hadh, Fad, glutaryl-CoA dehydrogenase (GcdH), glutaconyl-CoA decarboxylase (GcdA), Ech, 3-hydroxybutyryl-CoA dehydrogenase (PaaH), and acetyl-CoA acetyltransferase (AtoB). The genes for all these enzymes have been detected in Hermodarchaeota (Fig. 2 ; Supplementary Table 9 ). In gene fragments assembled from the above-mentioned metatranscriptome, two genes had high similarity to Hermodarchaeota bcrAC genes (>60% amino acid identity, ≥98% alignment length) (Supplementary Table 12 ). It is possible that Hermodarchaeota bcr- like genes can be transcribed in mangrove sediment. Collectively, the data suggest that Hermodarchaeota may be capable of using various aromatic hydrocarbons or alkanes as carbon and energy sources. Fig. 5 The gene composition in the contigs containing the benzoyl-CoA reductase operon in Hermodarchaeota genomes. Purple bocks indicate gene operon for the benzoyl-CoA reductase. Arrows show transcriptional orientation of the genes. To examine the distribution of the genes for Ass/Bss, Ass/Bss AE and Bcr in Asgard archaea, we analyzed all known Asgard genomes in nr database (Fig. 1a ). Among 118 genomes, nine contained the genes encoding both Ass/Bss and Ass/Bss AE, including one Heimdallarchaeota, five Lokiarchaeota, and three Hermodarchaeota; ten contained the genes for Bcr, including three Heimdallarchaeota, three Lokiarchaeota and four Hermodarchaeota; one (h02s_68 of Hermodarchaeota) harbored all the genes encoding the three enzymes. Statistical analysis of contigs comprising Hermodarchaeota MAGs revealed that the contigs containing genes for Ass/Bss, Ass/Bss AE, Bcr and the Wood–Ljungdahl pathway fell inside the 95 th percentile of a typical genome, supporting that these genes do belong to their respective genomes (Supplementary Figs. 5 and 6 ). These observations highlighted the important roles of members of Hermodarchaeota in the anaerobic degradation of alkanes and aromatic hydrocarbons. Some members of Lokiarchaeota and Heimdallarchaeota may also have the potential capability to anaerobically perform degradation of aromatics. Two Lokiarchaeota MAGs recovered from deep Costa Rica sediments were suggested to possibly utilize benzoate coupling with reduction of nitrate, nitrite and sulfite [ 15 ]. Compared to other Asgard archaea [ 62 ], Hermodarchaeota genomes possess more sophisticated energy-conserving complexes, including seven subunits of the F 420 H 2 dehydrogenase (encoded by fpo ), 11 subunits of NADH-quinone oxidoreductase, flavoprotein and FeS subunit of succinate dehydrogenase/fumarate reductase, group 4 [NiFe]-hydrogenase, the B and C subunits of the Rnf complex, and V/A-type adenosine triphosphate (ATP) synthase (Fig. 2 ; Supplementary Fig. 7 ; Supplementary Table 9 ). Group 4 [NiFe]-hydrogenase was only identified in Heimdallarchaeota and Odinarchaeota while the F 420 H 2 dehydrogenase and Rnf complex were not found in other Asgard archaea [ 62 ]. The presence of group 3b and group 3c [NiFe]-hydrogenases (Supplementary Fig. 7 ), together with the WLP suggest that Hermodarchaeota may be able to reduce CO 2 via the WLP using H 2 as electron donor. The membrane-bound Group 4 [NiFe]-hydrogenase [ 77 ], H + -translocating F 420 H 2 dehydrogenase [ 78 ], and Na + / H + -translocating Rnf complex [ 79 ] can couple H 2 oxidation with Na + / H + translocation across cytoplasmic membrane and further generate an electrochemical ion gradient that drives ATP synthesis. The coupling is absent in other Asgard phyla [ 62 ]. Of note, one to two copies of genes that encode D and E subunits of CoB-CoM heterodisulfide reductase (Hdr) were identified in h02s_80, h02s_68, h02m_117 and h02m_52 genomes (Fig. 2 ), which are absent in most methanogens and other Asgard archaea, but found solely in the Methanosarcinales [ 62 , 80 ]. HdrDE is an integral membrane complex, it accepts electrons from reduced methanophenazine (MPH 2 ), and assists in the production of a chemiosmotic gradient across the cell membrane (Fig. 2 ). In methanogens, the membrane-bound electron transport chain is more efficient than electron bifurcation [ 81 ]. It may be speculated that Hermodarchaeota have a higher growth yield than other Asgard archaea without HdrDE complex. In addition, hdrABC and mvhADG for methyl-viologen-reducing hydrogenase were also detected (Fig. 2 ), suggesting that the complex possibly leads to co-reduction of CoM-S-S-CoB heterodisulfide and ferredoxin by electron bifurcation mechanism as shown previously for Methanosarcina acetivorans [ 80 ]. Owing to an absence of MCR, the reaction forming a heterodisulfide of CoM and CoB by MCR is not present in Hermodarchaeota. Although Hermodarchaeota contained genes for cytoplasmic fumarate reductase (Supplementary Table 13 ), which has been shown to reduce fumarate using CoM-S-H and CoB-S-H in Methanobacterium thermoautotrophicum [ 82 ], the reaction is not accompanied by energy conservation. Thereby, like most Asgard archaea, it remains to be determined whether the cycle of CoM-S-S-CoB is operative in Hermodarchaeota. In addition to metabolic pathways for aromatic hydrocarbons and alkanes, Hermodarchaeota genomes also contain multiple peptidases, aminopeptidases, carboxypeptidases, and amino acid and oligopeptide transporters (Supplementary Fig. 8a ) that have been identified in other Asgard archaea [ 62 ], suggesting that these microorganisms can utilize peptides and proteins as their sources of carbon and nitrogen. Similar to the peptide fermentation of Pyrococcus furiosus [ 83 ], amino acids hydrolyzed by these peptidases can be oxidatively deaminated by glutamate dehydrogenase ( gdh ), aspartate aminotransferases ( aspC ), 2-oxoglutarate ferredoxin oxidoreductase (kor ), 2-ketoisovalerate ferredoxin oxidoreductase ( vor ), indolepyruvate ferredoxin oxidoreductase ( ior ), and pyruvate ferredoxin oxidoreductase ( por ) to generate acetyl-CoA and reduced ferredoxin (Fig. 2 ; Supplementary Table 9 ). Furthermore, the aliphatic and aromatic aldehydes produced by these oxidoreductases can be oxidized to carboxylic acids by multiple aldehyde ferredoxin oxidoreductases, producing reduced ferredoxin (Fig. 2 ) [ 84 ]. The resulting acetyl-CoA is further converted by acetyl-CoA synthetase to produce acetate (ADP-forming) (Fig. 2 ), with concomitant formation of ATP [ 85 ]. In addition, similar to other Asgard archaea [ 10 , 62 ], Hermodarchaeota may be capable of using carbohydrates as carbon or energy sources because they possess sugar transporters, sucrose transporters, and various carbohydrate-active enzymes (Fig. 2 , Supplementary Fig. 8b ). The resulting glucose can then be metabolized to generate intermediates for anabolism via glycolysis and an incomplete citric acid cycle, along with the formation of ATP and reduced nicotinamide adenine dinucleotide (NADH). Subsequently, the NADH is assumed to be oxidized by NADH-quinone oxidoreductase (complex I) to create a transmembrane proton gradient, or be applied to reduce ferredoxin via the Rnf complex (Fig. 2 ). Proper electron acceptors are pivotal to successful degradation of aromatic compounds and alkanes. We did not identify any dissimilatory sulfite reductase (Dsr) or anaerobic sulfite reductase (Asr) in Hermodarchaeota genomes. However, Hermodarchaeota genomes contain genes encoding nitrate transporter NrtD and NrtB, as well as homolog of molybdopeterin oxidoreducatase harboring molybdopterin guanine dinucleotide-binding (MGD) domain (Fig. 2 ; Supplementary Table 9 ; Supplementary Fig. 9 ) which is present in nitrate reductase alpha subunit (NarG) [ 86 ]. Phylogenetic analysis revealed that these molybdopeterin oxidoreducatases and nitrate reductases from Desulfovibrio and Chloroflexi formed a large cluster adjacent to the respiratory membrane-bound Nar clade (Supplementary Fig. 10 ). Several strains of Desulfovibrio have been reported to be capable of performing respiratory nitrate reduction [ 87 , 88 ]. Furthermore, among these molybdopeterin oxidoreducatase coding genes, two were found to be collocated with a gene encoding 4Fe-4S dicluster domain-containing protein (Supplementary Table 9 ), a gene arrangement also found in the E.coli respiratory Nar gene cluster [ 86 ]. It is deduced that the molybdopeterin oxidoreducatase and 4Fe-4S dicluster domain-containing protein in Hermodarchaeota may be analogous to NarG and NarH in the membrane-bound nitrate reductase of bacteria. The results suggest that members of Hermodarchaeota may be capable of utilizing nitrate as an electron acceptor during the oxidation of aromatic hydrocarbons as shown previously in denitrifying bacterium Thauera aromatica [ 89 ]. In addition to nitrate, Fe (III) has been found to serve as the sole electron acceptor of hyperthermophilic archaeon Ferroglobus placidus when oxidizing benzoate and phenol. In Methanosarcina acetivorans , electrons can be channeled to Fe (III) via HdrDE complex, which drives methane oxidation [ 90 ]. Thus, it is possible that Hermodarchaeota can couple oxidation of aromatic hydrocarbons with reduction of Fe (III) through the HdrDE complex (Fig. 2 ). Similar to Lokiarchaeota and Thorarchaeota, identification of reductive dehalogenase in Hermodarchaeota genomes implicates that these microorganisms are also capable of performing organohalide respiration using chlorinated ethenes/ethanes (Supplementary Fig. 11 ). We could not identify any pilus and extracellular cytochromes that mediate electron transfer across species, as shown previously in Ca. S. butanivorans and ANME archaea [ 7 , 91 ]. Therefore, it is unclear whether a syntrophic partner organism receiving reducing equivalents exists for members of Hermodarchaeota. Environmental distribution Based on full-length 16S rRNA genes assembled from the metagenomes using phyloFlash, we analyzed microbial community composition in the six mangrove sediment samples collected from Techeng Island (Fig. 6a ). The most abundant phyla were, in a reducing order, Chloroflexi, Bathyarchaeota, Desulfobacterota, Proteobacteria, and Euryarchaeota in these metagenomes. Four Asgard phyla were detected including Hermodarchaeota, Heimdallarchaeota, Lokiarchaeota and Odinarchaeota, with their abundance accounting for 0.81–10.78% of reads matching total full-length 16S rRNA genes in each metagenome. The relative abundance of Hermodarchaeota increased from 0.57% to 1.63% in H02 core with depth while from 0.34% to 1.47% in H03 core with depth. In H02 core, the abundance of Hermodarchaeota appeared to be associated with contents of benzene and methylbenzene in sediments (Supplementary Table 1 ), possibly further supporting utilization of aromatics by Hermodarchaeota. Fig. 6 Abundance and distribution of Hermodarchaeota in environments. a Microbial community composition from the six mangrove sediments on Techeng Island. The community composition was based on full-length 16S rRNA genes assembled from the metagenomes. b Global distribution of homologs of Hermodarchaeota Ass/Bss and 16S rRNA genes identified in metagenomes from various environments. Circles with different colors represent metagenomes from different habitats; the hexagons with different colors indicate the range of 16S rRNA gene count normalized to sequencing depth; the range of Ass/Bss gene count normalized to sequencing depth is indicated using squares with different colors; the numbers in the figure correspond to the ID in Supplementary Table 6 . To investigate the distribution and diversity of Ass/Bss sequences from archaea, Ass/Bss gene sequences from Hermodarchaeota were used to identify homologs in the six mangrove sediment samples in this study and across 1,000 publicly available metagenomes from around the world. A total of 394 nearly full-length Ass/Bss genes with archaeal GC motif were recovered from the six mangrove sediment samples (Techeng Island, China) (Supplementary Table 13 ), likely suggesting that they may be derived from phylogenetically diverse archaea. Only were 34 full-length Ass/Bss genes with bacterial AC/LC motif identified from these mangrove sediments (Supplementary Table 13 ). The results indicate that archaea may be major players in anaerobic degradation of alkanes in some mangrove sediments. Hermodarchaeota Ass/Bss -like gene fragments were also detected in various marine and freshwater environments, including marine bay sediments (Fagans, Australia), Guaymas Basin sediments enriched in hydrocarbon seeps (Gulf of California, USA), hot spring sediments (California, USA), deep-sea sediments with petroleum seeps (Eastern Gulf of Mexico), mangrove sediments (Yunxiao, China), Towuti Lake sediments (South Sulawesi, Indonesia), and formation water in coal beds (Qinshui Basin, China) (Fig. 6b , Supplementary Table 6 ). Furthermore, in these environments, a considerable number of 16S rRNA gene fragments were found to have high sequence similarity (≥90% identity) to that of Hermodarchaeota (Fig. 6b , Supplementary Table 6 ). Hermodarchaeota 16S rRNA and Ass/Bss -like genes were notably in higher relative abundance in the metagenomes generated from deep-sea sediments associated with petroleum seepage, mangrove sediments, Lake Towuti sediments of Indonesia, hydrothermal vent, and hot spring sediments where more hydrocarbons may be present, which was in accordance with attribution of Hermodarchaeota. The homologs of Ass/Bss were also identified in the thermophilic pure archaeon A. fulgidus isolated from a submarine hot vent [ 6 ] as well as in composite genomes of Thorarchaeota and Lokiarchaeota from deep seabed petroleum seeps [ 92 ]. These results suggest that members of Hermodarchaeota, and other archaea capable of performing oxidation of alkanes and aromatic hydrocarbons through addition to fumarate, may be ubiquitous in nature. In addition to utilization of alkane, Hermodarchaeota is able to perform anaerobic oxidation of aromatic hydrocarbon via addition to fumarate coupling with the benzoyl-CoA degradation pathway. The three subunits of the key enzyme Bcr for the benzoyl-CoA pathway are highly related to those of ATP-consuming class I Bcr of Anaerolineales bacterium of Chloroflexi (Supplementary Fig. 12 ), suggesting occurrence of horizontal gene transfers between Hermodarchaeota and Chloroflexi. The bcr genes and genes for downstream transformation were also identified in composite genomes of Thermoplasmata and Bathyarchaeota from deep-sea sediments with petroleum seeps [ 92 ] and Lokiarchaeota from deep Costa Rica sediments [ 15 ]. In addition, a new pathway for anaerobic oxidation of short-chain hydrocarbon via alkyl-coenzyme M formation has been proposed in Bathyarchaeota [ 8 ], Hadesarchaeota [ 9 ], Ca . Syntrophoarchaeum [ 7 ], and Helarchaeota [ 10 ]. These results demonstrate that metabolic processes of hydrocarbons in archaea may be more complicated than thought before. Such complexity likely suggests that utilization of hydrocarbons by archaea may have existed for a long time in the earth. The discovery of Hermodarchaeota and its ubiquitous distribution expands the domain of archaea and has crucial significance for understanding of the ecological functions and evolutionary history of the mysterious Asgard archaea."
} | 9,066 |
38753795 | null | s2 | 8,688 | {
"abstract": "Colloidal self-assembly allows rational design of structures on the micrometer and submicrometer scale. One architecture that can generate complete three-dimensional photonic bandgaps is the diamond cubic lattice, which has remained difficult to realize at length scales comparable with the wavelength of visible or ultraviolet light. In this work, we demonstrate three-dimensional photonic crystals self-assembled from DNA origami that act as precisely programmable patchy colloids. Our DNA-based nanoscale tetrapods crystallize into a rod-connected diamond cubic lattice with a periodicity of 170 nanometers. This structure serves as a scaffold for atomic-layer deposition of high-refractive index materials such as titanium dioxide, yielding a tunable photonic bandgap in the near-ultraviolet."
} | 199 |
25114551 | PMC4122557 | pmc | 8,689 | {
"abstract": "Commonly used in biotechnology applications, filamentous M13 phage are non-lytic viruses that infect E. coli and other bacteria, with the potential to promote horizontal gene transfer in natural populations with synthetic biology implications for engineering community systems. Using the E. coli strain TG1, we have investigated how a selective pressure involving elevated levels of toxic chromate, mimicking that found in some superfund sites, alters population dynamics following infection with either wild-type M13 phage or an M13-phage encoding a chromate reductase (Gh-ChrR) capable of the reductive immobilization of chromate (ie, M13-phage Gh-ChrR ). In the absence of a selective pressure, M13-phage infection results in a reduction in bacterial growth rate; in comparison, in the presence of chromate there are substantial increases in both cellular killing and biomass formation following infection of E. coli strain TG1with M13-phage Gh-ChrR that is dependent on chromate-reductase activity. These results are discussed in terms of community structures that facilitate lateral gene transfer of beneficial traits that enhance phage replication, infectivity, and stability against environmental change.",
"introduction": "Introduction Bacteriohage (phage), a bacterial virus, is about 1/40th the size of most bacteria and represent the simplest, most abundant organism on earth, thriving wherever bacteria grow—with an estimated 10 30 viral particles in the Earth’s oceans alone. 1 Metagenomic studies indicate that phage genes are widely present in bacterial genomes from groundwater samples, including those at major superfund sites (eg, Rifle, CO). 2 – 5 Phage are suggested to play an important role in the biogeochemical cycle by controlling marine and other bacterial and phytoplankton communities, thereby influencing pathways of matter and energy transfer within global ecosystems. 6 At the population level, phage-mediated bacterial lysis results in boom-bust cycles of virus and bacterial host abundance increases, and imposes a well-understood co-evolutionary fitness in which hosts evolve novel phage adaptations to avoid infection, while viruses evade host defenses to retain their infectivity. Such measurements form the basis for theoretical models that support what has become known as “kill-the-winner” hypothesis, 7 in which successful bacterial hosts that become abundant in the environment become targets of viral attack. This negative density-dependent selection leads to increased host diversity, and has been suggested to be critical to community stability. The co-evolutionary dynamics of the model are characteristic of the well known “Red Queen” effect, 8 whereby both viruses and hosts show continual evolutionary adaptation while maintaining broad constancy in relative fitness. While such models are broadly consistent with a large number of ecological theories that describe population dynamics, these models typically do not take into account positive selective pressures whereby non-lytic phage might act to provide an ability for host to exhibit enhanced fitness through lateral gene transfer, and the potential of population dynamics to allow shifts in the metabolic capacities of populations that enhance their fitness against environmental change. Prior measurements indicate that unlike lytic phages, which can dramatically disrupt microbial communities and the formation of biofilms through bacterial cell wall lysis, 9 that M13 and other temperate (filamentous) phages can enhance growth and biofilm formation. 10 , 11 Although the underlying mechanisms remain uncertain, it has been suggested that a major contribution to community stability involves the presence of extracellular DNA arising from cell death, which is thought to represent an important matrix element necessary for the formation of biofilms. Additional factors that enhance microbial growth may be related to the ability of many nonlytic phage, including M13, to promote horizontal gene transfer and the rapid acquisition of desired metabolic functionalities (eg, enzyme activities) that favor community stability. 12 – 14 Examples include the ability of microbial populations to mobilize natural genetic variation in response to environmental change that enhance fitness. While these latter mechanisms are commonly suggested to involve the presence of naked DNA that arises through natural mechanisms of cellular death unrelated to mechanisms of lateral gene transfer, recent data suggests a coordination between DNA release and uptake within the population. 14 To better understand the possible role of phage in promoting lateral gene transfer, and their relevance to possible applications involving bioremediation, we have investigated the community dynamics associated with the phage M13 and an E. coli host. In these measurements, a phagemid vector was constructed that encodes a chromate reductase (Gh-ChrR) (ie, M13-phage ChrR ), where Gh-ChrR has previously been demonstrated to efficiently reduce toxic chromate (Cr(VI)) in the presence of extracellular reductants naturally present within soils as humic compounds (such as quinones). 15 – 19 Infection only occurs in the presence of a helper phage, which is essential for phage maturation and assembly, permitting an understanding of possible differences between initial infection and subsequent transfer of Gh-ChrR within the bacterial population. Using these constructs, we have assessed how the presence of toxic chromate modifies E. coli growth and viability, focusing on the possible role of phage infection in promoting community stability. We find that in comparison to wild-type M13 phage (not expressing Gh-ChrR), that M13-phage Gh-ChrR infection of E. coli results in substantial increases in the population dynamics, resulting in enhanced bacterial growth, cell death, and total biofilm formation of E. coli strain TG1.",
"discussion": "Discussion We have demonstrated that the need for host tolerance to high concentrations of toxic chromate limits the ability of hosts to evade viral attacks and creates an evolutionary trade-off between growth rate maximization and defense that enhances microbial community co-evolution. In our experiments, the host E. coli bacterium challenged with infective phage encoding a functional chromate reductase experiences substantial amounts of both cellular killing and enhanced growth, leading to increases in biomass production and biofilm formation. These results indicate that phage infectivity can stabilize community structures, leading to more bacteria proliferation and initiation of a beneficial boom-bust cycle to produce more phages. Our measurements build on prior biodesign principles that indicate the ability of M13 and other temperate phages to enhance biofilm formation. 10 , 11 Central to this capability is the functional capacity of the M13-phage to express a function (ie, chromate reductase activity) that enhances fitness to a defined selective pressure (ie, chromate toxicity). Under these latter conditions, there are substantial increases in total biomass and biofilm thickness, despite substantial increases in phage-mediated cellular killing. These results indicate that the enhanced bioremediation of chromate and corresponding tolerance of toxic chromate by microbial communities represent a selectable pressure that can be engineered to promote community stabilization and enhanced remediation. In nature bacteria commonly form biofilms, which represent natural microbial communities that form on virtually any surface exposed to water. Such biofilms are a common target of antimicrobials that seek to minimize contamination in medical, industrial, and food processing. However, the establishment of beneficial microbial communities is now recognized to have considerable importance for human heath, and in natural environments may enhance stability to environmental change. In this respect, biofilm formation has been suggested to be critical for the environmental radionuclide waste bioremediation process, as observed following acetate injection experiments from US Department of Energy environmental subsurface bioremediation projects. 25 , 26 Indeed, subsurface environmental bacterial biofilms contribute to the long-term reductive immobilization of uranium (U(IV)) or chromium (Cr(III)) through formation of precipitates on sediment grain surfaces. 27 As a result, strategies that stabilize bacterial biofilms, such as those described here, are expected to improve the bioremediation efficiency in the subsurface environment. Our results suggest that bacteriophage may play an important role in the biogeochemical cycle by controlling bacterial and phytoplankton communities. As a result, strategies that enhance formation of bacterial biofilms are expected to improve the bioremediation efficiency in the subsurface environment. Bioremediation of chromate and uranyl toxic metals in contaminated soils remains a major technological challenge. Current solutions involve a range of biological approaches that include the targeted feeding of specific anaerobic microbes (eg, Geobacter sulfurrenducens ) whose specialized metabolism permits the biosorption, biosequestration, and reductive immobilization of extracellular chromate and uranyl metals, which can serve as terminal electron acceptors of cellular respiration. 28 – 34 Likewise, Shewanella oneidensis MR-1 contains specialized metal reductases located on the outer membrane that selectively associate with metal oxides to mediate their reduction. 35 , 36 The recent structural determination of these metal reductases and the understanding of how cellular machinery regulates the targeted assembly of a metal reductase complex on the outer membrane, all suggest possible synthetic biology approaches to enhance these pathways and promote more effective bioremediation of contaminated sites. 37 – 40 Likewise, identification of soluble enzymes capable of reductive immobilization of chromate and uranyl under both anaerobic and aerobic conditions offers a means to re-engineer microbes to enhance bioremediation. 18 In this latter respect, bacteriophage present at natural sites offer a potential means to serve as gene delivery vectors for these synthetic biology applications. 41 , 42 Additional mechanisms of bioremediation may take advantage of the ability to display catalytic protein moieties on the surface of bacteriophage, such as we describe, where the reducing potential of available humic compounds act as electron shuttles to allow transfer of reducing equivalents to extracellular catalysts. In this respect, it is necessary to further develop bioengineering strategies to retain viable phage in microbial populations responsive to environmental contaminates, permitting long-term immobilization of toxic metals such as chromate (Cr(VI))."
} | 2,716 |
36936286 | PMC10018498 | pmc | 8,690 | {
"abstract": "A novel contact–separation triboelectric generator\nconcept\nis proposed in this paper, which consists of a limestone-based mounting\nputty and a metallized polyester (PET/Al) sheet. This is an attempt\nto explore tacky materials for power generation and extend the operational\nfrequency bandwidth compared to existing TriboElectric NanoGenerators\n(TENGs). Moreover, the proposed design is very cost-effective and\neasy to build. Unlike traditional TENGs, which generate power solely\ndue to a charge developing on the surface, the putty also replies\non charge developed inside the material. Parametric study was conducted\nto determine the optimal putty thickness in a shaker test at 40 Hz.\nIt was found that a putty layer at 0.6 mm thick yielded maximum power\ngeneration. During the separation phase, the electrical breakdown\nbetween triboelectric layers allows most existing electrons to flow\nback from the ground due to rapid charge removal at the interface.\nWe are able to achieve a peak power of 16 mW in a shaker test at 40\nHz with an electrical load of 8 MΩ, which corresponds to a power\ndensity of 25.6 W/m 2 . A peak power of 120 mW in a manual\nprototype generator is achieved, which operates at approximately 2\nHz. Since putty material has less tackiness than double-sided tape,\nwe are able to expand the frequency bandwidth up to 80 Hz, which is\nsignificantly higher than a TENG (typically <10 Hz). The mounting\nputty material contains limestone with approximate 31 nm of mean grain\nsize mixed with synthetic rubber materials. Elasticity from rubber\nand the nanohardness of calcite crystallites allow us to operate a\nputty generator repeatedly without the concern of grain fracture.\nAlso, a durability test was conducted with up to 250,000 contact–separation\ncycles. In summary, comparable performance is achieved in the proposed\nputty generator to benefit energy harvesting and sensor applications.",
"conclusion": "Conclusions In this paper, we proposed a putty-based\ntriboelectric generator\nconcept, which is composed of an assembly of Al/PET–putty–PET/Al.\nPower generation is comparable to the state of the art of TENG devices.\nKey conclusions are summarized below: This is the first attempt to utilize the mounting putty\nmaterial in the triboelectric generator design. Under different putty thicknesses, power output changes\nsince PSC generates charges inside putty bulk materials, which is\ndifferent from surface charge generation as typically observed in\nTENGs. Putty generator has potential\nfor use at high frequencies\nsince it has less tackiness than a double-sided tape. We are able\nto extend the frequency bandwidth to 80 Hz, which is significantly\nhigher than a TENG (typically <10 Hz). Current simple design allows easy fabrication with only\na craft-level skill compared to the nanotechnology-based techniques\nused in TENG devices. Further investigation on putty-based generators is expected\nto\nexplore different minerals such as marble, sandstone, and lunar soil\nwith abundant amounts of Ca in plagioclase. It is also important to\nfully understand the charging mechanism at small-scale levels.",
"introduction": "Introduction Since its invention in 2012, the TriboElectric\nNanogenerator (TENG)\nhas been extensively investigated by many scientists and researchers\nbecause it has a great potential to power low-power profile sensors\nand electronics in many engineering applications without a major impact\non environments. 1 − 9 However, there are some challenges to overcome, primarily in improving\nperformance. In addition, spark generation during the separation significantly\nreduces the efficiency of power generation. 10 − 13 Therefore, external pumping devices\nand circuits must be introduced in TENGs, which add the complexity\nof the system. For example, in one study, voltage multiplying circuits\n(VMCs) were used to increase the output voltage. 14 The maximum power density of 38.2 W/m 2 was obtained\nat a load resistance of 4 MΩ, which operates at 4 Hz. A power\ndensity of 40 W/m 2 was achieved when using a ferroelectric\nP(VDF-TrFE) triboelectric layer and a VMC circuit. 14 TENG with an external charge excitation module was established\nwith a carbon/silicone gel electrode to avoid the air breakdown between\ntriboelectric layers. 10 The power density\nat 115.6 W/m 2 was reported. Although recent TENG developments\nshow enhanced performance in terms of power generation, the design\nand production of contact–separation-based TENGs must be simplified\nto reduce the system complexity and the environmental impact due to\nthe involved chemical evaporation process during the fabrication. Recently, we proposed a new way of fabricating triboelectric generators\nby exploring tacky materials. 15 A simple\nconfiguration of an assembly with double-sided tape and PET/Al sheets\nallows us to obtain a peak power of 25 mW in a shaker test at 20 Hz,\nwhich corresponds to a power density of 20.4 W/m 2 . The\nhighest peak power is 106 mW in a manually operated prototype, which\ncorresponds to a power density of 169.6 W/m 2 . Compared\nto existing TENGs, which often use nanotechnology-based fabrication\nmethods, this newly proposed triboelectric generator concept uses\ndouble-sided tape, making it far more cost-effective and simpler to\nbuild. The strong bonding nature of acrylic adhesive on the tape enables\na higher charge when contact. In addition, the electric breakdown\nat the interface makes the most existing electrons to flow back to\nthe ground during the separation, which contributes to additional\npower generation. The strong tackiness of a double-sided tape prevents\nfrom operating at higher frequencies. Small energy harvesting applications\nsuch as health monitoring and wearable exoskeleton systems require\na wider frequency bandwidth to harvest the energy from human motion. 16 − 18 Therefore, it is necessary to explore less tacky materials for higher-frequency\npower generation. In this paper, we propose a contact–separation\ntriboelectric\ngenerator concept with a higher-frequency operation and design flexibility,\nwhich consists of a less tacky mounting putty material and a metallized\npolyester (PET/Al) sheet. Commercial off-the-shelf (COT) removable\nmount putty is typically made by limestone mixed with rubber polymer\nmaterials. The physical phenomenon of this putty-based triboelectric\ngenerator is different during contact phase. Instead of surface charge\ngeneration on the interface between triboelectric layers as observed\nin TENGs 1 − 9 and the double-sided tape-based triboelectric generator, 15 a removable mounting putty-based triboelectric\ngenerator produces charge inside the putty body. Therefore, the thickness\nof a putty layer plays a major role for maximum power generation.\nIn geoscience, it is well known that minerals like limestone, marble,\nand sandstone have anomalous current generation when applying pressure\nto minerals due to a process known as pressure-stimulated current\n(PSC). 15 , 19 − 21 It has been proposed\nthat PSC occurs because of a plastic deformation in minerals. Once\nPSC signal is observed, tested minerals were broken into pieces. In\na limestone-concentrated putty material, the flexible nature of the\nputty allows power generation to be repeatable without any material\nfailure due to applied pressure. The generated power depends on the\nthickness of the putty, which indicates that the contribution of the\nputty bulk material is crucial for a power generation. Similar to\nthe double-sided tape-based triboelectric generator during the separation\nphase, the electrical breakdown between triboelectric layers allows\nmost existing electrons to flow back from the ground due to rapid\ncharge removal at the interface, which provides additional power output\ncompared to TENGs. A double-electrode configuration was adopted\nwith an assembly of\nAl/PET–putty–PET/Al to achieve maximum power generation\nduring contact-separation. Parametric study was conducted to determine\nthe optimal putty layer thickness during a shaker test at 40 Hz. A\nputty layer with 0.6 mm thickness produced maximum power generation.\nA peak power of 16 mW was achieved in a shaker test at 40 Hz with\nan electrical load of 8 MΩ, which corresponds to a power density\nof 25.6 W/m 2 . We can also evaluate the proposed putty-based\ntriboelectric generator up to 80 Hz in a shaker test. A durability\ntest was conducted up to 250,000 contact–separation cycles.\nIt is shown that the performance of power generation degrades slightly.\nA manual putty triboelectric generator produces a peak power of 120\nmW operating approximately at 2 Hz. In summary, this newly proposed\nputty-based triboelectric generator\nwas comprehensively evaluated. We are able to produce a comparable\namount of power to the double-sided tape generator and TENGs with\nmuch higher frequency.",
"discussion": "Results and Discussion The powder XRD pattern of the\nmounting putty is shown in Figure 1 . Calcite (CaCO 3 ) rhombohedral crystal peaks\nfrom limestone are identified\nin the plot. 22 , 23 Based on the Scherrer equation,\nthe mean size of calcite grain can be estimated using the following\nequation Here, K , λ, β,\nand θ are the shape factor, X-ray wavelength (Cu Kα: 1.5406\nÅ), the line broadening at FWHM (0.3°), and the Bragg angle\n(14.8°), respectively. The mean size of the calcite grain is\nfound to be 31 nm by applying the above equation. Therefore, it is\nplausible to state that the greater portion of the limestone putty\nconsists of calcite nanocrystallites. To investigate the effect\nof putty thickness on the power generation,\nparametric studies were conducted by varying the putty thickness in\na shaker test. Shaker putty generator with a double-electrode configuration\n(Al/PET/putty–PET/Al) is shown in Figure 2 a. The input frequency was set at 40 Hz.\nA 4 MΩ resistor was introduced to serve as an electric load\nin the circuit. Figures 3 a and 4 a show voltage and current time history\ncollected from the right Al electrode (PET/AL, shown in red) and left\nAl electrode (Al/PET/putty, shown in black), in which the putty thickness\nis 0.6 mm. Identical voltage and current profiles in the opposite\nphase are observed in both Al electrodes. Combined power history is\nshown in Figure 5 a.\nAn instantaneous peak power of 10 mW is achieved, which corresponds\nto a power density of 16 W/m 2 . The power density value\nwas slightly lower than our previous double-sided triboelectric generator15\n(17.3 W/m 2 at 20 Hz). Nevertheless, the removable mounting\nputty has less tackiness compared to a double-sided tape, which enables\nit to use for higher-frequency power generation. The collected voltage\ntime history for the case of 1.2 and 1.8 mm putty thicknesses is shown\nin Figure 3 b,c, respectively.\nThe associated current time history is shown in Figure 4 b,c. A similar trend is obtained for both\nvoltage and current as shown in Figures 3 a and 4 a when the\nputty thickness is 0.6 mm. However, there is a reduction on both voltage\nand current amplitude as the putty thickness increases. The corresponding\npower time history is shown in Figure 5 b,c. Apparently, as the putty thickness increases,\nthe power value decreases. This confirms that the charge generation\ndepends on the thickness of putty in our putty-based triboelectric\ngenerator design. Figure 6 shows the effect of putty thickness on power generation.\nNote that the combined peak power value from both electrodes is plotted.\nThe maximum power is observed when the putty thickness is 0.6 mm.\nAs the thickness increases from 0.6 mm, the peak power drops and levels\noff at a constant value. Figure 3 Putty thickness effect on the collected voltage:\n(a) 0.6 mm, (b)\n1.2 mm, and (c) 1.8 mm (left Al electrode in black and right Al electrode\nin red). Figure 4 Putty thickness effect on the collected current: (a) 0.6\nmm, (b)\n1.2 mm, and (c) 1.8 mm (left Al electrode in black and right Al electrode\nin red). Figure 5 Putty thickness effect on instantaneous power: (a) 0.6\nmm, (b)\n1.2 mm, and (c) 1.8 mm (4 MΩ resistor served as an electrical\nload). Figure 6 Average peak power as a function of putty thickness with\nan electrical\nload of 8 MΩ. If we assume that the power generation is solely\ncontributed by\nthe triboelectric effect, when the putty layer becomes thicker, the\ndistance between the putty and left Al electrode becomes too far for\ncharge attraction. The power production from the putty side (i.e., P = I putty side 2 R load ) will be negligible because the\ncharge flow ( I = d Q /d t ) in the Al electrode is minimized. On the other hand, the right\nPET side will produce the same amount of electric field to the Al\nelectrode since the charge is generated at the interface between putty\nand PET when contacting. Accordingly, the power generation will converge\nto half of the maximum value as the putty thickness increases. However,\nas shown in Figure 6 , the peak power value varies with the putty thickness and does not\nconverge to the expected half amount of peak value, which indicates\nthat the assumption is not valid. Therefore, it is plausible that\nputty bulk plays an important role in power generation. As a\nmatter of fact, in geoscience, researchers observed an electric\ncurrent emission with a positive voltage amplitude in some minerals\nincluding limestone when it was pressed. 20 , 21 , 24 It is called pressure-stimulated current\n(PSC). It was proposed that the plastic deformation (or fracture)\nof grains causes the electric pulse when limestone was pressed. Such\na fracture would normally prevent limestone from being useful for\na contact and separation-based energy harvesting operation. However,\ntwo reasons allow us to operate a putty generator repeatedly without\nthe concern of grain fracture. First, the rubber contents introduce\nelasticity in a putty material. Second, once the crystallites of calcite\nbecome small as nanoscale sizes, i.e., diameter less than 200 nm,\nthe nanohardness of the crystallites increases abruptly to 2.74 GPa\ncompared to the Vickers hardness number of 105–135 (1.03–1.334\nGPa). 25 Therefore, a substantial force\nis required to induce potential fracture damage in limestone minerals.\nIt is observed that power generation depends on the applied pressure\nduring contact. Less power generation is achieved when the contact\npressure decreases. Both 40 and 65.6 kPa were applied to our\nputty generator in a shaker\ntest at 40 Hz. The peak power of 10 mW was observed when the pressure\nis 65.6 kPa, which is higher than the peak power value of 6 mW in\nthe 40 kPa case. Not that those pressure values are nowhere close\nto the hardness value of the limestone whether it is in nanosize or\nnot. Therefore, the limestone putty generator can be operated for\na long period of time without material degradation concern. A durability\ntest was performed and will be discussed in a later section. As a rule of thumb, the electric field ( E ) on\nthe left electrode can be expressed as E = Q /(4πε d 2 ) in terms\nof permittivity (ε), total charge generated in putty layer ( Q ), and mean distance ( d ). Namely, if the\nputty thickness increases, the attraction from generated charges will\nbecome weaker to cause a drop in power generation. Also, a thicker\nputty will produce a less charge density per unit volume since total\ncharge Q remains the same throughout the putty bulk\nunder the same applied force. However, when the putty thickness is\nbelow 0.6 mm, the total charge Q generation is limited\nby a very thin layer of putty. Accordingly, the peak power decreases\nas shown in Figure 6 . Based on the above observation and discussion, the operational\nprinciple of a putty triboelectric generator is explained in Figure 7 a,b. In contact as\nshown in Figure 7 a,\nputty bulk generates positive charges throughout its thickness, while\nthe PET layer generates negative charges only on its surface. Since\nthe electron attraction in the Al electrode (left) inversely depends\non the square of the mean distance between the Al electrode and putty,\nwhich is approximated by the PET thickness plus half of putty thickness,\nthe power generation depends on the thickness of putty. On the right-hand\nside, negative charges in PET will attract positive charges in the\nAl electrode. In separation as shown in Figure 7 b, the air breakdown removes all charges\ninside the putty and on the surface of a PET. The attracted charges\nin Al electrodes will flow back to the ground. Figure 7 Schematics of the electric\ncharge generation in a putty triboelectric\ngenerator in (a) a contact and (b) a separation, respectively. Panels\n(c) and (d) show open circuit voltage from putty when interfaced with\nhuman skin and polypropylene material in a tapping test, respectively.\nPanels (e) and (f) show LEDs lighting up at a high-frequency shaker\noperation at 60 and 80 Hz, respectively. The behavior of the limestone putty in power generation\nis quite\nsimilar to the high entropy materials (HEMs). 26 For example, high entropy materials have a tendency to form nanotwins\nwith lower stacking fault energy when the force is applied to the\nsystem. 27 This distortion of lattice is\nresponsible for increasing the electron mean free path. 28 In limestone putty, PSC can be generated because\nof the plastic deformation of crystals under pressure. Therefore,\nit is plausible that the applied mechanical energy is possibly absorbed\nin a way of producing plastic deformations of limestone in putty.\nIt may lead to increase the electron mean free path like HEMs so that\nPSC can be observed in the energy harvester. To verify the charge\ngeneration by PSC instead of the triboelectric\neffect during a contact, a putty layer, which has a size of 25 mm\n× 25 mm × 1 mm, was interfaced with both human skin and\npolypropylene material in a tapping test, in which a Cu grid sheet\nis integrated inside the putty to serve as an electrode. Human skin\nand polypropylene are very strong positive triboelectric materials\nwith charge affinities of 45 and 55 nC/J, respectively. 29 If the charge is contributed by the triboelectric\neffect, a negative voltage will be collected during a contact since\nskin and polypropylene surfaces are strongly positive. If a positive\nvoltage is observed during a contact, this indicates that positive\ncharges are generated via PSC instead of triboelectrification. Figure 7 c,d shows the collected\nopen circuit voltage signals for human skin and polypropylene case,\nrespectively. Positive voltage is clearly demonstrated in both cases\nwhen contacting. Moreover, almost the same peak voltage amplitude\nis observed in both cases, which indicates that PSC contributes to\nthe charge developed inside the putty. Therefore, it is a direct proof\nthat the limestone putty has a different way of generating electricity\nduring a contact. Figure 7 e,f shows\n148 LEDs lighting up using a putty triboelectric generator in a shaker\ntest at 60 and 80 Hz, respectively (see Supporting Videos S1 and S2 ). It indicates\nthat the limestone putty generator extends the operational frequency\nfor broader applications. Figure 8 shows the\npeak power as a function of electrical loads for the putty generator\nwith the double-electrode configuration in a shaker test at 40 Hz,\nin which the putty thickness is 0.6 mm. Both voltage and current were\nmeasured separately from the Al electrode using the circuit as described\nin Figure 2 a to derive\nthe total harvested power. The combined highest peak power was 16\nmW under an electrical load of 8 MΩ, which corresponds to the\npower density of 25.6 W/m 2 . Figure 8 Peak power as a function\nof electric loads with a 0.6 mm putty\nthickness. As shown in Figure 9 , a durability test was performed for our putty generator\nin a shaker\ntest at 40 Hz, in which the putty thickness is 0.6 mm and a 4 MΩ\nelectrical load is connected to both electrodes. The peak power starts\nwith a value of 9.5 mW and slightly increases to 11 mW at 40,000 contact–separation\ncycles. Then, it decreases slightly to 8 mW at 250,000 cycles. It\nis shown that power generation degrades slightly. A manual putty generator\nis shown in Figure 10 a with an assembly of Al/PET–putty–PET/Al\nattached to the top and bottom plastic plates, in which polypropylene\nmaterials are used for springs on both sides. The contact-separation\nmotion can be introduced manually, which operates approximately at\n2 Hz. The collected voltage from both Al electrodes and combined power\nare shown in Figure 10 b,c, respectively. The peak power was 120 mW with an active area\nof 50 mm × 50 mm. The power density of the manual generator was\n48 W/m 2 . Therefore, the manual generator demonstrated higher\npower generation compared to the case in the shaker test because the\npolypropylene springs allow faster separation for quicker charge dissipation.\nA demonstration of a manual putty generator using 296 LEDs is shown\nin Figure 10 d (see\nSupporting Video S3 ). Figure 9 Durability test with\na 0.6 mm putty thickness and a 4 MΩ\nelectrical load as shown in Figure 2 . Figure 10 (a) Picture of a manual putty generator and assembly of\nAl/PET–putty–PET/Al\nattached to the top and bottom plastic plates and polypropylene springs\non both sides; (b) voltage amplitudes of the top Al electrode (black)\nand bottom Al electrode (red); (c) combined power; and (d) 296 LEDs\nlighting on by the manual putty generator operation with a hand. In summary, the proposed putty-based triboelectric\ngenerator shows\ncomparable performance in contrast to existing TENGs and our previous\ndouble-sided tape triboelectric generator. 15 Moreover, less tackiness allows us to operate it at a higher frequency,\nwhich could lead to a new sensor design configuration."
} | 5,404 |
29101358 | PMC5670136 | pmc | 8,692 | {
"abstract": "Since the early discovery of the antireflection properties of insect compound eyes, new examples of natural antireflective coatings have been rare. Here, we report the fabrication and optical characterization of a biologically inspired antireflective surface that emulates the intricate surface architectures of leafhopper-produced brochosomes—soccer ball-like microscale granules with nanoscale indentations. Our method utilizes double-layer colloidal crystal templates in conjunction with site-specific electrochemical growth to create these structures, and is compatible with various materials including metals, metal oxides, and conductive polymers. These brochosome coatings (BCs) can be designed to exhibit strong omnidirectional antireflective performance of wavelengths from 250 to 2000 nm, comparable to the state-of-the-art antireflective coatings. Our results provide evidence for the use of brochosomes as a camouflage coating against predators of leafhoppers or their eggs. The discovery of the antireflective function of BCs may find applications in solar energy harvesting, imaging, and sensing devices.",
"introduction": "Introduction Natural surfaces have demonstrated how different micro/nanoscale surface architectures can yield an array of distinct interfacial functions 1 – 9 . While many of these surface structures can now be manufactured using advanced manufacturing techniques 10 – 13 , scalable fabrication methods capable of producing a number of these natural structures have remained elusive. Among these natural structures are leafhopper-produced brochosomes (Fig. 1 ) 14 – 16 . Naturally occurring integumental brochosomes are microscale granules with nanoscale surface indentations arranged in a honeycomb pattern, making the geometry of a brochosome particle similar to those of a soccer ball 17 . Leafhoppers living in different regions create brochosomes with significantly varied structural geometries, with distinct diameters and numbers of pits 18 . In addition to their use as non-sticking coatings 19 , 20 , the intricate nanoscale architecture and three-dimensional periodicity of these BCs suggest they may have complex optical properties. Interestingly, Swain proposed in 1936 that these brochosomes might serve as a camouflage coating to hide the eggs of leafhoppers from their predators or parasites 21 , but no experimental evidence has been shown thus far. This is due to the fact that the optical functions of the BCs remained minimally understood as large quantities of brochosomes for systematic study are not readily producible. Until now, micro/nanomanufacturing techniques to create brochosomes of various geometries and material compositions have not been available. Fig. 1 Leafhopper and its brochosomes. a Optical image of a leafhopper, Alnetoidia alneti (Dahlbom), and b an electron micrograph showing its brochosomes. Both images are reprinted from ref. 19 , by permission of the Royal Society. Scale bars, 1 mm (leafhopper) and 200 nm (brochosomes) \n Here, we develop a fabrication concept for preparing BCs composed of closely packed artificial brochosomes, whose shape and geometry closely mimic the natural ones created by leafhoppers. We utilize double-layer colloidal crystal (DCC) templates combined with site-specific electrochemical growth to prepare artificial BCs comprising metals, metal oxides, polymers, or their hybrids on any conductive substrates. The structure of the synthetic BCs, defined by the diameter of the brochosomes, the inter-brochosome distance, as well as the size and depth of the pits within the brochosomes, can be precisely engineered, allowing us to systematically explore their structure–property relationships. Using silver (Ag) as a model material, we have shown that 2-µm thick Ag BCs (i.e., BCs comprise 2 µm diameter brochosomes) are capable of reflecting <~1% on average of any wavelength in the 250–2000 nm optical window. This reflectance is comparable to those of the state-of-the-art synthetic antireflective materials 22 . The superior antireflection is attributed to the unique structural geometries of the brochosomes, as demonstrated experimentally and numerically. Additionally, our experimental results suggest a possible use of BCs as a camouflage and protective layer for leafhoppers or their eggs against potential predators in their natural habitats 18 , 23 , 24 .",
"discussion": "Discussion The ultra-antireflective property of the synthetic BCs at the UV and visible light range may suggest that their natural counterparts could have been optimized for antireflective and camouflage functions against leafhoppers’ predators (e.g., birds or insects), whose active vision spectra are also in the UV and visible light range 34 . To demonstrate the possible camouflage function of the BCs, we placed the synthetic BCs next to various leaf species (i.e., Chrysanthemum, Lantana, Callicarpa, and Fushia), and compared their colors through the simulated visions of a ladybird beetle (a predator of leafhopper) 35 . Note that the active vision spectrum of a ladybird beetle is from 311 to 605 nm 36 . Based on the images generated by the simulated vision of a ladybird, the appearance of the BCs and the green leaves have very high level of similarity both qualitatively (Fig. 5a ) and quantitatively (Supplementary Fig. 23 ). To further support our hypothesis, we gathered the geometrical parameters of a number of representative natural integumental BCs, and found that their geometries (i.e., R \n b and R \n t ) are within the design parameters for strong omnidirectional antireflectance (<10%) of the synthetic BCs (Fig. 5b and Supplementary Fig. 24 ). This further suggests that the natural and synthetic BCs may possess similar antireflective properties, which could be important survival functions that protect leafhoppers or their eggs from being detected by their predators in their natural environments 18 , 23 , 24 . Fig. 5 Possible camouflage function of BCs and their fabrication in different materials compositions. a Simulated human and ladybird beetle visions of BCs and two different leaf species. b Comparison of the structural parameters of natural and synthetic brochosomes for their optical reflectance; here, the color bar represents the experimental reflectance measurements of various synthetic BC structures and the magenta lines represent the natural brochosomes (i.e., the visible spectrum of the ladybird over R \n t determines the line length and position along the x -axis). These natural brochosomes include a Oncometopic orbona , male. b Proconia esmeraldae , female. c Homalodisca coagulata , male. d Proconia esmeraldae , male. e Diestostemma stesilea , male (Supplementary Fig. 24 ). The observing (incident) angle is 45°. Note that the geometrical parameters of the aforementioned natural brochosomes are located at the low reflection region in the optical reflectance map obtained from the reflectance measurements of the synthetic brochosomes. The normalized plot allows one to identify the corresponding antireflection design parameters of the synthetic brochosome given a specific wavelength of light. c Scanning electron micrographs showing various BCs fabricated from nickel (Ni), gold (Au), manganese oxide (MnO 2 ), silver/manganese oxide (Ag/MnO 2 ), and polypyrrole (PPy). Scale bars: 2 µm (Ni, Au, MnO 2 , PPy) and 5 µm (Ag/MnO 2 , inset, 2 µm) \n Finally, the fabrication concept of synthetic BCs is very general and can be extended to other material systems compatible with electrochemical deposition technique (Fig. 5c ). For example, BCs of nickel (Ni), gold (Au), manganese oxide (MnO 2 ), and polypyrrole (PPy) can be prepared, where these materials have broad applications in energy and sensing applications 37 , 38 (Supplementary Figs. 25 – 29 ). In addition, BCs of hybrid materials, for instance, Ag/MnO 2 can be prepared by a step-by-step electrodeposition method (Supplementary Fig. 29 ). Translating the highly complex geometries of natural brochosomes into materials architectures of various compositions may lead to advanced applications in solar energy harvesting, imaging, and sensing devices 39 – 41 ."
} | 2,042 |
35515927 | PMC9060656 | pmc | 8,694 | {
"abstract": "A novel approach, combining a microbial fuel cell (MFC) with an integrated vertical flow constructed wetland (IVCW), was developed, and its ability to simultaneously produce electrical energy while treating swine wastewater was verified. The system combined the singular water flow path of a traditional vertical flow constructed wetland (upflow and downflow)-microbial fuel cell (CW-MFC), which demonstrates better characteristics in the aerobic, anoxic, and anaerobic regions. It not only enhanced the anti-pollution load ability and the organic compound removal effect, but also improved the gradient difference in the redox potential of the system. The results showed that the structure and substrate distribution in the device could both improve swine wastewater treatment and increase bioelectricity generation capabilities. The average chemical oxygen demand (COD) and ammonia nitrogen (NH 4 + –N) removal efficiencies were as high as 79.65% and 77.5%, respectively. Long-term and stable bioelectricity generation was achieved under continuous flow conditions. The peak values of the output voltage and power density were 713 mV and 456 mW m −3 . The activated carbon layer at the bottom of this system provided a larger surface for the growth of microbes. It showed significant promotion of the relative abundance of electrochemically active bacteria, which might result in the increase of bioelectricity generation in integrated vertical flow constructed wetland-microbial fuel cells (IVCW-MFCs). The electrochemically active bacteria, Geobacter and Desulfuromonas , were detected in the anodic biofilm by high-throughput sequencing analysis.",
"conclusion": "4. Conclusions By taking advantage of the structure and substrate distribution of the reactor and combining downflow and upflow effects, the combination of IVCW and MFC proved effective in simultaneously treating swine wastewater and generating bioelectricity. In the IVCW-MFC, the average removal rates of COD, NO 3 − –N, and NH 4 + –N were 79.65%, 75.13%, and 77.5%, respectively. The stable output voltage was in the range of 598–713 mV, the maximum power density was 0.456 W m −3 , and the maximum CE was 0.386%. Well-known electrogenic bacteria were detected in the anode surface biofilm under IVCW-MFC-C.",
"introduction": "1. Introduction Swine wastewater contains high concentrations of organic matter and other pollutants. Inadequate treatment or direct discharge of such wastewater into water bodies will result in serious water pollution and ecosystem damage. 1 With the rapid growth of large-scale pig breeding in China over the past few decades, the amount of pig farm pollutants has also increased. According to recent issues of the Survey Bulletin of China's Pollution Sources (2010), 2 the organic pollution loading derived from livestock and poultry breeding in China accounts for 41.9% of the total organic pollution loading (represented by COD) of water. Among these pollution sources, the discharge of swine wastewater is the main contributor, which in some areas accounts for more COD than industrial and domestic sewage. 2 Due to the environmentally benign characteristics, many bio-measures have been proposed to purify this kind of swine wastewater. The main techniques are: (i) anaerobic biological treatment technologies ( e.g. , biogas tanks, upflow anaerobic sludge blankets (UASB), anaerobic migrating blanket reactors (AMBR)), 3 (ii) aerobic biological treatment technologies ( e.g. , aerobic sequencing batch reactors and activated sludge), 4 (iii) natural methods ( e.g. , constructed wetlands and stabilization ponds). 5 Recently, most livestock and poultry breeding farm pollution treatment systems only contained anaerobic treatment installations, such as biogas tanks. Biogas is a key energy resource in the emerging global renewable energy resource market, and biogas technology is considered crucial for the transition away from fossil fuel dependence. 6 The organic concentration in the effluent of biogas tanks is still high. A few farms also built aerobic facilities to deal with biogas slurry, but were unable to consistently operate these facilities due to high energy consumption and operating costs. Constructed wetlands (CW) are an engineering technology that purify wastewater. The integrated vertical flow CW is a modification of the structure of the conventional constructed wetland. Compared with other biological treatment technologies, the integrated vertical flow constructed wetland possesses the advantages of lower investment and operating costs, better anaerobic and aerobic regions, better HRTs and anti-pollution load ability, and particularly better removal effects of the high nitrogen and phosphorus nutrients of livestock and poultry wastewater. These factors contribute to IVCW application in the treatment of swine wastewater. In the process of wastewater treatment, it has the potential of further energy conversion and recycling, and advanced treatment of organic pollutants. This system combined the singular water flow path of the traditional vertical flow CW-MFC (upflow and downflow), improved gradient difference in the redox potential of the system and reduction of the energy consumption in pumping influent into the upflow vertical subflow CW. Microbial fuel cells (MFCs) are newly emerging methods of purifying pollutants and producing energy simultaneously. With advantages like low-sludge production, no-tail gas treatment, and low operating cost, studies on MFC opportunities have received extensive attention in recent years. 7,8 In the anode, electrons and protons are generated by microbial oxidation and decomposition of organic compounds; the electrons are then transmitted to the cathode via an external circuit, and the produced protons are diffused into the cathode through the proton exchange membrane. 9 The electron acceptor (as O 2 ) in the cathode chamber is reduced after it receives the electrons and protons from the anode chamber. 10 With the degradation of organic substrates in the anode and the reduction reaction in the cathode. 11 The constructed wetland (CW) has structural advantages for creating an MFC. 12 The CW system contains better anaerobic regions, aerobic regions, and redox gradients, and its higher specific surface area in the stroma is favorable for the adsorption of an electron mediator, making it possible to couple MFCs with CWs. The coupling of an MFC with the widely used CW technology has great practical significance and application prospects. 13 However, in retrospect, previous works on CW-MFC revealed several limitations on the existing structures (concentrated in vertical and horizontal flow constructed wetlands). Excessive or unstable influent pollution will influence the treatment effect and power generation performance of the system. 14,15 At the same time, a certain amount of energy is consumed in the application of up-flow influent. In this study, we designed and constructed an IVCW-MFC integrated system that enhanced the wastewater treatment effect and realized the synchronous production of electricity. Actual swine wastewater was used in this system. The influent model of this IVCW-MFC differed from the conventional upflow vertical subflow constructed wetland. This system combined the singular water flow path of the traditional vertical flow CW-MFC (upflow and downflow), improved the gradient difference in the redox potential of the system and reduced the energy required to pump influent into the upflow vertical subflow CW. On one hand, the anode was placed deep in the system to eliminate the influence of atmospheric air, and the cathode was exposed to atmospheric air. This ensured maximum reduction of dissolved oxygen (DO) in the anode region and provided an anaerobic environment for the anode. On the other hand, the energy free influent mode coupled plant roots in front of the anode area, which contained the aerobic nitrification phase and enhanced the nutrient removal effects and anti-pollution load capacity. Furthermore, high or fluctuating pollution loads in the influent did not affect the treatment efficiency and the bioelectricity generation performance. It demonstrated good practicability and economy, and rendered the system more suitable for practical engineering operation in the future. In this study, the wastewater treatment performance was assessed via markers such as COD, NO 3 − –N, and NH 4 + –N in open- and closed-circuit systems. Additionally, its bioelectricity generation properties were assessed via the closed-circuit system's voltage output, power density, current density, and Coulomb efficiency (CE). Furthermore, the microbial community structure of electrogenic bacteria was tested by a high-throughput sequencing analysis. The feasibility, effectiveness and potential synergy of the integrated system for wastewater treatment and synchronous power generation was tested.",
"discussion": "3. Results and discussion 3.1. COD removal The swine wastewater was provided for 92 consecutive days. Fig. 2(a) depicts the stable COD removal by the IVCW-MFC system under long-term operation; the influent COD concentrations varied from 324–708 mg L −1 , and the average concentration of COD entering the IVCW-MFC was 505 mg L −1 . After stable treatment with a two-day HRT, the average effluent concentration of the closed- and open-circuit systems were 102.7 mg L −1 and 181.1 mg L −1 , respectively, and the average removal effect of COD was 79.65% and 64.71% in each system, respectively. Results showed that the COD removal of the closed-circuit system was 14.94% higher than for the open-circuit system, and the synchronous electric generation from the closed-circuit system could improve wastewater treatment. It may have resulted from the use of the anode of the CW-MFC as a temporary electron acceptor to contribute to the process of anaerobic treatment. 20 Fig. 2 (a) COD removal efficiency, (b) correlation between COD loading and its removal, and (c) variations in COD concentration with water flow. In Fig. 2(b) , the regression analysis between COD removal (g m −2 d −1 ) and COD loading (g m −2 d −1 ) can be seen. R 2 values of IVCW-MFC-C and IVCW-MFC-O were 0.9749 and 0.9030 respectively, which indicated that COD removal effect prediction in IVCW-MFC-C mode was more reliable in IVCW-MFC-C mode in the system over the 92 day study. The pollution load varied during the operation with swine wastewater, but the stable COD removal efficiency and the anti-load ability of the IVCW-MFC were confirmed. This may be a result of the overall aerobic–anaerobic–aerobic process in the IVCW-MFC. Especially, in the aerobic phase, the Canna indica roots formed a special microbial community and produced oxygen in rhizodegradation, both of which promote the degradation of nitrogen and organic matter. It is clear from Fig. 2(c) that, compared with an open-circuit system, the COD concentration in the closed-circuit system reduced more intensely from the inlet to S1–S3, followed by a gradual reduction from S4 to S9. The anodic region became the main area of COD removal, with a total of 58.93% COD reduction occurring. The microcurrent surroundings and exudates from plant roots could promote the growth and activity of microorganisms and wetland plants, 21 and some complex organic compounds can be hydrolyzed from even a small amount of oxygen released from roots. The hydrolytic products, such as acetic acid, propionic acid, butyric acid, and other low-molecular organic compounds, were used by electrogenic bacteria to promote the removal of pollutants in the swine wastewater, 22,23 and the unused organic matter would be removed further. The COD removal rate reached 73.3% in IVCW-MFC-C at S5 and S6 after the activated carbon layer treatment and was 22.9% higher than in IVCW-MFC-O. The concentration of organic matter was reduced greatly when reaching the cathode region. If the concentration of organic matter in the cathode was high, the oxygen would be consumed during the decomposition process, and the aerobic surroundings would be damaged in the cathode, affecting the reduction reaction necessary to form the complete circuit. This would cause mass propagation of heterotrophic bacteria, limit the mutual transmission between the reactant and the product on the electrode, and bring harmful effects to the bioelectricity generation ability. It is worth noting that COD decreased sharply in the anode (electricity producing) layer of IVCW-MFC-C from S2 to S3, and the removal amount was trending higher than the total amount in the pure activated carbon layer: S3–S6. The COD removal ratio reached 18.3%, which is 26.9% higher than for the same region of IVCW-MFC-O. While the anode layer occupied only 10.4% of the entire liquid volume, its role in removing organic pollutants was emphasized. Fang 13 developed similar results, in which the COD removal ratio was enhanced 12.7% by the anode action of the system. The anode provided a suitable growth environment to the biomembrane and was also an insoluble final electron receptor that increased the metabolic rate of anaerobic bacteria, 13 which contributed to the reduction of COD. The connection of external circuits also promoted the growth of electrogenic bacteria in the anode region and enhanced nutrient removal. 24 3.2. Nitrogen removal Swine wastewater is characterized by high nitrogen concentrations, which is a key index of treatment. The reactor was reformed in the traditional CW structure to enhance the denitrification effect, and it was separated into aerobic, anoxic, and anaerobic regions without power. The distributions of NH 4 + –N and NO 3 − –N were then analyzed in this system. As shown in Fig. 3(a) , the influent NH 4 + –N concentrations varied from 138–284 mg L −1 , with an average concentration of 215 mg L −1 . The pollution load varied greatly; however, the overall ammonia removal efficiency remained stable. The average removal rate of IVCW-MFC-C reached 77.5%, which was higher than that of IVCW-MFC-O by 57.6%, an improvement over the Knight 25 study of 135 wastewater treatment plants, whose CW denitrification effect was 48%. In addition, the ammonia nitrogen removal per unit area was 12 g m −2 d −1 , which was higher than that obtained in previous studies (8.5 g m −2 d −1 ). 1 These results indicate that the integration of MFCs in the CW system promoted the decomposition of the swine wastewater. Fig. 3 (a) NH 4 + –N removal efficiency and (b) the concentration varieties of NH 4 + –N and NO 3 − –N along the water flow. As shown in Fig. 3(b) , the NH 4 + –N concentration decreased drastically from the influent to S2, and the decrease in NH 4 + –N removal was met with an increase in NO 3 − –N concentration. The increased NO 3 − –N accounted for 49.02% (IVCW-MFC-C) and 46.12% (IVCW-MFC-O) of the NH 4 + –N removal in the same sample layer, indicating that nitrification occurred in front of the anode area. In this IVCW-MFC system the improvement of nitrification can be attributed to the advantage of the downflow in the anode and the oxygen supply from the plant roots, increasing the ammonia nitrogen removal rate. The NH 4 + –N reduction in the anode region alone accounted for 71.4% of the total reduction of the IVCW-MFC-C system. The NO 3 − –N concentration reduction was primarily due to the organic substrates and the plant absorption and denitrification. 26 The NO 3 − –N decreased from its highest concentration at 70.62 mg L −1 (IVCW-MFC-C) and 70.32 mg L −1 (IVCW-MFC-O) to its lowest concentration at 17.56 mg L −1 (IVCW-MFC-C) and 23.58 mg L −1 (IVCW-MFC-O), although a small amount of ammonia nitrogen nitrification occurred in the aerobic stage of the cathode, resulting in a slight increase in NO 3 − –N; the overall nitrate reduction, however, decreased up to 75.13% (IVCW-MFC-C) and 66.47% (IVCW-MFC-O). These results suggest that the IVCW-MFC-C mode promoted nitrogen removal, and the integrated system had a better removal effect on nitrate than does the traditional system. 27 The high concentration of wastewater in the anode region promoted the growth of biofilm in the anode area, and the oxygen diffusion was limited by the accumulation of organic matter, which increased the anoxic region at the bottom of the system. The sufficient carbon sources improved the nitrate removal. 28 Meanwhile Nguyen et al. 29,30 investigated nitrate removal in MFC by using abiotic and biotic cathode. With the increase of imposed cell potential, enhanced nitrate removal efficiency from 18% to 43%. On the other hand, the cathode region had a better reoxygenation effect under the double action of the plants and air cathodes. At the same time, sufficient organic matter and carbon sources could promote the growth of aerobic denitrifiers in the cathode region. These denitrifying bacteria could use both oxygen and nitrate as terminal electron acceptors to denitrify the system under aerobic conditions. 14,19 3.3. Bioelectricity generation To study the power production effect of the system in the purification of swine wastewater, the voltage values were recorded every six minutes for 92 days continuously in the IVCW-MFC-C mode, as shown in Fig. 4(a) . In the early stages of the experiment, the voltage reached a stable output after two increases. The swine wastewater was rich in dissolved organic matter, which was easily decomposed by microorganisms. Furthermore, the activated carbon layer in the anode region of this system provided a larger surface for the growth of microbes. Increasing the bacteria might result in the increase in electrons and protons and lead to a stable voltage output rapidly. The output voltage required approximately six days to reach the first stable phase. Then, the exclusive bacterial flora gradually formed in approximately the next twelve days, and under the effect of a closed external circuit, the output voltage achieved the second stable power generation phase. The biofilm structure on the substrate and electrode surface (including microorganism species, abundance, and other factors) was gradually stable, and more electrons and protons were produced by microorganism decomposition and oxidation of organic matter in the swine wastewater, which promoted electricity production. In the stable operation period, the output voltage of the system was in the range of 598–713 mV which was higher than the values of studied by Pratiksha et al. , 24 Wang et al. , 27 and José et al. 31 Based on the cathode half-cell reaction (O 2 + 4H + + 4e − → 2H 2 O) and oxygen as the final electron receptor, the air cathodes and plants photosynthesis provided high oxygen concentrations for the cathode. 32 The particular water flow path and the activated carbon layer at the bottom prevent the organic compounds from reaching the cathode. In this phase, the COD removal amount occupied 92.03% of the whole COD removal amount of the system. This effectively prevents the large amount of organic compound from consuming the oxygen in the cathode and promoted a cathodic redox reaction to reduce the cathode resistance and increase the cathode potential. At the same time, a continuous influx was used in this experiment; the wastewater flowed from anode to cathode while transporting protons, shortening the pH gradient between the two points and avoiding the small effect of electricity production caused by excess acidity at the anode or excess alkalinity at the cathode. 33 Fig. 4 (a) Electricity generation performance, (b) polarization and power density curves, and (c) the Coulomb efficiency variations with the COD removal amount. The electrochemical performance of IVCW-MFC was evaluated by polarization and power density curves obtained at the end of experiment. As shown in Fig. 4(b) , the maximum power density was 0.456 W m −3 (when the current density was 22.5 mA m −2 ), which was higher than the maximum power densities of 0.093 W m −3 from Oon et al. 16 and 0.302 W m −3 from Zhou et al. 32 The internal resistance was one of the limiting factors of the CW-MFC bioelectricity generation ability, which directly affected the voltage output of the system. 34 The internal resistance of the system measured by the polarization curve method was 463.66 Ω, which is theoretically close to the external resistance value, 450 Ω. The lower internal resistance was due to the high conductivity of the electrode and the advantages of the system structure, such as continuous water flow. 35 The internal resistance of CW-MFC was mainly composed of the activation internal resistance, ohmic resistance, and concentration resistance. The activation internal resistance in this study was mainly rooted in the activation energy needed for an electrochemical reaction on the surface of the carbon felt air cathode and the stainless-steel wire mesh coated granular active carbon anode. Research on MFCs has indicated that the catalytic converter in the electrochemical reaction could reduce the activation internal resistance when the microbes grow well. 36 The swine wastewater treated in this study contained a great deal of dissolved organic matter that could satisfy microbial metabolism nutrient needs, prevent the substrate transport from being limited (which reduces the internal resistance by increasing the electrical conductivity of the substrate), and improve the bioelectricity generation ability of CW-MFC. The output voltage varied with the COD concentration. The ohmic resistance in this study was mainly rooted in the conductivity of electrolytes, electrodes, and surface biofilms. The metal titanium wires, as perfect conductors, hardly corroded and were interwoven through the carbon felt and connected the outside circuit to increase the electrical conductivity of the cathode. The microbes attached to the electrode surface could also reduce the electrode resistance. 37 During the operation period, a total of 7137 C of electric charge were produced, with an average of 6.74 C d −1 L −1 . The maximum coulombic efficiency (CE) was 0.386%, which close to the values obtained by Doherty et al. , 18 Fang et al. , 13 and Zhou et al. 32 As indicated in eqn (5) , CE was closely related to the COD removal of the reactor; this was also confirmed in Fig. 4(c) , where the CE curve trend is opposite that of COD removal. The large number of microorganisms from the swine wastewater competed with the electrogenic bacteria in the reactor, which resulted in most organic compounds being used in the anaerobic digestion reaction e.g. , consumed in methanogenic or fermentation processes; Kim et al. 38 Only a small fraction of organic matter was used as a bioelectron donor. Charge transfer resistance due to the slow activation rate on the anode and cathode electrode constitute the dynamic transfer limit. This was also one of the main reasons for the low CE value in the IVCW-MFC and even in the general CW-MFC. 3.4. The influence of electrodes on electrogenic bacteria The microorganisms on the electrodes presented unique structural characteristics while simultaneously purifying swine wastewater and producing electricity. Swine wastewater was known to contain various kinds of useful bacteria, including exoelectrogens, hydrogen-producing bacteria, and methanogens for anaerobic digestion, 39 and the activated sludge from wastewater treatment that was inoculated as the seed sludge into all IVCW-MFCs was identical. To identify the microbial communities from the two operation modes, 223 528 raw sequences and 217 268 high-quality reads with an average length of ∼422 bp were obtained. The Shannon diversity index ( H ′) indicated both richness (the number of species present) and evenness (how each species is distributed), showing that the anode surface biofilm biodiversity ( H ′ = 5.32, from IVCW-MFC-C) was the highest among the four electrode samples while the biodiversity ( H ′ = 4.57) was lowest from the same sampling position with IVCW-MFC-O. The cathode surface biofilm biodiversity ( H ′ = 5.06 from IVCW-MFC-C; H ′ = 5.08 from IVCW-MFC-O) for both IVCW-MFCs was similar. The results revealed that the closed-circuit mode resulted in high biodiversity on the anode, which promoted the generation of bioelectricity and the degradation of organic compounds. The dendrogram cluster analysis showed dissimilarity in the bacterial community structure between the two modes ( Fig. 5(a) ). Two clear clusters were observed: samples collected together in the anode and cathode regions. This suggested a clear distinction in the microbial community structure between the anode and cathode regions. At the genus-level, Geobacter (with a relative abundance of 17.87% in the IVCW-MFC-C and 3.89% in the IVCW-MFC-O) was the typical anaerobic bacteria, which were detected only in the anaerobic region, but not in aerobic regions. This could be due to the unique structure of the reactor, which forms clear partitions in the anaerobic (anode) and aerobic (cathode) regions. Fig. 5 (a) Bray TREE Plot and (b) a heatmap of each genus. From left to right: CF-IVCW-C, CF-IVCW-O, GAC-IVCW-C, and GAC-IVCW-O. CF is short for carbon felt as the cathode, GAC is short for granular-activated carbon as the anode. \n Fig. 5(b) shows the relative bacterial community abundance characterized at the genus levels to compare the phylogenetic differences in the compositions of the bacterial community structures of the IVCW-MFC-O and IVCW-MFC-C modes. Many researchers have extrapolated that different substrate types could form different exoelectrogenic bacteria community structures. 39,40 The well-known electrogenic genera, Geobacter and Desulfuromonas , were detected by a genus-level analysis ( Fig. 5(b) ). Desulfuromonas (Proteobacteria), with a relative abundance of 1.59%, was only detected in the anode surface biofilm under the closed-circuit mode and was not detected in the other three samples. This may due to the IVCW-MFC structure, which provided an extra electron acceptor in the form of the anode to enhance the growth of anaerobia. Geobacter (Proteobacteria), a genus of Fe( iii )-oxide reducing bacteria that possesses autohydrogenotrophic denitrification capabilities, was found in anode regions under both open- and closed-circuit modes. It was obvious that the abundance of Geobacter in the IVCW-MFC-C mode was higher than in the IVCW-MFC-O mode. Reguera et al. 41 and Ganesh et al. 42 found that Geobacter could be demonstrated to generate currents by pathways involving direct electron transfer and pili. Many electrogenic bacteria, including Geobacter , can directly produce current from acetate without cooperation from other bacteria. 39 Other bacteria with significant differences in frequency between samples were also identified. Trichococcus (Firmicutes) and Thiobacillus (Proteobacteria) (with relative abundances of 11.59% and 13.51%, respectively) were more abundant in the anode of the IVCW-MFC-C mode than in the same region of the IVCW-MFC-O mode, and neither could really be found in cathode regions under both open- and closed-circuit modes. Nitrospira (Nitrospirae) and Hyphomicrobium (Proteobacteria) showed the opposite presentation, being more abundant in the cathode of the IVCW-MFC-C mode than in the same region of the IVCW-MFC-O mode and infrequently being found in anode regions under both open- and closed-circuit modes. Notably, Geobacter , Desulfuromonas , Thiobacillus , Hyphomicrobium belong to Proteobacteria, and Trichococcus belongs to Firmicutes. The two phyla Proteobacteria (43.01–57.66%), and Firmicutes (8.86–25.02%) were frequently observed in MFCs and could also be recognized in IVCW-MFC-C samples. These two phyla accounted for the majority of the bacterial community, with more than 66.5% in every sample. Trichococcus (Firmicutes) played an important role in consuming sugar compounds under higher current-producing conditions. 40 Thiobacillus (Proteobacteria) are Gram-negative betaproteobacteria and denitrifying bacteria using nitrate as electron acceptors, which may bring negative effects to the electrogenesis process in MFCs. Nitrospira (Nitrospirae) and Hyphomicrobium (Proteobacteria) are denitrifying bacteria and could be found in the aerobic/cathode regions. This indicates that various microbes could participate in organic oxidation and/or electricity generation."
} | 7,109 |
29883774 | null | s2 | 8,697 | {
"abstract": "The explosion of microbial genome sequences has shown that bacteria harbor an immense, largely untapped potential for the biosynthesis of diverse natural products, which have traditionally served as an important source of pharmaceutical compounds. Most of the biosynthetic genes that can be detected bioinformatically are not, or only weakly, expressed under standard laboratory growth conditions. Herein we review three recent approaches that have been developed for inducing these so-called silent biosynthetic gene cluster: insertion of constitutively active promoters using CRISPR-Cas9, high-throughput elicitor screening for identification of small molecule inducers, and reporter-guided mutant selection for creation of overproducing strains. Together with strategies implemented previously, these approaches promise to unleash the products of silent gene clusters in years to come. [Image: see text]"
} | 226 |
36099393 | PMC9733646 | pmc | 8,698 | {
"abstract": "Abstract Microbial destabilization induced by pathogen infection has severely affected plant quality and output, such as Anoectochilus roxburghii , an economically important herb. Soft rot is the main disease that occurs during A. roxburghii culturing. However, the key members of pathogens and their interplay with non‐detrimental microorganisms in diseased plants remain largely unsolved. Here, by utilizing a molecular ecological network approach, the interactions within bacterial communities in endophytic compartments and the surrounding soils during soft rot infection were investigated. Significant differences in bacterial diversity and community composition between healthy and diseased plants were observed, indicating that the endophytic communities were strongly influenced by pathogen invasion. Endophytic stem communities of the diseased plants were primarily derived from roots and the root endophytes were largely derived from rhizosphere soils, which depicts a possible pathogen migration image from soils to roots and finally the stems. Furthermore, interactions among microbial members indicated that pathogen invasion might be aided by positively correlated native microbial members, such as Enterobacter and Microbacterium , who may assist in colonization and multiplication through a mutualistic relationship in roots during the pathogen infection process. Our findings will help open new avenues for developing more accurate strategies for biological control of A. roxburghii bacterial soft rot disease.",
"conclusion": "CONCLUSIONS Here we showed that the onset of A. roxburghii bacterial soft rot disease may begin in the RZS, migrated into the plant roots, transferred to the stems, and finally induce soft rot disease (Figure 2A ). The pathogenic Pantoea invasion is associated with a drastic reduction in microbial diversity, abundance, and community composition, especially with the microbes in diseased plant stems. By changing community structure, invasive pathogenic microbes generate positive feedback that enhances both their own competitiveness and subsequent interactions with resident endophytic partners. These non‐detrimental bacteria benefit from promoting pathogens, which might lead to the assemblage of additional bacterial genera into plant roots and stems from RZS, eventually causing the outbreak of A. roxburghii soft rot disease. These results will provide more accurate guidelines for soft rot disease control.",
"introduction": "INTRODUCTION A balanced microbiome is important for humans, plants, and environmental health, while diseases are often associated with microbial community dysbiosis (Hooks & O'Malley, 2017 ). Plants grown in natural soils are colonized by a phylogenetically structured microbial consortium. And plant pathogens are diverse and ubiquitous in the environment. They are major causes of loss in vegetable, crop, and medicinal plant production (Lazcano et al., 2021 ). Destabilization caused by pathogen infection affects microbiome richness, evenness, and network complexity (Bello et al., 2018 ; Wang et al., 2021 ). Understanding the pathogen infection is a key challenge for medicinal plant cultivation which can exert significant impacts on the output and quality of herbal medicine (Huang et al., 2018 ; Tian et al., 2020 ). Biotic interactions, such as the network between pathogens and non‐detrimental resident microbes, have severe impacts on the infection process via resource utilization and competition (Wei et al., 2015 ). Therefore, deciphering the microbiota variation of the pathogen‐infected plants provides a crucial basis for estimating the severity and process of disease. It has been well documented that root zone soils (bulk soils, RZS) are the main reservoir for microorganisms colonizing the rhizosphere. The first step of infection is the colonization of the plant rhizosphere, where the plant drives the migration of microorganisms by depositing the root‐specific exudates that regulate bacterial gene expression and physiology at the soil‐root interface, which means that the pathogens must outcompete other microbial taxa for successful colonization (De Coninck et al., 2015 ; Rico‐Jiménez et al., 2022 ). Therefore, rhizosphere microbiota can function as the first line of defence against pathogen invasion (Bakker et al., 2018 ). Studies have also uncovered that endophytic microbes that colonize interior plant compartments without inducing disease may also contribute to host resistance against pathogens (Hu et al., 2020 ). Endophytes suppress pathogen infection via the induction of host resistance genes, competition, and the production of bioactive compounds (Wang et al., 2021 ). Surprisingly, the harmless endophytic microbes were found to assist pathogen invasion via a wide variety of mechanisms including cell–cell signalling (Gupta et al., 2021 ), metabolic interactions (Sun et al., 2021 ), evasion of the immune response (Ma et al., 2021 ), and a resident‐to‐pathogen switch (Venturi & Silva, 2012 ). These entirely opposite roles of endophytes call for serious consideration of pathogen–microbe interactions in the host for disease severity and control. Bacterial soft rot caused by Pantoea species is a devastating disease of plants with large‐scale crop losses worldwide, such as blackleg of potatoes, foot rot of rice, and bleeding canker of pears (Walterson & Stavrinides, 2015 ), and extensive efforts have been made to prevent and control this disease (Shin et al., 2019 ). \n Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae), a perennial herb grows under evergreen broad‐leaved forests or bamboo forests with relatively high humidity (Ye et al., 2017 ). It is widely used as a treatment booster and medicine because of its various beneficial properties, including the curative effects of heat dissipation and cooling of blood, elimination of dampness, detoxification, and immunity enhancement (Zeng et al., 2020 ). Its major chemical constituents are polysaccharides, flavonoids, glycosides, and Kinsenoside (Qi et al., 2018 ), which can be used for clinical treatment of hyperuricemia, type 2 diabetes, and chronic hepatitis B (Guo et al., 2019 ; Smoak et al., 2021 ). With a low reproduction rate and frequent forest management, the distribution ranges of A . roxburghii are sharply declining and artificial cultivation has been applied to improve its production (Li et al., 2019 ). However, bacterial soft rot occurs frequently during A . roxburghii cultivation (Shao et al., 2014 ). The outbreak of soft rot disease has led to a maximum of 70%–80% reduction in A . roxburghii yield, which severely hindered the development of the A . roxburghii medicinal industry and failed to keep up with the customers' increasing demand (Shao et al., 2014 ). However, the pathogen microbiota and their dynamics in the host plants remain largely uninvestigated. Microbial diseases occur as a result of multifarious host–pathogen interactions which could be explored by the newly developed co‐occurrence network approaches (Deng et al., 2012 ; Põlme et al., 2018 ). Within network structure, the configuration and distribution of links among plant soils and endophytic microbiota can provide strong predictions on the succession of pathogen infection (Deng et al., 2021 ; He et al., 2021 ; Wei et al., 2015 ). Network analysis could also identify keystone microbial members or other microorganisms that may function in the defence against pathogen invasion (Hu et al., 2020 ; Zamkovaya et al., 2021 ). Therefore, interactions both within the soil and endophytic communities and between the resident communities and invading pathogens are likely to be important for plant health and fitness (Michalska‐Smith et al., 2021 ). Here, we found that many A . roxburghii plants in the greenhouse were infected with stem rot when they were transplanted for 1 month in the soil. And other plants around it get sick when one plant gets sick. To investigate the mechanism of this disease occurrence and spreads, we studied the bacterial diversity, community composition, and dynamics in RZS, rhizosphere soils (RS), roots, and stems from healthy and soft rot infected A . roxburghii plants by high throughput sequencing. We then tracked the source of microbial migration from soils to endophytic communities during pathogen invasion. Furthermore, the interactions between pathogens and other host‐associated microbiota were investigated through network analysis.",
"discussion": "DISCUSSION \n Pantoea ananatis , which could symbiosis with plants, is better known as a phytopathogen affecting the yield of many economically important plants that causes blight and dieback of Eucalyptus (Arriel et al., 2014 ), maize leaf spot disease (Krawczyk et al., 2021 ) and brown stalk rot (Weller‐Stuart et al., 2014 ), leaf blight and bulb rot of onion (Weller‐Stuart et al., 2014 ), palea browning and stem necrosis of rice (Azizi et al., 2020 ), and fruit rot of netted melon (Özdemir, 2021 ). However, there are also many plant‐associated microbes that can perform a wide range of life‐beneficial functions, including plant nutrient acquisition, immune development, and plant tolerance of multiple stresses (Hu et al., 2022 ). The plant‐microbe balance keeps plant's normal growth. And higher microbial diversity increases community invasion resistance due to interactive effects on community stability (Gao et al., 2021 ; Ravi et al., 2022 ). Our results showed that the microbial diversity of soils, roots, and stems were higher in healthy samples than in diseased samples according to both Shannon diversity and PD indices (Figure 1 ). It could be explained by the fact that the dominance of pathogens depressed the resident bacteria growth during the onset of soft rot disease. Through the species classification, we found that OTUs assigned to potential pathogenic Pantoea were rarely observed in all healthy samples (HRZS, HRS, HES, and HER), but showed fairly high abundance in the diseased samples (DRZS, DRS, DES, and DER), consistent with field observations of plant soft rot. Correspondingly, the relative abundances of several bacteria were clearly altered after bacterial soft rot infection. There was a decline in the relative abundances of Ktedonobacter , Devosia , Enterobacter , Pseudolabrys , Sphingomonas , Mycobacterium , Streptomyces , Dokdonella , Mesorhizobium , Gemmatimonas , and Rhodanobacter in DRZS and DRS. These compositional changes could be a consequence of pathogen invasion. Streptomyces was report could induction of microbial autophagy by secrets rapamycin. The effect of rapamycin on TOR can promote the degradation of the histone acetyltransferase Gcn5, thereby reducing the acetylation level of Atg8 and promoting autophagy (Wang et al., 2021 ). In the endophytic compartments, the relative abundances of Enterobacter , Microbacterium , Pseudomonas , and Rhizobium showed significant increases in diseased samples compared to the healthy ones. It is worth noting that the abundance of Enterobacter exhibits a contrasting trend in soils and endophytic samples. Actually, some microbes have dual attributes. Genera like Pseudomonas , Streptomyces , Bacillus , Paenibacillus , Enterobacter , Pantoea , Burkholderia , and Paraburkholderia have been reported for their roles in pathogen suppression (Ma et al., 2021 ; Sun et al., 2021 ). Enterobacter is the most promising candidates against Gaeumannomyces graminis (Compant et al., 2019 ). However, soft‐rot Enterobacter were broad host‐range pathogens that cause wilt, rot, and blackleg diseases on a wide range of plants (Charkowski et al., 2011 ). Some species of Rhizobium are capable to fix nitrogen when in symbiosis with leguminous plants, but some were pathogenic bacteria to plants (Ji et al., 2010 ). The relatively high abundances of Rhizobium , Ktedonobacter , and Enterobacter , suggest they may be involved in the process of pathogen invasion and have mutualistic relationships with pathogenic members of Pantoea (Elsas et al., 2012 ), or they are opportunists, which take advantage of potential ecological niches opened by pathogen invasion (Lundberg et al., 2012 ). As the differences in microbial diversity and community composition between healthy and diseased plants have been observed, a further understanding of where the pathogens come from and how they interact with each other will provide a “road map” to disentangle the pathogen invasion process which contributes to disease control. Many studies have investigated that microorganisms can enter roots through the root tip and root hair or enter shoots via stomata and enter the seed during seed germination (Hugouvieux‐Cotte‐Pattat et al., 2014 ; Compant et al., 2021 ; Synek et al., 2021 ). Previous studies showed that soft rot pathogens invaded A. roxburghii via the roots and wounds, and then aggressively spread to the aerial compartments throughout the vascular system (Peeters et al., 2013 ; Walterson & Stavrinides, 2015 ). In line with this view, our SourceTracker analysis revealed that the bacteria communities in the stems were mainly derived from roots (55%), and the root endophytes largely derived from RS (48.5%), which come from RZS (62%). This is supported by the evidence that the RZS was the main source of microbial species richness in the plant rhizosphere (Jansson & Hofmockel, 2020 ), and plant endophytes were largely originated from soils and then transferred upwardly to the above‐ground tissues via the vascular system (Figure 2A ). Therefore, the soft rot pathogen invasion investigated in this study may begin in the RZS, break into the plant roots, colonize the stems, and finally induce soft rot disease. Recent studies have shown that harmless resident bacteria can be important to the incoming pathogens and in the outcome of the disease (Venturi & Silva, 2012 ). From the perspective of resource utilization and competition, plants and pathogens can have direct co‐evolutionary relationships (Friesen, 2020 ; Singh et al., 2018 ). To further investigate how the pathogens interact with each other and their impacts on neutral or beneficial microbes after infection. We performed network analyses on root and stem bacterial community interactomes of diseased and healthy plants, and revealed their topological features (Figure 2C,D ). Five modules were uncovered with each one matched to a specific sample type. For example, the soft rot disease‐infected samples were positively correlated with modules 1 and 3, while the healthy plants were correlated with modules 2 and 4, indicating that microbes in modules 1 and 3 were more likely to be pathogens or pathogen helpers, and microbes in modules 2 and 4 may contribute to the host plant's resistance to pathogen invasion. Crucially, highly connected and modular microbiota could prime the plant immune system for accelerated activation of defence against the pathogen (Burdett et al., 2019 ; Tzipilevich & Benfey, 2021 ). In this sense, by changing community structure, invasive pathogenic microbes generate positive feedback that enhances both their own competitiveness and subsequent interactions with their “resident” partners. In addition, we found that pathogenic Pantoea was positively associated with Enterobacter and Microbacterium (Figure 2E ). It has been reported that the highly connected and anomalously correlated nodes were either targets or helpers of diverse pathogens (Ahmed et al., 2018 ). Such as, microbes that positively interact with Ralstonia were the preferred helpers for pathogen attack in tobacco bacterial wilt disease (Wei et al., 2015 ). Similarly, the positively correlated Enterobacter and Microbacterium were also possible the preferred helpers during soft rot pathogen invasion. Certain species from Enterobacter are phytopathogens, causing bacterial palea browning of rice (Cao et al., 2020 ), sprouting decay, and seedling stunting of upland cotton (Nagrale et al., 2020 ), as well as affecting other plants including onion bulb (Weller‐Stuart et al., 2014 ), edible ginger (Liu et al., 2020 ), mulberry (Zhu et al., 2010 ), and papaya fruit (Keith et al., 2008 ). Another important finding was that an olive tree pathogen Pseudomonas savastanoi collaborated with a harmless resident endophyte and induced a significantly bigger tumour via interspecies quorum sensing cell–cell signalling (Hosni et al., 2011 ). The ecological functioning changes, like cellular processes and signalling, membrane transport, amino acid metabolism, carbohydrate metabolism were more abundant in diseased plants than in healthy ones. Taken together, we infer that the infection by pathogenic Pantoea members may be highly associated with positive interactions between them and non‐detrimental bacteria, including Enterobacter and Microbacterium , and that these non‐detrimental bacteria benefit from promoting pathogens, which might lead to the assemblage of additional bacterial genera into plant roots and stems from RZS, eventually causing the outbreak of A. roxburghii soft rot disease. These discoveries will open new avenues for developing more accurate strategies for A. roxburghii bacterial soft rot disease control. Further work is needed to confirm these findings."
} | 4,340 |
29878056 | PMC6247936 | pmc | 8,701 | {
"abstract": "Abstract Summary With the rapid accumulation of sequencing data from genomic and metagenomic studies, there is an acute need for better tools that facilitate their analyses against biological functions. To this end, we developed MetQy, an open–source R package designed for query–based analysis of functional units in [meta]genomes and/or sets of genes using the The Kyoto Encyclopedia of Genes and Genomes (KEGG). Furthermore, MetQy contains visualization and analysis tools and facilitates KEGG’s flat file manipulation. Thus, MetQy enables better understanding of metabolic capabilities of known genomes or user–specified [meta]genomes by using the available information and can help guide studies in microbial ecology, metabolic engineering and synthetic biology. Availability and implementation The MetQy R package is freely available and can be downloaded from our group’s website ( http://osslab.lifesci.warwick.ac.uk ) or GitHub ( https://github.com/OSS-Lab/MetQy ).",
"introduction": "1 Introduction The advent of molecular biology has made the characterization and analysis of genomic sequences a key part of all areas of life sciences research. In the case of single–cell organisms, identification of specific functions within the genome directly influences our ability to assess their fitness in a given environment and their potential roles in biotechnology. Particularly, we should theoretically be able to translate genomic data into physiological predictions. Genomic databases are a pre-requisite for making such predictions, but their full use also requires computational tools that allow easy access and systematic analyses of the data. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is one of the oldest and most comprehensive collections of databases. Its primary aim has been the digitising of current knowledge on genes and molecules and their interactions ( Kanehisa, 1997 ; Kanehisa and Goto, 2000 ) and it includes 16 databases and 3 sequence data collections ( Kanehisa et al. , 2017 ). While these data can be analysed via different tools on the KEGG website, the existing web interface allows only specific retrieval of information and analyses. Furthermore, although the whole of the data can be downloaded via (paid) FTP access, the systematic analysis of these data in a user–defined manner remains difficult and developing computational analysis tools for this purpose remains a niche expertise that is still not available in many research labs. There are several specific tools that make use of certain aspects of the KEGG data more available to a wider user-base. Examples include PICRUSt ( Langille et al. , 2013 ), BlastKOALA and GhostKOALA ( Kanehisa et al. , 2016 ), all of which focus on metagenomics data analysis. However, to our knowledge there are no tools that facilitate the analyses and information retrieval from KEGG with regards to studying the relationship between genomic data and physiological function. Therefore, we have developed MetQy, an open–source, easy–to–use and readily expandable R package for such analyses. MetQy uses the R –platform because it is commonly used among biologists, it is featured in undergraduate education, and it contains extensive statistical packages which are useful in subsequent data analyses. MetQy was developed to readily interface between the KEGG orthology, module and genome databases and perform automated cross–analyses on them. It consists of a set of functions that allow querying genes, enzymes and functional modules across genomes and vice versa, thereby enabling better understanding of genotype–phenotype mapping in single–celled organisms and providing guidance for cellular engineering in synthetic biology. MetQy can be used ‘as-is’, since the relevant components of the KEGG databases (downloaded on 20/02/2018) are included within the package. The included KEGG data constitutes only part of the entire encyclopedia and is ‘hidden’ in the package so that direct access to the data is not possible, complying with KEGG licence. Users with a paid KEGG subscription can use MetQy parsing functions to update the data that the package uses. The MetQy package and GitHub wiki contain extensive documentation and usage examples for each function."
} | 1,060 |
28420738 | PMC5395668 | pmc | 8,702 | {
"abstract": "ABSTRACT Deep-ocean regions beyond the reach of sunlight contain an estimated 615 Pg of dissolved organic matter (DOM), much of which persists for thousands of years. It is thought that bacteria oxidize DOM until it is too dilute or refractory to support microbial activity. We analyzed five single-amplified genomes (SAGs) from the abundant SAR202 clade of dark-ocean bacterioplankton and found they encode multiple families of paralogous enzymes involved in carbon catabolism, including several families of oxidative enzymes that we hypothesize participate in the degradation of cyclic alkanes. The five partial genomes encoded 152 flavin mononucleotide/F420-dependent monooxygenases (FMNOs), many of which are predicted to be type II Baeyer-Villiger monooxygenases (BVMOs) that catalyze oxygen insertion into semilabile alicyclic alkanes. The large number of oxidative enzymes, as well as other families of enzymes that appear to play complementary roles in catabolic pathways, suggests that SAR202 might catalyze final steps in the biological oxidation of relatively recalcitrant organic compounds to refractory compounds that persist.",
"conclusion": "Conclusions. This study of SAR202 genomes provided unexpected insights into the metabolic strategies of these deep-ocean bacteria. In these genomes, we discovered genes for an unusually rich assortment of enzymes implicated in the oxidation of recalcitrant organic compounds. These genes include multiple FMNO paralogs, P450, and enzymes predicted to function in sterol and aromatic compound catabolism. Due to the deep branching and high diversity of the SAR202 clade, as well as recent preliminary descriptions of SAGs from other subclades of SAR202 ( 89 ), it is probable that a number of these features are specific to the group III SAR202, and it is expected that there will be significant differences between the group III SAR202 and the other subclades. We also found in these genomes a key evolutionary signature of an ancient proliferation of FMNOs. Tree topologies place this event near the root of the Chloroflexi , possibly in a common ancestor of SAR202 and Ktedonbacter , who share a basal position in the phylum in our phylogenomic analysis. These results suggest an ancient origin of this metabolism. The findings we present and the current successful occupation of the dark-ocean niche by SAR202 lead us to hypothesize that SAR202 diverged soon after the atmosphere became oxidized and expanded into the niche of oxidizing semilabile and recalcitrant DOM by cleavage with powerful BVMOs and other oxidative enzymes. Kim et al. ( 90 ) demonstrated that a diversification of oxygen-consuming enzyme families occurred between 1.4 and 2.9 billion years ago, roughly coincident with the rise in atmospheric oxygen and the period through which oxygenation of the prehistoric oceans progressed ( 91 ). Their analysis also indicated that enzymatic reactions involving sulfonates only appeared following the initial increase in oxygen ( 90 ), which may help to explain the deep bifurcation between the alkanal monooxygenases associated with SAR202 and the alkanesulfonate monooxygenases in the global phylogeny of these enzymes ( Fig. 2 ). About 2 billion years ago, there was a massive sequestration of carbon into ocean sediment, followed by a correction, both of which are thought to be biologically mediated events ( 92 ). A scenario consistent with the observations we report is the expansion of FMNO-dependent catabolism in an ancestor of the SAR202 clade as a part of a larger bacterial proliferation into the ecological niche of consuming marine DOM, in the wake of the slow rise in global oxygen. The biochemistry of deep-ocean DOM oxidation is largely unexplored, but pioneering laboratory experiments with natural marine communities support the theory that the production of refractory DOM in the deep ocean is biologically mediated, with fresh substrates being transformed into heterogeneous mixtures of recalcitrant DOM in short-term incubations of less than 30 days ( 28 , 93 ). Experimental approaches such as this provide an avenue for studying biological processes mediated by communities composed of uncultivated cell types. The genome-based hypotheses we report are likely to prove useful for the design of future studies that aim to validate biochemical pathways of deep-ocean DOM oxidation. We propose the SAR202 group III clade be given the following class and order assignments: Monstramaria , classis nov., Monstramariales , ord. nov., Monstramariaceae , fam. nov. The root of the class, order, and family name stems from the Latin for “sea monster,” to reflect the nearly exclusive presence of members of the SAR202 clade to a number of marine and freshwater environments and represents the ancient divergence of the SAR202 from other Chloroflexi , its cryptic genomic and metabolic features, the low amino acid identity of its protein coding sequences to known orthologs, and, most importantly, the great depths in which it resides.",
"introduction": "INTRODUCTION The mass of the accumulated refractory dissolved organic matter (DOM) pool in the ocean is nearly equivalent to the carbon in atmospheric CO 2 , making it an important component of the global carbon budget ( 1 ). Deep-ocean DOM is composed of a heterogeneous mixture of carbon compounds that are thought mainly to originate from photosynthesis and carbon cycling activity in the upper ocean (the euphotic zone and the upper mesopelagic) ( 1 – 4 ). Persistent deep-ocean DOM, which can have a half-life of thousands of years, is thought to be either intrinsically refractory to biological oxidation ( 5 ) or to yield too little energy to benefit cells, presumably because the cellular cost of synthesizing the enzymes needed to catabolize it exceeds the value of the resource ( 6 – 8 ). Some data suggest that a fraction of the DOM, resistant to biological oxidation at low concentrations, can become labile at higher concentrations ( 8 ). In any case, the biological reactivity of DOM is a continuum, with labile DOM (operationally defined as having turnover times from hours to days) at one end of the spectrum, to recalcitrant compounds (defined as having turnover times from decades to centuries), and finally the refractory end members that persist in the environment for millennia ( 3 ). Both biotic and abiotic factors complicate DOM diagenesis: molecules can be altered by heterotrophic microbes that use the most reactive moieties to provide carbon and energy, and molecular heterogeneity can be increased by abiotic transformations, such as racemization. As labile DOM is converted to refractory DOM by heterotrophic marine bacteria, they produce inorganic carbon and new biomass, some fraction of which is also recalcitrant or refractory. The microbial carbon pump (MCP) describes the combined effect of these processes, which is a net sequestration of organic carbon resulting from the conversion of the labile organic carbon pool to a biologically recalcitrant mixture ( 2 , 4 , 8 , 9 ). However, the MCP is largely conceptual and is built from concepts that are illustrated by a few examples, with most microbiological and biochemical details unknown. SAR202 is a clade of uncultured bacteria that were first discovered in 1993 in the mesopelagic ocean (200 to 1,000 m) ( 10 ). Later studies confirmed these bacteria are rare in sunlit surface waters but comprise about 10% of all plankton cells in the dark ocean, where they increase from about 5% of mesopelagic communities to up to 30% in the bathypelagic (1,000 to 5,000 m) ( 11 – 14 ). They have since been found to be ubiquitous in the dark ocean, as well as common in subseafloor environments and deep lakes ( 15 – 17 ). The metabolism and geochemical role of the SAR202 clade are unknown, but their high abundance and evolutionary position in the phylum Chloroflexi have long fueled speculation they might play a role in the degradation of recalcitrant organic matter ( 10 , 14 ). The Chloroflexi have an ancient origin among the early diverging lineages of the domain Bacteria ( 18 – 21 ). Current estimates place the branching of the Chloroflexi at ~2.8 billion years ago (Ga), during the Early Proterozoic (2.5 to 0.5 Ga), a period that coincides with the rapid appearance of the first oxygen-consuming families of enzymes ( 22 ). SAR202 encompasses a diverse monophyletic group of lineages that are nearly all planktonic or associated with sediment in deep-ocean and deep-lake environments. Although there is great uncertainty about early events in bacterial evolution, a simple hypothesis that is consistent with the modern ecology of the SAR202 clade and insights from phylogenetic inference is that SAR202 diversified into dark-ocean niches sometime in the Precambrian eon (4.5 to 0.5 Ga). The phylum Chloroflexi encompasses a tremendously wide range of metabolic strategies, from anoxygenic photosynthesis to anaerobic reductive dehalogenation in Dehalococcoides , which has an extremely small genome size of 1.4 Mbp ( 23 ), to aerobic heterotrophy in the filamentous, sporeformer Ktedonobacter racemifer ( 24 , 25 ), which has one of the largest sequenced bacterial genomes (13 Mbp). Metabolic diversity within Chloroflexi has precluded predictions about the geochemical activity of the SAR202 clade from phylogeny alone, and thus far these cells have eluded cultivation. Although deep-ocean DOM is thought mainly to consist of refractory compounds ( 3 ) that have an estimated turnover time of ~30,000 years, it has been proposed that about 30% of deep-ocean DOM is semilabile, with a turnover time of <50 years ( 26 ). The slow oxidation of this DOM fraction is likely to be a source of energy for specialized deep-ocean microbial communities, along with chemolithotrophic metabolism that is based primarily on inorganic compounds that also originate from DOM. Although deep-ocean DOM is highly heterogeneous, a large fraction of it is thought to consist of cyclic alkanes, the best-documented example of which is known as carboxyl-rich alicyclic matter (CRAM) ( 27 ), a mixture of at least several hundred distinct aliphatic ring structures that are rich in oxygenated side groups ( 27 , 28 ). Nuclear magnetic resonance (NMR) analysis of CRAM indicated that sterol and hopanoid-like ring structures would satisfy the median chemical constraints provided by the NMR data and are likely precursors to some forms of deep-ocean DOM ( 27 ). These structures are thought to be extremely stable; hopanoids, for example, are some of the most abundant molecular “fossils” found on earth ( 29 , 30 ). The natural recalcitrance of cyclic alkanes is likely an additional factor that contributes to the slow oxidation of deep-sea DOM ( 27 , 29 ). In this study, we reconstruct SAR202 metabolism from genomes that were acquired from deep-ocean samples by single-cell genome sequencing. We sought to understand biogeochemical functions that could explain the extraordinary success of these cells in the dark ocean. These genome data are made more interesting by the deep-branching position of SAR202 in the tree of life. The early divergence of these cells and their modern distributions together suggest these organisms may have originated in ancient oceans. The unusual complement of genes in these genomes was resolved by reconstructing previously unknown metabolic pathways for DOM catabolism that we hypothesize are active in recalcitrant DOM oxidation. Given the ubiquity and abundance of the SAR202 clade throughout the deep oceans of the world, insight into the metabolism of this group of organisms has far-reaching implications, extending well into the realm of global biogeochemistry.",
"discussion": "RESULTS AND DISCUSSION SAG isolation, sequencing, and assembly. Four SAR202 single-amplified genomes (SAGs) from the group III SAR202 subclade ( 12 ) previously shown to be abundant in the mesopelagic zone were initially identified to determine the phylogenetic affiliation of the SAG sequences based on 16S ribosomal RNA sequences (see Fig. S1 in the supplemental material). Two of these SAGs, Chloroflexi bacterium SCGC AAA240-N13 and Chloroflexi bacterium SCGC AAA240-O15, were isolated from 770 m at the Hawaii Ocean Time-Series (HOTS) sampling site (22°45′N, 158°00′W). The remaining two, Chloroflexi bacterium SCGC AAA001-F05 and Chloroflexi bacterium SCGC AAA007-M09, were isolated from a depth of 800 m in the South Atlantic Gyre (12°29′S, 4°59′W). A fifth SAG ( Chloroflexi bacterium SCGC AB-629-P13), belonging to a novel group of SAR202, isolated from the North Atlantic at a depth of 511 m, was chosen for its distant position within the SAR202 clade and will be identified here as the group V SAR202 ( Fig. 1 ; Fig. S1 ), related to the previously unclassified clone SAR242 described by Morris et al. ( 12 ). Assemblies of The SAR202 group III SAGs had total assembly sizes ranging from 1,096,525 bp to 1,423,799 bp. The single group V SAR202 ( Chloroflexi bacterium SCGC AB-629-P13) had a total assembly size of 807,656 bp. All assemblies from the group III SAR202 SAGs had a consistent GC content of ~55%; the group V SAR202 had a GC content of 41%. Full assembly statistics, potential contamination, and completeness estimates from CheckM and crossover point (Cp) values for all SAGs are included in Table S1 in the supplemental material. 10.1128/mBio.00413-17.2 FIG S1 16S ribosomal RNA-based phylogenetic tree showing the position of the 5 SAR202 SAGs within the phylum Chloroflexi . The tree shows 5 distinct groups within the SAR202 clade, the first four of which correspond to the taxonomy put forth by Morris et al. ( 12 ). The majority of marine SAR202 members are represented by groups II and III, with the namesake of the clade being placed in group III. Group V represents a previously unreported group of SAR202 whose environmental relevance is yet to be determined. The SAR202 single-amplified genomes (SAGs) are marked in red, with 4 of the genomes belonging to the group III SAR202 and a fifth genome belonging to the group V scale. The tree is rooted on Streptomyces bikiniensis . The bar represents 0.2 nucleotide substitution/per site. Download FIG S1, PDF file, 0.1 MB . Copyright © 2017 Landry et al. 2017 Landry et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . 10.1128/mBio.00413-17.8 TABLE S1 Assembly statistics for 5 SAR202 single-amplified genomes (SAGs). The crossover point (Cp) represents the number of hours into the MDA reaction where the product reached half of its maximum double-stranded DNA (dsDNA) fluorescence. Only contigs larger than 2,000 bp were used in our analyses. Download TABLE S1, PDF file, 0.1 MB . Copyright © 2017 Landry et al. 2017 Landry et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . FIG 1 Phylogenomic tree of the Chloroflexi . This is a maximum likelihood tree based on a concatenated sequence alignment of 70 amino acid sequences and allowing for 20% missing data. Branches are color coded by class-level taxon assignments. In this tree, the SAR202 clade and Ktedonobacteria are the two deepest-branching classes within the phylum. The tree is rooted using Pirellula staleyi as an outgroup. Phylogenomic relationships. In maximum likelihood phylogenomic trees based on 17,003 informative amino acid positions, SAR202 and the Ktedonobacteria emerged as the lineages most closely related to the common ancestor of the phylum Chloroflexi ( Fig. 1 ). A basal phylogenomic position for Ktedonobacteria within the Chloroflexi has been observed previously in phylogenomic studies ( 20 , 21 ). Ktedonobacter racemifer was originally isolated from a compost heap using a buffered medium of humic acids and mineral salts ( 24 , 31 ). Subsequently, this and other Ktedonobacteria were shown to be aerobic carbon monoxide oxidizers ( 32 ) or chemoheterotrophs capable of utilizing a wide variety of organic carbon compounds ( 24 , 33 ). The SAR202 group V genome branched deeply within the SAR202 clade, as expected. The closest derived relatives of SAR202 in the tree were Dehalococcoidia . Cultured Dehalococcoidia are anaerobes that link the oxidation of hydrogen to the reduction of organohalogens ( 34 – 38 ) and also use aromatics and organosulfur and inorganic sulfur compounds as the substrates for energy and growth ( 39 , 40 ). Estimated genome sizes. Estimated genome sizes are shown in Table 1 . Estimation of genome sizes is challenging due to the large evolutionary distances between SAR202 genomes and their most closely related reference genomes. The estimated genome sizes varied widely among the SAGs, ranging from 1.4 to 13.2 Mbp, raising the possibility that these organisms have highly plastic genomes, a trait that has been alluded to in other members of the phylum ( 25 , 41 , 42 ). Genome sizes were estimated from a set of 71 single-copy gene clusters conserved in Chloroflexi . A power law regression that assumed the 25 fully sequenced Chloroflexi genomes are representative of the phylum indicated that the set of 71 conserved genes is on an asymptote approaching the true core genome set for Chloroflexi (see Fig. S2 in the supplemental material). Among the four SAR202 group III SAGs analyzed, estimated genome sizes ranged from ~3 to 13 Mbp. The completeness estimates from this analysis are slightly higher than size estimates generated using CheckM ( Table S1 ). These genome size estimates should be viewed with a degree of caution, as some may overestimate the actual genome size. Outliers in genome size estimation appear to be more prevalent, with both low and high numbers of recovered conserved genes (see Fig. S3 in the supplemental material). Since the genome size estimates for the group III SAR202 genomes appear to converge on 3 to 4 Mbp with increasing levels of completeness, we predict these genomes are most likely in this size range. 10.1128/mBio.00413-17.3 FIG S2 Mean, mode, standard deviation, and power law regression of the distribution of conserved gene set size for all possible combinations of a given number of the Chloroflexi genomes included in our analysis. The mode closely follows the values predicted by the power law regression, which approaches an asymptote at 71 conserved genes in the tail region. Download FIG S2, PDF file, 0.1 MB . Copyright © 2017 Landry et al. 2017 Landry et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . 10.1128/mBio.00413-17.4 FIG S3 Box plot distributions of genome recovery as a function of recovered Chloroflexi -conserved single-copy marker genes, based on a rolling starting point within the concatenated genome sequence for all Chloroflexi included in our analysis. The boxes represent the first and third quartiles of genome fractions recovered for a given number of recovered conserved single-copy genes. Whiskers represent the interquartile range × 1.5 (IQR 1.5) of genome fractions recovered for a given number of recovered conserved single-copy genes. Red bars represent the median, and “+” symbols represent outlier data points outside the IQR 1.5. Notice the increase in observed outliers, as well as the increase in skew for distributions in the tail ends of the range of recovered conserved single-copy genes. Download FIG S3, PDF file, 0.1 MB . Copyright © 2017 Landry et al. 2017 Landry et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . TABLE 1 Genome completion, estimated genome size, and metadata for 5 SAR202 SAGs a Site Depth (m) Group SAG name No. of identified conserved genes Estimated fraction complete Estimated genome size (bp) HOTS 770 3 AAA240-O15 33 0.47 3,032,068 ± 289,181 770 3 AAA240-N13 20 0.33 4,259,313 ± 234,134 South Atlantic 800 3 AAA001-F05 9 0.19 6,949,189 ± 161,421 800 3 AAA007-M09 3 0.08 13,153,458 ± 88,969 North Atlantic 511 5 AB-629-P13 42 0.56 1,436,137 ± 158,466 a Completion and size estimates are based on a set of 71 core genes found to be conserved in all Chloroflexi included in our analysis. Environmental distribution. Reciprocal best-BLAST recruitment of SAR202 SAGs against the Microbial Oceanography of ChemolithoAutotrophic planktonic Communities (MOCA) and HOTS depth profiles are shown in Fig. S4 and S5 and Table S2 in the supplemental material. Average nucleotide identities (ANIs) to bathypelagic metagenomic data were very high (≥90%), and the genomes were well represented in samples from 4,000 to 5,000 m of depth. Although this survey was limited to the few deep-ocean metagenomes that were available, the results indicate that the group III SAGs, which were isolated from depths of 770 to 800 m, likely represent abundant bathypelagic cells ( 14 ). SAR202 relative recruitment was highest in the deepest samples (3.8% [ Table S2 ]), but the real abundance of SAR202 DNA is expected to be significantly higher since, in proportion to incompleteness, SAG recruitment underestimates genome abundance. The group V SAR202 SAG, which was isolated from a depth of 511 m in the North Atlantic, contributed only 196 reads out of the 20,152 recruited reads from MOCA and 115 out of 12,293 reads from the HOTS depth profile. Therefore, the environmental relevance of this subclade in the open ocean remains unknown. 10.1128/mBio.00413-17.5 FIG S4 Reciprocal best-BLAST recruitment of SAR202 contigs to the MOCA North Atlantic depth profile. Depths of recruited fragments are color coded according to the key in the upper right. Download FIG S4, PDF file, 0.3 MB . Copyright © 2017 Landry et al. 2017 Landry et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . 10.1128/mBio.00413-17.6 FIG S5 Reciprocal best-BLAST recruitment of SAR202 contigs to the HOTS/ALOHA depth profile. Depths of recruited fragments are color coded according to the key in the upper right. Download FIG S5, PDF file, 0.2 MB . Copyright © 2017 Landry et al. 2017 Landry et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . 10.1128/mBio.00413-17.9 TABLE S2 Reciprocal best-BLAST recruitment of SAR202 single-amplified genomes against reads from two metagenomic depth profiles. Note that the highest levels of recruitment are from the deepest samples, indicating that these cells likely interact with chemical species found in the most remote depths of the ocean. Download TABLE S2, PDF file, 0.1 MB . Copyright © 2017 Landry et al. 2017 Landry et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . Global phylogeny of FMN/F420-dependent monooxygenases. The most salient feature of the SAR202 genomes was a high number of paralogous protein families, which was apparent in comparisons with other marine bacterial species ( Table 2 ; see Fig. S6 in the supplemental material). Proliferations of paralogs often accompany the evolutionary expansion of organisms into new niches, wherein the paralogs provide the new functions that are needed to adapt ( 43 , 44 ). We focused on the paralogs for clues that would help us understand what early events in evolution might have propelled SAR202 to its highly successful colonization of the dark-ocean habitat. 10.1128/mBio.00413-17.7 FIG S6 The fraction of protein-coding genes in the SAR202 genomes with discernible paralogs at a number of similarity thresholds compared with Ktedonobacter racemifer and a number of other representative marine bacteria. The SAR202 genomes and Ktedonobacter show consistently higher levels of paralogs than the other organisms. Download FIG S6, PDF file, 0.1 MB . Copyright © 2017 Landry et al. 2017 Landry et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . TABLE 2 The 10 most highly represented Sfam protein families from SAR202 SAG assemblies a a The 10 families shown here represent a number of paralogous protein sequences. This table shows remarkably high representation of some families, and there are a number of additional Sfam families present in the genomes with identical structural and functional annotations. Prominent among the paralogs were 152 sequences representing a large and highly diverse family of flavin mononucleotide(FM)/F420-dependent monooxygenase catalytic subunits (FMNOs) ( 45 – 47 ). hmmscan ( 48 ) with Sfams revealed that the five SAR202 genomes altogether harbored 48 genes related to Sfam 4832, which is closely related to the Sfam 8474 FMNO family of Baeyer-Villiger monooxygenases (BVMOs). An additional 104 sequences from the genomes were associated with 16 related Sfam models in the FMNO family. These 17 models were used to recruit 15,623 amino acid sequences from the Refseq65 bacterial protein set, as well as an additional 115 amino acid sequences from Swiss-Prot/UniProtKB. Dereplication of these sequences yielded consensus sequences for 990 gene lineages. Figure 2 is an approximate maximum likelihood tree made from the final FMNO alignment. SAR202 FMNOs spanned the full range of the FMNO tree, often branching with other Chloroflexi- derived sequences. FIG 2 Global phylogeny of bacterial FMNO proteins rooted on a consensus sequence from archaeal FMNO homologs. This tree shows that SAR202 FMNO paralogs branch deeply in trees, indicating ancient diversification of these genes in SAR202 and related Chloroflexi . Ancestry of SAR202, Ktedonobacter , and other Chloroflexi are indicated in descending priority by the color of tree segments according to the key in the upper right. Shimodaira-Hasegawa test values are represented by circles on the internal nodes of SAR202 and Chloroflexi , with radii proportional to confidence and coloring according to the key in the lower left. Terminal nodes are color coded by majority Sfam family; a color key and number of sequences recruited from databases (Swiss-Prot and RefSeq) for each family are shown in the upper left-hand corner. Radiations associated with enzyme functions are highlighted according to the key in the lower right. The majority of SAR202 sequences are in the radiation associated with Type II Baeyer-Villiger monooxygenase (BVMO) functionality. The arrow indicates the deep node at which BVMO enzymes diverged from the more common alkanesulfonate monooxygenases.\n FMNOs and other flavoenzymes have been classified into 8 classes (A to H) in the system devised by van Berkel ( 45 – 47 ): they catalyze a variety of oxidation reactions on an enormous range of substrates, including aromatic ring hydroxylation, desulfurization, alkane oxidation, Baeyer-Villiger oxidation, epoxidation, and halogenation. Predominant annotations for protein sequences recruited by the 17 FMNO Sfam hidden Markov models (HMMs) were “alkanesulfonate monooxygenase,” “hypothetical protein,” “monooxygenase,” “luciferase,” “ N 5, N 10-methylene tetrahydromethanopterin reductase and related flavin-dependent oxidoreductases,” or “alkanal monooxygenase.” All of these would be categorized as class C flavin-dependent monooxygenases in the nomenclature system for flavoenzymes devised by van Berkel ( 45 – 47 ). Bacterial luciferase is the canonical class C monooxygenase that oxidizes aldehydes to carboxylic acids. Alkanesulfonate monooxygenases (SsuD) are also well represented among class C FMNOs; they catalyze the cleavage of carbon-sulfur bonds in a wide range of sulfonated alkanes and have high affinities for sulfonated compounds with large conjugated R-groups ( 49 ). Additional class C monooxygenases include nitrilotriacetic acid monooxygenase, dibenzothiophene monooxygenase, and type II Baeyer-Villiger monooxygenases ( 46 ). The most common annotated function of FMNO sequences recruited from public databases is “alkanesulfonate monooxygenase,” but this functional annotation is largely associated with one protein family, Sfam 1831. While prevalent across the tree of life, this family is only represented by a single protein from SAR202. Sfam 1831 represented 296 nonredundant lineages and 8,695 of the recruited FMNO protein sequences, while the other 16 models were assigned to 694 of the lineages representing 7,195 amino acid sequences. Most of the Chloroflexi FMNO lineages are phylogenetically diverged from Sfam 1831 ( Fig. 2 ). While alkanesulfonate monooxygenases are structurally and phylogenetically related to the other FMNOs, they diverge at a deep-tree node, and most of the FMNO diversity represented by SAR202, Ktedonobacter and other Chloroflexi , is on the other side of this node, among the non-alkanesulfonate monooxygenase lineages. Genome annotation predicts that the majority of SAR202 FMNO enzymes are Baeyer-Villiger monooxygenases that break alicyclic rings by an oxidative mechanism. Interpreting these genes in the context of other genes found in SAR202 genomes leads us to propose that the function of the SAR202 BVMOs is to activate recalcitrant molecules for biodegradation. Closer examination of the Sfam 4832 family sequences (which contain the most sequences of the FMNO families represented in SAR202 [ Table 2 ]) using the PHYRE2 structural homology recognition server ( 50 ) showed high predicted structural similarity of these proteins to 3,6-diketocamphene 1,6-monooxygenase (template/fold identification c2wgkA). 3,6-Diketocamphene 1,6-monooxygenase is a type II Baeyer-Villiger monooxygenase that catalyzes the insertion of an oxygen atom into the alicyclic 3,6-diketocamphene molecule to convert a ketone group to an ester ( Fig. 3 ). In the camphor degradation pathway, this insertion is followed by the spontaneous decomposition of the unstable ring structure into a partially linearized carboxylic acid ( 51 ). Additional support for type II BVMO functionality is found in our phylogenetic data. The majority of the Sfam 4832 nonredundant lineages (for which there are no representative proteins in Swiss-Prot) share an ancestry with the Sfam 8474 lineages, branching together at a single deep node in the phylogenetic analysis ( Fig. 2 ). Sfam 4832 is uncommon across the bacterial tree, with only 64 other proteins recruiting to this family from the entire RefSeq database of bacterial genomes (mostly from a number of Actinobacteria ) and none from Swiss-Prot. The only sequences recruited to the closely related Sfam 8474 HMM from the manually curated Swiss-Prot database, however, were the type II BVMO enzymes 2,5-diketocamphene 1,2-monooxygenase and 3,6-diketocamphene 1,6-monooxygenase of Pseudomonas putida ( 51 ) and the limonene 1,2-monooxygenase of Rhodococcus erythropolis , which catalyzes the epoxidation of an alicyclic ring structure. While the most common type of FMNO found in the SAR202 genomes is these putative type II Baeyer-Villiger monooxygenases, divergent SAR202 sequences in the FMNO family may perform other canonical catalytic functions of class C FMNOs, such as the removal of sulfur and nitrogen groups from alkanesulfonates, or aminocarboxylic acids, respectively, yielding alkanes for assimilation or oxidation. The presence of a sulfite oxidase suggests that sulfite derived from organosulfur compounds might be used as an energy source; alternatively this enzyme might benefit cells by detoxifying organosulfur-derived sulfites. Alternatively, the presence of an alkanesulfonate monooxygenase may simply impart the ability to utilize organic sulfur to fulfill the cell’s sulfur requirements. FIG 3 Types of reactions catalyzed by group C flavin monooxygenase proteins and possible fates of reaction products (in boxes). Canonical reactions for enzymes in this protein family include (A) alkanal monooxygenases, which convert aldehydes to fatty acids; (B) alkanesulfonate monooxygenases, which catalyze the cleavage of carbon-sulfur bonds to produce an aldehyde and a free sulfite; (C) nitrilotriacetate monooxygenase, which catalyzes the removal of a glyoxalate group from an aminocarboxylic acid; (D) dibenzothiophenone monooxygenase, which catalyzes the cleavage of a carbon-sulfur bond to produce a substituted biphenyl; and finally (E) the type II Baeyer-Villager monooxygenases, which catalyze the insertion of a single oxygen adjacent to a ketone or aldehyde group. The resulting lactone is decyclized spontaneously or through an esterase-catalyzed reaction. red, reduced; ox, oxidized. Sequence diversity within the SAR202 FMNOs and the pattern of shared deep branches with Ktedonobacter FMNOs cannot be explained by horizontal gene transfer without accepting a remarkable amount of accidental coincidence. Rather, this pattern strongly suggests the early expansion of these paralogs in the common ancestor of Ktedonobacter and SAR202. In the absence of empirical experimental data, the substrates for these divergent type II BVMOs cannot be predicted with certainty; however, their annotations were consistent with predictions of alkane degradation functions elsewhere in the genomes, prompting us to conclude that the SAR202 FMNOs are most likely involved in the activation of long-chain or cyclic aliphatic molecules for degradation. The ancient origin of the SAR202 clade, its deep-branching diversity, and its current role in ocean ecology are compatible with the hypothesis that these FMNOs diversified in a progenitor of SAR202 that expanded into a niche that required the strong oxidative activity of FMNOs. Acyl-CoA:CoA transferases. In addition to the monooxygenase sequences, a number of other paralogous protein families were encoded in the SAR202 genomes. The most prominent of these are a family of acyl coenzyme A (acyl-CoA):CoA transferases (formerly bile acid-inducible protein f; Caib/Baif [Sfams 1517, 17645, 25993, 33148, 42237, and 67858]). The Baif form of this enzyme is thought to catalyze the release of deoxycholate, a sterol derivative, with the subsequent binding of cholate to CoA, and has been shown to be active over a wide range of substrates ( 52 ). The major advantage of these proteins may be that they allow the integration of new substrate into degradation pathways without the consumption of reductant or ATP. A subset of the putative acyl-CoA transferases encoded by SAR202 have top hits to the DddD enzyme of Marinomonas (UniProtKB, SP no. A6W2K8.1 ; NCBI GI no. 928589252 ) compared to the Swiss-Prot/UniProtKB database. The DddD of Marinomonas is a dimethylsulfoniopropionate (DMSP) transferase/lyase that has been shown to be homologous to Caib/Baif-type proteins, indicating the possibility of SAR202 using the common algal osmolyte DMSP, although DMSP as a substrate is unlikely to be present in the deep ocean ( 53 ). The lyase activity of the DddD protein also prompts us to believe that it is possible that some of these CoA transferase proteins may also encode novel lyase functions for additional substrates. Short-chain dehydrogenases. The five SAR202 genomes altogether contained 60 proteins belonging to Sfam 1639 ( Table 2 ), most of which are annotated as “oxidoreductase” or “short-chain dehydrogenase.” In comparisons to the Swiss-Prot/UniProtKB database, a number of these enzymes had high amino acid identities (AAIs [~50%]) to cyclopentanol dehydrogenase or 3-α (or 20-β)-hydroxysteroid dehydrogenase. There is a profound similarity between the reactions catalyzed by these enzymes. Cyclopentanol dehydrogenase and 3-α (or 20-β)-hydroxysteroid dehydrogenase, while active on seemingly different substrates, both convert an alicyclic-bound alcohol group to a ketone, producing reduced NADH as a product ( 54 – 56 ). The conversion of sterols to ketones is illustrated by the reaction catalyzed by 3-α (or 20-β)-hydroxysteroid dehydrogenase. The production of similar enzymes by SAR202 would help explain the documented vertical pattern of sterols and steroid ketones throughout the meso- and bathypelagic ocean ( 57 , 58 ). The conversion of alcohol groups to ketones is a priming mechanism for subsequent BVMO oxidation ( 59 ). Given the large number of putative Baeyer-Villiger monooxygenases in SAR202 genomes, we propose that SAR202 short-chain dehydrogenase paralogs prepare deep-ocean DOM species for oxygen insertion and ring opening through a lactone intermediate, partially linearizing the molecule and allowing the conversion of a recalcitrant alicyclic ring structure to a more labile carboxylic acid. The gene annotations described above lead us to hypothesize that SAR202 might participate in the oxidation of a variety of cyclic alkanes, including sterol and hopanoid-like structures. Sterols and hopanoids are chemically related triterpenoid molecules that play analogous roles in the cell membranes of eukaryotes and bacteria, respectively ( 27 ), and have been shown to be common in the water column ( 29 , 60 – 64 ). These molecules exhibit highest concentrations in the lower euphotic zone/upper mesopelagic and decline with depth. There is a corresponding appearance and subsequent slow disappearance of steroid ketone molecules with depth that is hypothesized to be the result of biotic conversion of sterols to steroid ketones ( 57 , 58 , 65 , 66 ). These patterns are consistent with the previously reported vertical range of SAR202 ( Fig. S4 and S5 ) throughout the mesopelagic ( 10 , 14 ). Transporter proteins. Across the five genomes, there were 129 proteins annotated as subunits of major facilitator superfamily 1 (MFS1) transporters. In comparisons to the Swiss-Prot/UniProtKB database, these proteins were most similar to proteins annotated as multidrug efflux transporters. MFS1 transport proteins are solute antiporters that have in the past been reported to be widely promiscuous in substrate, with individual enzymes capable of transporting a wide range of compounds in and out of the cell ( 67 – 69 ). The genomes also encoded 14 proteins (from Sfams 1548, 13968, 18128, 50865, and 54520) that encode tripartite ATP-independent periplasmic (TRAP) transporters. TRAP transporters are associated with a wide variety of compounds, including dicarboxylic acids and sugars ( 70 ). The five genomes additionally included a total of 182 proteins involved in ABC transport systems with affinities for a variety of substrates, including putative transporters for inorganic nitrogen and phosphate species as well as organic phosphonates. Other oxidative enzymes. Each of the genomes contained several versions of the promiscuous enzyme cytochrome P450, which catalyzes the formation of hydroxyl, carboxyl, or carbonyl groups from alkanes, including enzymes of the CYP125 type that has been previously implicated in sterol degradation ( 71 ). Also present were most enzymes for the oxoglutarate:ferredoxin oxidoreductase variant of the tricarboxylic acid (TCA) cycle, which oxidizes acetyl-CoA, yielding reduced ferredoxin. Bacterial cytochrome P450 enzymes are typically activated with reduced ferredoxin, which is a more electronegative carrier than the more common nucleotide cofactors NADH and NADPH. These synergistic genome features are further evidence of a metabolism equipped for the oxidation of a broad range of heterogeneous recalcitrant compounds. The assemblies also contained multiple families annotated as carbon monoxide dehydrogenase/xanthine dehydrogenase/aldehyde oxidases, including a total of 28 proteins associated with Sfam 1575. Some members of the Ktedonbacteria have been shown to be capable of oxidizing carbon monoxide ( 32 ), and low concentrations of carbon monoxide present (<1 nM) in the deepwater column, indicate that there may be a CO sink in the meso- and bathypelagic ( 72 ). However, given the other functions of enzymes in this protein family and the diverse alkane degradation genes contained in our assemblies, it seems more likely that many or most of these proteins have functions similar to those of xanthine dehydrogenase rather than carbon monoxide dehydrogenase. Xanthine dehydrogenase enzymes often additionally show a xanthine oxidase activity ( 73 ), and some versions of this enzyme are active across broad substrate ranges ( 73 , 74 ). We speculate these enzymes could be involved in the initial steps of degrading complex substrates to more tractable forms by the addition of hydroxyl groups to alkanes or the formation of ketones from alkene groups. Either of these reactions would initiate priming of an alkanal molecule for eventual oxygen insertion by a Baeyer-Villiger monooxygenase. Choline degradation and C 1 oxidation. The SAR202 genomes contained a large number of proteins related to choline dehydrogenases that likely have analogous functions, such as oxidizing alcohols to aldehydes (Sfams 108560, 1557, 163373, 2157, 27990, 38781, and round2_109). Sfam family round2_109 alone recruited 38 proteins from the five genomes. The SAR202 genomes also harbored a number of the other proteins that have predicted functions in the choline degradation pathway, including most of the subunits of multimeric sarcosine oxidase (EC 1.5.3.1) and serine hydroxymethyltransferase (EC 2.1.2.1). The genomes additionally included genes for C 1 (one-carbon) oxidation with components of the tetrahydrofolate-linked demethylation pathway and genes for formaldehyde oxidation (EC 1.2.1.46) and formate dehydrogenase (EC 1.2.1.2), which oxidizes formate to CO 2 . This suite of genes likely confers upon these cells the ability to demethylate compounds such as choline and to oxidize the methyl groups to CO 2 in an energy-yielding reaction. The presence of a multimeric sarcosine oxidase as well as a DMSP lyase in some of the SAR202 genomes also indicates that it can metabolize some low-molecular-weight, semilabile forms of carbon. If SAR202 does indeed utilize these smaller forms of organic carbon, it may contribute to a “priming effect” in some deepwater systems where refractory DOM is readily degraded when labile carbon is available to support baseline metabolism ( 5 , 75 , 76 ). 3-Hydroxypropionate cycle. The group III SAR202 genomes contained genes predicted to encode many enzymes of the 3-hydroxypropionate cycle (3HPC), including subunits of the key trifunctional malyl/methylmalyl/citramalyl lyase (see Table S3 in the supplemental material) ( 77 , 78 ). The 3-hydroxypropionate cycle was first described in the Chloroflexi phylum member Chloroflexus aurantiacus , where it was interpreted as a pathway of autotrophy in the absence of conventional carbon fixation pathways ( 77 , 78 ). While associated with carbon fixation, in the first experimental papers describing the cycle and its biochemical role, the authors stated that it likely is repurposed for this cause and that the original role of the 3-hydroxypropionate cycle was probably to facilitate the utilization and salvage of a wide range of carbon substrates ( 78 , 79 ). The SAR202 genomes harbored propionyl/acetyl-CoA carboxylases that are associated with the CO 2 fixation steps of the cycle, as well as subunits of a key enzyme, trifunctional malyl/methylmalyl/citramalyl lyase, that is associated with the full bi-cycle (identified by Phyre2 structural prediction server, 100% predicted structural similarity to the trifunctional lyase of Chloroflexus aurantiacus ). Due to the low estimated completeness of the genomes, we cannot rule out the possibility that the cycle plays a chemolithotrophic role in SAR202, but the lack of coding sequences definitively associated with the chemolithotrophic metabolism and the overwhelming presence of genes that appear to be involved in complex alkane degradation lead us to hypothesize that the primary role of the 3HP cycle in SAR202 is the assimilation of metabolic intermediates that result from the catabolism of complex deep-ocean DOM. 10.1128/mBio.00413-17.10 TABLE S3 Candidate protein sequences found within the SAR202 genomes that potentially code for enzymes or enzyme subunits required by the 3-hydroxypropionate cycle. Download TABLE S3, PDF file, 0.1 MB . Copyright © 2017 Landry et al. 2017 Landry et al. This content is distributed under the terms of the Creative Commons Attribution 4.0 International license . We propose that a full or partial version of this pathway may be used in SAR202 to facilitate absorption of varied metabolic intermediates produced by the degradation of recalcitrant DOM. Most 3HPC intermediates are short 3- to 4-carbon carboxylates or dicarboxylic acids, representative of the types of compounds that would remain following incomplete oxidation of modified, complex, or branched-chain fatty acid oxidation. Autotrophically grown Chloroflexus aurantiacus cells have also been shown to preferentially absorb organic carbon through some of these intermediate forms, despite an excess of CO 2 and H 2 available for carbon fixation ( 79 ). Segments of the 3HPC, involving one or more successive enzymes, are implemented in the metabolism of other marine microbes to assimilate compounds such as short carboxylic acids and aldehydes produced by catabolic pathways, such as in DMSP catabolism ( 79 , 80 ). Fatty acid degradation. Gene candidates for all of the central enzymes involved in fatty acid beta-oxidation were found among the five SAR202 genomes. All three genes of the propionyl-CoA pathway (overlapping with the 3-hydroxypropionate cycle) for odd-length and methylated fatty acid degradation were found in multiple members of the SAR202 genomes. This set includes propionyl-CoA carboxylase (EC 6.1.4.3), methylmalonyl-CoA epimerase (EC 5.1.99.1), and methylmalonyl-CoA mutase (EC 5.4.99.2). The presence of a full beta-oxidation pathway, and a propionyl-CoA degradation pathway, indicates that SAR202 is able to fully degrade both odd- and even-chain fatty acids. Putative genes for alpha-oxidation of branched alkanes, which are highly unusual among bacteria, were found among the genomes. Both phytanoyl-CoA dioxygenase and 2-hydroxyphytanoyl-CoA lyase were present. These genes indicate that SAR202 may be capable of degrading branched-chain alkanes that are resistant to beta-oxidation, including phytanyl compounds, such as the phytol side chains of chlorophyll molecules and the isoprenyl chains of archaeal lipids. Aromatic degradation. A pathway for catabolism of aromatic compounds could not be completely reconstructed, but the SAR202 SAGs harbored multiple genes encoding enzymes involved in aromatic carbon catabolism, including proteins with predicted functions in 4-hydroxyphenylacetate degradation, protocatechuate degradation, catechol degradation, and phenylpropionate degradation. Also present were ring-hydroxylating and ring-cleavage dioxygenases and isomerases that could be involved in degradation of aromatics. The diversity and prevalence of these genes lead us to propose that SAR202 cells are able to catabolize some aromatic compounds. Carboxylate degradation. Genes encoding proteins involved in carboxylic acid degradation pathways were abundant in the SAR202 genomes. Pathways for acetoacetate degradation and 2-oxobutanoate degradation were complete or nearly complete, and there were a large variety and number of acyl-CoA or formyl-CoA transferase homologs: in addition to the 62 aforementioned Caib/Baif-type proteins in Sfam 1517, there were 11 proteins from Sfam families 172716, 17645, 25993, 33148, 42237, and 67858. Coenzyme A can serve as a carrier for a wide variety of acyl compounds during biological oxidation, and we interpret the proliferation of CoA transferases in these cells as evidence they might be capable of metabolizing many different acyl compounds by forming CoA thioesters. Such acyl compounds are among the products predicted to form through the action of the strongly oxidative FMNO and P450 enzymes found elsewhere in the genome. The ability of acyl-CoA transferases to remove substrates that are easily oxidized without a carrier (such as formate) from CoA cofactor through substitution of a new ligand without the use of ATP is an energetically efficient mechanism used by cells to move substrates through oxidative pathways. This strategy seems well suited to the niche we propose for SAR202 of oxidizing some of biology’s least favorable organic substrates in the dark ocean. Sialic acid. Acetylated aminosugars, including sialic acid, have been shown to be a major component of marine DOM, accounting for up to 17 to 43% of high-molecular-weight DOM at depth, making them a large component of the heteropolysaccharide portion of deep-ocean DOM ( 5 , 27 , 81 , 82 ). Sialic acids often decorate glycoproteins and are abundant in S-layers, which are proteinaceous outer wall structures found in many bacteria and archaea. SAR202 genomes have multiple genes predicted to function in sialic acid biosynthesis. These include N -acetylneuraminate synthase, CMP- N -acetylneuraminate synthase, and candidates for polysaccharide biosynthesis protein CapD. While the existence of an S-layer has not been documented in cultured Chloroflexi , S-layers have been predicted for members of the phylum due to the presence of sialic acid biosynthetic enzymes observed previously in single-amplified genomes and assembled metagenomes ( 40 , 83 ). The presence of sialic acid biosynthetic pathways has ecological implications: S-layers provide protection against phage predation in bacteria ( 84 – 87 ), presumably by modifying or masking cell surface epitopes. These observations raise the possibility SAR202 S-layers might contribute to the high-molecular-weight fraction of deepwater DOM, through a mechanism consistent with ideas expressed by the microbial carbon pump hypothesis. Metabolic overview. A metabolic reconstruction based on annotated SAR202 gene functions is shown in Fig. 4 . The gene content and predicted metabolism of SAR202 genomes are distinct from any described previously and ostensibly well suited to the niche of oxidizing recalcitrant DOM in the deep ocean. The key to reconstructing this metabolism is the presence of multiple, anciently diverged families of paralogous enzymes, particularly the FMNOs, which are united by their strongly oxidative catalytic mechanism. In a general sense, the reaction mechanisms of these enzymes are especially suited to the oxidation of recalcitrant compounds and produce labile alkanyl intermediates that are favorable carbon or energy sources, but could be highly diverse, depending on the heterogeneity of the parent compounds. From a thermodynamic perspective, it seems likely that these compounds are recalcitrant because a powerful oxidation step is required to transform them into molecules that are tractable for further catabolism. The derivatives of the oxidation step are alkanal molecules that are potentially rich sources of energy. In the scenario we propose, paralogs diversified in response to evolutionary pressure to use a variety of substrates via a common pathway mechanism. Other enzymes likely contributing to the initial oxidation of marine DOM include cytochrome P450, short-chain alcohol dehydrogenases, enzymes associated with aromatic degradation, and purine dehydrogenases/oxidases. FIG 4 A proposed metabolic schema for cells of the SAR202 clade. Oxidative enzymes encoded in the SAR202 genomes provide semispecific initial oxidation of heterogenous recalcitrant DOM on the cell surface or upon initial transport of these compounds into the cell. Carboxylate degradation products are ligated into a pool of CoA-linked substrates via acyl-CoA transferases. CoA-linked substrates move to the beta- and possibly alpha-oxidation pathways. Partial degradation products of fatty acids and substituted alkanes released from the partial oxidation of recalcitrant DOM may provide intermediates for absorption via the 3-hydroxypropionate cycle (3HPC). Incompletely degraded substrates may be substituted for more active substrates using acyl-CoA transferase activity, with released products being exported from the cell via a number of MFS1 transport proteins with documented promiscuous transport activities, thus contributing to a more refractory environmental pool of marine dissolved organic matter. 3HPC pathway gaps for which no suitable gene candidates were found in the draft genomes are marked in red. While it is uncertain as to whether the enzymes of the 3HPC shown here participate in carbon fixation, even an incomplete 3HPC could provide a means of assimilating a number of small carbon intermediates into central metabolism. Several mechanisms for cleavage of sulfite from sulfur and organosulfur compounds are encoded by the genome and may provide a source of sulfur for growth or a substrate for sulfite oxidase. To illustrate, Fig. 5 uses predicted enzyme functions from the SAR202 genomes in a hypothetical pathway for degradation of the common phytoplankton lipid stigmastanol. Through a combination of the activities of cytochrome P450, alpha- and beta-oxidative enzymes, alcohol dehydrogenase, and a Baeyer-Villiger monooxygenase, reactive moieties, such as the side chain, can be removed and rings broken by the insertion of oxygen atoms, which results in decyclization through a lactone intermediate. Further degradation results in a final product resembling deep-sea DOM structures proposed by Hertkorn et al. ( 27 ). This is not to say that SAR202 is degrading sterols/stanols per se , but to demonstrate by example that annotated enzymes found within these genomes potentially encode pathways that are capable of partially oxidizing recalcitrant cyclic DOM compounds, resulting in the production of more refractory products. We propose that a variety of small CoA-linked derivatives that result from incomplete oxidation of branched alkanes are assimilated as intermediates via the 3HPC. In this scenario, CoA-linked compounds that are not substrates for the 3HPC assimilation or further oxidation could be eliminated from the CoA pool through the activity of the CoA transferase paralogs that are predicted to function without the consumption of ATP. In the proposed metabolic scheme, these enzymes link the ligation of newly imported or more labile substrates to the displacement of partially oxidized, unusable compounds, providing an entry point for new carbon compounds into degradation pathways, while simultaneously conserving energy. FIG 5 A possible alicyclic carbon degradation mechanism, with stigmastanol as an example, using reactions thought to be encoded in the SAR202 genome annotations. The side chain is activated through a CYP125 cytochrome P450. This is followed by beta- and alpha-oxidation of the side chain. The sterol is converted to a steroid ketone through the activity of a dehydrogenase, and a type II Baeyer-Villiger monooxygenase subsequently creates a lactone intermediate. The lactone ring can decompose spontaneously or through a lactonase- or esterase-catalyzed reaction. Alcohols can be oxidized to aldehydes through the action of alcohol dehydrogenases. Aldehydes can be further converted to carboxylic acids through the action of an aldehyde monooxygenase. Exposed carboxylic acid residues can be further degraded through beta-oxidation, leaving a carboxyl- and carbonyl-rich alicyclic fragment with limited opportunities for further degradation. The reconstructed pathways shown in Fig. 5 include steps that we predict could partially oxidize recalcitrant compounds, but do not explain their complete oxidation to CO 2 . In this reconstruction, we speculate that the multiple MFS1 transport proteins encoded in these genomes function to export substrate fragments that cannot be further oxidized in these cells. If this scenario is correct, then it seems plausible that group III SAR202 contributes to the accumulation of refractory DOM in the deep ocean through the exudation of partially degraded substrates that are resistant to further catabolism. Such a scenario would place SAR202 in a key position in the marine carbon cycle, catalyzing steps in the conversion of recalcitrant DOM to refractory compounds by partially oxidizing recalcitrant compounds and further lowering their chemical reactivity. Similar scenarios have been proposed for freshwater DOM ( 88 ). Conclusions. This study of SAR202 genomes provided unexpected insights into the metabolic strategies of these deep-ocean bacteria. In these genomes, we discovered genes for an unusually rich assortment of enzymes implicated in the oxidation of recalcitrant organic compounds. These genes include multiple FMNO paralogs, P450, and enzymes predicted to function in sterol and aromatic compound catabolism. Due to the deep branching and high diversity of the SAR202 clade, as well as recent preliminary descriptions of SAGs from other subclades of SAR202 ( 89 ), it is probable that a number of these features are specific to the group III SAR202, and it is expected that there will be significant differences between the group III SAR202 and the other subclades. We also found in these genomes a key evolutionary signature of an ancient proliferation of FMNOs. Tree topologies place this event near the root of the Chloroflexi , possibly in a common ancestor of SAR202 and Ktedonbacter , who share a basal position in the phylum in our phylogenomic analysis. These results suggest an ancient origin of this metabolism. The findings we present and the current successful occupation of the dark-ocean niche by SAR202 lead us to hypothesize that SAR202 diverged soon after the atmosphere became oxidized and expanded into the niche of oxidizing semilabile and recalcitrant DOM by cleavage with powerful BVMOs and other oxidative enzymes. Kim et al. ( 90 ) demonstrated that a diversification of oxygen-consuming enzyme families occurred between 1.4 and 2.9 billion years ago, roughly coincident with the rise in atmospheric oxygen and the period through which oxygenation of the prehistoric oceans progressed ( 91 ). Their analysis also indicated that enzymatic reactions involving sulfonates only appeared following the initial increase in oxygen ( 90 ), which may help to explain the deep bifurcation between the alkanal monooxygenases associated with SAR202 and the alkanesulfonate monooxygenases in the global phylogeny of these enzymes ( Fig. 2 ). About 2 billion years ago, there was a massive sequestration of carbon into ocean sediment, followed by a correction, both of which are thought to be biologically mediated events ( 92 ). A scenario consistent with the observations we report is the expansion of FMNO-dependent catabolism in an ancestor of the SAR202 clade as a part of a larger bacterial proliferation into the ecological niche of consuming marine DOM, in the wake of the slow rise in global oxygen. The biochemistry of deep-ocean DOM oxidation is largely unexplored, but pioneering laboratory experiments with natural marine communities support the theory that the production of refractory DOM in the deep ocean is biologically mediated, with fresh substrates being transformed into heterogeneous mixtures of recalcitrant DOM in short-term incubations of less than 30 days ( 28 , 93 ). Experimental approaches such as this provide an avenue for studying biological processes mediated by communities composed of uncultivated cell types. The genome-based hypotheses we report are likely to prove useful for the design of future studies that aim to validate biochemical pathways of deep-ocean DOM oxidation. We propose the SAR202 group III clade be given the following class and order assignments: Monstramaria , classis nov., Monstramariales , ord. nov., Monstramariaceae , fam. nov. The root of the class, order, and family name stems from the Latin for “sea monster,” to reflect the nearly exclusive presence of members of the SAR202 clade to a number of marine and freshwater environments and represents the ancient divergence of the SAR202 from other Chloroflexi , its cryptic genomic and metabolic features, the low amino acid identity of its protein coding sequences to known orthologs, and, most importantly, the great depths in which it resides."
} | 15,049 |
35200373 | PMC8869864 | pmc | 8,703 | {
"abstract": "In this study, the nitrogen-fixing, Gram-negative soil bacteria Rhizobium anhuiense was successfully utilized as the main biocatalyst in a bacteria-based microbial fuel cell (MFC) device. This research investigates the double-chambered, H-type R. anhuiense -based MFC that was operated in modified Norris medium (pH = 7) under ambient conditions using potassium ferricyanide as an electron acceptor in the cathodic compartment. The designed MFC exhibited an open-circuit voltage (OCV) of 635 mV and a power output of 1.07 mW m −2 with its maximum power registered at 245 mV. These values were further enhanced by re-feeding the anode bath with 25 mM glucose, which has been utilized herein as the main carbon source. This substrate addition led to better performance of the constructed MFC with a power output of 2.59 mW m −2 estimated at an operating voltage of 281 mV. The R. anhuiense -based MFC was further developed by improving the charge transfer through the bacterial cell membrane by applying 2-methyl-1,4-naphthoquinone (menadione, MD) as a soluble redox mediator. The MD-mediated MFC device showed better performance, resulting in a slightly higher OCV value of 683 mV and an almost five-fold increase in power density to 4.93 mW cm −2 . The influence of different concentrations of MD on the viability of R. anhuiense bacteria was investigated by estimating the optical density at 600 nm (OD 600 ) and comparing the obtained results with the control aliquot. The results show that lower concentrations of MD, ranging from 1 to 10 μM, can be successfully used in an anode compartment in which R. anhuiense bacteria cells remain viable and act as a main biocatalyst for MFC applications.",
"conclusion": "5. Conclusions Here, we have shown that the nitrogen-fixing, Gram-negative bacterium R. anhuiense could be successfully utilized as a main biocatalyst in the anode compartment by using modified carbon felt anode in an H-type microbial fuel cell setup. Our results demonstrated that R. anhuiense -based MFC performances could be enhanced by over 240% compared to control by re-feeding the anode bath with glucose after cultivation for 75 h. Moreover, the corresponding anode potential and power density can be improved if 5 μM menadione was added to the modified Norris medium. Notably, this amount of redox mediator does not significantly impact R. anhuiense bacteria growth; thus, it can be used without any side effects. In this case, the designed MFC’s maximal open-circuit voltage and power density were estimated to be 683 mV and 4.93 mW m −2 , respectively. Overall, this research opens a new avenue for the R. anhuiense bacteria to be exploited as a main biocatalyst in bacteria-based MFCs.",
"introduction": "1. Introduction In order to meet the growing demand for human food, the agriculture industry is intensifying production by new technologies, some of which involve the excessive use of nitrogen with other elemental fertilizers and alter chemical products. According to Robertson and Vitousek [ 1 ], the global application of nitrogen fertilizers has increased by more than ten times in the last 50 years [ 2 , 3 ]. Although adding chemical nitrogen to agricultural systems has major benefits, there are numerous unpleasant environmental impacts. Recently, some studies have revealed that the use of nitrogen in agriculture is one of the main triggers for coastal zone eutrophication processes [ 4 ]. This process leads to hypoxia in the coastal zone and other surface water bodies. Algae blooms are also triggered by nitrogen (N) uptake from agricultural land [ 5 , 6 ]. Therefore, intensive agricultural systems emit reactive nitrogen-based gases, particularly ammonia and various nitrogen oxides, which act as powerful greenhouse gases in the troposphere [ 7 , 8 , 9 ]. As an alternative for chemical nitrogen fertilizers, soil bacteria could be used, which can fix atmospheric nitrogen. They occur either as free-living soil bacteria (e.g., Azotobacter , Clostridium pasteurianum ) or in interaction with the roots of leguminous plants (e.g., Rhizobium , Bradyrhizobium ) [ 10 , 11 , 12 , 13 ]. This alternative is more environmentally friendly and has several positive aspects. For instance, soil bacteria increase the biodiversity of soil organisms as well as stimulate biogeochemical cycles [ 14 ]. All these aspects lead to better soil health. The agronomic approach for these bacteria has been widely analyzed and used in practice [ 15 , 16 , 17 ], and it is hypothesized that they could provide power to microbial fuel cells, and, after this process, return to the soil ecosystem and act symbiotically with legumes for atmospheric N 2 fixation. However, there is a lack of information about the use of this group of bacteria for microbial fuel cells and their potential to produce electrical power. At the beginning of the nineteenth century, the first article was published about electricity that was produced by bacteria. The main research object was Saccharomyces or bacteria and their metabolic pathways [ 18 ]. For the next hundred years, this capability was tested/applied only in the laboratory. Moreover, in the last decade, researchers focused on “green” renewable energy under growing energy requirements and climate change. One research area for the promising generation of green energy is microbial fuel cells. A fuel cell is usually defined as a cell that converts chemical energy into electrical energy without any direct combustion [ 19 , 20 ]. Several different types of microbial fuel cells were reported during the last decade. We can group them according to the kind of energy production: benthic microbial fuel cells (BMFC) [ 21 , 22 ]; photosynthetic microbial fuel cells (PhMFCs) [ 23 , 24 , 25 ]; plant microbial fuel cells (PMFC) [ 26 ]. Bacterial species that have the ability to transfer electrons extracellularly are referred as exoelectrogens [ 27 , 28 ]. Several lists of bacteria consortiums that can be used in the generation of electricity are provided in Table 1 . In all five reference lists, soil bacteria are included. Plant microbial fuel cell operation is based on the interaction of plant roots and microbes in the rhizosphere [ 26 ]. Rhizobium bacteria are classified as gram-negative and rod-shaped cells. Rhizobia–legume symbiosis is a well-documented example of symbiosis. Plants secrete flavonoids (pisatin, genistein) into the rhizosphere (active plant root zone) that activate rhizobial nod genes via the transcriptional activator NodD. Nod gene expression leads to the synthesis of the bacterial chemical signal, Nod factor, a lipochitin oligosaccharide. The Nod factor binds to specific plant kinases initiating a signalling pathway leading to root hair curling and trapping of rhizobia [ 29 , 30 ]. Microbial fuel cells employed by Rhizobium bacteria could provide a market for green energy. However, there is a lack of information on the design of MFC based on Rhizobium species bacteria. Electroactive bacteria strains are important for power generation in MFC devices. In order to enhance the performance of MFC, many recent studies have been focused on the chemical and genetic modifications of microorganisms [ 34 ]. Luo et al. [ 35 ] reported the additional treatment of K. rhizophila bacteria with lysozyme, which accelerats electron transfer about 1.75 times. However, chemical modification usually brings some disadvantages, such as reduced microorganism viability and long-term stability, thus making the species more susceptible to the environmental biota. Genetic engineering has a significant impact, increasing the performance of MFCs via the modification of biocatalysts cells. Nandy et al. [ 36 ] showed that genetically “improved” E. coli cells through cloning and expressing α-amylase gene leads to a high power density of 279.04 mW m −2 . Since the bacterium R. anhuiense belongs to the class of exoelectrogens, it was assumed that R. anhuiense could act as the main biocatalyst in an anode compartment to provide electrons and thus to generate electric power. R. anhuiense is known to be a bacterium that can survive under oxygen-containing or oxygen-free conditions (in cases when the bacteria are in symbiosis with legume plants). Furthermore, this advantage provides a reason to predict that this nitrogen-fixing bacteria could be used in both biofuel cell compartments (anode and cathode). Since this bacterium could be prescribed to the class of exoelectrogens, further investigations are required to show their capability to act as the main biocatalyst in MFCs. In this study, we have investigated the applicability of R. anhuiense bacteria as the main biocatalyst for constructing dual-chamber microbial fuel cells (MFCs). The carbon felt (CF) anode, used herein as biofilm-hosting electrode, was modified in acidic solutions to provide greater hydrophilicity and improved wetting properties. The bacterial growth kinetics, open-circuit potential variations, and power generation of the designed MFCs have been investigated. Besides, several soluble redox mediators, in particular menadione (MD), riboflavin (RF), and methylene blue (MB), have been applied to enhance the electron transfer from bacteria to solid electrodes.",
"discussion": "4. Discussion The efficiency of MFC depends on various aspects, including cell design, the electrodes used, and the biocatalyst, but mainly on the charge transfer efficiency towards conductive surfaces, which usually determine the whole device performances [ 65 ]. Bacteria cells are adapted to use various organic compounds, including carbohydrates, lipids, and proteins, as the main carbon sources. These organic nutrients act as electron donors for many complex redox reactions; thus, molecules of the energy carrier adenosine triphosphate (ATP) have been produced. Depending on the main carbon sources, the nutrients can be metabolized by bacteria through glycolysis and related processes into acetyl-CoA molecules, and further subjected to the citric acid cycle, as shown in the scheme in Figure 7 . At this stage, the redox reaction is coupled to the reduction of NAD + and FAD to their oxidized/reduced forms (NADH and FADH 2 ) [ 66 ]. In these cases, where the bacteria are weak exoelectrogens, the soluble redox shuttle that carries electrons to the solid electrodes is required to enhance or even detect the current densities provided by MFC [ 67 ]. Both forms of MD (oxidized and reduced) are neutral and lipophilic, with the molecular structure close to ubiquinone known as a membrane-bound redox mediator [ 68 ]. The electron transfer mechanism in such systems is mainly based on its permeation through the cell outer membrane and reduction by the redox enzymes to menadiol (MD red ) that are located in the cytosol or mitochondria and catalyzing the electron transfer from NAD(P)H to quinone substrates [ 63 ]. The MD red further diffuses outside the bacteria cell and interacts with the CF electrode, being oxidizing to the previous form of MD ox and completing the cycle as illustrated in the schematic in Figure 7 . Based on the power outputs generated by the MFC device in this study and by comparing them with previous research (see Table 2 ), it can be assumed that gram-negative R. anhuiense bacteria cannot be prescribed to the class of strong exoelectrogens. However, it was found that the menadione redox mediator could cause a 10-fold increase in MFC performance. Nevertheless, the obtained power density value (4.93 mW m −2 ) dictated that the electron transfer rate between R. anhuiense and CF electrode was not sufficient in comparison with today’s most powerful MFC devices, where the values of their power output range from several hundred to a few Watts per square meter [ 69 ]. It was assumed that an electron acceptor—molecular oxygen—could take a significant amount of electrons, making the whole device less efficient. To the best of our knowledge, the nitrogen-fixing R. anhuiense bacterium has never been used as a main biocatalyst in MFC devices. The obtained energy output values seem to be promising, boosted by the fact that these microorganisms naturally grow in aerobic conditions except the stage when participating in symbiosis with legume plants on their roots [ 70 ]. Although there is minimal information about the biochemical structure and possible electron transfer chains inside R. anhuiense cells, it was shown that by using menadione as a redox mediator, this soil bacterium could be successfully used as a main biocatalyst for the construction of MFC."
} | 3,139 |
32282803 | PMC7153904 | pmc | 8,704 | {
"abstract": "Ecological theories posit that heterogeneity in environmental conditions greatly affects community structure and function. However, the degree to which ecological theory developed using plant- and animal-dominated systems applies to microbiomes is unclear. Investigating the metabolic strategies found in microbiomes are particularly informative for testing the universality of ecological theories because microorganisms have far wider metabolic capacity than plants and animals. We used metagenomic analyses to explore the relationships between the energy and physicochemical gradients in Lake Fryxell and the metabolic capacity of its benthic microbiome. Statistical analysis of the relative abundance of metabolic marker genes and gene family diversity shows that oxygenic photosynthesis, carbon fixation, and flavin-based electron bifurcation differentiate mats growing in different environmental conditions. The pattern of gene family diversity points to the likely importance of temporal environmental heterogeneity in addition to resource gradients. Overall, we found that the environmental heterogeneity of photosynthetically active radiation (PAR) and oxygen concentration ([O 2 ]) in Lake Fryxell provide the framework by which metabolic diversity and composition of the community is structured, in accordance with its phylogenetic structure. The organization of the resulting microbial ecosystems are consistent with the maximum power principle and the species sorting model.",
"conclusion": "Conclusions Assessment of the gene family diversity and metabolic marker genes indicates that PAR and [O 2 ] control the distribution of potential metabolic strategies in Lake Fryxell. A multivariate statistical analysis of the relative abundance of metabolic marker genes shows that oxygenic photosynthesis, carbon fixation, and flavin-based electron bifurcation are the key metabolic strategies that differentiate mats growing in different environmental sub-habitats. Metabolic marker genes for anaerobic respiration likely result from spatial and temporal heterogeneity in [O 2 ] in Lake Fryxell. Further, the high relative abundance of btaA suggests that microbial mats in Fryxell appear to be phosphorus-, not nitrogen-limited in the anoxic portion of the lake, consistent with water column concentrations of nitrite, nitrate, and soluble reactive phosphorus. Attenuation of red light with depth may explain the dearth of anoxygenic photosynthesis genes. Finally, the pattern of gene family diversity through the mat layers and metabolic marker gene relative abundances of psbA , rbcL , and hdrB correlate strongly with PAR and [O 2 ] and point to the importance of their seasonal fluctuation. The spatial heterogeneity of PAR and [O 2 ] in Lake Fryxell provide the foundation for the organisms in Lake Fryxell to organize according to metabolic diversity and composition, similar to their phylogenetic structure [ 23 ], supporting the maximum power principle as applicable in this microbial ecosystem. More broadly, the species sorting model appears to be applicable to the metacommunity in Lake Fryxell as regards both phylogenetic lineages [ 23 ] and metabolic traits because niche selection (via the maximum power principle) governs which lineages and metabolic marker genes are found in which habitats.",
"introduction": "Introduction The microbial components of ecological communities (the microbiome) provide a large proportion of the genetic novelty and perform a large proportion of the functions of an ecosystem (for example, [ 1 – 3 ]). However, many of the methods that are used to explore microbiomes were developed by investigating plant- and animal-dominated ecosystems [ 4 ]. The composition, assembly, and function of microbiomes are considerably more complex than macroscopic processes because there are more individuals, more populations, and thus more possible interactions. Further, the phylogenetic relationships and metabolic capacity of microorganisms are often fundamentally different from plants and animals ( e . g ., horizontal gene transfer and mixotrophy). If the ecological theories developed from well-studied macroscopic ecosystems are universal, they should apply to microbial ecosystems. Therefore, we can gain insights into the general applicability of ecological theories by studying microbiomes [ 5 – 7 ]. In all ecosystems, heterogeneity in environmental conditions can greatly affect community membership. The extent to which this holds depends on the extent to which niche selection, drift, speciation or mutation, and dispersal affect taxonomic and functional richness, evenness, and composition [ 8 ]. The explicit effects of these processes have been developed into a metacommunity framework, applicable across spatial scales, and give rise to alternative models (species sorting, neutral theory, patch dynamics, and mass effects), depending on the degree to which each is relevant for a given ecosystem [ 9 , 10 ]. In the species sorting model [ 10 ], fitness advantages of existing community members limit the survival and growth of immigrants to only those that are most competitive. Therefore, environments with greater habitat heterogeneity have more diverse fitness landscapes and are thus inhabited by a more diverse community than homogenous habitats. Communities that conform to the species sorting model are ones in which species are distributed according to local environmental conditions and community heterogeneity matches environmental heterogeneity. Drift, speciation or mutation, and dispersal effects are damped out by niche selection. Thus, under the species sorting model, we expect to observe community composition changing in response to habitat features, and total habitat heterogeneity in a landscape directly influences community diversity. The species sorting model is the most widely cited as important in shaping microbial community dynamics, especially in aquatic ecosystems [ 11 – 13 ]. In contrast, the neutral theory model assumes that all organisms in a community are equally suited for their habitat. Therefore drift, speciation or mutation, and dispersal processes dominate over niche selection and community variations do not reflect variations in environment. Thus, neutral theory models produce a wide range of communities that are randomly distributed across heterogeneous habitats. Neutral dynamics have successfully described microbial community assembly in host-associated microbiome studies [ 14 – 16 ]. The patch dynamics model assumes homogeneous local environments where species coexist due to stochastic extinction and advantageous dispersal. In this model, community composition depends on early colonizers, which induce priority effects [ 17 ]. Differences in niche and fitness may exist among community members, but drift and dispersal dominate, leading to a community that does not depend on environmental heterogeneities. Under the patch dynamics model, populations are randomly distributed across relatively homogenous habitats, with greater diversity than expected from the homogenous landscape. Patch dynamics models are consistent with some studies of the human microbiome, which show significant diversity across individual patients or body sites that are interpreted as due to colonization history [ 18 ]. Finally, the mass effects model applies when dispersal overwhelms and masks selection, drift, and speciation or mutation, creating uniform communities composed of the same dominant organisms irrespective of environment. The mass effects model produces ecosystems in which community composition varies across different habitats and geography according to dispersal from parent communities. For example, the microbial composition of arctic streams are similar to the soils from which they originate near headwaters due to mass effects, but change as geographic distance and environmental heterogeneity increase [ 19 ]. The metacommunity framework explicitly describes community membership, but necessarily applies to functional aspects of communities as well. The means by which selection, drift, mutation, and dispersal affect community membership are through individuals' traits. The species sorting , neutral theory , patch dynamics , and mass effects models all hinge on the relative fitness of community members, which is determined by how well their phenotypes (functions) allow them to survive and reproduce in a given habitat or under specific environmental conditions. These metacommunity models can be used to understand ecological processes in microbial communities that lack macroscopic organisms. Specifically, microbial ecosystems in ice-covered lakes in the McMurdo Dry Valleys (MDVs), Antarctica, serve as natural laboratories to test the extent to which these models can explain community variations as a function of environmental gradients in photosynthetically active radiation (PAR) and oxygen concentration ([O 2 ]). The MDV lake environments are stable on decade-long timescales [ 20 , 21 ], containing well characterized PAR and slowly changing [O 2 ] gradients, which lead to predictable habitat heterogeneity [ 22 , 23 ]. PAR and [O 2 ] gradients are particularly prominent in Lake Fryxell, a perennially ice-covered, density-stratified lake in the Taylor Valley, Antarctica. Our prior investigations into the relationships between the phylogenetic structure and taxonomic composition of Lake Fryxell’s benthic microbial mats and local environmental conditions demonstrated that PAR and [O 2 ] affect local community membership differently at mm- and m-scales [ 23 ]. At the mm-scale, phototrophs dominate top mat layers where they maximize conversion of PAR into chemical energy and suppress α-diversity due to their high population [ 23 ]. The phylogenetic diversity of the underlying non-phototrophic layers increases with depth into the mat, consistent with the maximum power principle, which predicts that communities are structured to optimize energy consumption over time [ 23 , 24 ]. In mat layers where [O 2 ] was saturating, PAR structured the community. At the m-scale however, [O 2 ] positively correlated with diversity and affected the distribution of dominant populations across the three habitats. This suggests that meter-scale diversity is structured by PAR, as predicted by species-energy theory, which posits that areas with greater net primary productivity have more diverse habitats [ 4 , 25 ]. Because both the maximum power principle and species-energy theory require niche selection, prior results suggest that the species sorting model may be most appropriate for describing the benthic mat structure in Lake Fryxell across large- and small-scale PAR and [O 2 ] gradients. Neutral theory models are not appropriate because the communities systematically vary along environmental gradients. Similarly, the stratification of lake water means that the transport of organisms within Lake Fryxell is likely too low for populations to be controlled by mass effects. Finally, since the landscape features (PAR, [O 2 ]) are heterogeneous, the patch dynamics model does not apply [ 23 ]. Because species sorting was found to be an appropriate model for the phylogenetic diversity and taxonomic composition of Lake Fryxell’s benthic microbial mats, we tested whether patterns of metabolic capacity reflect the environmental conditions in Lake Fryxell across lake depth and through mat layers, also consistent with the species sorting model. Recent work has found that different ecological processes may influence phylogenetic and metabolic composition and diversity in microbial communities [ 26 ]. Indeed, due to the modular structure of cellular biochemistry [ 27 ], it may be the case that metabolic structure is more directly affected by environmental conditions than phylogenetic structure, which is additionally influenced by species-species interactions [ 28 ]. Application of the species sorting model to metabolic capacities would mean that the local distributions of PAR and [O 2 ] dictate the local metabolic capacity of the mats, similar to the distribution of species.",
"discussion": "Discussion Photosynthetically active radiation correlated with key metabolic genes in Lake Fryxell, specifically the capacity for oxygenic photosynthesis and carbon fixation. Oxygenic photosynthesis genes are most abundant in the top layers at each depth, consistent with greater PAR at mat surfaces and prior studies of phylogenetic data [ 22 , 23 ]. Photosynthesis requires PAR, so the decreasing relative abundances of psbA with layers into the mat and from 9.0 to 9.3 m is consistent with the utility of photosynthesis where there is light (Figs 1 and 3 ). However, the proportion of psbA in surface mat layers did not correlate directly with PAR across all lake depths. The amount of PAR reaching the mats growing at 9.8 m is just above the threshold for net photosynthetic production [ 22 , 34 ], yet samples from the film and top mat layers have the highest relative abundance of psbA of all depths ( Fig 3 ). The single Cyanobacterial lineage Phormidium pseudopristleyi dominates these samples [ 22 , 23 ]. The high population density of this organism likely explains the disproportionate representation of psbA . Energy capture and use: Photosynthesis, respiration, and flavin-based electron bifurcation The high relative abundance of the capacity for oxygenic photosynthesis overall supports previous studies indicating that oxygenic photosynthesis is the most ecologically important energy capture mechanism available to the communities in Lake Fryxell at depths where PAR is available [ 22 , 23 ]. The relative abundances of psbA and rbcL genes have a Pearson’s correlation coefficient of 0.998 ( Table 5 ), which is consistent with them being hosted in the same organisms, likely Cyanobacteria which are fixing the most carbon and generating the most biomass in the lake. The relative abundance of the capacity for polysaccharide hydrolysis ( amyA ) correlated with those for oxygenic photosynthesis and carbon fixation at only 9.0 m ( S4 Table ), where mats are oxygenated to a greater extent than at any other depth, and likely throughout the year [ 34 ]. Psychrophilic organisms that encode amyA are generally aerobes [ 58 – 60 ] and may be more efficient at polysaccharide hydrolysis in oxic environments [ 61 ]. The relative scarcity of genes encoding anoxygenic photosynthesis (absence of pscA and very low relative abundance of pufL ) is interesting in the context of previous work indicating that anoxygenic phototrophs are often abundant in low-light environments ( e . g ., [ 62 , 63 ]). Anoxygenic phototrophs that use pufL are part of the planktonic community in Lake Fryxell [ 64 , 65 ], and also have been detected in MDV Lake Vanda [ 66 ], but appear to be absent from MDV Lake Joyce [ 67 ]. The low relative abundances of pufL and absence of pscA may be related to the spectrum of light reaching the benthic surface of Lake Fryxell. The absorption spectrum of bacteriochlorophyll is near 700 nm [ 68 ] and the majority of light reaching the mats in ice-covered lakes is shorter wavelength due to increasing attenuation of longer wavelengths with depth [ 69 , 70 ]. The paucity of light at wavelengths suitable for anoxygenic phototrophs may render anoxygenic phototrophy an ineffective metabolic strategy, consistent with both the paucity of pufL and pscA genes in general, as well as their absence at 9.8 m. In Lake Joyce, the penetration of irradiance through the ice cover is also low, between approximately 0.4% and 4% [ 67 ]. In Vanda, approximately 16% of incident irradiance penetrates the ice cove[ 71 ]. Thus, it appears that PAR wavelength attenuation contributes to habitat suitability for anoxygenic phototrophs in MDV lakes. Where sufficient O 2 is available, aerobic respiration is the most efficient means of ATP generation for organisms. In Fryxell’s benthic mats, no statistically significant difference in the capacity for aerobic respiration, as measured by ccoNO relative abundance, exists between habitats where oxygen is constantly available, those where it is seasonally available, and those where it is constantly absent ( Fig 4 ). The widespread capacity for aerobic respiration across [O 2 ] in Fryxell mats may be attributable to the fact that bacteria can perform aerobic respiration at nanomolar concentrations of O 2 using terminal oxidases with a high-affinity for O 2 ( ccoNO ) [ 72 ]. Although the heterogeneity of anoxic environments has not been directly characterized in Fryxell mats, it is likely that micro-oxic and anoxic sub-habitats are more common as oxygen declines with depth in the lake and into the mats [ 34 ]. In such habitats, genes for both aerobic and anaerobic respiration are likely maintained because enough oxygen heterogeneity exists both spatially and temporally to make both strategies valuable. Anaerobic respiration using nitrate and sulfate appear to be viable strategies at all depths ( Fig 5 ). The greater relative abundance of nitrogen respiration genes over assimilatory nitrate reduction genes in Fryxell ( Table 4 ) may indicate the importance of nitrogen species as electron acceptors. Testing expression patterns of nitrogen cycling genes in shoulder and winter seasons would allow a better understanding of the effects of strong seasonality, especially availability of PAR and [O 2 ], has on these communities. While photosynthesis and aerobic respiration are the dominant energy metabolisms in Lake Fryxell, mats at 9.8 m show an interesting possible alternative metabolic strategy, as represented by the relative abundance of hdrB genes. hdrB encodes a subunit of a cytoplasmic complex that reduces two thiol coenzymes [ 73 ], which is crucial to methane production in methanogens that have been found in Fryxell’s planktonic community [ 74 , 75 ]. hdrB is strictly inhibited by oxygen [ 76 ]. However, in Fryxell mats, hdrB homologs were found in statistically higher relative abundance in the 9.8 m film sample type ( Table 6 ) where the mats are anoxic only during the winter months [ 34 ]. Phylogenetic markers of methanogens are absent in samples with high relative abundances of hdrB [ 22 , 23 ], suggesting hdrB is hosted in non-methanogens. Interestingly, hrdB is present in some sulfate reducing bacteria [ 77 – 79 ] and may be necessary for energy generation among diverse anaerobes [ 56 ]. In these organisms, hdrB is part of an enzyme complex called flavin-based electron bifurcation that acts as an alternative to both substrate level phosphorylation (fermentation) and electron transport [ 80 ]. In Fryxell mats, hdrB appears to mark capacity for flavin-based electron bifurcation in sulfate reducers rather than methane production, the first ecological evidence of this function of hdrB to our knowledge. Nutrient cycling and limitation Nitrogen fixation capacity in Lake Fryxell appears to be limited by local [O 2 ] as nifH is absent from mats continuously exposed to oxic water ( Fig 4 ). Typically in microbial mats, nitrogen fixation and ammonium and nitrate assimilation are performed by community members living near the surface of a mat that is illuminated and oxygenated [ 81 ], particularly by Nostoc spp. [ 82 ]. Many Antarctic mat ecosystems have a greater apparent capacity for nitrogen fixation than we found here, especially where Nostoc spp are in high abundance [ 81 ]. However, Nostoc spp . are rare in Fryxell’s mats [ 22 , 23 ]. Nitrogen fixation in non-heterocystous cyanobacteria occurs at night, when oxygen is no longer being generated and depleted from the cells [ 83 , 84 ]. The absence of dark conditions during the Antarctic summer leads to the continuous production of oxygen by cyanobacteria, which inhibits nitrogen fixation. Thus the polar latitude of Lake Fryxell may significantly limit nitrogen fixation above the oxycline even if the communities contained the capability to do so, consistent with previous metagenomic results [ 85 ]. Further, Fryxell's water column above the oxycline contains less than 1 μg / L nitrate or ammonium [ 22 ], leading to the hypothesis that the planktonic microbial community is also limited by nitrogen [ 20 ]. Given the low relative abundance of nifH , Lake Fryxell mats above the oxycline are also likely nitrogen limited, whereas water column nitrate and ammonium levels rise below the oxycline [ 22 ]. In contrast to the likely inhibition of nitrogen fixation in the O 2 supersaturated mats at 9.0 m, the absence of nifH in the top layer at 9.8, where mats are only weakly oxic seasonally, may be due to the high population density of the Phormidium , which often lacks the ability to fix nitrogen [ 86 ]. In the bottom layer at 9.8 m, where the capacity for nitrogen fixation could be attributable to heterotrophic bacteria [ 87 ], the absence of nifH is likely due to low availability of energy for nitrogen fixation, which requires an abundance of ATP [ 88 ]. In contrast, the low-light environment in the bottom layers at 9.3 m may provide enough PAR to support nitrogen fixation, and nifH is detectable in this layer ( Fig 7 ). Nitrogen and phosphorus cycling in planktonic communities in Lake Fryxell were recently investigated by [ 89 ], who found evidence that nitrogen and phosphorus are co-limiting. The relative availability of nitrogen versus phosphorus can affect the substitution of nitrogenous groups for phosphate groups in membrane lipids [ 57 ], a process that requires the gene btaA . The increased relative abundance of membrane phosphorus substitution genes at 9.8 m relative to samples with lower predicted nitrogen availability may indicate a switch in nutrient limitation from nitrogen to phosphorus at the oxycline. Mats growing below the oxycline in Fryxell have nitrogen available to them both through nitrogen fixation via nifH and water column nitrate and ammonium levels rise faster than dissolved reactive phosphorus below the oxycline [ 22 ]. Thus, variations in water column chemistry and the distribution of btaA indicate that there is likely spatial variability in nutrient availability. In contrast to nitrogen cycling, microbial sulfur cycling occurs across a range of oxygen concentrations, and sulfur oxidation and reduction are typically performed throughout microbial mats [ 83 ]. Assimilatory sulfate reduction is required for incorporation of sulfur into amino acids (biomass) in the absence of sulfide, whereas dissimilatory sulfate reduction is a means of anaerobic respiration. In general, dissimilatory sulfate reduction is an important anaerobic metabolism in microbial mats, especially where cyanobacteria generate low molecular-weight organics as substrates [ 84 ]. However, assimilatory sulfate reduction genes are found in greater relative abundance than dissimilatory sulfate reduction genes in Lake Fryxell (Figs 5 and 7 and Table 4 ). The difference in relative abundance of sulfate reduction genes in Fryxell mats may indicate that sulfate is primarily used for biomass generation rather than respiration. The species sorting model applied to metabolic composition and diversity Analyses of taxonomic composition and phylogenetic diversity suggested that the species sorting model is the most appropriate for describing benthic mat structure in Lake Fryxell across large- and small-scale PAR and [O 2 ] gradients [ 23 ]. Therefore, we expected the metabolic strategies of the mat communities to also closely match the local heterogeneity of PAR and [O 2 ] at the millimeter- and meter-scales. Understanding the metabolic capacity of the Fryxell's mat communities across the gradients of PAR input and [O 2 ] is crucial to understanding the processes driving community composition because fitness is dictated by individuals' traits. Gene family diversity trends support the hypothesis that the species sorting model can be appropriately applied to the communities in Lake Fryxell. We found that gene family diversity increased at the meter-scale across the lake floor and at the millimeter-scale through mat layers at 9.0 and 9.3 m, negatively correlating with PAR. Likely, the genes needed for oxygenic phototrophy, the dominant metabolic strategy in the top layers at 9.0 and 9.3 m ( Table 4 and Fig 3 ), suppress gene family diversity, which is relieved as phototrophy becomes less dominant through mat layers. This is consistent with phylogenetic and taxonomic results of these samples [ 22 , 23 ], and supports the interpretation that the communities through the layers at 9.0 and 9.3 m are organized to maximize energy capture [ 23 , 24 ]. The proportions of metabolic genes change as PAR decreases, indicating that the metabolic capacity of the mats at 9.0 and 9.3 m is structured by the local environmental conditions. In contrast, gene family diversity decreased through mat layers at 9.8 m, where [O 2 ] varies the most seasonally. Gene family diversity is also greatest in the film at 9.8 m. Samples from the top layer at 9.8 m show strong negative correlation between phylogenetic diversity and gene family diversity (Pearson correlation coefficient -0.790). The phylogenetic diversity in this habitat is quite low, likely due to the highly selective environmental conditions [ 23 ]. This implies that in this seasonally illuminated, seasonally oxic, low-energy, sulfidic environment, gene family diversity is important for survival as habitat conditions change throughout the year. Future investigation into how gene family diversity is distributed among community members in the film and top layers at 9.8 m will likely provide further insight into tradeoffs between fitness and diversity in this habitat. The metabolic marker genes that varied significantly between different local [O 2 ] and PAR input are those most important for optimization of energy capture. The relative abundances of genes encoding oxygenic photosynthesis ( psbA ) and carbon fixation ( rbcL ) at 9.8 m are greatest where high populations of Cyanobacteria capture the energy available at the mat surface. Cyanobacteria produce O 2 , which drives aerobic respiration and supports other, lower energy metabolisms when the mats become anoxic over winter. For example, organic carbon fixed by photoautotrophs likely supplies the substrates required by organisms using flavin-based electron bifurcation ( hrdB ), which is O 2 -inhibited and would be active only in the winter. The potential metabolic strategies of Fryxell mats across environments with different energy inputs suggest that they have maximized energy capture consistent with the maximum power principle [ 24 , 90 ] and the species sorting model. Alternative models within the metacommunity framework do not explain the patterns of metabolic diversity and composition in Fyrxell’s benthic mats. The patch dynamics model is inappropriate to Lake Fryxell because it requires local habitats conditions to be uniform, which does not conform to variability in PAR and [O 2 ] with depth in Lake Fryxell. The mass effects model would suggest that the metabolic composition of communities on the surface of the mats at each depth would be similar to that of the nearby lake water due to the settling of microorganisms. However, the benthic community is strikingly different from the planktonic community; specifically, the planktonic community contains abundant and diverse purple phototrophic bacteria [ 65 ], which are absent from the benthic microbial mats. The neutral model would be expected to produce communities that might vary in their metabolic diversity but without any relationship to environmental conditions, and therefore fails to explain the patterns of marker gene distribution along the PAR and [O 2 ] gradients in Lake Fryxell. Self-organizing systems such as these microbial communities are structured by their environment across both spatial and temporal scales; the relative abundances of species housing specific metabolic strategies adjust in population to achieve maximum power input given average energy availability throughout the year, with depth into the lake and through mat layers. Phototrophic and heterotrophic populations in Lake Fryxell’s benthic community likely change differently over the course of the annual PAR cycle because they occupy different niches. Phototrophs require PAR, and so likely increase in activity in the spring and summer. In the winter, phototrophs generally respond by a combination of entering dormant states, enduring reduced population abundances and loss of biomass via cell death, and shifting to heterotrophy or fermentation [ 91 ]; in MDV lakes, phototrophs may also be buried in mat over years rather than seasons [ 67 , 92 ]. Heterotrophs, and mixotrophs (seasonally), rely on organic carbon reservoirs built up over the years by the autotrophs. Heterotroph and mixotroph populations in the benthic mats likely shift according to organic carbon quality and quantity throughout the summer and winter, as do populations in the pelagic community [ 33 , 93 ]. Additionally, both phototrophic and heterotrophic populations living at 9.0 m likely change differently than those at 9.8 m. At 9.0 m, the O 2 saturation of the mats makes aerobic respiration available year-round. But at 9.8 m, the mats are predicted to become anoxic during winter, so other electron acceptors then become important. The increased relative abundance of extremely low-energy strategies such as flavin-based electron bifurcation via hdrB at 9.8 m ( Fig 6 ) are evidence that annual variation in PAR further affects the metabolic strategies found in the mats according to local environmental heterogeneity, in this case seasonal energy availability. The metabolic patterns uncovered here are consistent with the species sorting model because spatial and temporal heterogeneity of physicochemical characteristics (PAR, [O 2 ], nitrate, phosphorus, etc .) explain patterns of metabolic genes in Fryxell’s benthic mats. Independent evidence suggests that OTU abundances optimize energy capture in Fryxell’s planktonic community [ 75 ], and the same is true for Fryxell’s benthic community. An even more extreme example of the applicability of the species sorting model to microbial communities may be found in hot springs in Yellowstone National Park. The hot springs are considerably more constrained than Lake Fryxell, both phylogenetically and metabolically, where the dominant phylogenetic lineage may compose between 63 and 100% by SSU amplicon analyses and [O 2 ] limitation favors hydrogen metabolisms [ 94 , 95 ]. In contrast, the microbial mats growing in Guerrero Negro are phylogenetically stratified, likely according to PAR and geochemical gradients [ 96 , 97 ]. At Guerrero Negro, the chemical complexity of the habitat allowed the phylogenetic diversity to map onto environmental heterogeneity. The Guerrero Negro mats are therefore more similar to the stratified and stably heterogeneous environment of Lake Fryxell. These habitats differ in environmental conditions, but all demonstrate the applicability of the species sorting model, and metacommunity theory generally, to frame future research in extreme environments and microbial mat ecosystems."
} | 7,824 |
34302317 | null | s2 | 8,705 | {
"abstract": "Combining surface-initiated, TdT (terminal deoxynucleotidyl transferase) catalyzed enzymatic polymerization (SI-TcEP) with precisely engineered DNA origami nanostructures (DONs) presents an innovative pathway for the generation of stable, polynucleotide brush-functionalized DNA nanostructures. We demonstrate that SI-TcEP can site-specifically pattern DONs with brushes containing both natural and non-natural nucleotides. The brush functionalization can be precisely controlled in terms of the location of initiation sites on the origami core and the brush height and composition. Coarse-grained simulations predict the conformation of the brush-functionalized DONs that agree well with the experimentally observed morphologies. We find that polynucleotide brush-functionalization increases the nuclease resistance of DONs significantly, and that this stability can be spatially programmed through the site-specific growth of polynucleotide brushes. The ability to site-specifically decorate DONs with brushes of natural and non-natural nucleotides provides access to a large range of functionalized DON architectures that would allow for further supramolecular assembly, and for potential applications in smart nanoscale delivery systems."
} | 310 |
36248250 | PMC9540798 | pmc | 8,706 | {
"abstract": "Abstract Shallow coastal waters are dynamic environments that dominate global marine methane emissions. Particularly high methane concentrations are found in seasonally anoxic waters, which are spreading in eutrophic coastal systems, potentially leading to increased methane emissions to the atmosphere. Here we explore how the seasonal development of anoxia influenced methane concentrations, rates of methane oxidation, and the community composition of methanotrophs in the shallow eutrophic water column of Mariager Fjord, Denmark. Our results show the development of steep concentration gradients toward the oxic–anoxic interface as methane accumulated to 1.4 μ M in anoxic bottom waters. Yet, the fjord possessed an efficient microbial methane filter near the oxic–anoxic interface that responded to the increasing methane flux. In experimental incubations, methane oxidation near the oxic–anoxic interface proceeded both aerobically and anaerobically with nearly equal efficiency reaching turnover rates as high as 0.6 and 0.8 d −1 , respectively, and was seemingly mediated by members of the Methylococcales belonging to the Deep Sea‐1 clade. Throughout the period, both aerobic and anaerobic methane oxidation rates were high enough to consume the estimated methane flux. Thus, our results indicate that seasonal anoxia did not increase methane emissions.",
"introduction": "Introduction Atmospheric concentrations of methane—a major regulator of global climate—have increased nearly threefold from preindustrial levels (Saunois et al. 2020 ). Yet contemporary methane trends (1982–2020), including periods of stabilization (2000–2007) and renewed growth (2007–present), have thus far not been explained, demonstrating the need for resolving methane sources and sinks (Turner et al. 2019 ). Shallow coastal waters are estimated to contribute 50–80% of global marine methane emissions although they only cover 15% of the total ocean surface area (Weber et al. 2019 ). Particularly high concentrations of methane are observed in oxygen‐depleted waters (Reeburgh et al. 1991 ; Sansone et al. 2001 ; Capelle et al. 2019 ). Since the mid‐20 th century, declining oxygen levels have been recorded in coastal seas, bays, and estuaries such as the Baltic Sea, Chesapeake Bay, and the Danish coastal zone (Gilbert et al. 2010 ), where hypoxic and anoxic conditions are increasing in size, number, and frequency (Breitburg et al. 2018 ). Typically, coastal hypoxia and anoxia are recurrent seasonal phenomena owing to the seasonality of density stratification and algal growth (Diaz and Rosenberg 2008 ). In a fully oxygenated water column, methane concentrations are usually < 0.05 μ M (Reeburgh 2007 ), whereas the concentrations that accumulate under oxygen‐deficient conditions can vary widely between systems, from 0.1 μ M in the seasonally anoxic shelf waters of west India (Shirodkar et al. 2018 ) to 0.25 μ M in the hypoxic Boknis Eck in the Baltic Sea (Steinle et al. 2017 ) and 1.9 μ M in seasonally anoxic Saanich Inlet, British Columbia (Capelle et al. 2019 ), to reach 40 μ M in the highly eutrophic and seasonally anoxic Chesapeake Bay on the US East coast (Gelesh et al. 2016 ). In these systems, methane is primarily sourced from anoxic sediments, where it is produced by methanogenic archaea (Ferry 1992 ). The spatiotemporal variability in methane dynamics contributes to shallow coastal waters being the most uncertain term in the marine methane budget (Weber et al. 2019 ) and highlights the importance of understanding how methane cycling is regulated on a seasonal scale and under different oxygen conditions. In particular, little is known about the efficiency of methane oxidation in the water column under different oxygen conditions, and how the process responds to the development of hypoxic and anoxic conditions and to the associated increase in methane concentrations. In the presence of oxygen, methane is oxidized aerobically by methane‐oxidizing bacteria using the oxygen‐dependent particulate or soluble methane monooxygenases (for a review, see Trotsenko and Murrell 2008 ). Studies on pelagic methanotroph distributions are scarce, but members of the Gammaproteobacteria are suggested as important methane consumers in the ocean (Tavormina et al. 2010 ). Pelagic marine methanotrophs are capable of consuming methane to levels below atmospheric saturation concentrations (Reeburgh 2007 ) and thereby constitute an effective filter. Rates of aerobic methane oxidation from various marine water columns range several orders of magnitude (from 10 −5 to 10 3 nmol L −1 d −1 ; Mau et al. 2013 ), with much of the variability driven by differences in methane concentrations. Thus, the highest activities are measured in methane plumes (Steinle et al. 2015 ) and rates up to 150 nmol L −1 d −1 are reported from hypoxic methane‐enriched coastal waters (Steinle et al. 2017 ; Rogener et al. 2021 ). Methane oxidation can also proceed anaerobically in anoxic waters. Anaerobic methane oxidation was first observed in marine sediments where it is coupled to the reduction of sulfate and has been studied extensively (Knittel and Boetius 2009 ). Since then, anaerobic methane oxidation has been shown to couple to the reduction of nitrate, nitrite, iron, and manganese (Ettwig et al. 2010 ; Haroon et al. 2013 ; Ettwig et al. 2016 ), and there is growing evidence that the process may be of importance in both marine and freshwater systems. Recently, nitrate‐ or nitrite‐dependent anaerobic methane oxidation was proposed as a major pelagic methane sink in a marine oxygen minimum zone (OMZ; Thamdrup et al. 2019 ), and methane oxidation rates from anoxic depths in lakes have been proposed to be coupled to a variety of electron acceptors (van Grinsven et al. 2020 ; Roland et al. 2021 ). Still, the potential role of anaerobic methane oxidation in anoxic coastal waters remains to be explored. Nitrate and nitrite are the most energetically favorable electron acceptors after oxygen and potentially relevant in coastal and freshwater systems, where they have become increasingly available as a result of eutrophication (Galloway et al. 2008 ). Methane oxidation coupled to nitrate reduction has been described for “ Candidatus Methanoperedens nitroreducens” of the archaeal ANME‐2d clade (new family Methanoperedenaceae , Haroon et al. 2013 ), members of which to date are primarily reported from freshwater sediments. Nitrite driven methane oxidation is mediated by bacteria of the candidate NC10 phylum, which are hypothesized to dismutate NO from nitrite reduction into N 2 and oxygen, the latter of which is used for intra‐aerobic methane oxidation via the particulate methane monooxygenase pathway (Ettwig et al. 2010 ). Transcriptionally active “ Ca . Methylomirabilis oxyfera” of the NC10 clade have been observed in OMZs (Padilla et al. 2016 ) and its relative “ Ca . Methylomirabilis limnetica” was found to comprise up to 27% of the bacterial community in two anoxic lakes (Graf et al. 2018 ; Mayr et al. 2020 ), suggesting a major contribution to methane oxidation. Although considered to be obligate aerobes, gammaproteobacterial methanotrophs have also been shown to thrive in various anoxic waters, such as in seasonally anoxic Saanich Inlet (Walsh et al. 2009 ; Torres‐Beltrán et al. 2016 ), in anoxic lakes (Blees et al. 2014 ; Mayr et al. 2020 ), and in OMZs (Tavormina et al. 2013 ; Padilla et al. 2017 ). In some cases they may maintain their metabolism through in situ oxygen production by photosynthesis (Oswald et al. 2015 ). Yet members of the class are also capable of partial denitrification (Kits et al. 2015 ; Oswald et al. 2017 ; Padilla et al. 2017 ) and can oxidize methanol anaerobically (Dam et al. 2013 ). In Saanich Inlet, their depth distribution is highly suggestive of an involvement in anaerobic methane oxidation (Torres‐Beltrán et al. 2016 ). Still, the metabolic ability for complete anaerobic methane oxidation has yet to be demonstrated. In order to investigate how the seasonal development of water column stratification and anoxia influences methane oxidation and the methanotroph community in eutrophic coastal waters, we undertook monthly sampling during the development of anoxia in Mariager Fjord, Denmark. We combined biogeochemical measurements, experimental analysis of rates of aerobic and anaerobic methane oxidation, and analysis of the methanotrophic community through biomolecular analysis. Mariager Fjord is a brackish fjord on the northeastern coast of Jutland in Denmark, which due to nutrient loading from land combined with its topography has anoxic bottom waters between spring and late fall, but is typically flushed with oxygen‐rich seawater during winter (Fig. S1 ; Fenchel et al. 1995 ; Fallesen et al. 2000 ). Mariager Fjord thus serves as a good model system for investigating how biogeochemical processes respond to the seasonal development of an oxic–anoxic interface.",
"discussion": "Discussion By following the onset of anoxia in Mariager Fjord, our study provided the opportunity to investigate the roles of aerobic and anaerobic methane oxidation in methane removal during the gradual development of redox stratification over summer. We evaluate the efficiency of the observed microbial methane filter in mitigating the release of methane to surface waters and thus, ultimately, preventing the accumulating methane pool from contributing to increased emissions to the atmosphere. Development of seasonal anoxia and steep concentration gradients There was a clear seasonal progression in the redox stratification in Mariager Fjord in 2019, where the onset of anoxia in May preceded the development of steep redox gradients over summer and early fall (Fig. 2C ). Oxygen concentrations followed the typical pattern observed in previous studies in Mariager Fjord (Fenchel et al. 1995 ; Fallesen et al. 2000 ) and in the long‐term monitoring program carried out by the Danish Environmental Protection Agency (Fig. S1 ). However, compared to conditions in the 20 th century, when oxygenation events only occurred every 1–3 years (Fenchel et al. 1995 ; Ramsing et al. 1996 ; Fallesen et al. 2000 ), the winter oxygenation events now seem more regular, since bottom waters were oxygenated every winter between 2011 and 2020, except in 2013 (Fig. S1 ). Thus, in its present state, Mariager Fjord can be used as a model system for studying seasonal anoxia, with results that are relevant to various other coastal seasonally anoxic systems such as the Indian continental shelf (Shirodkar et al. 2018 ), estuaries like Chesapeake Bay (Gelesh et al. 2016 ), and fjords such as Saanich Inlet (Torres‐Beltrán et al. 2017 ; Capelle et al. 2019 ), as well as seasonally anoxic lakes that undergo a similar gradual development of redox gradients during their post‐turnover phase (e.g., Diao et al. 2017 ). The gradual steepening of redox gradients over summer and fall implies increasing fluxes of oxidized and reduced substrates toward the oxic–anoxic interface, since the specific density gradient across the interface weakened over the same time period ( dρ / dz from 0.37 to 0.29 kg m −3 m −1 between July and October, based on a 6‐m depth interval transecting the oxic–anoxic interface; no salinity data available from June). A higher substrate flux typically sustains a larger microbial community capable of efficient substrate conversions (Brune et al. 2000 ). However, in a seasonally anoxic system, where redox gradients develop gradually, the generation time of the microbial populations near the oxic–anoxic interface may potentially determine how rates respond to the accumulation of substrate. Generation times of methanotrophs in culture vary considerably, from hours or days in some aerobic gammaproteobacterial methanotrophs (Graham et al. 1993 ; Hirayama et al. 2013 ) to > 2 months for some anaerobic methane oxidizers (Knittel and Boetius 2009 ). Thus, if the growth rate of methanotrophs in the water column is slow, the accumulation of methane in anoxic bottom waters could result in an increased turbulent‐diffusive flux across the oxic–anoxic interface, or, if growth rates of methanotroph populations near the oxic–anoxic interface are high enough, they may continuously mitigate the release of methane from bottom waters. In Mariager Fjord, methane accumulated in the bottom water from < 0.1 μ M in May to the maximum of 1.4 μ M in August (Fig. 3A–E ). A study performed there in August 1994, when bottom waters had remained anoxic since winter 1992–1993, measured 40 μ M methane near the sediment–water interface (Fenchel et al. 1995 ), suggesting that methane may continue to accumulate if not interrupted by an oxygenation event. The rate of methane accumulation over time was similar to that seen in seasonally anoxic Saanich Inlet where concentrations typically increase from ~ 0.05 μ M in winter to ~ 1.3 μ M during summer (Capelle et al. 2019 ). However, since the anoxic water column in Mariager Fjord is about 10 times shallower than in Saanich Inlet (~ 10 m and ~ 100 m, respectively) the methane gradient in Mariager is steeper and the resulting flux correspondingly higher. The increase in methane concentrations with depth in Mariager Fjord is indicative of a sediment source. Indeed, Fenchel et al. ( 1995 ) estimated the methane efflux from anoxic sediments in Mariager Fjord to be 0.3–1.5 mmol m −2 d −1 . These values are in line with fluxes reported from other highly eutrophic coastal sediments, such as seasonally hypoxic Randers Fjord (up to 0.4 mmol m −2 d −1 ; Abril and Iversen 2002 ) and saline Lake Grevelingen (0.6–2.2 mmol m −2 d −1 ; Egger et al. 2016 ). In these environments it is expected that rapid sedimentation rates sustain the high methane fluxes, by providing abundant substrate for methanogenesis in surface sediments, as well as reducing the residence time of slow‐growing anaerobic methanotrophs in the sulfate–methane transition zone (Egger et al. 2016 ), emphasizing the importance of understanding the methane‐oxidizing capacity of the overlying water column. Efficient methane oxidation across an emerging redox boundary mediated by Methylococcales With the aim of investigating the seasonal dynamics of aerobic and anaerobic methane oxidation, we carried out two parallel sets of incubations with ~ 28 μ M oxygen and < 50 nM oxygen, respectively (Figs. 4A–E , S6A–E ). Several lines of evidence suggest that methane oxidation in incubations with < 50 nM oxygen occurred anaerobically. First, rates of methane oxidation in incubations from multiple depths (e.g., all anoxic depths in June, August, and October) were > 50 nmol L −1 d −1 . This is higher than what could have been sustained by the maximum possible oxygen concentration in the incubations, despite applying the conservative 1 : 1 oxygen to methane consumption ratio of carbon assimilating aerobic methanotrophs, as typically the ratio is closer to 2 : 1 (Naguib 1976 ). In addition, a system with high primary production like Mariager Fjord (Fallesen et al. 2000 ) will harbor an abundant heterotrophic community (Andersen and Sørensen 1986 ) and the methanotrophs would therefore face strong competition for the limited oxygen pool from oxygen‐respiring heterotrophs. A previous study in Mariager Fjord estimated oxygen consumption rates of 3.4 μ M d −1 just above the oxic–anoxic interface (Ramsing et al. 1996 ), which is in line with the oxygen drawdown of up to 5 μ M d −1 evident in our oxic incubations. These rates would deplete an oxygen pool of < 50 nM in less than 10 h, assuming half‐saturation ( K \n m ) values in the 66–259 nM range as estimated for various Danish coastal waters (Tiano et al. 2014 ). Incubations would thus be functionally anoxic for most of the 24 h incubation time. Since methane oxidation rates were linear over the full 24 h incubation time, there was no indication of a shift from aerobic to anaerobic metabolism. We also exclude photosynthesis as an internal oxygen source as incubations were carried out in the dark. We thus conclude that the methane oxidation activity observed in the incubations with < 50 nM oxygen was anaerobic. Therefore, in subsequent discussion, we refer to the two incubation types as oxic and anoxic and will henceforth refer to the activity observed in each of the incubations as aerobic and anaerobic methane oxidation, respectively. Overall, rate constants of methane oxidation in Mariager Fjord (0.09–0.8 d −1 , Fig. 4A–E ) correspond to the upper range of rate constants reported from hypoxic coastal waters (≤ 0.4 d −1 , Rogener et al. 2021 ; ≤ 0.084 d −1 , Steinle et al. 2017 ) and they agree well with values seen below the oxic–anoxic interface in stratified Lake Lugano (0.07–0.71 d −1 , Blees et al. 2014 ). In Mariager Fjord, rate constants in incubations with ~ 28 μ M and < 50 nM oxygen (aerobic and anaerobic methane oxidation, respectively) were very similar at each investigated depth (Fig. 4A–E ). This contrasts a previous report of methane oxidation in hypoxic coastal waters, where rates were about fivefold lower in incubations with 2–230 μ M oxygen compared to ~ 0.3 μ M oxygen (0.2–0.6 and 2 nmol L −1 d −1 , respectively; Steinle et al. 2017 ), and likewise differs from observations of anaerobic methane oxidation from an OMZ (incubations with < 0.1 μ M oxygen) where rates were up to 90% inhibited by oxygen concentrations of 1 μ M and higher (Thamdrup et al. 2019 ). Furthermore, since rate constants of both aerobic and anaerobic methane oxidation remained relatively constant with depth across the oxic–anoxic interface, the efficiency of either metabolism in the experiments did not appear regulated by the in situ concentration of oxygen. All three methanotrophic ASVs identified in Mariager Fjord belonged to the Deep Sea‐1 clade (Lüke and Frenzel 2011 ; Tavormina et al. 2015 ) of the Methylococcales (Fig. 4F–O ). The uniform relative abundance of the ASVs across the oxic–anoxic interface was consistent with our observations of similarly invariable rate constants, although relative abundances cannot be converted directly to absolute abundances. The increase in rate constants over time may still suggest that the methanotrophic community was growing. In August, we observed a shift in the dominating ASV from ASV 10 to ASV 23 corresponding to the highest observed rate constants. This could potentially be related to the intrusion of a different water mass indicated by hydrographic profiles in August (Figs. 2 , 3 ), although it is unclear how it would contribute to the observed population shift. Since we did not detect any known anaerobic methanotrophs such as “ Ca . Methylomirabilis” or ANME, we speculate that members of Deep Sea‐1 were responsible for both the aerobic and anaerobic methane oxidation activity observed in our incubations. Their involvement in aerobic methane oxidation is expected, based on genetic potential and observed substrate use of the close relative M. sedimenti (Tavormina et al. 2015 ), however, whether members of the Deep Sea‐1 clade are capable of switching from aerobic to anaerobic methane oxidation is not known. Phylogenetic analysis of the three ASVs demonstrated a close relatedness to sequences from other anoxic environments such as Saanich Inlet (Walsh et al. 2009 ) where the Deep Sea‐1 clade primarily occupies the anoxic part of the water column (Torres‐Beltrán et al. 2016 ). The depth interval sampled in Mariager Fjord (4–5 m) was likely too narrow to resolve any redox‐driven population distributions. Still, our anaerobic methane oxidation rate data, as well as previously observed depth distributions, strongly suggest that members of the Deep Sea‐1 clade are involved in anaerobic methane oxidation. Alternatively, the anaerobic activity could be conducted by as‐yet‐unrecognized organisms, although this would leave the role of Deep Sea‐1 in anoxic water columns unexplained. Indeed, a growing number of studies have identified populations of Methylococcales and other gammaproteobacterial methanotrophs that inhabit the anoxic hypolimnia of stratified lakes and likewise raised the question of the potential for anaerobic metabolisms within these groups (Oswald et al. 2017 ; Mayr et al. 2020 ; Rissanen et al. 2021 ). Molecular analysis of members of Methylococcales has demonstrated their ability to couple partial denitrification (to NO or N 2 O) with methane oxidation (Kits et al. 2015 ; Oswald et al. 2017 ; Padilla et al. 2017 ). Oxygen however still appears required for the first step of methane activation by the particulate methane monooxygenase enzyme, but subsequent oxidation of methanol can be carried out anaerobically (Dam et al. 2013 ). Whether organisms such as the Deep Sea‐1 can fully bypass oxygen by utilizing other electron acceptors like nitrate, nitrite, or other nitrogen intermediates, or otherwise obtain oxygen by alternative means similar to “ Ca . Methylomirabilis,” are questions that merit attention in future studies. Based on the existing evidence, we therefore hypothesize that the Deep Sea‐1 ASVs in Mariager Fjord are facultative anaerobes and conduct anaerobic methane oxidation when oxygen is exhausted. Thus, we predict that they possess enzymatic capabilities for both aerobic and anaerobic methane oxidation, as an adaptation to the dynamic environment near the oxic–anoxic interface in Mariager Fjord. A potential link between anaerobic methane oxidation and nitrogen respiration In all months with anoxic bottom waters, nitrate and nitrite penetrated below the oxic–anoxic interface and maintained a zone where nitrate and nitrite respiration could dominate (Fig. 3F–J ). This zone narrowed with time, which is consistent with a previous observation, where both compounds penetrated below the oxic–anoxic interface in July but were depleted at the oxic–anoxic interface in September and October (Jensen et al. 2009 ). To investigate whether anaerobic methane oxidation in Mariager Fjord could theoretically be sustained by denitrification, we measured rates of denitrification alongside methane oxidation (Fig. S6 ). Rates of denitrification at anoxic depths (0.2–7.7 μ mol N 2 L −1 d −1 ) were comparable to previous measurements in Mariager Fjord (0.5–2.8 μ mol N 2 L −1 d −1 , Jensen et al. 2009 ). Throughout the study, denitrification rates in Mariager Fjord were approximately ten times higher than rates of anaerobic methane oxidation (Fig. S6 ). Considering this order of magnitude difference along with the proposed 3 : 4 CH 4 to N 2 stoichiometry of methane oxidation coupled to denitrification from nitrite (Ettwig et al. 2010 ), it is reasonable to hypothesize that anaerobic methane oxidation in Mariager Fjord iscoupled to the denitrification pathway, e.g., to the production of NO or N 2 O as seen in some gammaproteobacterial methanotrophs (Kits et al. 2015 ; Oswald et al. 2017 ; Padilla et al. 2017 ), which are closely related to the ones observed here. While sulfate could potentially also serve as electron acceptor for AOM, we find it unlikely that this was the case in our incubations given the absence of ANME archaea, which so far appear critical for sulfate‐dependent AOM (Knittel and Boetius 2009 ). A robust methane filter composed of aerobic and anaerobic methane oxidation Although methane always constituted ≤ 2% of the total upward flow of electrons to the oxic–anoxic interface, which was dominated by ammonium and sulfide (Figs. 3F–J , S4A–E ), the overall methane gradient steepened between June and October (Fig. 3A–E ), suggesting an increased diffusive methane flux from bottom waters to the oxic–anoxic interface that might cause an increased release to surface waters. In order to evaluate whether methane oxidation near the oxic–anoxic interface could consume the upward methane flux, we estimated the vertical flux of methane due to eddy diffusion and compared it to depth integrated rates of methane oxidation over the 4–5‐m depth interval analyzed for rates near the oxic–anoxic interface (Table 1 ). The flux of methane from sediments and toward the depth interval analyzed for rates was calculated using the gradient from 24–25 m to the deepest sample collected for rate measurements (18–19 m in July–October). In June, when the oxic–anoxic interface was at 24‐m depth, the methane flux was calculated across the oxic–anoxic interface (23–25‐m depth). Estimated values of K \n \n z \n in bottom waters varied between 0.002 and 0.008 cm 2 s −1 across months in agreement with prior estimates in the system (0.0078 cm 2 s −1 , Zopfi et al. 2001 ; 0.003–0.014 cm 2 s −1 , Jensen et al. 2009 ). For a conservative estimate of the role of methane oxidation, we used K \n \n z \n = 0.008 cm 2 s −1 for our flux calculations. Table 1 Monthly comparison of methane fluxes and depth integrated methane oxidation rates. Rates were integrated over the 4–5‐m depth interval analyzed for rates near the oxic–anoxic interface and are shown for oxic (~ 28 μ M oxygen) and anoxic (< 50 nM oxygen) incubations, resulting in, respectively, an aerobic CH 4 sink and an anaerobic CH 4 sink. The vertical flux was estimated by multiplying the methane gradient in bottom waters with the vertical turbulent mixing coefficient, K \n \n z \n , calculated from the density gradient ( see “flux calculations” in methods). No methane flux was detected in May. Month Interval for depth integr. (m) CH 4 flux ( μ mol m −2 d −1 ) Aerobic CH 4 sink ( μ mol m −2 d −1 ) Anaerobic CH 4 sink ( μ mol m −2 d −1 ) May 20–25 ‐ 56 36 Jun 20–25 5.0 139 100 Jul 15–19 8.3 72 66 Aug 14–18 11.2 376 439 Oct 16–19 3.7 358 342 Estimated fluxes of methane ranged from 3.7 to 11.2 μ mol m −2 d −1 between months and were highest in August (Table 1 ). Fluxes were however always 1–2 orders of magnitude lower than the depth integrated rates of either aerobic or anaerobic methane oxidation (36–439 μ mol m −2 d −1 ), suggesting that methane oxidation near the oxic–anoxic interface in any month could consume the total upward methane flux. Still, the orders of magnitude imbalance between the calculated source (flux) and sink (integrated rates) implies uncertainties associated with one or both estimates. The transfer of activity observed in killed control incubations (Fig. S5 ) could result in an overestimate of rates by up to ~ 13%, however, such transfer rates (5–51 μ mol m −2 d −1 ) would only account for a minor part of the discrepancy. Another possibility is that the eddy diffusivity model does not capture the entire methane flux. This could be due to complex, intrusion‐driven mixing patterns at the oxic–anoxic interface, which were indicated by short‐term fluctuations in salinity and temperature profiles resulting in nonsteady state conditions in a previous study in Mariager Fjord conducting multiple CTD casts per day (Zopfi et al. 2001 ). Given such conditions, which are not visible in a single CTD profile as obtained here, our fluxes would most likely be underestimated. Likewise, a substantial non‐diffusive contribution to the methane flux from gas ebullition as reported from the near‐by Eckernförde Bay (Lohrberg et al. 2020 ) cannot be excluded, as the potential occurrence of shallow gas and rising bubbles in Mariager Fjord remains to be investigated. Nevertheless, the observation that each respective methane sink, namely aerobic and anaerobic, exceeded the methane source by factors between 10 and 100 (Table 1 ) strongly suggests that aerobic and anaerobic methane oxidation near the oxic–anoxic interface, potentially mediated by members of Methylococcales belonging to the Deep Sea‐1 clade, can effectively prevent the escape of methane to surface waters and thereby mitigate the effect of the accumulating methane on emissions to the atmosphere. The effectiveness of the filter is further witnessed by the methane concentration minimum, which persisted just above the oxic–anoxic interface (Fig. 3 ). Thus, we conclude that methane oxidation near the oxic–anoxic interface serves as an effective methane filter independent of ambient oxygen concentrations, which is a feature of particular importance in a shallow and dynamic system such as Mariager Fjord, where oxygen conditions can change rapidly."
} | 7,105 |
26819628 | PMC4728756 | pmc | 8,708 | {
"abstract": "Background Utilization of lignocellulosic feedstocks for bioenergy production in developing countries demands competitive but low-tech conversion routes. White-rot fungi (WRF) inoculation and ensiling are two methods previously investigated for low-tech pretreatment of biomasses such as wheat straw (WS). This study was undertaken to assess whether a combination of forced ensiling with Lactobacillus buchneri and WRF treatment using a low cellulase fungus, Ceriporiopsis subvermispora , could produce a relevant pretreatment effect on WS for bioethanol and biogas production. Results A combination of the ensiling and WRF treatment induced efficient pretreatment of WS by reducing lignin content and increasing enzymatic sugar release, thereby enabling an ethanol yield of 66 % of the theoretical max on the WS glucan, i.e. a yield comparable to yields obtained with high-tech, large-scale pretreatment methods. The pretreatment effect was reached with only a minor total solids loss of 5 % by weight mainly caused by the fungal metabolism. The combination of the biopretreatments did not improve the methane potential of the WS, but improved the initial biogas production rate significantly. Conclusion The combination of the L. buchneri ensiling and C. subvermispora WRF treatment provided a significant improvement in the pretreatment effect on WS. This combined biopretreatment produced particularly promising results for ethanol production. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0437-x) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions This study demonstrated that a substantial colonization of C. subvermispora in WS is facilitated by a combination of ensiling and a washing step as preconditioning, resulting in a pronounced pretreatment effect. The combination of ensiling and washing removed extractives such as waxes, fats and toxic compounds, which in turn, permitted the subsequent fungal growth and delignification. The combination of ensiling, washing and fungal growth produced efficient pretreatment of WS as assessed by enzymatic convertibility and ethanol yields. Furthermore, the results were achieved with only a minor TS loss due to fungal metabolism. When assessed for biogas production, the initial biogas production rate was significantly increased by the combination of biopretreatments, while the biomethane potential of the WS was not significantly improved. In the present study, both the ensiling and the WRF pretreatment were done under controlled conditions employing defined cultures for each step. In the context of use in developing countries, it is likely necessary to establish some microbiological routines to maintain the cultures pure and/or at least maintain a pure stock. Both the lactic ensiling culture and the C. subvermispora are easy to cultivate. A particular advantage of using microbial pretreatments is that a fraction of the microbial inoculum from the previous batch can be reused to initiate the next round of treatment (back-slopping), similar to classical food fermentations. In conclusion, the presented combination of biopretreatments can be a viable future alternative to high-tech methods, applicable in cases where decreased scale and low plant complexity is more important than relatively long pretreatment time.",
"discussion": "Results and discussion White-rot fungal pretreatment WRF pretreatment with C. subvermispora was carried out on untreated WS and on ensiled WS (EWS). In order to study the effect of extractives, also washed WS (w WS) and washed EWS (w EWS) samples were included in the set-up. After WRF pretreatment of WS, EWS, w WS and w EWS, it was visually apparent that the w EWS F sample (i.e. ensiled plus fungally pretreated material, which had been washed between the ensiling and the WRF treatment) exhibited the most substantial, uniform and fastest colonization with C. subvermispora (Fig. 1 ). Fig. 1 Wheat straw after ensiling and fungal pretreatment: a w WS F (visually similar to EWS F and w WS F), b w EWS F In general, the WS F, EWS F and w WS F biomass samples were only colonized by the fungus to a very limited extent. On the colonized w EWS F material yellowish droplets were moreover visible. These droplets presumably contained secreted enzymes and metabolized water, and their appearance might be linked to the rapid colonization, since the amounts of secreted enzymes generally correlate to the extent of growth of the fungal biomass [ 24 , 25 ]. After the WRF treatment, the w EWS F samples had a significantly brighter colour than the WS F, EWS F and w WS F samples. This brighter colour could indicate partial lignin degradation. During the WRF pretreatments, the total solids (TS) contents remained constant at around 20 % by weight, and none of the samples exceeded a TS level of 23 % at any time, based on gravimetrical measurements. The total TS losses observed during pretreatment were low, the highest TS loss being 5 % by weight in the w EWS F, while being 0, 0 and 2 % in the WS F, EWS F and w WS F, respectively. The untreated WS generally had very low extractives levels, except that the level of total phenols (0.41 g/100 g TS) was at the same level as that in the other samples (Table 1 ). Washing of the WS changed the extractives levels very little, although phenols levels decreased. In the EWS extractives, the levels of lactic acid and acetic acid increased as expected and reached 2.1 and 1.5 g/100 g TS, respectively, and also a low amount (0.5 g/100 g) of xylo-oligosaccharides and about 2 g/100 g TS of free xylose were recovered (Table 1 )—the free xylose was presumably a residual from the xylose added prior to the addition of the L. buchneri preparation used for ensiling (see methods). The washing of the EWS, as expected, reduced the lactic acid and acetic acid levels, and removed about half of the free xylose and about 2/3 of the low amount of free xylo-oligosaccharides (Table 1 ). It was possible to recover these substances in the washing water (Additional file 1 : Table S1). The analysis of the extractives also revealed that the extractives contents of the EWS F sample were similar to those of the EWS sample, underlining that hardly any fungal growth occurred on the unwashed EWS F. By contrast, the w EWS F sample contained relatively high levels of free xylo-oligomers (1.9 g/100 g TS), some gluco- and arabino-oligosaccharides, a very low amount of free glucose, and virtually no free xylose, free lactate or acetate (Table 1 ). The data, particularly the elevated xylo- and arabino-oligomer levels, indicated that the growth of C. subvermispora on the washed ensiled WS was accompanied by liberation of both cellulosic and hemicellulosic oligomers, and thus suggest that the latter might have constituted a significant carbon-source for the fungus during growth. Interestingly, the w EWS and the w EWS F had the lowest levels of soluble phenols among all the samples (Table 1 ). Table 1 Soluble carbohydrates, acids and phenols in water extracts of the differently pretreated samples (the contents of xylitol and formic acid were below 0.1 g/100 g TS in all samples, data not shown) g/100 g TS WS EWS w WS w EWS WS F EWS F w WS F w EWS F Free Sugars Glucose 0.16 ± 0.02 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.09 ± 0.14 0.24 ± 0.02 Xylose 0.07 ± 0.06 2.10 ± 0.04 0.02 ± 0.00 0.95 ± 0.05 0.01 ± 0.01 1.89 ± 0.13 0.08 ± 0.08 0.01 ± 0.02 Arabinose 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.19 ± 0.02 0.00 ± 0.01 0.00 ± 0.00 Oligomers Glucan 0.12 0.11 0.13 0.05 0.26 0.22 0.49 0.45 Xylan 0.25 0.46 0.14 0.16 0.72 0.63 0.97 1.89 Arabinan 0.03 0.06 0.02 0.03 0.18 0.12 0.23 0.53 Acids Lactic acid 0.00 ± 0.00 2.10 ± 0.08 0.01 ± 0.00 0.60 ± 0.02 0.01 ± 0.01 1.91 ± 0.04 0.00 ± 0.00 0.00 ± 0.00 Acetic acid 0.16 ± 0.02 1.49 ± 0.04 0.09 ± 0.00 0.37 ± 0.01 0.03 ± 0.02 1.22 ± 0.09 0.02 ± 0.01 0.05 ± 0.00 Total phenols 0.41 ± 0.05 0.22 ± 0.02 0.23 ± 0.02 0.11 ± 0.03 0.57 ± 0.05 0.39 ± 0.03 0.57 ± 0.05 0.14 ± 0.02 Standard deviations are presented when applicable The analysis of the solid fractions, after the different pretreatments, showed that the w EWS F samples had a lignin content of only 13.5 ± 0.6 g/100 g TS, while the lignin contents in all other samples were 19–22 g/100 g TS (Table 2 ). The latter data indicate that there was a lignin-degrading effect of the C. subvermispora WRF pretreatment of the ensiled, washed, WS. The mass balances for the TS of the pretreated samples did not all close at 100 % (Table 2 ) making it difficult to directly estimate the significance of this apparent delignification. However, when assessing the resulting effect on the relative glucan enrichment of the biomass, i.e. by comparing the ratio glucan:[glucan + xylan + arabinan + lignin] in the samples, it is evident that the observed decrease in lignin in the w EWS F samples, produced a relative enhancement of the glucan, by increasing the glucan:[glucan + xylan + arabinan + lignin] ratio to 0.56 in the biomass, whereas this ratio was ≤0.51 in all the other samples (Table 2 ) (Compared with the glucan enrichment on the basis of glucan/[Sum of all components], i.e. including ash and extractives in the denominator, the glucan enrichment in the w EWS F samples was 0.48 vs. a value of ≤0.42 in the other pretreated samples). This relative increase in glucan in the solid fibre fraction is presumed conducive to achieving improved enzymatic conversion of the carbohydrates in the fibre fraction (see next section). A delignification effect has also been seen in previous studies using C. subvermispora for WRF pretreatment [ 6 , 15 , 26 ], but the results have not always been unequivocal. For instance, Cianchetta et al. [ 15 ] investigated lignin reduction of WRF pretreated WS with C. subvermispora , but did not observe any significant lignin reduction after 4 weeks. However, after 10 weeks of the fungal growth, a lignin reduction similar to the one in our study was observed, but the lignin reduction came with a TS loss of 20 % [ 15 ]. Table 2 Composition of ensiled and WRF-pretreated biomasses and controls g/100 g TS Glucan Xylan Arabinan Lignin Ash Extractives Sum G/G + X + A + L WS 38.0 ± 1.1 c \n 20.1 ± 0.5 b \n 2.4 ± 0.3 cd \n 20.7 ± 0.2 ab \n 5.0 ± 0.1 a \n 3.4 ± 2.1 b \n 89.5 0.47 EWS 36.0 ± 1.7 cd \n 20.1 ± 0.6 b \n 2.6 ± 0.2 bc \n 19.5 ± 0.6 ab \n 3.6 ± 0.2 ab \n 7.1 ± 1.4 a \n 89.0 0.46 w EWS 47.8 ± 1.5 ab \n 26.4 ± 1.0 a \n 3.3 ± 0.3 a \n 19.5 ± 0.4 ab \n 3.3 ± 0.2 ab \n 1.3 ± 2.5 b \n 101.7 0.49 w WS 49.5 ± 0.3 a \n 25.1 ± 0.5 a \n 3.1 ± 0.3 ab \n 20.3 ± 1.1 ab \n 3.7 ± 0.2 ab \n 3.0 ± 1.4 b \n 104.8 0.51 WS F 34.1 ± 3.0 cd \n 17.0 ± 1.9 cd \n 2.0 ± 0.3 de \n 20.5 ± 0.3 ab \n 4.9 ± 0.4 ab \n 2.6 ± 0.9 b \n 81.1 0.46 EWS F 31.6 ± 3.0 d \n 17.1 ± 1.2 cd \n 2.1 ± 0.2 cde \n 19.3 ± 0.3 b \n 4.2 ± 0.8 ab \n 5.9 ± 2.8 ab \n 80.1 0.45 w WS F 32.3 ± 0.3 d \n 15.4 ± 0.1 d \n 1.6 ± 0.0 e \n 21.8 ± 1.3 a \n 4.2 ± 0.5 ab \n 3.6 ± 1.2 b \n 79.0 0.45 w EWS F 43.6 ± 1.4 b \n 19.1 ± 0.7 bc \n 2.1 ± 0.3 cde \n 13.5 ± 0.6 c \n 3.4 ± 0.9 b \n 8.3 ± 1.3 a \n 90.1 0.56 Data are given as average values ± standard deviation. Different roman superscript letters indicate significantly different average values in the same column ( p < 0.05). G/G + X + A + L is an estimate of relative glucan enrichment of the biomass, calculated from the g/100 g TS values as Glucan/(Glucan + Xylan + Arabinan + Lignin) The quantitative data for the extractives of the solid fractions came with relatively high standard deviations, and the absolute values were low, ranging from 1.3–8.3 g/100 g TS (Table 2 ). It is nevertheless tempting to speculate that in addition to the ensiling treatment itself, presumably inducing a mild pretreatment effect of the glucan by the acids produced [ 9 ], the removal of the soluble matter/extractives from the EWS by the washing facilitated the WRF growth. The w EWS samples thus had the lowest measured level of extractives (1.3 g/100 g TS, Table 2 ), whereas the levels in the EWS samples were higher (7.1 g/100 g, Table 2 ). Apparently, the fungal growth in turn also produced some extractives, since the levels in the w EWS F were higher than in the other samples (8.3 g/100 g, Table 2 ). Analyses of hydrophilic extractives in WS have shown that phenolic substances, fatty acids and sugar alcohols are dominant substances [ 23 ], but sterols, waxes, steryl esters and triglycerides have also been reported to be present in hot-water extractions of WS [ 27 ]. For several hardwood species, it has been found that the extractives content are related to durability against fungal attack [ 28 ], and although no direct correlation has been reported, hardwood extractives are known to inhibit the growth of some WRF under laboratory conditions [ 29 ]. However, the response of C. subvermispora to phenolics, including those likely encountered during lignin modification/degradation, is highly complex, and appears to be compound specific with respect to both growth and enzyme production. C. subvermispora has thus been found to produce increased lignin-modifying enzyme levels (laccase and manganese peroxidase) in response to, e.g. syringic acid and other di-methoxylated compounds that might occur as soluble substances during white-rot growth on lignocellulosic biomass [ 30 ]. Enzymatic convertibility The efficiency of the pretreatment was evaluated by enzymatic convertibility. The enzymatic convertibility was determined on each of the triplicates of the WRF pretreatments. The results obtained for w EWS F were comparable for the triplicates (not different, p >0.05 % for both glucan and xylan), which confirmed the reproducibility of the pretreatment method (Fig. 2 ). The enzymatic convertibility of the samples that were not subjected to fungal pretreatment (WS and EWS) are comparable to results from our previous work on ensiling of WS [ 9 ]. Fig. 2 Enzymatic convertibility of carbohydrates of WRF-pretreated material in triplicates (a, b and c) and raw material. Dark grey glucan, light grey : xylan, arabinan were below 0.5 g/100 g TS in all samples. The results are grouped according to significance ( p = 0.05 %), where ‘a’ is significantly higher than ‘b’ and so forth. Error bars represents standard deviations It is clear that w EWS F produced significantly higher enzymatic convertibility of both glucan and xylan, compared to the remaining fungal pretreatment as well as the raw materials. On average 23.4 ± 1.1 g glucan and 7.4 ± 0.4 g xylan were enzymatically converted in w EWS F per 100 g TS. The convertibility of the w EWS F was approximately twice as much as that achieved in the other samples and three times as much as that obtained for the untreated straw (Fig. 2 ) (For the w EWS F the convertibility was equivalent to a ratio of glucan:xylan conversion of 3.2, whereas the converted glucan:xylan ratio of untreated WS was 2.9 as calculated from the data in Fig. 2 ). Apparently the washing step helped support the colonization of C. subvermispora in the w EWS F biomass. Furthermore, there was no corresponding sugar release of the w WS, i.e. washed straw, but not subjected to ensiling. The fact that the enzymatic convertibility of both xylan and glucan was improved in w EWS F corroborated that there was a true and significant pretreatment effect and not only an effect of an up-concentration of glucan (Fig. 2 ). The attained level of 23.4 ± 1.1 g glucan converted/100 g TS is comparable to previously published results of 22.8 ± 1.9 g glucan converted/100 g TS obtained for classically hydrothermally pretreated WS (180 °C for 10 min) [ 9 ]. Ethanol fermentation The pretreatment effect was confirmed in a simultaneous saccharification and fermentation (SSF) assessment where w EWS F produced an increase in ethanol yield of 278 % compared to untreated WS (Fig. 3 ). A final yield of 13.1 ± 0.8 g ethanol/100 g TS was reached, corresponding to 66 % of the theoretical maximum based on glucan content (Fig. 3 ). This ethanol yield is comparable with published results on high-tech pretreatment of WS [ 9 , 31 , 32 ]. For example, Thomsen et al. [ 31 ] reached similar ethanol levels when using a three-step hydrothermal treatment reactor system in pilot plant scale. Thereby, the validity of the combined ensiling and fungal biotreatment approach as potential low-tech pretreatment method is verified, especially when taking into account that neither the enzymatic conversion nor the fermentation were optimized. Zeng et al. [ 33 ] have shown that the biological pretreatment performance on WS can be greatly enhanced in the presence of inorganic salts. A similar approach might increase the effect of the tested WRF pretreatment in future set-ups. Fig. 3 Simultaneous saccharification and fermentation of pretreated materials and controls. The results are grouped according to significance ( p = 0.05 %), where ‘a’ is significantly higher than ‘b’ and so forth. Error bars represents standard deviations The fermentations showed no sign of inhibition in the gravimetrical monitoring (data not shown), not even in the very early stages of fermentation. Formation of inhibitors is linked to elevated pretreatment temperatures commonly employed in biomass pretreatment, where a wide range of sugar degradation products may form [ 34 ]. The avoidance of such inhibitor formation effects is an additional positive trait of WRF pretreatment. Furthermore, after WRF pretreatment, the pH of the wet pretreated biomass was nearly optimal for SSF (pH ~ 5) (data not shown). This pH is more optimal for further enzymatic processing than that obtained after common pretreatments such as hydrothermal (pH ~ 3–4) or alkali pretreatment (pH > 9). Therefore, there is no need for using large quantities of chemicals (such as lime) for pH adjustment prior to the fermentation when using the presented combination of biopretreatment. Even though there are perspectives for the presented combination of biopretreatments as a low-tech pretreatment option, there are still important issues to address: sterilization of the biomass prior the fungal inoculation was needed in order to be able to differentiate between the effect of the fungal treatment and that of other microbes for the research. In a previous study, low doses (in the order of ppms) of inexpensive chemicals such as sodium sulphate were used to delay microbial growth while C. subvermispora colonized the biomass [ 35 ]. Alternatively, a short steaming procedure at ≤100 °C has previously been sufficient to allow C. subvermispora colonization without compromising its delignification abilities [ 36 ]. Similarly, it is possible to design the washing step in-between the ensiling and WRF inoculation to favour C. subvermispora colonization. In general, this work has addressed the use of a low-tech, inexpensive pretreatment for small-scale units. Scale-up of the procedure is an obvious challenge, which has not yet been accomplished on WS. However, within the pulping industry, biopulping trials (with WRF) have been executed with wood chips on 50-ton pilot scale with satisfactory outcomes [ 21 , 37 ], suggesting a potential for successful scale-up of the WRF pretreatment of WS as well as other lignocellulosic feedstock. Therefore, the presented approach offers opportunities for biomass pretreatment in cases where external factors require low process complexity and small-scale implementation as, e.g. in the developing world. Biogas production The positive effect of WRF pretreatment seen in enzymatic convertibility and fermentation for the w EWS F samples did not produce higher increases in the biomethane potential (BMP) than the other ensiled samples after anaerobic digestion (Fig. 4 a). The ensiled samples, EWS, EWS F, w EWS and w EWS F tended to have higher BMP than the non-ensiled samples, but the EWS F and EWS samples reached the same BMP level as the w EWS F (Fig. 4 a). Presumably, the reason for the similarities among the ensiled, but otherwise differently pretreated samples, may be related to the anaerobic digestion process, which utilizes a large range of organic compounds, i.e. sugars as well as fatty acids and proteins. This utilization means that the conversion is not necessarily limited by the accessibility of the glucan. The overall higher BMP of the ensiled samples can be explained by the availability of organic acids resulting from the ensiling process, but may also relate to any possible physical changes of the biomass resulting from the acidic ensiling, such as decreased hydrophobicity enabling better suspension of solids, despite no profound chemical compositional effects being evident. Fig. 4 \n a BMP of pretreated samples and controls. b Biogas production rate first week of production. The results are grouped according to significance ( p = 0.05 %), where ‘a’ is significantly higher than ‘b’ and so forth. Error bars represents standard deviations In contrast, evaluation of the biogas production rate during the first week of production clearly showed that the initial conversion rate of the w EWS F sample was higher than that of the other samples, a result that we interpret as being ascribable to the organic compounds of the biomass likely being more accessible to the fungus after ensiling and in turn enabling faster growth and conversion (Fig. 4 b). Thus, the higher initial biogas production rate and the relatively high BMP of the w EWS F sample corroborated the efficiency of the w EWS F pretreatment compared to the w EWS treatment. Based on the anaerobic digestion alone, it can be argued that the combination of biopretreatments presented in this study has an insignificant effect on the BMP of WS. Nevertheless, the faster conversion may potentially translate into options for using smaller, and thus cheaper, digesters."
} | 5,481 |
27104979 | null | s2 | 8,709 | {
"abstract": "Scale invariance refers to the maintenance of a constant ratio of developing organ size to body size. Although common, its underlying mechanisms remain poorly understood. Here, we examined scaling in engineered Escherichia coli that can form self-organized core-ring patterns in colonies. We found that the ring width exhibits perfect scale invariance to the colony size. Our analysis revealed a collective space-sensing mechanism, which entails sequential actions of an integral feedback loop and an incoherent feedforward loop. The integral feedback is implemented by the accumulation of a diffusive chemical produced by a colony. This accumulation, combined with nutrient consumption, sets the timing for ring initiation. The incoherent feedforward is implemented by the opposing effects of the domain size on the rate and duration of ring maturation. This mechanism emphasizes a role of timing control in achieving robust pattern scaling and provides a new perspective in examining the phenomenon in natural systems."
} | 255 |
26178309 | PMC4648426 | pmc | 8,710 | {
"abstract": "The microbiome’s underlying dynamics play an important role in regulating the behavior and health of its host. In order to explore the details of these interactions, we created an in silico model of a living microbiome, engineered with synthetic biology, that interfaces with a biomimetic, robotic host. By analytically modeling and computationally simulating engineered gene networks in these commensal communities, we reproduced complex behaviors in the host. We observed that robot movements depended upon programmed biochemical network dynamics within the microbiome. These results illustrate the model’s potential utility as a tool for exploring inter-kingdom ecological relationships. These systems could impact fields ranging from synthetic biology and ecology to biophysics and medicine.",
"discussion": "Discussion Although interconnectivity between commensal bacterial physiology and host behavior has been experimentally observed 5 , the underlying biochemical interactions 38 have yet to be fully understood. Here, we have created a unique in silico tool that enables us to explore this relationship with synthetic biology. Much in the way that synthetic gene circuits allows the exploration of genetic pathways and relationships in a single organism 39 , this tool could be used to augment and examine the interconnected networks that drive host-microbiome interactions. Crucially, we explored two different topologies of information flow critical for host-microbiome interactions. First, by simulating the toggle switch, we examined information flow from the environment to the microbiome, and then to the robotic platform. This system design ( Fig. 3 ) allowed us to establish an initial behavior theme: host alternation between nutrient sources (i.e., lactose and arabinose carbon depots) resulting from a repeatedly toggled, bistable gene network. We then demonstrated that a translational parameter, RBS strength, could serve as a tunable component for modifying the robot’s affinity for these nutrient sources. Thus, we were able to use both genetic topology and parameter strength to prescribe a range of robot behaviors ( Figs 4 and 5 ). However, host-microbiome systems in nature are not limited solely to microbiome-to-host communication. They also include mechanisms for host-to-microbiome information flow 40 . By adding the additional P lux-λ driven circuit and subroutine 6, we included this feature in our robotic system. In doing so, we simulated a system capable of mimicking host-microbiome interactions found in nature ( Fig. 6 ). The addition of this circuit resulted in robot behavior analogous to stalk-pause-strike vertebrate predation 37 . Furthermore, performing a one-dimensional parameter walk (i.e., varying the RBS strength driving cI expression) within this genetic topology showed that multiple distinct robot behaviors could be modulated by this single parameter ( Fig. 7 ). In addition to predation-like movement, these behaviors ranged from alternating between carbon source depots to permanently stalling. Our results demonstrate that small changes in biochemical parameters can result in the emergence of very different host robotic behaviors. Our model system provides a useful system for exploring host-microbiome interactions with synthetic biology. By integrating an engineered microbiome, a microfluidic chemostat mimicking the microbiome’s environment within an organism, and a robotic conveyance, we have designed, modeled, and simulated a biomimetic system that allows us to explore natural phenomena through both synthetic biological and robotic programming. We expect this model system will have implications in fields ranging from synthetic biology and ecology to mobile robotics."
} | 932 |
19859707 | PMC2804790 | pmc | 8,711 | {
"abstract": "Pentanol isomers such as 2-methyl-1-butanol and 3-methyl-1-butanol are a useful class of chemicals with a potential application as biofuels. They are found as natural by-products of microbial fermentations from amino acid substrates. However, the production titer and yield of the natural processes are too low to be considered for practical applications. Through metabolic engineering, microbial strains for the production of these isomers have been developed, as well as that for 1-pentanol and pentenol. Although the current production levels are still too low for immediate industrial applications, the approach holds significant promise for major breakthroughs in production efficiency.",
"conclusion": "Conclusions Pentanols are a useful class of compounds that have garnered increasing attention due to recent pressure to find alternatives to petroleum. While a lot of this attention is toward the fuel applications of petroleum, it should be remembered that petroleum-derived products permeate modern society. Biologically produced pentanols can be a part of the solution to moving beyond petroleum, and this review has summarized current methods of pentanol production (Table 1 ) and discussed many of the diverse ways that pentanols are useful.\n Table 1 Summary of highest titers and yields for engineered pentanol production Pentanol Titer (g/L) Yield (g/g) Reference 2-Methyl-1-butanol 1.25 0.17 Cann and Liao 2008 \n 0.37 N/A a \n Fukuda et al. 1993 \n 0.25 N/A a \n Kielland-Brandt et al. 1979 \n 3-Methyl-1-butanol 1.28 0.11 Connor and Liao 2008 \n 0.41 0.008 Watanabe et al. 1990 0.28 0.002 Suzzi et al. 1998 \n 1-Pentanol 0.75 0.038 Zhang et al. 2008 \n 0.08 N/A a \n Mauricio et al. 1997 \n Pentenol 0.11 0.006 Withers et al. 2007 \n Not able to be determined from available data \n As far as engineered production in biological systems, production of pentanols is just beginning. While long known as minor fermentation products, engineered production of pentanols has only taken place within the last 2 years. This means that much more research is required before biologically produced pentanols will be industrially relevant. The next major challenge likely to be faced in the microbial production of pentanols is the issue of toxicity. Pentanols are basically short-chain fatty alcohols and thus have a hydrophobic region of significant size. While the large hydrophobic region translates to a low solubility (about 2%) and thus easier separation from water, it also means that these compounds are more fat-soluble and thus tend to associate in the cellular membranes of microbes. There is already much research into the toxicity of hydrophobic compounds in E. coli and other organisms (Okolo et al. 1987 ; Aono et al. 1994 ; Sikkema et al. 1995 ), and some solutions have been studied (Roffler et al. 1987 ; Aono 1998 ; Izak et al. 2008 ). As biological production of pentanols is pushed further, the issue of its toxicity will become more prominent. There are several directions for future research into biologically engineered pentanol production. Thus far, E. coli has been the organism of choice for this production, though many of the key enzymes utilized are derived from other important and useful organisms such as yeast strains and lactobacilli. These microbes along with others can potentially offer advantages to make them better hosts for pentanol production. Further desirable research might be to include cellulose degradation directly to pentanols or even incorporate CO 2 for pentanol production. The area of biologically engineered pentanol is certainly still a nascent field.",
"introduction": "Introduction Modern society relies heavily on energy, especially the energy required for the transportation of goods, services, and people. For the past century, most of our transportation energy has come from fuels derived from petroleum. However, petroleum is a non-renewable resource, and recent efforts have pushed for alternatives. A heavily produced alternative to gasoline is ethanol primarily because ethanol production is a long-established art. Yet as a fuel, ethanol does not compare well to gasoline because it has a much lower energy density (only 21 MJ/L versus about 32 MJ/L for gasoline) and a high hygroscopicity. Pentanol isomers, as well as other higher alcohols, have a low affinity for water and an energy density of about 28 MJ/L and fit well into the current transportation infrastructure. Beyond their potential use as fuel, pentanol isomers also have a multitude of other applications. Several reviews about microbial production of biofuels in general have been written in the past 2 years (Antizar-Ladislao and Turrion-Gomez 2008 ; Atsumi and Liao 2008 ; Fortman et al. 2008 ; Connor and Liao 2009 ). These reviews address a wide range of topics but are limited in their discussion about the exciting new research on pentanol production. In this paper, we take a more in-depth look at the pentanols: how they are biologically produced, how that can be improved upon, and what applications they can serve."
} | 1,258 |
26135212 | null | s2 | 8,712 | {
"abstract": "CdiB/CdiA proteins mediate inter-bacterial competition in a process termed contact-dependent growth inhibition (CDI). Filamentous CdiA exoproteins extend from CDI(+) cells and bind specific receptors to deliver toxins into susceptible target bacteria. CDI has also been implicated in auto-aggregation and biofilm formation in several species, but the contribution of CdiA-receptor interactions to these multi-cellular behaviors has not been examined. Using Escherichia coli isolate EC93 as a model, we show that cdiA and bamA receptor mutants are defective in biofilm formation, suggesting a prominent role for CdiA-BamA mediated cell-cell adhesion. However, CdiA also promotes auto-aggregation in a BamA-independent manner, indicating that the exoprotein possesses an additional adhesin activity. Cells must express CdiA in order to participate in BamA-independent aggregates, suggesting that adhesion could be mediated by homotypic CdiA-CdiA interactions. The BamA-dependent and BamA-independent interaction domains map to distinct regions within the CdiA filament. Thus, CdiA orchestrates a collective behavior that is independent of its growth-inhibition activity. This adhesion should enable 'greenbeard' discrimination, in which genetically unrelated individuals cooperate with one another based on a single shared trait. This kind-selective social behavior could provide immediate fitness benefits to bacteria that acquire the systems through horizontal gene transfer."
} | 368 |
38576863 | PMC10993153 | pmc | 8,714 | {
"abstract": "Utilizing renewable lignocellulosic resources for wastewater remediation is crucial to achieving sustainable social development. However, the resulting by-products and the synthetic process characterized by complexity, high cost, and environmental pollution limit the further development of lignocellulose-based materials. Here, we developed a sustainable strategy that involved a new functional deep eutectic solvent (DES) to deconstruct industrial xylose residue into cellulose-rich residue with carboxyl groups, lignin with carboxyl and quaternary ammonium salt groups, and DES effluent rich in lignin fragments. Subsequently, these fractions equipped with customized functionality were used to produce efficient wastewater remediation materials in cost-effective and environmentally sound manners, namely, photocatalyst prepared by carboxyl-modified cellulose residue, biochar-based adsorbent originated from modified lignin, and flocculant synthesized by self-catalytic in situ copolymerization of residual DES effluent at room temperature. Under the no-waste principle, this strategy upgraded the whole components of waste lignocellulose into high-value-added wastewater remediation materials with excellent universality. These materials in coordination with each other can stepwise purify high-hazardous mineral processing wastewater into drinkable water, including the removal of 99.81% of suspended solids, almost all various heavy metal ions, and 97.09% chemical oxygen demand, respectively. This work provided promising solutions and blueprints for lignocellulosic resources to alleviate water shortages while also advancing the global goal of carbon neutrality.",
"introduction": "Introduction Because of the rapid industrialization development and global population explosion, the contamination and scarcity of drinkable water resources have seriously plagued human survival and development worldwide [ 1 ]. Approximately 4.5 billion individuals currently reside in proximity to impaired water sources, and this situation is anticipated to deteriorate further in the foreseeable future (Fig. 1 A) [ 2 ]. Hence, the recovery of drinkable water from wastewater is an essential way to achieve the United Nations Sustainable Development Goal 6 (Clean Water and Sanitation), which has a substantial socioeconomic impact [ 3 , 4 ]. A wide range of water remediation materials, such as polyacrylamide [ 5 ], polyaluminum chloride [ 6 ], graphene [ 7 ], and metal nanoparticles [ 8 ], are used in various water purification processes. However, all these materials mentioned above depend on fossil resources and have several disadvantages, such as high cost and adverse environmental toxicity [ 1 , 9 , 10 ]. Thus, a more cost-effective and sustainable strategy, such as the utilization of renewable, nonedible, and cheap lignocellulosic biomass resources for wastewater remediation, is highly desirable, which can liberate us from the dependence on fossil resources [ 11 – 13 ]. The value-added application of lignocellulose in the preparation of water remediation materials is an economically viable solution with a low-carbon footprint, facilitating the solution of human drinkable water problems and promoting carbon neutrality. Fig. 1. Valorization of industrial XR for relieving water crisis. (A) Gridded wastewater production at 5 arc min of spatial resolution. Data taken from [ 4 ]. (B) Schematic showing the harms of mineral processing wastewater to the ecological environment. (C) Schematic demonstrating the whole composition of XR for sequential purifying the lead–zinc mineral processing wastewater into drinkable water. First, the functional DES deconstructed XR efficiently while giving various components customized functionality. Then, 3 wastewater remediation materials were produced in simple, cost-effective, and eco-environmentally manners based on the customized structural features of various components. Finally, 3 wastewater remediation materials could sequentially purify actual lead–zinc mineral processing wastewater containing suspended solids, harmful microorganisms, heavy metal ions, and various organic pollutants, obtaining safe drinkable water. It is difficult to effectively convert lignocellulosic resources into high-value products due to the firmly compact structure of plant cell walls and a protective bulwark constructed by its chemical compositions [ 14 ]. The structural advantages of various components in untreated lignocellulose cannot be fully utilized, so directly preparing wastewater remediation materials usually have the characteristics of high energy consumption, low utilization rate, and inferior effect. Therefore, deconstructing lignocellulosic biomass to prepare scalable building blocks is a top priority for upgrading biomass into wastewater remediation materials, but the inherent heterogeneity of plant cell walls makes this goal elusive [ 15 , 16 ]. Developing a low-cost, environmental, and sustainable pretreatment technique has always been a beacon pursued by researchers [ 17 , 18 ]. Recently, deep eutectic solvents (DESs) have gained marked interest due to simple synthesis, recyclability, environmental friendliness, and selective solubility of biomass components [ 19 ]. More importantly, functional DES can efficiently deconstruct the lignocellulose while modifying its principal constituents to bestow targeted functionalities, thereby enhancing the economic and environmental advantages of the resulting material for wastewater remediation [ 20 ]. However, DES pretreatment also faced several challenges: (i) The incomplete regeneration of lignin fragments from the DES limits the recycling capacity of DES [ 21 ]. The ultimate disposal of DES effluents after multiple use cycles is also a cause for concern as it may lead to environmental contamination and resource waste [ 22 ]; (ii) the comprehensive utilization of all components of biomass resources under the no-waste principle is still an onerous problem [ 23 ]. These challenges above restrict the potential for attaining enduring industrial-scale implementations of DES in biomass processing [ 24 ]. Therefore, deconstructing lignocellulosic biomass and comprehensive utilization of various components to create effective materials for the collaborative elimination of diverse pollutants from wastewater not only aids in producing drinkable water but also alleviates environmental and resource strains. Herein, for the first time, we developed a cost-effective, sustainable, and holistic strategy that enables the nature of each biomass component to be unlocked for multistage industrial wastewater purification under the principle of zero waste (Fig. 1 C). Wasted lignocellulose [e.g., xylose residue (XR), a by-product of the xylose industry] was chosen as raw material for the proof-of-concept demonstration due to its low cost, abundance, and underutilization. This work synthesized a novel and efficient functional DES [methyl acryloyloxyethyl trimethylammonium chloride (MTAC)/acrylic acid (AA)/aluminum chloride] for deconstructing waste lignocellulose and enhancing functionalities by introducing customized functional groups into various biomass components in situ (Fig. 2 A). Subsequently, leveraging the structural features of the resulting components, a suite of wastewater remediation materials was synthesized using a simple, mild, and sustainable strategy. Biomass-based wastewater remediation materials could progressively treat suspended solids, bacteria, heavy metals, and organic pollutants in wastewater, ultimately transforming the unsuitable wastewater into potable domestic water. By utilizing functional DES to deconstruct and modify wasted lignocellulose, this study provides a systematic and feasible paradigm for producing efficient wastewater remediation materials, contributing significantly to the sustainable acquisition of drinkable water from industrial wastewater. Fig. 2. The deconstruction and functionalization effects of DES pretreatment on XR. (A) Schematic illustration of DES deconstructing XR and functionalizing the various components. (B) The delignification rate as a function of pretreatment temperature. (C) The FT-IR spectra of XR and R-110. (D) Quantitative analysis of hydroxyl and carboxyl groups in DEL and L-110 (carboxyl group, –COOH; phenolic hydroxyl group, Ph–OH; alcoholic hydroxyl group, Al–OH). (E) Cycle efficiency of DES (pretreatment temperature at 110 °C).",
"discussion": "Discussion Briefly, we successfully developed a new sustainable strategy to sequential purify wastewater into drinkable water using the whole composition of industrial by-product XR. This work efficiently deconstructed the main components of XR while endowing them with customized functionalization, and then produced 3 wastewater remediation materials based on the distinctive structural features of these components, including flocculants, adsorbents, and photocatalysts. In contrast to other corresponding material preparation methods, these production processes in this study avoid the use of a large number of harmful reagents, high temperature, sophisticated equipment, and lengthy processing times, demonstrating promising environmental and economic benefits. In terms of effects, the 3 materials not only demonstrate superior purification capabilities but also have outstanding universality. In particular, these 3 materials have quite compatible and complementary properties with each other and can carry out sequential purification of most actual wastewater. Taking the mineral processing wastewater with high hazard and difficult-to-treat characteristics as an example, the 3 materials successfully purified the mineral processing wastewater step by step into safe and drinkable water. It is roughly estimated that the annual output of XR in China alone can purify approximately 74% of the nation’s total yearly wastewater discharge for consumption. This study contributes solidly to the global-water-renewable resource nexus and provides promising solutions and blueprints for alleviating ever-mounting challenges posed by the water crisis and resource scarcity while achieving sustainable development."
} | 2,538 |
36289523 | PMC9608927 | pmc | 8,715 | {
"abstract": "Background Microalgae can absorb CO 2 during photosynthesis, which causes the aquatic environmental pH to rise. However, the pH is reduced when microalga Euglena gracilis (EG) is cultivated under photoautotrophic conditions. The mechanism behind this unique phenomenon is not yet elucidated. Results The present study evaluated the growth of EG, compared to Chlorella vulgaris (CV), as the control group; analyzed the dissolved organic matter (DOM) in the aquatic environment; finally revealed the mechanism of the decrease in the aquatic environmental pH via comparative metabolomics analysis. Although the CV cell density was 28.3-fold that of EG, the secreted-DOM content from EG cell was 49.8-fold that of CV ( p -value < 0.001). The main component of EG’s DOM was rich in humic acids, which contained more DOM composed of chemical bonds such as N–H, O–H, C–H, C=O, C–O–C, and C–OH than that of CV. Essentially, the 24 candidate biomarkers metabolites secreted by EG into the aquatic environment were acidic substances, mainly lipids and lipid-like molecules, organoheterocyclic compounds, organic acids, and derivatives. Moreover, six potential critical secreted-metabolic pathways were identified. Conclusions This study demonstrated that EG secreted acidic metabolites, resulting in decreased aquatic environmental pH. This study provides novel insights into a new understanding of the ecological niche of EG and the rule of pH change in the microalgae aquatic environment. Supplementary Information The online version contains supplementary material available at 10.1186/s13068-022-02212-z.",
"conclusion": "Conclusion This study demonstrated that EG caused a decrease in aquatic environmental pH by secreting many acidic metabolites. These metabolites were humic acids composed of N–H, O–H, C–H, C=O, C–O–C, and C–OH. The metabolomic analysis confirmed the critical acidic metabolites secreted by EG and determined its potential metabolic pathways, especially itaconic acid biosynthesis, which was first discovered in microalgae. These studies provide a new understanding of the mechanism of pH changes in the aquatic environment of microalgae culture and the role of microalgae in secreting metabolites to the aquatic environment.",
"discussion": "Results and discussion Photoautotrophic growth and potential maximum photosynthetic capacity of E. gracilis The results showed that the biomass and cell density of E. gracilis (EG) and C. vulgaris (CV, as a control group) gradually increased under the photoautotrophic conditions (Fig. 1 A–C). However, the accumulated biomass of EG was significantly lower than that of CV, especially the CV biomass (OD 750 = 1.9, 1.3 g L −1 ) was 2.7 (1.5)-fold than that of EG (OD 750 = 0.7, 0.8 g L −1 ) (Fig. 1 A, B, P < 0.01) at day 6, respectively. Similarly, the CV cell density (73.6 × 10 6 cells mL −1 ) was 28.3-fold than that of EG (2.6 × 10 6 cells mL −1 ) on the 6th day, suggesting that the EG cell density was significantly lower than that of CV ( P < 0.001) (Fig. 1 C). The potential maximum photosynthetic capacity ( F v / F m ) reflects the ability of microalgae to dissipate, absorb, and transmit light energy. It is a valuable parameter that indicates physiological state and growth rate and is also an internal probe of the relationship between microalgae and their environment [ 20 , 25 , 26 ]. The EG F v / F m was lower than CV ( p < 0.01). For instance, the CV F v / F m (0.8) was 1.6-fold that of EG (0.5) on day 6 (Fig. 1 D, p < 0.01), suggesting that EG potential carbon fixation capacity was lower than that of CV, which may also explain why the biomass of EG was lower than CV. Usually, the chloroplast is the place where cells carry out photosynthesis. According to literature reports, EG chloroplasts were derived from secondary endosymbiotic green microalgae [ 25 ]. Its chloroplasts were easily permanently lost after being treated by the external chemicals, such as erythromycin [ 27 ], and ofloxacin [ 28 ], thereby reducing the photosynthetic efficiency of EG. Moreover, it can trigger EG trophic type from the original photoautotrophic type (CO 2 as a carbon source) to heterotrophic (organic matter, e.g., glucose as a carbon source) type. In addition, E. gracilis \n F v / F m can be reduced via secreted humic acid under recycled-cultured conditions [ 22 ], suggesting that a part of the biomass accumulated by E. gracilis may be converted into secreted metabolites through photosynthesis so that the accumulated biomass becomes lower. Especially when these secreted metabolites were secreted into the aquatic environment and then became humic acid, which led to a decrease in EG F v / F m , which may further inhibit EG photosynthetic efficiency. Therefore, we speculated that EG might secrete more growth-inhibiting organics than CV, reducing EG photosynthetic efficiency. Fig. 1 Growth and F v / F m of E. gracilis under photoautotrophic conditions compared to C. vulgaris . EG represents E. gracilis ; CV represents C. vulgaris, as the control group; A OD 750 ; B dry weight; C cell density; D \n F v / F m ; F v / F m reflects the maximum quantum efficiency of photosystem II (PSII) photochemistry in microalgae. ** represents p < 0.01, *** represents p < 0.001; the values represent mean ± S.D. n = 3 E. gracilis secretes a large number of humic acids Generally, microalgae photosynthetic efficiency (e.g., F v / F m ) is gradually increased, resulting in a large amount of biomass accumulation under photoautotrophic conditions. Most microalgal species can absorb a large amount of CO 2 dissolved in the aquatic environment, increasing the pH of the aquatic environment [ 3 – 5 , 29 ]. This phenomenon was again verified in the CV as the control group in this study, but not in the aquatic environmental pH where EG was cultivated (Fig. 2 A). For example, even if the initial pH was set as the same at 7.6, the CV aquatic environmental pH increased from 7.6 to 10.9 on the 6th day of cultivation. In contrast, EG aquatic environmental pH gradually decreased to 2.9. This similar phenomenon of EG has been confirmed by our previous research [ 9 ]. This study cultured EG under aseptic and normal photoautotrophic growth conditions (Fig. 1 , Additional file 1 : Fig. S1). It confirmed that the EG aquatic environmental pH gradually decreased, which was different from the aquatic environmental pH of most microalgae. Therefore, we preliminarily speculated that EG cells were likely to secrete acidic substances that cause the aquatic environmental pH to decrease. Fig. 2 pH and characterization of DOM in the aquatic environment from EG compared to CV. A pH value; B (quantification by volume) and C (quantification by single cells) represent the content of the dissolved organic matter dissolved (DOM) in EG and CV on day 6, respectively; D , E represent the characterization of DOM via three-dimensional fluorescence excitation-emission matrix (3D-FEEM) spectra (I and II, aromatic proteins, III, fulvic acid-like, IV, soluble microbial by product-like material; V, HA, humic acid) in EG and CV at day 6, respectively; EG represents E. gracilis ; CV represents C. vulgaris ; black triangle represents the location of the peak; ** represents p < 0.01, *** represents p < 0.001; The values represent mean ± SD, where n = 3 Although EG’s biomass and cell density were lower than CV’s (Fig. 1 A, B), the DOM content in the aquatic environment was significantly higher than that of CV. For example, the DOM content of EG (78.5 mg L −1 ) was 1.9-fold that of CV (41.5 mg L −1 ) on day 6 of culture (Fig. 2 B, quantification by volume, p < 0.01). What is more, the DOM content of EG (29.9 mg cell −1 × 10 –6 ) was 49.8-fold that of CV (0.6 mg cell −1 × 10 –6 ) (Fig. 2 C, quantification by single cells, p < 0.001), indicating that a large amount of DOM was secreted from EG cells. The analysis of 3D-EEM spectroscopy showed that these DOM were mainly composed of humic acid, and EG had a significantly higher fluorescence intensity than that of CV (Fig. 2 D, E), indicating that EG secreted a large number of humic acids than that of CV. We used FTIR analysis to reveal their differences to further study the difference in the composition of humic acid secreted by EG and CV. The firm peaks of EG’s aquatic environmental DOM could be detected at wavelengths were 3148.62 (3100–3500 cm −1 , N–H, O–H, and C–H [ 18 , 21 ] and 959.73, 1120.02 (900–1200 cm −1 , C=O, C–O–C, and C–OH [ 30 ]) ranges (Fig. 3 B) compared to that of CV (Fig. 3 A), indicating that these functional groups secreted from EG could readily polymerize to form macromolecular humic acids. These DOM may cause the pH to be reduced in the EG’s aquatic environment. Since EG was a single cell alga without a rigid cell wall like other microalgae, so it may be affected by changes in the external environment and cause the cell to rupture. The substances inside the cell are released into the aquatic environment [ 9 , 22 ]. However, according to the growth of EG (Fig. 1 A–C) and the previous microscopic observations (data not shown), it was unlikely that all metabolites were released into the aquatic environment due to algal cell rupture. Therefore, we need to confirm further which critical metabolites were secreted from the intracellular EG cells. Fig. 3 FTIR spectra of the aquatic environment from E. gracilis ( B ) compared to C. vulgaris ( A ) on day 6 pH was reduced by acidic metabolites secretion To confirm which acidic metabolites were secreted from EG and to analyze how the EG's aquatic environmental pH decreased, the secreted-differential metabolites from EG cells were screened and analyzed via comparative metabolomics methods. 9156 and 10,496 metabolite peaks (Additional file 2 : Table S1-NEG-ORG, Table S1-POS-ORG) were detected in negative ion (NEG) and positive ion (POS) modes, respectively. EG and CV could be clearly distinguished under orthogonal-projections-to-latent–structures discriminate (OPLS-DA) analysis (Fig. 4 A, Additional file 1 : Fig. S2A), indicating that the metabolites were different at the different cultured stages. The metabolites that could be determined in the control group CV in NEG and POS mode were 192 and 239 (Additional file 2 : Table S1-NEG-CV, Table S1-POS-CV), and EG were 184 and 294 (Additional file 2 : Table S1-NEG-EG, Table S1-POS-EG), respectively. In the NEG mode, these metabolites can be divided into nine categories. Especially, lipids and lipid-like molecules (LLM), organoheterocyclic compounds (OHC), and organic acids and derivatives (OAD) account for the largest proportion of EG and CV metabolites. However, the proportion of EG and CV metabolites was not significantly different (Fig. 4 B, C). On the contrary, the proportion of three types of EG metabolites was significantly larger than that of CV in the POS mode (Additional file 1 : Fig. S2). It suggested that the three types of metabolites might be secreted from EG cells, causing the aquatic environmental pH to decrease. Fig. 4 OPLS-DA analysis and composition of DOM in the aquatic environment from EG compared to CV in the negative ion mode . \n A The OPLS-DA analysis; B the composition of DOM from CV aquatic environment; C the composition of DOM from EG aquatic environment; DOM, dissolved organic matter. As the test group, EG1, 3, 6 represents the total metabolite peaks from EG aquatic environment on the 1, 3, and 6 days of cultivation; as the control group, CV1, 3, 6 represents the total metabolite peaks of CV aquatic environment on the 1, 3, and 6 days of cultivation. EG: E. gracilis ; CV: C. vulgaris To confirm the difference between the critical metabolites secreted from EG cells and the CV in the aquatic environment, we selected the critical metabolites selected by VIP > 1, P < 0.05 for cluster analysis. After cluster analysis of the heat map, we found that the relative concentrations of some EG extracellular differential metabolites (EEs) were higher than that of intracellular EG (IEE) (Fig. 5 A, Additional file 1 : Fig. S3A). Also, the relative concentrations of these EEs were higher than that of extracellular CV (Fig. 5 B, Additional file 1 : Fig. S3B), indicating that these were candidate biomarker metabolites (BKs), secreted by EG cells. Fig. 5 Heat map of differential metabolites. A The heat map of E. gracilis (EG) differential metabolites between intracellular (IEG) and extracellular (EE); B the heat map of differential metabolites from the aquatic environment between C. vulgaris (CV) and EG; all metabolites were detected in negative ion mode (NEG mode); BKs, represent candidate biomarkers In addition, it revealed that the distribution of BKs under different pH conditions was different, such on the first day of culture (pH ≈ 5.0), there were 4 metabolites, on the 3rd day (pH ≈ 4.0), there were 12 metabolites, and 4 on the 6th day when the pH is 2.9 (Fig. 5 ). Similarly, there were similar results in the POS mode (Additional file 1 : Fig. S3). It indicated that EG secretes different organic matter under different culture stages and pH conditions. Yoshioka et al. [ 31 ] found that when EG was protected from light and under anaerobic conditions, the pH in the medium would affect the secretion of EG metabolites, such as succinic acid, glutamic acid, glutamine, and other substances under acidic conditions (the yield was higher in the pH 3–5). In this study, a similar phenomenon was also found under photoautotrophic conditions. Therefore, it is speculated that EG secretes acidic metabolites, which leads to the pH decrease in the aquatic environment, and negative feedback affects EG secreting metabolites. It is interesting to study the effects of different pH on secreting metabolites of EG under photoautotrophic conditions in the future. After all metabolites from POS and NEG modes were analyzed, 24 candidate biomarkers (BKs) were confirmed and secreted from EG cells into the aquatic environment (Table 1 ). Except that the NNP-pKa value of 6-deoxyfagomine was 15.02, slightly higher than that of the control group H 2 O 14. The pKa value of BKs was lower than the acidity value of H 2 O, indicating that the BKs were acidic substances. In addition, BKs were mainly distributed in OAD, LLM, and OHC, which were in line with the results of Fig. 4 mentioned above. In contrast, Zerveas et al. found that the photosynthetic process of microalgae induces pH increase by protons (H + ) uptake independently in aquatic environment [ 5 ], indicating that EG may secreted a lot of H + in the aquatic environment. Finally, it confirmed that the secretion of the acidic metabolites by EG led to the pH decrease in the aquatic environment. Table 1 Summary of 24 candidate biomarkers in the aquatic environment of E. gracilis BK Super class XBP-pKa NNP-pKa EXP-pKa 1-Deoxy- d -xylulose 5-phosphate OOC 1.56 1.70 – 2,3-Dihydroxybutanedioic acid OOC 3.13 2.86 – Fumaric acid OAD 3.31 3.09 3.02 trans -Aconitic acid OAD 2.87 3.16 – Ethyl glucuronide OOC 3.65 3.45 – Benzoic acid BZO 4.15 3.54 4.21 Itaconic acid LLM 3.48 3.65 3.9 N -Acetylglutamic acid OAD 3.42 3.69 – Methylsuccinic acid LLM 3.92 3.86 – l -Homocysteic acid OAD 3.58 3.90 – Homogentisic acid BZO 4.24 4.17 4.4 3-Hydroxycapric acid OAD 4.68 4.55 – Dihydrolipoate LLM 4.82 4.86 – 3-Methoxyanthranilate BZO 4.99 4.86 – Ochratoxin A PPP 3.54 5.38 – cis -Coutaric acid OAD 2.48 5.63 – Palmitic acid LLM 7.02 5.81 – 16-Methylheptadecanoic acid LLM 7.08 6.33 – Imidazoleacetic acid OHC 5.44 6.56 – l -Gulonolactone OHC 7.04 8.42 – Uracil OHC 9.40 8.95 9.42 N -Methylsalsolinol OHC 9.92 9.34 – Levoglucosan OHC 12.37 10.64 – 6-Deoxyfagomine OHC 11.63 15.02 – H 2 O (control) a 14.00 The lower the pKa value, the stronger the acid; XBP- and NNP-pKa represent pKa predicted via XGBoost and Neural Network, respectively; EXP-pKa represents experimental pKa coming from the sub-database of iBonD. For more details, please click http://ibond.nankai.edu.cn . a the pKa value of H 2 O is 14.00 instead of 15.70, according to Silverstein and Heller [ 46 ] BK candidate biomarkers, OOC organic oxygen compounds, OAD organic acids, and derivatives, LLM lipids and lipid-like molecules, OHC organoheterocyclic compounds, BZO benzenoids, PPP phenylpropanoids, and polyketides; pKa = − log 10 Ka, at 25 °C EG could secrete succinic acid, lactic acid, and amino acids into the aquatic environment [ 14 , 15 ]. In addition, E. mutabilis selectively secreted large amounts of amino acids, polyamine compounds, urea, and some sugars, other organic substances in mine drainages, without fatty acids [ 13 ]. However, this study found that EG secreted fewer fatty acids into the aquatic environment under photoautotrophic conditions. In particular, fatty acids account for a relatively large proportion (Fig. 4 B, C, Additional file 1 : Fig. S2). Therefore, it is speculated that different algae species could selectively secrete metabolites into the aquatic environment under different conditions. Five hundred million years ago, EG appeared on the Earth. It became highly adaptable in different harsh conditions, such as high UV radiation, heavy metal pollutants, acid mine water, and nutrient deprivation [ 32 , 33 ]. Halter et al. [ 13 ] found that E. mutabilis lived in acidic aquatic environments, like acid mine drainages. It could synthesize sufficient oxygen and secrete many metabolites into bacterial communities in the aquatic environment. In addition, Ouyang et al. [ 34 ] found that both EG and Vibrio natriegens co-cultured in an acidic medium (pH 3.6) promoted the growth and paramylon content of EG. It found that EG secreted a large number of metabolites into the medium. EG provides nutrients for bacteria. In turn, bacteria provide growth-promoting factors for EG. According to these studies, EG may have maintained the genetic characteristics of secreting acidic metabolites to the aquatic environment after long-term evolution, resulting in a decrease in pH, which could selectively inhibit harmful bacteria in an aquatic environment, while acid-resistant, beneficial bacteria may absorb secreted metabolites of EG. Then it provides particular nutrients (such as vitamins B 1 and B 12 that could not be synthesized by EG, stably preserved under acidic conditions) to EG, resulting in the balance of the energy and nutrients in the particular aquatic ecosystem. Heterotrophic yeast acid tolerance mainly relied on combined efforts of Pma1p [ 35 ] and V-ATPases [ 36 ] to regulate H + permeability. However, the secreted acidic metabolites led to a higher H + concentration in the medium, suggesting that EG lived in an acidic aquatic environment with its cell membranes that were more temporarily impermeable for H + . This phenomenon has also been demonstrated in acidophilic algae Cyanidium caldarium and Galdieria sulphuraria [ 37 , 38 ]. However, it was still never previously reported whether photosynthetic microalgae, mainly algae EG, had a similar molecular mechanism to regulate intracellular pH, allowing them to live in extreme environment and maintain extreme ecosystems, which deserves future research. Taken together, this study proposed for the first time that EG could produce organic acids under photoautotrophic conditions verified by comparative metabolomics analysis. Meanwhile, it found that EG can produce acidic substances, of which the BKs were identified to cause the pH decrease in the aquatic environment. At the same time, we have a new understanding of the ecological niche of EG in the aquatic environment. Metabolic pathways of critical acidic metabolites As EG was cultivated to the 6th day, the aquatic environmental pH was 2.9, significantly lower than that of CV (pH = 10.9) (Fig. 2 A). Therefore, it was meaningful to further study the metabolic pathways of the BKs at this time. In addition to uracil and 6-deoxyfagomine, which have higher pKa values, the relative concentrations of eight critical BKs of EG were significantly different compared to that of CV. Especially, cis -coutaric acid, itaconic acid, fumaric acid, and trans -aconitic acid (Fig. 6 ). It indicated that these critical BKs with low pKa values were secreted from EG cells. Those BKs were the most critical factor that caused the aquatic environmental pH to decrease. KEGG analysis showed that the critical BKs were involved in the six critical metabolic pathways, of which itaconic acid biosynthesis was particularly critical (Fig. 7 ). Fig. 6 Volcano plot analysis of the BK in E. gracilis ’ aquatic environment at day 6. BK, candidate biomarkers. The biomarkers were determined via log 2 (FC) > 1.5, − log 10 ( p ) > 1 Fig. 7 The underlying metabolic pathway of the pH drop in E. gracilis aquatic environment. The solid line represents a one-step chemical reaction, and the dotted line represents a multi-step chemical reaction. Red words represent acidic metabolites secretion Itaconic acid is an unsaturated, dicarboxylic acid with a wide range of applications in the polymer industry and as building blocks for fuels, solvents, and pharmaceuticals [ 39 ] and immune activity [ 40 ]. Itaconic acid biosynthesis metabolic pathways were only found in fungi [ 39 , 41 ], marine bivalves [ 42 ], and mammalian immune cells [ 43 ]. Especially, a large amount of secreted acidic itaconic acid was produced by Aspergillus terreus or Ustilago maydis with glucose as a carbon source under the heterotrophic fermentation conditions, resulting in a pH decrease [ 39 , 41 ]. However, this study found for the first time that the photosynthetic autotrophic microalga EG cells using CO 2 as the carbon source can also secrete relatively high concentrations of itaconic acid into the aquatic environment, that causes the pH to decrease. More interestingly, EG could synthesize organic carbon through photosynthesis and convert it into itaconic acid, reducing growth costs. In the future, we need to elucidate this metabolic pathway, which may provide a new way for environmentally friendly production of itaconic acid. In addition, the biological significance of itaconic acid secretion by EG was also worthy of in-depth study. This study combined 3D-EEM spectra, FTIR, and metabonomics at different levels to analyze that EG-secreted acidic substances into the aquatic environment under photoautotrophic conditions, resulting in a pH decrease. Consequently, these acidic substances might aggregate together to form humic acid with enormous molecular weight. These findings fill the gaps in scientific research on the secretion of acidic substances from photosynthetically grown EG. Meanwhile, we put forward a new point of view: the increase in the pH of the photosynthetic autotrophic aquatic environment of microalgae was due to not only the high photosynthetic efficiency of microalgae and the absorption of CO 2 in the aquatic environment but also the microalgae cells not secreting a lot of acidic metabolites. It changed our previous understanding of this discovery. These findings have potential use-value for the control of pH regulation in large-scale microalgae cultivation. For example, EG can be mixed culture with other microalgae to balance the pH of the medium, reduce the cost of artificial pH control, and enhance microalgae biomass. In addition, we have a new understanding of the ecological function of EG in the aquatic environment."
} | 5,873 |
29358665 | PMC5778128 | pmc | 8,716 | {
"abstract": "Thawing submarine permafrost is a source of methane to the subsurface biosphere. Methane oxidation in submarine permafrost sediments has been proposed, but the responsible microorganisms remain uncharacterized. We analyzed archaeal communities and identified distinct anaerobic methanotrophic assemblages of marine and terrestrial origin (ANME-2a/b, ANME-2d) both in frozen and completely thawed submarine permafrost sediments. Besides archaea potentially involved in anaerobic oxidation of methane (AOM) we found a large diversity of archaea mainly belonging to Bathyarchaeota , Thaumarchaeota , and Euryarchaeota . Methane concentrations and δ 13 C-methane signatures distinguish horizons of potential AOM coupled either to sulfate reduction in a sulfate-methane transition zone (SMTZ) or to the reduction of other electron acceptors, such as iron, manganese or nitrate. Analysis of functional marker genes ( mcrA ) and fluorescence in situ hybridization (FISH) corroborate potential activity of AOM communities in submarine permafrost sediments at low temperatures. Modeled potential AOM consumes 72–100% of submarine permafrost methane and up to 1.2 Tg of carbon per year for the total expected area of submarine permafrost. This is comparable with AOM habitats such as cold seeps. We thus propose that AOM is active where submarine permafrost thaws, which should be included in global methane budgets.",
"introduction": "Introduction Terrestrial permafrost landscapes, which developed during glacial cold periods, are known to be a large reservoir of organic carbon (~1300 Pg) 1 . Permafrost thaw and the following microbial production of carbon dioxide and methane from liberated organic matter may act as a positive feedback to climate warming 2 . Methane has 34 times higher global warming potential over a 100 year period 3 and is a more critical greenhouse gas than carbon dioxide. The amount and the release rates of methane are not well constrained although they are critical for evaluating future climate change. Several Arctic sources of methane have been identified, including methane bursts during soil freezing 4 , thermokarst lakes 5 , lakes and ponds 6 , wetlands 7 , gas hydrates 8 and submarine permafrost 9 . Submarine permafrost on continental shelves of the Arctic Ocean is a consequence of the inundation of terrestrial permafrost by sea water during the Holocene marine transgression 10 , 11 . Coastal submarine permafrost froze under subaerial terrestrial conditions in alluvial/fluvial settings and has remained frozen since then 12 , 13 . Submarine permafrost is much more susceptible to thawing than permafrost on land, because of overlying warm marine water causing diffusion of salt water into the sediments from the top, and because of geothermal heat flux from below 9 . Based on thermal modeling of permafrost development over glacial/interglacial cycles, submarine permafrost is likely to have persisted in deep sediment layers for hundreds of millennia 14 . Over those millennial timescales submarine permafrost reaches its freezing point, between −2 and −1 °C, after which it slowly starts degrading due to the influence of high salt concentration 10 , 15 . Our knowledge of evolving carbon pools and carbon turnover in submarine permafrost is, however, scarce. Arctic submarine permafrost regions have the potential to emit large amounts of methane to the atmosphere 16 , however flux measurements are controversial. First estimates suggest a release of 8.0 Tg of methane per year from the East Siberian Arctic Shelf (comprising Laptev Sea, East Siberian Sea and the Russian part of the Chukchi Sea), which would equal the release of methane from the entire world ocean 9 . In contrast, recent estimates for the Laptev and East Siberian Seas are almost three times lower (2.9 Tg y −1 ) 17 . Methane is mainly released from the sediment but the source is unclear 18 . It is expected that permafrost degradation can create pathways for the release of gas captured within submarine permafrost sediment or within gas hydrates underneath 16 . Low δ 13 C-CH 4 values indicate a biogenic or thermogenic origin of methane released from submarine permafrost 13 , 19 . A previous study demonstrated a drop of methane concentrations at the permafrost thaw front deep inside the sediments, which coincided with increasing δ 13 C-CH 4 values from −70‰ to −35‰ that points to anaerobic oxidation of methane (AOM) 13 . Sulfate penetrating from the seabed into the sediment created a deep sulfate-methane transition zone (SMTZ) similar to other geological formations in the subsurface 20 , with sulfate being a potential electron acceptor for AOM 21 . Most studies on AOM in the marine system have focused on ANME archaea that couple the oxidation of methane to the reduction of sulfate via a syntrophic lifestyle with sulfate-reducing bacteria (SRB) 22 – 24 . Three main marine clades, ANME-1a/b, ANME-2a/b/c, and ANME-3 have been identified so far 25 . These clades are associated with sulfate-reducing bacteria of the genera Desulfosarcina , Desulfococcus , Desulfobulbus and Desulfofervidus , which belong to the class Deltaproteobacteria 25 and of the phylum Thermodesulfobacteria 26 . Besides the marine ANMEs there is evidence of terrestrial AOM driven by the ANME-2d clade 27 , 28 . ANME-2d sequences were detected in wetland and permafrost habitats 29 – 31 so they might mitigate the release of methane in these environments. A recent study on wetlands showed that even low amounts of sulfate (µM) are sufficient to couple AOM with sulfate reduction in terrestrial environments 32 . In addition, alternative electron acceptors for AOM such as iron 33 , 34 , manganese 33 as well as nitrate 27 , 35 and humic substances 36 , 37 have been suggested. To date, the microbial mitigation of methane emissions from permafrost environments is only evident in aerobic soils and sediments 38 , 39 , while communities that oxidize methane anaerobically in thawing permafrost in the deep subsurface remain unexplored 8 . We hypothesize that active marine and terrestrial ANME clades exist in thawing submarine permafrost similar to microorganisms found in terrestrial enrichments 27 , 35 or marine SMTZ 40 , 41 , and that these clades could function as an efficient methane filter for a diffusive methane release. We investigated AOM in two deep submarine permafrost cores (48 m and 71 m) from the Siberian Shelf with different stages of submarine permafrost thaw 42 that were inundated for about 540 and 2,500 years, respectively 11 , 13 . To identify microbial assemblages involved in AOM, we performed high-throughput sequencing of archaeal and bacterial 16S rRNA genes and of the functional marker gene methyl co-enzyme reductase ( mcrA ) that is encoded by both methanogens and anaerobic methanotrophs. We quantified mcrA and dissimilatory sulfate reductase ( dsrB ) gene markers by quantitative PCR (qPCR) and correlated their abundance with the 16S rRNA gene abundance of AOM-related microorganisms. We visualized AOM-related microorganisms by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and constructed ANME-specific 16S rRNA gene clone libraries to resolve their diversity in submarine permafrost sediments. Pore water geochemistry i. e., concentrations of methane, nitrate, sulfate, iron, and manganese, was analyzed to verify potential horizons of methane consumption. Analysis of stable carbon isotope geochemistry of methane and microbial membrane lipid biomarker were performed to evaluate the activity of AOM communities in situ .",
"discussion": "Discussion Atmospheric methane concentration has been increasing again for the last decade, but the sources and mechanisms for this increase are not fully understood 51 . One potentially large but highly controversial source of methane is submarine permafrost, which faces drastic changes due to global temperature increase and associated Arctic sea ice reduction 52 . Our study provides multiple lines of evidence that ANME communities are present in submarine permafrost layers where methane is being consumed. We thus propose the microbial mitigation of methane release from thawing deep submarine permafrost on the Siberian Arctic Shelf. We detected both marine and terrestrial ANME clades likely involved in the AOM process at various depths, not only at the permafrost thaw front, but also in still ice-bonded permafrost that undergoes degradation. Our study indicates that AOM occur at temperatures below 0 °C 42 (Table S2 ). Pore water methane concentrations in the two cores were in the typical range of deep sediments and soils 20 , 53 . The permafrost thaw front at the BK2 site, inundated about 540 years ago reflects a deep SMTZ. We identified a steep decline of methane concentration above the SMTZ. This is in accordance with core (IID-13) 54 in close proximity that also showed a large decrease (10 6 to 10 3 nM) in methane concentrations at the thaw boundary while δ 13 C-values of methane from unfrozen layers above (~15 to 4 m) of this core are not reported. Altogether the study of Sapart et al ., (2017) 54 finds no evidence for AOM, which might be due to regional influences such as river water, land surface run off and warming by river discharge. Our δ 13 C-values of methane in the unfrozen part clearly show a large shift that can only be explained by microbial oxidation. The calculated potential rates based on methane release and the fraction of methane oxidized (1.7–2.1 nmol cm −3 d −1 ± 0.9–1.2 nmol cm −3 d −1 13 ) 13 are typical for margin SMTZ and exceed those of subsurface SMTZ 25 . At the permafrost thaw front of BK2, we detected marine and terrestrial ANME clades that potentially mitigate the methane release into overlying sediment layers. The upper unfrozen part of the SMTZ represents a typical marine sulfate-dependent AOM community of ANME-2a/b (Fig. 2C ) affiliated with SEEP-SRB1/seep-associated Desulfobacterium anilini -group (Fig. S7A ). These sulfate-dependent AOM communities are of marine origin and are often found in mud vulcanoes, and in methane and hydrocarbon seeps 55 . Visualization of ANME-2a in situ using CARD-FISH further supports their occurrence at the permafrost thaw front of BK2.The molecular detection of associated SRB is, however, not conclusive. This might be due to SEEP-SRB partners that are not targeted by the DSS685 probe 56 or by yet unidentified bacterial partners 57 . The lower part of the SMTZ was characterized by a clear transition to a terrestrial AOM community closely related to Candidatus Methanoperedens nitroreducens (ANME-2d) that might be capable of using alternative TEA such as nitrate and iron 27 , 47 , although no information on the oxidative state of iron is available. A recent genomic study proposed that these organisms have the genetic repertoire for an independent AOM process without a bacterial partner 46 . Besides the analysis of AOM assemblages via a DNA approach we also found high ether lipid MI values in the SMTZ of BK2 that further supports a potential involvement of AOM communities 50 . The high abundances of marine-derived ANME-2a/b sequences in several depths above the SMTZ of BK2 with BIT indices towards an exclusively terrestrial origin show that terrestrial and marine sediments were only slightly mixed in the upper meters of BK2 (Fig. 2 ). The terrestrial sediments were thus influenced by sea water penetrating down to the permafrost table and thereby transport of marine organisms into deeper layers occurred (Fig. 2 ). This is consistent with profiles of other environmental parameters (pH, temperature, isotopes and ion concentrations) and the stratification and composition of the bacterial communities 42 . In core C2, the layer between 29 and 43 mbsf is characterized by alternating frozen and partly thawed sediments (Fig. 1 ) and showed high fluctuations in the concentrations of nitrate and manganese. These fluctuations indicate degradation of thawing permafrost (shaded area, Fig. 1 ) and an increase in microbial activity resulting in the consumption of labile carbon pools 58 . Active processes in ice-bonded permafrost close to thawing (mean temperature: −1.2 ± 0.2 °C, Table S2 ) can occur in liquid water films surrounding mineral particles, which form a network in which microbial activity is expected 59 . Microbial activity may thereby be supported through sulfate as additional electron acceptor 60 penetrating into the ice-bonded sediment. Indeed, sulfate concentrations decreased and dsrB copy numbers increased with depth pointing towards active sulfate reduction and effective anaerobic organic matter decomposition 61 . In C2, the SMTZ occurred below the actual permafrost thaw front but inside the ice-bonded permafrost and was characterized by high manganese and nitrate concentration. Nitrate and manganese concentrations at the SMTZ and in the lower thaw boundary were in the higher µM to mM ranges that exceeded those typically reported for subsurface environments 20 by an order of magnitude ( http://publications.iodp.org/ ). So far it is not clear why nitrate as favorable electron acceptor is not quickly consumed under anaerobic conditions. Nevertheless, the high concentrations of nitrate and manganese could promote AOM with alternative TEA 33 , 35 and shows ongoing degradation of ice-bonded permafrost. Also, the detection of ANME-2a/b in this layer shows their migration into permafrost by downward marine water intrusion 42 . Unlike in core BK2, the ice-bonded layer of C2 was almost free of methane. At the same time ANME-2d occurred almost entirely throughout this layer. The relatively low copy numbers of ANME-2d detected with specific mcrA primers and compared with total cell counts 42 are still in the range of North Sea and River sediment 48 . The highest abundance of ANME-2d coincided with the highest methane concentration, which we consistently detected by several molecular approaches (16S rRNA, mcrA and ANME-2d-specific mcrA ). Taken all this together we suggest that ANME-2d members are responsible for AOM in the ice-bonded permafrost before it completely thaws. An alternative explanation for the low methane concentrations and the low abundance of ANMEs in most of the ice-bonded layers are a relic of AOM communities that were active under different environmental conditions in the past. Whether methane was trapped in the permafrost during freezing or it was produced by microbial degradation of organic matter under recent in situ conditions cannot be resolved since radiocarbon analysis would give similar results in both cases. Even though sulfate reduction might be relevant for organic matter mineralization 61 , links to sulfate-dependent AOM were not observed in the core C2. While C2 exhibited high copy numbers of dsrB and of SRB-related sequences, sulfate reducers were almost exclusively linked to Desulfosporosinus that have not been observed in AOM consortia so far. This genus belongs to the phylum Firmicutes and has been found in natural terrestrial environments such as peatlands, aquifer and permafrost 62 , 63 . Other TEA than sulfate, such as nitrate, iron and manganese, could also be related to AOM, and showed relatively low concentrations at the highest occurrence of ANME-2d sequences at 52 mbsf. This serves further as an indication of methane consumption during the process of AOM as known from physiological studies of ANME-2d enrichments 27 , 47 but direct evidence is missing. Finally, besides the detected electron acceptors, other TEA such as humic acids could serve in the AOM process. Humic acids were shown to be involved in AOM 36 , 37 in peatlands where they were detected in high concentrations. Humic acids are produced during organic matter degradation and soil formation 64 and could thus play a role in thawing permafrost, too. Two clades, which we named DSPEG I and DSPEG II, mainly occurred in submarine permafrost layers that showed relatively high concentrations of iron and manganese in the pore water (Fig. 1B,C ). This could point towards an involvement in iron (III) and manganese (IV) reduction within the anaerobic oxidation of organic matter, in addition to sulfate reduction 62 . Since the oxidative state of both metals has not been determined it is not clear whether these metals are used as TEA. Future analysis should focus on oxidative states of metals in these environments to further clarify a potential role in organic matter degradation and AOM. Nevertheless, Spearman rank analysis showed significant correlations between DSPEG I and DSPEG II and manganese concentrations (R = 0.58, p < 0.006 and R = 0.53, p < 0.02, respectively) and negative correlations with methane concentrations (R = −0.57, p < 0.02 and R = −0.64, p < 0.004, respectively) as illustrated in the CCA (Fig. 5 ). We propose that these two groups reflect indicator taxa (Table S3 ) for degrading permafrost. This is also supported by high occurrence of DSPEG I and DSPEG II (~19 to 28%, Fig. 1C ) at the lower boundary of the ice-bonded permafrost, representing bottom-up permafrost thaw. Here, high concentrations of sulfate, nitrate, and manganese show an upwardly directed thaw process (Fig. 1B ). Both groups were also dominant (47%) in the deepest sample of BK2, while degradation and an upward thaw cannot be concluded from our data. Environmental sequences affiliated with the DSPEG groups were mainly retrieved from cold environments 65 , 66 and pristine aquifers 67 , which further support an active role in low temperature habitats. Taken together our molecular and biogeochemical data from two submarine permafrost cores indicate several microbial assemblages that have the potential to prevent the release of trapped or recently produced methane into the overlying unfrozen sediment following submarine permafrost thaw. Therefore, we challenge the assumption that high methane emissions reported for the Siberian Arctic Shelves originate from degrading submarine permafrost itself 9 and suggest different mechanisms to be responsible, such as diffusion or ebullition through discontinuities in permafrost or the release from gas hydrates 8 , 68 at a limited spatial scale. Microbial assemblages in deep permafrost environments are usually associated with slow growth rates 69 and low abundances 70 , and their activity is difficult to measure. New approaches such as BONCAT-FISH 57 have the potential for more direct detection of active microorganism and the analysis of their genomic potential. The calculated fraction of methane that was oxidized in the SMTZ of BK2 showed high efficiencies, pointing towards an effective biological methane filter. While methane oxidation within the intact ice-bonded permafrost section of BK2 is unlikely, C2 showed several layers with heavier stable isotopes (≥−45‰) and high fractions (72 to 86%) of methane that were oxidized. Actual methane oxidation rates may be even higher, since methane production of freshly available organic material is not taken into account. Still, on a global scale the estimated submarine permafrost area (~3 million km²) 2 , could consume 0.0001 to 1.1889 Tg y −1 of methane from newly degraded permafrost assuming AOM activities similar to those in our cores. This only account for the methane released, since it is difficult to determine the total sediment volume in which AOM activity takes place. Submarine permafrost is thus comparable to AOM in other environments with high methane fluxes such as seep sites (<10 Tg C y −1 ) 71 , while AOM in wetlands (200 Tg C y −1 ) 32 and marine SMTZ (<50 Tg C y −1 ) 71 clearly show higher consumption rates. The latter two span areas that are 6 to 40 times larger than those of submarine permafrost. Our study provides first molecular evidence of microbial communities in thawing submarine permafrost that are likely involved in AOM processes. In addition, many archaeal taxa such as the newly designated DSPEG groups, a large diversity of Bathyarchaeota , and Thaumarchaeota closely related to nitrogen cycling organisms are detected. Their function is unknown and need further investment to understand their contribution in organic matter degradation of permafrost thaw processes."
} | 5,073 |
26434553 | null | s2 | 8,717 | {
"abstract": "Biofilm formation by Bacillus subtilis is largely governed by a circuit in which the response regulator Spo0A turns on the gene for the anti-repressor SinI. SinI, in turn, binds to and inactivates SinR, a dedicated repressor of genes for matrix production. Mutants of the genes ylbF, ymcA and yaaT are blocked in biofilm formation, but the mechanism by which they act has been mysterious. A recent report attributed their role in biofilm formation to stimulating Spo0A activity. However, we detect no measurable effect on the transcription of sinI. Instead, we find that the block in biofilm formation is caused by an increase in the levels of SinR and of its mRNA. Evidence is presented that YlbF, YmcA and YaaT interact with, and control the activity of, RNase Y, which is known to destabilize sinR mRNA. We also show that the processing of another target of RNase Y, cggR-gapA mRNA, similarly depends on YlbF and YmcA. Our work suggests that sinR mRNA stability is an additional posttranscriptional control mechanism governing the switch to multicellularity and raises the possibility that YlbF, YmcA and YaaT broadly regulate mRNA stability as part of an RNase Y-containing, multi-subunit complex."
} | 300 |
40275130 | PMC12023398 | pmc | 8,718 | {
"abstract": "The Cuatro Cienegas Basin (CCB) in Mexico, represents a unique ecological habitat, characterized by extreme and fluctuating conditions, providing a window into ancient evolutionary processes. This basin, characterized by hypersalinity and phosphorus scarcity, harbors diverse microbial communities that exhibit remarkable adaptations to oligotrophic conditions. Among these, Halobacterium salinarum , a halophilic archaeon known for its polyploid genome and metabolic versatility, has been extensively studied as a model for extremophile survival. However, only a limited number of H. salinarum strains have been successfully cultured and characterized to date. Here, we report the isolation and genomic analysis of a novel Halobacterium salinarum strain, AD88, from microbial mats at the Archaean Domes site in the CCB. This strain displays unique genomic features, including smaller plasmid sizes and distinctive metabolic pathways for phosphorus and sulfur utilization. Comparative analyses with other Halobacterium strains revealed genetic innovations, such as genes involved in sulfolipid biosynthesis, enabling membrane stability in phosphorus-depleted environments, and adaptations for horizontal gene transfer, which facilitate genomic flexibility in response to environmental pressures. This study reveals that H. salinarum AD88 is the first recorded diploid strain of Halobacterium , a feature previously undocumented in this genus. Phylogenomic reconstruction positioned AD88 tightly within the Halobacterium clade, reflecting its evolutionary history within the genus. Pangenome analysis further highlighted the open nature of the Halobacterium genus, with AD88 contributing novel accessory genes linked to ecological specialization. These findings emphasize the evolutionary significance of the CCB as a natural laboratory for studying microbial adaptation and expand our understanding of archaeal genomic diversity and functional innovation under extreme conditions. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-025-11550-9.",
"conclusion": "Conclusion Using an approach that includes culture-dependent and culture-independent techniques, we describe the genomic characteristics of the Halobacterium salinarum AD88 strain. Striking differences compared to other Halobacteria were uncovered, as its genomic architecture is diploid and with smaller novel plasmids. Additionally, genomic analysis suggests that AD88 may compensate for phosphorus scarcity through an enhanced reliance on sulfur metabolism, including sulfolipid biosynthesis pathways that replace phospholipids in membrane composition. This shift toward sulfur-based adaptations would support the hypothesis that nutrient availability plays a fundamental role in shaping the evolutionary trajectory of extreme halophiles. Phylogenomic analysis confirmed AD88’s placement within the Halobacterium clade, revealing its close evolutionary relationship with other H. salinarum strains. While its positioning in the tree suggests a high degree of similarity, nucleotide sequence identity levels reveal specific genetic differences that distinguish it from closely related strains. These findings highlight AD88 as a genetically distinctive strain and raise questions about the evolutionary forces that shaped its divergence, prompting a reevaluation of our understanding of genome dynamics in halophilic archaea. While phylogenomic methods provide a useful framework for distinguishing species-level differences in archaea, they fail to capture the full complexity of genomic variations. Comparative genomic studies across diverse archaeal taxa have revealed patterns of conservation and divergence in core genes, genes of unknown function, and mobile genetic elements, shaped by both phylogenetic and ecological pressures. Investigating the role of conserved and accessory genes in facilitating ecological specialization and maintaining population structures across evolutionary gradients could illuminate the mechanisms regulating archaeal speciation, offering new insights into how genomic diversity and functional adaptations arise and are maintained in extreme environments. Further exploration of these dynamics through integrative approaches, combining genomics, transcriptomics, and environmental data would advance our ability to refine archaeal taxonomy and deepen our understanding of their evolutionary trajectories.",
"introduction": "Introduction Halobacterium salinarum , a gram-negative extremophilic archaeon, is an emerging model organism for studying halophiles due to its ability to persist and grow in high-salinity environments [ 1 , 2 ]. Its sophisticated mechanisms for ion regulation, osmoregulation, and phototrophic growth exemplify versatility for stress resistance and energy acquisition [ 3 , 4 ]. Characterized by a distinct red pigmentation attributed to bacteriorhodopsin [ 5 ], H. salinarum withstands high salt concentrations and exhibits extraordinary resistance to extreme temperature and radiation [ 6 – 8 ]. Its discovery and successful isolation have facilitated studies of their unique genomic and physiological properties, improving our understanding of archaeal adaptation strategies. Despite its frequent detection in hypersaline environmental samples, easy culturing, and tractability, only a few different H. salinarum isolates have been successfully cultured and maintained under laboratory conditions. Currently, there are only five distinct isolates available in culture collections: H. salinarum NRC 34,001 [ 9 ], H. salinarum R1 [ 10 ], H. salinarum NRC-1 [ 11 ], H. salinarum 91-R6 [ 12 ], and H. salinarum KBTZ01 [ 13 ]. These isolates exhibit variations in their gene content due to adaptation to specific environments and their unique evolutionary contingencies, contributing to the accessory genome of each one of them. Overall, the genome of Halobacterium salinarum is highly dynamic and polyploid, typically possessing between 10 and 30 copies of its genome per cell [ 14 , 15 ]. With an average genome size of ~ 2.0 Mbp and high G + C content (60%) [ 2 , 16 ], Halobacterium sp. NRC-1, generally holds two 191 and 365 kbp-containing mega-plasmids, pNRC100 and pNRC200, respectively, which encode several genes essential for the organism’s survival in saline environments [ 17 – 19 ]. Traditional pan-genome analyses employed extensively in well-researched taxa showcase the biogeography of diversity and gene distribution across phylogenetic and environmental correlations [ 20 ]. Applying these analyses to species with only a few cultured members, such as H. salinarum , can reveal genetic novelty and potential responses to environmental cues and resource availability. An example of such a response has been observed in the genome of Bacillus coahuilensis from the Cuatro Ciénegas Basin (CCB) [ 21 ]. The genomic analysis of B. coahuilensis revealed its ability to produce sulfolipids instead of phospholipids, as it lacks the genes for producing phosphorus-rich teichoic acids and polyanionic teichuronic acids [ 21 ]. The Cuatro Cienegas Basin (CCB) within the Chihuahuan Desert, Mexico, is a unique and ecologically significant region featuring extreme and fluctuating conditions, such as low phosphorus, high radiation, alkaline conditions, and high mineral-pool content, resulting in high salinity levels [ 22 – 25 ]. Recent studies have shown that a particular site at CCB harbors an extensive archaeal diversity. This site, named Archaean Domes (AD) given the microbial mats that form dome-like structures under wet conditions, is particularly rich in members of the Euryarchaeota phylum, which includes halophiles and methanogens, showing a relative abundance of approximately 14% over seven years [ 25 – 28 ]. Despite reports of a relative abundance of over 30% Euryarchaeota at the site in 2019 [ 27 ], obtaining an axenic culture of most organisms within this domain has remained elusive. We hypothesize that Halobacterium salinarum AD88, isolated from CCB, has acquired genetic adaptations absent in previously studied strains, given the distinct ecological pressures present in the Cuatro Cienegas Basin (CCB) and limited number of cultured H. salinarum strains. Specifically, we expect that the extreme and fluctuating conditions of the CCB, such as low phosphorus availability and high salinity, have driven genomic innovations in AD88, including the evolution of novel metabolic pathways, such as secondary responses to phosphorus depletion, and structural genomic changes. To test this hypothesis, we conducted a genomic analysis of H. salinarum AD88 and compared its genome to the twelve available Halobacterium strains and twenty-three additional genomes from the Halobacteriales order. Distinctive adaptations to oligotrophy, particularly to phosphorus limitations, were found, much as smaller plasmid sizes and strong evidence of a single genome duplication event, becoming the first diploid Halobacterium salinarum recorded to date.",
"discussion": "Results and discussion A needle in a haystack The newly discovered ecotype, Halobacterium salinarum AD88, was isolated from a microbial mat (Fig. 1 a) found within the Cuatro Cienegas Basin (CCB), in an extensive culturing assay spanning five years. The basin is home to one of the few examples of living stromatolites and microbial mats, holding a broad spectrum of microbial taxa [ 53 ]. In the Archaean Domes (AD), a specific vernal pond within the CCB measuring 35 m x 10 m x 20 cm (L x W x D), microbial communities exhibit dynamic structural and functional adaptations in response to water availability [ 25 ]. Under hydrated conditions, these communities form a dome-like structure, encapsulating methane gas within the central cavity. Conversely, during dry conditions, the dome-like structures collapse, becoming flattened. Further analysis of their microbial composition highlighted the complexity and diversity of these communities, in which a significant presence of archaea and a diverse array of viruses were identified. Archaea historically accounted for less than 2% of the community, yet recent studies have identified a large archaeal diversity holding not only Euryarchaeota phylum and TACK superphyla, but also Asgard and DPANN superphyla. Halobacterium salinarum , in particular, have been identified within these communities with a 0.03969% relative abundance [ 25 ]. The isolation of H. salinarum AD88 exemplifies the power of combining synthetic and experimental approaches, key to study ancient taxa. Phenotypic characterization Colonies grown in agar plates were small, smooth and round, displaying the typical red coloration after incubation for 7 days at 37ºC. Most isolates of extremely halophilic archaea form red-colored colonies as a result of C-50 carotenoid pigments secretion, which include bacteriorhodopsins and betaines [ 54 ]. Their synthesis is triggered by high-intensity light and osmotic stress, respectively. Under conditions of low oxygen availability, haloarchaea increase the production of bacteriorhodopsins to exploit light energy for generating a proton gradient across the cell membrane, which is then used to synthesize ATP. Scanning Electron Microscopy Imaging revealed characteristic pleomorphic rod-shaped cells (Fig. 1 b and c), and standard microscopic analysis showed flagellum-mediated motility, supporting the identification of two flagella coding genes found in one of the assembled plasmids. \n Fig. 1 Microbial Structures in CCB. (a) A CCB microbial mat characterized by stratified, colorful layers used for isolation of H. salinarum AD88. The upper most salt crust, composed primarily of halite crystals, often harbors halophilic microorganisms, including extremophilic archaea such as Halobacterium spp. and some halophilic bacteria. Beneath the crust, stratified microbial layers exhibit distinct colors due to the presence of pigments like chlorophylls (green layer), carotenoids (orange-red), and phycobiliproteins, reflecting the diversity of cyanobacteria, purple sulfur bacteria, and other anaerobes in deeper strata, (b) SEM image of a cluster of Halobacterium salinarum AD88 cells. The cells exhibit pleomorphic morphology, including variations in size and shape, a characteristic feature of haloarchaea. This structural adaptability aids survival in extreme saline conditions, facilitating aggregation and biofilm formation, which enhance resource acquisition and stress resistance, (c) Scanning Electron Micrograph (SEM) of a single rod-shaped cell of Halobacterium salinarum AD88, with a measured length of approximately 3.09 μm \n Halobacterium salinarum AD88 has smaller plasmid sizes The assembly of the draft genome obtained 60 scaffolds with an N50 = 374,983, an L50 = 3, a GC content of 66.1% and an estimated size of 2,520,761 bp. The genome has a 99% of completeness and less than 1% of contamination according with CheckM standards (a microbial genome quality assessment tool) [ 37 ]. Functional annotation yielded 47 tRNAs, a single copy of the 5 S , 16 S , and 23 S ribosomal genes, and 2,587 coding sequences. We further identified and assembled two plasmids present in the H. salinarum AD88 genome, displaying smaller plasmid sizes than those that have been reported previously for this species, possibly reflecting adaptation to the CCB environment (Fig. 2 ) [ 55 ]. Plasmid I with a GC content of 63.93%, contains a single contig of 22,589 bp and encodes 25 coding sequences. Plasmid II, consisting of 12 contigs with a total length of 115,924 bp, GC %= 56.82, encodes 118 coding sequences, of which only 50.84% are non-hypothetical proteins. Environmental pressures shape plasmid content, at least as much as they influence plasmid size. However, there is no direct evidence that plasmid size alone is an adaptation mechanism to extreme environments. Plasmid gene composition varies with habitat, carrying different accessory genes reflecting local selective needs [ 56 ]. Organisms in long-term stable or nutrient-poor extreme habitats often exhibit genome reduction, loosing non-essential genes to conserve energy. Despite predictions that costly plasmids should be lost, mechanisms like host-plasmid coadaptation (Bouma and Lenski 1988) and gene transfer can maintain them. Thus, smaller plasmid sizes in extreme environments may result from a combination of adaptation and relaxed selection rather than direct selective pressure alone [ 57 ]. Studies of plasmids isolated from various archaeal species have shown a great diversity in gene content and innovation in replication strategies [ 58 , 59 ]. The role of plasmid-borne genes in haloarchaea species goes beyond acquisition of beneficial genes, as these plasmids usually encode genes essential for survival [ 60 ]. Host cells are more likely to retain plasmid-encoded genes when these genes provide a higher fitness by positive frequency-dependent selection, despite the natural tendency for plasmids to be lost during cell division (segregation loss) [ 61 , 62 ]. In this study, the genetic content of H. salinarum AD88 plasmid I reveals potential adaptive responses that are consistent with observed trends in haloarchaeal plasmids, encoding essential elements such as a cysteine desulfurase ( sufS) and an accessory transpersulfurase protein (s ufE) , which form an iron-sulfur carrier complex (EC 2.8.1.7) [ 63 , 64 ], playing a role in respiration, gene regulation, DNA repair and replication [ 65 ]. It also facilitates proper assembly and transfer of Fe-S clusters, which are critical to the function of redox-active enzymes and electron transport chains, particularly in environments where energy acquisition is challenging [ 66 , 67 ]. A notable aspect of H. salinarum AD88 is that a single copy of sufS gene, the catalytic subunit, is located in Plasmid I, whereas in other H. salinarum strains, these genes are typically found in multiple copies within the chromosome and plasmids (Supplementary Material Table 4 ). Given that Fe-S cluster biosynthesis is essential for cellular survival in extreme environments, the localization of these genes in AD88 plasmid may represent an adaptive strategy, potentially acquired through horizontal gene transfer. This unique genomic arrangement could provide regulatory advantages or increased genomic plasticity, reinforcing the role of plasmids as reservoirs of adaptive genes in haloarchaea [ 68 ]. Additionally, the plasmid contains fla/Che operon genes, key in the response to environmental stimuli through chemotaxis and typically located in the main chromosome [ 69 ]. H. salinarum strains are light energy-transducing systems and the encoded proteins enable the microorganism to process and respond to this stimulus through its flagellar motor [ 70 , 71 ]. CheA regulates the signal transmission, while CheW connects it to sensory receptors that detect light [ 72 ]. The methylation system involving CheB and CheR allows H. salinarum to modulate its sensitivity to changing light conditions, ensuring appropriate movement towards light sources [ 73 , 74 ]. Plasmid II is characterized by a high density of IS (Insertion Sequence) and ISH (Insertion Sequences of Halophiles) elements, the latter are unique to halophilic archaea [ 75 , 76 ], which vary in sequence and structure but share the common feature of having inverted repeats at their ends and a gene encoding a transposase enzyme [ 75 , 77 ]. The clustering of IS elements creates hotspots of genetic variability, influencing the expression and regulation of neighboring genes and facilitating horizontal gene transfer, thereby enhancing the organism evolutionary adaptability by inducing mutations and genomic rearrangements [ 78 ]. Other Halobacterium strains, such as NRC-1, also display an accumulation of frequent ISH insertions possibly mediating rapid adaptation to environmental stresses [ 79 ]. Interestingly, a potential homolog of the COQ5 gene was found within the largest plasmid. This gene catalyzes the only C-methylation involved in the biosynthesis of coenzyme Q in humans and the yeast Saccharomyces cerevisiae [ 80 ]. An additional homologue has been reported in E. coli , named UbiE [ 81 ]. Both UbiE and COQ5 are members of a family of methyltransferases involved in the biosynthesis of menaquinone and ubiquinone [ 81 ]. Members of this clade are widely distributed among bacteria and eukaryotes but are absent in archaea [ 82 ]. Future research should address specifically whether this gene performs a similar function as UbiE in E. coli and COQ5 in S. cerevisiae. Noteworthy, the reduction in plasmid size in AD88 , coupled with a high GC content, suggests an evolutionary response to the selective pressures of the CCB environment, where low nutrient availability, particularly the scarcity of phosphorus, could favor smaller, less metabolically demanding plasmids. Larger plasmids, which often carry additional genes, impose metabolic costs that make them energetically costly to maintain under nutrient-limited conditions. These costs can lead to a selective disadvantage, reducing the microorganism’s fitness, as resources that would otherwise be used for growth and survival are diverted to maintain and replicate larger plasmids. A reduction in the reproductive success of individuals harboring larger plasmids, could lead to a decrease in their frequency within the population over time and drive the fixation of smaller plasmid variants. Consequently, smaller plasmids would enable AD88 ecotype to distribute more energy towards essential cellular functions, such as, protein synthesis and replication in response to CCB specific environmental conditions [ 83 , 84 ]. \n Fig. 2 Genomic and functional characterization of plasmids in the Halobacterium salinarum AD88 genome. The figure displays circular maps of two plasmids, Plasmid I (22,589 bp) and Plasmid II (115,924 bp), highlighting their genomic structure and annotated genes. The outermost rings represent annotated genes categorized by their functions. Genes associated with antibiotic resistance are labeled in red (e.g., tetracycline resistance gene tet , β-lactam resistance gene OXA , and macrolide resistance genes erm and msr ). Mobile genetic elements, such as transposases and insertion sequences, regions involved in horizontal gene transfer, replication and plasmid stability are highlighted in purple, including ori regions and partitioning systems. The concentric circles indicate GC content (black) and GC skew (innermost), visualizing nucleotide composition patterns for each plasmid \n Ploidy reduction in Halobacterium salinarum AD88 The genomic analysis showed consistent patterns in the distribution of read coverage and k-mer frequencies, suggesting a ploidy of two in the genome of Halobacterium salinarum AD88 (Fig. 3 ). The observation that AD88 possesses only two copies of its genome is intriguing given the conventional understanding that H. salinarum strains typically exhibit high levels of genome copy number [ 85 ]. The standard expectation within halophiles is the maintenance of multiple copies of their genomes, often ranging from 10 to 25 copies per cell, to cope with the extreme osmotic stress in their natural saline environments [ 14 ]. For other strains of H. salinarum , the cellular ploidy undergoes dynamic changes throughout the growth phases. During the exponential phase, fast-growing cells exhibit an average of approximately 25 copies of the chromosome, which decreases to 15 copies during the early stationary phase [ 14 ]. This reduction in ploidy is not influenced by variation in strain growth rate, as cultures with a twofold lower growth rate maintain the same chromosome copy number. Similarly, in Haloferax volcanii , the genome copy number remains high during the exponential phase, with an average of 18 copies per cell, but decreases to 10 copies upon entering the stationary phase [ 14 ]. The high ploidy level has been attributed to various factors, including the need for increased gene dosage to counteract the deleterious effects of high salt concentrations on DNA stability and replication fidelity [ 86 ]. \n Fig. 3 Smudge plot for the genome of H. salinarum AD88 reveals consistent read coverage and k-mer frequency patterns, suggesting a ploidy of two \n Polyploids can have several evolutionary advantages in comparison to monoploid species, such as gene redundancy, allowing mutations to occur in one copy of a gene without losing the original wild-type gene sequence, resistance against conditions that induce double strand breaks (DSB’s), and generally having lower spontaneous mutation rates [ 87 ]. A single genome duplication in AD88 strain could be linked to specific adaptations or niche preferences of this particular isolate. For genome duplication to be sustained by natural selection, its phenotypic benefits must outweigh the significant energetic and resource costs [ 88 ]. It is possible that the strain has evolved alternative mechanisms to mitigate stress, allowing it to succeed with fewer genome copies compared to other strains or species within the same genus. However, the reduced genome copy number in AD88 raises questions about the trade-offs between genome duplication and cellular fitness in extreme environments. While high genome ploidy may confer advantages regarding genomic robustness and adaptation to saline conditions, it also incurs metabolic costs associated with DNA replication, maintenance, and resource allocation [ 89 , 90 ]. Therefore, the observed low ploidy level in our strain may reflect a balance between the benefits of genome duplication and the metabolic demands imposed by maintaining multiple copies of the genome. Metabolic adaptations in response to phosphorus scarcity Amidst this genetic dynamism, there is a deeper narrative of ecological interconnectedness. Microbial life in CCB is partly a collective eco-evolutionary response to a complex web of abiotic interactions [ 53 ]. The scarcity of phosphorus and high mineral content in CCB appear to be the principal factors shaping the genetic landscape, where beneficial mutations are fixed, and the selection of genes involved in nutrient acquisition and metabolic versatility confers new traits that enhance fitness. While comparing metabolic capabilities of AD88 with other Halobacterium strains, we identified 20 orthologous gene families (Fig. 4 ). The identification of gene families shared between AD88, and a limited number of reference genomes suggests selective pressure driving the retention of these genes in response to similar environmental conditions. However, evaluating the homology of pathway genes using a 70% sequence identity threshold, did not detect a fully conserved presence of those pathways in Halobacterium sp. GSL 19, H. salinarum NRC 34001, and Halobacterium sp. NRC 1. Nevertheless, the absence of full pathway integrity at this threshold does not necessarily indicate that these strains lack the pathway entirely. It is possible that alternative genes or functionally analogous pathways fulfill the same metabolic role, or that sequence divergence in these strains reduces homology detection despite functional conservation. Notably, AD88 shared more metabolic similarities to H. salinarum 91-R6 strain than any other, particularly in genes that code for phosphonate and phosphate transporters, and those involved in sulfolipid biosynthesis. Given that the CCB is characterized by persistent phosphorus limitation, it provides an ideal environment where sulfolipid substitution could occur as an adaptive strategy to reduce cellular phosphorus demand while maintaining membrane integrity. Sulfolipids can replace phospholipids in membrane structures, reducing cellular phosphorus demand and offering a selective advantage in environments where phosphorus is scarce [ 91 , 92 ]. Genetic studies have demonstrated that sulfolipid biosynthesis is upregulated under phosphate limitation and that mutants lacking sulfolipids exhibit growth defects, confirming their essential role in maintaining membrane function [ 93 , 94 ]. This metabolic adaptation is reminiscent of the findings in Bacillus coahuilensis [ 21 ], an endemic bacterial species uniquely adapted to the extreme and oligotrophic conditions encountered in the Cuatro Ciénegas Basin (CCB), where the presence of sulfolipid-related genes was also identified as a critical adaptation for survival. The ability to utilize sulfolipids as a substitute for phospholipids suggests a convergent evolutionary strategy among microorganisms in the basin. Further experimental validation is needed to confirm whether Halobacterium salinarum AD88 substitutes phosphate forms with sulfolipids under phosphorus limitation. Lipidomic analyses comparing membrane composition under different phosphorus conditions, along with transcriptomic and mutant studies, could determine if sulfolipid biosynthesis is actively regulated in response to phosphorus stress. \n Fig. 4 Maximum-likelihood phylogenetic tree of 39 conserved ribosomal protein families from the core genome. The tree was inferred using the WAG + F + I + R3 substitution model, selected as the best-fit model via the Bayesian Information Criterion (BIC). Branch support values, derived from the ultrafast bootstrap algorithm with 1,000 replicates \n Further functional annotation revealed genes associated with a complete and specialized pathway for assimilatory sulfate reduction, including PAPSS (3’-phosphoadenosine 5’-phosphosulfate synthase), sat (sulfate adenylyltransferase), cysNC (bifunctional enzyme CysN/CysC), cysH (phosphoadenosine phosphosulfate reductase), and cysJ (sulfite reductase - NADPH flavoprotein alpha-component) (Fig. 5 ). This set of genes encode a complete enzymatic process that spans from the initial activation of sulfate to its final reduction to sulfide, allowing the synthesis of essential sulfur-containing molecules. Unlike dissimilatory reduction, which is primarily for energy production, assimilatory sulfate reduction is an energy-dependent process that requires ATP. This system’s primary function is biosynthetic, incorporating reduced sulfur into cellular components and supporting anabolic processes [ 95 ]. To investigate the uniqueness of the assimilatory sulfate reduction genes found in Halobacterium salinarum AD88, we performed BlastP alignments of all sulfur pathway genes against the 12 Halobacterium species used in this study (Supplementary Material Table 3 ). Our results confirm that AD88 harbors specific distinctive genes, with no full-length homologs identified in other Halobacterium species. While no homologs were found with 100% sequence identity across complete genes, we observed 100% identity in shorter sequence segments (4–5 AA). These findings suggest that while these genes may be unique to AD88 in their entirety, some conserved sequence motifs exist within related species. The absence of full-length homologs does not preclude the possibility of functional or distant homologs within the genus or other haloarchaea. These unique genetic features represent a distinct sulfur-reduction system, which, along with genes coding for resistance to toxic ions such as arsenic and heavy metals, are thought to be additional strategies to cope with phosphorus depletion. Arsenic reduction systems have been previously identified in other archaeal genomes isolated from CCB, such as Halorubrum sp. , found in co-cultured with Marinococcus luteus [ 96 ]. Phosphorus and arsenic are chemical analogs commonly found in oligotrophic environments, and they can substitute for each other in biological processes [ 97 ]. The ability of proteins to interact with multiple substrates, known as promiscuity, often serves as a foundation for the evolution of new protein functions. Thus, the chemical similarities between arsenate and phosphate allow for cross-reactivity between the two, which could facilitate an evolutionary shift. The cross-reactivity might enable organisms to transition from relying primarily on phosphate for metabolic processes to using arsenate, or the reverse, as environmental conditions demand. This flexibility in substrate use may have driven evolutionary changes, allowing AD88 to adapt to an environment with varying levels of these two compounds [ 98 , 99 ]. Overall, these adaptations represent significant evidence of the evolutionary pressures that shape the microbial communities in CCB in general and the AD site in particular, facilitating their survival through genetic flexibility, and demonstrating a remarkable example of how environmental stress can drive genomic innovations and diversification. \n Fig. 5 Sulfur metabolism pathways in a Halobacterium salinarum AD88. The figure illustrates sulfate uptake via the CysPUWA transporter and its subsequent activation to APS and PAPS. Assimilatory sulfate reduction converts sulfate to sulfide for amino acid biosynthesis (e.g., L-cysteine and L-serine), while dissimilatory pathways reduce sulfate to sulfide for energy generation. Intermediates like thiosulfate and trithionate are processed by specific enzymes, integrating sulfur metabolism with broader pathways, including carbon fixation and amino acid synthesis. The oxidation states of sulfur compounds are highlighted throughout the pathways \n Evolutionary relationships within Haloarchaea We conducted a phylogenomic reconstruction to position the newly identified strain within an evolutionary context and assess its relationship to established Halobacteriales taxa (Fig. 4 ). Analyzing their evolutionary distances provided crucial insights into the genetic divergence and relationships among halophilic archaea. The phylogenomic reconstruction reveals that Halobacterium salinarum AD88 clusters tightly alongside laboratory strains like NRC-1 and R1 [ 10 ], displaying ultrashort branches and reflecting recent divergence from a common ancestor. The close genetic relationships within Halobacterium clade suggest that recent evolutionary pressures have maintained high genetic similarity, likely due to similar environmental conditions and adaptive strategies. We propose that their evolutionary adaptations have been driven more by microenvironmental pressures not involving large-scale genetic changes within a relatively recent evolutionary timeframe, which might explain their close phylogenetic clustering. This highlights the importance of integrating phylogenetic, genomic, and ecological data to better understand microevolution in halophilic organisms. Meanwhile, the deeper evolutionary divergences observed with other species points to the long-term evolutionary isolation and specialization of lineages that have adapted to distinct ecological niches, like Haloarcula , Halococcoides and Halodesulfurarcheum genera. From a broader haloarchaeal evolution perspective, the clear separation of Halobacterium from other genera (like Haloferax , Haloarcula ,) reinforces that haloarchaea have diversified into multiple lineages that, despite sharing extreme halophily, have long independent evolutionary histories. Such insights can inform how haloarchaeal species may have radiated into different ecological niches, since some clusters consist of strains isolated from similar habitats (salt mines, salterns, etc.), indicating that geographic or environmental factors played a role in their divergence [ 100 ]. The tree topology also identifies Halobacterium zhouii as one of the more basal (early-branching) members of the genus, indicating it diverged earlier relative to others, this pattern is consistently observed in other phylogenies [ 101 , 102 ]. This aligns with our nucleotide identity analysis, which shows that H. zhouii exhibits the lowest synteny levels compared to AD88, further supporting its early divergence within the genus. A more detailed analysis of nucleotide identity levels, aimed refine the genetic relationships among Halobacterium strains, is discussed later in this study. Pangenome openness provides evidence of ongoing genetic diversification The genome comparison between 13 Halobacterium strains, including H. salinarum AD88, suggests that this genus has an open pangenome (Fig. 6 ) [ 103 ]. We observe that new gene families are continuously incorporated as more strains or species are discovered, with each new genome within Halobacteria bringing ~ 137 new genes to the pool of pan-genes [ 104 ]. In halophilic archaea, such as Halobacterium salinarum , the open pangenome structure may be an adaptive mechanism, enabling the species to prosper in hypersaline environments through extensive gene acquisition and loss. This characteristic is often seen in organisms exposed to dynamic and heterogeneous environments, as genetic flexibility allows for continuous adaptation and survival [ 105 ]. Pan genome analysis identified 3,744 homologous gene groups, of which 1,072 genes form part of the core genome , including those necessary for fundamental biological processes, indicating a shared evolutionary foundation [ 106 , 107 ]. Within the core (%) genome we found genes related to DNA replication and a photoreactivation repair system, such as photolyases coding genes phr1 and phr2 , which directly reverse UV-induced DNA damage, specifically pyrimidine dimers, ensuring the maintenance of genome integrity [ 108 ]. Additionally, 1,325 genes were categorized as softcore , defined as a set of genes found in 95% of the genomes [ 109 ]. Meanwhile, 1,182 were identified as shell genes , which are remaining moderately conserved genes, and present at intermediate frequencies in 15–95% of the strains [ 38 , 110 ]. Both softcore and shell genes show the genetic variability within the genus, suggesting that different species have evolved unique traits for adaptation to the specific environments they were isolated from [ 111 ]. Surprisingly, ars operon genes arsA , arsD , arsR2 , and arsM were identified as part of the shell genome, and not in any of the extrachromosomal replicons, as they usually occur in pNRC100 in Halobacterium sp. NRC-1 [ 112 ]. Last, we found 1,236 cloud genes, which appear in less than 15% of the strains and could indicate a reservoir of genetic diversity that may be involved in niche specialization [ 111 , 113 ]. As part of the cloud genome, we found the pleiotropic regulator bat gene, which controls light-sensing mechanisms and bacteriorhodopsin and retinal chromophore synthesis. We also observed copA and copB genes involved in metal homeostasis and regulate copper transport, sufS involved in the biosynthesis of iron-sulfur clusters and contributes to survival under varying redox conditions, and CRISPR-Cas associated genes, including cas1 , cas2 , and cas6. The high accessory genome in Halobacterium species shows the adaptive importance of genetic exchange mechanisms, particularly horizontal gene transfer (HGT), which is well-documented in haloarchaea and facilitated by genomic island mobility and homologous recombination [ 105 , 114 ]. These mechanisms have been pivotal in haloarchaeal evolution, enabling transitions from autotrophic anaerobes, as seen in methanogenic ancestors [ 115 ] to aerobic lifestyles through gene acquisitions from bacterial sources like Deinococcus radiodurans and Bacillus species [ 11 , 114 ]; Moreover, HGT has played a continuous and critical role in adaptation by providing haloarchaea access to new genetic variation, enabling adaptations that overcome evolutionary limitations. While cumulative mutations may trap species in local adaptive peaks, unable to explore better fitness options, HGT between haloarchaeal species introduces new genes far more rapidly than would occur via mutation without HGT [ 116 ]. HGT opens up evolutionary pathways that would be inaccessible, offering new adaptive opportunities and helping reverse deleterious mutations that may accumulate during periods of purifying selection [ 116 ]. \n Fig. 6 Pangenome and core-genome analysis of the studied strains. The pangenome was determined using the OrthoMCL algorithm, and the distribution of orthologous families is visualized in the Upset plot. The core-genome, observed in the first bar of the Upset plot, consists of 1,073 orthologous families. It was defined as the set of genes present in all strains and identified through the consensus of three algorithms: OrthoMCL (OMCL), Bidirectional Best Hits (BDBH), and COGtriangles \n Given the close phylogenomic relationship between Halobacterium salinarum AD88 and other Halobacterium strains, we conducted this comparative analysis to gain deeper insights into their genetic divergence and evolutionary proximity. This approach allowed us to further resolve their relationships at a finer scale, identifying potential genomic variations that contribute to their differentiation within the genus. When comparing the genomes of different Halobacterium strains, distinct patterns emerge at the nucleotide identity levels (Fig. 7 a). This comparative analysis illustrates how genomic data align with evolutionary and taxonomic distinctions, emphasizing that average nucleotide identity (ANI) values can serve as reliable indicators of genetic relatedness and evolutionary history. ANI values above 95% indicate strains are thought to be part of the same species, and intra-species ANI values tend to cluster closely around this threshold, especially in stable environments. Comparisons between Halobacterium salinarum strains show high ANI values ranging between 98.6 − 99.9%, indicating a high degree of genomic conservation. Within the same species, H. salinarum AD88 shows the highest similarity to H. salinarum NRC 34001, an isolate from a Canadian salted buffalo hide, with an ANI of 99.4%. The high synteny is represented by the nearly continuous alignment of homologous regions, with minimal interruptions or translocations (Fig. 7 b). Such conserved synteny suggests that these strains have undergone limited structural genomic changes since diverging from a common ancestor, likely due to similar environmental pressures that shaped their evolutionary trajectories and preservation of gene function and regulatory networks crucial for survival. At the interspecies level, AD88 shows high similarity to Halobacterium sp. NRC-1 (ANI of 99.1%) isolated from a stable, rich salt flat of Utah, where consistent high salinity and nutrient availability differ markedly from CCB’s dynamic ecosystem. The high similarities suggests shared adaptations for thriving in salt-rich habitats, likely contributing to their genomic overlap. However, it remains intriguing that despite their different isolation sources, they maintain such a degree of nucleotide identity. This similarity emphasizes how shared environmental stressors, such as high salinity, can lead to parallel adaptations through convergent evolution, even when the specific ecological contexts differ. In contrast, AD88 displays significant nucleotide differences when compared to Halobacterium zhouii XZYJT26, isolated from saline soil of Mangkang ancient solar saltern in Tibet, China, showing only 80% identity, and fragmented synteny characterized by multiple breaks, rearrangements, and scattered homologous regions (Fig. 7 b). The nucleotide differences can be attributed to distinct geographic and climate conditions; while the Tibetan region has a high-altitude, cold, and arid climate, CCB is characterized by a warm, semi-arid environment, fostering unique evolutionary divergences that distinguish microbial strains. The observed evolutionary divergence, rooted in environmental and taxonomic differences, is closely tied to broader mechanisms of genome evolution, such as, changes in gene organization. As microbes evolve, their genomes undergo rearrangements, such as inversions, transpositions, and duplications [ 117 ]. These processes disrupt the original order of genes. As a result, only a small fraction of genes retain the same arrangement (synteny) between distantly related organisms, which tend to increase with greater sequence divergence [ 118 ]. However, certain gene clusters maintain synteny even across distantly related organisms, which can signal that these genes are functionally conserved to ensure efficiency in specific niches. When synteny is preserved over large evolutionary distances, it likely reflects strong selection pressure to maintain the arrangement, indicating the importance of these genes, as their order and proximity are critical for their function, due to co-regulation, participation in the same metabolic pathways, and physical interaction in protein complexes [ 119 ]. \n Fig. 7 Average Nucleotide Identity (ANI) Analysis Among Halobacterium Strains. (a) The heatmap shows the ANI values between various strains of Halobacterium genus. Strains of the same species, such as Halobacterium salinarum exhibit high ANI values, reflecting significant genomic conservation, while comparisons between strains from different species, indicates greater evolutionary divergence, (b) The synteny plots of H. salinarum AD88 and related strains illustrate a continuum of evolutionary divergence, where conserved synteny and rearranged regions mirrors the dual demands of stability and innovation. While conserved synteny between H. salinarum AD88 and H. salinarum NRC 34001 are important for maintaining functional core pathways, rearranged regions between H. salinarum AD88 and H. zhouii XZYTJ26 give the evolutionary flexibility required for niche specialization"
} | 10,957 |
31457974 | PMC6644827 | pmc | 8,720 | {
"abstract": "Here,\nwe control the surface hydrophobicity and the adhesion of\nwater droplets by electrodeposition of poly(3,4-ethylenedioxypyrrole)\n(PEDOP) and poly(3,4-propylenedioxypyrrole) (PProDOP) with branched\nalkyl chains placed preferentially on the bridge to favor the formation\nof nanofibers. Branched alkyl chains of various sizes from very short\n(C 3 ) to hyperbranched (C 18 ) are studied because\nthey have lower surface hydrophobicity than long alkyl or fluoroalkyl\nchains (preferable for parahydrophobic properties). The electrodeposition\nis much more favored with the PEDOP derivatives because the ProDOP\nfilms are more soluble. However, the formation of nanoparticles is\nfavored with the PEDOP polymers in contrast to the formation of fibers,\nresembling the wax nanoclusters observed on lotus leaves, with the\nProDOP polymers. With both these PEDOP and PProDOP derivatives, it\nis possible to reach parahydrophobic properties characterized by a\nsticking behavior toward water droplets. This kind of surfaces could\nbe used in the future in water harvesting systems, for example.",
"conclusion": "3 Conclusions Here, we chose to investigate the electrodeposition\nof PEDOP and\nPProDOP films with branched alkyl chains [from very short (C 3 ) to hyperbranched (C 18 )] grafted on the 3,4-alkylenedioxy\nbridge with the aim to favor the formation of nanofibers with parahydrophobic\nproperties. It was easier to obtain insoluble polymer films with PEDOP\npolymers, whereas the polymer insolubility increased with the branched\nalkyl chain length. The formation of nanoparticles was more favored\nwith the PEDOP polymers, whereas with the ProDOP polymers, it was\npossible to obtain nanofibers, resembling wax nanoclusters observed\non lotus leaves. Moreover, with both these PEDOP and PProDOP polymers,\nit was possible to reach parahydrophobic properties with extremely\nhigh water adhesion. These surface properties are extremely interesting\nand could be applied in the future for water harvesting systems, for\nexample.",
"introduction": "1 Introduction The\nneed to control surface-wetting properties is very important\nfor various applications, where interfaces are present such as separation\nmembranes, water harvesting apparatus, nonstick pans, anti-icing windows,\nor antifouling paints. 1 − 4 There are numerous examples in nature, where species have special\nwettability. 5 For example, the superhydrophobic\n(both high hydrophobicity and low water adhesion) properties of lotus\nleaves are a combination of surface structuration at both micro and\nnanoscale and also the surface energy because the nanoclusters present\non these surfaces are composed in a part of hydrophobic waxes. 6 − 8 Other species are also capable to attract small water droplets and\neven fog in hot environments. 9 − 12 These properties are known in the literature as parahydrophobic\nproperties, and these surfaces have both high hydrophobicity and high\nwater adhesion. These species include red roses and geckos, for example.\nFor parahydrophobic properties, it is preferable to have surface structuration\nat only one scale (micro or nano) and also materials of higher surface\nenergy. In particular, one-dimensional (1D) nanostructures such as\nnanofibers or nanotubes are excellent candidates to control the surface\nhydrophobicity and water adhesion because these properties are dependent\non their length, diameter, orientation to the substrates, and their\nspacing, for example. 12 − 17 Conducting polymers are often used to obtain 1D nanostructures. 18 − 20 In the literature, the polyaniline has been extensively studied\nas a polymer model for the understanding of nanostructure formation,\nthanks to intra- and intermolecular interactions such as hydrogen\nbonds and π-stacking. 21 − 25 In particular, Wang et al. used a theoretical model from aniline\ntrimers formed at the beginning of aniline oxidation. This trimer\nbeing asymmetric can lead to various assemblies including nanofibers\n(1D-growth), nanosheets (2D growth), flowerlike or urchinlike structures\n(3D growth), depending on the experimental conditions (pH, reaction\ntime, etc.). 25 For various applications,\nit is necessary for the growth of nanostructures\ndirectly on substrates. 14 In the case of\nconducting polymers, different processes are possible such as vapor-phase\npolymerization, plasma polymerization, electropolymerization, or electrografting. 26 , 27 Among them, the electropolymerization, also called electrochemical\npolymerization or conducting polymer electrodeposition, was demonstrated\nas a fast technique with fine tuning of the surface nanostructures\nby playing with both electropolymerization parameters (solvent, substrate,\nconcentrations, electrolyte, and deposition method) and the monomer\nchemical structures. 28 − 31 In this process, a monomer is electrochemically oxidized on a working\nelectrode to induce the polymerization and the growth of polymer nanostructures.\nThe electropolymerization is performed on a conductive working electrode\nnot easily oxidizible such as gold, platinum, stainless steel, titania,\nor transparent indium tin oxide (ITO)-coated glass. For example, aluminum\nis not a suitable substrate because of the presence of an oxide layer.\nThe polymer growth can also be controlled with the deposition charge/time\nor the number of deposition scans. Another advantage is that the process\nis possible whatever the shape of the substrate such as mesh, textile,\nfoam, or tube. Hydrophobic substituents can also be grafted to change\nthe surface energy even if the substituent can also affect the surface\nstructures. For example, fluorinated and linear alkyl chains were\nused to obtain superhydrophobic properties, whereas branched alkyl\nchains and aromatic groups (substituents of lower hydrophobicity)\nto obtain parahydrophobic properties. 32 The monomers of the 3,4-alkylenedioxypyrrole family such as\n3,4-ethylenedioxypyrrole\n(EDOP) and 3,4-propylenedioxypyrrole (ProDOP) were now sufficiently\nstudied in the literature. 33 − 35 The resulting polymer films are\nexpected to have exceptional optoelectronic properties such as high\nconductivity, electrochromism, and rapid redox switching. For\nthe formation of nanofibers, it is preferable to keep amino\n(NH) groups free, favoring hydrogen bonds. Indeed, it is possible\nto place the substituent on the 3,4-ethylenedioxy bridge. 36 , 37 Fortunately, it was reported that a very interesting way to synthesize\nboth EDOP and ProDOP derivatives with hydroxyl groups on the bridge\nis through the reaction with epibromohydrin. 38 The formation of nanofibers was found to be possible using fluorinated\nchains. Here, to obtain parahydrophobic nanofibers, branched\nalkyl chains\nof various sizes from very short (C 3 ) to hyperbranched\n(C 18 ) have been grafted on the 3,4-alkylenedioxy bridge\nof EDOP and ProDOP. Figure 1 shows the different EDOP- br -R and ProDOP- br -R monomers studied, where different Rs are shown in this\nfigure. The aim is to study their influence on both the formation\nof nanofibers and the resulting hydrophobic properties. Such materials\nhave potential applications in water harvesting or in water/oil separation. 39 In water-harvesting systems, for example, it\nis important to have as possible both the highest apparent contact\nangle while having the strongest water adhesion. The aim is to capture\nwater droplets and to release them such that their size becomes higher. Figure 1 Original\nmonomers studied in this article (EDOP- br -R and ProDOP- br -R).",
"discussion": "2 Results\nand Discussion 2.1 Electrodeposition First of all, the\nmonomer oxidation potential ( E ox ) was\ndetermined by cyclic voltammetry and is between 0.90 and 1.08 V versus\nsaturated calomel electrode (SCE) for the EDOP derivatives and between\n1.08 and 1.14 V for the ProDOP derivatives, as a function of the substituent. Then, cyclic voltammograms were performed to have information of\nthe polymer growth. The cyclic voltammograms are given in Figures 2 and 3 . The most significance is the intensity of the peaks, which\nis much more important using the EDOP monomers, indicating especially\nhigher polymer insolubility and as a consequent a higher amount of\npolymer on the working electrode (confirmed later in the article).\nHowever, the oxidation potentials of the polymers are very low (below\n0 V vs SCE), which shows that the polymer chain lengths are long. Figure 2 Cyclic\nvoltammograms of the EDOP derivatives (0.01 M) in anhydrous\nacetonitrile with Bu 4 NClO 4 (0.1 M); scan rate:\n20 mV s –1 . Figure 3 Cyclic voltammograms of the ProDOP derivatives (0.01 M) in anhydrous\nacetonitrile with Bu 4 NClO 4 (0.1 M); scan rate:\n20 mV s –1 . To better characterize our polymers, we performed UV–vis\nabsorption spectra. For that, the polymers were electrodeposited on\n2 cm 2 ITO substrates but at a constant potential ( E = E ox ) because it is a better\npolymerization method for these monomers. A deposition charge of 200\nmC cm –2 was selected for this characterization.\nOnly the EDOP series was chosen because it was not possible to obtain\npolymer films with all of the ProDOP monomers. For the UV–vis\nmeasurement, it was also necessary to dedope the polymers (for 15\nmin at −1 V vs SCE) before measurement. The spectra are given\nin Figure 4 . This figure\nshows that the maximum absorption wavelength (λ max ) is approximately the same λ max ≈ 414–416\nnm for the polymers with branched alkyl chain from C 3 to\nC 11 , whereas it increases a little to ≈432 nm for\nthe longest branched alkyl chains (C 15 and C 18 ), which may indicate longer polymer chains. Moreover, all of the\nspectra display also strong absorption in the long wavelength region,\nwhich indicates that these polymers were still in the heavily doped\nstate. This is not surprising because it is known that the polymers\nof the poly(3,4-alkylenedioxypyrrole) and poly(3,4-alkylenedioxythiophene)\nare extremely stable in their doped state, and as a consequence, it\nis extremely difficult to fully reduce them. 33 − 35 Figure 4 UV–vis absorption\nspectra of the dedoped PEDOP polymers. For the surface characterization, the polymers were electrodeposited\non 2 cm 2 gold plates at a constant potential ( E = E ox ) but using different deposition\ncharges ( Q s from 12.5 to 400 mC cm –2 ) to control the polymer growth. With the ProDOP\nmonomers, the deposition was possible only with\nultralong branched alkyl chains (ProDOP- br -C 15 and ProDOP- br -C 18 ). Hence, the\nhigh hydrophobicity of these long branched alkyl chains facilitates\ntheir deposition even if they induce important steric hindrance. Hence,\nusing ProDOP- br -C 3 , even if a peak was\npresent in the cyclic voltammetry curve, the polymer is soluble and\ncannot form insoluble film on the working electrode. By contrast,\nusing ProDOP- br -C 18 , a thin film is formed\nbut is less soluble. With the EDOP derivatives, all of the polymers\ncould be deposited\nbecause of the higher polymer insolubility, as observed by cyclic\nvoltammetry. To better understand their solubility, solubility tests\nin acetonitrile were performed ( Table 1 ). It was observed that all of the PEDOP polymers are\nmore insoluble than the PProDOP polymers whatever the branched alkyl\nchain. By contrast, the increase in the branched alkyl chain increases\nthe polymer insolubility. Table 1 Polymer Solubility\nin Acetonitrile polymer solubility PEDOP- br -C3 slightly soluble PEDOP- br -C5 slightly soluble PEDOP- br -C7 slightly\nsoluble PEDOP- br -C11 highly insoluble PEDOP- br -C15 highly insoluble PEDOP- br -C18 highly\ninsoluble PProDOP- br -C3 highly soluble PProDOP- br -C5 highly soluble PProDOP- br -C7 highly\nsoluble PProDOP- br -C11 highly soluble PProDOP- br -C15 soluble PProDOP- br -C18 soluble Scanning electron microscopy\n(SEM) images are given in Figures 5 and 6 . For the EDOP derivatives,\na very significant change was\nobserved in the surface morphology from smooth to spherical nanoparticles\nbecause the branched alkyl chain increases ( Figure 5 ). Moreover, this change is observed from\nC 15 branched alkyl chain. This change in the surface morphology\nconfirms previous works showing that it is possible to change the\nsurface morphology from smooth to rough, if there is a huge change\nin the polymer solubility. 40 Compared to\nprevious works, 37 it should also be noticed\nthat when linear alkyl chains are used, this change takes place with\na shorter chain, and even shorter using fluorinated chains showing\nthat the main parameter is the substituent hydrophobicity. Figure 5 SEM images\nof the polymer films obtained with the PEDOP derivatives\nwith a deposition charge of 400 mC cm –2 . Figure 6 SEM images of the polymer films obtained with the PEDOP\nderivatives\nwith a deposition charge of 400 mC cm –2 . Here, the presence of nanoparticles induces a high\nincrease in\nthe surface hydrophobicity with θ w up to 135°\nfor Q s = 400 mC cm –2 ( Table 2 ). Moreover,\nthese surfaces are completely sticky (parahydrophobic), 11 as shown in Figure 7 . Water droplets placed on these surfaces\nremained completely stuck on them whatever the surface inclination,\nas observed on rose petals. 10 Indeed, for\napplications in water-harvesting systems, the best is to have the\nhighest apparent contact angle while being completely sticky. Figure 7 Picture of\na water droplet on a surface inclined to 90° obtained\nwith EDOP- br -C 18 and with a deposition\ncharge of 400 mC cm –2 . The droplet has a volume\nof 6 μL. Table 2 Roughness\n( R a and R q ) and Wettability Data\nfor the PEDOP Polymers polymer deposition charge [mC cm –2 ] R a [nm] R q [nm] θ w [deg] PEDOP- br -C3 12.5 28 34 83 25 20 25 89 50 25 31 73 100 136 175 79 200 122 171 72 400 154 218 72 PEDOP- br -C5 12.5 34 52 68 25 25 32 64 50 24 29 82 100 110 140 74 200 140 174 78 400 250 312 68 PEDOP- br -C7 12.5 30 37 85 25 95 123 63 50 169 221 85 100 200 257 45 200 210 274 73 400 402 595 68 PEDOP- br -C11 12.5 84 105 92 25 61 79 97 50 148 197 92 100 155 203 96 200 169 219 98 400 620 838 79 PEDOP- br -C15 12.5 46 61 92 25 62 78 88 50 67 78 97 100 55 65 98 200 133 171 117 400 165 208 132 PEDOP- br -C18 12.5 38 45 98 25 38 46 96 50 65 80 108 100 115 145 118 200 124 155 112 400 275 402 135 In\nthe case of ProDOP derivatives, crystal-like structures are\nobtained with ProDOP- br -C 15 ( Figure 6 ). Interestingly,\nthe crystal-like structures look similar to the structures observed\non lotus leaves. 6 This is not surprising\nbecause the structures are in part composed of hydrocarbon waxes,\nwhich are composed of esters with very long alkyl chains. Using ProDOP- br -C 18 , both large wrinkles and spherical nanoparticles\nare observed leading also to a high increase in θ w up to 128° for Q s = 400 mC cm –2 ( Table 3 ). Table 3 Roughness ( R a and R q ) and Wettability Data\nfor the PProDOP Polymers polymer deposition charge [mC cm –2 ] R a [nm] R q [nm] θ w [deg] PProDOP- br -C15 12.5 58 75 95 25 48 58 99 50 44 56 88 100 132 167 100 200 152 204 109 400 395 612 105 PProDOP- br -C18 12.5 52 73 97 25 66 76 98 50 52 61 98 100 92 121 104 200 160 210 119 400 374 494 128"
} | 3,782 |
34233870 | PMC8262818 | pmc | 8,722 | {
"abstract": "Conformal polymer coating leads to damage-tolerant architected ceramic structures with high strength and toughness.",
"introduction": "INTRODUCTION Ceramic materials are widely used in structural applications because of their outstanding environmental resistance, low density, and high strength properties. Also, the remarkable biocompatibility of ceramics has attracted them in many biomedical applications such as bone substitutes, tissue engineering scaffolds, dentals, surgical tools, and instruments. However, they display near-zero plastic deformation and low toughness due to a limited ability to resist fracture. Even the slightest defects or flaws introduced during processing can substantially compromise the strength and toughness of the ceramic. Thus, this inherent brittleness or poor toughening mechanism limits ceramic materials application in many structural components even at ambient conditions. Nature, on the other hand, overcomes such limitations by developing ceramic-based composites through multiple length-scale complex architectures with internalized designs, where the optimized composition of hard minerals is packaged with soft organic phases in a layer-by-layer assembly. There are many examples of lightweight ceramic-based composite structures with excellent strength and toughness in nature made of components that have relatively low mechanical properties. For example, nacre from mollusk shell is composed of ~95 volume % of fine layered brick-like aragonite (CaCO 3 ) platelets (<900 nm) bonded by biopolymers (5 volume %) in a three-dimensional (3D) brick-and-mortar assembly and has a fracture toughness roughly three orders of magnitude higher than its constituents ( 1 – 3 ). Similarly, bone is a hierarchical architected nanocomposite of a soft matrix (collagen fibrils, ~20 to 30%) and hard mineral nanocrystals (plate-shaped hydroxyapatite, ~60%) arranged in a periodic, staggered array along the fibrils ( 4 – 6 ). Despite very high mineral content of these ceramic-based composites, they can arrest crack propagation and avoid catastrophic failure through a combination of various toughening and strengthening mechanisms at many size scales ( 7 – 9 ). Typically, the microstructure of these natural composites is designed in a different architected orientation, while the hard ceramic surface layer provides high fracture strength and a soft proteinaceous subsurface allows large deformation. By mimicking the length scales and hierarchy of these biological materials, several research efforts have focused on developing architected damage-tolerant lightweight engineered ceramic structures at the nano- and microscale ( 10 – 16 ). Typically, synthetic ceramics have been developed using biomimetic mineralization, layer-by-layer deposition, solution casting, self-assembly, freeze casting or ice templating, and additive manufacturing to enhance the toughness of engineered materials ( 17 – 20 ). However, most of these processes are time consuming and are only capable of developing nano- and microscale ceramic-based composite structures. While ice templating ( 21 ) and additive manufacturing ( 22 – 26 ) are promising methods to develop scale-independent structures, there are many challenges that must first be addressed, such as materials limitations, controlled assembly, and surface quality. Hence, development of damage-tolerant ceramic-based architected structures at the macroscale still remains challenging. The bioinspired concept has been implemented for designing other engineered materials such as laminated glass and double-network hydrogel ( 27 – 30 ). In these structures, the primary disadvantage of the stiff and/or brittle phase (low resistance to fracture under loads) has been overcome by forming interlinked polymer-ceramic structures. As a result, combinatorial benefit of high strength and toughness has been achieved, allowing these structures to be used in several applications such as auto windshields, hurricane-proof building windows, blast-resistant windows, and synthetic connective tissues ( 27 , 28 , 30 ). Nevertheless, this particular concept can be further stretched for the development of innovative material design in terms of structural arrangement and/or configuration. Specifically, damage tolerance in ceramic-based structures by simply and cost-effectively wrapping a thin polymer film around, while ensuring no infiltration or composite formation, has not been realized yet. Although molten glass glaze in traditional ceramics has been used with the intention of filling surface cracks and achieving a smooth surface, such coating layers do not improve, if not deteriorate, the inherent brittleness of the structure. Here, we report a far simpler fabrication of damage-tolerant architected ceramic structures via stereolithography (SLA) 3D printing followed by a conformal polymer micro-coating that externalizes the soft phases entirely from the ceramic structures. A well-known architected geometry called schwarzite ( 31 ) has been developed using commercial silica-filled preceramic polymer, the polymer was completely pyrolyzed to create a fully ceramic structure, and then the ceramic structure was conformally coated with a thin layer of flexible epoxy polymer. The mechanical behavior of the coated architected ceramic has been analyzed by a uniaxial compression test and compared to the behavior of uncoated ones. The crack initiation, propagation, and arresting of these architected structures have been investigated by in situ micro–computed tomography (micro-CT) under different compression loads. Then, finite element method (FEM) based on the continuum plasticity-based damage model was performed to understand the damage propagation of the architected structure in compression load due to the conformal coating. Furthermore, we carried out atomistic modeling to reveal the strengthening and toughening mechanism of the epoxy-coated ceramic under uniaxial compression loading. Last, we analyzed different architectures and geometries to reveal the effect of coating for the general applicability regardless of the core ceramic structures.",
"discussion": "DISCUSSION In summary, we have printed the architected complex ceramic structure via SLA 3D printing, coated it with a thin polymer coating (~70 to 100 μm), and characterized its mechanical properties. The uniaxial compression test shows multifold improvement in strength and toughness of the coated ceramic geometry as compared to uncoated ceramic one. By in situ micro-CT imaging, we have demonstrated that the polymer micro-coating plays a crucial role in avoiding crack interconnection and propagation. Also, FEM analysis shows the strain accommodation and crack trapping due to the polymer coating. While the effect of micro-coating in crack initiation is not substantial, it considerably delays the damage propagation and catastrophic failure of the ceramic structures. While the effect of the polymer coating is shown to be independent of the structure of the underlying ceramic, the efficacy of the method is more pronounced in architected porous structures compared with their solid dense counterparts in terms of toughness due to their higher surface area. We envisage that our simple approach of externalizing soft phases unlike natural ceramic composite could be extended to many structural applications, where simultaneous optimization of weight and mechanical performance of the ceramic is required."
} | 1,859 |
33271798 | PMC7760959 | pmc | 8,724 | {
"abstract": "Over the past few decades, bioengineered cyanobacteria have become a major focus of research for the production of energy carriers and high value chemical compounds. Besides improvements in cultivation routines and reactor technology, the integral understanding of the regulation of metabolic fluxes is the key to designing production strains that are able to compete with established industrial processes. In cyanobacteria, many enzymes and metabolic pathways are regulated differently compared to other bacteria. For instance, while glutamine synthetase in proteobacteria is mainly regulated by covalent enzyme modifications, the same enzyme in cyanobacteria is controlled by the interaction with unique small proteins. Other prominent examples, such as the small protein CP12 which controls the Calvin–Benson cycle, indicate that the regulation of enzymes and/or pathways via the attachment of small proteins might be a widespread mechanism in cyanobacteria. Accordingly, this review highlights the diverse role of small proteins in the control of cyanobacterial metabolism, focusing on well-studied examples as well as those most recently described. Moreover, it will discuss their potential to implement metabolic engineering strategies in order to make cyanobacteria more definable for biotechnological applications.",
"introduction": "1. Introduction In nature, proteins are one of the most versatile classes of biological compounds. They serve multiple purposes as structural components, enzymes, membrane transporters, signaling molecules or regulatory factors. Given their tremendous variability in fulfilling tasks in all aspects of life, it is not surprising that proteins come in a manifold of sizes and shapes. For example, they can be single domain proteins or a part of huge protein complexes. The biggest so-far known example that is not part of a multiunit structure is the protein Titin, which is part of vertebrate muscles [ 1 ]. Depending on the splice variant, Titin has a size of 27,000–35,000 amino acids and contains over 300 domains [ 2 ]. On the contrary, the protein Tal, which was found in Drosophila melanogaster is composed of only 11 amino acids [ 3 ]. Albeit being so small, it is involved in controlling gene expression and tissue folding and hence, is the shortest functional protein described so far. It is known that the mean protein length of bacteria is 40–60% shorter than of eukaryotes [ 4 ]. Moreover, it was found that up to 16% of all proteins in a prokaryotic organisms might be actually smaller than 100 amino acids [ 5 ]. Consequently, more and more studies are suggesting that likely hundreds of small proteins are synthesized in bacterial cells and serve important structural and regulatory functions [ 6 ]. Of course, some of these small proteins are known for decades and well-studied, such as, for example, thioredoxins, which play important roles as antioxidants in almost all organisms, not only prokaryotes [ 7 , 8 , 9 ]. However, genes encoding small proteins are likely to be overlooked even in modern genome annotations because the minimal cutoff for small open reading frames is typically set to 100 amino acids [ 10 , 11 ]. In turn, this indicates the existence of a whole, unexplored universe of small proteins to be discovered in bacteria, which is especially exemplified by the phylum of cyanobacteria. Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis. To conduct and maintain their complex photosynthetic machinery, which is composed of several functionally related protein complexes, cyanobacteria use a plethora of small proteins. Some examples like Psb27 have been shown to be important in photosystem II (PSII) repair [ 12 ], while others like PetP are involved in stress adaptation of the photosynthetic electron transport chain [ 13 ]. In fact, more than 10 proteins smaller than 50 amino acids have been characterized to be important for the function of PSII alone [ 14 , 15 ]. Additionally, 293 candidate-genes for proteins smaller than 80 amino acids have been identified in the cyanobacterial model organism Synechocystis sp. PCC 6803 (hereafter Synechocystis ), indicating that cyanobacteria provide a paradigm for the utilization of small proteins and hence, the functional characterization of bacterial micro-proteomes [ 16 ]. This review highlights further prominent examples of small proteins in cyanobacteria beyond the photosynthetic apparatus, i.e., those exercising a regulatory function related to primary metabolism. However, in the literature, different definitions for the term ‘small protein’ exist. Some studies limit the term to proteins ≤85 amino acids [ 17 ], while others also include proteins up to a size of 200 amino acids [ 18 ]. Some authors also use the term ‘microproteins’ which is typically defined as proteins up to a size of 80 amino acids [ 16 ]. In this review, we did not set a specific cut-off for the size of considered proteins, but focused on those candidates that regulate metabolic pathways via protein–protein interaction, among which various truly small proteins of only a few kDa are found. Finally, the potential of small proteins as an add-on for the currently existing molecular toolbox for metabolic engineering in cyanobacteria is discussed."
} | 1,317 |
22563465 | PMC3341377 | pmc | 8,725 | {
"abstract": "Marine cyanobacteria of the genus Trichodesmium occur throughout the oligotrophic tropical and subtropical oceans, where they can dominate the diazotrophic community in regions with high inputs of the trace metal iron (Fe). Iron is necessary for the functionality of enzymes involved in the processes of both photosynthesis and nitrogen fixation. We combined laboratory and field-based quantifications of the absolute concentrations of key enzymes involved in both photosynthesis and nitrogen fixation to determine how Trichodesmium allocates resources to these processes. We determined that protein level responses of Trichodesmium to iron-starvation involve down-regulation of the nitrogen fixation apparatus. In contrast, the photosynthetic apparatus is largely maintained, although re-arrangements do occur, including accumulation of the iron-stress-induced chlorophyll-binding protein IsiA. Data from natural populations of Trichodesmium spp. collected in the North Atlantic demonstrated a protein profile similar to iron-starved Trichodesmium in culture, suggestive of acclimation towards a minimal iron requirement even within an oceanic region receiving a high iron-flux. Estimates of cellular metabolic iron requirements are consistent with the availability of this trace metal playing a major role in restricting the biomass and activity of Trichodesmium throughout much of the subtropical ocean.",
"conclusion": "Conclusion Relative to other diazotrophs [28] , [41] , enhanced iron demand resulting from coordination of photosynthesis and nitrogen fixation may contribute to Trichodesmium being particularly sensitive to iron availability. In turn, high iron requirements have likely led to the evolution of both mechanisms for acclimating to reduced availability [19] and novel acquisition strategies [89] . Using a combination of laboratory and field experiments we suggest that natural Trichodesmium populations from the subtropical North Atlantic display molecular characteristics representative of a reduction in metabolic Fe-metaloenzyme requirements relative to iron-replete cultures. It thus appears that iron may influence Trichodesmium ecophysiology even in some regions receiving relatively high dust inputs. The current study adds further molecular level understanding to a growing body of evidence supporting the role of iron as a control on oceanic N 2 fixation.",
"introduction": "Introduction Open-ocean diazotrophic cyanobacteria, such as Trichodesmium spp., are of particular importance due to their contributions to the carbon (C) cycle through primary production and to the nitrogen (N) cycle through the fixation of atmospheric N 2 \n [1] – [3] . Trichodesmium spp. are thought to comprise the most abundant diazotrophic cyanobacteria in the oceans [3] , often forming widespread blooms over subtropical and tropical regions and potentially contributing a larger fraction of total marine nitrogen fixation than any other organism [3] – [6] . While marine phytoplankton are N-limited through much of the tropical and subtropical oceans [7] – [9] , diazotrophic growth is likely limited by the availability of other nutrients such as phosphorous (P) and iron (Fe) [10] – [15] . Within the photosynthetic apparatus, the chlorophyll-binding membrane protein complexes photosystem II (PSII) and photosystem I (PSI) both have an absolute requirement for iron. Photosystem I (PSI) represents the largest photosynthetic requirement for iron, each PSI trimer containing 36 Fe atoms in 9 [4Fe-4S] clusters [16] . Compared with other autotrophs, photosynthetic diazotrophs, including Trichodesmium , have a significant further iron requirement due to the abundance of iron-containing enzymes in the nitrogen-fixation apparatus [17] – [19] . The nitrogenase complex (the key enzyme in N 2 fixation) is composed of two Fe-proteins encoded by the nifH gene, each containing 4 Fe atoms [20] and a dimeric MoFe protein encoded by nifD and nifK genes, which contains a total of 30 Fe atoms [20] , [21] , making it one of the most iron-rich enzymes in nature [17] , [22] . As a consequence, the availability of iron in marine systems appears to greatly influence N 2 fixation in cyanobacteria [10] , [23] – [26] . Models predict that the distribution of nitrogen fixation in the modern ocean may be constrained by the availability of iron [27] – [29] . Moreover, oceanographic distributions are consistent with the availability of iron affecting N 2 fixation and, consequently, the biogeography of diazotrophic organisms including Trichodesmium \n [14] , [15] . The North Atlantic Ocean has some of the highest N 2 fixation rates in the global ocean [15] , with waters characterized by high dissolved Fe concentrations tightly linked to high atmospheric dust inputs [14] , [30] . However, evidence for enhanced N fixation rates following Fe addition to North Atlantic waters suggest that enhanced Fe may influence Trichodesmium growth even in this ocean basin [12] , [31] . Diazotrophy is a significant challenge for oxygenic photoautotrophic microorganisms because O 2 is inhibitory to the N 2 reduction enzyme nitrogenase [32] , [33] . Diazotrophs have developed specific molecular and physiological strategies to protect nitrogenase from the O 2 evolved during photosynthesis [28] , [34] , [35] . Some diazotrophs have adapted to fix nitrogen during the dark period to avoid photosynthetic oxygen inhibition of the nitrogenase complex (temporal separation) [36] – [39] , while others have terminally differentiated cells, termed heterocysts, with thickened cell walls and reduced photosynthetic activity (spatial separation) [40] . The non-heterocystous Trichodesmium uniquely undertakes both CO 2 and N 2 fixation during the day in the same cell through a complex combination of apparently reversible temporal and intracellular-spatial separation of these processes [4] , [34] , [35] , [41] . The simultaneous occurrence of two iron-rich metabolic processes in Trichodesmium may also suggest a higher specific iron requirement per cell than for other diazotrophic organisms [28] . While the impacts of iron on growth, C and N 2 fixation, O 2 production and dark respiration in Trichodesmium in culture have been well documented [24] , [25] , [42] – [44] , relatively little is known of the molecular adaptation of this organism to iron availability in the environment. Although transcriptomic and protein level responses to iron stress have been observed in culture [19] , [45] – [47] , information from in situ natural populations is limited [48] – [50] . Here we report the responses of photosynthetic and nitrogen fixing proteins to iron availability in laboratory cultures of Trichodesmium IMS101 and compare these with natural populations of Trichodesmium from the subtropical North Atlantic. We quantified peptides indicative of the abundance of the major iron-binding proteins involved in nitrogen fixation and photosynthesis, including the iron protein of nitrogenase (NifH) and the major chlorophyll-binding proteins in photosynthesis, the D1 protein of PSII (PsbA) and a core subunit of PSI (PsaC). In addition we assessed the presence and accumulation of the iron-stress-induced chlorophyll-binding protein IsiA [51] , [52] . Through absolute quantification of these enzymes we characterized the molecular acclimation strategy of Trichodesmium to iron availability and hence estimate the iron supplies required to sustain natural populations.",
"discussion": "Discussion In cultures of Trichodesmium IMS101, the onset of iron starvation was indicated by a decline in photochemical efficiency (F v /F m ), as previously shown [25] , [41] . Although comparison of absolute values of F v /F m will be complicated by a range of other growth factors, in situ values measured on natural populations of Trichodesmium were consistent with those previously reported in the literature [34] and most comparable to iron-starved cultures. The photosynthetic/nitrogen-fixation protein profiles of Trichodesmium either in culture or in the field show a high ratio of NifH to PsbA and PsaC proteins ( Figure 5B and 5C ), potentially reflecting a lower enzymatic rate for nitrogenase compared to photosynthetic proteins [48] . Our results reveal a small decrease in abundance of photosystem I and II complexes, but a much more significant decline in nitrogenase in iron-starved cultures. These results corroborate the trends observed at a gene-expression level by [19] who documented an early decline in nifH transcripts during the development of iron stress followed by a later decline in transcripts encoding the photosynthetic apparatus. Consequently it appears that the metabolic process of N 2 fixation may be more sensitive to iron availability than photosynthesis [19] . In addition, PSII appears to be less sensitive to iron limitation than PSI at both gene and protein levels [19] , [48] . Our results indicate that the nitrogenase enzyme is the major metabolic sink for iron in Trichodesmium cells. Transfer of iron from the energetically and iron-expensive processes of nitrogen fixation to maintain energy production via the photosynthetic apparatus [19] , [47] could thus be speculated to act as a mechanism for surviving in oligotrophic subtropical and tropical areas characterized by fluctuations of iron supply [71] . Nitrogen fixation has been studied extensively in the tropical and subtropical North Atlantic [4] , [5] , [14] , [67] . Due to the proximity of African deserts, most significantly the Sahara, the area is subject to high rates of dust deposition [71] – [75] , which likely result in the observed high surface iron concentrations [14] . Indeed, the low latitude North Atlantic has the highest known Fe concentrations of any of the global subtropical basins [15] . Despite this relatively high iron availability, we still found potential protein level evidence of reduced iron requirements within natural Trichodesmium populations based on comparisons with the cultured strain IMS101. Significant caveats clearly need to be acknowledged when comparing culture results from a starvation experiment, run on a specific strain, under a limited set of other culture conditions, to field populations experiencing variable additional environmental forcings. Thus, although N 2 -fixing enzyme abundances were in similar range in both Trichodesmium natural populations and in iron-starved cultures, phosphate is also severely depleted in the subtropical North Atlantic gyre, likely as a result of enhanced N 2 fixation due to dust deposition [14] , [76] . Consequently, natural Trichodesmium colonies from the region frequently display evidence of phosphate stress [13] , [50] , [77] – [78] , which may thus contribute to any reduced capacity for N 2 fixation [11] . The IsiA protein was also present in all the natural populations sampled ( Figure 5C ) and is expressed [19] or accumulates ( Figure 5A ) under development of iron stress in Trichodesmium cultures. Although IsiA may be expressed under other growth conditions, including high light [79] , acclimation to iron stress appears to be the primary functional role for this chlorophyll binding complex [47] , [80] – [83] , which can act as a light-harvesting antenna for PSI [52] , [82] . In the present study, the IsiA protein constitutes a significant portion of the chlorophyll-binding protein in iron-starved cultures of Trichodesmium ISM101 and in natural populations ( Figure 6A ), with up to 4 and 6 times more IsiA than PSI and PSII proteins in starved cultures and natural populations, respectively. The observed maintenance of PSI electron transport throughout the photoperiod in natural communities, in contrast to the down-regulation of electron transport through oxygen-evolving PSII ( Figure 3D ), supports the suggestion that PSI electron transport may act to consume cellular O 2 in order to protect nitrogenase from inactivation in Trichodesmium \n [33] . A subsequent requirement for maintenance of cellular PSI concentrations may further enhance the iron requirements of this organism compared with other diazotrophs [41] . For example, Crocosphera watsonii separates N 2 fixation and oxygenic photosynthesis over the diel period [34] and hence can effectively share cellular Fe between these molecular processes [28] . Maintenance of PSI electron transport may also provide a rationale for increasing the light-harvesting cross-section of PSI in Trichodesmium under conditions of reduced iron availability through the expression of IsiA and the synthesis of IsiA-PSI supercomplexes [52] , [84] . Similar to previous calculations [25] , [66] estimation of iron within the molecular mechanisms of N 2 fixation and photosynthesis provides a means of extrapolating to the natural environment. Our estimate of 80–90% of the cellular Fe pool being associated with nitrogenase is consistent with theoretical calculations suggesting that 35–78% of the cellular Fe pool would be associated with N 2 fixation under optimal catalytic conditions [18] . Kustka et al. [18] estimated that cellular Fe:C ratios of 28–40 µmol:mol would be required to maintain a moderately iron-limited growth rate of around 0.1 d -1 , consistent with both laboratory studies [41] and our observations of natural populations ( Table 4 ). Combining observed nitrogen specific N 2 fixation rates with metabolic iron standing stocks, natural populations of Trichodesmium in the region sampled during D326 would hence require 50–100 nmol Fe m -2 d -1 ( Table 4 ). Cautious comparison with culture data ( Table 3 ) further indicates that iron requirements could potentially be an order of magnitude higher under fully iron replete conditions. Observed ten-fold higher standing stocks of Trichodesmium in other regions of the North Atlantic [67] , would also require correspondingly higher Fe turnover. Although iron is known to be readily recycled in the upper ocean, knowledge of the differential cycling of nutrients (i.e. N or Fe) in oligotrophic systems is incomplete [85] . Given that the N 2 fixed by diazotrophs represents a source of ‘new’ nitrogen ( sensu \n [86] ) to first order it is reasonable to postulate that new iron inputs [85] may be required to balance much of the daily diazotrophic requirement. Given our estimated metabolic Fe demands, atmospheric dust related inputs of dissolved iron to the subtropical North Atlantic would thus be sufficient to satisfy the requirements of the large standing stocks of Trichodesmium observed in this region ( Table 4 ). However, inputs would potentially be insufficient to satisfy the requirements of a metaloenzyme composition equivalent to that of an iron-replete culture. Simple ecological theory predicts that the rate of supply of a limiting resource will control the organism standing stock, while the ecophysiological characteristics of these organisms will determine the ambient concentration of the resource [29] , [87] , [88] . We thus suggest that the response of Trichodesmium to atmospheric dust inputs to the North Atlantic [14] may lead to population growth and subsequent iron uptake reducing bioavailable levels to the point necessitating some reduction of cellular requirements. Such biological control of iron availability by the diazotrophic population would, however, be unlikely to result in severe stress and heavily reduced growth, a scenario which is consistent with the lack of expression of biomarkers of high iron stress in the region [46] , [50] . Other factors will clearly complicate this simple scenario, including non-biological influences on surface iron bioavailability [84] and, particularly in the North Atlantic, depletion of phosphorous [11] , [12] , [50] , [76] . However, consistent with previous experimental work [12] , [50] , it appears that a limited degree of diazotrophic iron stress may potentially develop even in some regions of high input. In contrast to the North Atlantic, other subtropical oceanic regions receive much lower iron inputs, which may thus represent a severe constraint on the accumulation of high standing stocks of Trichodesmium \n [15] , [25] , [71] . For example, Trichodesmium biomass [14] and hence estimated iron requirements are 3 orders of magnitude lower in the subtropical South Atlantic ( Table 4 ). Our molecular characterization of the metabolic pools within natural populations of Trichodesmium hence provides further evidence of an important role for iron in dictating the large scale biogeography of this important diazotrophic taxon [15] , [25] , [27] , [29] . Conclusion Relative to other diazotrophs [28] , [41] , enhanced iron demand resulting from coordination of photosynthesis and nitrogen fixation may contribute to Trichodesmium being particularly sensitive to iron availability. In turn, high iron requirements have likely led to the evolution of both mechanisms for acclimating to reduced availability [19] and novel acquisition strategies [89] . Using a combination of laboratory and field experiments we suggest that natural Trichodesmium populations from the subtropical North Atlantic display molecular characteristics representative of a reduction in metabolic Fe-metaloenzyme requirements relative to iron-replete cultures. It thus appears that iron may influence Trichodesmium ecophysiology even in some regions receiving relatively high dust inputs. The current study adds further molecular level understanding to a growing body of evidence supporting the role of iron as a control on oceanic N 2 fixation."
} | 4,429 |
38632040 | PMC11075768 | pmc | 8,726 | {
"abstract": "Abstract Aquatic ecosystems are large contributors to global methane (CH 4 ) emissions. Eutrophication significantly enhances CH 4 -production as it stimulates methanogenesis. Mitigation measures aimed at reducing eutrophication, such as the addition of metal salts to immobilize phosphate (PO 4 3− ), are now common practice. However, the effects of such remedies on methanogenic and methanotrophic communities—and therefore on CH 4 -cycling—remain largely unexplored. Here, we demonstrate that Fe(II)Cl 2 addition, used as PO 4 3- binder, differentially affected microbial CH 4 cycling-processes in field experiments and batch incubations. In the field experiments, carried out in enclosures in a eutrophic pond, Fe(II)Cl 2 application lowered in-situ CH 4 emissions by lowering net CH 4 -production, while sediment aerobic CH 4 -oxidation rates—as found in batch incubations of sediment from the enclosures—did not differ from control. In Fe(II)Cl 2 -treated sediments, a decrease in net CH 4 -production rates could be attributed to the stimulation of iron-dependent anaerobic CH 4 -oxidation (Fe-AOM). In batch incubations, anaerobic CH 4 -oxidation and Fe(II)-production started immediately after CH 4 addition, indicating Fe-AOM, likely enabled by favorable indigenous iron cycling conditions and the present methanotroph community in the pond sediment. 16S rRNA sequencing data confirmed the presence of anaerobic CH 4 -oxidizing archaea and both iron-reducing and iron-oxidizing bacteria in the tested sediments. Thus, besides combatting eutrophication, Fe(II)Cl 2 application can mitigate CH 4 emissions by reducing microbial net CH 4 -production and stimulating Fe-AOM.",
"introduction": "Introduction Aquatic ecosystems are responsible for half of the global CH 4 emissions (Rosentreter et al. 2021 ). CH 4 release from organic sediments is strongly driven by eutrophication (Davidson et al. 2018 ), which remains a significant and ongoing worldwide problem (Beaulieu et al. 2019 ). Eutrophication increases autochthonous organic matter production, which precludes substrate limitation and promotes methanogenesis, and hence is estimated to increase CH 4 emissions by 30%–90%, making eutrophic waters hotspots for CH 4 emission (Attermeyer et al. 2016 , Beaulieu et al. 2019 ). Besides nutrient-load reduction, geo-engineering techniques are increasingly used to combat eutrophication. These include techniques using PO 4 3− (P)-binding compounds such as Fe(II)Cl 2 , lanthanum-modified bentonite clay, and aluminum-modified zeolite (Jančula and Maršálek 2011 ). However, there is little insight into how eutrophication remediation strategies affect aerobic and anaerobic CH 4 -cycling microorganisms, and how these affect CH 4 emissions (Jančula and Maršálek 2011 , Nijman et al. 2022 ). Therefore, in this study, we tested the effect of the P-binding agent Fe(II)Cl 2 on microbial CH 4 -cycling in field experiments and batch incubations. In oxic layers of Fe-rich sediments the majority of the phosphorus (P) is bound to ferric iron (Fe(III)) (Parsons et al. 2017 ). Once the bound Fe(III)-P reaches the anoxic zone of the sediment, bound P is released from the Fe(III)-P complex, as a consequence of the reduction and accompanying dissolution of the Fe particles (Cooke et al. 1993 ). Subsequently, in the anoxic zone of aquatic sediments, in the presence of Fe(II), P is known to form vivianite (Fe(II) 3 (PO 4 ) 2 •8 H 2 O) through authigenesis. Vivianite is a hydrated ferrous phosphate mineral that immobilizes phosphate (Walpersdorf et al. 2013 , Rothe et al. 2014 , Liu et al. 2018 , Heinrich et al. 2021 ). However, when exposed to alternative electron acceptors (e.g. O 2 , NO 3 − , NO 2 , SO 4 2− ) up to 50% of the Fe(II) present in vivianite can be oxidized to poorly crystalline mixed-valence or ferric Fe(III)-P molecules, increasing the bioavailable Fe(III) in the sediment (Nielsen and Nielsen 1998 , Miot et al. 2009 , Kusunoki et al. 2015 , Rothe et al. 2016 ). Additionally, ferrous Fe-salts like Fe(II)Cl 2 that are directly applied in the sediments can be directly oxidized with O 2 or NO 3 − (Benz et al. 1998 , Nielsen and Nielsen 1998 , Oikonomidis et al. 2010 ). Fe(II)Cl 2 application could therefore, through direct Fe(II)Cl 2 oxidation or through vivianite oxidation, substantially increase the bioavailable Fe(III) concentrations in the sediment. CH 4 formation (methanogenesis) is a microbial process commonly taking place in anoxic sediments (Segers 1998 ). CH 4 can be oxidized to CO 2 by methanotrophic microorganisms, either aerobically, using O 2 as electron acceptor, or anaerobically, using different alternative electron acceptors (e.g. NO 3 − , Fe(III), Mn 4+ or SO 4 2− ) depending on their availability (Segers 1998 ). Anaerobic oxidation of CH 4 (AOM) can oxidize up to 50–90% of the CH 4 formed in natural aquatic sediments and is suggested to be among the main processes involved in lowering the natural aquatic CH 4 emissions (Norði et al. 2013 , Segarra et al. 2015 , Weber et al. 2016 , Vigderovich et al. 2022 ). In Fe(III) rich freshwater sediments, Fe-dependent anaerobic CH 4 -oxidation (Fe-AOM) (Equation 1 ), has been found to oxidize up to 15% of the produced CH 4 (Sivan et al. 2011 , Ettwig et al. 2016 , Scheller et al. 2016 ), by exploiting soluble and nanophase Fe(III) as electron acceptors (Norði et al. 2013 ). \n (1) \n \\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym}\n\\usepackage{amsfonts}\n\\usepackage{amssymb}\n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n\\begin{eqnarray*}\n{\\mathrm{C}}{{\\mathrm{H}}}_4 + {\\mathrm{\\ }}2{{\\mathrm{H}}}_2{\\mathrm{O}} + 8{\\mathrm{F}}{{\\mathrm{e}}}^{3 + } \\to {\\mathrm{\\ C}}{{\\mathrm{O}}}_2 + 8{{\\mathrm{H}}}^ + + 8{\\mathrm{F}}{{\\mathrm{e}}}^{2 + }\n\\end{eqnarray*}\\end{document} \n Fe-AOM is mediated by anaerobic CH 4 -oxidizing archaea (ANME), which belong to three distinct clades (ANME 1–3) (Weber et al. 2017 ). Members of the ANME-1 clade are highly diverse, and fall in the order of Methanophagales . ANME-2 is comprised of three families, all in the order of Methanosarcinales , of which one is observed in freshwaters; the Methanoperedenaceae (ANME-2d), and two are observed in marine systems: Methanocomedenaceae and Methanogasteraceae . ANME-3 is closely related to known methanogens, and represents a novel genus within the family of Methanosarcinaceae (Chadwick et al. 2022 ). Since Fe(II)Cl 2 addition potentially leads to elevated Fe(III) concentrations in the sediment, it is hypothesized that this could lead to a significant change in the contribution of Fe-AOM to the CH 4 cycle in the long term, lowering both internal PO 4 3− concentrations and in situ CH 4 emissions. Here we aim to unravel the effects of Fe(II)Cl 2 addition on the aquatic carbon cycle. With this study we generate new insights into how bioremediation techniques -besides combating eutrophication- can help in reducing aquatic CH 4 emissions, contributing to climate-smart water management.",
"discussion": "Results and discussion Fe(II)Cl 2 addition effectively lowered PO 4 3- concentrations in the enclosures, lowering the PO 4 3- concentration from 2.2 ± 0.4 to 1 ± 0.4 µM in the surface water, and considerably boosted Fe concentrations in sediment and water (Table 1 ). This is in line with previous findings, where under favorable Fe: P ratios and redox conditions Fe addition successfully locked P into lake sediments (Wang and Jiang 2016 ). Here, in Fe(II)Cl 2 -treated enclosures, up to 91% of the Fe was present in the form of Fe(III) (Kang et al. 2023 ). Table 1. Average nitrate and trace metal concentrations in surface water and the sediment top layer (5 cm) for control and Fe(II)Cl 2 -treated enclosures at the end of the enclosure experiment (Nov 2021). Water Sediment Enclosure PO 4 3− µM NO 3 − µM Mn µM Total Fe µM SO 4 2− µM Mn mM kg −1 DW Total Fe mM kg −1 DW Control 2.2±0.42 0.3±0.25 8±1.3 61±15 14.5±6 5.1±2.3 180±52 Fe(II)Cl 2 1±0.38 0.1±0.05 10±5.6 280±153 21±17 2.4±1.7 207±137 CH 4 emissions of the Fe(II)Cl 2 -treated enclosures were on average 3.5 times lower than those of unamended ( P <0.001, r 2 =0.43, df=34, F=6.9, LM; Fig. 2 ), where control CH 4 emissions averaged 52 ± 78 mg CH 4 m −2 d −1 and Fe(II)Cl 2 -treated enclosures 15 ±14 CH 4 m −2 d −1 , suggesting that Fe(II)Cl 2 addition lowered CH 4 emissions. Phosphorus control has previously been found to lower CH 4 emissions through indirect effects of oligotrophication on the aquatic foodweb (Nijman et al. 2022 ). Additionally, we observed a general descending CH 4 emission intensity over time (Fig. 2 ), which is likely due to seasonality, where in colder months CH 4 emissions decreases. Here, we test if the effects on CH 4 emission can also result from direct effects on microbial CH 4 -production and oxidation in the sediment. Figure 2. Water-atmosphere CH 4 emissions from the enclosures in the field Y-axis is on log10 scale. Boxes show the median and first and third quartiles, whiskers indicate upper and lower quartiles, and dots indicate individual sampling points. CH 4 emissions from the Fe(II)Cl 2 -treated enclosures (N=4) were significantly lower compared to controls (N=4) based on a linear model (LM) ( P <0.001, r 2 =0.43, df=34, F=6.9). The aerobic CH 4 -oxidation rates of Fe(II)Cl 2 -treated sediments (average: 2.4 ± 1.2 µmol CH 4 g −1 d −1 ) were not significantly different than rates observed in the control-enclosure sediments (average: 3.6 ± 0.7 µmol CH 4 g −1 d −1 ; P =0.34, df=5, t=1.1; T-test) (Fig. 3A ), implying that aerobic CH 4 -oxidation played a minor role in lowering CH 4 emissions from the Fe(II)Cl 2 -treated enclosures. However, sediments from the Fe(II)Cl 2 -treated enclosures showed ∼3 times lower net CH 4 -production rate compared to the control sediments (Fe(II)Cl 2 average: 0.53 ± 0.13 µmol CH 4 g −1 d −1 , control average: 1.4 ± 0.52 µmol CH 4 g −1 d −1 , P =0.05, df=3.5 t=2.8, T-test, Fig. 3B and S1A ). Hence, given that: (i) sediment and water samples from Fe(II)Cl 2 -treated enclosures showed enrichment in Fe(III) content, (ii) other potential electron acceptors were only found in low concentrations (Table 1 , and in Kang et al. 2023 ), and (iii) potential aerobic CH 4 -oxidation rates were comparable between control and Fe(II)Cl 2 -treated enclosures, while in contrast net CH 4 -production rates—including potential anaerobic methane oxidation—were different, we hypothesized that Fe-AOM played an important role in lowering the in situ CH 4 emissions. Figure 3. Batch incubation of sediments from the Fe(II)Cl 2 -treated enclosures (blue) and the control enclosures (red). (A) Aerobic CH 4 -oxidation potential (calculated from the linear part of the trend) per g. dry weight sediment per day (µmol CH 4 g −1 d −1 ). No significant difference was observed between treatments (T-test, P =0.34, df=5, t=1.1). Boxes show the median and first and third quartiles, whiskers indicate upper and lower quartiles, dots indicate individual sampling points, and ‘x’ indicates the mean. (B) The net potential CH 4 -production rate of the sediment originating from different enclosures, expressed as µmol CH 4 per g. dry weight sediment per day (µmol CH 4 g −1 d −1 ). Sediments from control enclosures have a marginally significant higher net CH 4 -production rate, compared to the sediments from the Fe(II)Cl 2 -treatment (T-test, P =0.05, df=3.5, t=2.8). We monitored AOM rates and Fe(III) reduction in incubations of enclosure sediments with and without Fe(III) addition. We found clear indications that the AOM-potential was higher in sediments originating from the Fe(II)Cl 2 -treated enclosures than in sediments originating from untreated controls, as seen by their significantly higher 13/12 CO 2 ratio after incubation with 13 CH 4 (average 13/12 CO 2 ratio at the end of the incubation period: control 0.22 ± 0.001, Fe(II)Cl 2 0.29 ± 0.002) (Fig. 4A and S1B , Table S1 ). Moreover, the simultaneous addition of 13 CH 4 and Fe(III) to control sediments led to higher AOM-rates compared to the 13 CH 4 -only addition, and also significantly boosted Fe(II)-production, suggesting that Fe-AOM is a key pathway (Fig. 4B and S1C ). This finding is in line with a study on anaerobic methanotroph bioreactors inoculated with paddy soil, where ferrihydrite and 13 CH 4 addition also resulted in a rapid onset of 13 CO 2 production, indicating the occurrence of Fe-AOM (He et al. 2021 ). Figure 4. Batch incubation of sediments from the Fe(II)Cl 2 -treated enclosures (blue) and the control enclosures (red). (A) Increase in 13/12 CO 2 , with a significant difference between Fe(II)Cl 2 -treated sediments and control based on a LM ( P <0.0001, r 2 =0.88, df=90, F=69.5). Shaded areas show the 95% confidence interval. (B) Concentrations of dissolved Fe(II) (mM) at the end of the experiment (day 129). Boxes show the median and first and third quartiles, whiskers indicate upper and lower quartiles, dots indicate individual sampling points, and X indicates the mean. Letters depict which groups significantly differ from each other based on a two-way ANOVA ( P< 0.01, df=2, F=6.3) combined with a Tukey post-hoc test ( Table S2 ) (C) 16S rRNA gene sequencing data, “in situ” refers to the microbial community originating from the original enclosure sediment, whereas the other three samples are taken from the incubations at the end of the experiment from each serum bottle (day 130). Microbial community analysis revealed the presence of Fe-cycling and CH 4 -cycling microorganisms in the control and Fe(II)Cl 2 -treated sediments, implying there is Fe-AOM-potential (Fig. 4C ). The presence of Fe(II)-oxidizing bacteria supports the hypothesis that Fe(II) originating from Fe(II)Cl 2 can be oxidized to Fe(III), providing an electron acceptor for Fe-AOM (Benz et al. 1998 , Nielsen and Nielsen 1998 , Oikonomidis et al. 2010 ). The archaeal community mainly consisted of methanogens, explaining the observed high CH 4 emissions. Known CH-producing taxa such as Methanosarcinales and Methanomicrobia also include members capable of CH 4 -oxidation via reverse methanogenesis, possibly involved in Fe-AOM (Oni and Friedrich 2017 ). \n Methanoperedens species known to mediate both nitrate-dependent AOM and Fe-AOM (Legierse et al. 2023 ) were also present, albeit at low abundance (below 0.06% in both treatments). Additionally, in both control and Fe(II)Cl 2 -supplemented enclosure sediments, the methanogen Methanomassiliicoccaceae , was abundant (control 5%, Fe(II)Cl 2 7%), suggesting its involvement in Fe-AOM (He et al. 2021 ). Although the Fe(II)Cl 2 -treated sediments had a slightly higher abundance of Fe-cycling bacteria, the control sediments already contained Fe-oxidizers and Fe-reducers, indicating the functional capacity for Fe-AOM within the indigenous microbial community. This also explains the rapid—compared to Weber et al. ( 2017 )—onset of Fe-AOM in our incubations. Furthermore, the relative abundance of sulphide-oxidizing phototrophic bacteria such as Chlorobium was substantially lower in the Fe(II)Cl 2 -treated sediments (2.5%, compared to the control 5.7%) which was likely related to the lower availability of sulphides that may have precipitated in the form of iron sulphide minerals. At the end of our 130-day incubation experiment the microbial community did not differ much from the in-situ community composition. This implies that the structure and composition of bacterial and archaeal communities was persistent and represented environmental conditions. Additionally, we found that the microbial community in both sediments had the potential to reduce Fe(III), as suggested by the 16S rRNA sequencing, and indicated by the increase in Fe(II) throughout the incubation experiment (Fig. 5A ) . The Fe(II)-production rates were similar between H 2 O and 13 CH 4 treatment; which may be due to the active CH 4 supply caused by the sediments’ high methanogenesis rates. Fe(III)-reducers, mainly affiliating with Geobacteraceae were present at similar abundances in both types of sediment (2.2% in the control and 2.6% in the FeCl 2 -treated enclosure), however, irrespective of the incubation treatment, in all the Fe(II)Cl 2 -treated sediments, the higher availability of Fe(III) minerals, resulted in a significantly higher concentration of total Fe(II) over time (LM, P =0.003, r 2 =0.80, df=91, F=37.7). However, part of the Fe(II) formation may also arise from anaerobic respiration of organic substrates. Additionally, we found that the headspace CH 4 concentration correlated with total Fe(II) ( P= <0.0001) concentrations, which is a proxy for Fe(III) reduction. Moreover, irrespective of the incubation treatment, total headspace CH 4 concentration was lower in Fe(II)Cl 2 -treated sediments (Fig. 5B , LM, P =0.008, r 2 =0.72, df=91, F=25.8). This suggests that less CH 4 is released when there is more Fe(III) reduction. This hypothesis is furthermore strengthened by the significant correlation between 13/12 CO 2 and total Fe(II) concentration, caused by Fe(III) reduction (LM, P =<0.0001). Fe(II)Cl 2 -treated sediments had a marginally higher 13/12 CO 2 compared to control sediments (Fig. 5C , LM, P =0.05, r 2 =0.76, df=90, F=30.1), as a result of more 13 CH 4 oxidation . Figure 5. Batch incubation of sediments from the Fe(II)Cl 2 -treated enclosures (blue) and the control enclosures (red). Shaded areas show the 95% confidence interval and the dots indicate individual sampling points (A) Increase in total Fe(II) concentrations (mM) throughout different times during the incubation experiment. The Fe(II)Cl 2 treated sediments had significantly higher total Fe(II) concentrations (mM) over time (LM, P =0.003, r 2 =0.80, df=91, F=37.7). (B) The headspace CH 4 concentrations correlated to total Fe(II) concentrations (mM) ( P =<0.0001). Total headspace CH 4 concentration were lower in Fe(II)Cl 2 -treated sediments (LM, P =0.008, r 2 =0.72, df=91, F=25.8). (C) The increase in 13/12 CO 2 correlated to total Fe(II) concentrations ( P = <0.0001). Fe(II)Cl 2 -treated sediments had a marginally higher 13/12 CO 2 compared to control sediment (LM, P =0.05, r 2 =0.76, df=90, F=30.1). In conclusion, we show that Fe(II)Cl 2 -treatment of eutrophic sediments in an urban pond led to significantly decreased aquatic CH 4 emissions, which was likely facilitated by a rapid onset of Fe-AOM, without substantially changing the native microbial community. This highlights that Fe-AOM can be important in freshwater sediments. Therefore, in sediments where Fe(III) is the main alternative electron acceptor, Fe(II)Cl 2 application has the potential to combat eutrophication as well as CH 4 emissions, contributing to climate-smart water management."
} | 4,753 |
28680072 | PMC5498576 | pmc | 8,727 | {
"abstract": "Intriguing, yet uncultured ‘ARMAN’-like archaea are metabolically dependent on other members of the microbial community. It remains uncertain though which hosts they rely upon, and, because of the lack of complete genomes, to what extent. Here, we report the co-culturing of ARMAN-2-related organism, Mia14, with Cuniculiplasma divulgatum PM4 during the isolation of this strain from acidic streamer in Parys Mountain (Isle of Anglesey, UK). Mia14 is highly enriched in the binary culture (ca. 10% genomic reads) and its ungapped 0.95 Mbp genome points at severe voids in central metabolic pathways, indicating dependence on the host, C. divulgatum PM4. Analysis of C. divulgatum isolates from different sites and shotgun sequence data of Parys Mountain samples suggests an extensive genetic exchange between Mia14 and hosts in situ. Within the subset of organisms with high-quality genomic assemblies representing the ‘DPANN’ superphylum, the Mia14 lineage has had the largest gene flux, with dozens of genes gained that are implicated in the host interaction.",
"introduction": "Introduction Deep metagenomic analysis of environmental samples from acidic environments across our planet has demonstrated the existence of previously neglected uncultured archaea that are only very distantly related to recognised phyla 1 . Initially detected at Iron Mountain (California, USA), these archaeal lineages were subsequently confirmed to occur in various acid mine drainage (AMD) systems 2 . This enigmatic group of archaea (the so-called ‘Archaeal Richmond Mine Acidophilic Nano-organisms’, or ‘ARMAN’ was initially found in the fraction of cells filtered through 0.22 μm membrane filters 1 . Metagenomic assemblies suggested average genome sizes of these organisms to be relatively small for free-living organisms (approximately 1 Mbp) 1 . An interesting observation documented by electron microscopy was that some cells of a small size (<500 nm) interact through pili-like structures with larger cells that lacked cell walls. Comolli and colleagues 3 suggested the ‘ARMAN’ organisms were the ‘small’ cells, whereas cell wall-deficient larger cells were attributed to some members of the order Thermoplasmatales, a group of organisms known to be widely represented in AMD systems 4 . Emerging findings from metagenomic data sets of ARMAN-like archaea and especially their ubiquity suggest that this group plays important roles in the environment, although the exact roles have yet to be established 2 .The phylogenomic placement of archaea from this group still represents a matter for discussion 5 – 7 . The known example of small-sized cultured archaea is represented by Nanoarchaeum equitans , currently the only validly described member of the phylum Nanoarchaeota. Cells are about 500 nm (or smaller) in diameter and exhibit atypical archaeal ultrastructure 8 . Nanoarchaeum equitans exists only in association with the host, Ignicoccus hospitalis , which supplies certain organic compounds (lipids and amino acids), growth factors and likely ATP to N. equitans \n 9 . Other nanoarchaeota-related examples include an Nst1 archaeon forming an association with its host, the Sulfolobales- related organism 10 , and ‘ Candidatus Nanopusillus acidilobi’, thriving in a partnership with Acidilobus spp. 11 . These nanoarchaeota are hyperthermophilic marine and terrestrial organisms with extremely compact genomes that likely are not of an ancestral nature, but rather probably resulted from massive gene loss 6 . Nanoarchaeota-related organisms (including those known only by metagenomics-resolved genomes) are phylogenetically clustered within the ‘DPANN’ candidate superphylum (abbreviated after candidate divisions ‘Diaphetotrites’, ‘Parvarchaeota’ , ‘Aenigmarchaeota’, ‘Nanohalarchaeota’ and the only validly described phylum Nanoarchaeota) 12 . Recently, a number of uncultured ‘DPANN’ archaea with almost complete genomes were predicted by Castelle and co-authors 13 to be symbiotic and/orto have a lifestyle based on fermentation. To summarise, all experimentally validated examples of interactions between co-cultured small (or ‘nanosized’) archaea and their partners are limited to Crenarchaea being the hosts. All of them (except Ignicoccus sp.) are acidophiles, while so far no associations have been co-cultured or characterised for Euryarchaeota, except those from the recent report on a four-member consortium containing a fungus, two strains of Thermoplasmatales and ARMAN-1-related organism with, due to the complexity of this enrichment culture, only a partially sequenced genome 14 . Here, we report the co-cultivation and analysis of the ungapped genome of an ARMAN-like organism, the ‘ Candidatus Mancarchaeum acidiphilum’ Mia14, which was enriched in the laboratory binary culture with Cuniculiplasma divulgatum PM4, a recently described representative of the family Cuniculiplasmataceae within Thermoplasmata 15 . After additional sampling campaigns and de novo metagenome sequencing of the microbial community of the acidic streamer of Mynydd Parys/Parys Mountain, we revealed possible in situ interactions of these organisms with other microbial community members. Furthermore, we analysed the voids in its metabolic pathways (and thus dependencies on potential hosts) and mapped its phylogenetic position. Finally, using data on arCOGs gains and losses, we reconstructed its evolutionary trajectory starting from the last archaeal common ancestor (LACA), which pointed at Mia14 having the greatest known extent of gene fluxes within the ‘DPANN’ superphylum.",
"discussion": "Discussion In the present work, the enrichment culture from Parys Mountain AMD system was set-up to grow acidophilic members of the order Thermoplasmatales. The culture was eventually highly enriched in archaea from the genus Cuniculiplasma , and incidentally, with the significant (ca. 10% genomic reads or 20% of total population) community component belonging to yet uncultured archaea distantly related with ARMAN-2. Due to its high numbers in the enrichment, we were able to produce the fully assembled genome of the ‘ARMAN’-related organism. Based on the genome annotation and experimental data (co-existence in an enrichment culture and fluorescence microscopy), we inferred that the metabolic needs of this sentinel of Cuniculiplasma spp. termed ‘ Ca . Mancarchaeum acidiphilum’ resemble to some extent those of other archaea co-occupying the environment (e.g., reliance on external proteinaceous compounds and amino acids). However, the incompleteness or absence of the central metabolic pathways (e.g., TCA, glycolysis, quinone biosynthesis, etc.) and reduced genome size support an obligate partner-dependent (or ‘ectoparasitic’) lifestyle. Our data (Fig. 3 ) further suggest that sizes of Mia14 cells (and likely other ARMAN-related archaea) have a broad range, usually larger than the diameter of membrane filter pores (0.22 μm) used to enrich for these organisms. The penetration of cells through the 0.22 μm pores of membrane filters observed previously 3 may also be explained by the lack of rigid cell walls in these organisms. For example, the majority of 1–2 µm, cell wall-deficient Thermoplasmatales may squeeze through pores of this diameter. The occurrence of laterally transferred genes and GIs from Cuniculiplasma spp. in Mia14 highlights the relative connection between these organisms co-existing in one environment. It is, furthermore, likely that extracellular structures such as pili or pili-like organelles might be present in Mia14. One may also speculate on massive exchange of DNA through some cell pores or by using the Type IV pili system and numerous membrane proteins encoded within GIs, the likely conjugative elements. Under our experimental conditions, the preferred partner of Mia14 was Cuniculiplasma divulgatum (previously known as ‘G-plasma’ 16 ), which is an abundant inhabitant in AMD. However, the distribution of archaea related to Mia14 (or to ARMAN-2 cluster) in diverse, sometimes non-acidic environments, emphasises their higher plasticity and ability to adapt to the broader range of environmental conditions. This broader distribution of ‘ARMAN’-related organisms in other environments also suggests that Cuniculiplasma spp. may not necessarily be the exclusive partner (host) for ARMAN-2-like organisms. Mia14 is characterised by a very rudimentary metabolic capability. It is even devoid of minimal sets of enzymes required for biosynthesis of both types of nucleotides (purine and pyrimidine) and of 12 out of 20 amino acids (lysine, methionine, arginine, asparagine, alanine, aspartate, leucine, isoleucine, threonine, phenylalanine, tyrosine and tryptophan). Biosynthetic pathways for vitamins and cofactors (B1, B2, Coenzyme A, Coenzyme PQQ, B6, B12, heme, methanopterin, andubiquinone/menaquinone) are incomplete. In Mia14, all glycolytic enzymes are missing. The majority of enzymes for the pentose-phosphate pathway and the entire TCA cycle are also absent. On the other hand, the non-phosphorylating ED pathway of glucose oxidation is present. Additionally, fatty acid metabolism and beta-oxidation, folate cycle, phospholipid biosynthesis, aminosugar metabolism, glycine and serine catabolism pathway, urea cycle and amino group metabolism, nicotinamide, pyruvate metabolism and interconversion of pyruvate and acetyl-CoA, trehalose biosynthesis, glycogen metabolism and biosynthesis, propionate metabolism, heme biosynthesis, pentose-phosphate pathway (non-oxidative phase) and lipopolysaccharides (LPS) synthesis are absent. Furthermore, we have not found any substrate-level phosphorylation pathways. The Mia14 respiratory chain is also absent; no Complex I (NADH:ubiquinone oxidoreductase) 33 , Complex II (succinate:quinone oxidoreductase) 34 , Complex III (either cytochrome bc \n 1 complex 35 or ACIII 36 ) or Complex IV (heme-copper oxygen reductases) 37 proteins-coding genes were found in the genome. However, the presence of H + -translocating V-type ATP synthase in the organism suggests the activity of PMF-generating complexes. The only candidate complex for this role is the cytochrome bd quinol oxidase, which was found in the genome. The lack of appropriate endogenous electron donors for this complex in Mia14, which is deficient in isoprenoid quinone biosynthesis, could be compensated by exogenous quinones from the membrane of Cuniculiplasma sp., considering the assumption of mutualistic interactions between Mia14 and this organism. Indeed, the QH 2 oxidising cytochrome b \n 558 in the Mia14 cytochrome bd complex is localised on the surface of the cell membrane, as inferred from topology prediction and alignment of MIA14_0653 amino-acid sequence with its extensively characterised homolog from E. coli \n 31 . As both Cuniculiplasma species 15 lack cell walls and their cells are usually found in tight contact with Mia14 (Fig. 3 ), we can speculate that the latter organism utilises a broad diversity of Cuniculiplasma membrane quinones (either from living or dead cells) as electron donors for energy conservation. However, no genes of canonical heme biosynthesis, heme import pathways 38 or an alternative pathway for the formation of heme 39 have been found in the Mia14 genome. Besides the possibility of a completely novel heme biosynthesis pathway in this archaeon, the only way for proper assembly of the cytochrome bd complex is the incorporation of exogenous hemes. Accumulation of exogenous hemes in the membrane, which is capable of complementing the growth of heme-deficient organisms, has been demonstrated for pathogenic bacteria 40 . Considering that hemes b and d bind covalently to apoproteins and that the heme-binding amino acids are localised close to the surface of the cell membrane in cytochrome bd complexes 31 , it seems possible for Mia14 to acquire exogenous hemes from Cuniculiplasma spp. to assemble its only PMF-generating complex. It should be noted that the complete set of genes for canonical or non-canonical heme biosynthesis pathways is also absent in Cuniculiplasma strains PM4 and S5, although these aerobically respiring organisms possess heme-containing enzymes of the electron transfer chain 16 . It, therefore, seems possible that Cuniculiplasma , and probably Mia14, possess yet unknown mechanisms of heme biosynthesis. In many archaea, the surface layer is the only cell envelope component providing all functions normally associated with a cell wall, i.e., acting as the protective barrier and maintaining the cell shape. However, in some cases the surface layer proteins may also help in cell–cell association 41 , 42 . The Mia14 surface layer likely possesses a very complex and unique architecture, consisting of at least eight strain-specific secreted surface proteins. It is noteworthy that only four of these surface proteins (MIA14_0152, _0331, _0793 and_0946) require almost 2.5% of the whole genome. We identified two domain types in surface layer proteins displaying the PKD superfamily fold and beta-propeller Kelch and YVTN β-repeat domains fused to CARDB (cell adhesion related domain found in bacteria)-like adhesion module. Six of these surface layer proteins are predicted to be gained from various methanogenic and acidophilic euryarchaea and the members of ‘TACK’ superphylum. As previously hypothesised 42 , the expansion of proteins containing PKD and YVTN domains indicates their function in cell–cell interactions. Thus, we propose that the very rudimentary metabolic capability of Mia14 indicates a Cuniculiplasma- associated lifestyle and that numerous systems such as type IV pili, surface proteins and membrane channels provide an interface for the exchange of metabolites, energy, macromolecules including DNA between Mia14 and its host."
} | 3,456 |
29253871 | PMC5749889 | pmc | 8,728 | {
"abstract": "The majority of life on Earth depends directly or indirectly on the sun as a source of energy. The initial step of photosynthesis is facilitated by light-harvesting complexes, which capture and transfer light energy into the reaction centers (RCs). Here, we analyzed the organization of photosynthetic (PS) complexes in the bacterium G . phototrophica , which so far is the only phototrophic representative of the bacterial phylum Gemmatimonadetes. The isolated complex has a molecular weight of about 800 ± 100 kDa, which is approximately 2 times larger than the core complex of Rhodospirillum rubrum . The complex contains 62.4 ± 4.7 bacteriochlorophyll (BChl) a molecules absorbing in 2 distinct infrared absorption bands with maxima at 816 and 868 nm. Using femtosecond transient absorption spectroscopy, we determined the energy transfer time between these spectral bands as 2 ps. Single particle analyses of the purified complexes showed that they were circular structures with an outer diameter of approximately 18 nm and a thickness of 7 nm. Based on the obtained, we propose that the light-harvesting complexes in G . phototrophica form 2 concentric rings surrounding the type 2 RC. The inner ring (corresponding to the B868 absorption band) is composed of 15 subunits and is analogous to the inner light-harvesting complex 1 (LH1) in purple bacteria. The outer ring is composed of 15 more distant BChl dimers with no or slow energy transfer between them, resulting in the B816 absorption band. This completely unique and elegant organization offers good structural stability, as well as high efficiency of light harvesting. Our results reveal that while the PS apparatus of Gemmatimonadetes was acquired via horizontal gene transfer from purple bacteria, it later evolved along its own pathway, devising a new arrangement of its light harvesting complexes.",
"conclusion": "Conclusions Light-harvesting complexes in G . phototrophica harbor approximately 60 BChl a molecules arranged in 2 concentric rings surrounding the type 2 RC. This unique and elegant organization offers high efficiency of light absorption and excitation transfer as well as high structural stability. Our results also demonstrate that while the PS apparatus of Gemmatimonadetes was likely acquired via horizontal gene transfer from purple bacteria, it later evolved along its own trajectory devising a novel organization for its light-harvesting complexes.",
"introduction": "Introduction Photosynthetic (PS) microorganisms play an important role in many of Earth’s ecosystems due to their ability to harvest light and convert it to metabolic energy [ 1 ]. So far, phototrophic species were found in 7 bacterial phyla: Cyanobacteria, Proteobacteria, Chlorobi, Chloroflexi, Firmicutes, Acidobacteria, and Gemmatimonadetes [ 2 ]. The conversion of light into metabolic energy occurs in reaction centers (RCs) that carry out charge separation. Based on the terminal electron acceptor, the RCs can be divided in two groups [ 3 ]. Type 1 RCs, which use Fe-S clusters, are present in Chlorobi, Firmicutes, and Acidobacteria. Type 2 RCs, which use quinones, are possessed by Chloroflexi, Proteobacteria, and Gemmatimonadetes. Cyanobacteria are the only phototrophic prokaryotes that can evolve oxygen and possess both RC types. The latest group found to contain phototrophic representatives is the phylum Gemmatimonadetes [ 4 , 5 ]. This phylum was formally established in 2003, with G . aurantiaca as a type species [ 6 ]. Its only cultured phototrophic representative is G . phototrophica , which was recently isolated from a freshwater lake in the Gobi Desert [ 7 , 8 ]. G . phototrophica contains bacteriochlorophyll (BChl) a as a main light-harvesting pigment and a large quantity of carotenoids. Its photosynthesis genes are organized in a 42.3-kb photosynthesis gene cluster (PGC) whose organization closely resembles that of Proteobacteria [ 7 ]. Also, phylogenetic analysis of the PS genes confirmed their homology to Proteobacteria. Based on these facts, it was suggested that phototrophy in Gemmatimonadetes originated from an ancient horizontal gene transfer event of a complete PGC from a purple PS bacterium [ 7 ]. If true, G . phototrophica represents the first known example of horizontal gene transfer of a complete set of photosynthesis genes between phototrophic and nonphototrophic representatives of distant bacterial phyla [ 2 , 7 ]. The environmental significance and distribution of phototrophic Gemmatimonadetes is not completely clear. These organisms are photoheterotrophic species, which require organic carbon for their metabolism and growth, but they can supplement a large part of their energy requirements using light-derived energy. Based on the analyses of available metagenomes, the highest proportion of phototrophic Gemmatimonadetes was found in wastewater treatment plants, soils, lake waters and sediments, estuarine waters, biofilms, plant-associated habitats, estuaries, and intertidal sediments. In contrast, no sequences from phototrophic Gemmatimonadetes were found in marine waters [ 9 ]. Little is known about the PS apparatus of G . phototrophica . The presence of the puf operon in its genome indicates the presence of type 2 RCs homologous to RCs of phototrophic Proteobacteria. The in vivo absorption spectrum of G . phototrophica reveals 2 main bands (819, 866 nm) in the near infrared region (NIR) [ 7 ]. This resembles the spectra of many phototrophic Proteobacteria that possess two types of light-harvesting complexes, which serve to both increase cross-section and expand spectral range of the RCs [ 10 , 11 ]. The inner antenna LH1 subunits encircle the RC, forming together the LH1-RC core complex [ 12 – 14 ]. The outer antenna light-harvesting complexes 2 (LH2) are organized in small rings placed in physical contact with the core complex. Interestingly, the genome of G . phototrophica does not contain any LH2 genes [ 7 ], so the identity of its 2 NIR absorption bands is unknown. The second characteristic of G . phototrophica is the presence of a large amount of carotenoids responsible for a strong absorption in the blue-green spectral region [ 7 ]. The light harvesting role of these pigments is uncertain since the heterotroph G . aurantiaca contains a similar set of carotenoids. In order to elucidate the organization of the PS apparatus in G . phototrophica , we purified its PS complexes and performed a detailed biochemical and spectroscopic characterization.",
"discussion": "Results and discussion Characterization of the PS complexes from G . phototrophica The released PS membranes from G . phototrophica contained 2 clearly visible absorption bands in the NIR and a large amount of carotenoids ( S1 Fig ). The PS complexes were purified by a combination of anion-exchange and size-exclusion chromatography (for details see Materials and methods ). During chromatography, the majority of carotenoids eluted differently from the PS complexes ( S2 Fig ). This indicates that most of the carotenoids present in G . phototrophica’s membranes are not bound to PS complexes and do not serve for light harvesting. Based on the retention time during the size-exclusion chromatography and native gel electrophoresis, we estimated that the G . phototrophica PS complex has a molecular weight of approx. 800 ± 100 kDa ( S3A Fig ), which is about 2 times larger than the LH1-RC core complex in R . rubrum (approximately 400 kDa). Further separation of G . phototrophica PS complex by sodium dodecyl sulfate (SDS)-electrophoresis have identified only 6 main protein bands in the range between 2 and 40 kDa. The 2 most intense bands, with apparent molecular masses approximately 5 kDa, most likely originated from light-harvesting antenna subunits ( S3B Fig ). The purified complex contained 62.4 ± 4.7 BChl a molecules (mean ± SD, n = 4). Both results showed that the PS complexes in G . phototrophica are much larger than the core complexes of R . rubrum . The activity of the purified complex was verified by flash photolysis. The flash-induced difference spectrum was highly similar ( S4 Fig ) to the spectra previously recorded in purple PS bacteria [ 15 ], confirming that the RC of G . phototrophica is of the purple-bacterial type. The functionality was further confirmed using variable fluorescence measurements, which documented the high efficiency of primary charge separation ( F V /F M approximately 0.62) and active electron transfer ( S5 Fig ). Interestingly, the isolated complex retained fully functional photochemistry up to 60°C, which far exceeded G . phototrophica’ s growth optimum of 25−30°C [ 8 ]. Such high thermal stability indicates a robust architecture of the studied PS complexes. To obtain information about the overall structure of the light-harvesting systems of G . phototrophica , we analyzed the purified PS complexes using single particle analysis. The raw transmission electron microscopy (TEM) image revealed a large quantity of circular complexes ( S6 Fig ). The averaged image of the PS complex revealed a roughly circular structure with an apparent outer diameter of 198 Å ( Fig 1 ). Assuming a 10 Å layer of detergent, one can estimate the net dimension of the PS complex to approximately 18 nm. The side-view projection showed elongated structures, frequently with a bulge on 1 side of the complex (thickness including the bulge of approximately 72 Å), probably representing the attached cytochrome ( pufC gene product). For comparison, we also performed single particle analysis of the purified RC-LH1 complex of R . rubrum , which has an outer diameter of 13 nm. This means that the PS complex of G . phototrophica occupies an approximately 2 times larger membrane area when compared to the RC-LH1 complex of R . rubrum . 10.1371/journal.pbio.2003943.g001 Fig 1 Top view projection maps of the PS complexes of G . phototrophica (A) and R . rubrum (B). Side view of an individual PS complex of G . phototrophica (C). Symmetric cross-sectional profile of the PS complexes of G . phototrophica (red line) and R . rubrum (grey line) super imposed on R . rubrum structural model (D). The position of the cross sections is indicated by small triangles on the projection maps A and B. PS, photosynthetic. Steady-state spectra The isolated complexes were further characterized by steady-state spectroscopy ( Fig 2 ). The UV part of the spectrum was dominated by the Soret band of BChl a peaking at 370 nm ( Fig 2A ). Carotenoids cover an absorption range between 430–570 nm with the maximum at 515 nm. The vibrational sub-bands of the carotenoid spectrum were not well resolved. The minor absorption band at 575–595 nm seems to originate from the overlapping carotenoid 0–0 transition and the Q x transition of BChl a . In the NIR region, the spectrum was characterized by BChl a bands peaking at 816 and 868 nm; in the following, these spectroscopic species will be denoted as B816 and B868, respectively. The ratio of amplitudes B816:B868 was approximately 1.7. Lowering of the temperature to 77 K led to the narrowing of both BChl a absorption bands and a shift of their maxima to longer wavelengths ( Fig 2A ). The shift was much more pronounced in the case of B868 (12 nm versus 2 nm of B816). Such a large decrease of the transition energy upon cooling is characteristic of the excitonically coupled pigment pools (B870 and B850) of LH1 and LH2 complexes [ 16 ]. At 77 K, the blue edge of the B816 resolved into a well-defined shoulder at approximately 800 nm. 10.1371/journal.pbio.2003943.g002 Fig 2 Steady-state spectra of purified PS complexes from G . phototrophica . (A) Absorption spectra recorded at room temperature (red line) and at 77 K (blue line). (B) The thick line shows the LD ( LD = A H — A V ) spectra of the PS complex embedded in polyacrylamide gel. A H and A V correspond to absorbance of horizontally and vertically polarized light, respectively. For a flat, disk-like particle in a vertically compressed gel, the horizontal direction is parallel with the particle plane, vertical with particle normal. The thin line shows the reduced LD, LD / Abs ., where Abs . is isotropic absorbance. (C) Circular dichroism spectrum of PS complexes in solution. All dichroic spectra were measured at room temperature. Abs, absorbance; CD, circular dichroism; LD, linear dichroism; mdeg, millidegree; PS, photosynthetic; RT, room temperature. Linear dichroism (LD) spectrum of the PS complex embedded in the vertically compressed gel is shown in Fig 2B . Assuming that under compression, the preferential orientation of the plane of the flat, disk-like complex was horizontal (normal vertical), the Q y transitions of both the B816 and B868 were oriented predominantly parallel to the plane of the complex. The larger value of the LD/Absorbance ratio of B816 compared to B868 suggested that the Q y dipole moments of the B816 BChls were slightly more in-plane than those of B868 in contrast to the B800 BChl a of LH2 and the B808 of B808-B866 from Chloroflexi [ 17 – 19 ]. The Q x peak was observed at 583 nm and suggested that the corresponding dipole was oriented along the complex normal (vertically in the present geometry). The carotenoids exhibited very low LD. The circular dichroism (CD) spectrum was dominated by BChl a . Carotenoids contributed only a minor broad positive band in the 430–550 nm region ( Fig 2C ) similar to CD of whole PS membranes of R . rubrum [ 20 ]. In contrast, CD spectra of isolated antenna complexes (LH1, LH2, B808-866) typically exhibit large, often nearly conservative carotenoid bands [ 19 , 21 – 23 ]. The BChl a contribution consisted of positive peaks at approximately 582 nm, 795 nm, and 855 nm, and negative bands peaking at 820 and 880 nm. Although the CD spectrum of the B868 region could be easily interpreted as a LH1-like BChl a aggregate, the B816 region deserves more attention. The large asymmetry between the positive and negative lobe of the CD spectrum, accompanied with a large, 13 nm blue-shift of the zero-crossing point with respect to the maximum of the absorption band are not typical of LH2 or B808-866 complexes [ 19 , 21 , 22 ]. However, both features are present in the CD spectrum of the structural unit of the LH1 complex, B820, an excitonically coupled dimer of BChl a bound to α and β helices [ 20 , 24 ]. Femtosecond transient absorption spectroscopy To explore energy transfer between the B816 and B868 nm bands, we excited the complex at 820 nm and recorded transient absorption spectra in the 700–970 nm spectral window. Fig 3A shows kinetics at the wavelengths corresponding to the ground-state bleaching of the both bands. The kinetics clearly demonstrate the energy transfer process: as the signal at 820 nm decays, the signal at 880 nm (red-shifted with respect to steady-state absorption because of the contribution from the stimulated emission and overlap with excited-state absorption) appears. Fig 3B shows the complementary transient absorption spectra, which provide information about the spectral evolution of the system. 10.1371/journal.pbio.2003943.g003 Fig 3 (A) Kinetics measured at 816 and at 880 nm after excitation of the complex at 820 nm. Kinetics are normalized to the bleaching maximum. Lines represent the fits obtained from global fitting. The inset shows the 880 nm kinetics over a longer timescale. (B) Representative transient absorption spectra recorded after excitation at 820 nm. OD, optical density; rel., relative. Global fitting of the whole spectro-temporal dataset revealed time constants of 2 ps and 210 ps ( S6 Fig ). The first time constant obviously characterizes the energy transfer between the B816 and B868 bands because it is associated with the decay of the B816 band and concomitant rise of the B868 band ( Fig 3A and S7 Fig ). The B816-B868 energy transfer time of 2 ps is slightly longer but comparable to the B800-B850 energy transfer in LH2 complexes: Rhodobacter sphaeroides (0.7 ps) [ 25 ], Rhodopseudomonas acidophila (0.8–0.9 ps) [ 26 , 27 ], Thermochromatium tepidum (0.8–0.9) [ 28 ], Rhodospirillum molischianum (1.0 ps) [ 29 ]. A similar situation was found in Chloroflexi, which contain type 2 RCs surrounded by a circular antenna, which in this case binds 2 different pools of pigments [ 19 , 21 ]. Here, the energy transfer times in the core complex of Chloroflexus aurantiacus [ 30 ] and Roseiflexus castenholzii [ 31 ] were almost the same as in G . phototrophica . The slower kinetics, observed in the B868 band, populated by energy transfer from B816, have a lifetime of 210 ps ( S7 Fig ), which thus characterizes B868-RC energy transfer ( Fig 3A , inset). The shape of transient absorption spectra can also provide some information about arrangement of BChl a molecules within the PS complex of G . phototropica . The ground-state bleaching signals of both B816 and B868 bands are accompanied by positive, blue-shifted excited-state absorption bands. This pattern is well-known from systems containing excitonically coupled BChls, such as LH1 [ 32 ] or LH2 [ 33 ] complexes of purple bacteria. The G . phototrophica light-harvesting complex is thus likely a system in which both B816 and B868 bands exhibit signatures of excitonic coupling. It is also worth mentioning that the zero-crossing point in the transient absorption spectrum at approximately 810 nm hardly moved with time ( Fig 3B ). Essentially the same behavior was recorded for the B820 complex, whereas in the LH1 complex the zero-crossing point shifted over time due to equilibration among LH1 subunits [ 32 ]. Thus, as for the CD spectra described above, the dynamic behavior of the transient absorption spectra also points to the B816 band as being composed of BChl a dimers with no or slow energy transfer between them. Working model of the G . phototrophica ’s PS complex All the collected structural and spectroscopic data provide evidence for some unique features of G . phototrophica ’s PS complex. It is an approximately circular aggregate with an outer diameter of approximately 18 nm. The complex contains 62.4 ± 4.7 BChl a molecules per RC. This number is almost identical to the value determined previously from the whole cell extracts [ 7 ], which indicates that the number of BChl a molecules in the complex is fixed and is not dependent on growth conditions. This number also far exceeds the pigment pools of 30–36 BChl a molecules per RC observed in LH1–RC complexes of Proteobacteria [ 14 , 34 ]. These considerations led to a double concentric ring organization of the G . phototrophica PS complex with a densely packed inner part, similar in dimension to LH1 for B868 and a loosely spaced outer shell of B816. To determine the number of subunits, we analyzed the angular distances of the subunits observed on the peripheral part of the complex. The mean angular distance of the apparent subunits was 24 degrees, which corresponds to the 15-meric symmetry ( S8 Fig ). We assume that the BChl a molecules are divided into 3 pools—4 molecules as a part of the RC, 30 molecules forming an inner (LH1-like) ring around the RC, and 30 molecules forming the second peripheral ring (for details see the discussion in S1 Text ). The structural unit of both pigment pools can be assumed to be a 2-helix–2BChl a complex. This predicted organization with 2 concentric rings composed of 15 dimers each harboring 2 BChl a molecules translates in total to 60 BChl a molecules, which is consistent with the number of BChl a determined by the liquid chromatography. To verify our theoretical prediction, we calculated the steady-state NIR spectra (absorbance, CD, LD) using a point-dipole approximation [ 35 ] for such pigment geometry and compared the simulated spectra with the measured ones ( Fig 4A–4C ). The simulation started from a 2-helix–2BChl a building block (Protein Databank Identifier:2FKW) repeated so as to produce the required 2 rings with 15-meric symmetry, in a similar experiment to that done by Georgakopoulou et al. [ 23 ]. Parameters used in the cited work to simulate the LH1 spectra were used as a starting point. The dipole directions and transition energies were then adjusted to match the measured spectra of G . phototrophica PS complex, first manually and then fine-tuned using a genetic algorithm. The full set of parameters used to compute the steady-state spectra is presented in S1 Table . 10.1371/journal.pbio.2003943.g004 Fig 4 Model of G . phototrophica light-harvesting complex. Panels on the left show (A) absorption, (B) CD, and (C) LD spectra. The recorded NIR spectra (in color) are compared with the simulated steady-state spectra (in black). Blue area in (C) depicts the spectral region having LD > absorbance, red area marks the region with LD < absorbance (after normalization to B868 maximum). (D) Arrangement of BChl molecules in the complex used to calculate the steady-state spectra in (A-C). Yellow ring indicates the size of the LH1 complex from R . rubrum for comparison. (E) Detail of the section of the complex showing the hypothetical position of protein helices (red and blue dots) and BChl a molecules in green. These are taken directly from the structure of light-harvesting complexes. BChl, bacteriochlorophyll; CD, circular dichroism; LD, linear dichroism; LH1, light-harvesting complex 1; mdeg, millidegree; NIR, near infrared region; rel. u., relative units. As seen in Fig 4A–4C , the majority of the features of the experimental steady-state spectra are quantitatively accounted for by the given parameters, including the blue-shift of the B816 crossing point ( Fig 4B ), the slightly higher orientation of the B816 dipoles compared to B868 ( Fig 4C ), and a decrease of polarization in the blue edge of the B816 band ( Fig 4C ), due to the overlap of several excitonic components. On the other hand, the model failed to predict the extent of the nonconservative nature of the CD signals. However, this was expected because it was shown before (e.g., ref. [ 23 ]) that the inclusion of the interaction of BChl a Q y with higher energy transitions, such as Soret bands, Q x and carotenoids is necessary to produce the required degree of asymmetry in the CD bands. The relative difference of the intensity of the B816 and B868 bands is accounted for by less than 20% of the relative increase of the transition dipole moment of BChl a bound to B816 compared to B868, which is well within the values used to simulate spectroscopic properties of LH1 [ 23 ]. The above considerations led us to propose the model shown in Fig 4D . As expected, the dominant pigment–pigment interactions were found within the BChl a dimers (289 cm −1 and 220 cm −1 for B868 and B816 subunits, respectively). The strongest computed interaction between neighboring dimers was 55 cm −1 in the B868 ring. This is more than 5 times lower compared to typical inter-dimer interactions of both LH1 or LH2 complexes. This can be partially accounted for by the fact that the simulation was performed for a 15-meric ring with a diameter corresponding to the standard 16-meric LH1, leading to the larger separation between closest BChls of neighboring dimers, but it also likely indicates a difference in the detailed geometry of the pigments. The strongest inter-dimer interaction within the B816 ring was less than 7 cm −1 due to the large distance between the dimeric subunits; hence, B816 consists effectively of isolated dimers. The strongest predicted coupling between B868 and B816 pigments was −12 cm −1 . This is lower but comparable to the theoretically predicted B800–B850 couplings in LH2 [ 36 , 37 ] and in agreement with the observed excitation transfer times. In addition, because the present model of the G . phototrophica PS complex assumes concentric arrangement of dimeric subunits with the B868 forming an (approximately) LH1-like core surrounded by an external B816 antenna, it is of interest to compare it also to the functioning of the PS unit of LH2-containing purple bacteria. Here, the fastest LH2-LH1 transfer times were found in the range 3–5 ps [ 38 , 39 ] for the theoretically predicted electronic coupling between donor and acceptor states in the range of approximately 2–10 cm −1 [ 40 ]. For completeness, in Fig 4E we suggest the organization of the protein helices corresponding to the above described pigment geometry. Conclusions Light-harvesting complexes in G . phototrophica harbor approximately 60 BChl a molecules arranged in 2 concentric rings surrounding the type 2 RC. This unique and elegant organization offers high efficiency of light absorption and excitation transfer as well as high structural stability. Our results also demonstrate that while the PS apparatus of Gemmatimonadetes was likely acquired via horizontal gene transfer from purple bacteria, it later evolved along its own trajectory devising a novel organization for its light-harvesting complexes."
} | 6,277 |
30691532 | PMC6350386 | pmc | 8,731 | {
"abstract": "Background The current view suggests that in low-temperature acidic environments, archaea are significantly less abundant than bacteria. Thus, this study of the microbiome of Parys Mountain (Anglesey, UK) sheds light on the generality of this current assumption. Parys Mountain is a historically important copper mine and its acid mine drainage (AMD) water streams are characterised by constant moderate temperatures (8–18 °C), extremely low pH (1.7) and high concentrations of soluble iron and other metal cations. Results Metagenomic and SSU rRNA amplicon sequencing of DNA from Parys Mountain revealed a significant proportion of archaea affiliated with Euryarchaeota, which accounted for ca. 67% of the community. Within this phylum, potentially new clades of Thermoplasmata were overrepresented (58%), with the most predominant group being “E-plasma”, alongside low-abundant Cuniculiplasmataceae , ‘Ca. Micrarchaeota’ and ‘Terrestrial Miscellaneous Euryarchaeal Group’ (TMEG) archaea, which were phylogenetically close to Methanomassilicoccales and clustered with counterparts from acidic/moderately acidic settings. In the sediment, archaea and Thermoplasmata contributed the highest numbers in V3-V4 amplicon reads, in contrast with the water body community, where Proteobacteria , Nitrospirae , Acidobacteria and Actinobacteria outnumbered archaea. Cultivation efforts revealed the abundance of archaeal sequences closely related to Cuniculiplasma divulgatum in an enrichment culture established from the filterable fraction of the water sample. Enrichment cultures with unfiltered samples showed the presence of Ferrimicrobium acidiphilum , C . divulgatum , ‘ Ca . Mancarchaeum acidiphilum Mia14’, ‘ Ca . Micrarchaeota’-related and diverse minor (< 2%) bacterial metagenomic reads. Conclusion Contrary to expectation, our study showed a high abundance of archaea in this extremely acidic mine-impacted environment. Further, archaeal populations were dominated by one particular group, suggesting that they are functionally important. The prevalence of archaea over bacteria in these microbiomes and their spatial distribution patterns represents a novel and important advance in our understanding of acidophile ecology. We also demonstrated a procedure for the specific enrichment of cell wall-deficient members of the archaeal component of this community, although the large fraction of archaeal taxa remained unculturable. Lastly, we identified a separate clustering of globally occurring acidophilic members of TMEG that collectively belong to a distinct order within Thermoplasmata with yet unclear functional roles in the ecosystem. Electronic supplementary material The online version of this article (10.1186/s40168-019-0623-8) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions Our metagenomic and metabarcoding amplicon sequencing analyses have unambiguously pointed at the predominance of archaea in the low-to-moderate temperature environment of Parys Mt, in contrast to earlier considerations of the prevalence of bacteria in such environments. One particular group of uncultured Thermoplasmatales , dubbed ‘E-plasma’ was present in a clear majority. The overwhelming archaeal numbers in this natural acidic microbiome implies their pivotal functional and ecological role in the community. Our sample pre-treatment and cultivation attempts were successful in obtaining enrichment cultures with very high proportion of Cuniculiplasma spp., which, however were represented in natural samples as a minor component. An important finding is related to the TMEG archaeal group previously overlooked in acidophilic communities. Our analysis showed their presence in the microbial community of Parys Mt and suggested that at a global scale, there is a strong phylogenetic clustering of TMEG sequences from the low pH settings that form a separate clade at the level of the Order within Thermoplasmata . Considering the majority of microbiome constituents were represented by yet uncultured organisms, this environment is a good resource for isolation of apparent new archaeal taxa within the order Thermoplasmatales and class Thermoplasmata . Its exploration may also further expand the list of highly ranked taxa previously unknown to accommodate acidophilic members.",
"discussion": "Results and discussion Chemical composition of sediment and water samples Chemical analysis was performed on two types of samples, the sediment and the overlying water (Table 1 , Additional file 1 : Table S1). Overall, both contained high contents of iron and sulphur, respectively ca. 66.7 and 217 g/kg in sediment and 1.93 and 80.8 g/L in water sample. Al, K, Pb and Ti in the sediment and Zn, Cu, Pb and Ca for water fraction were also present in high concentrations. Total C and N values in the sediments were detected at 2.8% and 0.3% ( w / w ), respectively. In situ measurements for pH indicated 1.74 for both sample types. Upon arrival to laboratory, the values of pH showed a slight increase to 2.84 for the sediment and 1.90 for the aqueous fraction. The redox measured in the laboratory was found to be + 272 mV in the sediment and + 577 mV in the water sample. Electrical conductivity was found to be very high in the water sample (10.3 mS/cm). Total chemical data are shown for the particulate fraction in Table 1 and for the water fraction in Additional file 1 : Table S1. Table 1 Chemical data obtained in the samples of sediment and water of the acidic stream of Parys Mt. Values represent means ± SEM Parameter Unit Sediment Water Electroconductivity (mS/cm) 7.41 ± 0.66 10.26 ± 0.09 pH 2.75 ± 0.14 1.90 ± 0.05 Redox (mV) 272 ± 38 577 Total Al (mg/kg) 12,375 ± 2092 < 1 Total S (mg/kg) 1932 ± 543 80,754 ± 2260 Total K (mg/kg) 8966 ± 1146 204 ± 33 Total Ca (mg/kg) 574 ± 189 1112 ± 38 Total Ti (mg/kg) 1642 ± 151 < 1 Total V (mg/kg) 245 ± 34 < 1 Total Cr (mg/kg) 309 ± 71 660 ± 74 Total Mn (mg/kg) 191 ± 58 < 1 Total Fe (g/kg) 66.7 ± 10.6 216.5 ± 5.2 Total Ni (mg/kg) 11 ± 1 < 1 Total Cu (mg/kg) 495 ± 108 8477 ± 213 Total Zn (mg/kg) 632 ± 102 10,041 ± 294 Total As (mg/kg) 828 ± 51 244 ± 23 Total Sr (mg/kg) 38 ± 5 51 ± 2 Total Pb (mg/kg) 4122 ± 1111 1491 ± 129 Stone content (%) 5.4 ± 2.2 Dry bulk density (g/cm 3 ) 0.87 ± 0.16 Water filled porosity (%) 58.7 ± 0.9 Air filled porosity (%) 8.58 ± 5.47 Total C (%) 2.8 Total N (%) 0.30 C:N ratio 9.9 Microbial community composition in sediment and water samples Analysis of V3-V4 rRNA region profiling data revealed 123 significantly presented ASVs, corresponding to 12 different phyla (Fig. 1 ). Analysis of microbial diversity in the samples showed that while the water communities were similar between replicates, the sediment was much more heterogeneous: both alpha-diversity measures in the two sediment samples were significantly different from each other (Additional file 1 : Figure S2). This is a similar finding to samples taken from the Los Rueldos mine [ 7 ], and represents the physical non-uniformity of the sediment. Fig. 1 Abundance of SSU rRNA barcoded amplicon reads in water and sediment samples of acidic stream. Figure was performed under R programing environment using basic R packages Sediment-associated archaea In both analysed sediment samples, euryarchaeal class Thermoplasmata was the most abundant group constituting 31.7–65.5% of all amplicon reads. Within class Thermoplasmata , the Thermoplasmatales representatives were present in the highest abundance, with a dominance of ‘E-plasma’ reads (up to 43.5%, MH574490). The second abundant archaeon (ASV MH574492) (5.9–17.2% of total reads) was similar to the uncultured Thermoplasmatales archaeon ‘B_DKE’, previously reported in stable acidophilic enrichment of samples from a pyrite mine in the Harz Mountains, Germany [ 23 ]. The uncultured member of Thermoplasmatales (ASV MH574497), which is common in extreme acidophilic sites, was also represented constituting between 3.4 and 12.0% of total reads. The members of the latter group were detected in the abandoned mercury mine in Los Rueldos [ 7 ], those are almost equally related to Thermoplasmatales archaeon ‘A-plasma’ (94% 16S rRNA gene sequence identity) and Cuniculiplasma divulgatum (93% identity). Another novel Thermoplasmatales archaeon was detected in minor quantities (< 1.7% reads) and was represented by ASV MH57451 and is phylogenetically positioned between ‘A-plasma’ and ‘E-plasma’ (both 94% identity). Archaeal Cuniculiplasma- related taxa with cultivated representatives, earlier been isolated from this same mine site [ 10 ] were found only in minor quantities (0.2–0.5%). Small numbers of reads were also apparent for TMEG (0.2–4.5%) and for ‘ Ca . Micrarchaeota’ (0.3–0.4%), which resembles the earlier reports on the low TMEG numbers in Los Rueldos acidic ecosystem [ 1 ]. Planktonic archaea In surface water samples, archaea constituted a much lower proportion in comparison to sediment (8.1–11.7% of the total reads). ‘E-plasma’ and ‘B_DKE’ were also detected in moderate numbers (6.4–9.4% and 0.8–1.6%, respectively). Cuniculiplasma and TMEG-related reads were only present in very small quantities (0.7–0.3% and 0.2–0.08%, correspondingly) as was ‘ Ca . Micrarchaeota’ (1.7–1.4%). In relation to this, an increase of the content of ‘ Ca . Micrarchaeota’ organisms in water was seen, albeit their overall total representation was below 2%. The member of ‘ Ca . Micrarchaeota’, namely ‘ Ca . Mancarchaeum acidiphilum’ originated from this environment was reported recently to be co-cultured in vitro with Cuniculiplasma divulgatum [ 24 ]. Sediment-associated bacteria Barcoded V3-V4 amplicon sequencing analysis revealed that sediment-associated groups encountered for 14.9–29.9% reads derived from Actinobacteria and 12.3–21.8% from Proteobacteria , the largest proportion of which was attributed to yet uncultured organisms. Other groups were found in lower quantities. Actinobacteria can constitute a large proportion of organisms in nutrient-limited environments [ 25 ]. Thus, actinobacterial species Ferrimicrobium acidiphilum was isolated from the mine site in North Wales and was described as a mesophilic heterotrophic iron oxidiser/reducer and as the predominant bacteria in Parys Mt AMD systems [ 18 , 26 ]. The phylum Proteobacteria is known to include the most common acidophilic bacteria [ 1 ] accounted for 12.3–21.8% reads affiliated with γ- Proteobacteria Subgroup 1 (2.4–4.6%) and Metallibacterium spp. (family Xanthomonodaceae ) (1.1–9.4%) detected alongside unclassified proteobacterial sequences, followed by Acidithiobacillales ‘ cluster RCP 1-48’ (5.7–6.3%). Metallibacterium schleffleri , the cultured representative of metallibacteria, was described as acid-tolerant and organotrophic bacterium, sequences of related organisms were found to be broadly represented in mine-impacted environments being found in both biofilms and sediments [ 27 ]. Acidithiobacillales ‘cluster RCP 1-48’ was previously found to be associated with anaerobic sediments of Rio Tinto and mercury mine streamers [ 7 , 28 ]. Furthermore, the reads from candidate phylum AD3 (3.6–6.2%) and Nitrospiraceae (1.6–5.8%) were also identified. Additionally, a few cyanobacterial signatures were also determined in the sediments. Planktonic bacteria The principal bacterial members inhabiting the water fraction were Leptospirillum spp. ( Nitrospirae ) (22.9–30.6% of total reads), that are renowned iron oxidising colonisers of acidic environments and are considered as the one of the most low pH-resistant bacterial species [ 1 ]. These were followed by Ferrimicrobium spp. ( Actinobacteria ) (11–12.2 %) and other Acidimicrobiia from the same phylum . These patterns have previously been detected and reflect the predominance of Leptospirillum in mine waters and of Actinobacteria in filamentous growth matrix [ 18 ]. Similarly, the elevated presence of Thermoplasmata has been observed in sediment columns of Rio Tinto with Leptospirillum prevailing in the planktonic biomass [ 29 ]. Other important groups were Proteobacteria (19.6–22.3%) with detectable number of reads related to γ-proteobacterial genus Metallibacterium (5.9–6%), Acidithiobacillus (family Acidithiobacillaceae ) (3.4–4.6%) and α-proteobacterial Acidiphilium (family Acetobacteraceae ) (3.6–3.9%). Above taxa contain a number of well-studied organisms. Thus, chemolitotrophic, mesophilic, moderately thermophilic and low temperature-adapted Acidithiobacillus spp. were the first isolated acidophiles and possibly the best-studied species from acidic environments [ 1 ]; heterotrophic iron-reducing Acidiphilium are also known to be widely represented in low pH settings [ 1 ]; significant quantities of Acidobacteria (13.2–17.3%) were also revealed, mainly affiliated to a Subdivision 1 cluster. It was reported that the highest relative abundance of subdivision 1 cluster of Acidobacteria was present in ecosystems with moderately acidic pH (about 4), with the only acidobacterial species known to inhabit AMD environments being the mesophilic and obligately heterotrophic Acidobacterium capsulatum [ 1 , 30 ]. Other bacterial taxa were present in only minor quantities (< 2% of the total). Interestingly, Actinobacteria , whose cultured representatives were shown to be heterotrophic, were among the most abundant bacterial counterparts in both fractions. Other groups of bacteria Acidithiobacillales , Xanthomonodales ( Proteobacteria ) and Chloroflexi were present in larger proportions in sediment as compared to acidic water fractions (Fig. 1 ). Metagenomic sequencing of AMD microbiome Metagenomic analysis of water interface and sediment samples revealed that the microbial community consisted of Archaea (67%) and Bacteria (33%) (Fig. 2 , Additional file 1 : Figure S3). Among the archaea, Euryarchaeota accounted for 64%, ‘ Ca . Micrarchaeota’ for 3% and ‘ Ca . Parvarchaeota’ for 0.2%, both belonging to the candidate superphylum ‘DPANN’ (‘ Diaphetotrites - Parvarchaeota - Aenigmarchaeota - Nanohaloarchaeota - Nanoarchaeota ’), with the rest of the archaea contributing 0.5% of total prokaryotic reads. Thermoplasmata (64% from all prokaryotes) were the most abundant within euryarchaea, the majority of the former was represented by Thermoplasmatales (62%), specifically by unclassified Thermoplasmatales (58% of total sequencing reads) and Cuniculiplasmataceae (4%). Unclassified Thermoplasmatales were affiliated mainly with ‘E-plasma’ clonal variant. A better understanding the physiology and in situ function of this group could be achieved after its successful cultivation. Phylogenetic positioning of archaeal sequences deduced by V3-V4 amplicon sequencing and metagenome shotgun sequencing is presented in Fig. 3 . The indigenous Thermoplasmatales archaea showed high SSU rRNA gene sequence identities with counterparts from other acidic environments: Iron Mountain [ 8 ], Tongling AMD samples (see Accession Numbers in Fig. 3 ), floating macroscopic filaments from Rio Tinto [ 28 , 31 ], Pb/Zn mine tailing [ 32 ] and Frasassi cave system/acidic snottite biofilm [ 33 ]. All these Thermoplasmatales -related sequences belong to organisms still evading isolation. Since the archaea of the order Thermoplasmatales are known scavengers of proteinaceous substrates from microbial biomass [ 5 ], we hypothesise that the metabolic functioning Parys Mt archaea may be considered as heterotrophic to a large extent. The substantial archaeal proportions in samples with low total C and N values (Table 1 ) and lesser numbers of prokaryotic primary producers ( Leptospirillum sp. or Acidithiobacillus spp.) may be associated with green algae populating the surface ( Chlamydomonas spp.). Accordingly, Chlamydomonas is a renowned inhabitant of AMD systems [ 34 ] and is capable of exudation of low molecular weight organic compounds [ 35 ] that can further be utilised by other members of the microbial consortia. Altogether, these archaea, due to their elevated numbers, can be considered as significant contributors to the community functioning and to elemental cycling (carbon and probably iron) in this low-temperature ecosystem. Another factor favouring the elevated numbers of Thermoplasmatales is the high acidity of this environment. Preliminary analysis of the Parys Mt. microbial community showed that a large number (almost 57%) of total Illumina reads, resulting from shotgun sequencing of Parys Mt metagenome, were related to Thermoplasmatales archaea [ 24 ]. Fig. 2 Taxonomic classification of microbial community from metagenomic DNA sequencing data. Taxonomic classification was obtained using 1 GraftM, classifying short reads belonging to marker genes from metagenomic datasets. Graphical representation was performed under R programing environment using basic R packages. https://github.com/geronimp/graftM (This program does not have a publication yet, but they suggest to cite their github page) Fig. 3 16S rRNA gene sequence-based phylogenetic reconstruction of Thermoplasmatales clones from Parys Mt samples obtained in the course of this work. V3-V4 hypervariable region amplicon sequences and sequences extracted from metagenome contigs are highlighted by green. Number of amplified sequence variants, corresponding to each branch indicated in brackets. Reference sequences (brown) were taken from the Silva SSU 132 Ref NR 99 database (see the methods section). ‘Plus’ sign denotes presence of corresponding sequences in 16S V3-V4 amplicon, environmental and enrichment metagenomes Bacteria (33% of the total prokaryotic reads) showed affiliation to Proteobacteria (11%), with γ-Proteobacteria (8%) and α-Proteobacteria (2%) being the major classes. The closest γ-proteobacterial reads were similar to Xanthomonodaceae , with a significant number of Lysobacter spp. (2% of all prokaryotic and 7% of all bacterial reads). These microorganisms inhabit neutral soil and freshwater environments and are known to be a rich source of antibiotics [ 36 ]. α -Proteobacterial sequences (2% from total reads) were affiliated with Rhodospirillales with other groups present in minor numbers. Representatives of Rhodospirillales ( Acidiphilium spp., Acidicaldus organivorans , Acidocella spp. and Acidisphaera spp.) are known acidophilic heterotrophic bacteria able to use exudates and lysates from primary producers and were detected previously at Parys Mt site Dyffryn Adda streamers [ 18 ]. Other bacterial phyla were identified as Actinobacteria (6%), Nitrospirae (4%), Bacteroidetes (3%), Acidobacteria (2%), Firmicutes (2%) and others, present in minor proportions. Acidithiobacillales were represented in lesser quantities (0.3%). Unassigned bacterial sequences comprised 13% of total bacterial reads. The Shannon and Simpson diversity indices were calculated as 1.97 and 0.64, respectively (Additional file 1 : Figure S3). Earlier studies of Parys Mt sites using PCR-based methods revealed the dominance of bacteria with only a minor presence of archaea [ 22 ]. In addition, analysis of the Parys Mt Dyffryn Adda AMD community proposed that Acidithiobacillus ferrivorans (order Acidithiobacillales ), ‘ Ferrovum myxofaciens ’ ( Proteobacteria ) and Acidithrix ferrooxidans ( Actinobacteria ) were the dominant bacteria in this environment [ 18 ]. The ratios of bacteria to archaea have not been assessed in the study; however, archaeal sequences were distantly related to cultured members of Euryarchaeota and were much less diverse than bacteria. In addition, the authors concluded that the presence of archaea in other North Wales acidic mine sites is low [ 18 ]. In that context, our results found archaea to be in the clear majority. We suggest that this disparity between different studies may be connected with general limitation of PCR-based methods used and the different physico-chemical conditions in both sites, including higher pH values in Dyffryn Adda streamers (2.5 vs 1.8), notable increase in Eh (+ 669 vs + 577 and + 272) and significantly low concentrations of metals [ 18 ]. TMEG and Methanomassilicoccales -related archaea Additionally, 2% of the sequences were identified as TMEG-related ( Thermoplasmata ). The phylogenetic position of TMEG-related sequences suggested this group had Methanomassiliicoccus luminyensis strain B10 as the nearest cultured representative with the 16S rRNA gene sequence identity of 83% (Fig. 4 , Additional file 2 : Table S2). The phylogenetic tree (Fig. 4 ) clearly shows clustering of Parys Mt TMEG-related reads with other clones identified in acidic and moderately acidic environments discriminative to sequences from other places. Fig. 4 16S rRNA gene sequence-based phylogenetic relationships of microorganisms from the Terrestrial Miscellaneous Euryarchaeotal Group (TMEG). The tree was constructed using a total of 82 16S rRNA sequences that were assigned and documented in the literature to TMEG, including the clone described in this research (referred as PM clone ) and two sequences from Methanosarcina sp. and Methanomassiliicoccus luminyensis , respectively. Environmental pH is shown by coloured dots from acidic (purple) to neutral-low basic (green). Sequences from environments with unknown pH are shown in black. Two distinct clusters of sequences can be identified, corresponding to sequences from acidic (red cluster) and non-acidic environments (green cluster). The list of analysed sequences is presented in the Additional file 2 : Table S2 ‘TMEG Data’ These archaea are considered to be minor constituents of the communities, but were shown to be more associated with sediment rather than with the water fraction in this particular environment. Recent metagenomic studies indicated that this group might be linked to anoxic sulfate- and methane-containing settings. For instance, it was revealed that the highest number of clones associated with TMEG was detected in sediments with low amounts of SO 4 2− (2 mM) and moderate concentrations of CH 4 (3.5 mM) [ 37 ]. Another study [ 38 ] reported on the presence of TMEG in methane hydrate-bearing sediments of freshwater Baikal Lake comprising ca. 15% of the total microbial community in an upper level and at about 10% in sediments. Interestingly, the athalassohaline inland aquatic system, Salar de Huasco, located in the Chilean Altiplano and characterised by low temperatures showed a predominance of this group in a water sample with the highest sulfate concentration (40 mM) [ 39 ]. Thus, the physiology and functions of TMEG group still remain completely unexplored either in aquatic environments or sediments [ 40 ]. It was predicted that they have a potential to undertake fatty acid oxidation and anaerobic respiration via reduction of sulphite and/or sulfonate (bin Bg1) [ 41 ]. In relation to TMEG, an important point was recently made about a strong phylogenetic separation of gut-associated and free-living clades of Methanomassilicoccales -related archaea [ 42 ]. Furthermore, it was noted that the latter ‘environmental clade’ of Methanomassilicoccales was previously assigned to TMEG [ 43 ]. We detected the presence of 16S rRNA gene sequences with the identity of about 81–83% to those of M . luminyensis in sediments of the stream of Parys Mt in barcoded 16S rRNA gene amplicon sequences (V3-V4 region) and by shotgun metagenomic sequencing. Almost identical to Parys Mt sediment, TMEG-derived SSU rRNA gene sequences were earlier detected in La Zarza, Perrunal acid mine effluent (Iberian Pyritic Belt, Spain, HM745465 [ 44 ], Rio-Tinto, DQ303248 [ 31 ], terrestrial acidic spring AB600345 [ 45 ] and many other acidic or slightly acidic environments [ 7 , 46 , 47 ]). In some cases, the affiliation of these sequences to the order Thermoplasmatales has been erroneously considered. Our analysis pointed at a compact co-clustering of TMEG-related sequences from a wide range of acidic and moderately acidic ecosystems that were very distinct from the cluster of their counterparts from neutral environments. These acidophilic/acidotolerant TMEG archaea form a group on the level of a new order within Thermoplasmata , and are only distantly related to M . luminyensis . T-RFLP and clone library analyses conducted earlier identified similar organisms in other Parys Mt settings: in impounded water (corresponding microorganisms were referred as a ‘methanogens’) [ 21 ] and in filamentous growth streamers in the Dyffryn Adda mine adit [ 18 ]. The presence of a group of rRNA gene sequences with about 80% identity to M . luminyensis was also mentioned in a study of arsenic-rich creek sediments of Carnoulès Mine, France [ 48 ], in Los Rueldos acidic streamers ( [ 7 ] and elsewhere). Enrichment cultures An attempt to enrich and isolate archaea from this environment was undertaken. For this, we applied various treatments of samples, followed by enrichment and consequent shotgun sequencing of these variants (see ‘ Methods ’ section). After 25 days of cultivation of enriched samples (NN 4 and 5), certain diversification between variants was observed (Fig. 5 ). The EP culture containing sediment matrix was dominated by Ferrimicrobium acidiphilum , showing 99% 16S rRNA nucleotide identity with Fm . acidiphilum strain T23 T previously isolated from the Cae Coch sulphur mine [ 26 ]. Archaea were represented by Cuniculiplasma divulgatum and ‘ Ca . Mancarchaeum acidiphilum Mia14’, corresponding to 14.4% and 2.3% of classified metagenomic reads, respectively. Fig. 5 Abundance of archaea and bacteria according to the results of metagenomic sequencing of DNA isolated from enrichment cultures with differential pre-processing methods (samples EP, ES and EF). Proportion of classified (pink) vs. unclassified (light green) reads is shown by small pie-charts on top right corner of each bar The ES culture was established using the supernatant of the above centrifugate as an inoculum to reduce the numbers of large algal cells and solid particulate matter. Upon incubation, Fm . acidiphilum was the predominant organism with a lower abundance of Cuniculiplasmataceae than in EP culture. Nevertheless, ES culture was significantly more diverse than EP. Small numbers or ‘ Ca . Micrarchaeota’-related reads, with 16S rRNA gene sequence almost identical to that of ‘A_DKE’ organism from the enrichment culture of ‘Drei Kronen und Ehrt’ pyrite mine microbial consortium [ 23 ] and ‘ Ca. Mancarchaeum acidiphilum’ Mia14, corresponding to 0.4% and 0.9% of classified metagenomic reads, respectively, were detected. Minor bacterial taxa were represented by uncultivated species of Leptospirillum (0.6%) and Aciditerrimonas (1.3%). Closest 16S rRNA gene sequences for the latter were reported in clones from acid mine effluent of La Zarza, Perrunal mine located in the Iberian Pyritic Belt [ 44 ]. Finally, an enrichment culture (EF) was set up using the filtrate which had passed through a 0.45 μm pore membrane. This was used as an inoculum but required a longer cultivation time (45 days). It showed a predominance of C . divulgatum with only a minor fraction of bacteria (0.7%) of Leptospirillum and Ferrimicrobium spp . In contrast to the other enrichment cultures, no ‘ Ca . Micrarchaeota’ signatures were detected in the EF culture. However, one needs to consider that similar culturing conditions have been previously optimised for Cuniculiplasma divulgatum [ 9 ] and may be less favourable to other Parys Mt archaea."
} | 6,914 |
26650467 | PMC4692740 | pmc | 8,733 | {
"abstract": "MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.",
"introduction": "Introduction In the past decade, the assembly of artificial lipid bilayers has been substantially advanced through the development of the droplet interface bilayer method. Known as stable and robust, DIBs imposed themselves as alternative model systems to the classical painted (Mueller) and folded (Montal-Mueller) planar bilayers 1 . Although the idea of using droplets to create lipid bilayers dates back to the 1960s 2 , it has not gained popularity until recently. The first successful attempt was reported by the Takeushi group 3 , followed by several studies demonstrating bilayer formation using a network of droplets by the Bayley group 4-6 . More recently, encapsulation techniques were proposed by the Leo group 7-9 , who pioneered the concept of using DIBs as building blocks of novel stimuli-responsive material systems 10 . In previous studies, DIBs have proved their ability to respond to electrical 9,11 , chemical 10,12 , and optical stimuli 13 . Various biomolecules with different stimuli-responsive functionalities have been effectively stimulated when reconstituted in the DIB 10,14 . In light of these successful attempts an important question is raised: could the DIB respond to mechanical stimulus when appropriate biomolecules are incorporated? The interfacial forces acting on a DIB differ from those in other bilayer system 15,16 . Therefore, the tension in the bilayer held by the droplets could be controlled by regulating tension at the water-lipid-oil interfaces; a concept not applicable with the painted or folded bilayer systems. MscL channels, widely known as osmolyte release valves and fundamental elements of the bacterial cytoplasmic membrane, react to increased membrane tension 17,18 . In the event of hypo-osmotic shocks, several channels residing in the membrane of a small cell 19 can generate a massive permeability response to quickly release ions and small molecules, saving bacteria from lysis 20 . Biophysically, MscL is well studied and characterized primarily through the prominent patch clamp technique 21-23 . Reliable structural models explaining MscL's gating mechanism 24,25 are proposed based on its homolog's crystal structure 26,27 , modeling 28 , and results of extensive experimentation 24,29-31 . Under an applied tension of ~10 mN/m, the closed channel which consists of a tight bundle of transmembrane helices, transforms into a ring of greatly tilted helices forming a ~28 Å water-filled conductive pore 21,24,32 . It has also been established that the hydrophobicity of the tight gate, positioned at the intersection of the inner TM1 domains, determines the activation threshold of the channel 33 . Correspondingly, it was found that by decreasing the hydrophobicity of the gate, the tension threshold could be lowered 22 . This property of MscL made possible the design of various controllable valves 34 , primarily for drug delivery purposes. For all the aforementioned properties and based on its fundamental role of translating cell membrane excessive tensions into electrophysiological activities, MscL makes a great fit as a mechanoelectrical transducer in DIBs. In this article, we present an original micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels under mechanical stimulation. We report for the first time, the response of DIBs to mechanical stimulus and the functional reconstitution of the V23T low-threshold mutant of MscL in DIBs 35 . The experimental system consists of lipid encased aqueous droplets anchored to the tips of two opposing borosilicate glass micropipettes. When droplets are brought into contact a lipid bilayer interface is formed. This technique offers control over the chemical composition and size of each droplet (bulk), as well as the dimensions of the bilayer interface. In addition, asymmetric membranes with various lipid compositions in each leaflet could be easily formed. Having one of the micropipettes attached to a harmonic piezoelectric actuator, provides the ability to apply a pre-programmed single-cycle or oscillatory stimulus. Tension is delivered to the artificial membrane through the compression of both droplets supporting it. As a result of droplet deformation, the areas of water-lipid-oil interfaces increase, and simultaneously the angle between the droplets decreases, causing an increase in membrane tension and transient MscL activation. Through analysis of the shapes of the droplets during deformation, the tension created at the interface could be estimated. Even though the focus in this article is on the mechano-transduction properties of the DIB, we also emphasize that other types of biomolecules, such as alamethicin, can be activated by this multi-functional platform. We present here, all the technical aspects of preparing, assembling, and taking measurements with this new method in a step-by-step manner.",
"discussion": "Discussion Mechanosensation signifies one of the first sensory transduction pathways that evolved in living organisms. Using this phenomenon for studying and understanding the mechano-electrical properties of the DIB, is a crucial step toward functional stimuli-responsive materials. It involves the incorporation and activation of a mechanosensitive channel, MscL, in the DIB as a mechanoelectrical transducer and a strain gauge to detect tension increase in the lipid bilayer interface. On another note, the function of MS channels could be regulated through the basic material properties of lipid bilayers including thickness, intrinsic curvature, and compressibility. In light of the aforementioned, the micropipette-based technique provides a valuable tool allowing the researcher the ability to study MS channels in DIBs and provides insights into the structure of the lipid bilayer, as well as the lipid-protein interactions. Over the past three decades, patch-clamp was the primary method to study MS channels, since it allows clamping of both voltage and tension. However, patch-clamp requires bulky equipment and not suitable for miniaturization, a property required for the engineering of sensory and conversion devices. DIBs due to their simplicity, stability, and compactness represent a suitable environment to study the activity of MscL. Here, we extend previous advances in the DIB formation techniques by proposing a micropipette-based technique, with the ability to control the size of droplets and bilayer interface, the chemical composition of each droplet, and the tension at the interface through dynamic stimulation. The technique consists of anchoring aqueous droplets, containing proteoliposomes, to the tips of coaxially opposing glass capillaries. The droplets are placed in a bath of organic solvent and when brought in contact a lipid bilayer forms at the interface. The micropipettes are attached to piezoelectric oscillators, allowing horizontal displacement of the droplets. Dynamically compressing the droplets, results in an increase of interfacial tension at the water oil interface and therefore an increase in bilayer tension. Two major aspects differentiate this method from the similar and recently published contact bubble bilayer (CBB) technique 37 . Using the technique presented herein, the size of the bilayer is controlled using micromanipulators and thus the volumes of the droplets remain constant, unlike in the CBB method. In addition, the CBB technique calls for pressure pumps, which are not needed in the method presented in this paper making it simpler and easier to build. We are able to incorporate and stimulate bacterial MscL for the first time without the use of a patch pipette or chemical modifications 38 . Since the system facilitates the formation of robust asymmetric lipid bilayer membranes, it more closely mimics the lipid asymmetry found in biological membranes. This allows us to study the effects of controlled membrane composition or asymmetry on the activity of MscL. Additionally, through image processing techniques, this method helps estimate the tension at the bilayer interface. This technique assists in understanding the principles of interconversion between bulk and surface forces in the DIB, facilitates the measurements of fundamental membrane properties, and improves the understanding of MscL response to membrane tension. Although this method takes us a step closer toward a biomolecular stimuli-responsive material system and to a different physiological environment to study MscL, there are limitations to the system. Tension in this system cannot be clamped due to the presence of the lipid reservoir in the form of liposomes in each droplet, which tends to relieve tension at the oil/water interface. Therefore, at present mechanosensitive channels can be stimulated in DIBs only in a dynamic regime. The presence of air bubbles in the system significantly affects the precision and reproducibility of the experiments. Air bubbles present in the hydrogels could result loss if electrical connection. While we describe the use of the micro-pipette based method for the stimulation of MscL, the technique could be used to study other types of MS channels and has the potential to be used by researchers to study a variety of biomolecules. For instance, similar setup has been used in our lab to study the mechanoelectrical response of a channel-free droplet interface bilayer membrane. Various proteins could be reconstituted and activated using this highly controlled setup, taking in consideration that the reconstitution environments of each biomolecule vary. The method described in this article touches on a considerably wider application potential that is only limited to the imagination of the researcher."
} | 2,890 |
22371825 | PMC3286854 | pmc | 8,734 | {
"abstract": "Reservoir computing is a recently introduced, highly efficient bio-inspired approach for processing time dependent data. The basic scheme of reservoir computing consists of a non linear recurrent dynamical system coupled to a single input layer and a single output layer. Within these constraints many implementations are possible. Here we report an optoelectronic implementation of reservoir computing based on a recently proposed architecture consisting of a single non linear node and a delay line. Our implementation is sufficiently fast for real time information processing. We illustrate its performance on tasks of practical importance such as nonlinear channel equalization and speech recognition, and obtain results comparable to state of the art digital implementations.",
"discussion": "Discussion We have reported the first demonstration of an opto-electronic reservoir computer. Our experiment has performance comparable to state of the art digital implementations on benchmark tasks of practical relevance such as speech recognition and channel equalization. Our work demonstrates the flexibility of reservoir computers that can be readily reprogrammed for different tasks. Indeed by re-optimizing the output layer (that is, choosing new readout weights W k ), and by readjusting the operating point of the reservoir (changing the feedback gain α , the input gain β , and possibly the bias ϕ ) one can use the same reservoir for many different tasks. Using this procedure, our experimental reservoir computer has been used successively for tasks such as signal classification, modeling a dynamical system (NARMA10 task), speech recognition, and nonlinear channel equalization. We have introduced a new feature in the architecture, as compared to the related experiment reported in 18 . Namely by desynchronizing the input with respect to the period of the reservoir we conserve the necessary coupling between the internal states, but make a more efficient use of the internal states as the correlations introduced by the low pass filter in 18 are not necessary. Our experiment is also the first implementation of reservoir computing fast enough for real time information processing. (We should point out that, after the submission of this manuscript, related results where reported in 28 ). It can be converted into a high speed reservoir computer simply by increasing the bandwidth of all the components (an increase of at least 2 orders of magnitude is possible with off-the-shelf optoelectronic components). We note that in future realizations it will be necessary to have an analog implementation of the pre-processing of the input (digitisation and multiplication by the input mask) and of the post-processing of the output (multiplication by output weights), rather than the digital pre- and post-processing used in the present work. From the point of view of applications, the present work thus constitutes an important step towards building ultra high speed optical reservoir computers. To help achieve this goal, in the supplementary material we present guidelines for building experimental reservoir computers. Whether optical implementations can eventually compete with electronic implementations is an open question. From the fundamental point of view, the present work helps understanding what are the minimal requirements for high level analog information processing."
} | 846 |
29729665 | PMC5935971 | pmc | 8,735 | {
"abstract": "Background Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo -inositol-1-phosphate synthase (Ino1), myo -inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway. Results A more stable miox4 from Arabidopsis thaliana was chosen in the present study. In addition, high copy delta-sequence integration of miox4 into the S. cerevisiae genome was performed to increase its expression level further. Enzymatic assay and quantitative real-time PCR analysis revealed that delta-sequence-based integrative expression increased MIOX4 activity and stability, thus increasing glucaric acid titer about eight times over that of episomal expression. By fed-batch fermentation supplemented with 60 mM (10.8 g/L) inositol, the multi-copy integrative expression S. cerevisiae strain produced 6 g/L (28.6 mM) glucaric acid from myo -inositol, the highest titer that had been ever reported in S. cerevisiae . Conclusions In this study, glucaric acid titer was increased to 6 g/L in S. cerevisiae by integrating the miox4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes. Delta-sequence-based integrative expression increased both the number of target gene copies and their stabilities. This approach could be used for a wide range of metabolic pathway engineering applications with S. cerevisiae . Electronic supplementary material The online version of this article (10.1186/s12934-018-0914-y) contains supplementary material, which is available to authorized users.",
"conclusion": "Conclusions In this study, glucaric acid titer in S. cerevisiae was increased by expressing a more stable MIOX4 from A. thaliana and integrating the target genes into the delta sequence of the genomes. Delta-sequence-based constitutive expression increased both the number of target gene copies and their stability and can be used for a wide range of metabolic pathway engineering projects in S. cerevisiae . The final strain produced 6.0 g/L (28.6 mM) glucaric acid, which is the highest titer reported in S. cerevisiae .",
"discussion": "Discussion Cell factories created by metabolic engineering and synthetic biology to produce fine chemicals have emerged as an increasingly popular alternative to chemical synthesis [ 40 ]. Ever since the glucaric acid biosynthetic pathway was constructed in E. coli [ 1 ], great efforts have been expended to increase target product titer. Gupta et al. [ 8 ] reported that MIOX activity and myo -inositol availability were rate-limiting in glucaric acid production, not only in E. coli , but in S. cerevisiae . Therefore, the strategy to increase glucaric acid production was to improve MIOX activity and stability while increasing the myo -inositol flux to glucaric acid. In S. cerevisiae , myo -inositol can be produced by converting d -glucose-6-phosphate by native inositol-1-phosphate synthase (Ino1), which is tightly regulated by Opi1 [ 36 ]. In this study, the OPI1 gene was deleted to remove its negative regulation of myo -inositol synthesis. Although more myo -inositol was produced by S. cerevisiae , exogenous supplementation of myo -inositol was still necessary to increase MIOX4 activity (Fig. 2 a) and glucaric acid accumulation. In addition, the constructed strain supplemented with 60 mM (10.8 g/L) myo -inositol apparently produced more glucaric acid than 10 mM (1.8 g/L) myo -inositol (Fig. 5 a), which indicated that efficient production of glucaric acid at high titer needs a high concentration of myo-inositol might because the low affinity activity of the Itr1/2 myo-inositol transporter in S. cerevisiae [ 41 ]. However, only a small proportion of the supplemented myo -inositol was transported into the cell (Additional file 1 : Figure S2). When the myo -inositol residue was fed 60 mM (10.8 g/L) myo -inositol to a shake flask culture, only about 20% (2.16 ± 0.11 g/L) of myo -inositol was consumed (Additional file 1 : Figure S2A). At least 3.26 g/L myo -inositol was required to produce 3.8 g/L glucaric acid in the shake flask culture. This means that at least 1.1 g/L myo -inositol was endogenously synthesized from glucose; the yields were 1.76 ± 0.08 g glucaric acid/g myo -inositol and 0.037 ± 0.003 g myo -inositol/g glucose. In the fed-batch fermentation of glucaric acid, about 3.6 ± 0.18 g myo -inositol was consumed (Additional file 1 : Figure S2B), and about 5.14 g/L myo -inositol was required to produce 6.0 g/L glucaric acid. Therefore, at least 1.54 g/L myo -inositol came from glucose, and the yield was 1.67 ± 0.08 g glucaric acid/g myo -inositol, or 0.051 ± 0.006 g myo -inositol/g glucose. Robinson reported that the uptake activity of the Itr1p myo -inositol transporter was affected by growth phase in S. cerevisiae because it would reach a maximum at exponential and then decrease rapidly to minimum in the stationary phase, and this regulation was independent of OPI1 [ 41 ]. Improving the transport ability of Itr1p through protein engineering may be very important. Besides myo -inositol availability, low activity or instability of myo -inositol oxygenase is the key bottleneck in the biosynthetic pathway to glucaric acid. Given that the codon-optimized mouse-derived MIOX gene had low activity and instability according to Moon et al. [ 1 ], the miox4 gene from A. thaliana was selected for this study. MIOX4 showed relatively high stability compared with mouse MIOX, but the specific activity of episomal plasmid expression of miox4 was still very slow. To overcome this problem, delta-sequence-based integrative expression of miox4 and udh genes was carried out. A delta sequence is a kind of long-end repeated DNA sequence located in the reverse transcription transposon Ty of chromosome DNA in S. cerevisiae . Because there are approximately 425 delta sequences, delta sequence-based homologous recombination is more efficient than traditional methods for exogenous DNA integration into the S. cerevisiae genome, and the target gene was expressed more stably in the delta sequence because it avoided plasmid loss in the episomal expression strain [ 17 ]. The miox4 transcription level as determined by quantitative real-time PCR was an indirect characterization of gene copies compared with that of the episomal plasmid. The transcription level of miox4 in integrative expression was 5.43 times that of episomal expression (Fig. 6 ). These results indicated that this multi-copy integrative expression method is efficient for overexpression of miox4 genes, resulting in obviously enhanced MIOX4 activity and stability and high glucaric acid titer. The authors believe that the delta-sequence integration method can be used in a wide range of low-copy genes to remove the bottleneck in metabolic engineering of S. cerevisiae ."
} | 1,822 |
36336839 | null | s2 | 8,737 | {
"abstract": "One important direction of synthetic biology is to establish desired spatial structures from microbial populations. Underlying this structural development process are different driving factors, among which bacterial motility and chemotaxis serve as a major force. Here, we present an individual-based, biophysical computational framework for mechanistic and multiscale simulation of the spatiotemporal dynamics of motile and chemotactic microbial populations. The framework integrates cellular movement with spatial population growth, mechanical and chemical cellular interactions, and intracellular molecular kinetics. It is validated by a statistical comparison of single-cell chemotaxis simulations with reported experiments. The framework successfully captures colony range expansion of growing isogenic populations and also reveals chemotaxis-modulated, spatial patterns of a two-species amensal community. Partial differential equation-based models subsequently validate these simulation findings. This study provides a versatile computational tool to uncover the fundamentals of microbial spatial ecology as well as to facilitate the design of synthetic consortia for desired spatial patterns."
} | 300 |
28303187 | PMC5306006 | pmc | 8,738 | {
"abstract": "Abstract Access to resources depends on an individual's position within the environment. This is particularly important to animals that invest heavily in nest construction, such as social insects. Many ant species have a polydomous nesting strategy: a single colony inhabits several spatially separated nests, often exchanging resources between the nests. Different nests in a polydomous colony potentially have differential access to resources, but the ecological consequences of this are unclear. In this study, we investigate how nest survival and budding in polydomous wood ant ( Formica lugubris ) colonies are affected by being part of a multi‐nest system. Using field data and novel analytical approaches combining survival models with dynamic network analysis, we show that the survival and budding of nests within a polydomous colony are affected by their position in the nest network structure. Specifically, we find that the flow of resources through a nest, which is based on its position within the wider nest network, determines a nest's likelihood of surviving and of founding new nests. Our results highlight how apparently disparate entities in a biological system can be integrated into a functional ecological unit. We also demonstrate how position within a dynamic network structure can have important ecological consequences.",
"introduction": "1 Introduction An individual's access to resources is strongly influenced by its position in the environment relative to that resource. This can have important behavioral consequences; for example, optimal foraging strategies have evolved to make best advantage of available resources, given an individual's position in the environment (Ydenberg, 2007 ). This is particularly true in species such as social insects, which form nests that are spatially fixed (at least in the short term). The position of a nest in the environment is likely to affect access to resources and ultimately the fitness of the individuals within the nest (McGlynn, 2012 ). Many ant species inhabit multiple spatially separated, but socially connected nests, a strategy called polydomy (Debout, Schatz, Elias, & Mckey, 2007 ; Robinson, 2014 ). Nests within a polydomous system often exchange resources (e.g., Buczkowski, 2012 ; Ellis, Procter, Buckham‐Bonnett, & Robinson, in press ; Hoffmann, 2014 ). A nest's access to resources will depend not only on its location within the foraging environment but also on its position relative to other nests. For example, in polydomous wood ant ( Formica lugubris ) colonies food and other resources are transported through the colony by workers traveling along trails between nests (Ellis, Franks, & Robinson, 2014 ; Ellis & Robinson, 2015 , 2016 ). The combined nests and trails of a polydomous wood ant colony therefore act as a resource redistribution network: food resources are transferred along the trails between pairs of nests, resulting in colony‐level redistribution of resources organized at a local level (Ellis & Robinson, 2016 ; Ellis et al., 2014 ). Wood ants’ major source of food is honeydew, a spatially and temporally stable resource (Domisch, Risch, & Robinson, 2016 ). For a worker, therefore, access to food will depend not only on their nests’ location within the stable foraging environment but also on their nests’ position in the nest network structure. Workers from the same colony, but inhabiting different nests, therefore have different access to resources. However, the ecological consequences of this differential access to resources of nests within the network, and the effect that this differential access has on the structure of the colony, are unclear. In a polydomous colony, there are several possible ecological consequences of a nest's access to resources. For example, a nest's survival, i.e., its continued inhabitation, is likely to depend on its ability to access enough resources to sustain the ants within the nest. Similarly, workers within a nest may be influenced by access to resources when founding new nests. In polydomous wood ant colonies, new nests are often established by budding: During budding, workers and queens leave a nest on foot to found a new nest (Bourke & Franks, 1995 ; Ellis & Robinson, 2015 ). It would be expected that the decision of ants within a nest to bud a new nest is influenced, positively or negatively, by their nest (the founder nest's) access to resources (Holway & Case, 2000 ; Lanan, Dornhaus, & Bronstein, 2011 ; Sorvari & Hakkarainen, 2005 ). It is important to note that both of these traits: survival and budding, are inherently time dependent and need to be studied in a dynamic framework. The ecological interdependence of nests will define the nature of the polydomous system. In a monodomous colony (a colony inhabiting a single nest), the survival and budding of a nest are affected only by properties inherent to that nest, such as its size and location in the environment. Nests within a polydomous system may similarly survive and bud based only on their inherent properties, with no ecological consequences of the nest network structure. Survival and budding based only on inherent properties of a nest would suggest that there is a low level of integration between nests in the system and that a polydomous colony is simply a cluster of mutually nonaggressive nests and not part of a single cooperative and functional unit. In contrast, if the nests of a polydomous system are part of the same functional unit, the survival and budding of each nest will be affected not only by inherent nest properties, but also by either its position in the colony nest network or more general colony‐level effects. In this study, we investigate how the survival and budding of nests in polydomous colonies are affected by three levels of organization: (i) attributes of the individual nest, (ii) position of the individual nest within the network, and (iii) properties common to the whole network. The ecological consequences of differential access to resources within a polydomous colony will give important insights into how polydomous colonies are structured and, more generally, the potential importance of an individual's position within a dynamic network.",
"discussion": "4 Discussion In this study, we found that the position of a nest within the network of polydomous Formica lugubris colonies has important ecological consequences for that nest and the structure and integration of the colony. Nests with a higher flow of resources, even if this comes indirectly via other nests, have an increased chance of surviving and founding new nests than nests with a lower flow of resources; nest size is also accounted for and does not eliminate this effect. Distance to the nearest foraging tree does not affect nest survival. Resource flow through a nest depends on its connections to the other nests and how it fits into the broader structure of the network. The survival and budding of a nest is dependent on its relationship with other nests and the wider pattern of interaction between the nests in the polydomous colony. Our results show that, despite being spatially separated, the interconnected nests of a polydomous colony can be considered a single ecological unit, at least in terms of resource acquisition. We also demonstrate that dynamic network position can have important ecological consequences. The view of the nests of polydomous wood ant colonies as forming a single ecological unit, supported by our results, suggests that the factors influencing the fitness of individuals in a given nest are likely to be strongly linked to the fitness of individuals in other nests. The ability of a nest to survive and bud depends, in part, on its position in the colony nest network. This dependence shows that the resource movement through the colony has an important ecological influence. Changes in the environment near any given nest have the potential to affect the survival and budding of nests throughout the network. However, it is important to note that the survival and reproduction of nests are driven by proximate process, namely the access of nests via the workers within them, to resources. This finding supports other work that has detected no evidence for top‐down, colony‐level effects on the structure of polydomous colonies (Ellis et al., 2014 ). The extent to which nests can be considered as part of the same colony, super‐colony, or super‐organism is an important consideration when assessing, for example, the level at which selection acts in a colony (Helanterä, Strassmann, Carrillo, & Queller, 2009 ; Kennedy, Uller, & Helanterä, 2014 ; Moffett, 2012 ). Resources are often distributed heterogeneously in the environment; polydomy may be a way to more efficiently exploit these dispersed resources (Cook et al., 2013 ; Holway & Case, 2000 ; Lanan et al., 2011 ; Schmolke, 2009 ). The nest and foraging network of polydomous colonies can be viewed as a transportation network to move resources from food sources to the nests and then between nests (Cook et al., 2014 ; Latty et al., 2011 ). Transport efficiency refers to the ease with which resources can flow through a network. In the polydomous nest system, nests with a high resource flow are at points in the network important for colony‐level resource redistribution and therefore colony‐level transport efficiency (e.g., Croft et al., 2008 ; Perna & Latty, 2014 ). If network transport efficiency is being retained within a colony, nests and trails with a higher resource flow, and therefore greater importance for efficiency, may be more likely to survive than those with a lower betweeness. We found that nests with a high resource flow are more likely to survive than nests with a lower betweeness. However, trails with a higher resource flow are not more likely to survive than trails with a lower betweeness. Efficient transport structures are therefore not preferentially being retained in the nest network. Additionally, the process of nest foundation will also degrade efficient transport structures. As new nests are founded by nests with a high transport value (a high flow of resources), this will alter the structure of the colony around that nest. This establishment of new nests and trails will change the previously existing, efficient, transport structures. In a system which is under strong selective pressure for efficiency, it is expected that highly effective transport structures will be retained. As this is not the case in the red wood ant polydomous colonies, it may be that transport efficiency is not under strong selective pressure. The flow of resources through a particular nest can change over time due to other nests in the network being gained and lost. The integrated nature of the system means that a given nest could maintain the same connections to neighboring nests and trees but still undergo a change in the amount of resources available to it (and therefore its chances of surviving and reproducing), due to nests being abandoned or founded elsewhere in the colony. Nests in unprofitable areas, and therefore with a low resource flow, are more likely to be abandoned than nests in profitable areas. These dynamics will result in the colony moving toward resources and away from unprofitable areas. For a spatially embedded network, such as a polydomous network, this movement is physical movement of nodes. In networks which are not spatially embedded, such as social networks, this process could result in a network clustering around certain nodes, for example individuals with information. The reverse could also occur; a network could cluster away from specific nodes, for example diseased individuals in a social network. These changes in the network structure are self‐organized, resulting from selective pressure based on an individual's position in the network. The nest networks of polydomous ant colonies are, in some ways, analogous to the social networks of individual organisms. Like individuals, ant nests can survive and reproduce (in the sense of founding new nests). There are, however, crucial differences. For example, the death of an individual animal in a social network has direct fitness consequences. In contrast, although abandoning a nest will result in the loss of the resources invested in constructing the nest (which may be considerable), it is unlikely to result in the death of the ants in the nest; they will simply join other nests in the colony. Despite these important differences, polydomous ant colonies may be useful models of social networks. Similar to the ant nest networks, the position of an individual in a social network can have important consequences for their access to, for example, information (e.g., Blonder & Dornhaus, 2011 ; Farine, Aplin, Sheldon, & Hoppitt, 2015 ) and disease (e.g., Cross et al., 2004 ; Otterstatter & Thomson, 2007 ). However, linking these network position effects to the life history of individuals is challenging, due to the difficulties in collecting sufficiently high‐quality temporal data to allow the networks to be examined dynamically (Croft et al., 2008 , 2011 ; Kurvers et al., 2014 ; Whitehead, 2008 ). Using the polydomous nest networks, we have demonstrated that network position can have an important influence on the survival, population change, and budding in a dynamic system. This provides a useful basis for examining the importance of network position in other biological systems such as social systems. The network dynamics observed in these polydomous colonies illustrate the potential feedback between the individual level and the system level in biological networks. The position of an individual within a biological system can affect that individual's exposure to, for example, food, mates, information, and disease (e.g., Aplin, Farine, Morand‐Ferron, & Sheldon, 2012 ; Christley et al., 2005 ; Oh & Badyaev, 2010 ). The structure of the network is, in turn, affected by the nodes within the network. For example, the overall pattern of interactions between individuals in a system can be influenced by a variety of biotic and abiotic factors such as food availability, sex demographics, and season (Brent, MacLarnon, Platt, & Semple, 2013 ; Darden, James, Ramnarine, & Croft, 2009 ; Foster et al., 2012 ). The nests within polydomous colonies highlight how these effects can be reciprocal in a dynamic system. Differential survival and reproduction of nodes in a system will change the structure of the network as new nodes appear and others disappear. This will, in turn, change an individual's relative position within the network, altering its chances of surviving and reproducing. The network, therefore, will be continually restructuring, resulting in a dynamic system which is not stable through time. Dynamic processes will react differently to static systems when facing ecological and environmental changes (Kurvers et al., 2014 ). In conclusion, we found that the survival and budding of nests within polydomous Formica lugubris colonies are related to their position in the trail network. These results highlight how apparently disparate entities in a biological system can be integrated into a functional ecological unit. It also shows how indirect access to resources, through others in a resource exchange system, can have important ecological consequences."
} | 3,846 |
39924486 | PMC11808958 | pmc | 8,741 | {
"abstract": "Background Hemarthria compressa, a widely cultivated forage grass, is critical for supporting livestock production and maintaining the ecological balance in grassland ecosystems. Enhancing its stress resistance and productivity is crucial for sustainable grassland utilization and development. Silicon (Si) and Selenium (Se) are recognized as beneficial nutrients that promote plant growth and stress tolerance, and modulate of plant-microorganism interactions. However, the intricate linkages between the endophytes shifts and host grass growth induced by Si/Se amendments are poorly understood. In this study, a pot experiment was conducted to examine the effects of foliar-applied Si/Se on the growth and nutritional quality of H. compressa grass, as well as the composition, diversity and potential functions of endophytic bacteria in leaves. Results Both Si and Se treatments significantly improved grass biomass by approximately 17%. Nutritional quality was also improved, with Si application increased plant Si and neutral detergent fiber contents by 25.6% and 5.8%, while Se significantly enhanced the grass Se content from 0.055 mg kg −1 to 0.636 mg kg −1 . Furthermore, Si/Se amendments altered the structure of the leaf endophytic bacterial community, resulting in an increased alpha diversity and a more modularized co-occurrence network. Moreover, both Si and Se treatments enriched plant growth-promoting bacterial genera such as Brevundimonas and Truepera . Metabolic function analysis revealed that Si application promoted chlorophyllide biosynthesis by 152%, several carbon metabolism pathways by 35–152%, and redox-related pathways by 57–93%, while the starch biosynthesis pathway was downregulated by 79% of the endophytic bacterial community. In contrast, Se application mainly enhanced starch degradation, CMP-legionamine biosynthesis by 71% and TCA cycle-related pathways by 23–58%, while reducing L-threonine metabolism by 98%. These specific functional changes in the endophytic bacteria induced by Si/Se amendments were closely linked with the observed growth promotion and stress resistance of the host H. compressa grass. Conclusions Si and Se amendments not only enhanced the growth and nutritional quality of H. compressa grass, but also altered the community structure and functional traits of endophytic bacteria in grass. The enrichment of beneficial endophytes and the modification of community metabolic functions within the endophytic community may play important synergistic effects on improving grass growth.",
"conclusion": "Conclusion In summary, Si and Se application significantly improved the growth and nutrient quality of Hemarthria compressa , while also altering the community structure of its endophytic bacteria. The putative functions of plant growth-promoting and stress resilience of the endophytic community were upregulated, and the beneficial endophytes such as Brevundimonas and Truepera were enriched by Si or Se amendment. Furthermore, the diversity of endophytic bacteria and their interactions were enhanced by Si and Se amendments. These findings underscore the critical role of endophyte responses to Si/Se amendments in promoting forage growth and productivity. Future research could further explore the mechanisms underlying these interactions and their long-term impacts on grassland ecosystems.",
"introduction": "Introduction Grasslands, covering 37% of the Earth's terrestrial surface, serve as a critical resource by providing essential forage and habitat for grazing livestock [ 1 ]. Hemarthria compressa (L.f.) R.Br., a perennial grass belonging to the Poaceae family, is widely cultivated in southern Asia for its use as forage [ 2 ]. H. compressa has great adaptability, high regrowth capacity and good feed palatability, which makes it pivotal in supporting local livestock and ecological environment [ 2 , 3 ]. However, many grasslands are situated in marginal areas characterized by low-quality soil, insufficient water resources, and poor fertility [ 4 ]. These stressful conditions limit grass growth and further constrain the overall productivity and ecological services provided by grassland ecosystems [ 5 ]. Therefore, enhancing the growth and stress resistance of forage grasses is crucial for grassland ecosystem maintenance. The beneficial elements silicon (Si) and selenium (Se) have been suggested to play important roles in plant growth promotion and stress resistance under both normal and stress conditions [ 6 , 7 ]. As a bioactive metalloid, Si interacts with various fiber components in plant cell walls, and its content can account for up to 10% of the dry weight of grasses [ 8 ]. Silicification of plant tissues could enhance the structural stability of cell walls and increase the mechanical strength of aerial parts, helping to prevent pathogen and pest infestation [ 8 – 10 ]. Si supplements have been reported to promote the growth of crop plants such as rice and maize by regulating the uptake of essential nutrients (e.g., nitrogen, phosphorus, boron) [ 11 – 13 ], improving photosynthetic activity [ 14 ], and modulating hormonal signals [ 15 ]. Additionally, with a certain amount of Si amendments (mainly through 1% sodium silicate), plants showed more resistant to adverse environments like drought, saline, heavy metal and herbivore stresses through the activities of defense and antioxidant systems [ 16 – 18 ]. Se is considered an essential trace element for animals and humans, as well as some prokaryotes [ 19 ]. It has also been widely reported that Se application can not only increase the Se content in a variety of plants, such as rice, tomato, and grape, but also improve their growth and nutritional quality. Additionally, Se amendment could mitigate oxidative stress through reactive oxygen species scavenging and cell detoxification [ 20 ]. Although Si and Se could act as beneficial elements for most crop plants, it is unclear whether they have a similar positive effect on grass plant growth, and the mechanisms underlying the observed changes in plants due to Si and Se application are still under investigation. Endophytes are ubiquitous in a variety of plant species, they latently or actively colonize every accessible plant tissue (roots, stems, and leaves) and can develop a range of different relationships with their host plants [ 21 ]. Endophytic bacteria are currently receiving considerable attention due to their diverse positive impacts on plant growth, disease resistance, and tolerance to environmental stresses. As the “second genome” of plants, endophytic microorganisms have been documented to be sensitive to agricultural practices such as fertilization [ 22 , 23 ]. For instance, it has been reported that nitrogen application could enrich some endophytic microbes in rice seeds, such as Pseudomonadaceae and Stenotrophomonas , which possess beneficial traits including nitrogen fixation, indole acetic acid (IAA) production, and anti-fungal properties [ 24 ]. Furthermore, studies have shown that Si or Se application could significantly alter the composition, biodiversity and functional characteristics of the rhizosphere microbes of plants such as wheat, rice and soybean, thereby contributing to stress resistance [ 17 , 25 – 27 ]. It has also been revealed that three endophytes ( Paraburkholderia megapolitana , Alcaligenes faecalis and Stenotrophomonas maltophilia ) isolated from soybeans under Se-treatment conditions could augment the plant’s tolerance to drought and salt stress while simultaneously stimulating growth [ 35 ]. Hence, it is reasonable to hypothesize that the endophytic communities may also be shifted by Si/Se application, as well as their functional roles in supporting host plant growth and resilience against adverse conditions. In this study, a pot experiment was conducted to investigate the effects of foliar-applied Si and Se on the grass growth performance and nutritional quality. High-throughput sequencing was used to study the changes in the community structure and potential functionalities of the leaf endophytic bacteria under Si and Se treatments. The objective of this study was to assess how the endophytes in H. compressa respond to Si and Se application, and whether the changes in the endophytic community have synergistic effects on plant growth.",
"discussion": "Results and discussion Impact of Si and Se application on grass biomass and nutritional traits Foliar application of silicon (Si) and selenium (Se) significantly increased the accumulation of beneficial elements in Hemarthria compressa (Table 1 ). In comparison with control (CK), Si supplementation significantly increased the Si concentration in grass tissues by 25.5% and the Se concentration by 34.5% ( p < 0.05). Similarly, foliar Se application significantly increased the plant Se content from 0.055 to 0.636 mg Se kg −1 ( p < 0.05) but only slightly increased the Si content by 11%. Both Si and Se treatments led to a significant 17% increase in grass dry weight (DW) ( p < 0.05). These findings revealed that the application of Si or Se not only increased the nutrient content of H. compressa but also enhanced its yield. The observed increases in plant biomass induced by Si or Se application are consistent with previous studies on several forage grasses and crops [ 44 – 50 ], which may be closely related to the optimized energy supply and enhanced metabolic activity [ 51 ]. For example, Si could improve plant photosynthetic efficiency through increased chlorophyll content, which in turn promotes overall plant growth and development [ 52 ]. Se could enhance antioxidant metabolism, thereby mitigating oxidative stress and promoting plant growth [ 20 ]. Moreover, the elevated Se content in the leaves could indirectly supply livestock with essential Se nutrients.\n Table 1 Effects of foliar Si and Se application on biomass and leaf nutritional characteristics of Hemarthria compressa plants Group DW (g per pot) Si (g kg −1 ) Se (mg kg −1 ) CP (g kg −1 ) NDF (%) ADF (%) CK 16.8 ± 1.2b 37.16 ± 1.76b 0.055 ± 0.003c 189.67 ± 5.93a 45.13 ± 1.40b 36.93 ± 1.46a Si 19.7 ± 1.1a 46.66 ± 0.74a 0.074 ± 0.013b 186.50 ± 4.01a 47.75 ± 0.96a 35.08 ± 0.73a Se 19.7 ± 0.7a 41.09 ± 2.60ab 0.636 ± 0.016a 182.97 ± 3.41a 46.57 ± 0.62ab 35.33 ± 1.30a Different letters indicated significant difference between treatments ( p < 0.05), and values are means ± standard deviations ( n = 4) DW dry weight, CP crude protein, NDF neutral detergent fiber, ADF acid detergent fiber Meanwhile, Si fertilization significantly increased the neutral detergent fiber (NDF) content by 5.8% compared to CK ( p < 0.05), whereas Se fertilization increased the NDF content by 3.2% with no statistically significant. However, neither Si nor Se fertilizers had a significant effect on the plant crude protein (CP) or acid detergent fiber (ADF) content ( p > 0.05). According to previous studies, the increased fiber and Si contents are often closely related to the mechanical strength, which is likely due to the decomposition of silica in the cell wall [ 8 , 53 ]. This enhancement could improve the ability of plants to defend against pathogen invasions. Hence, it can be speculated that Si or Se could improve the H . compressa grass growth and resistance under abiotic and biotic conditions."
} | 2,831 |
27620972 | PMC5216901 | pmc | 8,742 | {
"abstract": "Summary \n Photosystem I (PSI) is a pigment protein complex catalyzing the light‐driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana , showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type‐R light‐harvesting complexes (LHCr4‐8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained.",
"introduction": "Introduction In organisms performing oxygenic photosynthesis, Photosystem I (PSI) is responsible for the light‐driven electron transport from plastocyanin/cytochrome 6 to ferredoxin (Raven et al ., 1999 ; Nelson & Yocum, 2006 ; Croce & van Amerongen, 2013 ; Bernal‐Bayard et al ., 2015 ). In eukaryotes, PSI is organized as a supercomplex composed of two moieties, the core and the peripheral antenna system. The PSI core is composed of 12–14 subunits (Busch & Hippler, 2011 ). PsaA and PsaB bind the reaction center P700 as well as most of the cofactors involved in electron transport and c . 100 chlorophylls (Chls) active in light harvesting (Jordan et al ., 2001 ; Qin et al ., 2015 ). PsaA and PsaB are highly conserved in cyanobacteria, algae and plants (Allen et al ., 2011 ). Other core subunits are instead differently distributed in various phylogenetic groups. For instance PsaG and PsaH are only found in plants and green algae (Vanselow et al ., 2009 ; Busch & Hippler, 2011 ) where they serve as docking site for the association of the peripheral antenna (Lunde et al ., 2000 ; Ben‐Shem et al ., 2003 ). The peripheral antenna is composed of light‐harvesting complexes (LHC) whose sequences vary in different organisms: LHCa1–6 are present in plants (Jansson, 1999 ), LHCa1–9 are present in the green alga Chlamydomonas reinhardtii (Mozzo et al ., 2010 ), whereas LHCR proteins are believed to comprise the peripheral antenna in red algae and diatoms (Busch et al ., 2010 ; Busch & Hippler, 2011 ; Thangaraj et al ., 2011 ). The number of LHC associated with PSI is also variable: four subunits are associated with the PSI core in higher plants , in the moss Physcomitrella patens , and in some red algae and diatoms (Ben‐Shem et al ., 2003 ; Veith & Büchel, 2007 ; Busch et al ., 2010 , 2013 ). Differently, nine antenna subunits are found associated with PSI in other red algae (Gardian et al ., 2007 ; Thangaraj et al ., 2011 ) as in the green alga C. reinhardtii (Drop et al ., 2011 ). Finally, the oligomeric state of PSI also varies in different organisms. Cyanobacterial PSI often has been reported to be a trimer (Boekema et al ., 1987 ; Jordan et al ., 2001 ), although PSI tetramers have been identified in a growing number of species (Watanabe et al ., 2011 , 2014 ; Li et al ., 2014 ). However, PSI was found to be monomeric in all eukaryotes analyzed so far, including plants (Ben‐Shem et al ., 2003 ; Kouril et al ., 2005a ), diatoms (Veith & Büchel, 2007 ; Ikeda et al ., 2013 ), green and red algae (Gardian et al ., 2007 ; Drop et al ., 2011 ). A peculiar feature of PSI is the presence of Chls absorbing at energy lower than the primary electron donor P700, called red forms (Croce & van Amerongen, 2013 ). Although these far red‐absorbing Chls account only for a small fraction of the total absorption, they have a strong influence in excitation energy transfer and trapping, slowing down the trapping time as they introduce uphill steps in the energy transfer process (Gobets et al ., 2001 ; Jennings et al ., 2003 ; Engelmann et al ., 2006 ; Wientjes et al ., 2011b ). The presence of low energy absorbing Chls is ubiquitous in PSI, but their energy appears to be highly species‐dependent (Gobets & van Grondelle, 2001 ; Croce & van Amerongen, 2013 ). In plants, most red forms are associated with the outer antenna complexes and in particular with LHCA3 and LHCA4 (Schmid et al ., 1997 ; Castelletti et al ., 2003 ), although the core also contains low energy forms (Croce et al ., 1998 ; Gobets & van Grondelle, 2001 ). In the present work, we investigated the structural and functional properties of the Photosystem I supercomplex (PSI‐LHC) of the eustigmatophycea Nannochloropsis gaditana . This microalga belongs to the phylum Heterokonta, which also includes diatoms and brown algae (Cavalier‐Smith, 2004 ; Riisberg et al ., 2009 ), that originated from a secondary endosymbiotic event where an eukaryotic host cell engulfed a red alga (Archibald & Keeling, 2002 ). In the last few years, species belonging to the Nannochloropsis genus have gained increased attention not only for their evolutionary position, but also for their ability to accumulate a large amount of lipids (Rodolfi et al ., 2009 ; Bondioli et al ., 2012 ; Simionato et al ., 2013 ). The N. gaditana photosynthetic apparatus presents distinct features with respect to other algae such as the presence of only Chl a and an atypical carotenoid composition with violaxanthin and vaucheriaxanthin esters as the most abundant xanthophylls (Sukenik et al ., 1992 , 2000 ; Basso et al ., 2014 ). A deeper characterization of this organism thus contributes to a better understanding of the variability of photosynthetic organisms in an evolutionary context.",
"discussion": "Discussion Structural organization and function of PSI ‐ LHC in the heterokont Nannochloropsis gaditana \n In this work the combination of structural, proteomic and functional analysis provides a comprehensive picture of the structure and composition of Photosytem I light‐harvesting complexes (PSI‐LHC) from Nannochloropsis gaditana . EM analysis of PSI purified from N. gaditana shows that this is a supercomplex composed of a core complex and a peripheral antenna system, as in all eukaryotes analyzed thus far (Fig. 1 ). At variance with plants and other algae (Drop et al ., 2011 ; Thangaraj et al ., 2011 ; Qin et al ., 2015 ), however, five LHCs, identified by MS analysis as LHCr4‐8, are found to be associated with N. gaditana PSI‐LHC (Table 2 ; Fig. 3 c). MS analysis detected three more LHCr‐type LHCs, eight LHCf, three LHCx, thus covering the entire LHC superfamily identified in the genome of N. gaditana (Table 2 ). These additional LHCs, however, were not specifically enriched in PSI‐LHC suggesting that they are not strongly associated with this supercomplex. It is, however, not possible to exclude the possibility that some additional antenna are loosely associated with PSI in vivo but lost during purification. The superimposition of the N. gaditana PSI supercomplex EM map with the high‐resolution structures of plant PSI evidenced a peculiar arrangement of antenna complexes with two LHCs bound in a highly conserved position, the same occupied by LHCa2/3 in plants (Fig. 2 ). Three additional LHCs are instead found at the other side of the core complex, where PsaL is located. As schematized in Fig. 6 , this peculiar structural organization correlates with differences in the composition of the PSI core complex derived from genome and proteome analysis (Tables 1 , 2 ; Fig. 3 ). In plants the association of LHCa1/4 with the core complex is partly mediated by PsaG (Ben‐Shem et al ., 2003 ; Qin et al ., 2015 ). PsaG is absent in N. gaditana and indeed no LHC was observed in the position corresponding to plant LHCa1/4. PsaF and J have been suggested to mediate interactions with LHCa2/3 in plants (Qin et al ., 2015 ). Consistent with the conservation of PsaF and PsaJ in N. gaditana , two LHCs are found in the same position also in this species. Considering that the core complex subunits are well conserved in different organisms (Table 1 ) this observation also suggests that two LHCs are likely to be bound in this position in PSI from all photosynthetic eukaryotes, including diatoms. Figure 6 Schematic comparison of Photosystem I supercomplex (PSI‐LHC) structural organization in Nannochloropsis gaditana with plants and other algae. Letters from A to K indicate the predicted position of the corresponding Psa subunits. Dark green subunits are those conserved in all eukaryotes (see Tables 1 and 2 ), red subunits are present in plants, and light green subunits are only in N. gaditana . In plants, psak knockdown plants showed destabilized LHCa2/3 association (Jensen et al ., 2000 ) suggesting that PsaK contributes to their binding to PSI. The recent structure of plant PSI supercomplex, however, showed that PsaK does not interact directly with the peripheral antennae (Mazor et al ., 2015 ; Qin et al ., 2015 ). PsaK is not found in N. gaditana or in any other heterokont analyzed thus far (Table 1 ) (Le Corguillé et al ., 2009 ; Starkenburg et al ., 2014 ) likely because of a loss that occurred during or after the secondary endosymbiosis. In these organisms the absence of PsaK does not prevent the association of two LHCs on this side of the core, which confirms that PsaK is not strictly necessary for the association of peripheral antenna. Additional PSI core subunits identified in plants are not conserved in heterokonts. PsaN in plants is found to be associated with PsaF in the docking site for plastocyanin (Haldrup et al ., 1999 ), the soluble PSI electron donor. In N. gaditana and other heterokonts only PsaF is present, suggesting that PsaN is dispensable for efficient electron transfer to PSI, consistent with its absence in cyanobacteria. This is likely correlated with a difference in electron transport chain, because N. gaditana genome lacks a plastocyanin encoding gene (Corteggiani Carpinelli et al ., 2014 ) and the PSI lumenal electron donor is likely cytochrome c6 , as previously suggested for the red alga Galdieria sulphuraria (Vanselow et al ., 2009 ) and diatoms (Grouneva et al ., 2011 ). It is also worth underlining the absence of PsaH, a subunit essential for the association of LHCII during state transitions in plants (Lunde et al ., 2000 ). In N. gaditana other LHCs associate with the core complex in the region generally occupied by PsaH (Fig. 6 ). This picture suggests that state transitions, if present in N. gaditana , most likely involve different structural interactions between antenna and PSI complexes than those described in plants and green algae (Kouril et al ., 2005b ; Drop et al ., 2014 ). The functional data show that, despite this different organization, energy transfer and trapping in the PSI complex of N. gaditana is very fast. Indeed, the average decay time of the N. gaditana PSI‐LHC monomer is even shorter (32 ± 3 ps) than that of both Arabidopsis thaliana and Chlamydomonas reinhardtii PSI‐LHCI ( c . 50 ps; Wientjes et al ., 2011b ; Le Quiniou et al ., 2015b ). This difference can be due to a smaller number of pigments associated with the PSI of N. gaditana and/or to a difference in the red forms. It is well documented that the number and the energy of these forms influence the excitation energy migration towards the reaction center in PSI (i.e. more red forms, slower transfer) (Gobets et al ., 2001 ; Wientjes et al ., 2011b ; Le Quiniou et al ., 2015b ). Although the number of Chls associated with the LHC in N. gaditana is not known and we can thus not exclude that the antenna size of PSI in N. gaditana is smaller, it is very likely that part of the observed difference in trapping time is due to the diversity in red forms. Indeed, the red forms of PSI‐LHC of N. gaditana are at a higher energy than those of A. thaliana as indicated by their emission maximum (722 nm for N. gaditana vs 735 nm for A. thaliana ) and absorption maximum (697 nm in N. gaditana vs 705–710 nm in A. thaliana ; Morosinotto et al ., 2003 ; Croce et al ., 2007 ) and are then expected to have a smaller influence on the trapping time than the red forms of plants. Independently from the exact origin of this difference, the very fast trapping time observed for PSI‐LHC of N. gaditana also indicates that all of the LHC subunits are functionally well connected with the core, allowing for fast excitation energy transfer and high quantum efficiency of energy conversion. This high efficiency is common to all PSI complexes analyzed so far (Wientjes et al ., 2011b ; Le Quiniou et al ., 2015a , b ) and thus appears to be independent of the organization of the antenna around the core because, for example, the position of the additional LHC in N. gaditana differs from that of both LHCI and LHCII in plants and C. reinhardtii (Kouril et al ., 2005b ; Drop et al ., 2011 , 2014 ; Qin et al ., 2015 ). This suggests that the design of the PSI core allows the functional association of additional subunits to different part of the complex such that even a PSI core completely surrounded by antennae can maintain a very high quantum efficiency."
} | 3,463 |
39516195 | PMC11549363 | pmc | 8,743 | {
"abstract": "The endosymbiont Candidatus Azoamicus ciliaticola was proposed to generate ATP for its eukaryotic host, an anaerobic ciliate of the Plagiopylea class, fulfilling a function analogous to mitochondria in other eukaryotic cells. The discovery of this respiratory endosymbiosis has major implications for both evolutionary history and ecology of microbial eukaryotes. However, with only a single species described, knowledge of its environmental distribution and diversity is limited. Here we report four complete, circular metagenome assembled genomes (cMAGs) representing respiratory endosymbionts inhabiting groundwater in California, Ohio, and Germany. These cMAGs form two lineages comprising a monophyletic clade within the uncharacterized gammaproteobacterial order UBA6186, enabling evolutionary analysis of their key protein complexes. Strikingly, all four cMAGs encode a cytochrome cbb 3 oxidase, which indicates that these endosymbionts have the capacity for aerobic respiration. Accordingly, we detect these respiratory endosymbionts in diverse habitats worldwide, thus further expanding the ecological scope of this respiratory symbiosis.",
"introduction": "Introduction Host beneficial endosymbionts (HBEs) are widespread in eukaryotes, and often evolve from a parasitic or pathogenic ancestor 1 . The best studied HBEs are nutritional symbionts of insects that provide their hosts with essential molecules (such as vitamins or cofactors), which the hosts cannot synthesize or obtain from their diet 2 , 3 . Another example are defensive endosymbionts that synthesize compounds that protect the host or its offspring against predators or pathogens 4 , 5 . Beyond insect hosts, intracellular endosymbionts also perform a wide range of beneficial functions in protists, such as photosynthesis, scavenging of hydrogen or other metabolic products, or complementing catabolic pathways (reviewed in ref. 6 ). The recently discovered protist endosymbiont Ca . A. ciliaticola fulfills yet another function, using a denitrifying respiratory chain to generate ATP that can be supplied to its ciliate host 7 . The Plagiopylean ciliate host of Ca . A. ciliaticola was proposed to harbor only metabolically reduced organelles known as mitochondrion-related organelles (MROs) 8 , 9 , possibly in the form of hydrogenosomes. Thus the host is likely incapable of (aerobic) respiration, although it might still be able to generate ATP in the MROs, or through substrate level phosphorylation in the cytoplasm 7 , 10 . The endosymbiont Ca . A. ciliaticola also lacks the capability for aerobic respiration, restricting the ecological niche of its ciliate host to permanently anoxic habitats, such as the hypolimnion of a meromictic lake. Interestingly, virtually all organisms capable of denitrification are facultative anaerobes 11 , 12 , and therefore it was hypothesized that Ca . A. ciliaticola evolved from a predecessor that was capable of both aerobic respiration and denitrification 7 . To investigate this possibility, we searched for genomes of endosymbionts related to Ca . A. ciliaticola in publicly available environmental metagenomic sequencing datasets. In this work we describe the discovery for four additional closed genomes representing respiratory endosymbionts. The five genomes form a monophyletic clade and have strongly conserved gene content and genomic features, indicating a shared function in their host. Using the expanded genomic coverage, we confidently place this endosymbiont clade in the UBA6186 order, which we accordingly propose to rename to Azoamicales . We show that the four newly retrieved genomes encode the capability for aerobic respiration in addition to respiration of nitrogen oxides, and that the genes required for both were acquired horizontally. Finally, we use the expanded genome coverage to show that the respiratory endosymbionts are globally distributed.",
"discussion": "Discussion The four Azoamicaceae genomes described in this study greatly expand our understanding of the diversity and distribution of respiratory endosymbiosis. We show that Azoamicaceae are globally distributed, and have the genomic potential for both denitrification and aerobic respiration. Of the metabolic capacities encoded in the new endosymbiont genomes it is undoubtedly the capacity to respire oxygen that has the most profound physiological and ecological implications. Retention of the cbb 3 terminal oxidase in these extremely reduced genomes, as well as the comparatively high ccoNOP transcription in Ca . A. aquiferis, strongly implies an active role in endosymbiont and host physiology, by potentially conveying the capacity to respire oxygen to its host. Furthermore, the presence of the cbb 3 oxidase can explain the broad distribution of the symbiont in seasonally or permanently oxic environments, thus considerably expanding the ecological role for this symbiosis. In fact, the capacity for aerobic respiration (or oxygen detoxification) may have facilitated the dispersal of the (presumably anaerobic) host across oxic environments. Endosymbionts are common in anaerobic ciliates, and syntrophic endosymbionts have been proposed to play a role in the transition of ciliates to anaerobiosis 30 , 31 . Given their potential for aerobic respiration, the Azoamicaceae may represent an interesting example of a ciliate endosymbiont facilitating a secondary adaptation, this time to a (micro)aerobic lifestyle. We propose that Plagiopylean ciliates are the putative hosts of the groundwater Azoamicaceae. Indeed, based on a molecular analysis, Ciliophora were abundant in the wells of the Hainich CZE 32 , from which Ca . A. aquiferis was retrieved. We could reconstruct full length Plagiopylea 18S rRNA gene sequences from the metagenomes containing Ca . A. aquiferis as well as Ca . A. agrarius. A shorter Plagiopylea 18S rRNA gene fragment from the metagenome containing both Ca . A. viridis and Ca . A. soli could also be recovered. Interestingly, the phylogeny of 18S rRNA sequences (Supplementary Fig. 7 ) is congruent with the genome phylogeny of their respective putative symbionts (Fig. 1 ) and thus could be indicative of long-term vertical inheritance of the symbionts in the Odontostomatida order. This is further supported by the extremely reduced nature of the symbiont genomes that is consistent with an older, likely vertically inherited, symbiosis 6 . However, more data on the host identity are needed to confirm an exclusively vertical inheritance of the Azoamicaceae symbionts, as some protist hosts are able to replace their symbionts 33 , 34 . Additionally, while an apparent vertical inheritance of protist endosymbionts can be observed over short evolutionary time 35 , examples of long term vertical inheritance are more rare, possibly due to eventual replacement of protist symbionts 6 , or simply undersampling 36 . Our comparative genomics of the five Azoamicaceae genomes shows a varying degree of genome erosion, as observed in insect HBEs 37 , 38 . The Azoamicaceae seem to converge on a minimal gene set encoding the genes required for ATP generation using a denitrifying and oxygen respiring respiratory chain, with the conserved core genes comprising over 80% of the protein complement of the smallest genomes. The loss of biosynthetic pathways essential for the respiratory function, such as heme biosynthesis and cytochrome c maturation, strongly suggests that host proteins are targeted to the endosymbiont. Host protein targeting has previously been observed in rare cases in insect HBEs 39 , 40 in the trypanosomatid Angomonas deanei 41 , and more extensively in the chromatophore of Paulinella chromatophora 42 and in the nitroplast (formerly Candidatus Atelocyanobacterium thalassa) of Braarudosphaera bigelowii 43 . These observations have blurred the boundaries between HBEs and organelles 36 , 44 , and the extant Azoamicaceae may represent another snapshot of the transition between the two. While comparative genomic analyzes can provide a great insight into the evolutionary history and metabolic potential of these enigmatic endosymbionts, the establishment of an experimentally tractable laboratory culture of the host is essential to test these exciting hypotheses."
} | 2,064 |
28928205 | PMC5605933 | pmc | 8,746 | {
"abstract": "ABSTRACT The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms leading to similar phenotypes. Here, we establish the importance of the balance between these two factors for accurate regulation of the transition between the planktonic and sessile lifestyles. This balance is achieved by negative-feedback regulation of BolA and c-di-GMP. BolA not only contributes directly to the motility of bacteria but also regulates the expression of diguanylate cyclases and phosphodiesterases. This expression modulation influences the synthesis and degradation of c-di-GMP, while this signaling metabolite has a negative influence in bolA mRNA transcription. Finally, we present evidence of the dominant role of BolA in biofilm, showing that, even in the presence of elevated c-di-GMP levels, biofilm formation is reduced in the absence of BolA. C-di-GMP is one of the most important bacterial second messengers involved in several cellular processes, including virulence, cell cycle regulation, biofilm formation, and flagellar synthesis. In this study, we unravelled a direct connection between the bolA morphogene and the c-di-GMP signaling molecule. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in Escherichia coli . This work provides important clues that can be helpful in the development of new strategies, and the results can be applied to other organisms with relevance for human health.",
"introduction": "INTRODUCTION The ability of bacteria to sense and adapt to environmental changes is critical for survival. Under stress conditions, prokaryotic cells rapidly adjust their gene expression in order to induce the physiological and molecular adaptations needed. The Escherichia coli ( E. coli ) bolA gene is induced at the onset of stationary phase and in response to several stresses, leading to substantial changes in the cell ( 1 , 2 ). BolA expression is tightly regulated at the transcriptional and post-transcriptional levels ( 1 , 3 – 5 ). Under optimal growth conditions, bolA transcription is regulated by a constitutive promoter, bolAp2 , whose activity depends on the housekeeping sigma factor σ 70 ( 1 ). Under harsh environmental conditions, its expression is mostly driven by a gearbox promoter, bolAp1 , controlled by sigma factor σ S ( 1 , 2 , 6 ). Additionally, bolA expression is repressed by the histone-like protein H-NS ( 7 ) and by OmpR in its phosphorylated form ( 8 ). The post-transcriptional regulation of bolA mRNA levels involves both RNase III and poly(A) polymerase I (PAPI) ( 5 , 9 ). BolA and its homologues constitute a protein family that is widely conserved across prokaryotes and eukaryotes ( 10 ). Functional studies have associated BolA with a range of cellular processes, such as bacterial morphology, membrane permeability, motility, and biofilm formation (reviewed in reference 11 ). However, the biological details regarding its mechanism of action in the regulation of biofilm were only recently unravelled ( 12 ). BolA was shown to repress flagellar synthesis and induce TCA cycle-related genes, with consequences for bacterial motility ( 12 ). One of the most extensively studied factors involved in the transition from the motile state to the non-motile/biofilm state in bacteria is the bacterial second messenger c-di-GMP ( 13 ). This molecule is synthesized by diguanylate cyclases (DGCs), whose activity has been associated with the highly conserved GGDEF protein domain ( 14 ). On the other hand, its hydrolysis is carried out by phosphodiesterases (PDEs), enzymes with an EAL or HD-GYP domain ( 15 , 16 ). C-di-GMP was shown to be involved in several cellular processes, including cell differentiation ( 17 , 18 ), cell cycle progression ( 19 ), biofilm formation and dispersal ( 20 – 22 ), and cell motility ( 13 , 23 ). One of the most remarkable features of c-di-GMP is its ability to regulate the transition from the planktonic lifestyle to the sessile lifestyle ( 13 , 24 ). The increased production of c-di-GMP by certain DGCs has a negative effect on cell motility and strongly activates the production of adhesins and biofilm matrix components ( 13 ). In contrast, low levels of c-di-GMP, associated with the activity of PDEs, promote motility and increase bacterial dispersion ( 23 , 25 ). Thus, both BolA and c-di-GMP play key roles in the transition between the planktonic and the sessile lifestyles, repressing cell motility and promoting biofilm development ( 12 , 26 ). In this report, we have unravelled a direct connection between the bolA morphogene and the signaling molecule c-di-GMP. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in E. coli .",
"discussion": "DISCUSSION The molecular mechanism behind the bacterial transition from the planktonic lifestyle to the biofilm lifestyle has been a fascinating object of study. Despite significant advances in the topic, there are still several interconnected pathways that need to be clarified. Here, we are adding an important piece to the puzzle by showing that BolA can interfere with this transition by balancing the intracellular concentration of the bacterial second messenger c-di-GMP. Recently, we showed that BolA is a very important transcriptional switch, that is specifically involved in the transition between the planktonic stage and the attachment stage of biofilm formation processes ( 12 ). Elevated levels of BolA were shown to affect negatively the swimming of bacteria in semi-solid media ( 12 ). This was due to impaired flagellar assembly in a strain overexpressing BolA. Taking this into account and in agreement with the role of BolA in biofilm formation ( 27 ), it would be expected that bacteria would have improved swimming capacity in the absence of BolA. However, to our surprise, motility was impaired in the Δ bolA strain. The ability of bacteria to regulate the planktonic-to-sessile lifestyle transition is well known to involve the bacterial second messenger c-di-GMP ( 13 , 25 ). Its synthesis and degradation have received significant attention in recent years ( 43 ). The regulatory network of c-di-GMP is complex, partly due to the large number of enzymes which synthesize (DGCs) or degrade (PDEs) this molecule and which are encoded in the genome. In several previous studies ( 12 , 27 ), BolA protein was referred to as an important bacterial protein that stimulates biofilm formation. The fact that BolA and c-di-GMP have similar functions with respect to the different stages of the bacterial life cycle ( 12 , 25 ) and the fact that BolA represses flagellar synthesis, enhancing the curli biosynthetic pathway, led us to investigate if its expression had consequences for the regulation of c-di-GMP levels. Quantitative analyses of intracellular c-di-GMP revealed an increase in the concentration of this metabolite in Δ bolA cells. In fact, the reduced swimming seen with this strain might be associated with the 1.7-fold-increased levels of c-di-GMP. As mentioned above, there are large numbers of DGCs and PDEs encoded in the bacterial genomes. The balance between synthesis and degradation of c-di-GMP comes from a complex network of regulation that involves both types of enzymes ( 23 ). BolA was shown to directly interact with nucleic acids in order to activate or repress gene expression ( 12 , 38 , 39 ). In agreement, our results have revealed that BolA can play an active role in the regulation of c-di-GMP. Transcriptomic analyses, together with biolayer interferometry, allowed us to identify the BolA targets with regard to DGCs and PDEs. Among the differentially regulated genes, there are several that encode proteins still classified as putative DGCs or PDEs. Moreover, two of the most important enzymes involved in the synthesis and degradation of c-di-GMP are transcriptionally controlled by BolA. Those are the ydaM DGC and yhjH PDE. Both genes encode proteins that have been previously described as participating in the cascade of c-di-GMP regulation linked by the YciR trigger enzyme ( 36 ). YhjH is coregulated with flagellar genes and its levels reduce with the concomitant increase of c-di-GMP levels ( 35 ). At the same time, c-di-GMP impairs the activity of another PDE protein, YciR, in binding YdaM, which allows YdaM to generate c-di-GMP and to activate the curli biosynthetic pathway ( 36 ). Our data show downregulation of ydaM and upregulation of yhjH . Moreover, the BolA effect on c-di-GMP appeared to be fully dependent upon the presence of YdaM. BolA regulation of YhjH apparently had less influence on c-di-GMP. However, bolA and yhjH mutants showed additive effects on motility, suggesting yet other targets for BolA in the motility pathway. Thus, it is plausible to speculate about interference of BolA in the cascade referred to above, that would result in fine-tuned gradual regulation of c-di-GMP in the transition of bacteria to the sessile state. Interestingly, the interplay between BolA and c-di-GMP is not unidirectional. bolA mRNA transcription is also influenced by c-di-GMP. When this molecule is present in elevated amounts in the cell, there is a reduction in bolA mRNA levels. This strengthens the idea of the importance of the balance between these two molecules in achieving a correct adaptation of bacteria to growth conditions. C-di-GMP is known to bind proteins containing a PilZ domain ( 33 ). As a transcription factor, BolA is able to bind nucleic acids. We believe that the balance between these two players is of major importance for tight regulation of the complex flagellar and curli synthesis pathways. High levels of c-di-GMP are strongly linked to bacterial biofilm formation ( 13 ). Taking into consideration the fact that, without BolA, c-di-GMP levels are elevated but E. coli biofilm formation is nevertheless partially impaired, the present work underlines the importance of BolA for a proper bacterial stress response and consequent biofilm development ( Fig. 7 ). FIG 7 Cross-talk between the transcription factor BolA and the second messenger c-di-GMP in bacterial biofilm formation. BolA and c-di-GMP are known to be important players in biofilm development. ( A ) Our model suggests that a negative-feedback modulation, which leads to a balance between these two factors, is needed for a proper physiological response. ( B ) Additionally, the absence of BolA leads to less-robust biofilm formation, even in the presence of high levels of c-di-GMP, which evidences the determinant function of this protein in the regulation of biofilm formation. In bacteria, c-di-GMP is metabolized into pGpG, which is further hydrolyzed into GMP ( 44 ). When c-di-GMP was added to the media as a supplement, its characteristic effect in bacterial swimming was observed, suggesting the existence of a mechanism by which cells can sense or internalize this second messenger molecule. Additionally, it is plausible to hypothesize that bacteria that lyse can release different molecules to the media, including enzymes that metabolize c-di-GMP. When the medium was supplemented with pGpG, no response of the bacteria to this molecule was observed. However, cGMP and the precursors GTP and guanosine had an influence on the swimming of E. coli . Together, these observations may indicate not only that c-di-GMP can act via a sensor/receptor of bacteria but also that the degradation product cGMP and synthesis precursors may influence intracellular signaling mechanisms. Nevertheless, the specific mechanism by which c-di-GMP regulates bolA expression remains to be elucidated. Our results highlight the cross-talk that occurs between the BolA transcription factor and the second molecular messenger c-di-GMP during the transition between the planktonic and sedentary bacterial lifestyles. This finding underlines the complexity of bacterial cell regulation, revealing the existence of one additional tool for fine-tuning such an important cellular molecular mechanism. Whether the cross-talk between BolA and c-di-GMP connects to feed-forward or homeostatic regulation remains unclear. In a recent review, Caly and colleagues discussed strategies designed to control bacterial biofilm formation by targeting c-di-GMP ( 45 ). C-di-GMP is among the most important bacterial second messengers involved in many cellular processes, including differentiation, virulence, cell cycle regulation, biofilm formation, and flagellar synthesis ( 43 ). In this regard, the intricate relationship between BolA and c-di-GMP opens more options with the possibility to extend our studies to other organisms with relevance for human health."
} | 3,314 |
29946243 | PMC6006525 | pmc | 8,747 | {
"abstract": "Animals harbor an extensive, dynamic microbial ecosystem in their gut. Gut microbiota (GM) supposedly modulate various host functions including fecundity, metabolism, immunity, cognition and behavior. Starting by analyzing the concept of the holobiont as a unit of selection, we highlight recent findings suggesting an intimate link between GM and animal social behavior. We consider two reciprocal emerging themes: (i) that GM influence host social behavior; and (ii) that social behavior and social structure shape the composition of the GM across individuals. We propose that, throughout a long history of coevolution, GM may have become involved in the modulation of their host’s sociality to foster their own transmission, while in turn social organization may have fine-tuned the transmission of beneficial endosymbionts and prevented pathogen infection. We suggest that investigating these reciprocal interactions can advance our understanding of sociality, from healthy and impaired social cognition to the evolution of specific social behaviors and societal structure.",
"conclusion": "Conclusion Evidence emerging from biomedical and ecological studies suggests that GM plays an important role in shaping host sociality and that, vice versa, social organization and behavior influence the GM associated with individuals. The two processes appear to be linked, as microbes acquired through horizontal transmission by mothers can be transmitted vertically to the next generation. Gestation and infancy appear to be critical periods for the GM to influence the infant’s brain development, and may be critical in determining its future degree of sociability (Figure 1 ). These findings reinforce earlier hypotheses proposing that gregariousness, social structures, and social behaviors might in part have evolved because they enhance or fine-tune the beneficial transmission of endosymbionts (Troyer, 1984 ; Lombardo, 2008 ; Montiel-Castro et al., 2013 ). Simultaneously, some co-evolving gut bacteria might have gotten involved in modulating their host’s sociality because this would increase their own transmission between hosts. The mutual dependency between macroorganisms and symbionts, has been highlighted here by a focus on the reciprocal interactions between microorganisms and social processes at the macroorganism level. This evidence suggests that the holobiont may in fact be a “unit of selection”, understood both as a “replicator” or an “interactor” (Lloyd, 2017 ). While group living increases the risk of exposure to pathogens, the social transmission of beneficial GM may compensate for this risk if it increases the hosts resistance to infectious agents (Ezenwa et al., 2016b ). Findings in insects directly link socially transmitted GM to pathogen resilience (Koch and Schmid-Hempel, 2011b ); it will be interesting to see whether this generalizes to mammals. Beyond shedding light on the ecology and evolution of species, such findings may have major implications for human health. The fact that specific GM may shape social behavior could eventually translate into GM-based therapies for mental disorders associated with social deficits, i.e., psychobiotics (Vétizou et al., 2015 ). Also, closer examination of the long-term consequences of GM establishment during early life and how maternal diet, mode of delivery and feeding, and antibiotic treatments influence this process might optimize recommendations and nutritional interventions to promote a healthy brain development (Goyal et al., 2015 ). The sharing of GM through group living and social interactions may be relevant for human health as well, as group promoted or socially transmitted GM may support a healthy brain development and determine individual disease susceptibility. Industrialized societies have lower bacterial diversity within individuals, and less similar GM between individuals than non-industrialized societies, which may be mirrored in differences in the susceptibility to infectious and autoimmune diseases. This raises the question of to what extent differences in social structures and behaviors limit the horizontal transmission of potentially protective GM and, hence, contribute to these observations. Overall, it appears that the microbes, in particular GM, and host social behavior have coevolved to become virtually inseparable. The GM appear to shape the social behavior and structure of their hosts, and depend on them for transmission. In turn, GM offer benefits by protecting from several diseases. Further investigation of these fascinating reciprocal interactions may open avenues for the treatment of neurological disorders or the management of health and diseases with an evolutionary perspective. Moreover, investigating these reciprocal interactions may advance our understanding of sociality, from social cognition to the basis of society structures.",
"introduction": "Introduction Animals harbor a diverse community of microbes, in their gut and in almost any other site, both on and within, their bodies. Host and gut microbiota (GM) interact symbiotically (Sommer and Bäckhed, 2013 ). The GM contribute to host health and fitness, playing a major role in diverse host functions including development, fecundity, metabolism and immunity; for the microbes, animal intestines are a favorable niche (Shapira, 2016 ). By removing the need for culturing microorganisms for identification, next-generation sequencing methods have massively advanced the characterization of the human GM and are continuously expanding our knowledge about endosymbiotic microbes. We have just recently grasped that the human GM comprise hundreds of microbial species, with Firmicutes and Bacteroidetes as the dominant phyla (Lozupone et al., 2012 ), and that microbial life may thus have roles in multiple physiological processes including those related to mental health (Cryan and Dinan, 2012 ). This has opened the possibility of applying a “gestalt perspective” allowing us to understand physiological, behavioral and cognitive processes as part of an integrated whole (Koffka, 2013 ). Recent technological developments actually allow for the integration of data from various sources such as the genome, transcriptome, proteome, epigenome and microbiome in what has been termed “Gestaltomics”, as a useful approach to the understanding of psychiatric disorders at different levels of organization (Gutierrez Najera et al., 2017 ). Aware of the perils of our inference of adaptive significance from proximate control of behavior (Dewsbury, 1999 ), our review proceeds as follows: in view of the accumulating evidence linking biological processes between micro- and macroorganisms, first, we suggest that the use of the concept of a holobiont as a unit of selection should be applied to the conglomerate of organisms involved in such relationship. Second, we describe the neurobiology of social behavior highlighting the possible pathways through which microbiota and, particularly, GM may affect social behavior, including macroorganisms’ development. Even under the light of recent and excellent reviews on the topic (Ezenwa et al., 2012 ; Archie and Tung, 2015 ), knowledge of the mechanisms by which microbiota in different sites of a host’s body influence behavior is still lacking. In contrast, there are at least three purported pathways suggesting how the GM interact with an individual’s central nervous system (CNS), modifying behavior in general, and social interactions in particular (Sampson and Mazmanian, 2015 ). For this reason, we focus our review on GM and expand this perspective by describing three areas where social behavior can in turn influence individual microbial profiles: (1) due to stressing events in hosts’ social life; (2) because of differences between solitary and social life; and (3) due to social structure. Figure 1 introduces to key elements of these interactions. Figure 1 Reciprocal interactions between Gut microbiota (GM) and social structure illustrated for humans. Social interactions may allow for the horizontal GM transmission, presumably in direct relation to the strength of the social bonds (bold lines). Mothers can transmit their microbes vertically to the next generation. In reciprocity, gestation and infancy could be a critical period for the GM to influence infant brain development and future sociability. The nature and quantity of horizontally and vertically transmitted microbes may be influenced by external factors including diet, water, sanitation and hygiene, environment and antibiotic usage; vertical transmission is also influenced by the mode of delivery and method of feeding."
} | 2,160 |
36838386 | PMC9960345 | pmc | 8,748 | {
"abstract": "Biofilm formation can lead to the persistence of Salmonella Typhimurium (ST) and E. coli O157:H7 (O157). This study investigated the impact of meat processing surface bacteria (MPB) on biofilm formation by O157 (non-biofilm former; NF) and ST (strong biofilm former; BF). MPB were recovered from the contacting surfaces (CS), non-contacting surfaces (NCS), and roller surfaces (RS) of a beef plant conveyor belt after sanitation. O157 and ST were co-inoculated with MPB (CO), or after a delay of 48 h (IS), into biofilm reactors containing stainless steel coupons and incubated at 15 °C for up to 144 h. Coupons were withdrawn at various intervals and analyzed by conventional plating and 16S rRNA gene amplicon sequencing. The total bacterial counts in biofilms reached approximately 6.5 log CFU/cm 2 , regardless of MPB type or development mode. The mean counts for O157 and ST under equivalent conditions mostly did not differ ( p > 0.05), except for the IS set at 50 h, where no O157 was recovered. O157 and ST were 1.6 ± 2.1% and 4.7 ± 5.0% (CO) and 1.1 ± 2.2% and 2.0 ± 2.8% (IS) of the final population. Pseudomonas dominated the MPB inocula and biofilms, regardless of MPB type or development mode. Whether or not a pathogen is deemed BF or NF in monoculture, its successful integration into complex multi-species biofilms ultimately depends on the presence of certain other residents within the biofilm.",
"introduction": "1. Introduction Shiga toxin-producing Escherichia coli , in particular the serotype O157:H7, is a significant foodborne human pathogen, and beef is the most commonly identified vehicle of transmission in E. coli O157:H7 outbreaks in North America [ 1 ]. Cattle are asymptomatic carriers of E. coli O157:H7 [ 2 ]. Salmonella can be found not only in the gastrointestinal tract of cattle, but also in other organs, such as lymph nodes, without causing overt disease in the host [ 3 , 4 ]. Thus, healthy cattle can carry these two pathogens, with the prevalence varying by animal source and rearing practices. Recurrence of the same strain at the same processing facility and its meat products over an extended period of time has been reported for E. coli, including E. coli O157:H7 [ 5 , 6 , 7 ]. This persistence has largely been attributed to the biofilm-forming ability of strains originating from the animals that can adapt to and become established in the meat processing environment [ 8 , 9 ]. Similarly, strong biofilm-forming Salmonella strains were among those which were recovered from beef trim [ 10 ]. Biofilms are surface-attached, organized microbial communities of aggregated cells, embedded in a hydrated matrix of self-produced extracellular polymeric substances (EPS) [ 11 ]. The most significant characteristic of biofilms in relation to the food processing environment is their increased resistance to sanitizing treatments compared to their planktonic counterparts. This resistance is mediated through the physical barrier provided by EPS, efflux systems, differentiation of bacterial cells into a dormant state, and the modification of the microenvironment, which can render a particular sanitizer less effective [ 12 ]. The daily cleaning and sanitization of processing equipment in meat processing facilities is a multi-step process, aiming to achieve two main objectives: visibly clean equipment (the removal of food residues that support microbial growth) and a reduction in the number of bacteria to acceptable levels [ 13 ]. Effective sanitation can reduce indicator organisms by up to 3 log units for food-contacting surfaces and 1 log for non-food contacting surfaces of conveyor belts [ 14 ]. Inadequately cleaned surfaces may promote soil build-up, in addition to having higher surviving microbial load. Regardless, the cleaning and sanitization program is not intended to achieve sterility for surfaces, and, as such, various bacterial species can be found on cleaned surfaces [ 14 , 15 ]. The attachment and subsequent biofilm formation of bacteria depend on interactions between three main components: the bacterial cells, the attachment surface, and the surrounding medium [ 16 ]. During operations, the wetting of conveyor belts by meat juice and the adsorption of food residues to surfaces provide a conditioning layer, which modifies surface properties favorably for bacterial attachment and subsequent growth. The large body of information available on biofilm formation by E. coli O157:H7 and Salmonella in single-species cultures and, to a lesser extent in dual-species cultures, shows that biofilm formation by both species is highly strain-dependent and is impacted by companion species, with effects ranging from antagonistic to neutral to synergistic [ 10 , 17 , 18 ]. Impact of other bacteria on the biofilm formation of these pathogens has mainly been assessed in dual cultures using individual strains isolated from meat processing facilities. Published accounts on the interactions of the two pathogens with the equipment surface bacterial consortium surviving sanitation on the whole are largely lacking. In addition, it has been reported that the fate of a non-biofilm-forming E. coli strain can be altered by synergistic interactions and co-adhesion mechanisms with adherence-proficient bacteria in dual-species cultures [ 19 ]. Thus, a better understanding of the microbial dynamics in mixed biofilms by the bacterial population surviving sanitation, as well as Salmonella and E. coli O157:H7, and the ability of these two pathogens to insert into established mixed-species biofilms, could lead to the development of effective intervention strategies to control biofilms harboring these pathogens in the food processing environment. Therefore, the aim of the present work was to determine the dynamics of mixed-species cultures of post-sanitation equipment surface bacteria and E. coli O157:H7 and S . Typhimurium.",
"discussion": "4. Discussion The attachment of a bacterial cell to a surface, the first step of biofilm formation, is dictated by its ability to overcome the repulsive force of the surface [ 16 ]. The surface charge and appendages used for attachment play an important role in that regard and vary between species and even among different strains. In addition, environmental factors can alter the surface properties for a particular bacterium, including the presence of companion species. Thus, the population structure of mixed-species biofilms is the net outcome of the microbial composition, as well as the competitiveness of individual members in terms of surface attachment, their resistance to antimicrobial compounds produced by other residents in the population [ 29 ], and growth rate under the given conditions. In this work, we investigated the microbial dynamics of mixed-species biofilms containing bacteria that survived the routine cleaning and sanitation of a conveyor belt in a beef processing plant, along with a non-biofilm-forming E. coli O157:H7 strain [ 18 ] and a strong biofilm-forming S . Typhimurium strain [ 28 ]. In the present work, we identified 23 bacterial genera with a relative abundance ≥1% in the microbiota of MPB inocula, which was recovered from conveyor belt surfaces, and the dominant genus was found to be Pseudomonas. The other genera with notably high relative abundance included Comamonas , Acinetobacter , Flavobacterium , Pseudarcobacter , Bacteroides , Janthinobacterium , and Aeromonas . Four genera ( Pseudomonas , Comamonas , Acinetobacter , and Flavobacterium ) were also found on surfaces after sanitation in a previous study, where the bacterial community at the same processing facility was examined during cleaning of the conveyor belts [ 14 ]. In that study, Janthinobacterium and Aeromonas were found before cleaning started. In the present study, some minor constituents that were found among the MPB in the inocula or during growth were also found in the previous study. The large overlap in bacterial genera detected at the same facility over a six-year period may suggest the presence of a stable core resident microbiota within the plant itself. A study, which examined the microbiota of conveyor belts in a salmon processing facility post-sanitation, reported that Pseudomonas was the most prevalent and abundant genus, followed by Acinetobacter , both of which were found in more than 50% of samples [ 15 ]. Brightwell et al. [ 30 ] reported that 84% of the bacterial isolates recovered from cleaned and sanitized conveyor belts in a lamb boning room were also Pseudomonas spp. The other relatively abundant bacteria in both studies were mostly Gram-negative. The bacterial genera identified on post-sanitation conveyor belts in the present study were in agreement with those previously reported from protein processing facilities, where operating temperatures are low and humidity is high [ 31 ]. Six relatively abundant (≥1%) bacterial genera were detected in biofilms at 144 h for both co-development and insertion sets. Similarly, the number of bacterial genera found in naturally occurring biofilms in a meat processing plant ranged from four (conveyor belt) to 12 (drain), a comparatively small number considering the diversity of bacteria naturally present in such environments [ 32 ]. Pseudomonas dominated mixed-species biofilms formed from a cocktail of 14 bacterial isolates that survived sanitation of conveyor belts in salmon processing plants [ 15 ]. Pseudomonas spp. were also among the most frequently isolated bacteria in biofilms in a meat processing facility where pork, poultry, and beef were processed during operation and after sanitation [ 33 ]. Biofilms on food contact surfaces in two meat plants that processed ham, meatballs, and sausages mainly consisted of Pseudomonas and Acinetobacter [ 34 ]. The predominance of Pseudomonas spp. in the present study was not observed in our previous work, where mixed-species biofilms were developed using a cocktail of 41 MPB strains in equal proportions together with the same O157 or ST strains included here [ 20 ]. This difference could have resulted from differences in both the composition of the MPB and/or the relative abundance of Pseudomonas in the initial inoculum, since its apparent higher levels in the present study may have given it a competitive advantage. The introduction of O157 and ST affected the microbial dynamics in biofilms formed by MPB, especially during the early development stages. In the insertion cultures incubated for 48 h, the microbial composition of planktonic cultures and biofilms were largely similar and mainly consisted of three genera: Pseudomonas , Aeromonas, and Acinetobacter . The co-development biofilms at 2 h were dominated by ST and O157, even though Pseudomonas accounted for 50% of the total population in planktonic cultures. However, both pathogens were gradually displaced by Pseudomonas in co-development biofilms. This may suggest that the O157 and ST strains were able to adhere to the stainless steel surface more efficiently compared to the MPB, but they were less competitive when growing in biofilms under the conditions investigated. In meat plants, the air temperature in fabrication facilities where carcasses are fabricated to cuts and trimmings is maintained at 6–7 °C. Both Salmonella and E. coli are mesophilic organisms, and their growth at or below the operating temperature is limited [ 35 ]. During cleaning, the ambient temperature could rise to 15 °C [ 14 ]. Even so, the more psychrotrophic Pseudomonas , Aeromonas, and Acinetobacter may have a competitive edge at these temperatures. Compared to when they were initially introduced or six days after introduction, more variation was observed in the counts for the two pathogens at 48 h for ST in the co-development set and 144 h for both in the insertion set of planktonic cultures and in biofilms. Differences in the ability/opportunity to attach to different coupons/surfaces in the same bioreactor or in different trials may have contributed to this variability for biofilms. This variation may also be attributed to differences in the initial cell density and microbial population and the permissibility that affected the growth of O157 and ST. It is noteworthy that the relative abundance of both O157 and ST, as determined by 16S rRNA gene amplicon sequencing, was not always congruous with the culturing data. This discrepancy could have been caused by differences in DNA extraction efficiency and PCR amplification bias for different bacterial species [ 36 ]. For the co-development cultures at 2 h, both ST and O157 were among the major species in the total microbial population (>10% as determined by plating), with no difference ( p > 0.05) in their counts at this time or at any given time. These findings are in agreement with a previous study, which found that the two pathogens co-established in mixed-species biofilms, comparably, when each was co-inoculated individually with a mock community of 41 MPB strains [ 20 ]. The surface colonization of an E. coli O157:H7 strain that could not form biofilms on its own was greatly enhanced when co-cultured with an Acinetobacter calcoaceticus isolate recovered from a meat processing environment after disinfection [ 37 ]. A generic E. coli strain, PHL565, was not able to adhere to a glass surface on its own, but it could do so when co-cultured with Pseudomonas putida MT2 [ 19 ]. Here, biofilm formation in mixed-species cultures by the otherwise curli- and cellulose-deficient non-biofilm-forming O157 strain [ 18 ] was likely aided by the presence of companion strains, to a level comparable to the strong biofilm-forming ST strain. However, O157 did not insert into established biofilms as efficiently as ST, as reflected by the lack of recovery of O157 from any of the coupons from the insertion cultures at 50 h. Even so, the numbers of O157 and ST in insertion biofilms did not differ significantly by 144 h, regardless of surface type. In conclusion, the findings of this work demonstrated that Gram-negative bacteria were prevalent among the equipment post-sanitation microbial community, with Pseudomonas being most predominant, and Comamonas , Acinetobacter , Flavobacterium , Janthinobacterium , and Aeromonas were represented in sizable fractions. Despite the difference in biofilm-forming ability of the O157 strain and the ST strain in single-species cultures, they mostly did not differ in both mature co-development and insertion biofilms developed at temperatures relevant to meat processing facilities. Further work on gene expression levels would help to pinpoint the nature of the interactions in the mixed-species cultures. Nevertheless, the findings in this work underline the importance of considering the entire microbial community when developing strategies to control biofilms, rather than focusing on biofilm formation by individual strains/species."
} | 3,740 |
39745385 | PMC11774020 | pmc | 8,749 | {
"abstract": "ABSTRACT Prominent virulence traits of Candida albicans include its ability to produce filamentous hyphal cells and grow as a biofilm. These traits are under control of numerous transcription factors (TFs), including Brg1 and Rme1. In the reference strain SC5314, a brg1 Δ/Δ mutant has reduced levels of biofilm/filament production; a brg1 Δ/Δ rme1 Δ/Δ double mutant has wild-type levels of biofilm/filament production. Here, we asked whether this suppression relationship is preserved in four additional strain backgrounds: P76067, P57055, P87, and P75010. These strains represent diverse clades and biofilm/filament production abilities. We find that a rme1 Δ/Δ mutation restores biofilm/filament production in a brg1 Δ/Δ mutant of P76067, but not in brg1 Δ/Δ mutants of P57055, P87, and P75010. We speculate that variation in activities of two functionally related TFs, Nrg1, and Ume6, may cause the strain-limited impact of the rme1 Δ/Δ mutation. IMPORTANCE Candida albicans is a widespread fungal pathogen. The regulatory circuitry underlying virulence traits is well studied in the reference strain background, but not in other clinical isolate backgrounds. Here, we describe a pronounced example of strain variation in the control of two prominent virulence traits, biofilm formation and filamentation."
} | 329 |
40021823 | PMC11871072 | pmc | 8,751 | {
"abstract": "From LSTMs (Long Short-Term Memory) to Transformers, various networks have been used for runoff forecasting, though many complex structures may be unnecessary. This study introduces RR-TiDE, a simple model based on the Time Series Dense Encoder. RR-TiDE employs fully Multilayer Perceptron architecture for modeling and is specifically designed with understanding of hydrological processes. To manage non-stationarity in hydrological data, RR-TiDE incorporates Reversible Instance Normalization. The model was trained using the Catchment Attributes and Meteorology for Large-Sample Studies (CAMELS) dataset and evaluated on two tasks: (1) multi-basin runoff simulation; (2) prediction in data-sparse basins. In the first task, RR-TiDE outperformed both Transformer and LSTM-based models across all metrics for 7-day runoff predictions, which indicates that RR-TiDE is highly suitable for rainfall-runoff simulation. In the second task, it achieved a median NSE of 0.82 in 1-day runoff forecasting in 51 watersheds. This suggests that RR-TiDE possesses robust generalization capability, enabling spatial extrapolation. Comparisons were made between models with and without the feature projection layer and RevIN to further understand their individual contributions. Results indicate that the feature projection layer can effectively enhance the performance of RR-TiDE. Although RevIN provided limited overall improvements, it helped stabilize loss fluctuations during training, aiding model convergence.",
"conclusion": "Conclusions In this study, we proposed the RR-TiDE model, a novel rainfall-runoff simulation framework leveraging the Time Series Dense Encoder architecture. The RR-TiDE model is entirely based on a multi-layer perceptron (MLP) framework and is specifically designed to enhance understanding of rainfall-runoff processes. Using the CAMELS dataset, RR-TiDE was validated and applied to both multi-basin runoff simulation tasks and runoff prediction in data-scarce watersheds. Consistently, the model outperformed state-of-the-art Transformer-based and LSTM-based models in these applications. This demonstrates the promising potential of MLP-based approaches in integrating hydrological processes into neural network architectures. Furthermore, the integration of a feature projection layer significantly enhanced the model’s performance by effectively filtering noise, resulting in improved RMSE and NSE metrics across multiple basins. In addition, we incorporated Reversible Instance Normalization (RevIN), which played a crucial role in stabilizing the training process by mitigating loss fluctuations. This, in turn, accelerated model convergence, albeit with a limited impact on the final performance metrics. However, the RR-TiDE model has certain limitations. First, the model relies on an intuitive, non-explicit representation of hydrological processes, which results in limited interpretability. Additionally, its effectiveness in long-term runoff prediction still requires improvement. To address these limitations, future work will focus on incorporating runoff generation and concentration principles as prior knowledge within the MLP architecture. These advancements will enable more robust and interpretable rainfall-runoff modeling solutions.",
"introduction": "Introduction Runoff forecasting primarily supports water resource planning 1 , 2 , flood controlling, and shipping by delivering crucial information 3 . Transformer and LSTM have received widespread attention in runoff prediction in recent years. LSTM is generally considered the most suitable model for runoff prediction, and its structure has been widely promoted 4 – 6 . For example, utilizing a variant of LSTM for global runoff forecasting has outperformed existing large-scale forecasting systems 7 . Guo et al. 8 introduced a VMD-LSTM-Transformer hybrid model for monthly runoff prediction with a high prediction accuracy, indicating its superiority over traditional LSTM models. Wang et al. 9 created an Ia-LSTM model that integrates physical-based models with LSTM, enhancing the precision and reliability of runoff prediction. While being widely applied in various fields including runoff prediction 10 , 11 , LSTM exhibits certain limitations specifically in the context of hydrological modeling. LSTM models require large amounts of data to train properly, often leading to suboptimal performance in scenarios with limited or poorly monitored data sets 12 . This poses challenges for simulating streamflow in data-scarce or short-record basins. LSTM models also tend to overfit, especially when dealing with unsteady runoff, which performs worse in arid regions with short or incomplete time series data 13 . Therefore, more and more attention is paid to other models that may be more suitable for runoff prediction. Transformer 14 is a deep learning model wisely applied in natural language processing 15 – 17 and computer vision 18 – 20 , which is good at capturing the meaning of data in contexts. In recent years, an increasing number of Transformer-based models have been employed for various runoff prediction tasks. In transfer learning, Xu et al. 21 proposed a Transformer-based transfer learning framework named TL-Transformer for predicting flood events in data-sparse watersheds. For peak prediction, Li et al. 22 developed a Transformer model that integrates multiple loss functions to enhance the accuracy of peak urban river flood forecasting. However, recent research 23 suggests that Transformers may not always outperform simpler models such as linear regression, especially in specific time series forecasting tasks. This research highlights that the Attention operation, central to Transformer models, can overlook the temporal variability of input data, potentially leading to a loss of temporal information. This raises significant questions about the applicability of Transformer models in various domains 24 , including runoff prediction. Given this context, the field of time series forecasting has diverged into two primary approaches: one that continues to enhance Transformer architectures and another that investigates the efficacy of simpler models, such as Multiple Layer Perceptron (MLP), in achieving state-of-the-art performance. In the first approach, Zhang and Yan 25 identified that Transformers struggle with cross-dimensional dependencies in multivariate time series, proposing Dimension Segment Embedding (DSW) and Two-Stage Attention (TSA) to improve accuracy. Nie et al. 26 highlighted difficulties in capturing local semantic information and processing high-dimensional inputs, addressing these with patch design and channel independence methods. Liu et al. 27 suggested embedding variables separately to avoid meaningless attention maps, significantly enhancing generalization across variables. Among the approaches of second category, the Time Series Dense Encoder (TiDE) 28 model represents an enhancement over simple linear models. The TiDE model is constructed from residual blocks composed of simple MLP, which requires low computational loads during training. The TiDE model utilizes a feature projection layer to reduce dimensionality of dynamic features, and it well distinguishes between dynamic and static features, enabling effective exclusion of noise and extraction of useful information in historical data. To our knowledge, there has been little research exploring the second category of approaches in the context of rainfall-runoff simulation tasks. In addition, MLP may not be sufficient to address the non-stationary hydrological series caused by climate change and human activities. The non-stationary problem of hydrological data can be explained as the data distribution changing over time, and models calibrated based on the original distribution data perform poorly in other distribution data 29 . Almost all deep learning models predict future window data based on historical window data, and the distribution shift of data will make it unreasonable to use the same model weights for all window predictions. Because the model weights are only trained on the distribution of historical data, while the distribution of future data has changed. Reversible Instance Normalization (RevIN) is a method proposed by Kim et al. 29 to overcome distribution shifts in time series, and it has been widely applied in tasks related to time series. For example, Gong et al. 30 integrated RevIN with SCINet to enhance the accuracy of wind power forecasting. Baidya and Jeong 31 combined RevIN with Transformer for anomaly detection tasks. Liu et al. 32 combined LSTM with RevIN for fault detection and noted that RevIN effectively improved accuracy. But as far as we know, no research attempted to use RevIN to address the issue of non-stationarity in hydrological sequences. The novelty of this study lies in the first-ever application of the TiDE model for rainfall-runoff simulation and the innovative use of the RevIN method to address distribution shifts in hydrological data. TiDE represents a class of MLP-based models. Comparing TiDE with Transformer-based and LSTM-based models sheds light on different approaches within deep learning for rainfall-runoff simulation, highlighting whether simpler MLP-based models are sufficient to capture rainfall-runoff dynamics. We developed a novel hydrological model named RR-TiDE based on the TiDE model and further integrated the RevIN method to answer the following question: \n Is the RR-TiDE model suitable for rainfall runoff prediction tasks? Whether a simple MLP-based model can learn generalized rainfall-runoff patterns, compared to Transformer-based and LSTM-based models. Does RevIN help with non-stationary hydrological series? \n To address the first question, a large sample dataset (CAMELS) was used to test the 7-day runoff prediction capability of RR-TiDE, evaluating its suitability for the runoff forecasting task. For the second question, the TiDE model, representing MLP-based models, was compared with Transformer-based and LSTM-based models. State-of-the-art (SOTA) models from these two architectures on the CAMELS dataset were selected as benchmarks, providing a direct performance comparison for MLP-based models. Spatial generalization serves as a critical test of whether a model can truly capture the underlying mechanisms of rainfall-runoff processes. The model capable of predicting runoff in previously unseen basins demonstrates its ability to understand how basin characteristics shape runoff dynamics. To evaluate this, 51 basins from CAMELS were entirely excluded from model training (treated as data-scarce basins), and the performance of RR-TiDE was compared against benchmark models in these basins. For the third question, we examined the impact of RevIN on the performance of RR-TiDE. Since RR-TiDE includes a feature projection layer, its contribution to model performance was analyzed alongside that of RevIN.",
"discussion": "Discussion In deep learning, algorithm design typically relies on specific inductive biases 43 , which refer to prior constraints introduced under assumptions. By embedding domain-relevant prior knowledge into the architecture of models, inductive biases effectively constrain the solution space of neural network parameters, thereby driving the success of deep learning across numerous applications 33 . We propose that this concept provides a valuable analytical framework for applying deep learning to rainfall–runoff simulation. Theoretically, the functions of all neural networks can be fully represented by multilayer perceptrons (MLPs). For instance, convolutional neural networks (CNNs) can be regarded as localized MLPs, with their main inductive biases being locality and spatial invariance 44 . These characteristics align closely with the features of visual data, which is why CNNs excel in computer vision tasks 44 . Similarly, when unfolded along the temporal axis, recurrent neural networks (RNNs) can be interpreted as MLPs with shared weights across consecutive time steps. The inductive biases of RNNs lie in their sequential structure and temporal invariance 45 . Sequentiality implies that the state of the current time step depends only on the state of the previous time step and the current input, while temporal invariance means the model employs the same computational process across all time steps. Since many time series data adhere to the prior assumption of sequential dependency 46 , RNNs—particularly LSTMs—have achieved significant success in time-series modeling, including streamflow prediction 43 . However, from a hydrological perspective, the distribution of hydrological data is not stationary 47 and does not satisfy the assumption of temporal invariance. Without incorporating time-representing variables, using a single set of model weights for streamflow prediction across all time steps is bound to introduce systematic bias. Transformer models, on the other hand, break the sequential dependency inherent in RNNs. Transformers treat the relationship between the watershed state at the current time step and past time steps as unknown, relying on backpropagation to learn these dependencies 48 . Evidently, the data requirements for Transformers to capture such dependencies increase significantly 49 . We propose that developing deep learning models for rainfall-runoff simulation should be based on MLPs, with targeted improvements to accommodate the characteristics of the rainfall-runoff process. Following this principle, we designed RR-TiDE, a model entirely built upon MLPs with specific adaptations for rainfall-runoff simulation: (1) Since recent meteorological features often have a more significant impact on runoff, RR-TiDE incorporates a highway connection that directly routes recent meteorological features to the final layers. (2) Runoff frequently exhibits strong autoregressive characteristics. To address this, RR-TiDE introduces residual connections to directly link historical runoff to the current time step. (3) To mitigate the impact of distributional shifts in hydrological data, RR-TiDE implements two strategies. First, it incorporates temporal features, enabling the model to learn how rainfall-runoff dynamics evolve over time. Second, it employs RevIN to minimize the effects of data distribution shifts as much as possible. The results from multi-basin prediction tasks demonstrate that the RR-TiDE model is well-suited for rainfall-runoff simulations, achieving strong performance. In most forecast lead times, RR-TiDE consistently outperforms LSTMs and Transformers. These findings suggest a new perspective: rather than combining complex network structures from other fields, a simple MLP model, specifically designed with hydrological concepts, may yield better results. However, unlike Transformer and LSTM architectures, RR-TiDE does not explicitly account for establishing dependencies on historical runoff, which may explain its rapid decline in performance under long lead-time conditions. RevIN applies normalization to force all samples into a unified distribution. However, the results show that the performance improvement from this approach is quite limited. The size of the dataset and the model’s parameter capacity may already be sufficient to capture distribution shifts, as evidenced by the minimal difference in the final converged loss (Fig. 7 ). The oscillation in the loss in Fig. 7 for the model without RevIN may be attributed to the data not conforming to a consistent distribution, which makes it difficult for the model’s weights to generalize across temporal scales. In data-scarce basins, the limited amount of data may not be sufficient to overcome issues arising from distribution shifts, potentially making it difficult for the model to converge. In this respect, RevIN proves to be valuable in stabilizing the training process. Compared to RevIN, the feature projection layer had a more significant effect on improving the prediction accuracy of RR-TiDE. We further analyzed the cumulative distribution function (CDF) of NSE across the three models (the original, the one with only the feature projection layer, and the one with both the feature projection layer and RevIN), with the results for all 7-day lead times presented in Fig. 8 . Across all forecast periods, the feature projection layer led to a notable increase in NSE. Specifically, for the 7-day lead time, RR-TiDE without the feature projection layer recorded NSE values below 0.5 in approximately 30% of the watersheds, whereas this percentage dropped to 20% when the feature projection layer was added. For lead times of 1 to 3 days, the improvement in NSE was minor, but as the lead time extended, the positive impact of the feature projection layer became more pronounced. \n Fig. 8 CDFs of models with or without RevIN and features projection. \n We noted that RR-TiDE also exhibits significant limitations. First, the model often reports negative biases in both average and median values in our cases, suggesting that its forecasts tend to underpredict flood risks. Additionally, as a deep learning model, the RR-TiDE model lacks inherent interpretability, but the significance of each model input can be assessed using external interpreters such as SHAP 50 and LIME 51 . For instance, SHAP has been utilized by Wang et al. 52 to elucidate the predictive capabilities of the XGBoost model integrated with the SWAT forecasting framework. Furthermore, Maloney et al. 53 utilized interpretative machine learning tools, including LIME, to study temporal changes in biological stream conditions. In addition to using external interpreters, interpretability modules can also be added within the model to enhance the interpretability of RR-TiDE. For example, Temporary Fusion Transformer 54 has modified the standard multi head attention to obtain the weights of various attributes. An internal interpretability module for RR-TiDE is our future work."
} | 4,489 |
28777486 | PMC5639371 | pmc | 8,752 | {
"abstract": "Abstract A new class of hydrogels utilizing DNA (DNA quadruplex gel) has been constructed by directly and symmetrically coupling deoxynucleotide phosphoramidite monomers to the ends of polyethylene glycols (PEGs) in liquid phase, and using the resulting DNA‐PEG‐DNA triblock copolymers as macromonomers. Elongation of merely four deoxyguanosine residues on PEG, which produces typically ≈10 grams of desired DNA‐PEG conjugates in one synthesis, resulted in intelligent and biodegradable hydrogels utilizing DNA quadruplex formation, which are responsive to various input signals such as Na + , K + , and complementary DNA strand. Gelation of DNA quadruplex gels takes place within a few seconds upon the addition of a trigger, enabling free formation just like Ca + ‐alginate hydrogels or possible application as an injectable polymer (IP) gel. The obtained hydrogels show good thermal stability and rheological properties, and even display self‐healing ability."
} | 240 |
20686787 | PMC3021705 | pmc | 8,753 | {
"abstract": "Plant communities vary dramatically in the number and relative abundance of species that exhibit facilitative interactions, which contributes substantially to variation in community structure and dynamics. Predicting species’ responses to neighbors based on readily measurable functional traits would provide important insight into the factors that structure plant communities. We measured a suite of functional traits on seedlings of 20 species and mature plants of 54 species of shrubs from three arid biogeographic regions. We hypothesized that species with different regeneration niches—those that require nurse plants for establishment (beneficiaries) versus those that do not (colonizers)—are functionally different. Indeed, seedlings of beneficiary species had lower relative growth rates, larger seeds and final biomass, allocated biomass toward roots and height at a cost to leaf mass fraction, and constructed costly, dense leaf and root tissues relative to colonizers. Likewise at maturity, beneficiaries had larger overall size and denser leaves coupled with greater water use efficiency than colonizers. In contrast to current hypotheses that suggest beneficiaries are less “stress-tolerant” than colonizers, beneficiaries exhibited conservative functional strategies suited to persistently dry, low light conditions beneath canopies, whereas colonizers exhibited opportunistic strategies that may be advantageous in fluctuating, open microenvironments. In addition, the signature of the regeneration niche at maturity indicates that facilitation expands the range of functional diversity within plant communities at all ontogenetic stages. This study demonstrates the utility of specific functional traits for predicting species’ regeneration niches in hot deserts, and provides a framework for studying facilitation in other severe environments. Electronic supplementary material The online version of this article (doi:10.1007/s00442-010-1741-y) contains supplementary material, which is available to authorized users.",
"introduction": "Introduction Facilitation has been shown to play an important role in the assembly and dynamics of many plant communities (Callaway et al. 2002 ; Valiente-Banuet et al. 2006 ; Cavieres and Badano 2009 ; Butterfield et al. 2010 ) particularly those of low-productivity environments (Bertness and Callaway 1994 ; Callaway 1995 ). In plant communities where facilitation is common, species’ responses to neighbors can still vary from strongly positive (here termed “beneficiaries”) to strongly negative (“colonizers”) (McAuliffe 1988 ; Liancourt et al. 2005 ), with the number, relative abundance, and specialization of species with respect to facilitation having important consequences for community stability and dynamics (McAuliffe 1988 ; Verdu and Valiente-Banuet 2008 ; Butterfield 2009 ). While the proximate causes of facilitation have been studied extensively—for example, improved water relations, reduced herbivory, temperature buffering, etc. (Flores and Jurado 2003 ; Callaway 2007 )—the intrinsic biological characteristics of species that determine whether or not they engage in facilitative interactions are just beginning to be explored (Valiente-Banuet et al. 2006 ; Lopez and Valdivia 2007 ). Thus, an improved ability to predict the outcome of interactions based on species’ traits would be an important step forward in our understanding of the community and ecosystem-level consequences of facilitation. In many severe environments, the presence of a neighbor is generally considered to produce a more benign microenvironment than open, bare ground, thereby permitting less stress-tolerant species to persist within a community (Michalet et al. 2006 ; but see Maestre et al. 2009 ). Despite this apparently simple contrast, many factors may differ in complex ways in the presence versus absence of a neighbor. This is particularly true in arid ecosystems, in which local environmental differences in soil moisture (Tielbörger and Kadmon 2000 ), light (Valiente-Banuet and Ezcurra 1991 ), soil fertility (Turner et al. 1966 ), herbivory (McAuliffe 1986 ) and temperature extremes (Nobel 1980 ) between sub-canopy and open microsites may simultaneously drive facilitation. Terms such as “benign” and “stressful” may not accurately represent this complex variation, particularly since many species perform better outside the influence of established canopies (McAuliffe 1988 ; Verdu and Valiente-Banuet 2008 ), which has been attributed to shade intolerance and belowground competition from larger neighbors (Holmgren et al. 1997 ; Miriti et al. 1998 ). Clearly, many environmental variables differ between open and sub-canopy microsites, and act in concert to determine plant growth and survival. Understanding biological responses to complex environments can benefit from a trait-based approach (McGill et al. 2006 ). Measuring functional traits related to life-history strategies, resource economies and disturbance responses in contrasting environments provides a broad perspective on the selection pressures acting on plant populations (Westoby 1998 ; Westoby et al. 2002 ; Wright et al. 2004 ), all of which may be relevant to facilitation (Valiente-Banuet et al. 2006 ). The nature of trait relationships with facilitation may also vary through ontogeny (Valiente-Banuet and Verdu 2008 ), as seedling and mature trait values are often uncorrelated (Grime et al. 1997 ; Cornelissen et al. 2003 ). Seedling traits should be particularly important in desert perennial plant communities, since facilitation is most relevant during the regenerative phase of the perennial life-cycle (Yeaton 1978 ; McAuliffe 1988 ; Miriti 2006 ). However, ontogenetic trait conservatism may also result in the regeneration niche (in this case, sub-canopy or open microsite) influencing mature functional strategies (Valiente-Banuet et al . \n 2006 ; Poorter 2007 ). This is an important consideration, in that the functional traits of mature plants are much more likely to drive ecosystem processes than are those of seedlings, given their much larger cumulative biomass (Grime 1998 ). In addition, mature plant traits may determine if a species functions as a nurse plant at maturity, thereby influencing community structure both in terms of responses to and effects on microenvironmental conditions (Butterfield 2009 ). Thus, these broader consequences of regeneration niche selection should be assessed by measuring mature functional traits directly. A broad array of both seedling and mature functional traits may exhibit strong relationships with one another and with the regeneration niche across desert plant species. Seed mass is a particularly important trait in this context, due to possible relationships with dispersal and reproductive strategies as well as seedling energy reserves (Moles and Westoby 2006 ). Following germination, maximum relative growth rate (RGR max ) indicates a plant’s ability to acquire resources during brief windows of opportunity, which may come at a cost to survival under persistently stressful conditions (Arendt 1997 ). Biomass allocation to different tissues reveals tradeoffs in the acquisition of different resources, which may differ greatly in sub-canopy and open microsites. Root-to-shoot ratio represents the relative limitation of water or mineral nutrients versus light or carbon, whereas height and leaf mass fraction (LMF) are associated with light and carbon acquisition, respectively (Poorter and Nagel 2000 ). In addition to allocation, tissue quality is also important, indicating tradeoffs between resource turnover and efficiency. Specific root length (SRL) is positively correlated with root elongation and maximum uptake rates in saturated soils, but negatively correlated with root longevity and drought tolerance (Nicotra et al. 2002 ; Markesteijn and Poorter 2009 ). Conversely, leaf mass per area (LMA) is negatively correlated with maximum photosynthetic rates and stomatal conductance, while being positively associated with leaf lifespan (Wright et al. 2004 ). LMA may also be positively correlated with the cell wall content and rigidity of leaves, which may partially regulate wilting point (Niinemets 2001 ). In addition to LMA, photosynthetic rates are also related to leaf size (i.e., leaf dry mass; M D ) and several aspects of leaf composition (Niklas et al. 2007 ), including leaf dry matter content (LDMC), percent nitrogen (%N) and surface area (SA). These organ-specific traits related to water and carbon economies scale up to influence whole-plant water use efficiency (WUE), which is positively associated with average longevity of individuals in desert perennial plant populations (Schuster et al. 1992 ). Individual longevity is also positively associated with the total size or biomass of desert plants, which are negatively correlated with frequency of establishment and positively correlated with proportion of older plants in a population (Goldberg and Turner 1986 ). While the links between some of these life-history, allocation, construction and composition traits have been explored independently, their cumulative description of functional differentiation would provide a more comprehensive picture of functional strategies and an opportunity to quantitatively assess the functional basis of the regeneration niche in desert plants. In this study, we measured the broad suite of functional traits discussed above on a variety of colonizer and beneficiary species across three different hot desert biogeographic regions of the southwestern USA. Both seedlings and mature plants were studied in order to determine potential effects of the regeneration niche on multiple ontogenetic stages. With these data, we tested the hypotheses that (1) functional traits are highly inter-correlated across species (due to strong spatial autocorrelation in environmental factors between open and sub-canopy microsites), (2) beneficiary and colonizer species have different functional strategies (i.e., occupy different volumes of trait space), with the contrast being greatest during the seedling stage, and (3) seedling and mature functional strategies (i.e., multivariate trait axes) will be correlated due to ontogenetic conservatism, even if individual traits are poorly correlated through ontogeny.",
"discussion": "Discussion Functional strategies of beneficiaries and colonizers were significantly different, both for seedlings and mature plants. Taken together, the patterns of trait loadings on both seedling and mature principal components suggest that woody desert plants are strongly differentiated in their responses to environmental predictability, and can be arrayed along an axis from opportunistic to conservative strategies. These patterns reflect a tradeoff between resource turnover and efficiency that may be generally universal for vascular plants (Wright et al. 2004 ), and sheds an important light on the microenvironmental factors and corresponding functional strategies that shape plant life in severe environments. Several lines of evidence from the seedling data suggest that sub-canopy microsites represent relatively consistent, resource-limiting environments in contrast to open microsites that fluctuate between highly severe and resource-rich states. The high density tissues of beneficiary leaves (high LMA and LDMC) and roots (low SRL), as well as their large leaf dry mass suggest a slow rate of return on long-lived organs suited to persistent water and/or light limitation (Valladares and Niinemets 2008 ; Markesteijn and Poorter 2009 ). Similarly, the allocation of resources to root mass and height (high RMF and height) at the expense of leaves (low LMF) indicates that substantial initial investments are required to acquire sufficient water and light beneath canopies. An alternative but complementary explanation is that colonizers construct cheap tissues (low LMA, high SRL) to maximize photosynthesis and water uptake during brief soil moisture pulses. Greater height does not increase light interception in the open, nor does allocation to roots in briefly saturated soil improve water relations, thereby justifying the high LMF, short stature and low RMF of colonizers. Arguments from both perspectives suggest that the slow-turnover, high efficiency beneficiary strategies and high-turnover, low-efficiency colonizer strategies are beneficial in their respective microenvironments. Trait patterns related to resource turnover and efficiency may be largely explained by variation in seed size, RGR max and short-term drought survival. The comparatively larger seeds of beneficiaries could be related to differences in energy reserve requirements, dispersal patterns and vectors, and maternal investment (Pianka 1970 ). Greater seed reserves are advantageous in many low-light environments (Westoby et al. 1996 ), and larger seeds are often associated with deeper tap-roots, leading to greater long-term water access (Guerrero-Campo and Fitter 2001 ). In contrast, production of many small seeds may increase the odds of at least some colonizers becoming established in sufficiently wet conditions (Wright and Westoby 1999 ; Moles et al. 2004 ). High RGR max is critical under these circumstances, permitting seedlings to acquire sufficient carbon before going dormant (a phenological characteristic common to all of the colonizer species in this study) during subsequent inter-pulse periods. The patterns of RGR max and seed size provide functional support for the assertion that effective soil moisture pulses are more frequent but of smaller size and duration in open microsites, whereas soil moisture is more persistent (but often limiting) beneath canopies at an intra-annual scale (Huxman et al. 2004 ). When viewed as a whole, the above trait relationships suggest that colonizers and beneficiaries may not only be well-suited to their observed regeneration niche, but also maladapted to the other. Light-demanding colonizer species may not be able maintain a positive carbon balance in the low-light environment beneath shrub canopies, although the semi-substitutable nature of light and water can result in a shift toward facilitation in wet years (Butterfield et al. 2010 ). In contrast, beneficiaries may be excluded from the open for two primary reasons. First, many beneficiaries require protection from herbivory (McAuliffe 1984 , 1986 ), likely due to the high costs incurred by losing large, expensive leaves that cannot be quickly replaced. In a similar vein, while beneficiaries had greater short-term drought survival, this is most likely linked to seed reserves. Once these reserves are exhausted, the dense tissues and belowground allocation of resources exhibited by beneficiaries may result in negligible photosynthesis during extended dry periods in the field, coupled with the inability to fix sufficient carbon during brief wet periods in the open. Thus, short-term drought survival on a per-individual basis may not be directly relatable to establishment at the population level. Colonizers and beneficiaries maintained their general strategies of opportunism and conservatism through ontogeny. The only mature axis not correlated with the regeneration niche was PC M 1, with high scores indicating high %N, SA, M d and low LDMC, all of which are associated with high potential photosynthetic rates. The term “potential” refers to the role of water availability in determining how often, how long, and to what extent maximum photosynthetic rates are approached (Reynolds et al. 1999 ; Housman et al. 2006 ). Traits associated with PC M 2 likely indirectly regulate photosynthetic rate by determining leaf water potential. Species that were beneficiaries tended to have high scores on PC M 2 indicating high LMA and large plant size, with the loadings of SA and M d likely related to selection on LMA. Larger desert plants tend to have deeper root systems (Schenk and Jackson 2002 ), permitting more persistent use of deep soil moisture. High LMA may support this persistent water uptake via a more negative wilting point while simultaneously minimizing diffusive water loss (Poorter et al. 2009 ). However, high LMA may come at a cost to maximum photosynthetic rate through reduced internal CO 2 diffusion (Niinemets 1999 ). Smaller, more shallow-rooted species generally experience more pulsed soil moisture conditions (Schwinning and Ehleringer 2001 ), making cheap, leaky, low-LMA leaves viable during wet periods. Contrasting selection pressures on water conservation and carbon acquisition may explain why WUE is relatively independent of the other traits measured: a similar WUE may be attained by either minimizing water loss or maximizing carbon gain, with larger, higher LMA beneficiary species employing the former strategy and smaller, lower LMA colonizer species the latter. Beneficiaries did, however, maintain significantly greater WUE than colonizers, suggesting some coordination between WUE and unmeasured physiological or functional traits. Interestingly, the range of functional strategies employed by beneficiaries at maturity were more diverse than those of colonizers, suggesting that ontogenetic drift or altered selection pressures influence development despite correlations between the seedling and mature principal components related to the regeneration niche. Functional convergence through ontogeny might be expected for colonizers, with initial differences among seedlings of different species being reduced by strong selection for a common growth form in open microsites. In contrast, some beneficiaries experience a strong ontogenetic niche shift upon emerging above their nurse’s canopy whereas others remain perpetually beneath the canopy. This could lead to ontogenetic functional divergence among beneficiary species. Colonizer convergence and/or beneficiary divergence are hinted at by our results (Fig. 1 a, b). Alternatively, the greater age at reproductive maturity and longer lifespan of beneficiaries may lead to greater ontogenetic drift in the absence of any strong selection differences (Coleman et al. 1994 ). In general, ontogenetic shifts and phenotypic plasticity are two areas of future research that may provide important insights into the form and function of desert woody plants, as well as the causes and consequences of facilitation. The results of this study draw strong parallels to many existing theories regarding plant functional strategies, but with varying degrees of congruity. Traditionally, desert perennials have been classified as stress-avoiders or stress-tolerators (Noy-Meir 1973 ). This classification scheme unfortunately does not define “stress” well, particularly given the differences in the nature of resource and environmental fluctuations in open versus sub-canopy microsites. Similarly, “stress” is highly dependent upon a species’ functional strategy as is clear from the inability of many colonizers to establish beneath canopies despite conditions being highly favorable for beneficiaries. Perhaps most notably, r – K selection (MacArthur and Wilson 1967 ) bears a resemblance to the functional strategies and microsite successional dynamics of colonizers and beneficiaries (as does the similar R – S axis of CSR theory (Grime 1977 )). While the functional analogy is accurate, the community dynamics generated by these strategies in deserts differ from the successional paradigm in which r–K selection theory was developed. Establishment of colonizers in deserts is dependent upon temporal windows of opportunity during wet periods (Butterfield et al. 2010 ), with the availability of resources that make establishment possible regulated by exogenous supply rates (precipitation), rather than resources being freed up by removal of K -selected species (Goldberg and Novoplansky 1997 ). Second, replacement of colonizers by beneficiaries via facilitation is not a function of differences in intrinsic growth rates and competitive exclusion, as with r - and K -selection dynamics (Pianka 1970 ), but by alteration of the local environment (see Connell and Slatyer 1977 for definitions of successional mechanisms). Third, a self-replicating beneficiary vegetation state does not exist in deserts, likely due to direct and indirect negative effects of mature plants on conspecific establishment (McAuliffe 1990 ; Miriti 2006 ). This results in individual microsites cycling through semi-independent dynamics of colonization, replacement, and abandonment (McAuliffe 1988 ). While r–K selection theory clearly does not address the intricacies of desert plant communities, these comparative differences between arid and more mesic ecosystems provide important insights into shifts in the structure and dynamics of plant communities across broad gradients in precipitation and productivity. The functional causes and consequences of facilitation are just beginning to be studied, and should be critically compared to existing theories that do not explicitly consider positive interactions. Similarly, no single framework for studying the functional ecology of facilitation is likely to be sufficient. Facilitation occurs in many different biomes and ecosystems, with likely variation in the qualitative differences between open and sub-canopy microsites, as well as the traits that are most relevant to plant fitness in these contrasting microenvironments. Clearly more research is needed to determine the relationships between facilitation and functional strategies, however general concepts such as tradeoffs between resource turnover and efficiency should be used to make meaningful comparisons across biomes and taxa."
} | 5,439 |
34164103 | PMC8179378 | pmc | 8,754 | {
"abstract": "Bioelectrochemical approaches for energy conversion rely on efficient wiring of natural electron transport chains to electrodes. However, state-of-the-art exogenous electron mediators give rise to significant energy losses and, in the case of living systems, long-term cytotoxicity. Here, we explored new selection criteria for exogenous electron mediation by examining phenazines as novel low-midpoint potential molecules for wiring the photosynthetic electron transport chain of the cyanobacterium Synechocystis sp. PCC 6803 to electrodes. We identified pyocyanin (PYO) as an effective cell-permeable phenazine that can harvest electrons from highly reducing points of photosynthesis. PYO-mediated photocurrents were observed to be 4-fold higher than mediator-free systems with an energetic gain of 200 mV compared to the common high-midpoint potential mediator 2,6-dichloro-1,4-benzoquinone (DCBQ). The low-midpoint potential of PYO led to O 2 reduction side-reactions, which competed significantly against photocurrent generation; the tuning of mediator concentration was important for outcompeting the side-reactions whilst avoiding acute cytotoxicity. DCBQ-mediated photocurrents were generally much higher but also decayed rapidly and were non-recoverable with fresh mediator addition. This suggests that the cells can acquire DCBQ-resistance over time. In contrast, PYO gave rise to steadier current enhancement despite the co-generation of undesirable reactive oxygen species, and PYO-exposed cells did not develop acquired resistance. Moreover, we demonstrated that the cyanobacteria can be genetically engineered to produce PYO endogenously to improve long-term prospects. Overall, this study established that energetic gains can be achieved via the use of low-potential phenazines in photosynthetic bioelectrochemical systems, and quantifies the factors and trade-offs that determine efficacious mediation in living bioelectrochemical systems.",
"conclusion": "Conclusions In this study, we revealed that energetic gains in cyanobacterial anodes can be achieved using low-potential phenazine mediators, but mediator selection must be based on several criteria. An ideal diffusional electron mediator should: (i) be cell membrane permeable; (ii) be able to mediate electron transfer at concentrations that do not cause cytotoxicity; (iii) participate in minimal deleterious side reactions (for example with O 2 ); (iv) be robust against deactivation mechanisms such as molecule breakdown to minimize the need for regular replenishments (noting that endogenous production mitigates this need) and; (v) possess an appropriate midpoint potential to minimize energy loss. This study showed that a fine balance exists between point (v) and the other criteria. The more negative the mid-point potential of the mediator is (and less energy loss from the PETC), the more likely undesirable side reactions with O 2 will occur in addition to kinetics losses. Interestingly, an unexpected example of a deactivation mechanism (point iv) was observed, whereby cells acquired resistance to DCBQ mediation effect over time, which was not observed for PYO. Although phenazines have only partially fulfilled the aforementioned selection criteria, we show that they exhibit many advantages, including the ability to be endogenously produced by genetically modified cyanobacteria; this is likely to be needed to build low cost microbial photoelectrochemical systems with long lifetimes. Strategies to improve their efficacy could involve, for example, chemical alterations to shift their E m to slightly higher potentials to avoid oxygen reduction. Synthetic biology approaches could help overcome some of the limitations observed. Strategies for increased phenazine production have been mentioned, but temporal control, for example through coordination with the circadian clock, 59 could circumvent the issues with oxygen reactivity: phenazines could be released during the night to transfer reducing equivalents that have been generated during the day and stored in reserves of fixed carbon. Expression of enzymes to neutralise ROS ( e.g. superoxide dismutase) might decrease the cytotoxicity of phenazines to Synechocystis , allowing higher concentrations to be produced. There are many derivatives of biologically produced phenazine, some of which may satisfy more of the criteria listed above, and synthetic biology can be used to expand the variety of phenazine molecules even further. 60 Establishing long-term efficient wiring of biological systems to electrodes will be key to the success of bio-electrochemical technologies. This study starts to systemically unpick ways of achieving this using diffusional mediators.",
"introduction": "Introduction Oxygenic photosynthetic microorganisms can be ‘wired’ (electrically connected) to electrodes to harness light energy for electrical power generation in biophotovoltaic (BPV) systems ( Fig. 1A ), 1–4 and potentially for fuel generation in semi-artificial photosynthesis. 5 Cyanobacteria are particularly attractive biocatalysts for these biotechnologies since they are abundant, simple in cellular architecture, can reproduce and self-repair, have wide-ranging biosynthetic capabilities, and contain efficient photo-harvesting machineries for carrying out endergonic reactions. 1,6,7 However, although photosynthetic microorganisms are known to give rise to steady and long-lived photocurrents, 8 the output magnitudes are also much lower compared to isolated proteins and synthetic systems. 5,9 Artificially added exogenous electron mediators are therefore widely used for efficient wiring of the photosynthetic cells to electrodes. Fig. 1 (A) A schematic of a biophotovoltaic (BPV) system employing photosynthetic microorganisms at the anode. Under illumination, photosynthetic microorganisms oxidize H 2 O to O 2 and H + and transfer some of the resulting energized electrons to an anode. The electrons flow to the cathode, where O 2 is catalytically reduced to reform H 2 O, generating a current. At the anode, an indirect extracellular electron transfer (EET) pathway is shown, where electron transfer is mediated by a redox molecule diffusing between the intracellular electron donors and the electrode surface. (B) Energetic landscape of the photosynthetic electron transport chain (PETC; shown by solid arrows): electrons are derived from H 2 O oxidation by photosystem II (PSII), photoexcited by the reaction center, P680, 16,17 and exit via the terminal quinones, Q A/B . The plastoquinone pool passes electrons to the cytochrome b 6 f complex (Cyt b 6 f), which shuttles electrons via plastocyanin ( Pc ) to photosystem I (PSI). 18 The electrons are picked up by ferredoxin (Fd), and delivered to ferredoxin–NADP + reductase (FNR) 19 to reduce NADP + . The dashed arrows represent the points from which electrons can be intercepted by 2,6-dichloro-1,4-benzoquinone (DCBQ) 11 and phenazines. 20,21 All midpoint potentials ( E m ) correspond to pH 7. Benzoquinones are common electron mediators found in electron transport chains and synthetic derivatives are commonly used as exogenous electron medators. 8,10 A typical benzoquinone used as the exogenous mediator for both protein and cell-based photoelectrochemical systems is 2,6-dichloro-1,4-benzoquinone (DCBQ, midpoint potential: E m = 0.315 V vs. SHE 11 ). 5,12 During photosynthesis, electrons derived from water oxidation at photosystem II (PSII) are fed into the photosynthetic electron transport chain (PETC) in the thylakoid membranes. 8,13 In the absence of exogenous mediators, some electrons leave the PETC downstream of photosystem I (PSI) to participate in extracellular electron transfer, which then give rise to photocurrents if the electrons are collected by an electrode ( Fig. 1 ). 14 DCBQ is hypothesized to function similarly to the terminal photosystem II (PSII) electron acceptor, Q B , and extract electrons from the PETC downstream of PSII. 8,10 However, exogenous electron mediators such as DCBQ have poorly defined modes of toxicity, and have high scale-up costs. 8,10,15 Also, the positive E m value of DCBQ means that mediation comes with a significant thermodynamic cost ( Fig. 1B ), 11 where the potential difference between the terminal acceptor for PSI and DCBQ is approximately 700 mV. The identification of efficacious electron mediators capable of enhancing photocurrents at potentials more negative than mediators like DCBQ would allow more energy to be recovered from photosynthesis and be a breakthrough in the field. Further gains could be made by producing electron mediators endogenously. However, design principles for effective endogenous or exogenous mediators are currently lacking. Phenazines are a class of redox-active secondary metabolites produced by a few clades of bacteria, most notably pseudomonads, 20,22 but not cyanobacteria. 23 They have been shown to enhance current outputs from microbial fuel cells (MFCs) 24–28 but have not yet been explored as mediators for cyanobacteria or BPVs. Phenazines are suitable electron shuttles since they are small and lipid-soluble 6,21 and are therefore able to pass through bacterial membranes to facilitate EET. They have E m values more negative than DCBQ, but still positive enough to accept electrons from the PETC ( Fig. 1B ). 11,18–21,29 Additionally, they have considerable bioengineering potential as the genetics and biochemistry of their biosynthesis are well understood, 20,23 though cyanobacteria have not been engineered for their biosynthesis. Here, we assessed an approach for improving indirect mediation for cyanobacteria—using lower midpoint potential electron mediators to minimise energy loss, with phenazines as the model mediators. A library of phenazines was tested as electron mediators for Synechocystis sp. PCC 6803 (hereafter Synechocystis ), which is a model cyanobacterium for BPVs. We identified pyocyanin (PYO) as a promising low-potential candidate and tested the limits of its mediation ability in terms of energetics, permeability, concentration required for mediation, cytotoxicity, overall longevity and potential for endogenous production in cyanobacteria. We revealed benefits and limitations of both phenazines and benzoquinones relating to their modes of mediation and deactivation.",
"discussion": "Results and discussion Phenazine screening Four phenazines ( Fig. 2A ) were initially screened as potential electron mediators for BPVs: phenazine (PHZ), 1-hydroxyphenazine (1-OHP), phenazine-1-carboxylic acid (PCA) and PYO. 1-OHP, PCA and PYO were of interest as they can be endogenously synthesized by bacteria such as Pseudomonas aeruginosa , 2,6 and can potentially be introduced endogenously into microbial electrochemical systems either by genetic modification or through a co-culturing approach. 30 PHZ contains the basic phenazine backbone and was included as a control. Fig. 2 Photoelectrochemical screening of phenazines for their ability to mediate electrons between Synechocystis and an electrode. (A) Structures and midpoint potentials ( E m ) of the phenazines screened. The E m potentials of the phenazines were measured in BG11 media at pH 8.5 and are reported vs. SHE. (B) A typical chronoamperometry profile obtained of a Synechocystis -loaded ITO electrode under dark/light cycles at an applied potential of 0.3 V vs. SHE; the photocurrent is defined by the difference between the steady-state dark and light currents. (C) Chl a normalised photocurrent densities of Synechocystis -loaded electrode in the presence of four different phenazines (200 μM). All chronoamperometry measurements were recorded at an applied potential of 0.3 V vs. SHE, and under atmospheric conditions at 25 °C in BG11 medium electrolyte. Light conditions used: λ = 680 nm, 1 mW cm −2 (50 μE m −2 s −1 ). The mean was taken from three biological replicates and the error bars represent the standard error of the mean ( n = 3). Suitable electron mediators for photosynthetic organisms should exhibit E m values more positive than key components of the PETC (>−0.41 V vs. SHE, Fig. 1B ) to favour kinetics of electron transfer; however, those with very positive E m values will result in significant energy losses. The E m value of each mediator candidate was measured using cyclic voltammetry under typical BPV conditions (in BG11 medium at pH 8.5 in which the cyanobacterial cultures were grown to favour bicarbonate uptake into the cells, Fig. S1 † ). All candidates showed a single anodic and cathodic peak corresponding to a concerted two-proton two-electron oxidation and reduction, respectively. 31,32 The redox reactions of all mediator candidates considered in this study are shown in Fig. S3. † The E m values of the phenazines fell within the range −0.120 to −0.252 V vs. SHE, whereas DCBQ exhibited a more positive E m value of 0.250 V vs. SHE. As the redox reactions of phenazines and benzoquinones are proton coupled, 31,32 their electrochemical properties are pH dependent and the E m values observed for pH 8.5 were negatively shifted (by ca. 90 mV) compared to experimental and reported values obtained near pH 7 (Fig. S2 † ). 20,21,33 Thermodynamically, all four phenazines can accept electrons downstream of PSI. The P680 and P700 reaction centers presumably cannot directly reduce the phenazines as they are embedded deep within protein complexes and are difficult for mediators to access. 34,35 The more positive E m value of PYO suggests that it could also be reduced to a small extent by the terminal quinones ( Q B E m = −0.06 V vs. SHE 29 ). This is not expected for the other phenazines as their E m values are more negative than that of Q A/B . DCBQ is a commonly used electron mediator that targets PSII, 8,10 but its positive E m value suggests that it can also accept electrons from PSI. To test the ability of the phenazines to mediate electron transfer in microbial photoelectrochemical systems, a previously described three-electrode photoelectrochemical set-up using the model cyanobacterium Synechocystis was employed ( Fig. 2A ). 8 Synechocystis cells were first dropcast as a concentrated planktonic culture (150 nmol Chl a mL −1 ) onto a porous inverse opal indium tin-oxide (IO-ITO) electrode, allowing cell adsorption over 16 h in the dark. The Chl a loading was determined afterwards, with average Chl a loading 3.26 μg Chl a cm −2 (5 nmol Chl a per electrode). In a typical chronoamperometry experiment, illumination of the Synechocystis -loaded working electrode induces a photocurrent corresponding to a change in electron flux from the photosynthetic microorganism to the electrode ( Fig. 2B ). A diffusional mediator serves to enhance the photocurrent by increasing the flux of electrons from the microorganism to the electrode, but this will only take place when the electrode is at a potential that can oxidize the mediator and when the mediator is above a threshold concentration. Chronoamperometry experiments using a bare IO-ITO electrode confirmed that the observed photocurrents stemmed from the photosynthetic microorganisms (Fig. S4 † ). Previous studies by Grattieri et al. have shown that measuring the E m of quinone redox mediators in aqueous environments is not sufficient when trying to understand their ability to mediate EET in photosynthetic purple bacteria. 36 This is due to contributions by the lipophilic membranes that the mediators must traverse, and also the E m corresponds to a 2e − /2H + process that does not often accurately reflect the electron exchange processes occurring within biological contexts. The E m values discussed should therefore be treated only as a helpful guide for comparing thermodynamics between redox species. To determine the minimum applied potential needed for photocurrent enhancement by a phenazine electron mediator, PYO was introduced to the photoelectrochemical cell and stepped chronoamperometry experiments were performed (Fig. S5 † ). Competing charge transfer pathways in the form of photocathodic currents have been known to obfuscate the photocurrents at potentials <0.2 V vs. SHE. These are due to the reduction of oxygen (onset at 0 V vs. SHE, Fig. S4C † ) or reactive oxygen species (ROS) at the electrode. 29,37 Maximum photocurrents were observed at 0.3 V vs. SHE, which is 0.2 V more negative than the applied potential required to observe the maximum photocurrent mediated by DCBQ (0.5 V vs. SHE). 11 Since the other phenazines have more negative E m values than PYO, their onset potentials are expected to convey the same trend, and subsequent chronoamperometry experiments involving phenazines were carried out with an applied potential of 0.3 V vs. SHE. The photocurrent densities from Synechocystis -loaded electrodes in the presence of 200 μM of each of the phenazines at 0.3 V vs. SHE are shown in Fig. 2C . To take account of possible cytotoxicity effects and other sources of variation in cell numbers, the Chl a content of each electrode was quantified after each experiment and the photocurrent densities presented are normalised by Chl a content on the electrode. PYO gave rise to a 4-fold enhancement in photocurrent density; the other phenazines did not exhibit mediation. To understand whether the lack of mediation by the other phenazines may be attributed to poor cell permeability, clog D values were calculated at a range of pH values for each phenazine. clog D values are computationally determined partition coefficients, which have been developed to predict the permeability of drugs through mammalian plasma membranes. 38 A clog D value of greater than 0.5 is required for a 50% probability of the molecule being highly permeable to cell membranes. 38 Using this predictor, the oxidised and reduced states of PHZ, 1-OHP and PYO are expected to permeate into the cell due to their high clog D values at the media pH of 8.5, as well as out of the cell due to their high clog D values at the cytoplasmic pH of 7.5 (Fig. S6 † ). In contrast, PCA was predicted to be membrane impermeable in both its oxidised and reduced states and therefore unable to perform mediation. All phenazines had a sufficiently high clog D value at the thylakoid lumen pH of 4.6, suggesting mediators are not sequestered within this compartment. The poor mediation ability of PHZ and 1-OHP, despite their predicted membrane permeability, may be attributed to factors such as poor aqueous solubility or low driving force for electron transfer. PYO, having been found to be the most promising mediator from the screening, was taken forward and compared with the model benzoquinone mediator, DCBQ. Cytotoxicity versus mediation concentration The efficacy of a diffusional mediator depends on it being in excess at the bio-electrode interface. For efficient shuttling, bulk concentrations of >100 μM are typically employed. 8,10,14,39–41 However, an often-neglected trade-off is the toxicity of the mediator towards the living cells, which also scales with the concentration of the mediator present. Although DCBQ cytotoxicity is often observed 8,10 and a non-photochemical quenching effect of DCBQ has been described, 42 the effect of DCBQ concentration on the growth of photosynthetic microorganisms has not been tested to the best of the authors' knowledge. Spot assays were performed to probe the effect of different concentrations of PCA, PYO and DCBQ on Synechocystis growth to identify the highest concentration of mediator at which the cells could still grow ( Fig. 3A ) and the time course over which this concentration was non-toxic ( Fig. 3B ). PCA was included because it is the precursor from which PYO is derived, 23,43 so is likely to be present in anticipated PYO-producing cells. Fig. 3 Mediator concentrations giving rise to cytotoxicity and photocurrent enhancement. (A) Cytotoxicity spot assay to determine the effect of PYO, PCA and DCBQ on the growth of Synechocystis . Synechocystis cells (5 nmol Chl a ) were incubated with the mediators at different concentrations for 24 h under 1 mW m −2 (50 μE m −2 s −1 ) white light. The cells were resuspended in BG11 and their concentration standardized to an OD 750 of 0.5. Serial dilutions were prepared, spotted on agar and incubated for 1 week. Data from a single representative plate from 3 replicates is shown. (B) The spot assay was repeated for the highest concentration of mediators that did not inhibit cell growth after 24 h, with the incubation period extended to 3 days. (C) Chl a normalised initial photocurrent densities from Synechocystis in the presence of different concentrations of PYO and DCBQ (200 μM). Inset: initial photocurrent densities from Synechocystis in the presence of PYO (200 μM) and DCBQ (200 μM), without normalization by Chl a content. All chronoamperometry measurements were recorded at an applied potential of 0.3 V vs. SHE, except for measurements made using DCBQ, which were performed at an applied potential of 0.5 V vs. SHE under atmospheric conditions at 25 °C in BG11 medium. Light conditions used: 1 mW cm −2 (50 μE m −2 s −1 ) of 680 nm wavelength. The mean was taken from three biological replicates and the error bars represent the standard error of the mean ( n = 3). Equivalent amounts of Synechocystis cells to those that adhered to the IO-ITO electrodes for photoelectrochemical tests (5 nmol Chl a ) were incubated with PCA, PYO, and DCBQ at different concentrations for 1 or 3 days under constant light (1 mW m −2 (50 μE m −2 s −1 )). Concentration ranges of up to 0.5 mM were tested for PCA and PYO due to their limited aqueous solubility. It was possible to test concentration ranges of up to 1 mM for DCBQ, and this was done to parallel previous photoelectrochemical experiments. 8,33 After exposure to the mediators, the Synechocystis cells were re-suspended in fresh BG11 medium, spotted onto BG11 agar plates, and incubated for 1 week. Three control conditions were also assayed: BG11 only (no cells), cells in BG11 (no mediator), and cells in BG11 with 0.2% (v/v) DMSO (the highest concentration of DMSO solvent in the DCBQ cytotoxicity tests) as shown in Fig. 3A and S7. † The quantity of DMSO employed was confirmed to be non-toxic. As shown in Fig. 3A , Synechocystis incubated for 1 day with 200 μM PYO or 200 μM DCBQ showed growth comparable to the controls, indicating that exogenously added PYO and DCBQ were non-toxic at concentrations <200 μM. At higher concentrations of mediator, the cells showed significantly poorer growth than the controls, indicating cytotoxicity at concentrations >200 μM. Cells incubated with PCA showed comparable growth to the controls, indicating that exogenously added PCA is non-toxic to at least 500 μM even after 3 days of incubation; this is consistent with its predicted low cell membrane permeability (Fig. S6 † ). \n Synechocystis cells incubated for 3 days with 500 μM PCA or 200 μM DCBQ showed growth comparable to the controls ( Fig. 3B and S7 † ), indicating that exogenously added PCA and DCBQ at these concentrations are non-toxic for 3 days. Cells incubated for 3 days with 200 μM PYO exhibited noticeably poorer growth than the controls, indicating that exogenously added PYO at higher concentrations is toxic in the long-term. This may be attributed to the ability of PYO to produce harmful ROS upon interaction with oxygen. 20,44–47 To ascertain the minimum concentration of PYO needed for efficient electron shuttling, chronoamperometry with dark/light cycles using Synechocystis -loaded IO-ITO electrodes was performed in the presence of different concentrations of PYO. Based on the results of the cytotoxicity assay, 200 μM was chosen as the maximum concentration of PYO to be tested. Interestingly, when incubated with 10 μM PYO, the photocurrent diminished to less than 50% of the no-mediator condition ( Fig. 3C ); however, photocurrent enhancement was observed at concentrations >100 μM. A likely explanation for the different mediation behaviour of PYO with concentration change is that PYO (−0.120 V vs. SHE, pH 8.5) may inhibit EET analogous to methyl viologen ( E m = −0.42 V vs. SHE 14 ), which can intercept electrons from PSI in the PETC and reduce O 2 . 48,49 We hypothesize that after PYO is reduced, O 2 is in competition with the electrode to accept electrons from reduced PYO. At concentrations of PYO <200 μM under atmospheric conditions, O 2 (present at around 250 μM in aqueous solution) can intercept most electrons before PYO can shuttle them to the electrode, reducing the photocurrent observed. At higher concentrations of PYO, more reduced mediator can reach the electrode, giving rise to the photocurrent enhancement. To test this, the chronoamperometry experiments were repeated in 10 μM PYO with N 2 bubbling to purge O 2 from the electrolyte, though it is difficult to remove O 2 completely from an oxygenic photosynthetic system. Decreasing the O 2 concentration in the electrolyte restored the photocurrent to approximately 80% of that obtained in the absence of mediator (Fig. S8 † ), indicating that O 2 was competing with the electrode to accept electrons from reduced PYO. It can be concluded that for PYO to serve as an efficient electron mediator for Synechocystis , a concentration of between 100–200 μM should be used to enhance photocurrents whilst still minimizing acute cytotoxicity. It was previously demonstrated that this concentration range of PYO can be endogenously expressed by Pseudomonas aeruginosa, 50 hence the concentration needed for mediation in BPVs may be feasibly achieved with bio-engineering and/or a co-culturing approach. The initial photocurrents of Synechocystis -loaded IO-ITO electrodes mediated by 200 μM PYO or DCBQ were compared ( Fig. 3C inset). Synechocystis -loaded electrodes in the presence of PYO and DCBQ produced ca. 4-fold and 40-fold greater initial photocurrent densities, respectively, compared to electrodes with no exogenous mediator. It should be noted that the initial photocurrents reported here for DCBQ are an order of magnitude lower than those reported when higher, cytotoxic, concentrations of DCBQ were used. 8 Hence, PYO harvested a significantly smaller fraction of the cells' available reducing equivalents compared to DCBQ. There are several likely explanations for this. Firstly, PYO and DCBQ have different intracellular targets and the positive E m value of DCBQ means that it could be reduced by more components of the PETC. Additionally, the more positive E m value of DCBQ imparts it with a greater thermodynamic driving force for reduction, which also increases the electron transfer kinetics. Lastly, reduced PYO has a competing O 2 reduction side-reaction, which does not occur for DCBQ due to its higher E m value. Sites of mediator reduction To probe the site of reduction of PYO and DCBQ within the PETC, two PETC inhibitors were used in chronoamperometry experiments: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and methyl viologen ( Fig. 4A ). DCMU inhibits electron flow downstream of PSII by competing with Q B 29 and preventing electron transfer from PSII to the plastoquinone pool ( Fig. 4B ). 14 As previously described, methyl viologen competes for electrons downstream of PSI and transfers them to O 2 . 14 Combining PYO with methyl viologen completely ablated the photocurrents originating from Synechocystis , consistent with PYO accepting electrons downstream of PSI. Findings from a previous study suggested that phenazines can compete with NAD + for reduction by enzymes within the cell; in the photosynthetic context PYO may be able to accept electrons from the ferredoxin:NADP + reductase. 51 The addition of DCMU to PYO mediated systems almost completely eliminated any mediation effects (11 A [mol Chl a ] −1 cm −2 ). Small photocurrents (28 A [mol Chl a ] −1 cm −2 ) were still observed when DCMU was added to DCBQ mediated systems. This indicates that although water oxidation has been inhibited by the DCMU, electron flow downstream of PSII can still contribute towards EET. It is possible that some electron flow continues in the presence of DCMU because the respiratory and photosynthetic electron transfer chains in Synechocystis share the same plastoquinone pool. 14 Combining DCBQ with methyl viologen resulted in a 10-fold decrease in the photocurrent yielded by DCBQ mediation, but this photocurrent was still 4-fold greater than the no-mediator condition ( Fig. 2C ). This is consistent with DCBQ being able to accept electrons upstream of PSI even though electrons downstream of PSI are intercepted by MV. The observation that the presence of methyl viologen could significantly diminish the photocurrent mediated by DCBQ suggests that EET can stem from downstream of PSI. These results confirm that PYO and DCBQ have different intracellular targets and mechanisms of EET mediation. Fig. 4 Mediation mechanism and longevity. Chl a normalized initial photocurrent densities from Synechocystis in the presence of (A) PYO (200 μM) and (B) DCBQ (200 μM) and the PETC inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, 1 mM) or methyl viologen (MV, 1 mM). (C) Turn-over numbers (TON) of Synechocystis -loaded IO-ITO electrodes per PSII in the presence of PYO (200 μM) and DCBQ (200 μM) over 15 h of light exposure. (D) Rates of photocurrent decay of Synechocystis -loaded IO-ITO electrodes in the presence of PYO (200 μM) and DCBQ (200 μM) over 15 h of light exposure. (E and F) Photocurrent densities from Synechocystis : without the addition of mediators (BG11), after addition of 200 μM PYO (+PYO) and DCBQ (+DCBQ) and after 15 h light exposure with the mediator, after replacement with fresh BG11 media (fresh BG11), and after the addition of 200 μM fresh PYO (fresh PYO) or DCBQ (fresh DCBQ). All chronoamperometry measurements were recorded at an applied potential of 0.3 V vs. SHE under atmospheric conditions at 25 °C in BG11 medium electrolyte, except for measurements made with DCBQ, which were performed at 0.5 V vs. SHE. Light conditions used: 1 mW cm −2 (50 μE m −2 s −1 ) of 680 nm chopped light. The mean was taken from three biological replicates and the error bars represent the standard error of the mean ( n = 3). Mediator longevity It is well documented that the electron mediation effects of DCBQ drop off quickly over time and the decrease in effectiveness is accompanied by a poorly characterized non-photochemical quenching effect. 8,42 To study the longevity of the mediation by both PYO and DCBQ, chronoamperometry was carried out over 15 h under constant illumination. In the absence of exogenous mediators, the photocurrent was observed to remain constant for over 15 h, consistent with photocurrents previously observed for living cells. 8 In cases where mediators were introduced, immediately following mediator addition, a sharp rise in photocurrent could be observed, which decayed rapidly to a steady rate at ca. 80 min (Fig. S9 † ). The rapid initial decay in photocurrent could be attributed to the time needed for the electrode double layer to reach equilibrium and the decay in the concentration of reduced mediator species at the electrode surface over time, since the system was unstirred and mass transport was likely to be a limitation. The turn-over number (TON), which is proportional to the cumulative charge collected by the Synechocystis -loaded electrodes over 12 h is summarized in Fig. 4C and S9. † The turn-over frequency (TOF) relating to the rate of oxygen evolution per PSII was generated based on the assumption that the ratio of PSI : PSII was 1 : 1 in the PETC, and that the faradaic efficiency for oxygen evolution in relation to the photocurrent output is near unity (Fig. S9B † ). DCBQ was observed to give rise to greater than 10-fold higher TON and TOF numbers compared to PYO, but the rate of decay in the mediated photocurrent also vastly exceeded that of PYO. After the initial drop in photocurrent, the DCBQ mediated system continued to decay at ca. 100 A (mol Chl a ) −1 cm −1 h −1 after 6 h, whereas the PYO mediated system showed near-zero decay rates after 3 h ( Fig. 4D ). This implies that PYO mediation has a different deactivation mechanism from DCBQ, which has been reported to exert quenching of the photoexcited Chl a within the PETC upon close interactions. 42,52,53 After 15 h, the DCBQ mediated photocurrent decreased 12-fold ( Fig. 4 ); in contrast, the PYO-mediated system decreased 3-fold. In the absence of any mediators, Synechocystis -loaded IO-ITO electrodes gave rise to steady photocurrents with zero decay rate over time, consistent with previous reports. 8 Since the cytotoxicity assays showed that 200 μM DCBQ was non-toxic for 72 h ( Fig. 3A ), the significant decline in the photocurrent output over 15 h could also be attributed to cellular sequestration of the DCBQ molecules over time, which has been shown to occur in Chlamydomonas reinhardtii , 54 or molecular breakdown. To test this, the electrolyte of the photoelectrochemical cell was replaced with fresh BG11 medium alone after 15 h of illumination in the presence of DCBQ, and the photocurrent output was re-measured. The photocurrent densities observed were the same in magnitude as those of Synechocystis without DCBQ addition ( Fig. 4F ). This indicates that cells treated with 200 μM DCBQ are still viable and photoactive after prolonged exposure, consistent with the cytotoxicity results. Fresh DCBQ was further added to the same set-up and measurements of the photocurrents were taken. In the second DCBQ exposure, the initial mediated photocurrent produced by the Synechocystis was 10-fold that of a non-mediated system but was 6-fold lower than that observed during the first DCBQ exposure. The mediated photocurrent (with fresh mediator) eventually returned to the same current density as observed after the first 15 hours. The partial recovery of the mediation effect is consistent with the breakdown or cellular sequestration of DCBQ over time, but the lack of full recovery points to Synechocystis becoming resistant to the mediating effects of DCBQ over time. The analogous experiment was performed for Synechocystis exposed to 200 μM PYO and light for 15 h ( Fig. 4E ). In this case, the replacement of electrolyte with fresh BG11 medium gave rise to photocurrent densities that were 4-fold lower than those observed from cells without any exposure to mediators. This indicates that the cells have lost viability from the prolonged exposure to 200 μM PYO, consistent with the cytotoxicity assays ( Fig. 3B ). The subsequent addition of fresh PYO to the pre-exposed cells resulted in a 4-fold enhancement in the photocurrents, which implies that the decline in mediated photocurrent output over time was also partially due to a decrease in PYO concentration over time. This may be caused by the cellular sequestration of the mediator, which has been shown to occur for quinones in Chlamydomonas reinhardtii , 54 or to molecular breakdown. To probe the molecular stability of the PYO and DCBQ, solutions of PYO and DCBQ under light illumination in the presence and absence of redox cycling were studied using cyclic voltammetry and UV-visible spectroscopy (Fig. S10 and S11 † ). CVs recorded after 15 hours of redox cycling under light illumination for both PYO and DCBQ under atmospheric conditions showed that the redox peaks had almost completely disappeared, indicating that they had both broken down (Fig. S10 † ) under mediation conditions. UV-vis spectra of PYO before and after 15 hours of light illumination (with no redox cycling) showed no significant differences, indicating PYO is stable under light illumination alone (Fig. S11A † ), and the molecular breakdown was likely due to deleterious ROS interactions. UV-vis spectra of DCBQ after 15 hours illumination were significantly changed compared to the spectra recorded before the illumination (Fig S11B † ), and a colour change in the DCBQ solution was observed (Fig. S11C † ). Combined, these studies show that DCBQ is intrinsically less chemically stable than PYO. It can be summarized that both mediators break down over 15 hours, and the mechanism responsible for mediated photocurrent decline over time is different. Synechocystis does not become resistant to the mediation effects of PYO over time, though it cannot cope with the excess ROS generated as side products. Endogenous production of PYO by Synechocystis If the photosynthetic microorganism were able to (re)generate its own mediators endogenously, this would eliminate the need to replenish the microbial devices constantly with synthetic mediators that are lost due to molecular breakdown. Biological production of phenazines in nature is limited to a few bacterial clades, typically occupying soil-dwelling or plant-associated niches, though phenazines are also implicated in human pathogenicity as part of opportunistic Pseudomonas aeruginosa infections. 55 No photosynthetic microorganisms naturally produce phenazines, but two approaches for the endogenous expression of PYO in a microbial photoelectrochemical system are possible; co-culturing cyanobacteria with a PYO producing bacterial strain, or using genetic engineering to introduce the PYO synthesis pathway to cyanobacteria. As heterologous expression of phenazines has previously been achieved in E. coli 23,24 we attempted to express PYO as an endogenous electron mediator in Synechocystis . Expressing PYO as an endogenous mediator in cyanobacteria is challenging due to the large number of genes required. Biosynthesis of PYO is well characterized, proceeding via PCA, which is itself synthesized from the central metabolite chorismate by the seven-gene phzA-G operon. 23 Two accessory genes, phzS and phzM are then required for conversion of PCA to PYO. We built genetic constructs to express the genes as two transcriptional units, one containing the core phzA-G genes and the other the accessory phzSM genes, from constitutive promoters known to function in Synechocystis 56 (Fig. S12 † ). Successful expression of the cloned PYO biosynthesis genes from the standard pSB3K3 plasmid was first confirmed in E. coli. The production and export of PYO from E. coli TOP10 with pSB3K3-PYO was confirmed by UV-visible spectroscopy and mass spectrometry of spent medium from culture grown for four days (Fig. S13 † ), without any PCA intermediate detected, indicating full conversion. We initially hypothesized that a PYO producing pathway could be engineered into a previously engineered triple respiratory terminal oxidase (RTO) knockout strain of Synechocystis to enhance photocurrent outputs further. The RTO knockout strain lacks the electron sinks cytochrome c oxidase, bd -quinol oxidase, and the alternative respiratory terminal oxidase, and exhibits power output four-fold higher than the wild-type with ferricyanide as the mediator. 57 Unexpectedly, in initial screening experiments where PYO was added to an electrode loaded with the RTO knockout mutant, photocurrent diminishment was observed instead of the enhancement seen with wild type cells (Fig. S14 † ). This can be rationalized by the presence of a higher cellular O 2 concentration in the RTO knockout mutants as a result of the absence of the terminal oxidases, which serves to reduce O 2 back to water. This is consistent with PYO mediation being affected by cellular O 2 levels. Bioengineering efforts therefore focused on introducing the PYO biosynthetic pathway into wild-type Synechocystis . Wild-type Synechocystis was transformed with the PYO biosynthesis genes on a broad host range vector backbone (pDF-PYO), and the production and export of PYO was confirmed by mass spectrometry of spent medium from early stationary phase cultures at an OD 750 of ca. 1 (Fig. S15 † ). The intermediate PCA was not detected in the spent medium using mass spectrometry. The mass for PYO was detected only in the spent medium from Synechocystis transformed with pDF-PYO, and not in the negative control strain, which had been transformed with an empty plasmid (pDF-lac), confirming the phenazine is not naturally produced by this cyanobacterium. The amount of PYO produced and exported by Synechocystis with pDF-PYO was measured using UV-visible spectroscopy. A concentration calibration curve using the absorbance of PYO at 690 nm in BG11 medium was plotted and an extinction coefficient of 1.52 mM −1 cm −1 calculated (Fig. S16 † ). The concentration of PYO present in the spent medium of early stationary phase culture of Synechocystis with pDF-PYO was calculated to be 8.2 μM (average of 3 biological replicates) ( Fig. 5A ), whilst PCA was not detected. However, the concentrations of the phenazines inside of the cells may be significantly higher. Fig. 5 PYO can be endogenously expressed in Synechocystis . (A) A representative UV-vis spectrum of spent medium from continuous cultures of Synechocystis transformed with pDF-PYO. Spent medium was taken from mid-late stationary phase cells with an OD750 of ca. 1. The spectrum was recorded from 280 to 800 nm with spent medium from the negative control strain taken as the background. UV-vis spectra of PCA (50 μM) and PYO (80 μM) in BG11 medium, with BG11 taken as the backgrounds, are shown for comparison. (B) Photocurrent densities of the negative control Synechocystis strain and the PYO producing Synechocystis strain with the spent medium in which they were cultured as the electrolyte. All chronoamperometry measurements were recorded at an applied potential of 0.3 V vs. SHE under atmospheric conditions at 25 °C. Light conditions used: 1 mW cm −2 (50 μE m −2 s −1 ) of 680 nm wavelength. Data was collected from 3 biological replicates and the error bars represent SEM ( n = 3). The transformed cells were grown until the culture had reached OD 750 = 1, concentrated, dropcast on to the porous IO-ITO electrodes and allowed to adhere for 16 hours. The spent BG11 medium from the cultures was retained and used as the electrolyte in the chronoamperometry experiments. There was no significant difference between the photocurrents produced by the negative control strain and the PYO-producing strain ( Fig. 5B ), which indicates that the concentration of PYO produced is too low for current enhancement. Optimization of the bioengineering strategy is still needed to increase the production level to match the concentrations needed to enhance photocurrents. This may be achieved by codon optimization of the open reading frames and RBS sequences 58 and selection of appropriate promoters to increase expression levels of the biosynthesis gene."
} | 10,732 |
24920474 | PMC4071554 | pmc | 8,755 | {
"abstract": "Collective decisions in animal groups emerge from the actions of individuals who are unlikely to have global information. Comparative assessment of options can be valuable in decision-making. Ant colonies are excellent collective decision-makers, for example when selecting a new nest-site. Here, we test the dependency of this cooperative process on comparisons conducted by individual ants. We presented ant colonies with a choice between new nests: one good and one poor. Using individually radio-tagged ants and an automated system of doors, we manipulated individual-level access to information: ants visiting the good nest were barred from visiting the poor one and vice versa. Thus, no ant could individually compare the available options. Despite this, colonies still emigrated quickly and accurately when comparisons were prevented. Individual-level rules facilitated this behavioural robustness: ants allowed to experience only the poor nest subsequently searched more. Intriguingly, some ants appeared particularly discriminating across emigrations under both treatments, suggesting they had stable, high nest acceptance thresholds. Overall, our results show how a colony of ants, as a cognitive entity, can compare two options that are not both accessible by any individual ant. Our findings illustrate a collective decision process that is robust to differences in individual access to information.",
"introduction": "1. Introduction Comparative assessment is a powerful tool for choosing between options, and it is widely implemented by decision-making animals, including insects, crabs, birds, bears and primates [ 1 – 7 ] and is even used by slime moulds [ 8 ]. However, comparative assessments can have drawbacks, including the emergence of ‘economically irrational’ behaviour [ 6 – 8 ] and the potential for cognitive overload [ 9 – 11 ]. House-hunting social insects are a model system for studying decision-making, demonstrating the ability to make effective choices in a range of choice contexts [ 12 – 15 ]. In these collective decision processes, there are two levels at which comparative assessment can be performed: individual and group. Ant colonies choosing between potential new nest-sites seem to be immune from both irrational behaviours [ 16 , 17 ] and cognitive overload [ 16 , 18 ], which would seem to indicate that comparative assessment does not play a role in collective decisions. At the individual-level, ants are capable of making comparative assessments [ 19 ], but their decision-making behaviour during emigration to a new nest-site can be explained without invoking comparison [ 20 ]. A simple threshold model in which ants either reject nests as being unsuitable and continue searching, or accept nests and recruit nest-mates to them, but do not directly compare available nests, reproduces observed collective decision behaviour [ 21 ]. In this study, we ask which individual-level behavioural rules are important for robust collective-scale decisions. Specifically, we ask whether the ability of individual ants to make comparative assessments of available options plays an essential role in collective decisions and, if not, whether the actions of partially informed individuals are consistent with the predictions of a simple threshold model of decision-making [ 21 ]. To test the role of individual comparisons in collective decision-making, we used radio-tagged ants and an automated system of doors, so that without physically removing any ants from the colony, we could manipulate individual-level access to information [ 22 ]. Using individual tagging to manipulate, the information available to certain animals is a powerful tool in understanding the mechanisms of collective decision-making [ 22 , 23 ]. We presented ant colonies ( Temnothorax albipennis ) with a choice between two new nests: one good and one poor. Each colony went through two treatments: in the ‘no-comparison’ treatment, ants which had visited the good nest were automatically prevented from visiting the poor nest, and vice versa; in the control treatment, all ants could visit both nests. Thus, in the ‘no-comparison’ treatment, no single individual had sufficient data to make a comparison, but the colony as a whole did have this information. In this way, we tested whether colonies could make successful decisions even when individuals were prevented from making direct comparisons between options, and we also explored the individual-level mechanisms through which effective collective choice can emerge.",
"discussion": "4. Discussion Our results clearly show that preventing individual comparisons does not adversely affect accuracy or speed of collective decision-making in house-hunting ants, relative to paired controls in which individual comparisons were permitted. This is consistent with a threshold rule of decision-making in which ants make decisions about whether or not to commit to a nest based only on the quality of that nest, relative to an internal standard and not in relation to other nests [ 21 ]. These results are also consistent with the behaviour of pheromone trail-laying ant colonies that are able to collectively choose between differing options: the shorter of two paths or the better of two food sources. In these cases, the collective decisions emerge from the interactions of foragers mediated via the pheromone trail, and can be explained by models which do not invoke individual-level comparison [ 31 – 34 ]. Individual access to information has not been directly manipulated in this foraging system, but has in honeybee swarms choosing between new nest-boxes [ 35 ]. Removing bees that encountered both of the nest-boxes (about 18% of scouts) did not prevent or delay swarms from choosing a nest [ 35 ]; however, the boxes were equidistant and of equal quality, so comparison might be expected to be of limited importance. Our results show that even when options differ in attractiveness, preventing comparisons does not impair collective choice. At the individual-level, our results show that preventing scouts from having access to more than one nest does affect the behaviour of the ants during the decision-making process. In the ‘no-comparison’ treatment, ants that have visited the poor nest, then are recorded attempting (and failing) to enter the good nest, are subsequently more active at the entrances to both nests than in the control treatment, in which they would be able actually to enter the good nest. This behaviour matches what would be predicted by a simple threshold model of decision-making [ 21 ], in which ants that reject a poor nest search for alternatives. The individual-level data also suggest why preventing individual comparisons does not affect colony-level decision-making: both the number of ants visiting the good nest and the number of visits per ant to the good nest are similar between the treatments; it is the behaviour of the ants visiting the poor nest first that is most affected by our manipulation. So, in both treatments, it is possible for the colony to reach quorum (i.e. a number of ants sufficient to trigger nest-mate carrying behaviour and full emigration) in the good nest, even though in the ‘no-comparison’ treatment, the ants that first visit the poor nest cannot switch to the good nest and contribute to this quorum. This smaller number of available ants could be expected to slow down the process of reaching quorum in the ‘no-comparison’ treatment [ 28 ], but we do not see a significant difference in speed of decision-making. This may be because we are providing the colonies with a very simple choice challenge. We know that T. albipennis colonies can solve more complex problems and work over much greater scales [ 15 , 36 ]. In our experiment, the environment is very simple visually and the ants find the two nests easily, so the scouting population is not widely dispersed across many possible sites. This may mean that the time taken to make decisions in the simple context is close to the minimum possible decision time, and making a few extra scouts available therefore makes no appreciable difference to the speed of achieving quorum. This idea is supported by the increased level of splitting between the two new nests that was seen when the nests were brought closer together. This made the discovery of both nests easier, both because they may have combined to make an area of visual interest, and because of the slightly disrupted recruitment caused by the doors on the nests. These doors let only one ant enter at once, so even in the control treatment the following ant would have to wait a little, and was likely to discover the other nearby nest instead. House-hunting ants are subjected to a speed-cohesion trade-off, and rapid discovery of both nests by many individuals means that it is possible for both to reach quorum quickly and around the same time whereupon the colony will split. Given that individual ants do have the ability to make comparative assessments [ 17 ], why does this capacity seem to play no role in colony decisions? Would these ants in their natural habitat even have the opportunity to visit more than one nest, and therefore be in a position to make comparisons? There have been no field studies that address this directly, however, inter-nest distances in the wild can be low (frequently less than 30 cm; E. J. H. Robinson and N. R. Franks 1992–2013, personal observation) compared with the much greater distances over which laboratory colonies will readily emigrate, with scouts visiting pairs of nests that are separated by as much as 1.2 m [ 20 , 37 ]. This gives an indication that we would expect some ants to have the opportunity to encounter multiple sites in the wild, however, across the scouting population, the information obtained may be very variable. Our findings illustrate that the collective decision process is robust to differences in individual access to information and the resulting behavioural changes. This may be important in a more natural, heterogeneous and unpredictable environment, where decisions may need to be made so quickly that there is no time to rely on individuals interrogating a wide set of options. Even in the relatively simple environment of our experiments, when we placed the new nests far apart, few ants encountered both the nests before the colony reached quorum in one and began transport to that nest. The time costs and diminishing returns of collecting more information mean animals may benefit from truncating the information gathering process and making a quicker and sufficiently accurate (rather than maximally accurate) decision [ 38 , 39 ]. The ability of individual ants to make comparative assessments might play a more subtle role in colony organization. The emergency emigrations investigated here, analogous to the breaking open of the fragile rock cavities in which these ants nest, are likely to be driven by the necessity for speed. Ant colonies also emigrate simply to improve their nest conditions, while their old nest is still intact [ 40 ]. In this context, speed is much less critical, and comparative assessment might potentially have a role to play. For example, the ability to make comparisons could be used to update an ant's individual acceptance threshold very slowly, so that ants which experience only poor nests gradually reduce their threshold, thus adjusting to their environment. Some adjustment to the local environment is possible without threshold change, if there is a distribution of acceptance thresholds within the colony. In this case, colonies housed in a poor nest would have a larger proportion of dissatisfied ants who would be likely to search for a better option, whereas well-housed colonies would have few ants inclined to search [ 41 , 42 ]. Updating acceptance thresholds could add a level of fine-tuning to this: further investigation of the role of comparative assessment in other decision contexts is required. The existence of a distribution of acceptance thresholds remains a hypothesis [ 20 , 21 , 42 ], but the individual-level data from this experiment do shed some light on this area. Some individual ants were recorded as playing an active role in both of the emigrations performed by their colony. In general, individual activity levels were correlated across the two emigrations. Variation in activity level among ants is well known [ 25 ], but interestingly, in this experiment ants that switched between nests in one emigration were likely to switch in the other emigration and this could not be simply explained by high activity levels in these ants. The results are consistent with these ants being ‘high threshold ants’ which switch (or attempt to, in the ‘no-comparison’ treatment), because the nest they have encountered does not meet their acceptance threshold. A colony's threshold distribution would be predicted to influence its ability to respond to decision-making challenges and is an interesting subject for future study. By manipulating the access of individuals to parts of the information set, we have shown that collective comparisons can emerge from the interactions of poorly informed individuals. In this example, the ant colony cannot simply ‘copy’ a good decision made by a single ant, but must reach a conclusion that is evident only on the collective scale. Collective decision-makers can thus enjoy all the benefits of comparison, in terms of taking the full choice set into consideration, but without potential drawbacks such as irrational behaviours and cognitive overload."
} | 3,387 |
30078568 | PMC6783311 | pmc | 8,756 | {
"abstract": "Summary Benthic foraminifera are unicellular eukaryotes inhabiting sediments of\naquatic environments. Several species were shown to store and use nitrate for\ncomplete denitrification, a unique energy metabolism among eukaryotes. The\npopulation of benthic foraminifera reaches high densities in oxygen-depleted\nmarine habitats where they play a key role in the marine nitrogen cycle.\nHowever, the mechanisms of denitrification in foraminifera are still unknown,\nand the possibility of a contribution of associated bacteria is debated. Here,\nwe present evidence for a novel eukaryotic denitrification pathway that is\nencoded in foraminiferal genomes. Large-scale genome and transcriptomes analyses\nreveal the presence of a denitrification pathway in foraminifera species of the\ngenus Globobulimina . This includes the enzymes nitrite\nreductase (NirK) and nitric oxide reductase (Nor) as well as a wide range of\nnitrite/nitrate transporters (Nrt). A phylogenetic reconstruction of the\nenzymes’ evolutionary history uncovers evidence for an ancient\nacquisition of the foraminiferal denitrification pathway from prokaryotes. We\npropose a model for denitrification in foraminifera where a common electron\ntransport chain is used for anaerobic and aerobic respiration. The evolution of\nhybrid respiration in foraminifera likely contributed to their ecological\nsuccess, which is well documented in palaeontological records since the Cambrian\nperiod.",
"introduction": "Introduction Production of biologically inaccessible dinitrogen (N 2 ) is\nattributed to anaerobic oxidation of ammonium (NH 4 + ) and to\nthe anaerobic respiration of nitrate (NO 3 - ) to N 2 ,\nnamed denitrification[ 1 ]. These processes in\nthe oceans are considered of major importance in the global nitrogen cycle[ 2 ]. Indeed, oxygen-depleted environments that\nare densely populated by denitrifying organisms constitute major sinks for\nbioavailable nitrogen-species [ 1 ]. While the\nproduction of N 2 by denitrification is widespread among prokaryotes[ 3 , 4 ], in\neukaryotes it has been reported only in foraminifera[ 5 ]. Foraminifera are known to colonise a wide range of marine habitats where\nabiotic factors, especially fluctuation or depletion of oxygen availability, are key\nto species diversity and success[ 6 ]. Several\nbenthic species of the order Rotaliida show denitrification activity[ 5 , 7 ].\nRecent studies predicted that denitrifying foraminifera contribute up to 100 % of\ntotal benthic denitrification in the Peruvian oxygen minimum zone[ 8 ], where foraminifera reach abundances of\n>500 individuals per cm 2 [ 8 ,\n 9 ]. Furthermore, foraminifera were shown\nto have a NO 3 - storage that is suggested to accumulate in\nintracellular vacuoles[ 10 ] and sustains\ndenitrification activity for months[ 11 ]. Several foraminifera species (e.g., Buliminella tenuata , and\n Virgulinella fragilis ) were shown to harbour intracellular\nbacteria[ 12 – 15 ], and it has been suggested that these bacteria perform the\ndenitrification reported in those species[ 14 ,\n 16 ]. In contrast, the denitrifying\nforaminifera Globobulimina turgida [ 17 ] (previously determined as G. pseudospinescens [ 18 ]) harbours intracellular bacteria in a low\nabundance, such that the denitrification rates measured for this species cannot be\naccounted for a substantial bacterial contribution[ 5 ]. A eukaryotic denitrification pathway has previously been described\nin fungi[ 19 , 20 ]. However, the fungal denitrification is incomplete, where the end\nproduct is nitrous oxide (N 2 O) rather than N 2 . So far, only\nprokaryotes are known to encode the genetic repertoire to perform complete\ndenitrification. The commonly known denitrification pathway includes four enzymatic steps that\ncatalyse the reactions NO 3 - →\nNO 2 - → NO → N 2 O →\nN 2 . Dissimilatory reduction of NO 3 - to\nNO 2 - is facilitated by periplasmic or membrane-bound\nnitrate reductase (NapA or NarG, respectively). The second denitrification step is\ncatalysed by cd 1 -containing (NirS) or copper-containing\n(NirK) nitrite reductase. While NirS is exclusively found in prokaryotes, NirK\nhomologs are found in a few protists and fungi[ 21 ]. Further reduction of NO to the greenhouse gas N 2 O is\ncatalysed in prokaryotes by nitric oxide reductase (Nor), while an alternative\nenzyme (P450Nor) is documented in fungi. The last step of denitrification in\nprokaryotes is catalysed by nitrous oxide reductase (NosZ). Therefore, all\ndenitrifying organisms share a similar gene repertoire of the denitrification\npathway. Nonetheless, the denitrification gene set in foraminifera remains unknown.\nHere we investigate the genetic repertoire of the denitrification pathway in\nforaminifera. We analysed the whole genomes and transcriptomes of two\n Globobulimina species from the Gullmar Fjord (Sweden). We find\neukaryotic genes encoding for the denitrification enzymes in foraminifera and\ninvestigate their evolutionary origin.",
"discussion": "Discussion Our results demonstrate that the main denitrification enzymes are encoded in\nthe genome of Globobulimina , revealing a so far undescribed\neukaryotic denitrification pathway. The pathway origin is independent of known\neukaryotic enzymes and has a prokaryotic ancestry. Most of the eukaryotic genes of\nprokaryotic ancestry were shown to have been acquired by endosymbiotic gene transfer\n(EGT) from the mitochondrion ancestor at the origins of eukaryotes[ 29 , 30 ].\nThis process is still ongoing[ 31 ], and the\nmechanisms involved are beginning to be unravelled[ 32 ]. Eukaryotic gene acquisition from prokaryotic donors by lateral gene\ntransfer (LGT, as opposed to EGT) is frequently reported; however, these are often\nhampered by signatures of bacterial contamination, and, therefore, their\ninterpretation is often complicated[ 33 , 34 ]. We note that in our study we controlled\nfor possible prokaryotic contamination by analysing the genome and transcriptome of\n Globobulimina in parallel. Furthermore, gene origin in our\npipeline was determined by genomic binning approach, genomic context and\nphylogenetics. The phylogeny of Nrt supports an ancient origin of the nitrate metabolism in\nforaminifera. The foraminiferal NirK phylogeny and the absence of eukaryotic\nhomologs to the Nor found in foraminifera further indicate that the origin of the\nforaminiferal denitrification pathway is independent from the known fungal pathway.\nThe fungal NirK gene origin is likely an EGT[ 21 ], and indeed, it is clustering with other eukaryotic taxa in our\nphylogenetic analysis as expected for genes of endosymbiotic origin ( Figure 3C ; Data S2B ). The\n Globobulimina NirK and Nor are deep branching in all\nphylogenies, and the enzymes’ monophyly in foraminifera is supported by the\npresence of homologs in other rotaliids ( Data S2I-K ). These indicate an ancient origin of\ndenitrification enzymes in foraminifera by LGT from a prokaryotic donor. Our findings demonstrate that denitrification is performed by foraminifera\nrather than associated bacteria. Based on our results, we propose a denitrification\nmodel for foraminifera that draws upon known denitrification properties of fungi\n( Figure 5 ). Nitrate transport is a\nnecessary step preceding denitrification that can be facilitated by the Nrt. The Nrt\nfunction must not be limited to transport into the cell, it can also be integrated\ninto NO 3 - storage vacuoles and NO 3 - \ntransport into the mitochondria. In certain foraminifera species (family\nBolivinidae), mitochondria were observed to cluster near the tests’ pores in\noxygen-depleted environments[ 35 , 36 ]. Consequently, denitrification can be\nperformed inside the mitochondrion, as previously reported for fungi[ 37 ] and suggested for the foraminifera\n Bolivina spissa [ 38 ]. We\nhypothesise that enzymatic components of foraminifera localise similarly to their\nfungal and bacterial homologs. Therefore, NirK can be localised in the mitochondrial\nintermembrane space and Nor in the mitochondrial inner membrane[ 37 , 39 ].\nOur study is lacking evidence for foraminiferal genes performing the first and last\ndenitrification reactions. The Globobulimina sulfite oxidases\nhomologous to Nr ( Figure\nS3 ) could, hypothetically, catalyse the nitrate reduction step. However, it\nis also possible that so far uncharacterised or unrecognised proteins are catalysing\nthe missing denitrification reactions. Considering a tight association of\ndenitrifying enzymes with the respiratory chain, the putative dissimilatory nitrate\nreductase (dNr) and nitrous oxide reductase (Nos) enzymes are likely localised\ninside the mitochondrion. The proposed foraminiferal model suggests sharing of a common electron\ntransport chain between aerobic respiration and denitrification, permitting the use\nof both electron acceptors in parallel without the need of assembling new protein\ncomplexes as reported in the fungus Fusarium oxysporum [ 37 ]. We speculate that this ability lends\nforaminifera a substantial ecological advantage when exposed to hypoxia or in\nresponse to fluctuating oxygen levels, and explains their success in populating a\nwide range of marine habitats."
} | 2,277 |
36398563 | PMC10107152 | pmc | 8,758 | {
"abstract": "Abstract Engineering enzymes with novel reactivity and applying them in metabolic pathways to produce valuable products are quite challenging due to the intrinsic complexity of metabolic networks and the need for high in vivo catalytic efficiency. Triacetic acid lactone (TAL), naturally generated by 2‐pyrone synthase (2PS), is a platform molecule that can be produced via microbial fermentation and further converted into value‐added products. However, these conversions require extra synthetic steps under harsh conditions. We herein report a biocatalytic system for direct generation of TAL derivatives under mild conditions with controlled chemoselectivity by rationally engineering the 2PS active site and then rewiring the biocatalytic pathway in the metabolic network of E. coli to produce high‐value products, such as kavalactone precursors, with yields up to 17 mg/L culture. Computer modeling indicates sterics and hydrogen‐bond interactions play key roles in tuning the selectivity, efficiency and yield.",
"conclusion": "Conclusion In summary, we have successfully engineered 2PS enzyme to produce various C6‐functionalized TAL‐derivatized pyrone products under mild aqueous conditions with high chemoselectivity and reaction yield. We accomplished the goal by removing steric hindrance of Ile201, Leu202, Leu261 and Ile343 to smaller residues through I201V, L202T, L261G and I343S mutations, and by introducing potential hydrogen bonding interactions with I343S mutation. Computational modeling of the engineered mutants has revealed that properly enlarged active sites resulted in high yield for various substrates compared to WT‐2PS and good chemoselectivity for triketide generation. It also showed the importance of Glu197 in controlling the chemoselectivity of p ‐coumaroyl‐CoA. These gained insights make it possible to engineer one 2PS scaffold toward many other tetraketides production. Furthermore, we have developed a whole‐cell transformation process utilizing engineered enzyme with novel reactivity from modified substrate scopes to convert carboxylic acids directly into the final pyrone products in a complex metabolic network, resulting in comparatively high yields empowered by protein engineering and synthetic biology. Such system holds significant promise to generate more complex natural polyketide products from only one enzyme scaffold in metabolic engineering.",
"introduction": "Introduction Metabolic engineering utilizes the endogenous metabolic network of living organisms to offer a cost‐effective generation of valuable products. Traditional metabolic engineering approaches rely on natural enzymes’ reactivities to optimize naturally evolved pathways or redirect flux towards non‐natural products.[ \n 1 \n , \n 2 \n ] In addition, the space of possible metabolic pathways can be further expanded through high‐level metabolic engineering by incorporating novel reactions from enzymes of modified substrate scopes and even novel chemistry from de novo designed enzymes. However, implementation of novel synthetic pathways into metabolic networks is challenging since they often interfere with endogenous metabolism, leading to side reactions and dead‐end metabolites. \n [3] \n On the other hand, while engineered enzymes sometimes may have high catalytic efficiency outside the cells, they may not be compatible with other enzymes in vivo and thus become a bottleneck of biosynthetic pathways. Because of these limitations, higher‐level metabolic engineering is difficult when the active sites of the proteins are redesigned to exhibit novel or enhanced reactivities and then such engineered proteins are incorporated into metabolic networks to produce valuable products.[ \n 4 \n , \n 5 \n , \n 6 \n , \n 7 \n , \n 8 \n , \n 9 \n ] A primary example of such valuable products generated via metabolic engineering is the polyketide family. \n [10] \n Polyketides are a large class of biomolecules from bacteria, fungi and plants that have shown many clinically important biological properties, such as anticancer, antimicrobial, antioxidant and anti‐inflammatory activities.[ \n 11 \n , \n 12 \n , \n 13 \n ] Polyketides can be readily produced in vivo by incorporating key enzymes such as coenzyme A ligases and polyketide synthases (PKSs) in metabolic pathways without direct acyl coenzyme A supplement. Type I and type II PKSs are megaenzyme systems with multiple catalytic domains, which have been engineered to produce novel products by domain exchange and refinement in metabolic engineering,[ \n 14 \n , \n 15 \n , \n 16 \n , \n 17 \n ] and the type III PKSs are the simplest ones as homodimeric proteins of ∼40 kDa, with many type III PKSs sharing >50 % identity in both gene and protein sequences.[ \n 18 \n , \n 19 \n ] Although type III PKSs have been widely applied in whole cell transformation, triketide products, namely styrylpyrones (Figure 1A ), are usually generated as derailed side products,[ \n 19 \n , \n 20 \n ] which hampers the chemoselective production of target polyketides and complicates the separation processes. Moreover, PKSs suffer from inherently slow kinetics.[ \n 21 \n , \n 22 \n ] Therefore, to unlock the potential of PKSs, it is important to understand the structural characteristics of different type III PKSs and achieve increased chemoselectivity and catalytic efficiency for the target polyketides generation.\n Figure 1 TAL conversion pathway to value‐added products and 2PS‐catalyzed TAL generation. (A) TAL C6‐functionalized natural products. (B) Crystal structure of homodimer 2PS bound with intermediate acetoacetyl‐CoA. (C) TAL conversion pathway. Molecules which are used as food preservatives, additives, and fragrances are shown in blue, and molecules which are used as bifunctional intermediates and building blocks are shown in red. (D) 2PS‐catalyzed TAL generation pathway and mechanism. One of the simplest triketide products is triacetic acid lactone (6‐methyl‐4‐hydroxy‐2‐pyrone, TAL) that is generated by 2‐pyrone synthase from Gerbera hybrida (Figure 1B ). TAL can be produced via microbial fermentation of plant cell wall hydrolysates and further converted to high value‐added products, such as additives, fragrances, and pharmaceuticals, which fulfills the long‐term demand for carbon‐based products.[ \n 23 \n , \n 24 \n ] To obtain such products, however, TAL has to be derivatized through extra synthetic steps that include protection and deprotection under high pressure and long reaction time (Figure 1C ).[ \n 24 \n , \n 25 \n ] In contrast, C6‐functionalized TAL‐derived pyrone products widely exist in nature such as the styrylpyrone moiety (Figure 1A ). For instance, Katsumadain A, B and C, which were isolated from Alpinia katsumadain , are Chinese herbal drugs used as an anti‐emetic and stomachic agent, and kavalactones from Kava ( Piper methysticum ), which is an ethnomedicinal shrub with well‐established anxiolytic and analgesic properties.[ \n 11 \n , \n 26 \n ] Although styrylpyrones have shown prominent pharmaceutical properties, such products are mainly obtained by extraction from plants. \n [27] \n Recently, Weng and co‐workers revealed the first biosynthetic pathway of psychoactive kavalactones in kava and discovered styrylpyrone synthases (SPSs) involved in the process that exclusively produce triketide styrylpyrones as main products. Taking advantage of the discovery, about 2 mg/L pyrone product was produced in E. coli and yeast from p ‐coumaric acid, \n [11] \n but the potentials of heterologous production for styrylpyrones in high yields have not been fully explored, especially with engineered PKSs. Therefore, to overcome the limitations mentioned above and to advance both protein and metabolic engineering for high‐value products generation from renewable sources,[ \n 28 \n , \n 29 \n , \n 30 \n , \n 31 \n , \n 32 \n , \n 33 \n ] we herein report engineering of 2PS as a highly selective and efficient enzymatic system for the production of C6‐functionalized TAL‐derived pyrone products from acyl‐CoAs, as well as the construction of an E. coli whole‐cell transformation system that allows the use of the engineered enzymes to directly generate pyrones in vivo by feeding the corresponding carboxylic acids. We also apply computational modeling to gain insights into how engineered 2PS works to achieve high yields and chemoselectivity for different pyrone products.",
"discussion": "Results and Discussion Computational Design through Structural Homology Modeling 2PS catalyzes the formation of TAL from an acetyl‐CoA and two molecules of malonyl‐CoAs through the mechanism shown in Figure 1D . The nucleophilic attack from the thiolate of Cys169 to acetyl‐CoA triggers the decarboxylation of malonyl‐CoA to generate an enolate species, which is followed by Claisen condensation to produce a diketide intermediate, acetoacetyl‐CoA. One more round of such a process using the acetoacetyl‐CoA intermediate as the substrate would produce a triketide intermediate, which cyclizes to produce TAL as the final product. We commenced our study by searching for key structural features of several PKSs that produce aromatic polyketides and engineering 2PS by homology modeling. Besides SPS, chalcone synthase (CHS) \n [34] \n and stilbene synthase (STS) \n [35] \n accept aromatic primer CoAs and generate tetraketides. For all four type III PKSs, two phenylalanine residues (Phe220 and Phe270 in 2PS) act as gatekeepers that distinguish substrate specificity, and they share an identical catalytic triad Cys‐His‐Asn (Figure 2A ). \n [13] \n To generate C6‐functionalized TAL‐derivatized aromatic pyrones including styrylpyrones, a larger active site capable of accommodating aromatic primer CoAs rather than acetyl‐CoA is necessary. However, too large of a pocket would lead to potential side reactions, such as the generation of tetraketide intermediate, which would further lead to various side products. \n [35] \n Therefore, proper pocket size is important to achieve high reactivity while keeping good selectivity. To engineer 2PS to produce aromatic styrylpyrones, we aligned its structure with those of other type III PKSs that can generate similar products, aiming to identify the key residues in the active sites responsible for the functions. As shown in Table 1 and Supplementary Figure S1, SPS, CHS, and STS share more than 65 % sequence identity with 2PS. Moreover, all four enzymes share high‐level structural similarities with the pairwise C α root‐mean‐square deviation (RMSD) relative to 2PS below 1.0 Å. Although several key residues that can differentiate substrate and influence the chemoselectivity have been identified among type III PKSs,[ \n 18 \n , \n 19 \n , \n 34 \n ] residues exclusively responsible for styrylpyrone production in SPS have not been reported yet from the recently discovered SPS.\n Figure 2 2PS active site and homology modeling. (A) 2PS key active site residues (PDB: 1EE0). (B) Overlay of naringenin (CHS product, in magenta) and resveratrol (STS product, in green). (C) Structure of resveratrol and naringenin. (D) Key residues comparisons for homology modeling. Table 1 Similarity comparison among PKSs. \n Protein \n \n Original Product \n \n Protein Sequence Identity \n \n Cα RMSD [Å] \n \n 2PS (PDB: 1EE0) \n \n TAL \n \n 100 % \n \n 0 \n \n SPS (PDB: 6OP5) \n \n Styrylpyrone \n \n 68 % \n \n 0.932 \n \n CHS (PDB: 1CGK) \n \n Naringenin \n \n 70 % \n \n 0.637 \n \n STS (PDB: 1U0W) \n \n Resveratrol \n \n 66 % \n \n 0.658 \n Wiley‐VCH GmbH Despite the high similarities in protein sequence and structure among the four type‐III PKSs, differences in the active site residues can be identified by carefully comparing those structures. For example, by overlaying the structure of 2PS with those of product‐bound STS and CHS (Figure 2B ), we found that two bulky and hydrophobic residues, Leu261 and Ile343 in 2PS, may have strong steric clashes with resveratrol (product of STS) and naringenin (product of CHS) (Figure 2B and 2C ). At these two locations, two smaller and conserved residues (Gly261, Ser343) are present instead in STS, CHS and SPS, which allow the aromatic primer CoAs to bind (Figure 2D ). Therefore, we redesigned 2PS by introducing L261G/A/V and I343S mutations to make the two locations smaller, like those in STS, CHS and SPS. In addition, since Leu202 next to the above two residues is known to control the substrate specificity,[ \n 19 \n , \n 36 \n ] we replaced it with a smaller conserved threonine in CHS and STS through L202T mutation. To further reduce the steric conflict, we replaced Ile201 that is next to Leu202, with a smaller valine conserved in STS, CHS and SPS. Based on these designs, we have made a variant called 3AP (3‐triketide, Aromatic Primers) that contains I201V, L202T and I343S mutations. In addition, we have made further variants to study the steric influences where L261 is mutated to either alanine, glycine or valine and they are called 3AP‐L261A‐2PS, 3AP‐L261G‐2PS and 3AP‐L261V‐2PS, respectively. Condition Optimization and Kinetics All the designed variants were constructed, expressed and purified in E. coli (Supplementary Figure S2). We first tested the 3AP‐L261A‐2PS reactivity and compared it with wild type 2PS (WT‐2PS) and WT‐SPS under the conditions shown in Table 2 . Interestingly, 3AP‐L261A‐2PS generated styrylpyrone ( 1 ) in 60 % yield (Table 2 , entry 1) while WT‐2PS produced only 7 % yield (Table 2 , entry 2) and WT‐SPS displayed 23 % yield. (Table 2 , entry 3). This product ( 1 ) is an important precursor that is naturally generated by SPS and can be further converted to other natural products such as 5,6‐dehydrokavain by 4‐OH methylation chemically or enzymatically.[ \n 11 \n , \n 37 \n ] In the absence of any enzyme, no product was detected (Table 2 , entry 4), indicating that the enzymes are responsible for the observed product. We further screened the reaction condition to improve the yield of the transformation. When the malonyl CoA concentration was changed and the ratio between the starter CoA and extender CoA was screened (Table 2 , entry 5–6), a ratio of 1 : 3 still achieved the highest yield, while increasing malonyl CoA concentration did not improve the yield further. The yield increased from 60 % at pH=7 (Table 2 , entry 1) to 67 % at pH=8 (Table 2 , entry 8), whereas the yield was only 25 % at pH=6 (Table 2 , entry 7), indicating that the lower pH of the reaction buffer has a major impact on the reactivity of 3AP‐L261A‐2PS. The effect of reaction temperature was then tested (Table 2 , entry 9–10). The yield decreased from 60 % (Table 2 , entry 1) at 37 °C to 19 % at room temperature (Table 2 , entry 9) but didn't increase when the temperature was increased to 55 °C (58 %, Table 2 , entry 10).\n Table 2 Condition optimization of in vitro activity assay. \n \n \n \n \n Entry \n \n Catalyst \n \n pH \n \n Temp [°C] \n \n Malonyl‐CoA [μM] \n \n Yield [%] [a] \n \n \n 1 \n \n 3AP‐L261A‐2PS \n \n 7 \n \n 37 \n \n 150 \n \n 60±2.8 \n \n 2 \n \n WT‐2PS \n \n 7 \n \n 37 \n \n 150 \n \n 7±0.4 \n \n 3 \n \n WT‐SPS \n \n 7 \n \n 37 \n \n 150 \n \n 23±0.6 \n \n 4 \n \n No enzyme \n \n 7 \n \n 37 \n \n 150 \n \n n.d. [b] \n \n \n 5 \n \n 3AP‐L261A‐2PS \n \n 7 \n \n 37 \n \n 100 \n \n 43±2.2 \n \n 6 \n \n 3AP‐L261A‐2PS \n \n 7 \n \n 37 \n \n 200 \n \n 59±0.4 \n \n 7 \n \n 3AP‐L261A‐2PS \n \n 6 \n \n 37 \n \n 150 \n \n 25±1.3 \n \n 8 \n \n 3AP‐L261A‐2PS \n \n 8 \n \n 37 \n \n 150 \n \n 67±0.9 \n \n 9 \n \n 3AP‐L261A‐2PS \n \n 8 \n \n 25 \n \n 150 \n \n 19±0.3 \n \n 10 \n \n 3AP‐L261A‐2PS \n \n 8 \n \n 55 \n \n 150 \n \n 58±1.7 \n \n 11 \n \n 3AP‐L261G‐2PS \n \n 8 \n \n 37 \n \n 150 \n \n 90±1.9 \n \n 12 \n \n 3AP‐L261V‐2PS \n \n 8 \n \n 37 \n \n 150 \n \n 64±1.0 \n \n 13 \n \n 3AP‐Leu261‐2PS \n \n 8 \n \n 37 \n \n 150 \n \n 18±0.8 \n \n 14 \n \n 3AP‐L261G‐2PS \n \n 7 \n \n 37 \n \n 150 \n \n 86±1.7 \n \n 15 \n \n 1 mg 3AP‐L261G‐2PS cell lysate \n \n 7 \n \n 37 \n \n 150 \n \n 85±0.6 \n [a] Yield was determined by HPLC compared to a product standard curve (Supplementary Figure S3); three parallel experiments were conducted for each entry. [b] not detected. Wiley‐VCH GmbH After the initial reaction condition optimization, we investigated the reactivity of different 3AP mutants. When the steric hindrance at residue 261 was further decreased from 3AP‐L261A‐2PS to 3AP‐L261G‐2PS, a further increase in the yield to 90 % (Table 2 , entry 11) was observed. We further tested the reactivity of 3AP‐L261V‐2PS, and the increased sterics decreased the yield to 64 % (Table 2 , entry 12). When we kept the Leu261 in the 3AP mutant (I201V/L202T/I343S), the yield dropped drastically to only 18 % (Table 2 , entry 13). Therefore, increasing steric hindrance from Gly, Ala, Val to Leu261 in 3AP‐2PS dropped the overall yield correspondingly. We further tested the performance of 3AP‐L261G‐2PS at neutral pH because a high performance at neutral environment is important for further whole‐cell transformation using microorganisms like E. coli due to its optimal growth environment. \n [38] \n The yield remained at a similar level (86 %) at pH 7 (Table 2 , entry 14). As a result of such homology modeling, we have generated a 3AP‐L261G‐2PS mutant which displayed high reactivity toward product ( 1 ) generation with 13‐fold higher than WT‐2PS, 4‐fold higher than WT‐SPS under the optimal reaction conditions. With the successful design of a highly active mutant, we wonder whether such transformation can be directly achieved in a whole‐cell bioconversion system. To find out, we expressed the 3AP‐L261G‐2PS in BL21(DE3) E. coli , lysed the grown cell pellet, carried out the enzymatic transformation, and quantified the yield using the protocol described in Supporting Information. Product ( 1 ) was produced in 85 % yield under optimal conditions (Table 2 , entry 15). This whole‐cell transformation provides a convenient and straightforward method to generate styrylpyrone products from acyl‐CoA molecules. To characterize such a promising system, we further monitored the kinetics of different mutants toward product ( 1 ) production and calculated the Michaelis–Menten parameters using different concentrations of cinnamoyl‐CoA. While it took more than 2 hours for both WT‐2PS, WT‐SPS and 3AP‐L261A‐2PS to reach the maximum conversion, 3AP‐L261G‐2PS could achieve the reaction in only 15 minutes with a higher yield of 79 % vs. 1.3 % for WT‐2PS, 5 % for WT‐SPS and 15 % for 3AP‐L261A‐2PS (Supplementary Figure S4A‐S4B). We further compared the k \n cat and K \n M between 3AP‐L261G‐2PS and WT enzymes toward cinnamoyl CoA (Supplementary Figure S4C‐S4F). The k \n cat of 3AP‐L261G‐2PS was over 310‐fold greater than that of WT‐2PS (Table 3 ) and the overall catalytic efficiency ( k \n cat / K \n M ) was enhanced by around 70 folds. In comparison with WT‐SPS which is our mimicking target enzyme to produce styrylpyrone ( 1 ) in nature, The k \n cat of 3AP‐L261G‐2PS was over 58‐fold greater than that of WT‐SPS (Table 3 ) and the overall catalytic efficiency ( k \n cat / K \n M ) was improved by 30 folds. More importantly, the catalytic efficiency of the engineered 3AP‐L261G‐2PS is even higher than those of many native type III polyketide synthases’ reactivity (3600 s −1 M −1 for WT CHS and 4200 s −1 M −1 for WT STS) that have been frequently applied in metabolic engineering researches.[ \n 35 \n , \n 39 \n , \n 40 \n ] This provided a highly promising platform for higher level metabolic engineering where 2PS mutant could potentially produce styrylpyrone products naturally generated by SPS. Further kinetic experiments have been conducted on other 3AP mutants (Table 3 ) and the increased sterics at 261 position not only decreased the transformation yield, but also lowered the turnover number and catalytic efficiency (Supplementary Figure S5).\n Table 3 Michaelis–Menten parameters comparison using different concentrations of cinnamoyl‐CoA. \n Protein \n \n \n K \n M [μM] \n \n \n k \n cat [min −1 ] \n \n \n k \n cat / K \n M [s −1 M −1 ] \n \n WT‐2PS \n \n 1.6±0.5 \n \n (3.2±0.3)×10 −2 \n \n \n (3.4±0.6)×10 2 \n \n \n WT‐SPS \n \n 3.7±0.5 \n \n (17.2±0.6)×10 −2 \n \n \n (7.7±0.3)×10 2 \n \n \n 3AP‐L261G‐2PS \n \n 7.1±1.1 \n \n 9.9±0.6 \n \n (2.3±0.4)×10 4 \n \n \n 3AP‐L261A‐2PS \n \n 4.2±0.8 \n \n 1.87±0.14 \n \n (7.4±1.5)×10 3 \n \n \n 3AP‐L261V‐2PS \n \n 2.1±0.5 \n \n (1.35±0.1)×10 −1 \n \n \n (1.07±0.27)×10 3 \n \n \n 3AP‐L261‐2PS \n \n 1.4±0.3 \n \n (4.6±0.2)×10 −2 \n \n \n (5.68±1.16)×10 2 \n \n Wiley‐VCH GmbH Substrate Scope After designing the highly active variant and identifying optimal conditions, we examined the reaction scope to see if other larger acyl‐CoAs could fit into the pocket to produce TAL‐derived pyrone products (Table 4 ). When the sterics increased from malonyl‐CoA to methylmalonyl‐CoA, WT‐2PS failed to generate the corresponding methylated styrylpyrone ( 2 ), while WT‐SPS generated only 4 % yield. In contrast, 3AP‐L261G‐2PS still produced the methylated product with ∼58 % yield, suggesting that the designed variant can accommodate a bulkier substrate. In addition, the reactivity towards p ‐coumaroyl‐CoA as the substrate was compared among these enzymes. WT‐SPS generated the yangonin precursor product, bisnoryangonin 3 in 16 % yield, while the yield for 3AP‐L261G‐2PS and WT‐2PS was 7 % and 5 %, respectively. Notably, an HPLC peak close to triketide was observed in the 3AP‐L261G‐2PS catalyzed p ‐coumaroyl‐CoA system, but not in WT‐2PS or WT‐SPS system (Supplementary Figure S6A). We found this peak to be a tetraketide side product using LC–MS/MS (HPLC integration 1 : 3=tetraketide:target triketide product) (Supplementary Figure S6B‐D). Furthermore, we tested the reactivity of 3AP‐L261G‐2PS using CoAs containing middle length carbon chains such as C 6 hexanoyl‐CoA, C 8 octanoyl‐CoA and C 12 lauroyl‐CoA. Such CoAs could be enzymatically synthesized from middle chain fatty acids, and the acids could be extracted from biomass directly. \n [41] \n The generated 6‐alkylpyrone products are important natural product precursors such as pseudopyronines (Figure 1A ) \n [42] \n and can be used as a potent and selective G‐protein‐coupled receptor agonists. \n [43] \n Interestingly, all three enzymes can convert hexanoyl‐CoA to product 4 in >94 % yield. For octanoyl‐CoA, 3AP‐L261G‐2PS achieved the highest yield in 88 % with good chemoselectivity (<5% relative HPLC area for tetraketide side products, Supplementary Figure S7), while WT‐2PS and WT‐SPS showed yields of 80 % and 54 %, respectively. However, when the chain length was extended to lauroyl‐CoA, no product was detected for all three enzymes, suggesting that lauroyl‐CoA may be too long to fit into the binding pocket correctly. It is noteworthy that such 6‐alkylpyrone products would be even more challenging to be converted from TAL by post‐extraction synthetic derivatizations to elongate the C6 substituent chain. Finally, we compared the reactivity towards TAL production. WT‐2PS gave full conversion (100 % yield) in 30 minutes, while WT‐SPS achieved 16 % yield and 3AP‐L261G‐2PS generated 70 % yield with the fastest initial rate of 3.3 μM/min in the first 3 minutes (Supplementary Figure S8).\n Table 4 Substrate scope using various CoAs by in vitro enzymatic assays. \n \n \n \n \n Protein \n \n ( 2 ) Yield [%] [a] \n \n \n ( 3 ) Yield [%] [b] \n \n \n ( 4 ) Yield [%] [b] \n \n \n ( 5 ) Yield [%] [a] \n \n \n TAL Yield [%] [b] \n \n \n WT‐2PS \n \n n.d. [c] \n \n \n 5±0.5 \n \n 100±1.2 \n \n 80±1.1 \n \n 100±1.2 \n \n WT‐SPS \n \n 4±0.4 \n \n 16±2.7 \n \n 94±1.6 \n \n 54±3.0 \n \n 16±2.7 \n \n 3AP‐L261G‐2PS \n \n 58±1.2 \n \n 7±0.4 \n \n 100±0.7 \n \n 88±1.1 \n \n 70±1.5 \n [a] Yield was estimated using styrylpyrone ( 1 ) and alkylpyone ( 4 ) as the calibration‐curve standard for ( 2 ) and ( 5 ) correspondingly. [b] Yield was determined by HPLC compared to product standard curve (Supplementary Figure S3, S9–S10). Three parallel experiments were conducted for each reaction. [c] not detected by LC–MS. Wiley‐VCH GmbH Heterologous Production of Styrylpyrones in E. coli by Metabolic Engineering To establish more practical application platforms to produce these pyrone products from more available and cheaper carboxylic acids than CoAs, we explore supplementing the E. coli metabolic network with coenzyme A synthesis pathway to convert carboxylic acids into their corresponding CoAs in situ, which can then be further coupled with the engineered 2PS to produce novel polyketides. We successfully generated an E. coli Rosetta2(DE3) strain that contained the engineered 3AP‐L261G‐2PS gene in pET‐16b vector, as well as malonyl CoA synthetase gene (MCS) \n [44] \n and 4‐coumarate ligase (4CL) gene \n [45] \n in a pRSFDuet‐1 vector (Figure 3A ). MCS can convert malonic acid, ATP and coenzyme A to malonyl‐CoA, while 4CL catalyzes the formation of aromatic‐CoAs from cinnamic acid and phenylpropanoic acid with different substitutions, which would expand the substrate scope of the engineered 2PS.[ \n 46 \n , \n 47 \n ] Malonic acid is listed as one of the top 30 chemicals that are produced from biomass and aromatic acids can be generated from biomass‐derived carbon sources.[ \n 23 \n , \n 48 \n ] Such whole‐cell transformation not only offers a much more simplified and straightforward synthetic pathway of pyrone production from carboxylic acids, but also can be particularly useful to upgrade biomass‐derived products to enhance bioeconomy.\n Figure 3 \n E. coli strain construction for whole‐cell transformation. (A) plasmid information. (B) whole‐cell transformation titer. Products are quantified in product standard curve (Supplementary Figure S3, S9 and S11–S12). We first began with control experiments to test the engineered 2PS reactivities in vivo and the effect of intracellular thiolases in 2 mL small culture (Supporting Information Table S1), since it has been shown that some polyketoacyl‐CoA tholases in E. coli can produce polyketides including styrylpyrones without PKSs.[ \n 21 \n , \n 49 \n ] In this system, however, E. coli containing only the 4CL and MCS genes failed to generate styrylpyrone products that can be detected by HPLC from cinnamic acid or p ‐coumaric acid. When WT‐2PS was expressed together with MCS and 4CL, it produced trace amount (41.1 μg/L) of product ( 1 ) but no detectable product ( 3 ). Only when 3AP‐L261G‐2PS gene was expressed together with 4CL and MCS, it showed significant amount of product generation with 3.2 mg/L titer for product ( 1 ) and 17.3 mg/L titer for product ( 3 ). Although the yield for in vitro enzymatic experiments of 3AP‐L261G‐2PS showed higher reactivity for cinnamoyl‐CoA than p ‐coumaroyl‐CoA, the whole‐cell transformation titer did not follow this trend. This might result from the 4CL innate substrate preference and higher reactivity toward its native substrate p ‐coumarate over cinnamate. \n [50] \n \n We further tested 1‐liter large‐scale culture whole‐cell biotransformation (Figure 3B ) by feeding E. coli with 1 mM p ‐coumaric acid and 3 mM malonic acid. Product ( 3 ) was observed from HPLC with the calculated titer of 8.9 mg/L. As E. coli is known to produce a small amount of cellular malonyl‐CoA, we hypothesized that 3AP‐L261G‐2PS could use such intracellular malonyl‐CoA directly in E. coli without feeding it with malonic acid. \n [51] \n The HPLC titer remained at 8.9 mg/L (4.1 mg/L isolated yield) with feeding only 1 mM p ‐coumaric acid without malonic acid. Such phenomenon has been observed where MCS has been applied to increase intracellular malonyl‐CoA supply for TAL production. Although this strategy did increase the malonyl‐CoA supply, TAL production titer didn't improve, probably due to higher ATP requirements for MCS reactivity. \n [21] \n Previously native SPS with kava 4CL was applied to heterologously produce bisnoryangonin in E. coli using 1 mM p ‐coumaric acid and in S. cerevisiae using 2 mM p ‐coumaric acid, resulting in HPLC yields of 0.9–3.7 mg/L and 2–2.2 mg/L, respectively. \n [11] \n In comparison with these reported microorganism systems, our system showed at least 4‐fold increase in product yield in E. coli . When cinnamic acid was supplied, the product ( 1 ) was observed by HPLC, with calculated titer of 3.6 mg/L (2.0 mg/L isolated yield). In addition, phenylpropanoic acid and 3‐(4‐hydroxyphenyl)propanoic acid were tested to produce dihydro‐kavalactone precursors. The titer calculated by HPLC turned out to be 4.0 mg/L (2.0 mg/L isolated yield) and 17 mg/L (8.9 mg/L isolated yield), respectively. Interestingly, only <5 % (by HPLC area integration) tetraketide products could be detected from 3‐(4‐hydroxyphenyl)propanoic acid system by LC–MS/MS (Supplementary Figure S13), which indicates that both the rigidity and hydroxyl group are important for tetraketide production from p ‐coumaroyl‐CoA and p ‐coumaric acid. Besides aromatic acids, the middle‐chain fatty acid such as hexanoic acid was also tested in our system. Recently, PKS systems have been studied using hexanoyl‐CoA and malonyl‐CoA because the hexanoyl moiety may be produced by fatty acid synthase (FAS), which might provide novel metabolic pathways to generate novel products by combining FAS and PKS reactivities.[ \n 52 \n , \n 53 \n ] Our engineered E. coli system produced the triketide lactone product ( 4 ) from hexanoyl‐CoA and malonyl‐CoA with 2.0 mg/L titer. We also measured the cell weight used for 1‐liter biotransformation and around 4‐gram pellet in wet weight (1.25 gram in dry weight) was harvested and utilized after overnight cell growth. For the whole‐cell biocatalysis, the amount for each of the products per gram wet cell weight is shown in Figure 3B . Different whole‐cell transformation conditions were surveyed to enhance both substrate utilization and product titer (Supporting Information Table S2). Although the titer in 2 mL small cultures of cinnamate can be further increased to around 10 mg/L by providing 5–10 mM cinnamate and medium pH adjustment to optimal neutral pH, the substrate conversion toward the target product (0.47 %–0.93 %) was lower than when 1 mM cinnamate was used (1.68 %). Structural Explanations After demonstrating the reactivity both in test tubes and in E. coli , we tried to understand the influences of different mutations we have designed into 2PS by control experiments. When one out of the four mutations in 3AP‐L261G‐2PS (I201V/L202T/L261G/I343S) was restored to the original residue in WT‐2PS, the yield all decreased to different extent (Table 5 ). For example, when malonyl‐CoA was used as an extender for cinnamoyl‐CoA conversion, restoring Gly261 back to Leu261 resulted in significant decrease in the reactivity, from 86 % to only 18 % yield. When a larger extender such as methylmalonyl‐CoA was used, not only Leu261 (from 58 % to 6 %) but also Ile343 (from 58 % to 15 %) showed a major decrease in the product ( 2 ) production. These reactivity differences confirmed our initial designs to remove steric hindrance, especially the steric clashes at the Leu261 and Ile343 positions.\n Table 5 Control experiments on different mutants. \n \n \n \n \n Protein \n \n Malonyl‐ CoA [%] [a] \n \n \n Methylmalonyl‐ CoA [%] [b] \n \n \n WT‐2PS \n \n 7±0.4 \n \n n.d. [c] \n \n \n 3AP‐L261G‐2PS \n \n 86±1.7 \n \n 58±1.2 \n \n V201I/L202T/L261G/I343S‐2PS \n \n 66±1.0 \n \n 48±0.9 \n \n I201V/T202L/L261G/I343S‐2PS \n \n 67±1.6 \n \n 54±0.2 \n \n I201V/L202T/G261L/I343S‐2PS \n \n 18±0.8 \n \n 6±0.4 \n \n I201V/L202T/L261G/S343I‐2PS \n \n 73±3.1 \n \n 15±0.9 \n [a] Yield utilizing malonyl‐CoA as extender unit was determined by HPLC compared to product standard curve. Three parallel experiments were conducted for each reaction using biological replicates. [b] Yield utilizing methylmalonyl‐CoA as extender unit was estimated using styrylpyrone ( 1 ) as the calibration‐curve standard. Three parallel experiments were conducted for each reaction using biological replicates. [c] not detected by LC–MS. Wiley‐VCH GmbH Besides steric influences, Ser343 was known to be involved in key hydrogen bonding networks of both CHS and STS to control chemoselectivity of such enzymes. \n [35] \n For example, an inspection of the SPS crystal structure indicates that Ser339, the residue in SPS that corresponds to Ser343 in 2PS, has hydrogen bonding interactions with Glu192 and catalytic residue Cys164, which correspond to Glu197 and Cys169 respectively in 2PS (Supplementary Figure S14). To better understand the function of Ser343 of 3AP‐L261G‐2PS, a mutant of 2PS (I201V/L202T/L261G/I343G) without hydrogen bonding side chain at residue 343 was tested in both activity assay and kinetic studies. Although the sterics at 343 position was further decreased, this mutant could only produce product ( 1 ) in 63 % yield without the overproduction of tetraketide side products under standard activity assays. More importantly, it was shown that the reaction kinetics was slower than 3AP‐L261G‐2PS and the catalytic efficiency (5.1×10 3 s −1 M −1 ) was only 1/4 of 3AP‐L261G‐2PS (Supplementary Figure S15). All these evidence supports the function of Ile343Ser mutation in 3AP‐L261G‐2PS beyond sterics. Given observation of these results shown above, we used protein‐ligand docking and molecular dynamics simulations (Supplementary Figure S16–S17) to gain further insights. Styrylpyrone product ( 1 ) was docked into both the WT‐2PS and the predicted 3AP‐L261G‐2PS structural model (Figure 4A , 4B ). Although the predicted model of 3AP‐L261G‐2PS is similar to the structure of WT‐2PS, with the C α RMSD being only 0.44 Å, the styrylpyrone products such as ( 1 ) and ( 3 ) failed to be docked into the active site of WT‐2PS due to steric restrictions. In contrast, product ( 1 ) successfully entered the enlarged pocket of 3AP‐L261G‐2PS (Figure 4C ). This difference in the size of the pocket may account for the higher reactivity of 3AP‐L261G‐2PS toward styrylpyrone production than WT‐2PS.\n Figure 4 Protein‐ligand docking and molecular dynamics simulations. (A) Overlay between overall structures of WT‐2PS (in blue) and predicted and optimized 3AP‐L261G‐2PS structural model (in brown yellow). (B) Styrylpyrone product ( 1 , in red sphere) was docked into 3AP‐L261G‐2PS structure. (C) Interactions between ( 1 ) and active site residues of 3AP‐L261G‐2PS. (D) Interactions between ( 3 ) and active site residues of 3AP‐L261G‐2PS. (E) Interactions between ( 5 ) and active site residues of 3AP‐L261G‐2PS. (F) Interactions between product from C 12 aliphatic CoA and active site residues of 3AP‐L261G‐2PS. We also performed docking and molecular dynamics simulations with different substrates. Interestingly, the yield and selectivity of p ‐coumaroyl‐CoA were low, even if its structure is similar to that of cinnamoyl‐CoA. From the docking analysis (Figure 4D ), we could clearly see a strong hydrogen bonding interaction between Glu197 and 4‐hydroxyl substituent on the aromatic ring of p ‐coumaroyl‐CoA and such H‐bond persists along the simulation trajectory after it has been formed (Supplementary Figure S16B). Such a strong interaction may bury p ‐coumaroyl‐CoA deeper into the pocket, move it further away from the catalytic triad, and thus result in lower yield and selectivity than when cinnamoyl‐CoA is used. To test this hypothesis, we generated a new mutant called 3AP‐L261G‐E197D‐2PS to weaken the hydrogen bonding interaction between Glu197 and p ‐coumaroyl‐CoA by shortening the side chain of Glu197. Although the in vitro reactivity was not further enhanced, the selectivity was improved to generate bisnoryangonin 3 as the major product (HPLC integration 1 : 12=tetraketide:target triketide product) (Supplementary Figure S18), which showed the critical role of Glu197 for the chemoselectivity of p ‐coumaroyl‐CoA. To explain the reactivity among different lengths of the aliphatic carbon chain CoAs, the products of C 8 octanoyl‐CoA and C 12 lauroyl‐CoA were docked into 3AP‐L261G‐2PS (Figure 4E , F ). Products from octanoyl‐CoA could be docked in the correct orientation as pyrone ring interacts with the catalytic Cys‐His‐Asn triad. In contrast, the C 12 carbon chain of lauroyl‐CoA was too long to be placed into the active site with the correct orientation, which accounts for the inactivity of 3AP‐L261G‐2PS toward lauroyl‐CoA, but high activity toward octanoyl‐CoA."
} | 8,994 |
23148127 | null | s2 | 8,759 | {
"abstract": "This study examined the compression of solvated polymer brushes on bioengineered surfaces during the initial stages of Staphylococcus Aureus (S. aureus) adhesion from gentle flow. A series of PEG [poly(ethylene glycol)] brushes, 7 to 17 nm in height and completely non-adhesive to proteins and bacteria, were modified by the incorporation of sparse isolated ~10 nm cationic polymer \"patches\" at their bases. These nanoscale regions, which lacked PEG tethers, were electrostatically attractive towards negative bacteria or proteins. S. aureus drawn to the interface by multiple adhesive patches compressed the PEG brush in the remaining contact region. The observed onset of bacterial or fibrinogen capture with increases in patch content was compared with calculations. Balancing the attraction energy (proportional to the number of patches engaging a bacterium during capture) against steric forces (calculated using the Alexander-DeGennes treatment) provided perspective on the brush compression. The results were consistent with a bacteria-surface gap on the order of the Debye length in these studies. In this limit of strong brush compression, structural features (height, persistence length) of the brush were unimportant so that osmotic pressure dominated the steric repulsion. Thus, the dominant factor for bacterial repulsion was the mass of PEG in the brush. This result explains empirical reports in the literature that identify the total PEG content of a brush as a criteria for prevention of bioadhesion, independent of tether length and spacing, within a reasonable range for those parameters. Bacterial capture was also compared to that of protein capture. It was found, surprisingly, that the patchy brushes were more protein-than bacteria-resistant. S. aureus adhesion driven by patches within otherwise protein-resistant PEG brushes was explained by the bacteria's greater tendency to compress large areas of brush to interact with many patches. By contrast, proteins are thought to penetrate the brush at a few sites of PEO-free patches. The finding provides a mechanism for the literature reports that in-vitro protein resistance is a poor predictor of in-vitro implant failure related to cell-surface adhesion."
} | 557 |
19005565 | PMC2579483 | pmc | 8,760 | {
"abstract": "Disease epidemics have caused extensive damage to tropical coral reefs and to the reef-building corals themselves, yet nothing is known about the abilities of the coral host to resist disease infection. Understanding the potential for natural disease resistance in corals is critically important, especially in the Caribbean where the two ecologically dominant shallow-water corals, Acropora cervicornis and A. palmata, have suffered an unprecedented mass die-off due to White Band Disease (WBD), and are now listed as threatened under the US Threatened Species Act and as critically endangered under the IUCN Red List criteria. Here we examine the potential for natural resistance to WBD in the staghorn coral Acropora cervicornis by combining microsatellite genotype information with in situ transmission assays and field monitoring of WBD on tagged genotypes. We show that six percent of staghorn coral genotypes (3 out of 49) are resistant to WBD. This natural resistance to WBD in staghorn corals represents the first evidence of host disease resistance in scleractinian corals and demonstrates that staghorn corals have an innate ability to resist WBD infection. These resistant staghorn coral genotypes may explain why pockets of Acropora have been able to survive the WBD epidemic. Understanding disease resistance in these corals may be the critical link to restoring populations of these once dominant corals throughout their range.",
"introduction": "Introduction Disease epidemics have radically altered tropical coral reefs and are becoming more frequent and extensive because of climate change [1] – [3] . This is most apparent in the Caribbean where diseases have caused massive and widespread die-offs of the key herbivorous sea urchin Diadema anitillarum \n [4] , common Gorgonian sea fans [5] , [6] and the two ecologically dominant shallow-water corals–the staghorn coral Acropora cervicornis and the elkhorn coral A. palmata \n [7] , [8] . The Caribbean-wide mass die-offs of both the shallow-water Acropora corals and the keystone urchin D. antillarium , in particular, have been major contributors to the rapid decline of Caribbean coral reefs and the dramatic phase shift from coral to macroalgal dominance [7] , [9] , [10] . Reef-building corals, in general, have been susceptible to the global rise in marine diseases [1] , [11] , [12] . As foundation species on tropical reefs, the impacts of disease on corals can ripple throughout the ecosystem [7] , [11] . The effect of the White Band Disease (WBD) epidemic on the Caribbean Acropora corals demonstrates the ecosystem-level impacts of coral disease on tropical reefs [7] . Since it was first observed in the late 1970s [8] , WBD has caused unprecedented Caribbean-wide declines in its hosts A. cervicornis and A. palmata \n [7] , [13] , [14] , with losses of up to 95% of living acroporid cover common across the greater Caribbean [13] , [15] , [16] . Recovery of these formerly dominant shallow-water corals has been slow [7] , [17] , due in large part to poor larval recruitment [18] – [20] , highly restricted larval dispersal [21] , [22] and a heavy reliance on asexual (i.e. vegetative) propagation [23] – [25] . As a result, both species have recently been listed as threatened on the US Endangered Species Act [26] , [27] and listed as critically endangered under the International Union for the Conservation of Nature (IUCN) Red List criteria [28] . Yet, despite its dramatic impacts, much about the etiology and ecology of WBD remains poorly understood [7] , [11] . WBD draws its name from its appearance as a rapidly advancing white band of diseased tissue [8] , [29] ( Fig. 1A ). WBD appears to be host-specific, infecting only the Caribbean Acropora \n [11] . It has two forms–WBD type I which is ubiquitous throughout the Caribbean and WBD type II which has been described from the Bahamas [29] –and can be transmitted via direct contact and through vectors such as the corallivorous snail Corallophyllia abbreviata \n [30] . The WBD pathogen has not been isolated in pure culture, but histological and genetic data suggest that the pathogen is bacterial [29] , [31] – [33] . Recent genetic surveys indicate that a marine Rickettsia bacterium is associated with WBD type I [31] while the bacterium Vibrio charcharia appears to be associated with WBD type II [29] . Nothing is known about the potential for host resistance to WBD in the Caribbean Acropora species. 10.1371/journal.pone.0003718.g001 Figure 1 Resistance to White Band Disease (WBD) in the staghorn coral Acropora cervicornis . (A) WBD transmission to a coral fragment occurs rapidly as illustrated by the progress of the advancing white band of disease after three days of direct contact (grafting) with an infected coral fragment. (B) In situ transmission experiments identified five staghorn coral genotypes that did not contract WBD. (C) Field surveys of WBD prevalence identified ten genotypes that were not observed in the field with WBD. (D) Integrated field surveys and experimental transmission results show that three staghorn coral genotypes were resistant to WBD infection. Here we assess the potential for natural resistance to WBD in the threatened staghorn coral Acropora cervicornis . To do this, WBD resistance was assayed on 49 staghorn coral genotypes from four populations in Bocas del Toro, Panama using a series of in situ transmission experiments and field monitoring of WBD prevalence. We show that 3 out of the 49 staghorn coral genotypes assayed were naturally resistant to WBD.",
"discussion": "Discussion Data from our in situ transmission experiments and field surveys indicate that roughly six percent of staghorn genotypes (3 out of 49) from Bocas del Toro, Panama are resistant to WBD infection. This natural resistance to WBD in threatened staghorn corals provides the first evidence for host disease resistance in reef-building corals, and may explain why pockets of staghorn corals have survived the Caribbean-wide epidemic of WBD over the past thirty years. Natural resistance to WBD in staghorn corals has important evolutionary and ecological implications for how staghorn coral populations may be responding to the WBD epidemic. In an evolutionary scenario akin to G. C. Williams (1975) strawberry-coral model of genotype selection [34] , we predict that WBD resistant genotypes of staghorn coral will have a selective advantage over non-resistant genotypes, and thus should accumulate locally within populations over time via asexual, vegetative fragmentation. Staghorn corals are prolific vegetative fragmenters [19] , [24] , [35] , and thus asexual propagation of WBD resistant staghorn genotypes within populations (i.e. reefs) should provide an effective means for the local recovery and persistence of staghorn coral populations where WBD resistant genotypes occur. It is possible that differences in the numbers of naturally resistant genotypes between reefs may explain for why some staghorn coral populations have fared better than others over the course of the WBD epidemic. Ultimately, however, the broad-scale recovery of staghorn coral populations across the greater Caribbean will have to be achieved by the successful dispersal and recruitment of staghorn coral larvae; preferably those carry genetic variation for WBD resistance. This may prove to be the limiting step for staghorn coral recovery. Staghorn corals have historically been poor sexual recruiters [10] , [20] , [35] – [37] , relying predominantly on localized asexual fragmentation instead [19] , [23] – [25] , and staghorn coral recruits continue to be rare on most Caribbean reefs [18] , [38] . In addition, we know of no instances where a sexual recruitment pulse has resulted in the recovery of staghorn coral populations since the WBD epidemic. Instead, Caribbean Acropora populations appear to be experiencing recruitment failure [39] , possibly augmented by Allee effects resulting from WBD-induced population reductions [25] , [40] , [41] . Even if successful larval recruitment events were to occur, the scale of over which staghorn larvae could reseed downstream reefs will be limited by their restricted dispersal potential. Genetic data indicate that larval dispersal in A. cervicornis \n [22] and its congener A. palmata \n [21] is geographically restricted across the Caribbean over spatial scales less than 500 kilometers [21] , [22] , [42] , [43] . For A. cervicornis , the genetic data indicate that gene flow can be limited over spatial scales as small as adjacent reefs (i.e. 2–5 kilometers) [22] . The combination of poor sexual recruitment and geographically restricted gene flow in staghorn corals suggests that larval recruitment from healthy staghorn coral populations will not be sufficient to recover downstream reefs in the next few decades. Thus, the conservation and restoration of staghorn coral populations will have to be achieved through the local protection of remnant populations and aggressive strategies aimed at propagating and transplanting WBD resistant genotypes to reefs. Our data show that staghorn corals display a wide range of phenotypic variation in their response to WBD, ranging from highly resistant to highly susceptible coral strains. A number of environmental and genetic factors are likely contributing to this phenotypic variation. Yet, the occurrence of WBD resistant genotypes suggests that this disease resistance has an underlying genetic basis. Given the close evolutionary relationship between A. cervicornis and A. palmata [its congener and hybrid partner [44] ], it is likely that WBD resistance exists in A. palmata as well. Natural genetic variation for host disease resistance has been documented in a variety of animals and plants [45] – [48] , including marine shrimp [49] – [52] and oysters [53] – [57] . While nothing is known about which genes might confer disease resistance in reef corals, genetic surveys for resistance genes (or R-genes) in other taxa [46] , [47] indicate that the genetic basis of disease resistance often occurs on genes involved in pathogen recognition and innate immunity [46] , [47] . Reef corals do possess key components of the invertebrate innate immune pathway, including Toll/Toll-like receptors [58] , which might form the genetic basis for disease resistance in corals. Thus, future research on the genetic basis of disease resistance in corals has the potential to uncover the gene(s) involved in host-pathogen resistance and recognition and allow for surveys of resistant gene variants in disease impacted corals like the Caribbean Acropora . Evidence for natural disease resistance in a reef-building corals supports growing interest in the role that host resistance might play in buffering the impacts of the global rise in marine diseases on tropical coral reefs and elsewhere [1] , [6] , [59] , [60] . In addition to staghorn corals, disease resistance has been identified in a number of other marine taxa, including oysters [53] , shrimp [49] , [52] and abalone [61] . Rapid evolution of host genetic resistance has been evoked to explain sharp reductions in disease infections by the terrestrial fungus Aspergillus on Gorgonian soft corals in the Caribbean [6] . While it remains to be seen how important and pervasive disease resistance in reef corals might be, its existence demonstrates that some corals have the innate ability and adaptive genetic variation to respond to diseases and possibly other stressors, including coral bleaching. With coral bleaching, strong emphasis has been placed on the role of algal symbiont diversity as a means to respond to bleaching, i.e. the adaptive bleaching hypothesis [62] – [66] . Like host resistance to disease, we suggest that there is potential for adaptive genetic variation for bleaching resistance within the genomes of corals as well. Clearly, more research is needed to elucidate the genetic and environmental factors underlying natural disease resistance in reef-building corals. The approach taken here to identify naturally resistant strains of staghorn corals demonstrates that disease resistant corals can be identified using relatively simple means and provide a stepping-off point for further research on the genetic basis of disease resistance, pathogen recognition and innate immunity in reef-building corals. From a conservation standpoint, these simple transmission assays provide a means for identifying disease resistant coral strains, which in combination with coral farming and local replanting of diverse sets of disease resistant coral genotypes, would provide an effective means to restore threatened Acropora populations throughout the Caribbean."
} | 3,199 |
31128791 | null | s2 | 8,763 | {
"abstract": "Heterologous expression of natural product biosynthetic gene clusters (BGCs) is a robust approach not only to decipher biosynthetic logic behind natural product (NP) biosynthesis, but also to discover new chemicals from uncharacterized BGCs. This approach largely relies on techniques used for cloning large BGCs into suitable expression vectors. Recently, several whole-pathway direct cloning approaches, including full-length RecE-mediated recombination in Escherichia coli, Cas9-assisted in vitro assembly, and transformation-associated recombination (TAR) in Saccharomyces cerevisiae, have been developed to accelerate BGC isolation. In this chapter, we summarize a protocol for TAR cloning large NP BGCs, detailing the process of choosing TAR plasmids, designing pathway-specific TAR vectors, generating yeast spheroplasts, performing yeast transformation, and heterologously expressing BGCs in various host strains. We believe that the established platforms can accelerate the process of discovering new NPs, understanding NP biosynthetic logic, and engineering biosynthetic pathways."
} | 272 |
26078851 | PMC4461416 | pmc | 8,764 | {
"abstract": "Climate forecasts project further increases in extremely high-temperature events. These present threats to biodiversity, as they promote population declines and local species extinctions. This implies that ecological communities will need to rely more strongly on recovery processes, such as recolonization from a meta-community context. It is poorly understood how differences in extreme event intensity change the outcome of subsequent community reassembly and if such extremes modify the biotic environment in ways that would prevent the successful re-establishment of lost species. We studied replicated aquatic communities consisting of algae and herbivorous rotifers in a design that involved a control and two different heat wave intensity treatments (29°C and 39°C). Animal species that suffered heat-induced extinction were subsequently re-introduced at the same time and density, in each of the two treatments. The 39°C treatment led to community closure in all replicates, meaning that a previously successful herbivore species could not re-establish itself in the postheat wave community. In contrast, such closure never occurred after a 29°C event. Heat wave intensity determined the number of herbivore extinctions and strongly affected algal relative abundances. Re-introduced herbivore species were thus confronted with significantly different food environments. This ecological legacy generated by heat wave intensity led to differences in the failure or success of herbivore species re-introductions. Reassembly was significantly more variable, and hence less predictable, after an extreme heat wave, and was more canalized after a moderate one. Our results pertain to relatively simple communities, but they suggest that ecological legacies introduced by extremely high-temperature events may change subsequent ecological recovery and even prevent the successful re-establishment of lost species. Knowing the processes promoting and preventing ecological recovery is crucial to the success of species re-introduction programs and to our ability to restore ecosystems damaged by environmental extremes.",
"introduction": "Introduction Under climate change major regions on the planet will experience both gradual warming and an increase in temperature variability. In the past three decades, such variability has already emerged in the form summertime extremely hot outliers (Hansen et al. 2012 ). In the coming decades, hot extremes are expected to occur with increasing intensity, duration and frequency, in many locations, on a considerable fraction of the planet's surface (Karl and Trenberth 2003 ; IPCC 2007 ; Quesada et al. 2012 ; Fischer et al. 2013 ). Variability in terms of environmental extremes poses a greater threat to species and biodiversity than slow and gradual warming itself (Vasseur et al. 2014 ). Accordingly, research has started to not only focus on gradual increases in mean temperatures (see Bale et al. 2002 ; Brown et al. 2004 ) but also on the effects of increasingly severe extreme events (Jentsch et al. 2007 ) such as heat waves (e.g., Sentis et al. 2013 ) and other catastrophic climatic events such as floods (Thibault and Brown 2008 ), droughts (Mueller et al. 2005 ; Bogan and Lytle 2011 ) and storms (Pringle and Hamazaki 1997 ; Batista and Platt 2003 ). Some of the ecological consequences of extreme events are straightforward. Environmental extremes that exceed the tolerance limits of many species in a region enhance mortality and are likely to promote community-wide population declines that may result in local extinctions. This implies that ecological systems may need to increasingly rely on recovery via community reassembly assisted by meta-community dynamics, that is, by dispersal-mediated recolonization. However, how community reassembly will proceed is not easy to predict. Historical effects, such as the order in which species go extinct and are re-introduced, can be essential for the emerging patterns (Fukami et al. 2010 ). Ecological legacies may also be generated in other ways. For example, an extreme in the abiotic environment may not only drive a species locally extinct (by a breach of tolerance limits), but may also change the composition of the remaining community, that is, the biotic conditions, in ways that change the ability of that species to exist within it later on, when the abiotic conditions have returned to normal. Any process that creates such historical effects or ecological legacies may affect the outcome of community assembly or reassembly. To what extent extreme events of different intensities differ in their propensity to create alternative ecological legacies, which affect postevent recovery, is largely unknown. On the one hand, strong ecological forces may have canalizing effects that lead to rather reliable successional patterns and recovery (Pearson and Rosenberg 1978 ; Berlow 1997 ), but on the other hand, noise-enhancing mechanisms, alternative attractors, and historical contingencies may lead to severe unpredictability and a wide range of possible transients and outcomes of the recovery process (Drake 1990 , 1991 ; Berlow 1997 ; Fukami et al. 2010 ; Fukami and Nakajima 2011 ). It is presently not clear whether extreme events of different intensities tend to “reset” ecological succession in a similar way or will tend to create different ecological legacies that change subsequent recovery. It can, however, be expected that higher intensity extremes lead to higher mortality, stronger population declines, and hence to more frequent local extinctions. This “extraction” of ecological players could, in itself, create more room and degrees of freedom for noise enhancement. This in turn would result in higher postextreme community-level variability than would be the case following a lower intensity extreme event that caused fewer extinctions. Here, we focus on the consequences of differences in heat wave intensity for the dynamics, species loss, and reassembly of ectotherm communities. Ectotherms comprise the majority of all animal species on the planet and will be rather directly affected by extreme heat events as their metabolism, feeding rate, and overall activity is largely determined by ambient temperature (Wilson 1992 ; Deutsch et al. 2008 ). Very high temperature negatively affects ectotherms because it causes respiration to outpace resource intake, leading to a net loss of energy and to enhanced mortality (Walz 1993 ; Seifert et al. 2014 , in press 2015 ). An increasing intensity and frequency of heat waves may thus cause ectotherm population declines and lead to a higher rate of local species extinctions. This would lead to associated changes in the balance of competition and predation, which could prevent the re-establishment of some of the lost species, even if they were previously viable and successful as residents. Lundberg et al. ( 2000 ) appropriately coined the term “community closure” for this phenomenon. Community closure could prove to be a serious problem in a world experiencing a regime of increased environmental extremes as it could prevent successful ecosystem recovery through the “usual mechanism” of dispersal-mediated re-colonization, that is, through meta-community dynamics and the associated spatial insurance (Loreau et al. 2003 ). It is presently unknown whether extreme event intensity itself increases the likelihood of community closure. To study the effects of differences in heat wave intensity on subsequent community recovery, we tested the following hypotheses: (H1) Differences in heat wave intensity lead to differences in community dynamics including numbers of extinctions and relative abundances of the remaining species. As a consequence, (H2) differences in heat wave intensity then also lead to differences in the likelihood of community closure during the process of community reassembly. Finally, we hypothesize (H3) that a higher heat wave intensity, that is, more extreme heat, leads to a more variable outcome of the community reassembly process.\n\nDifferences in re-introduction success following species loss at 29 versus 39°C (H2) Brachionus havanaensis successfully re-established itself in all replicates after the 29°C heat wave. Re-introductions of the herbivores B. calyciflorus , B. havanaensis, and Lecane in the post-39°C heat wave community initially all seemed successful, but B. havanaensis , after maintaining a relatively stable level for 6 days, rapidly declined around day 25 and went extinct in all post-39°C heat wave replicates. We call this “delayed community closure”. In other words, B. havanaensis was unable to eventually establish itself in the post-39°C heat wave community, in contrast with the post-29°C heat wave community, where it always re-established itself. This difference in re-introduction success was significant (MWU test, P < 0.0001). This strongly supports Hypothesis 2.",
"discussion": "Discussion Recent climate change has seen an increasing frequency and intensity of extreme temperature events, including a new category of “extremely hot outliers” (Hansen et al. 2012 ). Projections of future climate extremes suggest that this trend will likely continue for a large fraction of the global land surface, although local trend variability is expected to be large, leading to great differences in the direction and nature of extremes among locations (Fischer et al. 2013 ). These extremely hot outliers may become increasingly ecologically important, as they could substantially increase local mortality among a range of species. However, such extremes in abiotic conditions could also have effects beyond inflicting direct mortality. They may fundamentally upset and change the biotic conditions that are essentially required for species persistence in the environment. This way an extreme event could leave an ecological legacy through changed conditions for life, even after the event itself is over. Preliminary mathematical food web model analyses confirm what is common sense and suggest that these conditions for life include not only sufficient resource availability but also survivable balances of competition and predation, next to abiotic conditions within species tolerance limits (M. Vos et al., unpubl. model analyses not shown). The experimental work we discuss here zooms in on such “hot outliers”. We consider both moderate and more intense extreme event intensity. In particular, we discuss the recovery of herbivorous freshwater plankton communities to which lost species were re-introduced following experimental heat waves of 29°C and 39°C. Responses to differences in extreme event intensity Our experimental heat waves did act as extreme events that led to animal species extinctions. The absence of extinctions in the control from days 8 to 14 shows that the extinctions occurring during that period in the two treatments were really caused by the heat wave. Under the 29°C heat wave, one of the established herbivore species, B. havanaensis , was lost. It went extinct in all replicates. Conversely, in the 39°C treatment, three of the four stably established herbivore species went extinct, also in all replicates. These extinctions included not only B. havanaensis , but also B. calyciflorus and Lecane . Cephalodella was a highly robust species as it survived both heat wave intensities. Nonetheless, it was strongly inhibited by the 39°C heat wave. The relatively short duration of the heat wave may have saved this species, preventing a full decline to actual extinction. Cephalodella recovered as soon as the heat stress was lifted. Its larger tolerance thus acted to “buy time”. Following the extreme event, the relative abundances of the remaining animal and algal species differed between heat wave intensities (Fig. 1 ). At the primary producer level, the 29°C treatment stimulated growth of Monoraphidium , at the expense of all other phytoplankton. This species became dominant. In contrast, no phytoplankton species developed such a degree of dominance in the 39°C treatment. Our results strongly support hypothesis (1) that differences in heat wave intensity lead to differences in community dynamics including numbers of extinctions and relative abundances of the remaining species. Trajectories of community reassembly Species re-introductions of the lost herbivore species ensured that all viable species that had been present before the heat wave had a chance to establish themselves in the postheat wave community. However, the different heat wave intensities had also led to significant shifts in relative algal abundances between the two heat wave treatments (Fig. 1E and F ). These differences in resource availability in the postheat wave community can be viewed as an ecological legacy. The observed differences in relative abundance among the different phytoplankton species may have acted in concert with species-specific differences in edibility of these algae for the different herbivore species to result in different food environments for re-introduced herbivores in the postheat wave communities. We speculate that these changes at the primary producer level are partly responsible for different outcomes in herbivore community reassembly between the different heat wave intensity treatments. These heat wave-mediated shifts in algal resources may have changed the competitive situation within the herbivore community, eventually leading to 100% re-invasion success of B. havanaensis in the post-29°C heat wave community and to 100% failure to re-establish in the post-39°C heat wave community. Such community closure under the highest intensity extreme event supports Hypothesis 2. Whether the observed community closure is a long-term phenomenon cannot be inferred from our experiment. Short-term disturbances can in principle lead to long-term changes in community composition, with serious implications for recovery success in natural ecosystems (Sorte et al. 2010 ; Eggers et al. 2012 ). Extreme event intensity and variability of recovery As hypothesized (H1, 2, 3), we found that the 39°C high-intensity extreme event led to a higher number of animal extinctions, to community closure for one of these species, and then to a higher variability of relative animal abundances during the recovery phase, as indicated by the Morisita similarity index and as visualized in the MDS plots. We considered variability at the primary producer level as an explanatory mechanism, but found a high similarity among the post-39°C final period replicates for algal abundances. We suggest that differences among replicates in the density of the surviving Cephalodella may have caused part of the variability in this treatment, as these could modulate the relative success of re-introduced competing herbivores. We thus hypothesize that recovery trajectories of these re-introduced herbivores were at least in part (1) governed by algal resource availability, and (2) modulated by differences in density of the competing herbivore that still existed at varying densities in the system after the heat wave. We intend to test this in manipulative follow-up experiments. In general, we expect a moderate heat wave to lead to fewer local extinctions than will be caused by a more intense heat wave. A smaller number of extinctions would allow a community to stay closer to its preexisting regime of ecological forces. This perspective is in line with the view that strong ecological forces tend to canalize patterns and hence decrease variability. Conversely, their disruption by (a large number of) local extinctions could enhance noise and magnify initially small differences among population densities. Our experimental results show that the consequences of a high-intensity extreme event (canalization vs. noise enhancement) may differ for different community-level phenomena. On the one hand, the most intense heat wave led to a highly repeatable number of herbivore extinctions (the same in all replicates) and to a highly repeatable community closure for one of these herbivores (also identical in all replicates). But on the other hand, the most intense heat wave led to a significantly higher variability of relative species abundances in the postre-introduction period. Community-level consequences of a high-intensity extreme event can thus seemingly include both elements of canalization and noise enhancement. We note that the latter only occurred in the herbivore community, which reassembled by means of species re-introductions, whereas it was absent in the algal community, where all species had continued to exist regardless of treatment. Implications for natural ecosystems Humankind strongly relies on resilient ecological processes and a reliable provision of ecosystem services in natural, rural, and urban landscapes. This all depends on the response traits (such as heat tolerance) of the involved organisms that through their effect traits contribute to these ecosystem functions and services. The importance of resilience and reliability implies the need for a better understanding of processes underlying ecological recovery and for appropriate management strategies to support natural restoration. Our study used controlled and replicated laboratory communities as a model system, that is, a simplification of reality. Such simplification is nonetheless useful in pointing to possible processes governing recovery from high-intensity extreme events. We conclude that high-temperature extremes can on the one hand generate strong ecological processes that deterministically cause extinctions and community closure, and on the other hand generate ecological legacies that increase variability and that make the details of subsequent recovery more difficult to predict."
} | 4,453 |
35539563 | PMC9077753 | pmc | 8,765 | {
"abstract": "Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA ( M n = 700 g mol −1 ) hydrogels are combined with a thiol-group presenting TMV mutant (TMV Cys ). By covalent coupling of TMV Cys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMV Cys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.",
"conclusion": "Conclusions The TMV Cys mutant was successfully functionalised with acrylate groups by coupling thiol-presenting CP Cys subunits with PEG-DA via the thiol-Michael reaction. The thus obtained TMV-PEG-A particles were integrated covalently into PEG-DA hydrogels, as evidenced by the increased G ′ of the hydrogels with increasing TMV-PEG-A concentration. On the other hand, wt-TMV did not have a significant effect on G ′ due to the missing covalent interactions. The presence of the TMV derivatives had no effect on the curing efficiency of PEG-DA or on the EWC. Due to the small mesh size of the hydrogels, both TMV derivatives were immobilised in the hydrogels. They were distributed quite homogeneously throughout the whole volume. The results give rise to the conclusion that TMV particles offer a way to add functionality into otherwise non-functional PEG-DA hydrogels, extending earlier findings for alginate and hyaluronan gels and adding a detailed characterisation to the initial promising results obtained for TMV-containing PEG-DA gels. 35 Especially the thiol groups of the TMV Cys mutant or functional groups of other TMV mutants not tested in this study might be used for future coupling of further functional moieties such as growth factors or enzymes into the hydrogels, e.g. for uses as cell culture or tissue engineering matrices.",
"introduction": "Introduction Hydrogels based on poly(ethylene glycol) (PEG) constitute an interesting class of materials for different applications such as drug delivery, 1,2 tissue engineering, 3,4 3D printing, 5–7 or sensing. 8,9 Hydrogels for these applications have to be functional materials with defined chemical, biological, or physical properties. For the preparation of PEG-based hydrogels, poly(ethylene glycol) diacrylate (PEG-DA) is frequently used as a building block due to its low toxicity 10 and excellent water-solubility. Additionally, the mechanical properties, the water content and the permeability for small molecular compounds of PEG-DA hydrogels can be easily adjusted by using different molecular weights and concentrations in the hydrogel precursor solutions. 11–13 Hydrogel formation using PEG-DA can be achieved e.g. by radical polymerisation of the acrylate end groups 14,15 or by Michael addition chemistry. 16,17 However, pure PEG-DA hydrogels provide rather limited chemical or biological functionality. The PEG backbone renders the hydrogels protein repellent and generally unsuitable for biological applications such as cell culture. 17,18 Chemical modification of cured PEG-DA hydrogels is also difficult without deteriorating the network structure due to the low reactivity of PEG. Therefore, functional PEG-DA hydrogels are often obtained by co-polymerisation of PEG-DA with functional building blocks 19 or by prior modification of one PEG-DA chain end by Michael addition of functional thiols 20–22 or amines. 23 Tobacco mosaic virus (TMV) is a versatile nano-scaled scaffold with the possibility to present more than 2000 functional groups per particle. This plant virus, composed of a genomic RNA and about 2100 coat protein (CP) subunits, has a tube-like structure 300 nm in length with inner and outer diameters of 4 nm and 18 nm, respectively. 24 TMV has been investigated as a carrier scaffold for example for enzymes, or as template for mineralisation for more than twenty years. 25,26 Virus-like particles (VLPs) can be obtained with artificially produced RNAs, 27,28 thus changing the length or form of the virus, as well as with altered CPs presenting genetically encoded specific coupling groups such as primary amines or thiols. 29 In addition, we have recently achieved to produce VLPs with highly defined longitudinal domains. 30 With regard to hydrogels, TMV by itself and in combination with alginate hydrogels has been shown to have a positive effect on osteogenesis without provoking adverse immune reactions in vivo . 31–33 For these alginate gels, the impact of TMV particles on their mechanical properties has been characterised, demonstrating that TMV rods induced non-linear responses to compression at high strains. 31 Additionally, Wang and co-authors have fabricated TMV-interlinked hydrogels from methacrylated hyaluronan (MeHA) and were able to demonstrate improved in vitro chondrogenesis of bone marrow mesenchymal stem cells (BMSCs) in these matrices, which have also been characterised for their internal structure, stability and TMV retention. 34 Lewis et al. produced hybrid hydrogel microparticles from PEG-DA ( M n = 700 g mol −1 ) and functionalized TMV and showed a regular distribution of TMV within the hydrogels at the microscale by fluorescence microscopy. 35 However, they did not investigate the impact of TMV on the mechanical properties of the hydrogels or virus distribution at the nanoscale. As described above, PEG-DA hydrogels can be adapted as a versatile platform for functional hydrogels. Therefore, we aimed at both covalent and non-covalent integration of TMV nanoparticles into PEG-DA hydrogels, and an in-depth physical characterisation of these hybrid materials. In particular, we wanted to assess the impact of covalent and non-covalent integration of TMV into PEG-DA hydrogels on the physical properties of the hydrogels, such as mechanical properties and equilibrium water content (EWC). For covalent integration, a cysteine-containing TMV mutant (TMV Cys ) was used. On the other hand, wildtype TMV (wt-TMV) was used for non-covalent integration. We hypothesised that due to the rod-like structure of TMV with the length of 300 nm, a covalent integration of TMV Cys into the PEG-DA hydrogel network would result in a stiffer hydrogel compared to a non-covalent integration of wt-TMV, provided that the TMV Cys is evenly distributed throughout the hydrogel volume.",
"discussion": "Results and discussion Reaction of TMV Cys and wt-TMV with PEG-DA In this study, two different TMV derivatives were applied for integration in PEG-DA hydrogels. A cysteine mutant, TMV Cys , 29 was used for covalent immobilisation of TMV particles in hydrogels and a wild-type, wt-TMV, was used for non-covalent immobilisation. The thiol groups available through the cysteine moiety in every CP subunit of TMV Cys should be able to participate in manifold chemical reactions due to their rich redox chemistry and extremely high nucleophilicity. 38 With PEG-DA, a chemical coupling with the thiols in aqueous media should be possible by the thiol-Michael reaction which is known to proceed in neutral to slightly basic conditions without any catalyst ( Scheme 1 ). 39 Thus, it should be possible to functionalise TMV Cys with acrylic double bonds, provided that one PEG-DA end group reacts with one TMV Cys CP, leaving the other PEG-DA end group unaltered. The so formed double bond functionalised TMV particles (TMV-PEG-A) should, in a second step, be able to participate in a subsequent radical cross-linking reaction which forms a PEG-DA hydrogel. A reaction of both PEG-DA end groups with CPs is to be avoided, if possible, as it would leave the resulting moiety with no residual acrylic end group for a subsequent hydrogel cross-linking reaction. For wt-TMV no chemical reaction is expected with PEG-DA. Scheme 1 Schematic representation of the coupling of PEG-DA to TMV Cys by thiol-Michael reaction resulting in double bond functionalised TMV particles (TMV-PEG-A, left) and subsequent photopolymerisation of the attached acrylate functionalities with more PEG-DA to hydrogels (right). Thus, covalent incorporation of TMV Cys into the hydrogels is possible. In the case of wt-TMV, no free cysteine moieties are present and no covalent coupling with PEG-DA is expected. In order to investigate the reaction of TMV Cys and wt-TMV with PEG-DA, we prepared mixtures of TMV Cys and wt-TMV, respectively, with PEG-DA in SPP buffer at pH 7.4 with different molar ratios of TMV CPs and PEG-DA acrylate end groups. The mixtures were then reacted for different times. In case of a coupling reaction between a TMV Cys CP and PEG-DA, an increase of molecular weight of the CP is expected. Therefore, the molecular weights of the reaction products were assessed by SDS-PAGE. With this method, the TMV particles are disassembled so that single CPs can be analysed. The resulting silver stained SDS-PAGE image is shown in Fig. 1 . Fig. 1 SDS-PAGE of the different coupling products between the CPs of TMV Cys with PEG-DA, and corresponding preparations with wt-TMV, at different reaction times and functional group ratios of TMV CP and PEG-DA, as indicated. For wt-TMV, no additional band of the wt-CP was observed, indicating that indeed no chemical reaction took place between wt-TMV and PEG-DA. This result can be explained by the absence of reactive groups on the wt-TMV surface. On the other hand for TMV Cys , depending on the reaction time and the ratio of functional groups, generally three different product species were identified after the reaction. One of them is the unmodified CP of TMV Cys , showing that reaction of PEG-DA with the CPs of TMV Cys did not proceed quantitatively. However, also one reaction product with a slightly larger molecular weight than the CP was observed. This was attributed to a coupling of one PEG-DA molecule ( M n = 700 g mol −1 ) with one CP, leading to CP-PEG-A adducts. This would result in TMV-PEG-A particles as depicted in Scheme 1 . Additionally, a reaction product was formed which had approx. twice the molecular weight of one CP. This can be explained by a reaction of two CPs with one PEG-DA molecule, stemming from CP-PEG-CP adducts. This can involve either two CPs of the same TMV Cys , resulting in a loop on the TMV Cys surface, or two CPs of two different TMV Cys , resulting in a coupling of two TMV Cys -containing particles. In these cases, both end groups of PEG-DA are converted and no residual acrylic end group can participate in a subsequent hydrogel cross-linking reaction. Therefore, this double reaction of PEG-DA is not desirable for the covalent integration of TMV Cys into PEG-DA hydrogels. The slight shift in migration distance of wt-CP and CP Cys in the presence of PEG-DA in comparison with CP Cys without PEG-DA is due to the influence of the polymer on the general migration behaviour, which also hinders the SDS-PAGE analysis of higher PEG-DA ratios to TMV ( e.g. 10 000 : 1). In order to have as many reactive acrylic groups as possible on the TMV Cys surface, we aimed to maximise the percentage of CP-PEG-A and to minimise the percentage of CP-PEG-CP. For this, we quantified the respective amounts under different reaction conditions by ImageJ analysis of Fig. 1 . 40 It was found that after one hour, at least one third of the CPs had reacted only once with PEG-DA corresponding to approx. 700 reactive acrylates per TMV particle. An increase in reaction time resulted especially for the lowest PEG-DA : TMV ratio in an increase of double-reacted PEG-DA ( Table 2 ). As a compromise between maximised CP-PEG-A content and minimised CP-PEG-CP content, for the hydrogel preparation experiments described below the coupling was allowed to proceed for one hour. Distribution of the different TMV Cys -CP/PEG-DA reaction products in % depending on educt ratio and time TMV : PEG-DA 1 : 3333 1 : 1000 Time [h] 3.00 1.00 0.25 3.00 1.00 0.25 Free CP, 17.6 kDa 34 39 56 41 57 68 CP-PEG-A, 18.3 kDa 44 42 33 35 30 23 CP-PEG-CP, 35.9 kDa 22 19 11 24 13 9 Hydrogel preparation and characterisation Next, both the TMV-PEG-A particles and wt-TMV were incorporated into respective PEG-DA hydrogels. The corresponding formulations contained PEG-DA, the photo-initiator Irgacure 2959 as well as wt-TMV or TMV-PEG-A, respectively. For all hydrogels prepared with one type of the respective TMV particles, the PEG-DA concentration c PEG-DA (20 wt%) and Irgacure 2959 concentration (0.1 wt%) were kept constant in order to evaluate the effect of TMV particle addition to the formulations on the hydrogel properties. For this, the respective TMV particles were simply added to the PEG-DA containing hydrogel formulations at concentrations of 0.1 wt%, 0.3 wt%, 1.0 wt%, and 2.0 wt% and were allowed to react with the PEG-DA for 1 h. With the molecular weight of one TMV CP of 17.6 kDa, the ratio of TMV CPs and PEG-DA acrylate groups thus was 10 000, 3333, 1000, and 500, respectively, which should lead mainly to TMV-PEG-A in the case of TMV Cys , whereas wt-TMV would remain unaltered. Subsequently, the formulations were cured by radical photo-polymerisation of the PEG-DA end groups induced by UV irradiation. As reference samples, pure PEG-DA hydrogels with solid contents of 20.1 wt%, 20.3 wt%, 21 wt%, and 22 wt% were prepared, thus having the same solid content as the TMV containing samples. The resulting compositions of all hydrogel formulations in this study are shown in Table 1 . The sample names denote which kind of compound is added to the basic hydrogel formulation as well as the additional amount of added compound in wt%. For example, sample TMV Cys 1.0 contained 20 wt% PEG-DA, 0.1 wt% Irgacure 2959 and 1.0 wt% TMV Cys in SPP buffer. For all samples, preparation of hydrogels by UV irradiation was possible with high efficiency. Upon curing, the mobile liquids were all converted into well-defined solids. Also with the eye, no differences between the hydrogels could be observed (photos in ESI † ). All hydrogels could be handled easily without breakage and were slightly opaque. In order to evaluate if the presence of wt-TMV or TMV Cys had an effect on the double bond conversion of the PEG-DA, ATR-FT-IR spectra of dried hydrogel samples were collected ( Fig. 2 ). Compared to unreacted PEG-DA, we observed the disappearance of the double bond stretching vibration at 1635 cm −1 and the in-plane scissoring vibration at 1408 cm −1 . 41,42 This was the case for all samples, indicating that all samples were fully cured and that the addition of wt-TMV or TMV-PEG-A to the hydrogel formulations did not hamper the radical polymerisation of the acrylate groups. Fig. 2 ATR-FT-IR spectra of unreacted PEG-DA and of dried hydrogel samples containing 20 wt% PEG-DA supplemented with either 1 wt% PEG-DA (PEG-DA1.0), TMV Cys (TMV Cys 1.0) or wt-TMV (wt-TMV1.0). Due to the high double bond conversion in all samples after curing, it can be expected that for the samples containing TMV-PEG-A, the TMV particles are covalently bound into the hydrogel polymer network as depicted in Scheme 1 . In order to investigate the effect of the TMV particle addition on the hydrogel properties, we assessed the mechanical properties of the hydrogels by oscillatory shear rheology. The values for the storage moduli G ′ and the loss moduli G ′′ are shown in Fig. 3 . For all samples, G ′ was two to three orders of magnitude larger than G ′′, revealing the mainly elastic behaviour of the hydrogels. This observation is in accordance with the ATR-FT-IR data confirming successful cross-linking. Looking at the storage moduli of pure PEG-DA hydrogels, a significant ( p < 0.01), nearly linear increase of G ′ with increasing c PEG-DA was observed from 225 kPa (PEG-DA0.1) to 234 kPa (PEG-DA0.3), 269 kPa (PEG-DA1.0), and 290 kPa (PEG-DA2.0). This behaviour can be explained by the increasing cross-link density with increasing PEG-DA content and is in accordance with the literature. 43 Fig. 3 Storage moduli ( G ′) and loss moduli ( G ′′) of PEG-DA hydrogels containing TMV Cys , wt-TMV or no TMV, respectively. The dashed lines are for the guidance of the eye only. Looking at the wt-TMV-containing samples, a different behaviour was observed. The addition of wt-TMV did not lead to a significant change of G ′, leading to G ′ values of 226 kPa (wt-TMV0.1), 229 kPa (wt-TMV0.3), 229 kPa (wt-TMV1.0) and 256 kPa (wt-TMV2.0). This observation is in accordance with the assumption that the wt-TMV particles are not covalently bound to the hydrogel polymer network upon photo-polymerisation and also do not interact with the polymer network significantly in any other way. This can be explained by the lack of functional groups on the wt-TMV surface which can participate in the radical cross-linking. This was not the case for hydrogels which contained TMV Cys . As described above, the thiols present on the TMV surface readily undergo thiol-Michael reactions to yield acrylate functionalized TMV-PEG-A. These can participate in the radical cross-linking and can be incorporated covalently into the hydrogel matrix. The effect can be seen from the G ′ values of the respective hydrogels which were generally higher than the G ′ of PEG-DA hydrogels with an identical solid content. Thus, with increasing concentration of TMV Cys , G ′ increased significantly ( p < 0.01) from 235 kPa (TMV Cys 0.1) to 243 kPa (TMV Cys 0.3), 321 kPa (TMV Cys 1.0), and 391 kPa (TMV Cys 2.0). All mean values were significantly different from the other mean values ( p < 0.01), except for the results of TMV Cys 0.1 and TMV Cys 0.3. The larger effect of TMV Cys addition compared to PEG-DA addition seems to confirm the different architecture of the network points resulting from TMV-PEG-A ( Scheme 1 ) as compared to the PEG-DA hydrogels. Comparing the different sample types (without TMV, with wt-TMV, with TMV Cys ) at the same solid content, the general trend in G ′ as a measure for hydrogel stiffness can be summarised as follows: G ′ TMV Cys > G ′ PEG-DA > G ′ wt-TMV . At smaller solid contents (20.1 wt%, 20.3 wt%), this trend is not significant, however at larger solid contents (21.0 wt%, 22.0 wt%), it becomes significant ( p < 0.05 and p < 0.01). Interestingly, the EWC of the hydrogels was practically constant for all formulations tested ( Fig. 4 ). All hydrogels contained between 81% and 82% of water. However, there was a small, significant effect ( p < 0.01) on the EWC with the solid content for all sample types which lead to a decrease of EWC with the solid content. Between the different sample types (without TMV, with wt-TMV, with TMV Cys ), no significant differences in EWC were observed. This leads to the conclusion that the water uptake capacity of the hydrogels was obviously dominated by the PEG-DA in all cases. This is due to the small fraction of TMV particles in the solid content of the hydrogels. Even at the highest concentrations tested, only 9.1% of the solid content were TMV particles, given by the ratio of 2 wt% TMV particles and the total solid content of 22 wt%. Since the wt-TMV particles do not interact strongly with the polymer network, a large influence on the EWC was not expected. However, also the covalent integration of TMV-PEG-A into the hydrogels showed only a very small effect on the EWC. Fig. 4 Equilibrium water contents (EWC) of PEG-DA hydrogels containing TMV Cys , wt-TMV or no TMV, respectively. In order to explain the different impact of TMV addition on G ′ and the EWC, a closer look at the molecular structure of the hydrogel networks is necessary. In order to estimate if unbound TMV particles can diffuse through the hydrogel networks, the EWCs of the pure PEG-DA hydrogels were used to calculate the molecular weight M c between cross-links in the hydrogels using the Flory–Rehner equation in its modified version by Merrill and Peppas. 44,45 With the Flory–Huggins interaction parameter with water (0.426), 46 the density (1.12 g cm −3 ), 47 and the number average molecular weight (700 g mol −1 ) of PEG-DA, an M c of approx. 200 g mol −1 is estimated for the pure PEG-DA hydrogels. Following the reasoning of Canal and Peppas, 48 with the average bond length (0.146 nm), 49 the molecular weight of the repeating unit (44.05 g mol −1 ), and the characteristic ratio of PEG (4.1), 49 this translates into average mesh sizes of approx. 2 nm. Therefore, it should be impossible for intact TMV particles with an outer diameter of 18 nm 50 to diffuse through the hydrogel network. As soon as the network is formed, the TMV particles are trapped inside the hydrogel independent of the presence of covalent bonds between the TMV particles and the network. Therefore, the amide I and amide II vibrations at 1655 cm −1 and 1545 cm −1 are visible in the ATR-FT-IR spectra both in the wt-TMV and TMV Cys containing hydrogels also after washing, without any notable difference between the two TMV particle types ( Fig. 2 ). The comparison between the average mesh sizes in the hydrogels and the size of the TMV particles with a length of 300 nm also gives insight into the impact of TMV-PEG-A addition to PEG-DA hydrogels on their molecular structure. The TMV nanorods penetrate through the length of approx. 150 meshes of the PEG-DA network. Due to the stiff nature of the TMV particles, this results in stiffening of relatively large regions of the hydrogel, also explaining the increase in G ′ with larger TMV-PEG-A concentrations. On the other hand, meshes adjacent to the TMV containing meshes and further away – which are the majority of meshes – are not changed much on the molecular level, resulting in very little impact on the EWC. The addition of wt-TMV therefore has no significant effect on the mechanical properties. However, it can be expected that the TMV particles occupy a large space compared to the average mesh size and thus cause defects in the polymer networks much bigger than the average mesh size. Without the covalent bonds which link TMV-PEG-A to the networks, with wt-TMV the PEG-DA network can obviously slide over the TMV surfaces, thereby overriding the stiffening effect on the hydrogel. However, these defects are not accessible to water due to the presence of the TMV particles, explaining the low effect of wt-TMV addition on the EWC. Interestingly, our results on the mechanical properties of the TMV containing PEG-DA hydrogels are not reflected by results published so far for hydrogels made from other polymers. Luckanagul et al. added 0.1% of wt-TMV into porous alginate hydrogels and found an increase of compressive modulus in unconfined uniaxial compression compared to samples containing no wt-TMV. 31 We assume that the reason for this different behaviour can be found in more non-covalent interactions which are possible between alginate and wt-TMV CP compared to the interactions between the rather unfunctional PEG backbone of PEG-DA and wt-TMV. On the other hand, Maturavongsadit et al. reported that in hyaluronan hydrogels prepared from methacrylated hyaluronan, the shear modulus of hydrogels in which covalently bound cysteine-containing TMV was present (0.1%) was slightly lower than for hydrogels without cysteine-containing TMV. 34 However, their samples were very soft with a G ′ in the range of 50 Pa to 100 Pa and the difference in sample properties was small. These data suggest a very low degree of cross-linking and a large mesh size in general, especially when compared to other hyaluronan hydrogels which were prepared by thiol-Michael addition cross-linking. 51 Additionally, the degree of hyaluronan conjugation to the cysteine-containing TMV was not determined in this study and thus the impact of TMV on the hydrogel structure cannot directly be compared. From our results it can be assumed that in their case the conjugation between hyaluronan and cysteine-containing TMV did not lead to substantially increased cross-linking density because they did not observe a stiffening effect. To further support the above-mentioned conclusion that TMVs cannot diffuse through the hydrogel network, we quantified the TMV concentrations in the three washing buffers of the hydrogels with a micro BCA assay. The washing buffer was exchanged every 24 h so that the three measurements allow determining the kinetics with which TMV is washed out of the hydrogels over 72 h. As a prerequisite for this experiment, we verified the possibility to quantify the concentration of wt-TMV and TMV Cys in suspension by measuring a calibration curve using TMV suspensions with defined concentrations (ESI † ). The micro BCA assay was then performed with the serial washing buffers, and the respective TMV concentration was calculated using the calibration function determined before. For both the wt-TMV and TMV Cys -containing hydrogels, only the first of the three washing buffers, collected after 24 h washing time, contained detectable concentrations of wt-TMV and TMV Cys , respectively. The relative amount of the TMV derivatives found in these washing buffers compared to the total amount of TMV in the hydrogels is shown in Fig. 5 . Fig. 5 Fraction of initially applied TMV released in the first washing step (24 h). In the subsequent two wash solutions, no further TMV was detected. It is evident that the washed out TMV varied between approx. 1% and 3% of the initial amount, independent of the TMV derivative used. No significant differences between the samples were observed. The fact that TMV was found only in the first washing buffer suggests that it derived mainly from TMV adsorbed on the hydrogel surface, but not from TMV diffused out of the interior of the hydrogel. Investigation of TMV structure in the hydrogels Depending on the solvent conditions, TMV particles can either be solvated as individual virus rods, or form nematic crystals, or aggregate to bigger virus bundles. 52 As PEG of higher molecular weight ( M n = 6000 g mol −1 ) was used for precipitating TMV, it cannot be completely excluded that a similar effect took place in PEG-DA solutions. Therefore, it was interesting to investigate the distribution of the virus particles within the hydrogels. So far most structural investigations on hydrogel preparations have been performed by (E-)SEM analysis of (freeze-)dried hydrogels with a resolution in the 100 nm to 1 μm range not sensitive enough to see features as small as TMV. 15 Also changes in hydrogel morphology can be expected to occur during the drying process. Therefore, the TMV containing hydrogels were embedded in epoxy resin, stained with uranyl acetate and sectioned into 60–100 nm thick slices. TMV particles in the hydrogels appear as 10 to 20 nm thick lines of different lengths (100–1000 nm, see Fig. 6 ). Fig. 6 TEM images of TMV containing hydrogels after resin embedding and sectioning. TMV particles can be seen as dark, uranyl acetate stained lines upon the cloudy hydrogel background. This variation in length is due to two effects; first, the virus particles are embedded with varying orientations and second, they tend to form so-called head-to-tail attachments so that five TMV attached in this way would result in a 1.5 μm long particle. 53 For both TMV variants, TMV particles could be detected at all concentrations. There was also no significant difference in the amount of TMV for wt-TMV in comparison to TMV Cys visible, supporting the conclusion that the viruses are confined within the hydrogels due to the small mesh size. The viruses were also relatively homogeneously distributed within the hydrogels, thus it can be concluded that PEG-DA does not have an aggregating effect on TMV. The absence of larger TMV aggregates in the hydrogels containing TMV Cys also indicates that the formation of CP-PEG-CP during the thiol-Michael reaction takes place prior to cross-linking preferentially by forming PEG loops on a single TMV particle rather than by linking CPs of two different TMVs to each other. This is in contrast to the results of Wu et al. , 54 they combined TMV at 1.5 wt% with alginate and observed immediate clouding of the solution while TMV-PEG-DA solutions stayed clear upon mixing. In addition, the corresponding TEM images showed clusters of laterally aligned TMV particles, oriented by the application of shear forces, within an unstained alginate background."
} | 7,344 |
38543411 | PMC10974445 | pmc | 8,767 | {
"abstract": "Thermal conductive coating materials with combination of mechanical robustness, good adhesion and electrical insulation are in high demand in the electronics industry. However, very few progresses have been achieved in constructing a highly thermal conductive composites coating that can conformably coat on desired subjects for efficient thermal dissipation, due to their lack of materials design and structure control. Herein, we report a bioinspired thermal conductive coating material from cellulose nanofibers (CNFs), boron nitride (BN), and polydopamine (PDA) by mimicking the layered structure of nacre. Owing to the strong interfacial strength, mechanical robustness, and high thermal conductivity of CNFs, they do not only enhance the exfoliation and dispersion of BN nanoplates, but also bridge BN nanoplates to achieve superior thermal and mechanical performance. The resulting composites coating exhibits a high thermal conductivity of 13.8 W/(m·K) that surpasses most of the reported thermal conductive composites coating owing to the formation of an efficient thermal conductive pathway in the layered structure. Additionally, the coating material has good interface adhesion to conformably wrap around various substrates by scalable spray coating, combined with good mechanical robustness, sustainability, electrical insulation, low-cost, and easy processability, which makes our materials attractive for electronic packaging applications.",
"conclusion": "4. Conclusions In summary, we have realized the scalable production of highly thermal conductive CNF/BN/PDA coating by mimicking the layered nanostructure of nacre. Taking advantage of the strong interfacial interaction and the strong mechanical shearing of a pan mill, efficient co-exfoliation and dispersion of CNF and BN can be achieved to produce processible slurry. The addition of PDA can largely increase the adhesion of the slurry to various substrate surfaces by simple spray coating. The resulting coating exhibits nacre-like layered structure with horizontally aligned BN nanoplates that are connected by CNFs. These interconnected networks guarantee ultrafast thermal conductive pathways for phonon transport, as well as multiple reinforcement mechanisms for energy dissipation. As a result, the composites coatings exhibit a high thermal conductivity of 13.8 W/(m·K), which is well beyond most of the previously reported thermal conductive coating, combined with good mechanical properties, low-cost, good adhesion and sustainability. We expect this material will find many real-world applications in the electronic, auto, and aerospace industries.",
"introduction": "1. Introduction The urgent requirement of miniaturized, densified, and multi-functional electronics significantly increases the power density of electronics, leading to fast heat accumulation in a limited or confined space [ 1 , 2 ]. This rapidly increased temperature inevitably affects the service life, safety, reliability, and speed of the electronics, even causing severe equipment damage and major fires. To address this issue, progress has been made to develop thermally conductive materials that can effectively dissipate the accumulated heat. Metallic and nanocarbon materials, including aluminum, MXene, graphene, and carbon nanotube have been intensively incorporated into polymeric matrix for constructing flexible, light weight composites with high-performance thermal management capabilities [ 3 , 4 , 5 ]. Although these resulting composites exhibit outstanding thermal conductivity, they usually also possess high electrical conductivity [ 6 , 7 , 8 ], which easily cause undesired short-circuit problem in the application of electronics. On the other hand, to achieve efficient thermal management, a seamless interface between electronics and thermal conductive composites is strictly needed. However, the conformal integration of these pre-formed thermal conductive composites onto the electronics is difficult due to the poor interface adhesion, especially for some irregular surfaces. The addition of glue could solve the adhesion problem but introduces additional thermal resistance and cost. Therefore, it is desirable to develop a thermal conductive but electrical insulating composites coating that can be seamlessly assembled on various irregular objects. Boron nitride (BN) not only has excellent thermal conductivity and electrical insulation property, but also has good chemical stability and oxidation resistance, making it a promising candidate as a nanofiller for constructing thermal conductive composite coating [ 9 , 10 ]. However, most BN-based composites coating still face the issues of low thermal conductivity and weak mechanical properties, due to the poor dispersion of conductive filler and lack of structure control, largely limiting their practical applications [ 11 , 12 ]. To realize high thermal conductivity and mechanical robustness, bioinspired structural hierarchy design is one of the most promising approaches to engineer the composites coating [ 13 ]. The most spectacular examples are nacre-like composites, using aligned micro/nanoplates in polymer matrix [ 14 , 15 ]. For instance, Han et al. report a strong nacre-mimetic BN/epoxy composite by using a bidirectional freezing technique, which can realize high thermal conductivity of 6.07 Wm −1 K −1 combined with good electrical insulation performance [ 15 ]. Pan et al., took advantage of hot-pressing technique to construct brick and mortar-structured Ag-Al 2 O 3 platelets/epoxy composites with a thermal conductivity of 6.71 Wm −1 K −1 [ 16 ]. However, these techniques usually need complex processes, which are difficult to be apply in composite coating and constructing nanostructured composites coating with a combination of high thermal conductivity, mechanical robustness, and good adhesion is still a big challenge. In nature, nacres utilize chitin nanofibers which are wrapped by proteins to glue CaCO 3 microplates to form hierarchical layered structures [ 17 ]. The highly ordered architecture and favorable interfacial strength enable nacre to have an amazing combination of mechanical strength and toughness, with added brilliant iridescence [ 18 ]. In this study, we report a bioinspired high-performance thermal conductive coating by exfoliation/dispersion of BN nanoplate and cellulose nanofibers (CNFs) and multiple interfacial interaction engineering. We take advantage of sustainable 1D CNF and dopamine as the building blocks to mimic the combination of chitin and proteins. CNFs have a similar structure to chitin, but it is more widely available and stronger [ 19 , 20 , 21 ], while polydopamine (PDA) is a well-known mussel-inspired adhesive that mimics the adhesive properties of mussels, which can adhere to a variety of surfaces under wet and dry conditions [ 22 ]. CNFs and BN nanoplates are integrated together to form viscose ink through the simultaneous exfoliation/dispersion induced by the strong shearing force of pan mill. The subsequent incorporation of dopamine not only significantly enhanced the interfacial strength of the composites, but also enabled strong adhesion to various substrates as conformal coating. The resulting coating demonstrates thermal conductivity of 13.8 W/(m·K) combined with good mechanical properties because the uniform layered structure provides prolonged phonon pathways. Thus, such an outstanding combination of thermal conductivity, mechanical properties, electrical insulation, adhesion, sustainability, and scalable process make our composites promising candidates for advanced electronic packaging technology applications.",
"discussion": "3. Results and Discussion The schematic representation shown in Figure 1 a–d illustrates the fabrication of the CNFs/BN/PDA thermal conductive composites ink. To facilitate the exfoliation and dispersion of CNFs, soft wood pulp has been pretreated using TEMPO oxidation, which selectively oxidizes the primary hydroxyl groups (C6) on the surface of cellulose fibers to negatively charged carboxyl groups [ 27 ]. The pretreated cellulose fibers were mixed with BN and subjected to pan mill to co- exfoliate and disperse CNFs/BN nanoplates mixture. During the process of co-exfoliation and dispersion, stacked large-size BN particles are peeled off into nanosheets, using the strong shear force of two pans. Meanwhile, the pretreated cellulose fibers also become nano fibers (CNFs). CNFs could inhibit the re-stacking and agglomeration of BN nanosheets through hydrogen bonding, hydrophobic interactions, and spatial site resistance, resulting in stable CNFs/BN slurries ( Figure S1 ) [ 23 ]. However, the resulting slurries are difficult to apply onto various substrates due to poor adhesion. To achieve a wide range of adaptability to different substrates, 5 wt % PDA were added into the above slurries to regulate the interactions. The strong multiple interactions, including hydrogen bonding, hydrophobic interaction, and π-π stacking interactions, would greatly enhance both adhesion with substrates and internal interactions [ 28 ]. As shown in Figure S2 , the Fourier Transform Infrared Spectrometer (FTIR) peak at 3350 cm −1 assigned to the hydrogen bonded –OH stretching vibrations increased after the addition of PDA, indicating the formation of additional hydrogen bonding by PDA. Taking advantage of evaporation induced self-assembly strategy, conformal dense coating with highly organized layered structure can form from the viscous CNFs/BN/PDA slurry. The slurry with different BN contents is coded to be BCNF10, BCNF30, BCNF50, and BCNF70 for 10 wt %, 30 wt %, 50 wt %, and 70 wt % BN, respectively. We investigate the morphological evolution of building blocks during the co-exfoliation and PDA formation. Pristine BN powder consists of nanoplates with a lateral size of 1–10 μm and a thickness of around 200 nm ( Figure 2 a), while original cellulose fibers from wood pulp have a diameter of around 10–15 μm ( Figure 2 d). Due to their large size and hydrophobic character, BN powders are difficult to disperse in water. After the pan mill treatment, the BN mixture and cellulose fibers are co-exfoliated into small sized particles to form a stable slurry ( Figure 2 b,e and Figure 3 a), where BN nanoplates are conformal wrapped by an elementary CNFs network with diameter of 3 nm ( Figure 2 c). We suggest that this co-exfoliation of CNFs/BN dispersion is facilitated by the amphiphilic property of CNFs, which consists of a hydrophobic and hydrophobic crystalline plane [ 29 ], while the –OH/-COOH groups of hydrophilic planes assist the dispersion of BN in water [ 30 ]. After the addition of PDA, numerous PDA nanoparticles are bonded on the CNFs and BN nanoplates’ surface due to strong adhesion ( Figure 2 f). This good exfoliation and good dispersion of building blocks is favorable for constructing thermal conductive coating materials. As shown in Figure 3 a, the obtained CNFs/BN/PDA slurry exhibits a homogeneous morphology with good stability up to several months ( Figure S3 ). This is because the amphiphilic character of CNFs allows them to firmly bond on the BN surface, while the high aspect ratio of CNFs forms a physical entanglement network to prevent aggregation through electrostatic repulsion [ 31 ]. As is already known, the rheological properties of dispersion play an important role in the coating processability, thus we systematically studied the rheological behavior of the resulting CNFs/BN/PDA slurries with different BN content. In the shear rate–viscosity curves, all the viscosity of slurries shows a similar downward trend with the increase in shear rate, indicating typical shear-thinning non-Newtonian behavior that is favorable for improving processability. This gradual decreased viscosity is caused by the orientation of CNFs/BN along the shear direction under low shear force [ 32 ]. Furthermore, with the increase in BN content, the viscoelastic characteristics of the suspensions decrease. The reason for this is that the BN dispersed in the slurry reduces entanglement between CNFs, so the higher the BN content, the less entanglement between the CNFs and the lower viscosity of the slurry [ 33 ]. Figure 3 c,d show viscoelastic storage modulus (G′) and loss modulus (G″) behavior of CNFs/BN/PDA slurries as a function of angular frequency (ω). For all the slurries, the G′ was much higher than G″ in all of the investigated angular frequency ranges, indicating the formation of a gel-like structure in the slurries. The G′ increases continuously with the decreasing addition of BN content, mainly due to the formation of a more entangled CNFs network with more CNF contents. Additionally, the G′ of slurries has a frequency dependent behavior, where G′ experiences continuously decrease when the frequency is unchanged and increase with the decreasing frequency. This behavior can be explained by the dynamic nanostructure change of slurries. As a high frequency is applied, the initial percolating network of slurries would be broken, leading to the decreased modulus. At relatively low frequency, the destruction and reconstruction of CNFs/BN entanglements can reach a balance at the plateau. Further lowering the frequency provides enough time to significantly reconstruct the percolating network, leading to an increase in elastic and viscous moduli [ 34 ]. Owing to the good processability and adhesion of the obtained slurry, conformal thermal conductive coating can be applied to various substrate materials. As shown in Figure 4 a, the BCNF70 slurries can uniformly coat the smooth steel, polytetrafluoroethylene (PTFE), and rough wood using a simple spray coating technique. The resulting coating exhibits a good smooth surface as illustrated in the 3D topographic images ( Figure S4 ). Also, the aligned texture on the wood is still maintained after the coating treatment, indicating that the coating tightly wraps around the substrate surface. The interface adhesion of the coating was measured to be 0.81, 0.61, and 0.38 MPa for steel, wood and PTFE, respectively, which is good enough to enable the practical applications ( Figure 4 c). Then, we investigated the structure of the coating with different BN loading. All of the surface has a dense and smooth morphology with uniform BN nanoplates distribution. Increasing BN loading leads to more BN nanoplates being exposed on the coating surface, but they are still wrapped by CNF network to prevent the leakage in the practical applications ( Figure S5 ). For the cross-section, all the coating has a nacre-like layered structure, where BN nanoplates are highly aligned along the substrate surface due to the strong capillary and gravity effect during drying. This uniform layered structure is not only favorable to connect BN nanoplates to form good thermal conductive networks for enhancing thermal conductivity, but also possesses multiple reinforcing mechanisms for mechanical enhancement. Additionally, the CNFs/BN/PDA coating is hydrophilic with a water contact angle of 82.4°. ( Figure S6 ). Next, the mechanical properties of the composites were investigated since it is critical for service life in practical applications. As illustrated in Figure 5 a–d, composites with 10 wt % BN can achieve good mechanical robustness with a strength of 100 ± 4 MPa, modulus of 17 ± 1 GPa and toughness of 1.3 ± 0.2 MJ/cm 3 . The increasing addition of BN nanoplates leads to the decrease in the mechanical properties, but the composites still maintain reasonable strength and toughness, even with 70 wt % BN loading. BN composites usually have limited strength and toughness because of the weak interfacial interactions between BN nanoparticles, while the superstrong CNFs can connect BN together to significantly enhance the interfacial strength [ 35 ]. To gain insight into the fracture mechanism of the composites, we investigated the fracture cross-section of the composites ( Figure 5 c). The SEM image of the cross-section exhibits hierarchical rough layered morphology where many nanofibers and nanoplates are pulled-out. We propose a multiple toughening mechanism response to the mechanical enhancement ( Figure 5 f). When applying force, the dynamic bonds are broken, followed by the stretching and slipping of CNFs and BN. With further loading, CNFs and BN reorient and align along the loading direction, which will further experience pull-out, delamination, and fracture. This process would largely dissipate fracture energy for achieving good mechanical properties [ 36 ]. The thermal conductivity of the composites coatings is illustrated in Figure 6 b. The thermal conductivity of composites with 10 wt % BN loading is around 3.1 W/(m·K), which is superior to most plastics. The thermal conductivity further increases linearly with the increase in BN loading and reaches a high value up to 13.8 W/(m·K) with 70 wt % BN incorporation. We suggest that this high thermal conductivity is owed to the highly ordered thermal conducting network of the composites. These well-exfoliated CNFs and BN nanoplates are closely packed together to form a dense layered structure, where CNFs can work as a thermal conductive bridge to connect BN nanoplates because of its high intrinsic thermal conductivity [ 37 ]. Additionally, the smooth and homogenous surface could also reduce the phonon scattering during thermal conduction [ 23 ]. The possible mechanism for the high thermal conductivity was schematically illustrated in Figure 6 a. The exfoliated BN nanosheets overlapped each other, and the CNFs attached around the BN nanosheets due to hydrophobic–hydrophobic interactions, forming a dense and oriented thermal conductivity network, which minimized the gaps between the BNs, reduced the thermal resistance between the composite interfaces of the BN and CNFs, and improved the thermal properties. When the CNFs/BN were heated, the heat flow diffused rapidly along the network of BN nanosheets and CNFs to the whole, due to the inherent high thermal conductivity, dense thermal network, and low thermal resistance of BN and cellulose, thus exhibiting excellent thermal conductivity [ 38 ]. To visually evaluate the heat transfer capability of our coating, we placed the coating films on a hotplate with a temperature of 80 °C. The heat dissipation performance is monitored by the temperature variations through infrared thermal imaging technology ( Figure 6 d). Obviously, the temperature of composites with higher BN loading increases faster, and can reach 75 °C within just 5 s due to their high thermal conductivity, exhibiting outstanding heat dissipation performance. Additionally, to highlight the heat dissipation performance, we compared the thermal conductivity of our coating materials with other reported thermal conductive coating materials ( Table S1 ) [ 39 , 40 , 41 , 42 , 43 , 44 ]. As shown in Figure 6 b, the thermal conductivity exhibited in this work is far beyond other thermal conductive coating materials. For instance, Xu et al., reported that the addition of 40 wt % BN into epoxy (EP) lead to conductivity of 1.5 W/(m·K), which can further increase to 2.4 W/(m·K) with the addition of graphene. Ligati et al. prepared graphene-loaded paint with a thermal conductivity of 1.6 W/(m·K). Clearly, the thermal conductivity of our work is far higher than these reported materials, even with the same BN loading ( Figure S7 )."
} | 4,849 |
34649602 | PMC8518188 | pmc | 8,770 | {
"abstract": "Background Long-read sequencing has shown its tremendous potential to address genome assembly challenges, e.g., achieving the first telomere-to-telomere assembly of a gapless human chromosome. However, many issues remain unresolved when leveraging error-prone long reads to characterize high-complexity metagenomes, for instance, complete/high-quality genome reconstruction from highly complex systems. Results Here, we developed an iterative haplotype-resolved hierarchical clustering-based hybrid assembly (HCBHA) approach that capitalizes on a hybrid (error-prone long reads and high-accuracy short reads) sequencing strategy to reconstruct (near-) complete genomes from highly complex metagenomes. Using the HCBHA approach, we first phase short and long reads from the highly complex metagenomic dataset into different candidate bacterial haplotypes, then perform hybrid assembly of each bacterial genome individually. We reconstructed 557 metagenome-assembled genomes (MAGs) with an average N50 of 574 Kb from a deeply sequenced, highly complex activated sludge (AS) metagenome. These high-contiguity MAGs contained 14 closed genomes and 111 high-quality (HQ) MAGs including full-length rRNA operons, which accounted for 61.1% of the microbial community. Leveraging the near-complete genomes, we also profiled the metabolic potential of the AS microbiome and identified 2153 biosynthetic gene clusters (BGCs) encoded within the recovered AS MAGs. Conclusion Our results established the feasibility of an iterative haplotype-resolved HCBHA approach to reconstruct (near-) complete genomes from highly complex ecosystems, providing new insights into “complete metagenomics”. The retrieved high-contiguity MAGs illustrated that various biosynthetic gene clusters (BGCs) were harbored in the AS microbiome. The high diversity of BGCs highlights the potential to discover new natural products biosynthesized by the AS microbial community, aside from the traditional function (e.g., organic carbon and nitrogen removal) in wastewater treatment. \n Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-021-01155-1.",
"conclusion": "Conclusions Overall, the iterative haplotype-resolved HCBHA approach developed addressed the challenge of HQ and high-contiguity (even complete) genome reconstruction from deeply sequenced, highly complex metagenomes. The high-contiguity MAGs greatly advance our capacity to capture the signals of some determinants, e.g., BGCs, allowing rapid and detailed screening of functional potential for the microbial community from complex environments. The wealth of identified SM biosynthetic potential reveals that the AS microbiome might be an untapped reservoir for discovering novel natural products, e.g., new antibiotics. Further experiments will be required to validate and decipher the functions of the SMs harbored in the AS microbiome.",
"introduction": "Introduction Rapid advances in long-read sequencing, also known as third-generation sequencing, achieved by Pacific Bioscience (PacBio) and Oxford Nanopore Technology (ONT), have demonstrated their ability to resolve genome assembly challenges [ 1 – 3 ], e.g., long repeat regions and structural variants [ 1 , 4 , 5 ]. Remarkably, the ONT platform can generate ultra-long reads of up to 2 Mb. On the one hand, the long read lengths could greatly improve the contiguity of genome assemblies [ 6 , 7 ]. On the other hand, contigs assembled from error-prone long reads have a much higher error rate at the nucleotide level than the short-read-based assemblies. Even with extensive short-read-based polishing, it is still challenging to generate a decent number of high-accuracy sequences, especially for metagenomes with high complexity [ 8 ]. Recently, select assemblers have been developed to take advantages of long reads, including the hybrid assemblers Unicycler (for bacterial isolates) [ 9 ] and OPERA-MS (for clinical metagenomics) [ 2 ], and long-read assemblers Canu [ 10 ] and Flye [ 11 ]. However, when implementing these assemblers to the complex environmental samples, limitations do apply, e.g., limited feasibility, accuracy, and demanding computational resources. Therefore, an urgent need to develop a new framework that could address the above issues and accurately characterize the high-complexity metagenome remains. Activated sludge (AS) is one of the primary engineered functional infrastructures associated with public health [ 12 ], harboring highly diverse but deficiently characterized microbial communities [ 13 ]. The microbial community in the AS is essential for wastewater treatment to remove pollutants (organic carbon, nitrogen, phosphorus, and other toxicants), playing a crucial role in building a sustainable modern society [ 13 ]. Therefore, it is important to enhance our understanding to decipher the ecology of the AS microbial community, which may provide new insights into future management strategies of the AS process. Many studies rely on 16S rRNA gene sequencing to capture AS microbial community profiles for linking the microbial composition to potential functions [ 14 – 16 ]. However, connecting the 16S rRNA gene-based microbial profile to the function is very limited due to the challenges of reconstructing HQ metagenome-assembled genomes (MAGs) that included 16S rRNA genes from the AS ecosystem [ 17 , 18 ]. AS is a diverse environment. Generally, more than 700 genera and a few thousands of operational taxonomic units (OTUs) are observed in the AS [ 14 , 15 , 19 ]. Besides, it is estimated that AS bacterial communities contain about 1 billion bacterial phylotypes, far more complex (at least one order of magnitude greater OTU richness) than those observed in the human gut microbiome [ 13 ]. Although sequencing and computational advances, e.g., high-throughput short-read sequencing and accurate assembly/binning tools are available [ 20 ], it is difficult to resolve the long repeat regions, as well as to recruit rRNA operons in the genome. These challenges result in the assembly of highly fragmented draft genomes [ 17 , 21 ], instead of HQ genomes containing full-length rRNA genes. Therefore, the high microbial diversity and complex genome characteristics hinder reference-quality genome reconstruction from AS ecosystems, which limited our capacity to obtain a comprehensive understanding of the AS microbiome. In this study, we employed a hybrid sequencing strategy and established a feasible iterative haplotype-resolved hierarchical clustering-based hybrid assembly (HCBHA) workflow for high-complexity ecosystems. Benchmarking results based on a mixed mock metagenomic dataset revealed that the average genome quality of the genome reconstructed using the HCBHA workflow achieved Q40, even better than that of recovered from a pure-culture dataset using the short-read-based method. Taking advantages of the high accuracy of Illumina short reads and the large span of ONT long reads, we reconstructed 557 bacterial and archaeal genomes, including 14 complete and 111 high-quality genomes containing full-length rRNA operons from a deeply sequenced, highly complex AS sample. The contiguity (in terms of N50) of these retrieved 557 genomes was improved by an order of magnitude when compared to a short-read-based method. Leveraging the high-contiguity MAGs (average N50 = 574 Kb), we reconstructed the keystone metabolic pathways and identified various biosynthetic gene clusters (BGCs) harbored in the AS microbiome from a wastewater treatment plant (WWTP). The wide range of BGCs highlights the potential versatility of the AS microbiome.",
"discussion": "Discussion The complexity of environmental samples, including the strain diversity and abundance of the microbial community, influences our ability to reconstruct genomes from metagenomes accurately. Currently, the high demand for computational resources and their large proportion in the total project budget for deeply sequenced, highly complex metagenomes are bottlenecks, limiting our capability to delineate the microbial communities in complex microbiome investigations, e.g., soil, marine, and human gut microbiomes [ 58 , 59 ]. To provide a workable solution, we proposed the iterative framework to reconstruct reference-quality genomes from highly complex samples. In addition to the much-improved genome reconstruction performance, the modular characteristic of our workflow enables the full integration of state-of-the-art tools. Furthermore, the haplotype-resolved workflow untangles the complexity of metagenomes by phasing reads into different lineages (candidate bins) and achieving hybrid assembly of microbial populations at the species or strain level. Our study and other works have demonstrated that long reads could empower complete genomes reconstruction from metagenomes [ 2 , 18 , 24 ]. The proposed iterative strategy [ 24 ] and haplotype-resolved method [ 60 ] could provide additional insights into launching the new era of “complete metagenomics” [ 61 ], enabling more circular, closed genomes to be deposited into reference databases [ 18 ]. The collected 125 HQ/complete MAGs harbored full-length rRNA genes, accounting for nearly 30% of the Shatin AS microbial community. Notably, full-length 16S rRNA genes were identified within 410 MAGs, representing 47.5% microbial community (Additional file 1 , S3). Linking 16S rRNA genes to the reconstructed MAGs enables precise prediction of potential functional profiles when relying on the cost-effective amplicon sequencing to monitor microbial communities in the local WWTP, especially considering the poor representation of high-quality AS genomes in public reference databases [ 62 ] and high diversity of the AS microbiome [ 13 ]. The reconstructed high-quality genomes will also help to improve functional prediction accuracy within similar lineages. We reconstructed multiple highly contiguous and accurate MAGs from the AS system, allowing us to profile the microbial community and potential metabolic traits in high resolution. Genome-centric analysis reveals the prevalence of truncated pathways within the reconstructed AS microbiome. For instance, microbes harboring the incomplete denitrification pathway are more than those encoding the complete pathway (Fig. 3 c). Cross-feeding benefits among microbes have been demonstrated in previous studies [ 63 – 65 ]; the synergistic network may promote the growth of relevant cohorts in the microbial community. Nonetheless, how the truncated and complete pathways affect the resistance and resilience of the microbial community under disturbance remains unclear. Additionally, although many genes encoded by MAGs are involved in the undesired process or intermediate products’ generation in the WWTP, e.g., the nitrite ammonification process and the release of N 2 O (Fig 4 b) [ 66 , 67 ], the expression and impact of these transformations require further investigation. To the best of our knowledge, the current study demonstrated the occurrence and distribution of BGCs harbored in the AS microbiome for the first time. The observed highly abundant and diverse BGCs identified in the reconstructed high-contiguity MAGs suggested that the AS microbiome might be a pristine treasure for the discovery of novel and valuable microbial bio-products. Besides, the lineage-specific analysis of BGCs provides potential phylogenetic targets for the generation of secondary metabolites of interest. However, the potential versatility of the AS microbiome was only demonstrated based on reconstructed MAGs and many BGCs are presumably inactive [ 68 , 69 ], confident links between diverse identified BGCs to the production of secondary metabolites require further metatranscriptomics and chemical screening (e.g., liquid chromatography-mass spectrometry) lines-of-evidence. Furthermore, induction mechanisms of these silent BGCs within the AS microbial consortia should also be discovered for developing new biotechnological and medical applications in the future. Considering that AS is a highly populated environment, microbial interaction may be more effective than in other dilute settings. Thus, investigation of BGCs in diverse environments may provide new insights into intracellular and intercellular roles of these secondary metabolites in nature [ 53 ]. Our iterative haplotype-aware approach recovered many (near-) complete MAGs from the highly complex AS metagenome; some previously known issues might remain, such as chimeric bins and inaccessible genome gaps. Thus, biological knowledge of specific populations and individual detailed manual curations might be required [ 70 ], although largely reduced. Besides, a significant proportion of microbial communities (~ 40%) were still not recovered; therefore, enrichment or isolation efforts should be considered to approach the comprehensive picture of the AS microbiome."
} | 3,224 |
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