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Methanogenic processes have great potential in the sustainable treatment of organic wastes with the production of methane as a renewable source of energy. However, the broader application of methan...

Theionarchaea is a recently described archaeal class within the Euryarchaeota. While it is widely distributed in sediment ecosystems, little is known about its metabolic potential and ecological features. Here, we used metagenomics and metatranscriptomics to characterize 12 theionarchaeal metagenome-assembled genomes, which were further divided into two subgroups, from coastal mangrove sediments of China and seawater columns of the Yap Trench. Genomic analysis revealed that apart from the canonical sulfhydrogenase, Theionarchaea harbor genes encoding heliorhodopsin, group 4 [NiFe]-hydrogenase, and flagellin, in which genes for heliorhodopsin and group 4 [NiFe]-hydrogenase were transcribed in mangrove sediment. Further, the theionarchaeal substrate spectrum may be broader than previously reported as revealed by metagenomics and metatranscriptomics, and the potential carbon substrates include detrital proteins, hemicellulose, ethanol, and CO2. The genes for organic substrate metabolism (mainly detrital protein and amino acid metabolism genes) have relatively higher transcripts in the top sediment layers in mangrove wetlands. In addition, co-occurrence analysis suggested that the degradation of these organic compounds by Theionarchaea might be processed in syntrophy with fermenters (e.g., Chloroflexi) and methanogens. Collectively, these observations expand the current knowledge of the metabolic potential of Theionarchaea, and shed light on the metabolic strategies and roles of these archaea in the marine ecosystems.

Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated ω-3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6-ndh, with high ethanol yield both under anaerobic and aerobic conditions, was taken as the genetic background for inactivation of levansucrase (sacB). SacB mutation eliminated the levan-forming activity on sucrose. The double mutant Zm6-ndh-sacB cells were immobilized in Ca alginate, and applied for syntrophic conversion of sucrose to DHA of C. cohnii, either taking the ethanol-containing fermentation medium from the immobilized Z. mobilis for feeding to the C. cohnii fed-batch culture, or directly coculturing the immobilized Zm6-ndh-sacB with C. cohnii on sucrose. Both modes of cultivation produced C. cohnii CCMP 316 biomass with DHA content around 2-3 % of cell dry weight, corresponding to previously reported results for this strain on glucose.

Direct interspecies electron transfer (DIET) between microbial species prevails in some key microbial consortia. However, the electron transfer mechanism(s) in these consortia is controversial due to lack of efficient characterization methods. Here, we provide an in situ anaerobic spectroelectrochemical coculture cell (in situ ASCC) to induce the formation of DIET coculture biofilm on the interdigitated microelectrode arrays and characterize the electron transfer directly. Two typical Geobacter DIET cocultures, Geobacter metallireducens and wild-type Geobacter sulfurreducens (G.m&G.s) and G. metallireducens and a G. sulfurreducens strain deficient in citrate synthase (G.m&G.s-ΔgltA), were selected. In situ Raman and electrochemical Fourier transform infrared (FTIR) spectroscopy indicated that cytochromes are abundant in the electric syntrophic coculture. Cyclic voltammetry and potential step experiment revealed a diffusion-controlled electron transfer process and the electrochemical gating measurements further demonstrated a cytochrome-mediated electron transfer in the DIET coculture. Furthermore, the G.m&G.s-ΔgltA coculture displayed a higher redox conductivity than the G.m&G.s coculture, consistent with the existence of an intimate and efficient electrical connection between these two species. Our findings provide the first report of a redox-gradient-driven electron transport facilitated by c-type cytochromes in DIET coculture, supporting the model that DIET is mediated by cytochromes and suggest a platform to explore the other DIET consortia.

The evolution of multicellularity paved the way for significant increases in biological complexity. Although multicellularity has evolved many times independently, we know relatively little about its origins. Directed evolution is a promising approach to studying early steps in this major transition, but current experimental systems have examined only a subset of the possible evolutionary routes to multicellularity. Here we consider egalitarian routes to multicellularity, in which unrelated unicellular organisms evolve to become a multicellular organism. Inspired by microbial syntrophies and lichens, we outline three such routes from a system of different species to an interdependent relationship that replicates. We compare these routes to contemporary experimental systems and consider how physical structure, the threat of invasion, division of labour and co-transmission affect their evolution.

How the acetate and propionate accumulation impact anaerobic syntrophy during methane formation is not well understood. To investigate such effect, continuous acetate (35 g/L), propionate (11.25 g/L) and bicarbonate (30 g/L) supplementation were used during mesophilic anaerobic digestion. The high throughput sequencing (16S rRNA and mcrA), Real-Time quantitative PCR, and stable carbon isotope fingerprinting were applied to investigate the structure and activity of microbial community members. The results demonstrated that the abundance of syntrophic acetate oxidizing bacteria exhibited a gradual decrease coupled with heavier stable carbon isotopic signature of methane (δ 13CH4) in the three reagents impacted reactors. The increased acetate and propionate concentrations exerted negative influence on biogas production but the relatively stable hydrogenotrophic methanogens together with syntrophic acetate/propionate oxidizing bacteria kept the stable methane formation facing acetate and propionate accumulation. The functional genes copy number of the hydrogenotrophic Methanocellaceae and Methanomicrobiaceae correlated significantly with δ 13CH4 (R2 > 0.74), but only the abundance of Methanocellaceae fitted well with δ 13CH4 (p

Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here, we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in coculture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in M. acetivorans grown in coculture versus pure-culture growth on acetate revealed that transcripts for the outer-surface multiheme c-type cytochrome MmcA were higher during DIET-based growth. Deletion of mmcA inhibited DIET. The high aromatic amino acid content of M. acetivorans archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based coculture as well as the archaellum-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based cocultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outer-surface electrical contacts for DIET in a methanogen. IMPORTANCE The conversion of organic matter to methane plays an important role in the global carbon cycle and is an effective strategy for converting wastes to a useful biofuel. The reduction of carbon dioxide to methane accounts for approximately a third of the methane produced in anaerobic soils and sediments as well as waste digesters. Potential electron donors for carbon dioxide reduction are H2 or electrons derived from direct interspecies electron transfer (DIET) between bacteria and methanogens. Elucidating the relative importance of these electron donors has been difficult due to a lack of information on the electrical connections on the outer surfaces of methanogens and how they process the electrons received from DIET. Transcriptomic patterns and gene deletion phenotypes reported here provide insight into how a group of Methanosarcina organisms that play an important role in methane production in soils and sediments participate in DIET.

Heavy metal and petroleum contaminated sites comprise a mixed culture of inherent microbial species yet the occurrence of competition for nutrients causes interference within species cooperating the biodegradation. Insights into the biodiversity of the contaminated sites and extreme conditions of oil reservoirs decipher the presence of both hydrocarbon degraders and biosurfactant-producing microbes. Biosurfactant-producing strains improves the bioavailability of hydrocarbons by emulsifying them or solubilize them using micellar structure formation, making it easy for the hydrocarbon degraders to metabolize them. Such inherent microbes can be designed into a stable microbial consortium harboring resilience, division of labor and resistance to extreme environmental drifts in order to improve the overall biodegradation efficiency. A top-down approach involves selectively enriching the active degraders within the inherent microbes. Yet, non-cultivable microbes are neglected in this approach reducing the overall biodegradation efficiency. Another bottom-up approach allows the inclusion of engineered microbes with equipped enzymes with the indigenous microbes for improving the overall consortium performance. Studies support the suitability of mutualism and commensalism as key interactions for consortium stability. Various phenomena such as syntrophy, biotransformation, detoxification and biofilm formation are the result of such interaction within a consortium. However, the culture conditions for these mutually interactive species need to be further optimized to improve the overall biodegradation and biosurfactant production. This chapter reports the various micro-consortium designs prepared to improve the overall biodegradation efficiency over axenic cultures.

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H(2), CO(2), or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO(2) to methane by electrons derived from β-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop–driven reduction of CO(2) by acyl–coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO(2) and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of β-oxidation in (methyl)menaquinone-containing organisms.

The origin of eukaryotes remains unclear1–4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as ‘Asgard’ archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon—‘Candidatus Prometheoarchaeum syntrophicum’ strain MK-D1—is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle–engulf–endogenize (also known as E3) model., Isolation and characterization of an archaeon that is most closely related to eukaryotes reveals insights into how eukaryotes may have evolved from prokaryotes.

In order to understand the subsurface microbial community and how it functions in a water-flooded reservoir, the whole metagenome of fluids from production and injection wells were sequenced using next-generation sequencing and then compared. Gene function was annotated by the Kyoto Encyclopedia of Genes and Genomes (KEGG), evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) and hydrocarbon degradation databases. More biomass was detected in fluid from the injection well than in the production well. As a typical water-flooded reservoir, injection and production communities were dominated by Proteobacteria. In the production metagenome, more genes involved in denitrification and hydrocarbons utilization pathways, reflecting the strict anaerobic and oil-rich conditions in reservoirs. The higher abundance of genes responsible for methyl chemotaxis, aerotaxis and flagella indicate that the community in reservoirs is motivated to access limited nutrients and electron acceptors. Combined with knowledge of porous flow, these metagenome results indicate that the microbial communities detected in produced fluids are actually derived from different habitats in the reservoir, which are established by the chromatographic effect of reservoir flooding. Furthermore, diverse flow patterns at pore scale could affect in situ proliferation and syntrophy between species, which could considerably shift the microbial composition in produced fluids.

In microbial fermentations at least 33% of the sugar-substrate carbon is lost as CO2 during pyruvate decarboxylation to acetyl-CoA, with the corresponding electrons lost in the form of H2. Previous attempts to reduce this carbon and electron loss focused on engineering of a single organism. In nature, most microorganisms live in complex communities where syntrophic interactions result in superior resource utilization. Here, we show that a synthetic syntrophy consisting of the solventogen Clostridium acetobutylicum, which converts simple and complex carbohydrates into a variety of chemicals, and the acetogen C. ljungdahlii which fixes CO2, achieved carbon recoveries into C2-C4 alcohols almost to the limit of substrate-electron availability, with minimal H2 and CO2 release. The syntrophic co-culture produced robust metabolic outcomes over a broad range of starting population ratios of the two organisms. We show that direct cell-to-cell interactions and material exchange among the two microbes enabled unforeseen rearrangements in the metabolism of the individual species that resulted in the production of non-native metabolites, namely isopropanol and 2,3-butanediol. This was accomplished by pathway-specific alterations of gene expression brought about by one organism on the other, and vice versa. While some of these gene-expression alterations can be explained by the exchange of metabolites that induce specific gene expression patterns, others, as demonstrated by co-culture setup in a transwell system, cannot. The latter, for now, would be attributed to complex direct physical interactions among the two organisms, thus providing a glimpse of the potential microbial complexity of simple or multicomponent microbiomes. Such direct material-transfer phenomena have not been documented in the literature. Furthermore, our study shows that syntrophic cultures offer a flexible platform for metabolite production with superior carbon recovery that can also be applied to electron-enhanced fermentations enabling even higher carbon recoveries.

We experimentally and theoretically investigated implications of H2 and a rate-limiting step in a mixed-culture biofilm anode fed with n-butyrate, one of the poorest substrates to exoelectrogens. Acetate and i-butyrate were formed as intermediates during anaerobic degradation of n-butyrate, which suggested oxidative acetogenesis of n-butyrate in syntrophy with H2 scavengers in the biofilm anode. Methane was not detected in an anode chamber, and no current was generated in the biofilm anode using H2 as the electron donor. These results indicated that acetogens would be a main H2 consumer in the biofilm. Pyrosequencing data showed dominance of Geobacter in the biofilm anode (83.6% of total sequences), along with Sphaerochaeta and Treponema, which supports the syntrophy between exoelectrogens and acetogens. Electrical conductivity of the butyrate-fed biofilm anode was as high as 0.67 mS/cm, demonstrating that EET does not limit current density in the biofilm. In-situ monitoring of dissolved H2 concentration proved H2 production (up to 12.4 µM) and consumption during current generation in the biofilm, which supports significance of H2–based syntrophy in the electrically conductive biofilm using n-butyrate as the primary electron donor.

Anaerobic oxidation of methane (AOM) microorganisms widespread in nature and they are able to utilize methane as electron donor to reduce sulfate, nitrate, nitrite, and high valence metals. However, whether persistent organic contaminants can also be degraded remains unknown. In this study, the organic pollutant methyl orange (MO) was used to address this open question. The initial concentration of MO affected its degradation efficiency and higher concentration (>100 mg/L) caused considerable inhibition. A 13CH4 isotope experiment indicated that methane oxidation was involved in MO degradation, which produced N, N-dimethyl-p-phenylenediamine, and 4-aminobenzenesulfonic acid corresponded stoichiometrically. During the long-term experiment, the maximum degradation rate was 47.91 mg/(L·d). The percentage of Candidatus Methanoperedens and Pseudoxanthomonas significantly increased after 30-d of MO degradation under CH4 conditions; moreover, Candidatus Methanoperedens dominated (46.83%) the microbial community. Candidatus Methanoperedens, either alone or in combination with Pseudoxanthomonas, utilized methane as the sole carbon source to degrade MO via direct interspecies electron transfer or the syntrophy pathway. This study will add to our understanding of the functions and applications of AOM microorganisms.

Interspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET) are two syntrophy models for methanogenesis. Their relative importance in methanogenic environments is still unclear. Our recent discovery of a novel species Candidatus Geobacter eutrophica with the genetic potential of IHT and DIET may serve as a model species to address this knowledge gap. To experimentally demonstrate its DIET ability, we performed electrochemical enrichment of Ca. G. eutrophica-dominating communities under 0 and 0.4 V vs. Ag/AgCl based on the presumption that DIET and extracellular electron transfer (EET) share similar metabolic pathways. After three batches of enrichment, Geobacter OTU650, which was phylogenetically close to Ca. G. eutrophica, was outcompeted in the control but remained abundant and active under electrochemical stimulation, indicating Ca. G. eutrophica’s EET ability. The high-quality draft genome further showed high phylogenomic similarity with Ca. G. eutrophica, and the genes encoding outer membrane cytochromes and enzymes for hydrogen metabolism were actively expressed. A Bayesian network was trained with the genes encoding enzymes for alcohol metabolism, hydrogen metabolism, EET, and methanogenesis from dominant fermentative bacteria, Geobacter, and Methanobacterium. Methane production could not be accurately predicted when the genes for IHT were in silico knocked out, inferring its more important role in methanogenesis. The genomics-enabled machine learning modeling approach can provide predictive insights into the importance of IHT and DIET.

Sulfate-coupled anaerobic oxidation of methane (AOM) is a major methane sink in marine sediments. Multiple lineages of anaerobic methanotrophic archaea (ANME) often coexist in sediments and catalyze this process syntrophically with sulfate-reducing bacteria (SRB), but the potential differences in ANME ecophysiology and mechanisms of syntrophy remain unresolved. A humic acid analog, anthraquinone 2,6-disulfonate (AQDS), could decouple archaeal methanotrophy from bacterial sulfate reduction and serve as the terminal electron acceptor for AOM (AQDS-coupled AOM). Here in sediment microcosm experiments, we examined variations in physiological response between two co-occurring ANME-2 families (ANME-2a and ANME-2c) and tested the hypothesis of sulfate respiration by ANME-2. Sulfate concentrations as low as 100 µM increased AQDS-coupled AOM nearly 2-fold matching the rates of sulfate-coupled AOM. However, the SRB partners remained inactive in microcosms with sulfate and AQDS and neither ANME-2 families respired sulfate, as shown by their cellular sulfur contents and anabolic activities measured using nanoscale secondary ion mass spectrometry. ANME-2a anabolic activity was significantly higher than ANME-2c, suggesting that ANME-2a was primarily responsible for the observed sulfate stimulation of AQDS-coupled AOM. Comparative transcriptomics showed significant upregulation of ANME-2a transcripts linked to multiple ABC transporters and downregulation of central carbon metabolism during AQDS-coupled AOM compared to sulfate-coupled AOM. Surprisingly, genes involved in sulfur anabolism were not differentially expressed during AQDS-coupled AOM with and without sulfate amendment. Collectively, this data indicates that ANME-2 archaea are incapable of respiring sulfate, but sulfate availability differentially stimulates the growth and AOM activity of different ANME lineages.

Syntrophy is a thermodynamically required mutualistic cooperation between fatty acid-oxidizing bacteria and methanogens that plays the important role in organic decomposition and methanogenesis in anoxic environments. In this study, three experiments were conducted to evaluate the cell-to-cell interaction in a thermophilic coculture consisting of Syntrophothermus lipocalidus and Methanocella conradii and a mesophilic coculture consisting of Syntrophomonas wolfei and Methanococcus maripaludis. First, syntrophs and methanogens were inoculated at different initial cell ratios to evaluate the growth synchronization. The quantitative PCR analysis revealed that the organism with a lower relative abundance at the beginning always grew faster, and the cell ratio converged over time to relative constant values in both the thermophilic and mesophilic cocultures. Next, intermittent ultrasound and constant shaking treatments were used to evaluate the influence of physical disturbance on microbial aggregation in the mesophilic coculture. The fluorescence in situ hybridization and scanning electron microscopy revealed that the tendency of syntrophic aggregation was not affected by the physical disturbances, although the activity was slightly depressed. Syntrophomonas dominated in the initial microbial aggregates, which, however, did not grow until Methanococcus was attached and increased to a significant extent, indicating the local growth synchronization during the formation and maturation of syntrophic aggregates. Last, microfluidic experiments revealed that whether or not Syntrophomonas or Methanococcus was loaded first, the second organism preferred moving to the place where the first organism was located, suggesting the cell-to-cell attraction between Syntrophomonas and Methanococcus. Collectively, our study demonstrated the growth synchronization and cell-to-cell attraction between the butyrate-oxidizing bacteria and methanogens for optimizing the syntrophic cooperation.

BackgroundAcetate is the major intermediate of anaerobic digestion of organic waste to CH4. In anaerobic methanogenic systems, acetate degradation is carried out by either acetoclastic methanogenesis or a syntrophic degradation by a syntrophy of acetate oxidizers and hydrogenotrophic methanogens. Due to challenges in isolation of syntrophic acetate-oxidizing bacteria (SAOB), the diversity and metabolism of SAOB, as well as the mechanisms of their interactions with methanogenic partners remain poorly understood.ResultsIn this study, we successfully enriched previously unknown SAOB by operating continuous thermophilic anaerobic chemostats fed with acetate, propionate, butyrate, or isovalerate as the sole carbon and energy source. They represent novel clades belonging to Clostridia, Thermoanaerobacteraceae, Anaerolineae, and Gemmatimonadetes. In these SAOB, acetate is degraded through reverse Wood-Ljungdahl pathway or an alternative pathway mediated by the glycine cleavage system, while the SAOB possessing the latter pathway dominated the bacterial community. Moreover, H2 is the major product of the acetate degradation by these SAOB, which is mediated by [FeFe]-type electron-confurcating hydrogenases, formate dehydrogenases, and NADPH reoxidation complexes. We also identified the methanogen partner of these SAOB in acetate-fed chemostat, Methanosarcina thermophila, which highly expressed genes for CO2-reducing methanogenesis and hydrogenases to supportively consuming H2 at transcriptional level. Finally, our bioinformatical analyses further suggested that these previously unknown syntrophic lineages were prevalent and might play critical roles in thermophilic methanogenic reactors.ConclusionThis study expands our understanding on the phylogenetic diversity and in situ biological functions of uncultured syntrophic acetate degraders, and presents novel insights on how they interact with their methanogens partner. These knowledges strengthen our awareness on the important role of SAO in thermophilic methanogenesis and may be applied to manage microbial community to improve the performance and efficiency of anaerobic digestion.

About 382 Tg yr−1 of methane rising through the seafloor is oxidized anaerobically (W. S. Reeburgh, Chem Rev 107:486–513, 2007, https://doi.org/10.1021/cr050362v), preventing it from reaching the atmosphere, where it acts as a strong greenhouse gas. Microbial consortia composed of anaerobic methanotrophic archaea and sulfate-reducing bacteria couple the oxidation of methane to the reduction of sulfate under anaerobic conditions via a syntrophic process. Recent experimental studies and modeling efforts indicate that direct interspecies electron transfer (DIET) is involved in this syntrophy. Here, we explore a fluorescent in situ hybridization-nanoscale secondary ion mass spectrometry data set of large, segregated anaerobic oxidation of methane (AOM) consortia that reveal a decline in metabolic activity away from the archaeal-bacterial interface and use a process-based model to identify the physiological controls on rates of AOM. Simulations reproducing the observational data reveal that ohmic resistance and activation loss are the two main factors causing the declining metabolic activity, where activation loss dominated at a distance of IMPORTANCE Anaerobic oxidation of methane is a globally important, microbially mediated process reducing the emission of methane, a potent greenhouse gas. In this study, we investigate the mechanism of how a microbial consortium consisting of archaea and bacteria carries out this process and how these organisms interact with each other through the sharing of electrons. We present a process-based model validated by novel experimental measurements of the metabolic activity of individual, phylogenetically identified cells in very large (>20-μm-diameter) microbial aggregates. Model simulations indicate that extracellular electron transfer between archaeal and bacterial cells within a consortium is limited by potential losses and suggest that a flexible use of electron donors can provide energetic advantages for syntrophic consortia.

Microbial syntrophy (obligate metabolic mutualism) is the hallmark of energy-constrained anaerobic microbial ecosystems. For example, methanogenic archaea and fermenting bacteria coexist by interspecies hydrogen transfer in the complex microbial ecosystem in the foregut of ruminants; however, these synergistic interactions between different microbes in the rumen are seldom investigated. We hypothesized that certain bacteria and archaea interact and form specific microbial cohorts in the rumen. To this end, we examined the total (DNA-based) and potentially metabolically active (cDNA-based) bacterial and archaeal communities in rumen samples of dairy cows collected at different times in a 24 h period. Notably, we found the presence of distinct bacterial and archaeal networks showing potential metabolic interactions that were correlated with molar proportions of specific volatile fatty acids (VFAs). We employed hypothesis-driven structural equation modeling to test the significance of and to quantify the extent of these relationships between bacteria-archaea-VFAs in the rumen. Furthermore, we demonstrated that these distinct microbial networks were host-specific and differed between cows indicating a natural variation in specific microbial networks in the rumen of dairy cows. This study provides new insights on potential microbial metabolic interactions in anoxic environments that have broader applications in methane mitigation, energy conservation, and agricultural production.

Anaerobic digestion encounters operational instability due to fluctuations in organic loading. Propionic acid (HPr) is frequently accumulated due to its unfavorable reaction thermodynamics. Here, ‘specific’ bioaugmentation using HPr enrichment cultures (three different injection regimes of quantity and frequency) was compared with ‘non-specific’ bioaugmentation using anaerobic sludge, and with non-biological supplementation of magnetite or coenzyme M. The specific bioaugmentation treatments showed superior recovery responses during continuous feeding after a peak overload. A ‘one-shot’ bioaugmentation with enrichment showed the best remediation, with ~25% recovery time and >10% CH4 conversion efficiency compared to the control. Consecutive bioaugmentation showed evidence of increased stability of the introduced community. Families Synergistaceae, Syntrophobacteraceae, and Kosmotogaceae were likely responsible for HPr-oxidation, in potential syntrophy with Methanoculleus and Methanobacterium. The different supplementation strategies can be considered to reduce the effect of start-up or overload in anaerobic digesters based on the availability of supplementation resources.

Methanogenesis is central to anaerobic digestion processes. The conversion of propionate as a key intermediate for methanogenesis requires syntrophic interactions between bacterial and archaeal partners. In this study, a series of methanogenic enrichments with propionate as the sole substrate were developed to identify microbial populations specifically involved in syntrophic propionate conversion. These rigorously controlled propionate enrichments exhibited functional stability with consistent propionate conversion and methane production; yet, the methanogenic microbial communities experienced substantial temporal dynamics, which has important implications on the understanding of mechanisms involved in microbial community assembly in anaerobic digestion. Syntrophobacter was identified as the most abundant and consistent bacterial partner in syntrophic propionate conversion regardless of the origin of the source culture, the concentration of propionate, or the temporal dynamics of the culture. In contrast, the methanogen partners involved in syntrophic propionate conversion lacked consistency, as the dominant methanogens varied as a function of process condition and temporal dynamics. Methanoculleus populations were specifically enriched as the syntrophic partner at inhibitory levels of propionate, likely due to the ability to function under unfavorable environmental conditions. Syntrophic propionate conversion was carried out exclusively via transformation of propionate into acetate and hydrogen in enrichments established in this study. Microbial populations highly tolerant of elevated propionate, represented by Syntrophobacter and Methanoculleus, are of great significance in understanding methanogenic activities during process perturbations when propionate accumulation is frequently encountered. Key points • Syntrophobacter was the most consistent bacterial partner in propionate metabolism. • Diverse hydrogenotrophic methanogen populations could serve as syntrophic partners. • Methanoculleus emerged as a methanogen partner tolerant of elevated propionate.

Fermentative chemoorganoheterotrophic bacteria (FCB) and purple photoorganoheterotrophic bacteria (PPB) are two interesting microbial guilds to process carbohydrate-rich wastewaters. Their interaction has been studied in axenic pure cultures or co-cultures. Little is known about their metabolic interactions in open cultures. We aimed to harness the competitive and syntrophic interactions between PPB and FCB in mixed cultures. We studied the effect of reactor regimes (batch or continuous, CSTR) and illumination modes (continuous irradiation with infrared light, dark, or light/dark diel cycles) on glucose conversions and the ecology of the process. In batch, FCB outcompeted (>80%) PPB, under both dark and infrared light conditions. In CSTR, three FCB populations of Enterobacteriaceae, Lachnospiraceae and Clostridiaceae were enriched (>70%), while Rhodobacteraceae relatives of PPB made 30% of the community. Fermentation products generated from glucose were linked to the dominant FCB. Continuous culturing at a dilution rate of 0.04 h-1 helped maintain FCB and PPB in syntrophy: FCB first fermented glucose into volatile fatty acids and alcohols, and PPB grew on fermentation products. Direct supply of carboxylates like acetate under infrared light enriched for PPB (60%) independent of reactor regimes. Ecological engineering of FCB- and PPB-based biorefineries can help treat and valorize carbohydrate-based waste feedstocks. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/444055v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1ee41forg.highwire.dtl.DTLVardef@12d74aorg.highwire.dtl.DTLVardef@f46351org.highwire.dtl.DTLVardef@a47706_HPS_FORMAT_FIGEXP M_FIG C_FIG

Profound biogeochemical responses of anoxic sediments to the fluctuation of dissolved oxygen (DO) concentration in overlaying water are often observed, despite oxygen having a limited permeability in sediments. This contradiction is indicative of previously unrecognized mechanism that bridges the oxic and anoxic sediment layers. Using sediments from an urban river suffering from long-term polycyclic aromatic hydrocarbons (PAHs) contamination, we analyzed the physicochemical and microbial responses to artificially elevated DO (eDO) in the overlying water over 9 weeks of incubation. Significant changes in key environmental parameters and microbial diversity were detected over the 0-6 cm sediment depth, along with accelerated degradation of PAHs, despite that eDO only increased the porewater DO in the millimeter subfacial layer. The dynamics of physicochemical and microbial properties coincided well with significantly increased presence of centimeter-long sulfide-oxidizing cable bacteria filaments under eDO, and were predominantly driven by cable bacteria metabolic activities. Phylogenetic ecological network analyses further revealed that eDO reinforced cable bacteria associated interspecific interactions with functional microorganisms such as sulfate reducers, PAHs degraders, and electroactive microbes, suggesting enhanced microbial syntrophy taking advantage of cable bacteria metabolism for the regeneration of SO42- and long-distance electron transfer. Together, our results suggest cable bacteria may mediate the impacts of eDO in anaerobic sediments by altering sediment physiochemical properties and by reinforcing community interactions. Our findings highlight the ecological importance of cable bacteria in sediments.

Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N2 fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A (Candidatus Atelocyanobacterium thalassa). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N2 fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems., Author summary Reduction of dinitrogen gas to biologically useful forms via nitrogen fixation is a key component of the biogeochemical cycle. In the marine environment, the cyanobacteria UCYN-A (Candidatus Atelocyanobacterium thalassa) has been found to be a primary contributor to biological nitrogen fixation at a global scale. UCYN-A exhibits a highly streamlined genome which lacks genes coding for essential cyanobacterial processes such as the energy-generating TCA cycle, oxygen-producing photosystem II, the carbon-fixing RuBisCo and de novo production pathways for numerous amino acids and nucleotides. Thus, it exists in a symbiosis with unicellular planktonic algae where it exchanges fixed nitrogen for fixed carbon with its host. However, both UCYN-A and its symbiotic partner remain uncultured under laboratory conditions. This necessitates implementing a computational approach to glean insights into UCYN-A’s unique physiology and metabolic processes governing the symbiotic association. To this end, we develop a constraints-based framework that infers all possible trophic scenarios consistent with the observed data. Possible mechanisms employed by UCYN-A to accommodate diazotrophy with daytime carbon fixation by the host (i.e., two mutually incompatible processes) are also elucidated. We envision that the developed framework using UCYN-A and its algal host will be used as a roadmap and motivate the study of similarly unique microbial systems in the future.

Extracellular electron transfer (EET) is an important biological process in microbial physiology as found in dissimilatory metal oxidation/reduction and interspecies electron transfer in syntrophy in natural environments. EET also plays a critical role in microorganisms relevant to environmental biotechnology in metal-contaminated areas, metal corrosion, bioelectrochemical systems, and anaerobic digesters. Geobacter species exist in a diversity of natural and artificial environments. One of the outstanding features of Geobacter species is the capability of direct EET with solid electron donors and acceptors, including metals, electrodes, and other cells. Therefore, Geobacter species are pivotal in environmental biogeochemical cycles and biotechnology applications. Geobacter sulfurreducens, a representative Geobacter species, has been studied for direct EET as a model microorganism. G. sulfurreducens employs electrically conductive pili (e-pili) and c-type cytochromes for the direct EET. The biological function and electronics applications of the e-pili have been reviewed recently, and this review focuses on the cytochromes. Geobacter species have an unusually large number of cytochromes encoded in their genomes. Unlike most other microorganisms, Geobacter species localize multiple cytochromes in each subcellular fraction, outer membrane, periplasm, and inner membrane, as well as in the extracellular space, and differentially utilize these cytochromes for EET with various electron donors and acceptors. Some of the cytochromes are functionally redundant. Thus, the EET in Geobacter is complicated. Geobacter coordinates the cytochromes with other cellular components in the elaborate EET system to flourish in the environment.

In this study, the microbiomes linked with the operational parameters in seven mesophilic full-scale AD plants mainly treating food waste (four plants) and sewage sludge (three plants) were analyzed. The results obtained indicated lower diversity and evenness of the microbial population in sludge digestion (SD) plants compared to food digestion (FD) plants. Candidatus Accumulibacter dominated (up to 42.1%) in SD plants due to microbial immigration from fed secondary sludge (up to 89%). Its potential activity in SD plants was correlated to H2 production, which was related to the dominance of hydrogenotrophic methanogens (Methanococcus). In FD plants, a balance between the hydrogenotrophic and methylotrophic pathways was found, while Flavobacterium and Levilinea played an important role during acidogenesis. Levilinea also expressed sensitivity to ammonia in FD plants. The substantial differences in hydraulic retention time (HRT), organic loading rate (OLR), and total ammonium nitrogen (TAN) among the studied FD plants did not influence the archaeal methane production pathway. In addition, the bacterial genera responsible for acetate production through syntrophy and homoacetogenesis (Smithella, Treponema) were present in all the plants studied.

Methanogens are microorganisms belonging to the Archaea domain and represent the primary source of biotic methane. Methanogens encode a series of enzymes which can convert secondary substrates into methane following three major methanogenesis pathways. Initially recognized as environmental microorganisms, methanogens have more recently been acknowledged as host-associated microorganisms after their detection and initial isolation in ruminants in the 1950s. Methanogens have also been co-detected with bacteria in various pathological situations, bringing their role as pathogens into question. Here, we review reported associations between methanogens and bacteria in physiological and pathological situations in order to understand the metabolic interactions explaining these associations. To do so, we describe the origin of the metabolites used for methanogenesis and highlight the central role of methanogens in the syntrophic process during carbon cycling. We then focus on the metabolic abilities of co-detected bacterial species described in the literature and infer from their genomes the probable mechanisms of their association with methanogens. The syntrophic interactions between bacteria and methanogens are paramount to gut homeostasis. Therefore, any dysbiosis affecting methanogens might impact human health. Thus, the monitoring of methanogens may be used as a bio-indicator of dysbiosis. Moreover, new therapeutic approaches can be developed based on their administration as probiotics. We thus insist on the importance of investigating methanogens in clinical microbiology.

How to feed an enzyme ethane When released from ocean floor seeps, small hydrocarbons are rapidly consumed by micro-organisms. Methane is highly abundant and is both produced and consumed by microbes through well understood biochemical pathways. Less well understood is how ethane, also a major natural component of gaseous hydrocarbons, is metabolized. To understand how microbes take advantage of this energy and carbon source, Hahn et al. solved the x-ray crystal structures of an enzyme they call ethyl coenzyme-M reductase, which converts ethane into the thioether ethyl-coenzyme M as the entry point for catabolism. They found an expanded active site and, using a xenon gas derivatization experiment, a distinctive tunnel through the protein that is proposed to permit access of the gaseous substrate. Science , abg1765, this issue p. 118

Background A bacterial consortium SCP comprising three bacterial members, viz. Stenotrophomonas acidaminiphila APG1, Pseudomonas stutzeri APG2 and Cellulomonas sp. APG4 was developed for degradation of the mono-azo dye, Reactive Blue 28. The genomic analysis of each member of the SCP consortium was done to elucidate the catabolic potential and role of the individual organism in dye degradation. Results The genes for glycerol utilization were detected in the genomes of APG2 and APG4, which corroborated with their ability to grow on a minimal medium containing glycerol as the sole co-substrate. The genes for azoreductase were identified in the genomes of APG2 and APG4, while no such trait could be determined in APG1. In addition to co-substrate oxidation and dye reduction, several other cellular functions like chemotaxis, signal transduction, stress-tolerance, repair mechanisms, aromatic degradation, and copper tolerance associated with dye degradation were also annotated. A model for azo dye degradation is postulated, representing the predominant role of APG4 and APG2 in dye metabolism while suggesting an accessory role of APG1. Conclusions This exploratory study is the first-ever attempt to divulge the genetic basis of azo-dye co-metabolism by cross-genome comparisons and can be harnessed as an example for demonstrating microbial syntrophy.

Introduction. Anaerobic digestion (AD) is considered one of the most beneficial technologies for the treatment of organic wastes, from which energy in form of biogas and digestate as a soil amendment can be obtained. However, AD often suffers from process instability due to high levels of inorganic toxic substances such as ammonium, phosphates, sulfates or metal ions, variations in operating parameters such as temperature and pH, as well as a lack of proper balance between microbial groups. One of the alternatives to combat these imbalances is bioaugmentation, which is defined as the addition of specific microorganisms (either pure cultures or microbial consortia) into the system to accomplish a certain function. In this work, hydrogenotrophic methanogens were used to improve the methane yield of a thermophilic AD process of the organic fraction of municipal solid wastes (OFMSW) in Mexico City. Methods. The OFMSW (substrate) and leachate (inoculum) were collected from a composting plant in Mexico City. The consortium containing the hydrogenotrophic methanogens was isolated from the leachate. A biochemical methane potential (BMP) test was performed to know if the consortium influenced the methane yield. A 1:1 volatile solids (VS) ratio substrate/inoculum was operated in 125 mL serum bottles with a working volume of 60 ml. The consortium was added to different volume ratios. The process lasted 32 days at 55° C and 60 rpm. The identification of the microbial communities of all treatment groups and consortium were done by high throughput DNA sequencing of bacterial and archaeal 16S rDNA libraries. Results. The highest yield >600 ml CH4/g VSadded was achieved when 25% of the consortium was added followed by 50%; however, between these two treatments, there was not a statistically significant difference. The rest of the treatments were under 600 ml CH4/g VSadded. Nevertheless, it should be noted that the treatment at 25% reached its methane peak after 10 days, meanwhile, the treatment at 50% reached it on approximately 8 days, suggesting in this last treatment, the generation of methane was more accelerated. The methane yield increased by approximately 7% and 5% compared to the control (0%), while using 25% and 50% of the consortium, respectively. Regarding the bacterial communities, it was found that the consortium was mainly composed of members of the phylum Synergistetes. Members of this phylum are known for their acetate oxidizing properties. In all the treatments the phyla Firmicutes, Thermotogae, and Synergistetes were dominant and as the concentration of the consortium in the treatments increased, the presence of the family Thermotogaceae also increased. Conclusions. In this research, we detected that the bioaugmentation using hydrogenotrophic methanogens improved the methane yield of the AD process by more than 5%. The bacterial communities present in the consortium have capacities to degrade cellulolytic compounds and act as acetate oxidants, which play a role in syntrophy with hydrogenotrophic methanogens, however, it is still necessary to know the dynamics of the methanogens. Acknowledgments. The project was supported by CIIEMAD IPN SIP 20172277, 20180942, 20195607, and CONACyT-163235 INFR-2011-01. AKGB was granted a CONACyT-700337 doctorate scholarship.

We report that two different bacterial organisms engage in heterologous cell fusion that leads to massive exchange of cellular material, including proteins and RNA, and the formation of persistent hybrid cells. The interspecies cell fusion observed here involves a syntrophic microbial system, but these heterologous cell fusions were observed even under nonstrict syntrophic conditions, leaving open the possibility that strict syntrophy may not be necessary for interspecies cell fusion and cellular material exchange. Formation of hybrid cells that contain proteins and RNA from both organisms is unexpected and unprecedented. Such fusion events are likely widely distributed in nature, but have gone undetected. The implications are profound and may shed light onto many unexplained phenomena in human health, natural environments, evolutionary biology, and biotechnology., Microbial syntrophy is universal in nature, profoundly affecting the composition and function of microbiomes. We have recently reported data suggesting direct cell-to-cell interactions leading to electron and material exchange between the two microbes in the syntrophy between Clostridium ljungdahlii and C. acetobutylicum. Here, transmission electron microscopy and electron tomography demonstrated cell wall and membrane fusions between the two organisms, whereby C. ljungdahlii appears to invade C. acetobutylicum pole to pole. Correlative fluorescence transmission electron microscopy demonstrated large-scale exchange of proteins. Flow cytometry analysis captured the extent and dynamic persistence of these interactions. Dividing hybrid cells were identified containing stained proteins from both organisms, thus demonstrating persistence of cells with exchanged cellular components. Fluorescence microscopy and flow cytometry of one species with stained RNA and the other tagged with a fluorescent protein demonstrated extensive RNA exchange and identified hybrid cells, some of which continued to divide, while some were in an advanced C. acetobutylicum sporulation form. These data demonstrate that cell fusion enables large-scale cellular material exchange between the two organisms. Although unanticipated and never previously reported, these phenomena are likely widely distributed in nature, have profound implications for species evolution and the function of microbial communities, and could find utility in biotechnology. They may shed new light onto little-understood phenomena, such as antibiotic heteroresistance of pathogens, pathogen invasion of human tissues, and the evolutionary trajectory and persistence of unculturable bacteria.

Antibiotic fermentation residue (AFR) is a kind of protein-rich biosolids that would be utilized as valuable substrates for biogas production. However, the effectiveness of conductive material supplementation and its effect on the microbiome in anaerobic digestion of AFR have not been fully elucidated. The objective of this study is to access the impact of Fe3O4 addition on anaerobic digestion of AFR and reveal its microbial mechanism. By adding Fe3O4 at concentration of 150 mg/gTS to two typical AFRs, the methane yield was decreased while the methane production rate was significantly enhanced by 48% and 21%, respectively. Genomic analyses revealed that the Fe3O4 addition altered the microbial community by selecting the unknown genus of Anaerolineae with the metabolic capacity of carbohydrate hydrolysis and proteolysis. Meanwhile, the hydrogenotrophic methanogenesis was the dominant pathway in the AFR digestion system, which was mostly contributed by the Methanoculleus and Methanolinea. More interestingly, Methanoculleus might possess fermentative capability involved in the versatile hydrolysis pathway. Although the genes related to electrically conductive pili were detected, the induction of direct interspecies electron transfer by Fe3O4 supplementation was not confirmed in this study. In summary, except the accelerated methane production rate, Fe3O4 addition functioned as biostimulator for Anaerolineae other than electron conduit in anaerobic methanogenesis in the AFR digestion system.

The class Dehalococcoidia within the Chloroflexi phylum comprises the obligate organohalide-respiring genera Dehalococcoides, Dehalogenimonas, and “Candidatus Dehalobium.” Knowledge of the unique ecophysiology and biochemistry of Dehalococcoidia has been largely derived from studies with enrichment cultures and isolates from sites impacted with chlorinated pollutants; however, culture-independent surveys found Dehalococcoidia sequences in marine, freshwater, and terrestrial biomes considered to be pristine (i.The class Dehalococcoidia within the Chloroflexi phylum comprises the obligate organohalide-respiring genera Dehalococcoides, Dehalogenimonas, and “Candidatus Dehalobium.” Knowledge of the unique ecophysiology and biochemistry of Dehalococcoidia has been largely derived from studies with enrichment cultures and isolates from sites impacted with chlorinated pollutants; however, culture-independent surveys found Dehalococcoidia sequences in marine, freshwater, and terrestrial biomes considered to be pristine (i.e., not impacted with organohalogens of anthropogenic origin). The broad environmental distribution of Dehalococcoidia, as well as other organohalide-respiring bacteria, supports the concept of active halogen cycling and the natural formation of organohalogens in various ecosystems. Dechlorination reduces recalcitrance and renders organics susceptible to metabolic oxidation by diverse microbial taxa. During reductive dechlorination, hydrogenotrophic organohalide-respiring bacteria, in particular Dehalococcoidia, can consume hydrogen to low consumption threshold concentrations (

SUMMARYFrequent fluctuations in sulfate availability rendered syntrophic interactions between the sulfate reducing bacteriumDesulfovibrio vulgaris(Dv) and the methanogenic archaeonMethanococcus maripaludis(Mm) unsustainable. By contrast, prolonged laboratory evolution in obligate syntrophy conditions improved the productivity of this community but at the expense of erosion of sulfate respiration (SR). Hence, we sought to understand the evolutionary trajectories that could both increase the productivity of syntrophic interactions and sustain SR. We combined a temporal and combinatorial survey of mutations accumulated over 1000 generations of 9 independently-evolved communities with analysis of the genotypic structure for one community down to the single-cell level. We discovered a high level of parallelism across communities despite considerable variance in their evolutionary trajectories and the perseverance of a rare SR+Dvlineage within many evolution lines. An in-depth investigation revealed that synergistic epistasis acrossDvandMmgenotypes had enhanced cooperativity within SR- and SR+ assemblages, allowing their co-existence asr- andK-strategists, respectively.

Authigenic carbonates represent a significant microbial sink for methane, yet little is known about the microbiome responsible for the methane removal. We identify carbonate microbiomes distributed over 21 locations hosted by 7 different cold seeps in the Pacific and Atlantic Oceans by carrying out a gene-based survey using 16S rRNA- andmcrA gene sequencing coupled with metagenomic analyses. These sites were dominated by bacteria affiliated to the Firmicutes, Alpha- and Gammaproteobacteria. ANME-1 and −2 clades were abundant in the carbonates yet their typical syntrophic partners, sulfate reducing bacteria, were not significantly present. Our analysis indicated that methane oxidizers affiliated to the ANME-1 and −2 as well as to theCandidatusMethanoperedens clades, are capable of performing complete methane- and potentially short-chain alkane oxidations independently using oxidized sulfur and nitrogen compounds as terminal electron acceptors. Gammaproteobacteria are hypothetically capable of utilizing oxidized nitrogen compounds in potential syntrophy with methane oxidizing archaea. Carbonate structures represent a window for a more diverse utilization of electron acceptors for anaerobic methane oxidation along the Atlantic and Pacific Margin.

International audience; The discovery of Asgard archaea, phylogenetically closer to eukaryotes than other archaea, together with improved knowledge of microbial ecology, impose new constraints on emerging models for the origin of the eukaryotic cell (eukaryogenesis). Long-held views are metamorphosing in favour of symbiogenetic models based on metabolic interactions between archaea and bacteria. These include the classical Searcy’s and Hydrogen hypothesis, and the more recent Reverse Flow and Entangle–Engulf–Endogenize models. Two decades ago, we put forward the Syntrophy hypothesis for the origin of eukaryotes based on a tripartite metabolic symbiosis involving a methanogenic archaeon (future nucleus), a fermentative myxobacterial-like deltaproteobacterium (future eukaryotic cytoplasm) and a metabolically versatile methanotrophic alphaproteobacterium (future mitochondrion). A refined version later proposed the evolution of the endomembrane and nuclear membrane system by invagination of the deltaproteobacterial membrane. Here, we adapt the Syntrophy hypothesis to contemporary knowledge, shifting from the original hydrogen and methane-transfer-based symbiosis (HM Syntrophy) to a tripartite hydrogen and sulfur-transfer-based model (HS Syntrophy). We propose a sensible ecological scenario for eukaryogenesis in which eukaryotes originated in early Proterozoic microbial mats from the endosymbiosis of a hydrogen-producing Asgard archaeon within a complex sulfate-reducing deltaproteobacterium. Mitochondria evolved from versatile, facultatively aerobic, sulfide-oxidizing and, potentially, anoxygenic photosynthesizing alphaproteobacterial endosymbionts that recycled sulfur in the consortium. The HS Syntrophy hypothesis accounts for (endo)membrane, nucleus and metabolic evolution in a realistic ecological context. We compare and contrast the HS Syntrophy hypothesis to other models of eukaryogenesis, notably in terms of the mode and tempo of eukaryotic trait evolution, and discuss several model predictions and how these can be tested.

In the seabed, gaseous alkanes are oxidized by syntrophic microbial consortia that thereby reduce fluxes of these compounds into the water column. Because of the immense quantities of seabed alkane fluxes, these consortia are key catalysts of the global carbon cycle. Due to their obligate syntrophic lifestyle, the physiology of alkane-degrading archaea remains poorly understood. We have now cultivated a thermophilic, relatively fast-growing ethane oxidizer in partnership with a sulfate-reducing bacterium known to aid in methane oxidation and have retrieved the first complete genome of a short-chain alkane-degrading archaeon. This will greatly enhance the understanding of nonmethane alkane activation by noncanonical methyl-coenzyme M reductase enzymes and provide insights into additional metabolic steps and the mechanisms underlying syntrophic partnerships. Ultimately, this knowledge could lead to the biotechnological development of alkanogenic microorganisms to support the carbon neutrality of industrial processes., Cold seeps and hydrothermal vents deliver large amounts of methane and other gaseous alkanes into marine surface sediments. Consortia of archaea and partner bacteria thrive on the oxidation of these alkanes and its coupling to sulfate reduction. The inherently slow growth of the involved organisms and the lack of pure cultures have impeded the understanding of the molecular mechanisms of archaeal alkane degradation. Here, using hydrothermal sediments of the Guaymas Basin (Gulf of California) and ethane as the substrate, we cultured microbial consortia of a novel anaerobic ethane oxidizer, “Candidatus Ethanoperedens thermophilum” (GoM-Arc1 clade), and its partner bacterium “Candidatus Desulfofervidus auxilii,” previously known from methane-oxidizing consortia. The sulfate reduction activity of the culture doubled within one week, indicating a much faster growth than in any other alkane-oxidizing archaea described before. The dominance of a single archaeal phylotype in this culture allowed retrieval of a closed genome of “Ca. Ethanoperedens,” a sister genus of the recently reported ethane oxidizer “Candidatus Argoarchaeum.” The metagenome-assembled genome of “Ca. Ethanoperedens” encoded a complete methanogenesis pathway including a methyl-coenzyme M reductase (MCR) that is highly divergent from those of methanogens and methanotrophs. Combined substrate and metabolite analysis showed ethane as the sole growth substrate and production of ethyl-coenzyme M as the activation product. Stable isotope probing demonstrated that the enzymatic mechanism of ethane oxidation in “Ca. Ethanoperedens” is fully reversible; thus, its enzymatic machinery has potential for the biotechnological development of microbial ethane production from carbon dioxide.

The productivity of a biological community often correlates with its diversity. In the microbial world this phenomenon can sometimes be explained by positive, density-dependent interactions such as cross-feeding and syntrophy. These metabolic interactions help account for the astonishing variety of microbial life and drive many of the biogeochemical cycles without which life as we know it could not exist. While it is difficult to recapitulate experimentally how these interactions evolved among multiple taxa, we can explore in the laboratory how they arise within one. These experiments provide insight into how different bacterial ecotypes evolve and from these, possibly new “species.” We have previously shown that in a simple, constant environment a single clone of Escherichia coli can give rise to a consortium of genetically and phenotypically differentiated strains, in effect, a set of ecotypes, that coexist by cross-feeding. We marked these different ecotypes and their shared ancestor by integrating fluorescent protein into their genomes and then used flow cytometry to show that each evolved strain is more fit than the shared ancestor, that pairs of evolved strains are fitter still, and that the entire consortium is the fittest of all. We further demonstrate that the rank order of fitness values agrees with estimates of yield, indicating that an experimentally evolved consortium more efficiently converts primary and secondary resources to offspring than its ancestor or any member acting in isolation. IMPORTANCE Polymicrobial consortia occur in both environmental and clinical settings. In many cases, diversity and productivity correlate in these consortia, especially when sustained by positive, density-dependent interactions. However, the evolutionary history of such entities is typically obscure, making it difficult to establish the relative fitness of consortium partners and to use those data to illuminate the diversity-productivity relationship. Here, we dissect an Escherichia coli consortium that evolved under continuous glucose limitation in the laboratory from a single common ancestor. We show that a partnership consisting of cross-feeding ecotypes is better able to secure primary and secondary resources and to convert those resources to offspring than the ancestral clone. Such interactions may be a prelude to a special form of syntrophy and are likely determinants of microbial community structure in nature, including those having clinical significance such as chronic infections.

Recent advances in microbial electrochemical technologies have revealed the existence of numerous and highly diverse microorganisms able to exchange electrons with electrodes. This diversity could reflect the capacity of microorganisms to release and/or retrieve electrons with each other in natural environments. So far, this interspecies electron transfer has been studied with a special focus on syntrophy and was successfully demonstrated for several couples of species. In this article we argue that electron exchange between microbes exists beyond syntrophy or mutualism and could also promote competitive and even parasitic behaviour. Based on three interesting case studies identified from the literature, we also highlight that such nonmutualistic interactions could be widespread and of particular significance for the survival of pathogens or the shaping of complex microbial communities.

Geobacter and Pseudomonas spp. cohabit many of the same environments, where Geobacter spp. often dominate. Both bacteria are capable of extracellular electron transfer (EET) and play important roles in biogeochemical cycling. Although they recently in 2017 were demonstrated to undergo direct interspecies electron transfer (DIET) with one another, the genetic evolution of this syntrophic interaction has not been examined. Here, we use whole-genome sequencing of the cocultures before and after adaptive evolution to determine whether genetic selection is occurring. We also probe their interaction on a temporal level and determine whether their interaction dynamics change over the course of adaptive evolution. This study brings to light the multifaceted nature of interactions between just two microorganisms within a controlled environment and will aid in improving metabolic models of microbial communities comprising these two bacteria., Interactions between microorganisms in mixed communities are highly complex, being either syntrophic, neutral, predatory, or competitive. Evolutionary changes can occur in the interaction dynamics between community members as they adapt to coexistence. Here, we report that the syntrophic interaction between Geobacter sulfurreducens and Pseudomonas aeruginosa coculture change in their dynamics over evolutionary time. Specifically, Geobacter sp. dominance increases with adaptation within the cocultures, as determined through quantitative PCR and fluorescence in situ hybridization. This suggests a transition from syntrophy to competition and demonstrates the rapid adaptive capacity of Geobacter spp. to dominate in cocultures with P. aeruginosa. Early in coculture establishment, two single-nucleotide variants in the G. sulfurreducens fabI and tetR genes emerged that were strongly selected for throughout coculture evolution with P. aeruginosa phenazine wild-type and phenazine-deficient mutants. Sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS) proteomics revealed that the tetR variant cooccurred with the upregulation of an adenylate cyclase transporter, CyaE, and a resistance-nodulation-division (RND) efflux pump notably known for antibiotic efflux. To determine whether antibiotic production was driving the increased expression of the multidrug efflux pump, we tested Pseudomonas-derived phenazine-1-carboxylic acid (PHZ-1-CA) for its potential to inhibit Geobacter growth and drive selection of the tetR and fabI genetic variants. Despite its inhibitory properties, PHZ-1-CA did not drive variant selection, indicating that other antibiotics may drive overexpression of the efflux pump and CyaE or that a novel role exists for these proteins in the context of this interaction.

The relationship between predominant physiological types of prokaryotes in marine sediments and propionate degradation through sulfate reduction, fermentation, and methanogenesis was studied in marine sediments. Propionate conversion was assessed in slurries containing sediment from three different biogeochemical zones of Aarhus Bay, Denmark. Sediment slurries were amended with 0, 3, or 20 mM sulfate and incubated at 25 &deg, C and 10 &deg, C for 514&ndash, 571 days. Methanogenesis in the sulfate zone and sulfate reduction in the methane zone slurries was observed. Both processes occurred simultaneously in enrichments originating from samples along the whole sediment. Bacterial community analysis revealed the dominance of Desulfobacteraceae and Desulfobulbaceae members in sulfate-amended slurries incubated at 25&deg, C and 10&deg, C. Cryptanaerobacter belonging to the Peptococcaceae family dominated sulfate-free methanogenic slurries at 25&deg, C, whereas bacteria related to Desulfobacteraceae were dominant at 10&deg, C. Archaeal community analysis revealed the prevalence of different genera belonging to Methanomicrobiales in slurries incubated at different temperatures and amended with different sulfate concentrations. Methanosarcinaceae were only detected in the absence of sulfate. In summary, Aarhus Bay sediment zones contain sulfate reducers, syntrophs, and methanogens interacting with each other in the conversion of propionate. Our results indicate that in Aarhus Bay sediments, Cryptanaerobacter degraded propionate in syntrophic association with methanogens.

Anaerobic digestion is a technology that simultaneously treats waste and generates energy in the form of biogas. Unfortunately, when a high organic loading rate is applied, anaerobic digestion can suffer from volatile fatty acid accumulation that results in pH drop and decreased biogas production. In particular, propionic acid has shown to inhibit biogas production even at a very low concentration. Therefore, the kinetics of biogas production in relation to propionic acid concentration needs to be investigated. In batch experiments on anaerobic co-digestion of food waste and dairy manure in the present study, cumulative biogas production showed little inhibition by propionic acid in the concentration range of 6.5–14.6 mM, but a lag phase of 9.4, 16.3 and 60.8 d was detected in the digesters with initial propionic acid concentrations of 22.7, 36.2, and 56.4 mM, respectively. After the lag phase, these digesters accelerated to specific biogas yields of 0.59–0.70 L g-VS−1. The similar specific biogas yields across all of the digesters at initial propionic acid concentrations of 6.5–56.4 mM indicated reversibility of the inhibition. The reversibility was made possible by microbial acclimation and the shift to hydrogenotrophic methanogenesis in syntrophy with acetogenic bacteria. Evidently, an increase of hydrogenotrophic Methanobacterium and Methanoculleus abundances was found at 36.2 and 56.4 mM. Batch digestion experiments must be extended beyond the lag phase in order to fully reveal the inhibition kinetics. This paper highlights the need for a standard protocol that experimentally evaluates inhibition in anaerobic digestion.

Across human populations, 16S rRNA gene-based surveys of gut microbiomes have revealed that the bacterial familyChristensenellaceae and the archaeal familyMethanobacteriaceaeco-occur and are enriched in individuals with a lean, compared to an obese, BMI. Whether these association patterns reflect interactions between metabolic partners remains to be ascertained, as well as whether these associations play a role in the lean host phenotype with which they associate. Here, we validated previously reported co-occurrence patterns of the two families, and their association with a lean BMI, with a meta-analysis of 1,821 metagenomes derived from 10 independent studies. Furthermore, we report positive associations at the genus and species level betweenChristensenellaspp. andMethanobrevibacter smithii,the most abundant methanogen of the human gut. By co-culturing threeChristensenellaspp. WithM. smithii,we show thatChristensenellaspp. efficiently support the ofM. smithiivia H2production, far better thanBacteroides thetaiotaomicron.C. minutaforms flocs colonized byM. smithiieven when H2is in excess. In culture withC. minuta, H2consumption byM. smithiishifts the metabolic output ofC. minuta’s fermentation towards acetate rather than butyrate. Together, these results indicate that the widespread co-occurrence of these microbiota is underpinned by both physical and metabolic interactions. Their combined metabolic activity may provide insights into their association with a lean host BMI.ImportanceThe human gut microbiome is made of trillions of microbial cells, most of which areBacteria, with a subset ofArchaea. The bacterial familyChristensenellaceaeand the archaeal familyMethanobacteriaceaeare widespread in human guts. They correlate with each other and with a lean body type. Whether species of these two families interact, and how they affect the body type, are unanswered questions. Here, we showed that species within these families correlate with each other across people. We also demonstrated that particular species of these two families grow together in dense flocs, wherein the bacteria provide hydrogen gas to the archaea, which then make methane. When the archaea are present, the ratio of bacterial products (which are nutrients for humans) is changed. These observations indicate when these species grow together, their products have the potential to affect the physiology of their human host.

Conductive iron oxides (CIO) have been proved recently to facilitate the anaerobic microbial syntrophy based on the direct interspecies electron transfer (DIET) in batch experiments. However, the effect of CIO was always insignificant in anaerobic digestion (AD) reactor especially when the DIET-based syntrophic partners were absent. In this study, the effect of magnetite on performance of AD system with sucrose as a sole carbon source was investigated, but limited enhancement was achieved during the first 36-day operation. The short-term effect of ethanol addition was further studied in the magnetite-amended AD reactor, and results showed that the AD reactor with 10gFe/L micro-sized magnetite (R3) achieved higher performance of COD removal and methane proportion compared with the other reactors (R1 without magnetite; R2 with 2gFe/L micro-sized magnetite; R4 with 2gFe/L nano-sized magnetite). Meanwhile, the pyridoxine in extracellular polymeric substances (EPS) and conductivity of anaerobic sludge from R3 increased more significantly than those of the others. Analysis of high-throughput sequencing indicated that the abundance of archaea increased in sludge from R3 and Methanosarcina responsible for DIET was dominant (63.64%). Additionally, the abundance of potential electroactive bacteria Chloroflexi in R3 was 7.57-fold, 3.61-fold and 7.37-fold as that of R1, R2 and R4, respectively. These results demonstrated that the electroactive microbes and methanogens could be enriched efficiently in anaerobic sludge via synergetic effect of magnetite addition and ethanol short-term stimulation.

Summary Interspecies electron transfer is a fundamental factor determining the efficiency of anaerobic digestion (AD), which involves syntrophy between fermentative bacteria and methanogens. Direct interspecies electron transfer (DIET) induced by conductive materials can optimize this process offering a significant improvement over indirect electron transfer. Herein, conductive graphene was applied in the AD of protein-derived glycine to establish DIET. The electron-producing reaction via DIET is thermodynamically more favorable and exhibits a more negative Gibbs free energy value (−60.0 kJ/mol) than indirect hydrogen transfer (−33.4 kJ/mol). The Gompertz model indicated that the kinetic parameters exhibited linear correlations with graphene addition from 0.25 to 1.0 g/L, leading to the highest increase in peak biomethane production rate of 28%. Sedimentibacter (7.8% in abundance) and archaea Methanobacterium (71.1%) and Methanosarcina (11.3%) might be responsible for DIET. This research can open up DIET to a range of protein-rich substrates, such as algae., Graphical Abstract, Highlights • Graphene led to an increase in peak bio-CH4 production rate from glycine by 28% • Kinetic parameters had linear correlations with graphene addition (0.25–1.0 g/L) • Direct interspecies electron transfer (DIET) contributed to the improved performance, Chemical Engineering; Environmental Chemical Engineering; Microbial Biotechnology; Nanomaterials

Methane oxidation coupled to denitrification (MOD) was tested in a membrane biofilm reactor (MBfR) using methane gas as the sole electron donor. Nitrate reduction to nitrite was rate limiting, and CH4 was present in the effluent. Slow kinetics of methane oxidation by bacteria were the factors that led to slow kinetics and incomplete removals. Methylocystaceae contained the largest fraction (21%) of bacterial SSU rRNA genes, and Archaea were nearly absent. The functional metagenome included all the genes essential for aerobic methane oxidation (pmo, mdh, mtdB, folD, and fdh) and nitrate reduction to dinitrogen (nap/nar, nir, nor and nos), but not for reverse methanogenesis (mcr). The functional metagenome supports that Methylocystaceae conducted MOD in syntrophy with heterotrophic denitrifiers (e.g., Comamonadaceae and Brucellaceae), suggesting aerobic MOD. DO measurements, serum-bottle tests, and calculation of O2 permeation bolster hypoxically aerobic MOD would mainly account for denitrification in the MBfR.

It remains elusive if direct interspecies electron transfer (DIET) occurs in canonical syntrophy involving short-chain fatty acids oxidation. In the present study, we determined the effects of carbon nanomaterials on syntrophic oxidation of butyrate in two lake sediment enrichments and a defined coculture comprising Syntrophomonas wolfei and Methanococcus Maripaludis. After four continuous transfers of enrichment cultivation, Syntrophomonas dominated the bacterial populations in enrichments, and the dominated methanogens comprised Methanosarcina and Methanospirillum in one enrichment (from Weiming Lake) and Methanoregula and Methanospirillum in another (from Erhai Lake). Butyrate oxidation and CH4 production was significantly accelerated by carbon nanotubes (CNTs) in both enrichments. Replacement of CNTs by magnetite caused similar stimulating effect. For the defined coculture, two carbon nanomaterials, CNTs and reduced graphene oxide (rGO), were tested, both showed consistently stimulating effects on butyrate oxidation. Addition of kaolinite, an electric nonconductive clay mineral, however, revealed no effect. The test on M. maripaludis in pure culture showed no effect by rGO and a negative effect by CNTs (especially at a high concentration). Fluorescence in situ hybridization (FISH) and scanning electron microscopy (SEM) revealed that microbial cells were interwoven by CNTs forming cell-CNT mixture aggregates, and in case of rGO, cells were attached to surface or wrapped-up by rGO thin sheets. Collectively, our data suggest that the presence of conductive nanomaterials likely induces DIET in syntrophic butyrate oxidation.