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Anaerobic digester has emerged as a technology of choice in management of waste and production of biogas. However, the microbial ecology of digesters utilizing various substrates are very poorly understood. The ecology of anaerobic digester utilizing Citrullus lanatus fruit waste was analyzed using metagenomics. Slurry substrate sample was collected from a functional digester aseptically and anaerobically. Metagenomic DNA was extracted using ZYMO DNA extraction Kit (Model D 6001, Zymo Research, USA) following manufacturer’s instruction. Extracted DNA was amplified using the 16S rRNA gene amplicon PCR primers set and sequenced using Illumina MiSeq platform. Taxonomic analysis of the reads was performed using NCBI-BLAST-2.2.24 and CLC bio Genomics workbench v7.5.1. Taxonomic classification of the sequences revealed that bacteria and archae were the top two kingdoms with reads counts of 57,554 and 80, respectively. The top 7 phyla were Unknown, Firmicutes, Proteobacteria, Planctomycetes, Bacteroidetes, Actinobacteria and Euryarchaeota in decreasing order of counts. A total of 30 microbial classes, 43 orders, 79 families and 210 species were further classified. Over half of the 210 species detected were not routinely cultured species in the laboratory, indicating that there is still a relatively wide gap between culturable and non-culturable species in an anaerobic digester. The Acidogens captured in this study were Clostridium, Uncultured rumen bacteria and Bacteroidetes species. Furthermore, we also detected uncultured syntrophic Acetogens such as Syntrophomonas species and the hydrogenotrophic thermophile, Methanothermobacter sp. The Syntrophomonas species is known to breakdown short chain fatty acids, like propionate and butanoate in concert with hydrogenotrophic Methanogens indicating methane generation was via the hydrogenotrophic route. However, the main representative hydrogenotrophic methanogens detected were Methanoculleus bourgensis and Methanoculleus marisnigri, with the former being more abundant. In addition to the aforementioned species, other species captured were largely classified as unknown or uncultured species and they include Uncultured species of Clostridium, Syntrophomonas, Synergistetes, Synergistaceae Anaerobic sp, Ruminococcaceae, Rumen sp, Thermomonas, Thermoanaerobacteriales, Bacterium, Compost, Firmicutes, Bacteroidetes, Chloroflexi, Clostriaceae, Acetobacter. Eubacterium, Alpha and Aacteroides. The results of the study revealed that culture-independent approach is better able to capture the anaerobes including both culturable and unknown that dominate anaerobic digesters and are responsible for the bioconversion of organic waste into biogas.

Syntrophic anaerobic consortia comprised of fatty acid-degrading bacteria and hydrogen/formate-scavenging methanogenic archaea are of central importance for balanced and resilient natural and manufactured ecosystems: anoxic sediments, soils, and wastewater treatment bioreactors. Previously published studies investigated interaction between the syntrophic bi-cultures, but little information is available on the influence of fermentative bacteria on syntrophic fatty acid oxidation, even though fermentative organisms are always present together with syntrophic partners in the above-mentioned ecosystems. Here, we present experimental observations of stimulated butyrate oxidation and methane generation by a coculture of Syntrophomonas wolfei with any of the following methanogens: Methanospirillum hungatei, Methanobrevibacter arboriphilus, or Methanobacterium formicicum due to the addition of a fermentative Trichococcus flocculiformis strain ES5. The addition of T. flocculiformis ES5 to the syntrophic cocultures led to an increase in the rates of butyrate consumption (120%) and volumetric methane production (150%). Scanning electron microscopy of the most positively affected coculture (S. wolfei, M. hungatei, and T. flocculiformis ES5) revealed a tendency of T. flocculiformis ES5 to aggregate with the syntrophic partners. Analysis of coculture’s proteome with or without addition of the fermentative bacterium points to a potential link with signal transducing systems of M. hungatei, as well as activation of additional butyryl coenzyme A dehydrogenase and an electron transfer flavoprotein in S. wolfei. IMPORTANCE Results from the present study open doors to fascinating research on complex microbial cultures in anaerobic environments (of biotechnological and ecological relevance). Such studies of defined mixed populations are critical to understanding the highly intertwined natural and engineered microbial systems and to developing more reliable and trustable metabolic models. By investigating the existing cultured microbial consortia, like the ones described here, we can acquire knowledge on microbial interactions that go beyond “who feeds whom” relations but yet benefit the parties involved. Transfer of signaling compounds and stimulation of gene expression are examples of indirect influence that members of mixed communities can exert on each other. Understanding such microbial relationships will enable development of new sustainable biotechnologies with mixed microbial cocultures and contribute to the general understanding of the complex natural microbial interactions.

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.

Butyrate is one of the most important intermediates during anaerobic digestion of protein wastewater, and its oxidization is considered as a rate-limiting step during methane production. However, information on syntrophic butyrate-oxidizing bacteria (SBOB) is limited due to the difficulty in isolation of pure cultures. In this study, two anaerobic chemostats fed with butyrate as the sole carbon source were operated at different dilution rates (0.01/day and 0.05/day). Butyrate- and acetate-oxidizing bacteria in both chemostats were investigated, combining DNA-Stable Isotope Probing (DNA-SIP) and 16S rRNA gene high-throughput sequencing. The results showed that, in addition to known SBOB, Syntrophomonas, other species of unclassified Syntrophomonadaceae were putative butyrate-oxidizing bacteria. Species of Mesotoga, Aminivibrio, Acetivibrio, Desulfovibrio, Petrimonas, Sedimentibacter, unclassified Anaerolineae, unclassified Synergistaceae, unclassified Spirochaetaceae, and unclassified bacteria may contribute to acetate oxidation from butyrate metabolism. Among them, the ability of butyrate oxidation was unclear for species of Sedimentibacter, unclassified Synergistaceae, unclassified Spirochaetaceae, and unclassified bacteria. These results suggested that more unknown species participated in the degradation of butyrate. However, the corresponding function and pathway for butyrate or acetate oxidization of these labeled species need to be further investigated.

Anaerobic digestion (AD) of waste activated sludge (WAS) has been widely used, while it poses problems including low methane yield and production rate. Hydrochar is produced by hydrothermal liquefaction of biomass; however, little is known about the role of hydrochar in promoting AD of WAS. The present study showed that hydrochar increased the methane production rate by 30.8% and yield by 31.4% of hydrothermal pretreated dewatered WAS. Hydrochar increased the methane production rate and yield by enhancing the acidification and methanogenesis processes. Genomic-centric metatranscriptomics were used to identify the metabolic activities and transcriptomic response of individual metagenome-assembled genomes that were enriched by hydrochar. Although Methanosarcina sp. FDU0106 had been shown unable to used H2, it had the complete pathway for the reduction of CO2 to methane. Syntrophomonas sp. FDU0164 expressed genes for extracellular electron transfer via electrically pili, suggesting that Syntrophomonas sp. FDU0164 and Methanosarcina sp. FDU0106 were exchanging electrons via direct interspecies electron transfer. The expression of pili was decreased, indicating that hydrochar could replace its roles. Additionally, Firmicutes sp. FDU0048, Proteiniphilum sp. FDU0082, and Aminobacterium mobile FDU0089 were related to the degradation of organics, which could be related to the enhanced methane yield.

Scum is formed by the adsorption of long-chain fatty acids (LCFAs) onto biomass surface in anaerobic digestion of oily substrates. Since scum is a recalcitrant substrate to be digested, it is disposed via landfilling or incineration, which results in biomass washout and a decrease in methane yield. The microbes contributing to scum degradation are unclear. This study aimed to investigate the cardinal microorganisms in anaerobic scum digestion. We pre-incubated a sludge with scum to enrich scum-degrading microbes. Using this sludge, a 1.3-times higher methane conversion rate (73%) and a faster LCFA degradation compared with control sludge were attained. Then, we analyzed the cardinal scum-degrading microbes in this pre-incubated sludge by changing the initial scum-loading rates. Increased 16S rRNA copy numbers for the syntrophic fatty-acid degrader Syntrophomonas and hydrogenotrophic methanogens were observed in scum high-loaded samples. 16S rRNA amplicon sequencing indicated that Syntrophomonas was the most abundant genus in all the samples. The amino-acid degrader Aminobacterium and hydrolytic genera such as Defluviitoga and Sporanaerobacter became more dominant as the scum-loading rate increased. Moreover, phylogenic analysis on Syntrophomonas revealed that Syntrophomonas palmitatica, which is capable of degrading LCFAs, related species became more dominant as the scum-loading rate increased. These results indicate that a variety of microorganisms that degrade LCFAs, proteins, and sugars are involved in effective scum degradation.

Microbial populations responsible for arsenite [As(III)] detoxification were examined in aged refuse treated with 75 μM As(III) under semi-aerobic conditions. As(III) was rapidly oxidized to As(V) via microbial activity, and substantial As was fixed in the solid phase. The abundance of arsenite oxidase genes (aioA) was about four times higher in the moderate As(III) stressed treatment than in the untreated control. Network analysis of microbial community 16S rRNA genes based on MRT (random matrix theory) further illuminated details about microbe-microbe interactions, and showed six ecological clusters. A total of 166 “core” taxa were identified by within-module connectivity and among-module connectivity values. When compared with the control treatment without As(III), 12 putative keystone operational taxonomic units were positively correlated with As(III) oxidation, of which 10 of these were annotated to genera level. Eight genera were associated with As(III) detoxification: Pseudomonas, Paenalcaligenes, Proteiniphilum, Moheibacter, Mobilitalea, Anaerosporobacter, Syntrophomonas and Pusillimonas. Most of those putative keystone taxa were rare species in landfill, which suggests that low-abundance taxa might significantly contribute to As(III) oxidation.

Long-chain fatty acids (LCFA) are common contaminants in municipal and industrial wastewater that can be converted anaerobically to methane. A low hydrogen partial pressure is required for LCFA degradation by anaerobic bacteria, requiring the establishment of syntrophic relationships with hydrogenotrophic methanogens. However, high LCFA loads can inhibit methanogens, hindering biodegradation. Because it has been suggested that anaerobic degradation of these compounds may be enhanced by the presence of alternative electron acceptors, such as iron, we investigated the effect of sub-stoichiometric amounts of Fe(III) on oleate (C18:1 LCFA) degradation by suspended and granular methanogenic sludge. Fe(III) accelerated oleate biodegradation and hydrogenotrophic methanogenesis in the assays with suspended sludge, with H2-consuming methanogens coexisting with iron-reducing bacteria. On the other hand, acetoclastic methanogenesis was delayed by Fe(III). These effects were less evident with granular sludge, possibly due to its higher initial methanogenic activity relative to suspended sludge. Enrichments with close-to-stoichiometric amounts of Fe(III) resulted in a microbial community mainly composed of Geobacter, Syntrophomonas, and Methanobacterium genera, with relative abundances of 83&ndash, 89%, 3&ndash, 6%, and 0.2&ndash, 10%, respectively. In these enrichments, oleate was biodegraded to acetate and coupled to iron-reduction and methane production, revealing novel microbial interactions between syntrophic LCFA-degrading bacteria, iron-reducing bacteria, and methanogens.

Anaerobic digestion (AD) has been commercially operated worldwide in full scale as a resource recovery technology underpinning a circular economy. However, problems such as a long start-up time, or system instability, have been reported in response to operational shocks. These issues are usually linked to the dynamics of the functional microbiota in AD. Exploring the microbiota-functionality nexus (MFN) could be pivotal to understand the reasons behind these difficulties, and hence improving AD performance. Here we present a systematic MFN study based on 138 samples taken from 20 well-profiled lab-scale AD reactors operated for up to two years. All the reactors were operated in the same lab within the same period of time using the same methodology to harvest physio-chemical and molecular data, including key monitoring parameters, qPCR, and 16S sequencing results. The results showed a core bacterial microbiota prevailing in all reactor types, including Bacillus, Clostridium, Bacteroides, Eubacterium, Cytophaga, Anaerophaga, and Syntrophomonas, while various methanogens dominated different communities due to different inocula origins, reactor temperatures, or salinity levels. This core bacterial microbiota well correlated with biogas production (Pearson correlation coefficient of 0.481, p < 0.0001). Such strong correlation was even comparable to that between the biogas production and the methanogenic 16S rRNA gene content (Pearson correlation coefficient of 0.481, p < 0.0001). The results indicated that AD performance only modestly correlated with microbial diversity, a key governing factor. AD microbiota was neither functionally redundant nor plastic, and a high variety in communities can exhibit a strong difference in reactor performance. Our study demonstrates the importance of a core bacterial microbiota in AD and supports inspiring considerations for design, bioaugmentation, and operational strategies of AD reactors in the future.

The understanding and enrichment of consortia formed by syntrophic butyrate-oxidizing bacteria and methanogens in the complex environment are crucial for effectively degrading butyrate and preventing acid inhibition. In this study, the better butyrate-tolerated and highly efficient microbial consortia were domesticated and enriched through adding butyric acid ranging from 0.2 to 4.4 g/(L·d). The volumetric biogas production continuously increased to 1.65 L/(L·d). Microbial community diversity showed that a dramatic shift of bacterial structure occurred at BAL of 1.6 g/(L·d) and the structure presented better stability at high BAL. The syntrophic consortia and the main metabolic pathways were revealed through combination of the 16S rDNA and metagenome sequencing analyses. Syntrophomonas was the major butyrate-oxidizing bacterium and oxidized butyrate mainly through β-oxidaiton. Synergistaceae and Mesotoga acted as the main acetate-oxidizing bacteria. IHT and methanogenesis pathways were strongly enhanced by DMER64 and Methanosarcina as the main H2 carrier and dominant methanogen, respectively.

The underlying mechanisms of biochar enhance high-solid anaerobic digestion (HSAD) of food waste were investigated with a focus on the cell viability, microbial community, and methanogenic pathways. This study assessed the effects of different dosages of biochar in HSAD. Optimal biochar dosage was found to be 25 g/L, which produced accumulative methane yields of up to 251 mL CH4/g VS significantly promote volatile fatty acid degradations, especially in butyric acid concentrations. Effects of biochar with a dosage of 25 g/L on the cell viability showed that viable cells based on cell membrane integrity increased from 2.9% to 6.4%. Meanwhile, intact and highly active cells with high DNA content were probably involved in direct interspecies electron transfer (DIET) via membrane-bound electron transport proteins. Further analysis demonstrated that Syntrophomonas and methanogens Methanosarcina & Methanocelleus were selectively enriched by biochar, which resulted in the methanogenic pathways shifting from acetoclastic/hydrogenotrophic methanogenic pathways to more metabolically diverse methanogenic pathways. Accordingly, biochar-mediated DIET was possibly established between Syntrophomonas and Methanosarcina species due to those viable cells. In conclusion, biochar is a feasible additive in enhancing HSAD methanogenic performance.

The compound p-cresol is the major off-odor and toxic component of strong aroma-type Chinese liquor. To trace its origin, the p-cresol contents in the liquor-making process were detected by gas chromatography-mass spectrometry. The prokaryotic communities involved in the process were revealed by 16S rRNA gene MiSeq sequencing. The results showed that the microbial diversity and concentration of p-cresol in pit mud significantly increased with increased pit depth. Canonical correspondence analysis further revealed that p-cresol was positively correlated with the genera Aminobacterium, Clostridium, Sedimentibacter and Syntrophomonas. On investigating potential p-cresol producers, we obtained 11 species from pit mud samples using selective culture media. Clostridium butyricum, Clostridium tyrobutyricum, Clostridium aminovalericum and Eubacterium contortum were confirmed to possess the capacity for p-cresol production. Moreover, based on volatile compounds data, these strains were assigned to the same clusters characterized by the high abundance of butanoic acid and p-cresol. Furthermore, 24 pairs of significant correlations were identified from 14 genera using co-occurrence network analysis. Clostridium was the hub in pit mud and could be inhibited by increasing levels of Lactobacillus. These findings represented a step forward for controlling p-cresol in a complex microbial community of Chinese liquor.

Municipal solid waste (MSW) landfills are the most prevalent waste disposal method and constitute one of the largest sources of anthropogenic methane emissions in the world. Microbial activities in disposed waste play a crucial role in greenhouse gas emissions; however, only a few studies have examined metagenomic microbial profiles in landfills. Here, the MiSeq high-throughput sequencing method was applied for the first time to examine microbial diversity of the cover soil and stored waste located at different depths (0-150cm) in a typical MSW landfill in Yangzhou City, East China. The abundance of microorganisms in the cover soil (0-30cm) was the lowest among all samples, whereas that in stored waste decreased from the top to the middle layer (30-90cm) and then increased from the middle to the bottom layer (90-150cm). In total, 14 phyla and 18 genera were found in the landfill. A microbial diversity analysis showed that Firmicutes, Proteobacteria, and Bacteroidetes were the dominant phyla, whereas Halanaerobium, Methylohalobius, Syntrophomonas, Fastidiosipila, and Spirochaeta were the dominant genera. Methylohalobius (methanotrophs) was more abundant in the cover layers of soil than in stored waste, whereas Syntrophomonas and Fastidiosipila, which affect methane production, were more abundant in the middle to bottom layers (90-150cm) in stored waste. A canonical correlation analysis showed that microbial diversity in the landfill was most strongly correlated with the conductivity, organic matter, and moisture content of the stored waste.

This study investigated the impacts of long-chain fatty acid (LCFA) feeding frequencies on microbial community structure, bioconversion kinetics, and process stability during anaerobic codigestion. Parallel laboratory-scale anaerobic codigesters fed with dairy cattle manure were either pulse-fed every two days or continuously-fed daily, respectively, with oleate (C18:1) in incremental step increases over 200 days up to 64% of the influent chemical oxygen demand (COD). The effluent acetate concentration exceeded 3000 mg/L in the continuous-fed codigester at the highest oleate loading rate, but remained below 100 mg/L in the pulse-fed codigester at the end of its 48-hr oleate feed cycle. Maximum substrate conversion rates of oleate (qmax, oleate) and acetate (qmax, acetate) were significantly higher in the pulse-fed codigester compared to the continuous-fed codigester. 16S rRNA gene amplicon sequencing showed that Bacteria and Archaea community profiles diverged based on the codigester LCFA feeding pattern and loading rate. LCFA-degrading Syntrophomonas bacteria were significantly enriched in both LCFA codigesters relative to the control digester. The pulse-fed codigester had the highest community fraction of Syntrophomonas 16S rRNA genes by the end of the experiment with 43% of Bacteria amplicon sequences. qmax, oleate and qmax, acetate values were both significantly correlated to absolute concentrations of Syntrophomonas and Methanosaeta 16S rRNA genes, respectively. Multiple-linear regression models based on the absolute abundance of Syntrophomonas and Methanosaeta taxa provided improved predictions of oleate and acetate bioconversion kinetics, respectively. These results collectively suggest that pulse feeding rather than continuous feeding LCFA during anaerobic codigestion selected for higher microbial bioconversion kinetics and functional stability, which were related to changes in the physiological diversity and adaptive capacity of syntrophic and methanogenic communities.

Co-digestion of food waste with sewage sludge is widely applied for waste stabilization and energy recovery around the world. However, the effect of solid retention time (SRT) on the microbial population dynamics, metabolism and interspecies interaction have not been fully elucidated. Here, the influence of SRTs (5-25 days) on the performance of the co-digestion system was investigated and state-of-the-art genome-centric metagenomic analysis was employed to uncover the dynamics and metabolic network of the key players underlying the well-functioned and poorly-functioned co-digestion microbial communities. The results of the microbial analyses indicated that SRT largely shaped microbial community structure by enriching the syntrophic specialist Syntrophomonas and CO2/H2 ( formate)-using methanogen Methanocorpusculum in the well-functioned co-digester operated at SRT of 25 days, while selecting acid-tolerant populations Lactobacillus at SRT of 5 days. The metagenome assembled genomes (MAGs) of key players, such as Syntrophomonadaceae, Methanocorpusculum, and Mesotoga, were retrieved, additionally, the syntrophic acetate oxidation plus hydrogenotrophic methanogenesis (SAO-HM) were proposed as the dominant pathway for methane production. The metabolic interaction in the co-digestion microbial consortia was profiled by assigning MAGs into functional guilds. Functional redundancy was found in the bacterial groups in hydrolysis step, and the members in these groups reduced the direct competition by niche differentiation.

Predicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in situ metabolic activities of uncultured species may enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in situ function of mixed microbiomes by combining stable-isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C4 fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve “who is doing what” by obtaining confirmatory assimilation of the labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes., Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable-isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in 13C after growing on [13C]butyrate. Differential abundance analysis of recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in heavy [13C]DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (

Due to long-chain fatty acids (LCFAs) and acidification, rapeseed oil as a typical lipid in kitchen waste is difficult to be biodegraded by anaerobic digestion. It has been reported that incorporation of some conductive materials into reactors treating complex organic matter could enhance reactor performance. In this study, the aim was to study this possibility of application of granular activated carbon (GAC) in anaerobic digestion of rapeseed oil. As expected, the GAC-amended reactor could significantly improve methane yield and reduce acidification. Besides, the GAC-amended broth could efficiently degrade palmitate into methane. Microbial community analysis showed that bacteria (Syntrophomonas) and methanogens (Methanosarcina) were greatly enriched on the GAC surface in GAC-amended system. These results, and the kwon of easy enrichment of Syntrophomonas on conductive materials or current-harvesting electrodes in methanogenic and/or electrogenic systems, suggest that Syntrophomonas could participate in direct interspecies electron transfer with Methanosarcina species, when GAC is available as an electron transfer mediator. Hence, the addition of GAC could efficiently, stably and environmentally enhance the methanogenic metabolism of rapeseed oil.

Linking the genomic content of uncultivated microbes to their metabolic functions remains a critical challenge in microbial ecology. Resolving this challenge has implications for improving our management of key microbial interactions in biotechnologies such as anaerobic digestion, which relies on slow-growing syntrophic and methanogenic communities to produce renewable methane from organic waste. In this study, we combined DNA stable isotope probing (SIP) with genome-centric metagenomics to recover the genomes of populations enriched in 13C after feeding 13C-labeled butyrate. Differential abundance analysis on recovered genomic bins across the SIP metagenomes identified two metagenome-assembled genomes (MAGs) that were significantly enriched in the heavy 13C DNA. Phylogenomic analysis assigned one MAG to the genus Syntrophomonas, and the other MAG to the genus Methanothrix. Metabolic reconstruction of the annotated genomes showed that the Syntrophomonas genome encoded all the enzymes for beta-oxidizing butyrate, as well as several mechanisms for interspecies electron transfer via electron transfer flavoproteins, hydrogenases, and formate dehydrogenases. The Syntrophomonas genome shared low average nucleotide identity (< 95%) with any cultured representative species, indicating it is a novel species that plays a significant role in syntrophic butyrate degradation within anaerobic digesters. The Methanothrix genome contained the complete pathway for aceticlastic methanogenesis, indicating that it was enriched in 13C from syntrophic acetate transfer. This study demonstrates the potential of stable-isotope-informed genome-resolved metagenomics to elucidate the nature of metabolic cooperation in slow-growing uncultured microbial populations, such as syntrophic bacteria and methanogens, that are important to waste treatment as well as global carbon cycling.ImportancePredicting the metabolic potential and ecophysiology of mixed microbial communities remains a major challenge, especially for slow-growing anaerobes that are difficult to isolate. Unraveling the in-situ metabolic activities of uncultured species could enable a more descriptive framework to model substrate transformations by microbiomes, which has broad implications for advancing the fields of biotechnology, global biogeochemistry, and human health. Here, we investigated the in-situ function of mixed microbiomes by combining DNA-stable isotope probing with metagenomics to identify the genomes of active syntrophic populations converting butyrate, a C4 fatty acid, into methane within anaerobic digesters. This approach thus moves beyond the mere presence of metabolic genes to resolve ‘who is doing what’ by obtaining confirmatory assimilation of labeled substrate into the DNA signature. Our findings provide a framework to further link the genomic identities of uncultured microbes with their ecological function within microbiomes driving many important biotechnological and global processes.

Degradation of long-chain fatty acids (LCFAs) in methanogenic environments is a syntrophic process involving the activity of LCFA-degrading bacteria and hydrogen-utilizing methanogens. If methanogens are inhibited, other hydrogen scavengers are needed to achieve complete LCFA degradation. In this work, we developed two different oleate (C18:1 LCFA)-degrading anaerobic enrichment cultures, one methanogenic (ME) and another in which methanogenesis was inhibited (IE). Inhibition of methanogens was attained by adding a solution of 2-bromoethanesulfonate (BrES), which turned out to consist of a mixture of BrES and isethionate. Approximately 5 times faster oleate degradation was accomplished by the IE culture compared with the ME culture. A bacterium closely related to Syntrophomonas zehnderi (99\% 16S rRNA gene identity) was the main oleate degrader in both enrichments, in syntrophic relationship with hydrogenotrophic methanogens from the genera Methanobacterium and Methanoculleus (in ME culture) or with a bacterium closely related to Desulfovibrio aminophilus (in IE culture). A Desulfovibrio species was isolated, and its ability to utilize hydrogen was confirmed. This bacterium converted isethionate to acetate and sulfide, with or without hydrogen as electron donor. This bacterium also utilized BrES but only after 3 months of incubation. Our study shows that syntrophic oleate degradation can be coupled to desulfonation.IMPORTANCE In anaerobic treatment of complex wastewater containing fat, oils, and grease, high long-chain fatty acid (LCFA) concentrations may inhibit microbial communities, particularly those of methanogens. Here, we investigated if anaerobic degradation of LCFAs can proceed when methanogens are inhibited and in the absence of typical external electron acceptors, such as nitrate, iron, or sulfate. Inhibition studies were performed with the methanogenic inhibitor 2-bromoethanesulfonate (BrES). We noticed that, after autoclaving, BrES underwent partial hydrolysis and turned out to be a mixture of two sulfonates (BrES and isethionate). We found out that LCFA conversion proceeded faster in the assays where methanogenesis was inhibited, and that it was dependent on the utilization of isethionate. In this study, we report LCFA degradation coupled to desulfonation. Our results also showed that BrES can be utilized by anaerobic bacteria., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004), funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. We also acknowledge Project MultiBiorefinery (SAICTPAC/0040/2015 [POCI-01-0145-FEDER-016403]), funded by Sistema de Apoio à Investigação Científica e Tecnológica (SAICT), Programas de Atividades Conjuntas (PAC), and the financial support of the European Research Council under the European Union Seventh Framework Programme (FP/2007-2013)/ERC (grant agreement 323009), info:eu-repo/semantics/publishedVersion

In this study, batch experiments were conducted to compare the effect of temperature and substrate source on microbial communities in the anaerobic digestion of kitchen waste. The results showed that the microbial communities of anaerobic digestion were not sensitive to varied sources of waste, but shifted with the change in operating temperatures. In the reactors operated at mesophilic conditions, Levilinea, Syntrophomonas, Methanothrix, and Methanosphaerula, etc. were the dominant microbes during the process. While in thermophilic reactors, Levilinea, Ornatilinea, Methanosphaerula and Methanomassiliicoccus, etc. prevailed. Meanwhile, an enrichment in Coprothermobacter, Defluviitoga, Defluviitalea, Tepidimicrobium, Lutispora and Fonticella were observed as the temperature changed from mesophilic to thermophilic, suggesting these genera could be selectively enriched at thermophilic conditions. The results provided fundamental understanding of the microbiology that could support the scale up of food waste anaerobic digestion.

The effects of four conductive nanomaterials (nano-carbon powder, nano-Al2O3, nano-ZnO, nano-CuO) on sludge anaerobic digestion (AD) performance and microbial community were investigated through a 36-day fermentation experiment. Results showed that biogas production enhanced by 16.9% and 23.4% with nano-carbon powder and nano-Al2O3 added but decreased by 90.2% and 17.3% with nano-ZnO and nano-CuO. Total solids (TS) removal efficiency was increased by 38.73% and 27.11% with nano-carbon powder and nano-Al2O3 added but decreased by 70.67% and 43.70% with nano-ZnO and nano-CuO. Kinetic analysis indicated four conductive nanomaterials could shorten the lag phase of AD sludge with an average rate of 51.75%. 16S rRNA amplicon sequencing results demonstrated microbes such as Syntrophomonas and Methanosaeta were enriched in nano-carbon powder and nano-Al2O3 reactors. However, microbial community diversity and richness were both inhibited by adding nano-ZnO and nano-CuO. Redundancy analysis (RDA) revealed that genera belong to Firmicutes and Chloroflexi could conduce to methanogenesis process.

Syntrophic oxidation of butyrate is catabolized by a few bacteria specialists in the presence of methanogens. In the present study, a highly enriched butyrate-oxidizing consortium was obtained from a wetland sediment in Tibetan Plateau. During continuous transfers of the enrichment, the addition of magnetite nanoparticles (nanoFe3O4) consistently enhanced butyrate oxidation and CH4 production. Molecular analysis revealed that all bacterial sequences from the consortium belonged to Syntrophomonas with the closest relative of Syntrophomonas wolfei and 96% of the archaeal sequences were related to Methanobacteria with the remaining sequences to Methanocella. Addition of graphite and carbon nanotubes for a replacement of nanoFe3O4 caused the similar stimulatory effect. Silica coating of nanoFe3O4 surface, however, completely eliminated the stimulatory effect. The control experiment with axenic cultivation of a Syntrophomonas strain and two methanogen strains showed no effect by nanoFe3O4. Together, the results in the present study support that syntrophic oxidation of butyrate is likely facilitated by direct interspecies electron transfer in the presence of conductive nanomaterials.

The impacts of ZVI on microbial community diversity in an anaerobic assay with high organic loading were investigated. The relative abundance of bacteria, archaea, and the functional methyl coenzyme-M reductase (mcrA) gene were investigated using high-throughput sequencing, and variations in their quantity were determined by qPCR. The results showed that ZVI significantly increased both the relative abundance and quantity of Methanobacteriales and Methanosarcinales during hydrogenotrophic and acetoclastic methanogenesis. The relative abundance of syntrophic Methanobacteriales at the hydrolysis and acidogenesis stages resulted in H2 partial pressure decrease through an interspecies hydrogen transfer (IHT) network, which further induced butyric conversion to acetic by Syntrophomonas. The primary microbial metabolism then converted to acetoclastic methanogensis in the assay with ZVI addition. The short duration of this process and high relative abundance of Syntrophomonas, Clostridium butyricum and Methanosarcinales potentially indicated the existence of a novelty syntrophic mechanism for extracellular electron transfer, which promoted CH4 generation.

In literature, the microbial diversity of hot spring waters has been the focus of extensive research, while there is a paucity of studies on thermal water distribution network of spa centres and, as yet, no studies have been carried out on the bacterial population of thermal muds. In this context, the aim of our study is to characterize the microbial community of sulphurous-bromine-iodine thermal water and mud within an Italian spa complex using Next Generation Sequencing (NGS) technologies. This is the first report of microbiome characterization along a water supply network from the spring to points of use within a spa. According to the 16S rRNA gene sequences analysis, our data highlight the presence of a typical microbial community, mainly composed of sulphur-cycling bacteria belonging to Desulfomonile, Thermodesulfovibrio, Geothermobacterium, Thermus, Thiofaba and Syntrophomonas genera. In addition, the characterization and evolution of the bacterial community in thermal muds during the maturation process is investigated for the first time. Interestingly, the microbiome of mature mud resulted dominated by bacteria capable of lipid biosynthesis, suggesting that these bacteria may play a role in the anti-rheumatic properties of thermal mud.

Syntrophomonas wolfei syntrophically oxidizes short-chain fatty acids (four to eight carbons in length) when grown in coculture with a hydrogen- and/or formate-using methanogen. The oxidation of 3-hydroxybutyryl-coenzyme A (CoA), formed during butyrate metabolism, results in the production of NADH. The enzyme systems involved in NADH reoxidation in S. wolfei are not well understood. The genome of S. wolfei contains a multimeric [FeFe]-hydrogenase that may be a mechanism for NADH reoxidation. The S. wolfei genes for the multimeric [FeFe]-hydrogenase ( hyd1ABC ; SWOL_RS05165, SWOL_RS05170, SWOL_RS05175) and [FeFe]-hydrogenase maturation proteins (SWOL_RS05180, SWOL_RS05190, SWOL_RS01625) were coexpressed in Escherichia coli , and the recombinant Hyd1ABC was purified and characterized. The purified recombinant Hyd1ABC was a heterotrimer with an αβγ configuration and a molecular mass of 115 kDa. Hyd1ABC contained 29.2 ± 1.49 mol of Fe and 0.7 mol of flavin mononucleotide (FMN) per mole enzyme. The purified, recombinant Hyd1ABC reduced NAD + and oxidized NADH without the presence of ferredoxin. The HydB subunit of the S. wolfei multimeric [FeFe]-hydrogenase lacks two iron-sulfur centers that are present in known confurcating NADH- and ferredoxin-dependent [FeFe]-hydrogenases. Hyd1ABC is a NADH-dependent hydrogenase that produces hydrogen from NADH without the need of reduced ferredoxin, which differs from confurcating [FeFe]-hydrogenases. Hyd1ABC provides a mechanism by which S. wolfei can reoxidize NADH produced during syntrophic butyrate oxidation when low hydrogen partial pressures are maintained by a hydrogen-consuming microorganism. IMPORTANCE Our work provides mechanistic understanding of the obligate metabolic coupling that occurs between hydrogen-producing fatty and aromatic acid-degrading microorganisms and their hydrogen-consuming partners in the process called syntrophy (feeding together). The multimeric [FeFe]-hydrogenase used NADH without the involvement of reduced ferredoxin. The multimeric [FeFe]-hydrogenase would produce hydrogen from NADH only when hydrogen concentrations were low. Hydrogen production from NADH by Syntrophomonas wolfei would likely cease before any detectable amount of cell growth occurred. Thus, continual hydrogen production requires the presence of a hydrogen-consuming partner to keep hydrogen concentrations low and explains, in part, the obligate requirement that S. wolfei has for a hydrogen-consuming partner organism during growth on butyrate. We have successfully expressed genes encoding a multimeric [FeFe]-hydrogenase in E. coli , demonstrating that such an approach can be advantageous to characterize complex redox proteins from difficult-to-culture microorganisms.

ZVI was reported to enrich H2-utilizing methanogens that enhanced interspecies H2 transfer, while Fe(III) oxide served as a conductive material to promote direct interspecies electron transfer (DIET). However, the interaction of these two modes in anaerobic digestion has not been clarified yet. In this study, when adding Fe3O4 and ZVI simultaneously into an anaerobic digester, the abundance of hydrogenotrophic methanogens decreased drastically compared to ZVI-added digester and Fe-free digester. However, the methane production of ZVI + Fe3O4 added digester were 68.9% higher than Fe-free digester and 20.0% higher than ZVI-added digester, respectively. Sludge reduction rate of these three digesters also showed similar results. These indicated that hydrogenotrophic methanogenesis was not the main reason for methanogenesis in Fe3O4-added digester. Instead, Syntrophomonas and Methanosaeta were specially enriched in Fe3O4-added digesters, which implied that the potential DIET between Syntrophomonas and Methanosaeta was likely a crucial reason for accelerating anaerobic digestion of waste sludge.

The process stability of biogas plants is often deteriorated by the accumulation of Long Chain Fatty Acids (LCFA). The microbial community shifts due to LCFA disturbances have been poorly understood as the molecular techniques used were not able to identify the genome characteristics of uncultured microorganisms and additionally, the presence of limited number of reference genomes in public databases prevented the comprehension of specific functional roles characterizing these microorganisms. The present study is the first research which deciphers by means of high throughput shotgun sequencing the dynamics of the microbial community during an inhibitory shock load induced by single pulses of unsaturated LCFA at two different concentrations (i.e. 2 g/L-reactor and 3 g/L-reactor). The metagenomic analysis showed that only the microbes associated with LCFA degradation could encode proteins related to “chemotaxis” and “flagellar assembly”, which promoted the ability to move towards the LCFA sources so as to degrade them. Moreover, the syntrophic interactions found between Syntrophomonas sp. together with Methanosarcina sp. were possibly assigned to the menaquinone-electron transfer. Finally, it was proven that a previously exposed to LCFA inoculum is more efficient in the degradation process of LCFA due to the specialization of the microbial consortium.

In the present study, RNA-sequencing was used to elucidate the change of anaerobic digestion metatranscriptome after long chain fatty acids (oleate) exposure. To explore the general transcriptional behavior of the microbiome, the analysis was first performed on shotgun reads without considering a reference metagenome. As a second step, RNA reads were aligned on the genes encoded by the microbial community, revealing the expression of more than 51 000 different transcripts. The present study is the first research which was able to dissect the transcriptional behavior at a single species level by considering the 106 microbial genomes previously identified. The exploration of the metabolic pathways confirmed the importance of Syntrophomonas species in fatty acids degradation, and also highlighted the presence of protective mechanisms toward the long chain fatty acid effects in bacteria belonging to Clostridiales, Rykenellaceae, and in species of the genera Halothermothrix and Anaerobaculum. Additionally, an interesting transcriptional activation of the chemotaxis genes was evidenced in seven species belonging to Clostridia, Halothermothrix, and Tepidanaerobacter. Surprisingly, methanogens revealed a very versatile behavior different from each other, even among similar species of the Methanoculleus genus, while a strong increase of the expression level in Methanosarcina sp. was evidenced after oleate addition.

International audience; Ammonia inhibition represents a major operational issue for anaerobic digestion (AD). In order to get more insights into AD microbiota resistance, anaerobic batch reactors performances were investigated under a wide range of Total Ammonia Nitrogen (TAN) concentrations up to 50.0 g/L at 35 C. The half maximal inhibitory concentration (IC50) value was determined to be 19.0 g/L. Microbial community dynamics revealed that above a TAN concentration of 10.0 g/L, remarkable modifications within archaeal and bacterial communities occurred. 16S rRNA gene sequencing analysis showed a gradual methanogenic shift between two OTUs from genus Methanosarcina when TAN concentration increased up to 25.0 g/L. Proportion of potential syntrophic microorganisms such as Methanoculleus and Treponema progressively raised with increasing TAN up to 10.0 and 25.0 g/L respectively, while Syntrophomonas and Ruminococcus groups declined. In 25.0 g/L assays, Caldicoprobacter were dominant. This study highlights the emergence of AD key phylotypes at extreme ammonia concentrations.

The number of bacterial phyla has greatly increased in the past decade. Among them, a candidate division named 'Synergistes' was proposed in a phylogenetic study on the global diversity of bacteria. We previously described the genus Jonquetella and suggested that it belonged to this not yet well-delineated candidate phylum. 16S rRNA gene based-phylogeny studies were conducted using four reconstruction methods and 599 sequences forming five datasets were used in an alternative treeing approach. These analyses indicated that the genera Aminiphilus, Aminobacterium, Aminomonas, Anaerobaculum, Dethiosulfovibrio, Jonquetella, Synergistes, Thermanaerovibrio and Thermovirga should be grouped in the same high-level taxon. This taxon was shown to be a phylum-rank lineage in the domain Bacteria and, because of the prior use of the name Synergistes for a genus, the name 'Synergistetes' is proposed for this candidate phylum. We also propose an emended delineation of the phylum 'Deferribacteres', which is now only represented by the family Deferribacteriaceae. The emended family Syntrophomonadaceae is limited to the genera Pelospora, Syntrophomonas, Syntrophothermus and Thermosyntropha.

The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities.

A spore-forming, anaerobic, syntrophic fatty-acid-oxidizing bacterium, strain 19J-3T, was isolated from a distilled-spirit-fermenting cellar in Hebei Province, China. The cells were slightly curved rods with a spore at the end of the cell. The optimal temperature for growth was around 37 °C and growth occurred in the range 25–45 °C. The pH range for growth was 6.5–8.5 and the optimum pH was 7.0–7.5. Crotonate was the only substrate that allowed the strain to grow in pure culture. However, the strain could oxidize saturated fatty acids with four to nine carbon atoms syntrophically in co-culture with Methanobacterium formicicum DSM 1535T. The strain was not able to utilize sulfate, sulfite, thiosulfate, DMSO, nitrate, fumarate or Fe(III) as electron acceptor. The DNA base composition was 48.8 mol% G+C. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain 19J-3T was related to members of the family Syntrophomonadaceae and most closely to Syntrophospora bryantii DSM 3014T (94.3 % similarity) and Syntrophomonas wolfei subsp. wolfei DSM 2245T (93.6 % similarity). Considering the phylogenetic relationship and phenotypic features, strain 19J-3T (=CGMCC 1.5041T=JCM 13582T) is designated as the type strain of a novel species of the genus Syntrophomonas, Syntrophomonas cellicola sp. nov. Based on the close phylogenetic relationship between the genera Syntrophospora and Syntrophomonas, the presence of sporulation-specific genes in the genome of Syntrophomonas wolfei subsp. wolfei DSM 2245T and the description of a spore-forming member of Syntrophomonas, ‘Syntrophomonas erecta subsp. sporosyntropha’, we propose the assignment of Syntrophospora bryantii to the genus Syntrophomonas as Syntrophomonas bryantii comb. nov.

Two obligate anaerobic bacterial strains (5-3-Z(T) and Y4-1) were isolated from river sediment and rice field mud, respectively. They degraded straight-chain fatty acids with 4-8 carbon atoms in syntrophic association with methanogens, however, neither tested branch-chain fatty acids nor could benzoate be degraded. The strains formed spores when cocultured with methanogens on butyrate, or when grew on butyrate plus dimethyl sulfoxide (DMSO) in pure culture. The cells were slightly curved rods with Gram-negative cell wall structure, and contained small amount of poly beta-hydroxyalkanoate. The strains could not degrade butyrate alone, nor could use fumarate, sulfate, thiosulfate, sulfur or nitrate as electron acceptors except DMSO for butyrate degradation. The generation time of strain 5-3-Z(T) was about 12h when growing on crotonate at 37 degrees C. The growth of the new strains occurred in the range of pH 5.5-8.4, and of temperature 20-48 degrees C, and at NaCl concentration of 0-700 mM. The G+C content of the genomic DNA of strain 5-3-Z(T) was 40.6mol%. Phylogenetic analysis based on 16S rRNA gene similarity showed the two strains to be a member of species Syntrophomonas erecta (98.4-98.9% sequence similarity), however they differed from the existing strains in both phenotypic and genetic characteristics. Therefore, a new subspecies of S. erecta, S. erecta subsp. sporosyntropha was proposed. The type strain was 5-3-Z(T) (=CGMCC1.5032(T)=JCM13344(T)).

To improve the stability of anaerobic reactors, more knowledge is required about how the different communities react against operating perturbations and which specific ones respond better. The objective of this work was to monitor the changes in microbial community structure of an anaerobic digester during a temperature drop by applying different complementary molecular techniques. Temperature decrease led to an increase of Bacteroidales order, Porphyromonadaceae family and Bacteroides genus and a decrease in Syntrophomonas and Clostridium genera. Once the temperature was restored, the reactor recovered the steady state performance without requiring any modification in operational conditions or in the microbiome. During the recovery period, Sedimentibacter genus and Porphyromonadaceae family played an important role in the degradation of the accumulated volatile fatty acids. The hydrogenotrophic methanogens appeared to be the keystone archaeal population at low temperatures as well as in the recovery period. This study stands out that the understanding of microbial community dynamics during temperature drop could be utilized to develop strategies for the mitigation of temperature change consequences and speed up the recovery of stable reactor performance.

The family Syntrophomonadaceae comprises the genera Syntrophomonas, Pelospora, Syntrophothermus, and Thermosyntropha. All these bacteria are strictly anaerobic and depend on reducing conditions for growth.They areGram-positivewith low DNA content, but in most cases the murein layer is thin and an outer membrane appears, resembling the cell wall architecture of Gram-negative bacteria. Also in Gram-staining, these bacteria mostly behave Gram-negative. Except for Pelospora, all members of this family degrade fatty acids of four carbon atoms or more by beta oxidation, in close association with hydrogenor formate-utilizing partner organisms, and depend on this association for thermodynamic reasons. Most representatives of this family can be grown in pure culture with crotonate which is dismutated to acetate and butyrate. Pelospora sp. grows by decarboxylation of glutarate or succinate.

Gan and colleagues ([1][1]) recently observed that upon the application of [13C]propionate to anoxic paddy soil, label was concurrently incorporated in Smithella spp. and Syntrophomonas spp. Smithella spp. utilize propionate in a nonrandomizing pathway in which propionate is first dismutated to