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In this study, strategies for recovery of ammonia-stressed AD reactors were attempted, by addition of preserved bioaugmentation consortium in gel (BioG), fresh consortium in liquid medium (BioL), woodchip biochar (BW), and straw biochar (BS). In comparison to control group with ammonia, effective treatments, i.e., BioG, BioL, BW and BS raised the maximum methane production rate by 77%, 23%, 35%, and 24%, respectively. BW possibly acted as interspecies electrical conduits for Direct Electron Transfer based on conductivity and SEM analysis. BioG facilitated slow release of bioaugmentation inocula from gel into the AD system, which protected them from a direct environmental shock. According to microbial analysis, both BioG, BioL and BW resulted in increased relative abundance of Methanothermobacter thermautotrophicus; and BS induced selective raise of Methanosarcina thermophila. The increase of methanogens via these strategies led to the faster recovery of the AD process. publishedVersion

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.

Anna Detman, Michał Bucha, Laura Treu, Aleksandra Chojnacka, Łukasz Pleśniak, Agnieszka Salamon, Ewa Łupikasza, Robert Gromadka, Jan Gawor, Agnieszka Gromadka, Wojciech Drzewicki, Marta Jakubiak, Marek Janiga, Irena Matyasik, Mieczysław K. Błaszczyk, Mariusz Orion Jędrysek, Stefano Campanaro, Anna Sikora., Background: During the acetogenic step of anaerobic digestion, the products of acidogenesis are oxidized to substrates for methanogenesis: hydrogen, carbon dioxide and acetate. Acetogenesis and methanogenesis are highly interconnected processes due to the syntrophic associations between acetogenic bacteria and hydrogenotrophic methanogens, allowing the whole process to become thermodynamically favorable. The aim of this study is to determine the influence of the dominant acidic products on the metabolic pathways of methane formation and to find a core microbiome and substrate-specific species in a mixed biogas-producing system. Results: Four methane-producing microbial communities were fed with artificial media having one dominant component, respectively, lactate, butyrate, propionate and acetate, for 896 days in 3.5-L Up-flow Anaerobic Sludge Blanket (UASB) bioreactors. All the microbial communities showed moderately different methane production and utilization of the substrates. Analyses of stable carbon isotope composition of the fermentation gas and the substrates showed differences in average values of δ13C(CH4) and δ13C(CO2) revealing that acetate and lactate strongly favored the acetotrophic pathway, while butyrate and propionate favored the hydrogenotrophic pathway of methane formation. Genome-centric metagenomic analysis recovered 234 Metagenome Assembled Genomes (MAGs), including 31 archaeal and 203 bacterial species, mostly unknown and uncultivable. MAGs accounted for 54%–67% of the entire microbial community (depending on the bioreactor) and evidenced that the microbiome is extremely complex in terms of the number of species. The core microbiome was composed of Methanothrix soehngenii (the most abundant), Methanoculleus sp., unknown Bacteroidales and Spirochaetaceae. Relative abundance analysis of all the samples revealed microbes having substrate preferences. Substrate-specific species were mostly unknown and not predominant in the microbial communities. Conclusions: In this experimental system, the dominant fermentation products subjected to methanogenesis moderately modified the final effect of bioreactor performance. At the molecular level, a different contribution of acetotrophic and hydrogenotrophic pathways for methane production, a very high level of new species recovered, and a moderate variability in microbial composition depending on substrate availability were evidenced. Propionate was not a factor ceasing methane production. All these findings are relevant because lactate, acetate, propionate and butyrate are the universal products of acidogenesis, regardless of feedstock.

The aim of this work was to provide solid proofs regarding the achievement of “steady-state conditions”, which means that the performance of the anaerobic digester is representative of the applied environmental conditions. For this reason, we investigated how, starting from different inoculum sources (i.e., municipal wastewater treatment, bio-waste treatment, and agricultural waste biogas plant), the microbial community adapted to the operational parameters and led to stable biogas production in thermophilic digesters treating the same influent feedstock. The results revealed that the different system achieved similar process performance and microbial community structure after a period that was equal to four hydraulic retention times, approved by a constant pH of 7.89 ± 0.08, 7.92 ± 0.05 and 7.85 ± 0.08, respectively, and stable TAN concentration of 1500 mg/L. Moreover, it was found that the microbial composition of the inocula was a key factor for the speed of achieving stable process performance; thus, a pre-adapted to the influent feedstock inoculum can shorten the stabilization process. On the contrary, after long term reactor operation, the microbial structure was shaped according to the chemical composition of the influent feedstock. The results of the study can also be used as a guide in future researches on anaerobic degradation, particularly in determining the time interval of an experiment to reflect changes in the microbial community of anaerobic digester.

Co-digestion with lipid-rich substrates is a likely strategy in biogas plants, due to their high energy content. However, the process stability is vulnerable to inhibition due to the sudden increase of fatty-acid concentration. Therefore, techniques that promote the adaptation of the microorganisms to the presence of lipids have been proposed. In this frame, the initial hypothesis of the work was that a gradual change in feedstock composition would enable us to elucidate the microbial organisation as a result of deterministic (i.e. chemical composition of influent) and stochastic (e.g. interspecies interactions) factors. This study investigates the response of the biogas microbiome to gradual increment of the Organic Loading Rate by supplementing the influent feedstock with Na-Oleate. The results showed that as a response to the feedstock shifts three clusters describing microbes behaviours were formed. The dynamics and the functional role of the formed microbial clusters were unveiled, providing explanations for their abundance and behavior. Process monitoring indicated that the reactors responded immediately to lipid supplementation and they managed to stabilize their performance in a short period of time. The dominance of Candidatus Methanoculleus thermohydrogenotrophicum in the biogas reactors fed exclusively with cattle manure indicated that the predominant methanogenic pathway was hydrogenotrophic. Additionally, the abundance of this methanogen was further enhanced upon lipid supplementation and its growth was supported by syntrophic bacteria capable to metabolize fatty acids. However, with the shift back to the original feedstock (i.e. solely cattle manure), the microbial dynamicity significantly altered with a remarkable increment in the abundance of a propionate degrader affiliated to the order of Bacteroidales, which became the predominant microorganism of the consortium.

In this study, the effects of bioaugmentation of typically dominant hydrogenotrophic methanogens to CSTR co-digesting cheese whey and manure, under in-situ biomethanation operations were investigated. Reactors working at mesophilic (37 °C) and thermophilic (55 °C) conditions were independently treated and examined in terms of microbial composition and process dynamics. Addition of Methanoculleus bourgensis in the mesophilic reactor led to a stable biomethanation, and an improved microbial metabolism, resulting in 11% increase in CH

Methanogenesis, a biological process mediated by complex microbial communities, has attracted great attention due to its contribution to global warming and potential in biotechnological applications. The current study unveiled the core microbial methanogenic metabolisms in anaerobic vessel ecosystems by applying combined genome-centric metagenomics and metatranscriptomics. Here, we demonstrate that an enriched natural system, fueled only with acetate, could support a bacteria-dominated microbiota employing a multi-trophic methanogenic process. Moreover, significant changes, in terms of microbial structure and function, were recorded after the system was supplemented with additional H2. Methanosarcina thermophila, the predominant methanogen prior to H2 addition, simultaneously performed acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis. The methanogenic pattern changed after the addition of H2, which immediately stimulated Methanomicrobia-activity and was followed by a slow enrichment of Methanobacteria members. Interestingly, the essential genes involved in the Wood-Ljungdahl pathway were not expressed in bacterial members. The high expression of a glycine cleavage system indicated the activation of alternative metabolic pathways for acetate metabolism, which were reconstructed in the most abundant bacterial genomes. Moreover, as evidenced by predicted auxotrophies, we propose that specific microbes of the community were forming symbiotic relationships, thus reducing the biosynthetic burden of individual members. These results provide new information that will facilitate future microbial ecology studies of interspecies competition and symbiosis in methanogenic niches.

Anaerobic digestion is a key biological process for renewable energy, yet the mechanistic knowledge on its hidden microbial dynamics is still limited. The present work charted the interaction network in the anaerobic digestion microbiome via the full characterization of pairwise interactions and the associated metabolite exchanges. To this goal, a novel collection of 836 genome-scale metabolic models was built to represent the functional capabilities of bacteria and archaea species derived from genome-centric metagenomics. Dominant microbes were shown to prefer mutualistic, parasitic and commensalistic interactions over neutralism, amensalism and competition, and are more likely to behave as metabolite importers and profiteers of the coexistence. Additionally, external hydrogen injection positively influences microbiome dynamics by promoting commensalism over amensalism. Finally, exchanges of glucogenic amino acids were shown to overcome auxotrophies caused by an incomplete tricarboxylic acid cycle. Our novel strategy predicted the most favourable growth conditions for the microbes, overall suggesting strategies to increasing the biogas production efficiency. In principle, this approach could also be applied to microbial populations of biomedical importance, such as the gut microbiome, to allow a broad inspection of the microbial interplays.

Ammonia released from the degradation of protein and/or urea usually leads to suboptimal anaerobic digestion (AD) when N-rich organic waste is used. However, the insights behind the differential ammonia tolerance of anaerobic microbiomes remain an enigma. In this study, the cultivation in synthetic medium with different carbon sources (acetate, methanol, formate, and H2/CO2) shaped a common initial inoculum into four unique ammonia-tolerant syntrophic populations. Specifically, various levels of ammonia tolerance were observed: consortia fed with methanol and H2/CO2 could grow at ammonia levels up to 7.25 g NH+-N/L, whereas the other two groups (formate and acetate) only thrived at 5.25 and 4.25 g NH+-N/L, respectively. Metabolic reconstruction highlighted that this divergent microbiome might be achieved by complementary metabolisms to maximize biomethane recovery from carbon sources, thus indicating the importance of the syntrophic community in the AD of N-rich substrates. Besides, sodium/proton antiporter operon, osmoprotectant/K+ regulator, and osmoprotectant synthesis operon may function as the main drivers of adaptation to the ammonia stress. Moreover, energy from the substrate-level phosphorylation and multiple energy-converting hydrogenases (e.g., Ech and Eha) could aid methanogens to balance the energy request for anabolic activities and contribute to thriving when exposed to high ammonia levels.

The bioaugmentation is crucial to improve the energy-efficient process for anaerobic digestion of organic wastes at high ammonia levels. Genomic insights into the intricate microbial networks at a high ammonia level remain underexplored. The present study showed that the addition of Methanoculleus sp. DTU887 remarkably enhanced the methane production yield of organic fraction of municipal solid waste by 21% and decreased the volatile fatty acids by 10% when compared to the period before bioaugmentation. Genome-centric metagenomics reports the functional contribution of microbial members during organic waste degradation under the extremely high level of 13.5 g NH4+-N/L. Specifically, metabolic reconstruction revealed that these organisms have the potential to perform fermentative and acetogenic catabolism, a process facilitated by energy conservation-related with H2/CO2 metabolism. Peptococcaceae spp. (DTU903, DTU900, and DTU895). and Tissierellales sp. DTU879 could degrade the organic waste hydrolysis product, i.e., glucose to acetate and H2. Tissierellales sp. DTU879 and Syntrophaceticus sp. DTU783 could degrade the derived acetate. The H2 scavenging Methanoculleus sp. DTU887 performs complementary metabolic reactions with Peptococcaceae spp., Tissierellales sp. and Syntrophaceticus sp., indicating syntrophic glucose and acetate degradation. This research offers the first insight that the key organisms form a syntrophy-supported food web in response to the bioaugmentation with ammonia tolerant methanogens performed in an AD system subjected to severe ammonia inhibition.

Gas fermentation for the production of building block molecules and biofuels is lately gaining attention as a means to eliminate the greenhouse gases emissions. Especially CO2 capture and recycling are in focus. Thus, the biological coupling of CO2 and H2 is of high interest. Therefore, the focus of the present work was to evaluate the performances of two up-flow reactors for CO2 and H2 assimilation. Process monitoring showed that the gas-liquid H2 transfer was highly affected by reactor design. A reactor filled with Raschig rings could lift up gases utilization leading to a CH4 content of 81% at 6 h gas retention time and 8.8 L/LR.h gas recirculation rate. In contrast, limited biomethanation was achieved in the absence of Raschig rings highlighting the positive role of packing material to the performance of up-flow-reactors. Additionally, high-throughput 16S rRNA sequencing revealed that the microbial community was ultimately resided by Methanothermobacter methanogens.

Background Microorganisms in biogas reactors are essential for degradation of organic matter and methane production. However, a comprehensive genome-centric comparison, including relevant metadata for each sample, is still needed to identify the globally distributed biogas community members and serve as a reliable repository. Results Here, 134 publicly available metagenomes derived from different biogas reactors were used to recover 1,635 metagenome-assembled genomes (MAGs) representing different biogas bacterial and archaeal species. All genomes were estimated to be >50% complete and nearly half ≥90% complete with ≤5% contamination. In most samples, specialized microbial communities were established, while only a few taxa were widespread among the different reactor systems. Metabolic reconstruction of the MAGs enabled the prediction of functional traits related to biomass degradation and methane production from waste biomass. An extensive evaluation of the replication index provided an estimation of the growth rate for microbes involved in different steps of the food chain. The recovery of many MAGs belonging to Candidate Phyla Radiation and other underexplored taxa suggests their specific involvement in the anaerobic degradation of organic matter. Conclusions The outcome of this study highlights a high flexibility of the biogas microbiome, allowing it to modify its composition and to adapt to the environmental conditions, including temperatures and a wide range of substrates. Our findings enhance our mechanistic understanding of the AD microbiome and substantially extend the existing repository of genomes. The established database represents a relevant resource for future studies related to this engineered ecosystem. Keywords Anaerobic digestion, Metagenome-Assembled Genomes, Biogas, Microbial community structure, Functional reconstruction

BackgroundMicroorganisms in biogas reactors are essential for degradation of organic matter and methane production through anaerobic digestion process. However, a comprehensive genome-centric comparison, including relevant metadata for each sample, is still needed to identify the globally distributed biogas community members and serve as a reliable repository.ResultsHere, 134 publicly available datasets derived from different biogas reactors were used to recover 1,635 metagenome-assembled genomes (MAGs) representing different bacterial and archaeal species. All genomes were estimated to be >50% complete and nearly half were ≥90% complete with ≤5% contamination. In most samples, specialized microbial communities were established, while only a few taxa were widespread among the different reactor systems. Metabolic reconstruction of the MAGs enabled the prediction of functional traits related to biomass degradation and methane production from waste biomass. An extensive evaluation of the replication index provided an estimation of the growth rate for microbes involved in different steps of the food chain. The recovery of many MAGs belonging to Candidate Phyla Radiation and other underexplored taxa suggests their specific involvement in the anaerobic degradation of organic matter.ConclusionsThe outcome of this study highlights a high flexibility of the biogas microbiome. The dynamic composition and adaptability to the environmental conditions, including temperatures and a wide range of substrates, were demonstrated. Our findings enhance the mechanistic understanding of anaerobic digestion microbiome and substantially extend the existing repository of genomes. The established database represents a relevant resource for future studies related to this engineered ecosystem.

This study investigates the efficiency in methane production of lab-scale mesophilic (37 °C) and thermophilic (54 °C) continuous stirred tank reactors fed with cheese whey at different operational conditions. Results showed that whey mono-digestion was feasible at mesophilic conditions, while at thermophilic conditions frequent acidification incidents were recorded. The limited buffer capacity of the influent feedstock was responsible for the unstable anaerobic digestion process. The co-digestion of cheese whey with cattle manure maintained the pH levels higher than 7.0, and therefore, stable methane production rates were achieved without any significant accumulation of volatile fatty acids. An additional enhancement of the methane productivity was achieved by in-situ H2 dispersion. Microbial community composition was investigated using high-throughput 16S rRNA gene amplicon sequencing and results were correlated with process parameters. Hydrogenotrophic methanogens were the dominant archaea during the whole experiment at mesophilic and thermophilic conditions.

In typical anaerobic digestion (AD) systems, the microbial functional assertion is hampered by synchronised versatile metabolism required for heterogeneous substrates degradation. Thus, the intricate methanogenic process from organic compounds remains an enigma after decades of empirical operation. In this study, simplified AD microbial communities were obtained with substrate specifications and continuous reactor operation. Genome-centric metagenomic approach was followed to holistically investigate the metabolic pathways of the AD and the microbial synergistic networks. In total, 63 metagenome assembled genomes (MAGs) were assembled from 8 metagenomes acquired in specific methanogenic niches. The metabolic pathways were reconstructed from the annotated genes and their dynamicity under experimental conditions. The results show that the methanogenic niches nourish unique metabolism beyond current knowledge acquired from cultivation-based methods. A novel glucose mineralization model without acetate formation was proposed and asserted in a pair of syntrophs: Clostridiaceae sp. and Methanoculleus thermophilus. Moreover, the catabolic pathway was elucidated in uncharacterized syntrophic acetate oxidizers, Synergistaceae spp. A remarkable evolutionary insight is the discovery that electron transport and energy conservation mechanisms impose selective pressure on syntrophic partners. Overall, the functional roles of the individual microbes tightly rely on the catabolic pathways and cannot always be physiologically defined in accordance with conventional four-step AD concept. The substrate-specific systems provided a traceable microbial community to dissecting the AD process. The genome-centric metagenomics successfully constructed genomes of microbes that have not been previously isolated and illustrated metabolic pathways that beyond the current knowledge of AD process. This study provides new perspectives to unravel the AD microbial ecology and suggests more attention should be paid on uncharacterized metabolism specifically harboured by AD microbial communities.

Background The expansion of renewable energy produced by windmills and photovoltaic panels has generated a considerable electricity surplus, which can be utilized in water electrolysis systems for hydrogen production. The resulting hydrogen can then be funneled to anaerobic digesters for biogas upgrading (biomethanation) purposes (power-to-methane) or to produce high value-added compounds such as short-chain fatty acids (power-to-chemicals). Genome-centric metagenomics and metatranscriptomic analyses were performed to better understand the metabolic dynamics associated with H2 injection in two different configurations of anaerobic digesters treating acidic wastes, specifically cheese manufacturing byproducts. These approaches revealed the key-genes involved in methanation and carbon fixation pathways at species level. Results The biogas upgrading process in the single-stage configuration increased the CH4 content by 7%. The dominant methanogenic species responsible for the upregulation of the hydrogenotrophic pathway in this reactor was Methanothermobacter wolfeii UC0008. In the two-stage configuration, H2 injection induced an upregulation of CO2 fixation pathways producing short-chain fatty acids, mainly acetate and butyrate. In this configuration, the abundant species Anaerobaculum hydrogeniformans UC0046 and Defluviitoga tunisiensis UC0050 primarily upregulated genes related to electron transport chains, suggesting putative syntrophisms with hydrogen scavenger microbes. Interestingly, Tepidanaerobacter acetatoxydans UC0018 did not act as an acetate-oxidizer in either reactor configurations, and instead regulated pathways involved in acetate production and uptake. A putative syntrophic association between Coprothermobacter proteolyticus UC0011 and M. wolfeii UC0008 was proposed in the two-stage reactor. In order to support the transcriptomic findings regarding the hydrogen utilization routes, an advanced bioconversion model was adapted for the simulation of the single- and two-stage reactor setups. Conclusions This is the first study investigating biogas reactor metatranscriptome dynamics following hydrogen injection for biomethanation and carbon fixation to short-chain fatty acids purposes. The same microbes showed different patterns of metabolic regulation in the two reactor configurations. It was observed an effect of the specialized acidogenic reactor on the overall microbial consortium composition and activity in the two-stage digester. There were also suggested the main species responsible for methanation, short-chain fatty acids production, and electron transport chain mechanisms, in both reactor configurations. Electronic supplementary material The online version of this article (10.1186/s40168-018-0583-4) contains supplementary material, which is available to authorized users.

Microbial dynamics in an upgrading biogas reactor system undergoing a more than two years-period at stable operating conditions were explored. The carbon dioxide generated during biomass degradation in the first reactor of the system was converted to methane into the secondary reactor by addition of external hydrogen. Considering the overall efficiency, the long-term operation period resulted in an improved biogas upgrading performance (99% methane content). However, a remarkable accumulation of acetate was revealed, indicating the enhancement of homoacetogenic activity. For this reason, a shift in the anaerobic digestion microbiome was expected and evaluated by 16S rRNA amplicon analysis. Results demonstrated that the most abundant archaeal species identified in the first time point, Candidatus Methanoculleus thermohydrogenotrophicum, was replaced by Methanothermobacter thermautotrophicus, becoming dominant after the community adaptation. The most interesting taxonomic units were clustered by relative abundance and six main long-term adaptation trends were found, characterizing functionally related microbes (e.g. homoacetogens).

The aim of this work was to elucidate the microbial ecology in twelve mesophilic and thermophilic full-scale biogas plants using a genome-centric metagenomic approach. In this study both biogas plants treating manure and those treating sludge from waste water treatment plants were considered. The identification of 132 Metagenome-Assembled Genomes (MAGs) and analysis of their abundance profile in different samples allowed the identification of the most abundant core members of the anaerobic digestion microbiome. Canonical correspondence analysis was used to determine the influence of biotic and environmental factors on MAGs abundance and to investigate the methanogenic performance of the biogas plants. Prediction of the functional properties of MAGs was obtained analyzing their KEGG pathways and their carbohydrate active domains. Network analysis allowed investigation of species-species associations and shed light on syntrophic interactions between members belonging to the anaerobic digestion dark matter (phylum Fermentibacteria). By stratifying and comparing different levels of information, it was predicted that some MAGs have a crucial role in the manure-supplemented thermophilic biogas plants and it was highlighted the importance of the glycine cleavage system in complementing the "truncated" Wood-Ljungdahl pathway.

Biogas upgrading via carbon dioxide hydrogenation is an emerging technology for electrofuel production. The biomethanation efficiency is strongly dependent on a balanced microbial consortium, whose high- resolution characterization along with their functional potential and interactions are pivotal for process optimization. The present work is the first genome-centric metagenomic study on mesophilic and thermophilic biogas upgrading reactors aiming to define the metabolic profile of more than 200 uncultivated microbes involved in hydrogen assisted methanogenesis. The outcomes from predictive functional analyses were correlated with microbial abundance variations to clarify the effect of process parameters on the community. The operational temperature significantly influenced the microbial richness of the reactors, while the H2 addition distinctively alternated the abundance of the taxa. Two different Methanoculleus species (one mesophilic and one thermophilic) were identified as the main responsible ones for methane metabolism. Finally, it was demonstrated that the addition of H2 exerted a selective pressure on the concerted or syntrophic interactions of specific microbes functionally related to carbon fixation, propionate and butanoate metabolisms. Novel bacteria were identified as candidate syntrophic acetate oxidizers (e.g., Tepidanaerobacter sp. DTU063), while the addition of H2 favored the proliferation of potential homoacetogens (e.g., Clostridia sp. DTU183). Population genomes encoding genes of Wood-Ljungdahl pathway were mainly thermophilic, while propionate degraders were mostly identified at mesophilic conditions. Finally, putative syntrophic interactions were identified between microbes that have either versatile metabolic abilities or are obligate/facultative syntrophs.

The mechanisms by which specific anaerobic microorganisms remain firmly attached to lignocellulosic material, allowing them to efficiently decompose organic matter, have yet to be elucidated. To circumvent this issue, microbiomes collected from anaerobic digesters treating pig manure and meadow grass were fractionated to separate the planktonic microbes from those adhered to lignocellulosic substrate. Assembly of shotgun reads, followed by a binning process, recovered 151 population genomes, 80 out of which were completely new and were not previously deposited in any database. Genome coverage allowed the identification of microbial spatial distribution in the engineered ecosystem. Moreover, a composite bioinformatic analysis using multiple databases for functional annotation revealed that uncultured members of the Bacteroidetes and Firmicutes follow diverse metabolic strategies for polysaccharide degradation. The structure of cellulosome in Firmicutes species can differ depending on the number and functional roles of carbohydrate-binding modules. In contrast, members of the Bacteroidetes are able to adhere to and degrade lignocellulose due to the presence of multiple carbohydrate-binding family 6 modules in beta-xylosidase and endoglucanase proteins or S-layer homology modules in unknown proteins. This study combines the concept of variability in spatial distribution with genome-centric metagenomics, allowing a functional and taxonomical exploration of the biogas microbiome. IMPORTANCE This work contributes new knowledge about lignocellulose degradation in engineered ecosystems. Specifically, the combination of the spatial distribution of uncultured microbes with genome-centric metagenomics provides novel insights into the metabolic properties of planktonic and firmly attached to plant biomass bacteria. Moreover, the knowledge obtained in this study enabled us to understand the diverse metabolic strategies for polysaccharide degradation in different species of Bacteroidetes and Clostridiales. Even though structural elements of cellulosome were restricted to Clostridiales species, our study identified a putative mechanism in Bacteroidetes species for biomass decomposition, which is based on a gene cluster responsible for cellulose degradation, disaccharide cleavage to glucose, and transport to cytoplasm.

Four full-scale anaerobic digesters with a long history of stable operation were characterized in terms of active microbiome and metagenome. Isotopic fractionation of biogas demonstrated that acetotrophy was rather prevalent in reactors operated at 6 gTAN L−1, suggesting that syntrophic acetate oxidizing bacteria (SAOB) played a significant role in the latter. These results were generally coherent with the observed active bacterial and archaeal communities but no known SAOB were observed. Metagenome descriptions yielded 73 assembled population genomes, of which only 7 could be assigned at the species level. Gene annotation and association to relevant metabolic pathways indicated that the phyla Chloroflexi and Bacteroidales might encompass new, currently undescribed, SAOB/formate producing species that would metabolize acetate via the glycine cleavage system. The predominant hydrogenotrophic counterpart at a high ammonia content belonged to the genus Methanoculleus, which could also grow on acetate to a certain extent.

The anaerobic digestion process is often inhibited by alteration of substrates and/or organic overload. This study aimed to elucidate changes of microbial ecology in biogas reactors upon radical changes of substrates and to determine their importance to process imbalance. For this reason, continuously fed reactors were disturbed with pulses of proteins, lipids and carbohydrates and the microbial ecology of the reactors were characterized by 16S rRNA gene sequencing before and after the imposed changes. The microbial composition of the three reactors, initially similar, diverged greatly after substrate change. The greatest increase in diversity was observed in the reactor supplemented with carbohydrates and the microbial community became dominated by lactobacilli, while the lowest corresponded to the reactor overfed with proteins, where only Desulfotomaculum showed significant increase. The overall results suggest that feed composition has a decisive impact on the microbial composition of the reactors, and thereby on their performance.

CO is a main component of syngas, which can be produced from the gasification of organic wastes and biomass. CO can be converted to methane by anaerobic digestion (AD), however, it is still challenging due to its toxicity to microorganisms and limited knowledge about CO converting microorganisms. In the present study, anaerobic granular sludge (AGS) was used for the simultaneous biomethanation of wastewater and CO. Batch experiments showed that AGS tolerated CO partial pressure as high as 0.5 atm without affecting its ability for synthetic wastewater degradation, which had higher tolerance of CO compared to suspended sludge (less than 0.25 atm) as previously reported. Continuous experiments in upflow anaerobic sludge blanket (UASB) reactors showed AGS could efficiently convert synthetic wastewater and CO into methane by applying gas-recirculation. The addition of CO to UASB reactor enhanced the hydrogenotrophic CO-oxidizing pathway, resulted in the increase of extracellular polymeric substances, changed the morphology of AGS and significantly altered the microbial community compositions of AGS. The microbial species relating with CO conversion and their functions were revealed by metagenomic analysis. It showed that 23 of the 70 reconstructed genome bins (GBs), most of which were not previously characterized at genomic level, were enriched and contained genes involved in CO conversion upon CO addition. CO-converting microorganisms might be taxonomically more diverse than previously known and have multi-functions in the AD process. The reductive tricarboxylic acid (TCA) cycle in combination with the oxidation of the CO was probably crucial for CO utilization by the majority of the GBs in the present study.

Upflow anaerobic sludge blanket (UASB) reactor is one of the most applied technologies for various high-strength wastewater treatments. The present study analysed the microbial community changes in UASB granules during the transition from mesophilic to thermophilic conditions. Dynamicity of microbial community in granules was analysed using high-throughput sequencing of 16S ribosomal RNA gene amplicons, and the results showed that the temperature strictly determines the diversity of the microbial consortium. It was demonstrated that most of the microbes which were present in the initial mesophilic community were not found in the granules after the transition to thermophilic conditions. More specifically, only members from family Anaerolinaceae managed to tolerate the temperature change and contributed in maintaining the physical integrity of granular structure. On the contrary, new hydrolytic and fermentative bacteria were quickly replacing the old members in the community. A direct result from this abrupt change in the microbial diversity was the accumulation of volatile fatty acids and the concomitant pH drop in the reactor inhibiting the overall anaerobic digestion process. Nevertheless, by maintaining deliberately the pH levels at values higher than 6.5, a methanogen belonging to Methanoculleus genus emerged in the community enhancing the methane production.

Recently, a first comprehensive catalogue of microbial genomes populating biogas reactors treating manure and agro-industrial residues was determined by sequencing samples collected from 22 biogas reactors including laboratory and full scale. Among the archaeal community, one of the most abundant methanogens belongs to Methanoculleus genus and for this reason it was provisionally named Methanoculleus sp. DTU006. Its full length 16S rRNA sequence is 97% similar to Methanoculleus marisnigri JR1 and to Methanoculleus palmolei DSM 4273. Despite the high similarity of the 16S gene sequence, Average Nucleotide Identity calculation (ANI) calculated on all protein encoding genes indicated that the two most similar species, Methanoculleus bourgensis MS2 T and Methanoculleus sp. MAB1, are divergent enough to define Methanoculleus sp. DTU006 as new archaeal species. Its genome (2.15 Mbp) has an estimated completeness around 93%. Analysis of the metabolic pathways using KEGG confirmed that it is a hydrogenotrophic methanogen and therefore it is proposed the Candidatus status by naming it as “ Candidatus Methanoculleus thermohydrogenotrophicum”.

The anaerobic decomposition of organic substrates leads to the generation of gases, such as methane, which can either be a valuable energy carrier in industrial applications or can be considered as a main greenhouse gas when it is naturally emitted. In this study we investigated in vitro the effect of dietary compounds, such as starch and proteins, on the microbial community present in the rumen fluid. High throughput shotgun sequencing, followed by metagenomic assembly and binning allowed the extraction of 18 genome bins. A composite bioinformatic analysis led to the prediction of metabolic pathways involved in the degradation of dietary compounds and in the biosynthesis of crucial products like propionate, methane and ammonia. The identification of genomes belonging to poorly characterized phyla such as Thermoplasmata and Elusimicrobia shed light on their putative role. The high abundance of methylotrophic archaea in the inoculum suggests a relevant role in methane production.

Upflow anaerobic sludge blanket (UASB) technology refers to reactor technology where granules, i.e. self-immobilised microbial associations, are the biological catalysts involved in the anaerobic digestion process. During the start-up period, UASB reactors operate at relatively long HRT and therefore the liquid phase of the reactor becomes a favourable environment for microbial growth. The current study aimed to elucidate the dynamicity of the suspended microbial community in UASB reactors, during the transition from mesophilic to thermophilic conditions. High throughput 16S rRNA amplicon sequencing was used to characterize the taxonomic composition of the microbiome. The results showed that the microbial community was mainly composed by hydrolytic and fermentative bacteria. Results revealed relevant shifts in the microbial community composition, which is mainly determined by the operational conditions and the reactor performance. Finally, shared OTUs between the microbial consortia of the suspended and the granular sludge showed that planktonic microbiota is significantly influencing the granule microbial community composition.

Biogas upgrading is envisioned as a key process for clean energy production. The current study evaluates the efficiency of different reactor configurations for ex-situ biogas upgrading and enhancement, in which externally provided hydrogen and carbon dioxide were biologically converted to methane by the action of hydrogenotrophic methanogens. The methane content in the output gas of the most efficient configuration was >98%, allowing its exploitation as substitute to natural gas. Additionally, use of digestate from biogas plants as a cost efficient method to provide all the necessary nutrients for microbial growth was successful. High-throughput 16S rRNA sequencing revealed that the microbial community was resided by novel phylotypes belonging to the uncultured order MBA08 and to Bacteroidales. Moreover, only hydrogenotrophic methanogens were identified belonging to Methanothermobacter and Methanoculleus genera. Methanothermobacter thermautotrophicus was the predominant methanogen in the biofilm formed on top of the diffuser surface in the bubble column reactor.

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.

Background Biogas production is an economically attractive technology that has gained momentum worldwide over the past years. Biogas is produced by a biologically mediated process, widely known as “anaerobic digestion.” This process is performed by a specialized and complex microbial community, in which different members have distinct roles in the establishment of a collective organization. Deciphering the complex microbial community engaged in this process is interesting both for unraveling the network of bacterial interactions and for applicability potential to the derived knowledge. Results In this study, we dissect the bioma involved in anaerobic digestion by means of high throughput Illumina sequencing (~51 gigabases of sequence data), disclosing nearly one million genes and extracting 106 microbial genomes by a novel strategy combining two binning processes. Microbial phylogeny and putative taxonomy performed using >400 proteins revealed that the biogas community is a trove of new species. A new approach based on functional properties as per network representation was developed to assign roles to the microbial species. The organization of the anaerobic digestion microbiome is resembled by a funnel concept, in which the microbial consortium presents a progressive functional specialization while reaching the final step of the process (i.e., methanogenesis). Key microbial genomes encoding enzymes involved in specific metabolic pathways, such as carbohydrates utilization, fatty acids degradation, amino acids fermentation, and syntrophic acetate oxidation, were identified. Additionally, the analysis identified a new uncultured archaeon that was putatively related to Methanomassiliicoccales but surprisingly having a methylotrophic methanogenic pathway. Conclusion This study is a pioneer research on the phylogenetic and functional characterization of the microbial community populating biogas reactors. By applying for the first time high-throughput sequencing and a novel binning strategy, the identified genes were anchored to single genomes providing a clear understanding of their metabolic pathways and highlighting their involvement in anaerobic digestion. The overall research established a reference catalog of biogas microbial genomes that will greatly simplify future genomic studies. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0441-1) contains supplementary material, which is available to authorized users.

Metagenomic sequencing is a fundamental tool to identify the functional potential of the prokaryotic species present in microbial communities, particularly for the unculturable microbes. Recent advances in software dedicated to metagenomic assembly allow nowadays to generate collections of scaffolds comprehensive of thousands genome sequences, but the binning of these scaffolds into OTUs representative of microbial genomes is still challenging. In the attempt to obtain a deep characterization of the anaerobic digestion microbiome, different metagenomic binning approaches were integrated into a new tool. To facilitate the binning process, this tool integrates two strategies; the taxonomic assignment of scaffolds and the clustering based on coverage values. By applying this procedure, 373 high quality genomes involved in the anaerobic digestion process have been extracted and annotated using COG, KEGG, SEED and Pfam. These high throughput approaches pose nowadays other basic challenges related to the computational effort needed for the taxonomic assignment of hundreds new microbial genomes. It is also mandatory to verify if other DNA sequences deriving from the same species are already present in public databases. Metagenomics raise new fundamental questions regarding the definition of what a microbial species is and how it can be defined solely considering its genome. In order to address these issues we have developed a collection of scripts to check the presence of the same genome sequence not only in different assemblies, but also in public databases and, finally, to simplify its functional annotation.

This research aimed to better characterize the biogas microbiome by means of high throughput metagenomic sequencing and to elucidate the core microbial consortium existing in biogas reactors independently from the operational conditions. Assembly of shotgun reads followed by an established binning strategy resulted in the highest, up to now, extraction of microbial genomes involved in biogas producing systems. From the 236 extracted genome bins, it was remarkably found that the vast majority of them could only be characterized at high taxonomic levels. This result confirms that the biogas microbiome is comprised by a consortium of unknown species. A comparative analysis between the genome bins of the current study and those extracted from a previous metagenomic assembly demonstrated a similar phylogenetic distribution of the main taxa. Finally, this analysis led to the identification of a subset of common microbes that could be considered as the core essential group in biogas production.

Mechanical pretreatment is considered to be a fast and easily applicable method to prepare the biomass for anaerobic digestion. In the present study, the effect of mechanical pretreatment on lignocellulosic silages biodegradability was elucidated in batch reactors. Moreover, co-digestion of the silages with pig manure in continuously fed biogas reactors was examined. Metagenomic analysis for determining the microbial communities in the pig manure digestion system was performed by analysing unassembled shotgun genomic sequences. A comparative analysis allowed to identify the microbial species firmly attached to the digested grass particles and to distinguish them from the planktonic microbes floating in the liquid medium. It was shown that the methane yield of ensiled grass was significantly increased by 12.3% due to mechanical pretreatment in batch experiments. Similarly, the increment of the methane yield in the co-digestion system reached 6.4%. Regarding the metagenomic study, species similar to Coprothermobacter proteolyticus and to Clostridium thermocellum , known for high proteolytic and cellulolytic activity respectively, were found firmly attached to the solid fraction of digested feedstock. Results from liquid samples revealed clear differences in microbial community composition, mainly dominated by Proteobacteria . The archaeal community was found in higher relative abundance in the liquid fraction of co-digestion experiment compared to the solid fraction. Finally, an unclassified Alkaliphilus sp. was found in high relative abundance in all samples.

Foam formation can lead to total failure of digestion process in biogas plants. In the present study, possible correlation between foaming and the presence of specific microorganisms in biogas reactors was elucidated. The microbial ecology of continuous fed digesters overloaded with proteins, lipids and carbohydrates before and after foaming incidents was characterized using 16S rRNA gene sequencing. Moreover, the microbial diversity between the liquid and foaming layer was assessed. A number of genera that are known to produce biosurfactants, contain mycolic acid in their cell wall, or decrease the surface tension of the media, increased their relative abundance after foam formation. Finally, a microorganism similar to widely known foaming bacteria (Nocardia and Desulfotomaculum) was found to increase its relative abundance in all reactors once foam was observed, regardless of the used substrate. These findings suggest that foaming and specific microorganisms might have direct association which requires to be further investigated.