Evaluation of the optimal sewage sludge pre-treatment technology through continuous reactor operation: Process performance and microbial community insights.

This study investigated the impact of two low-temperature thermal pre-treatments on continuous anaerobic reactors' performance, sequentially fed with sludge of different total solids content (∼3 % and ∼6 %) and subjected to progressively increasing Organic Loading Rates (OLR) from 1.0 to 2.5 g volatile solids/(LReactor⋅day). Assessing pre-treatments' influence on influent sludge characteristics revealed enhanced organic matter hydrolysis, facilitating sludge solubilization and methanogenesis; volatile fatty acids concentration also increased, particularly in pre-treated sludge of ∼6 % total solids, indicating improved heating efficiency under increased solids content. The reactor fed with sludge pre-treated at 45 °C for 48 h and 55 °C for an extra 48 h exhibited the highest methane yield under all applied OLRs, peaking at 240 ± 3.0 mL/g volatile solids at the OLR of 2.5 g volatile solids/(LReactor⋅day). 16S rRNA gene sequencing demonstrated differences in the reactors' microbiomes as evidence of sludge thickening and the different pre-treatments applied, which promoted the release of organic matter in diverse concentrations and compositions. Finally, the microbial analysis revealed that specific foam-related genera increased in abundance in the foam layer of reactors' effluent bottles, dictating their association with the sludge foaming incidents that occurred inside the reactors during their operation at 2.0 g volatile solids/(LReactor⋅day).

The effect of anaerobic digestate as an organic soil fertilizer on the diversity and structure of the indigenous soil microbial and nematode communities.

Anaerobic digestate is a popular soil additive which can promote sustainability and transition toward a circular economy. This study addresses how anaerobic digestate modifies soil health when combined with a common chemical fertilizer. Attention was given to soil microbes and, a neglected but of paramount importance soil taxonomic group, soil nematodes. A mesocosm experiment was set up in order to assess the soil's microbial and nematode community. The results demonstrated that the microbial diversity was not affected by the different fertilization regimes, although species richness increased after digestate and mixed fertilization. The composition and abundance of nematode community did not respond to any treatment. Mixed fertilization notably increased potassium (K) and boron (B) levels, while nitrate (NO3-) levels were uniformly elevated across fertilized soils, despite variations in nitrogen input. Network analysis revealed that chemical fertilization led to a densely interconnected network with mainly mutualistic relationships which could cause ecosystem disruption, while digestate application formed a more complex community based on bacterial interactions. However, the combination of both orchestrated a more balanced and less complex community structure, which is more resilient to random disturbances, but on the downside, it is more likely to collapse under targeted perturbations.

Decoding Microbial Responses to Ammonia Shock Loads in Biogas Reactors through Metagenomics and Metatranscriptomics.

The presence of elevated ammonia levels is widely recognized as a significant contributor to process inhibition in biogas production, posing a common challenge for biogas plant operators. The present study employed a combination of biochemical, genome-centric metagenomic and metatranscriptomic data to investigate the response of the biogas microbiome to two shock loads induced by single pulses of elevated ammonia concentrations (i.e., 1.5 g NH4+/LR and 5 g NH4+/LR). The analysis revealed a microbial community of high complexity consisting of 364 Metagenome Assembled Genomes (MAGs). The hydrogenotrophic pathway was the primary route for methane production during the entire experiment, confirming its efficiency even at high ammonia concentrations. Additionally, metatranscriptomic analysis uncovered a metabolic shift in the methanogens Methanothrix sp. MA6 and Methanosarcina flavescens MX5, which switched their metabolism from the acetoclastic to the CO2 reduction route during the second shock. Furthermore, multiple genes associated with mechanisms for maintaining osmotic balance in the cell were upregulated, emphasizing the critical role of osmoprotection in the rapid response to the presence of ammonia. Finally, this study offers insights into the transcriptional response of an anaerobic digestion community, specifically focusing on the mechanisms involved in recovering from ammonia-induced stress.

Comparative study on packing materials for improved biological methanation in trickle Bed reactors.

Packing materials improve biological methanation efficiency in Trickle Bed Reactors. The present study, which lies in the field of energy production and biotechnology, entailed the evaluation of commercial pelletized activated carbon and Raschig rings as packing materials. The evaluation focused on monitoring process indicators and examining the composition of the microbial community. Activated carbon resulted in enhanced methane purity, achieving a two-fold higher methane percentage than Raschig rings, maintaining a stable pH level within a range of 7-8 and reducing gas retention time from 6 h to 90 min. Additionally, the digestate derived from biogas plant was found to be a sufficient nutrient source for the process. Fermentative species with genes for ß-oxidation, such as Amaricoccus sp. and Caloramator australicus could explain the production of hexanoic and valerate acids during reactor operation. Based on the physical properties of packing materials, the efficiency of biological methanation could be maximized.

Integrating metagenomic binning with flux balance analysis to unravel syntrophies in anaerobic CO2 methanation.

BACKGROUND: Carbon fixation through biological methanation has emerged as a promising technology to produce renewable energy in the context of the circular economy. The anaerobic digestion microbiome is the fundamental biological system operating biogas upgrading and is paramount in power-to-gas conversion. Carbon dioxide (CO2) methanation is frequently performed by microbiota attached to solid supports generating biofilms. Despite the apparent simplicity of the microbial community involved in biogas upgrading, the dynamics behind most of the interspecies interaction remain obscure. To understand the role of the microbial species in CO2 fixation, the biofilm generated during the biogas upgrading process has been selected as a case study. The present work investigates via genome-centric metagenomics, based on a hybrid Nanopore-Illumina approach the biofilm developed on the diffusion devices of four ex situ biogas upgrading reactors. Moreover, genome-guided metabolic reconstruction and flux balance analysis were used to propose a biological role for the dominant microbes. RESULTS: The combined microbiome was composed of 59 species, with five being dominant (> 70% of total abundance); the metagenome-assembled genomes representing these species were refined to reach a high level of completeness. Genome-guided metabolic analysis appointed Firmicutes sp. GSMM966 as the main responsible for biofilm formation. Additionally, species interactions were investigated considering their co-occurrence in 134 samples, and in terms of metabolic exchanges through flux balance simulation in a simplified medium. Some of the most abundant species (e.g., Limnochordia sp. GSMM975) were widespread (~ 67% of tested experiments), while others (e.g., Methanothermobacter wolfeii GSMM957) had a scattered distribution. Genome-scale metabolic models of the microbial community were built with boundary conditions taken from the biochemical data and showed the presence of a flexible interaction network mainly based on hydrogen and carbon dioxide uptake and formate exchange. CONCLUSIONS: Our work investigated the interplay between five dominant species within the biofilm and showed their importance in a large spectrum of anaerobic biogas reactor samples. Flux balance analysis provided a deeper insight into the potential syntrophic interaction between species, especially Limnochordia sp. GSMM975 and Methanothermobacter wolfeii GSMM957. Finally, it suggested species interactions to be based on formate and amino acids exchanges. Video Abstract.

Optimization of supercritical carbon dioxide explosion for sewage sludge pre-treatment using response surface methodology.

The present work was conducted to assess whether the implementation of Supercritical Carbon dioxide Explosion (SCE) is an efficient approach for sewage sludge pre-treatment. In this context, SCE was optimized with the aim to develop a method attempting to increase the biodegradability of sewage sludge's organic matter content, and thus, to enhance the subsequent anaerobic digestion and methane production. The statistical tool of response surface methodology was applied to evaluate the effects of the main pre-treatment parameters (i.e. temperature and time) and their interactions on methane yield, which was defined as the response. Temperature was found to be the most significant variable, having the greatest effect on methane yield. Following this, an optimum set of pre-treatment conditions corresponding to a temperature of 115 °C and time of 13 min, was determined. Under these optimum conditions, the predicted response value was 300 mL CH4/g of volatile solids. The corresponding experimental value obtained from the validation experiment fitted well with this value, clearly demonstrating the effective use of response surface methodology in optimizing SCE. Additionally, under optimum conditions, the methane yield presented a statistically significant increment of 8.7%, compared to untreated sludge. This revealed the impact of SCE as an effective and alternative way for the efficient pre-treatment of sewage sludge. Finally, thermal pre-treatment, alkaline and acidic hydrolysis were also applied to the already pre-treated sludge. It was concluded that the combined pre-treatment techniques contributed to a further increase of methane production compared to raw (untreated) substrate.

Valorization of household food wastes to lactic acid production: A response surface methodology approach to optimize fermentation process.

Lactic acid is a valuable compound used in several industrial processes such as polymers, emulsifiers manufacturing, pharmaceutical, and cosmetic formulations. The present study aims to evaluate the potential use of food waste to produce lactic acid through fermentation, both by indigenous microbiota and by the bio-augmentation with two lactic acid bacteria, namely Lactobacillus plantarum BS17 and Lactobacillus casei BP2. Fermentation was studied both in batch and continuously fed anaerobic reactors at mesophilic conditions and a Response Surface Methodology approach was used to optimize the bioprocess performance and determine the environmental parameters (namely pH and time) that lead to the enhancement of lactic acid production during the batch fermentation by indigenous microorganisms. Results revealed an optimum set of conditions for lactic acid production at a pH value of 6.5 and a fermentation period of 3.5 days at 37 °C. Under these conditions lactic acid production reached a value of 23.07 g/L, which was very similar to the mathematically predicted ones, thus verifying the accuracy of the experimental design. This optimum set of conditions was further employed to examine the production of lactic acid under continuous fermentation operation. Furthermore, concentrations of volatile fatty acids and ethanol were monitored and found to be relatively low, with ethanol being the dominant by-product of fermentation, indicating the presence of heterofermentative bacteria in the food wastes. A final step of downstream process was performed resulting in the successful recovery of lactic acid with purity over 90%.

Thermophilic anaerobic digestion of olive mill wastewater in an upflow packed bed reactor: Evaluation of 16S rRNA amplicon sequencing for microbial analysis.

Olive mill wastewater, a by-product of olive oil production after the operation of three-phase decanters, was used in a thermophilic anaerobic digester targeting efficient bioconversion of its organic load into biogas. An active anaerobic inoculum originating from a mesophilic reactor, was acclimatized under thermophilic conditions and was filled into a high-rate upflow packed bed reactor. Its performance was tested towards the treatment efficacy of olive mill wastewater under thermophilic conditions reaching the minimum hydraulic retention time of 4.2 d with promising results. As analysis of the microbial communities is considered to be the key for the development of anaerobic digestion optimization techniques, the present work focused on characterizing the microbial community and its variation during the reactor's runs, via 16S rRNA amplicon sequencing. Identification of new microbial species and taxonomic groups determination is of paramount importance as these representatives determine the bioprocess outcome. The current study results may contribute to further olive mill wastewater exploitation as a potential source for efficient biogas production.

Evolution of the microbial community structure in biogas reactors inoculated with seeds from different origin.

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.

Microbial dynamics in biogas digesters treating lipid-rich substrates via genome-centric metagenomics.

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.

New insights from the biogas microbiome by comprehensive genome-resolved metagenomics of nearly 1600 species originating from multiple anaerobic digesters.

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 1635 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 dynamics for microbes involved in different steps of the food chain. 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.

Metabolic dependencies govern microbial syntrophies during methanogenesis in an anaerobic digestion ecosystem.

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. Video abstract.

Metagenomic insights into bioaugmentation and biovalorization of oily industrial wastes by lipolytic oleaginous yeast Yarrowia lipolytica during successive batch fermentation.

The lipolytic oleaginous yeast Yarrowia lipolytica was used in the bioaugmentation and biovalorization of oily industrial wastes during successive-batch fermentation. Five cycles of nonsterile successive batch fermentation with 70% medium replacement achieved the highest oil removal of 68.1 ± 5.60% and produced biomass and lipid yields of 0.213 ± 0.07 g/g-COD and 146.2 ± 46.5 mg/g-COD, respectively. The cell-bound lipase activity observed in the system was 170.74 ± 32 U/L. The auto-flocculation efficiency of the biomass was >90% within 60 Min. The microbial community changes between Y. lipolytica and indigenous microorganisms were monitored by metagenomic next-generation sequencing of internal transcribed spacer rDNA regions for yeasts and 16S rRNA gene for bacteria. Ylipolytica lipolytica was retained in the consortium together with other indigenous strains until the fifth cycle. Other minor oleaginous yeasts such as Kodamaea ohmeri and Candida tropicalis as well as polyhydroxyalkanoate-accumulating bacteria were found and are likely to have participated in lipid production. This study has shown the robustness of Y. lipolytica in nonsterile successive batch fermentation and its use could contribute greatly to the practical valorization of industrial wastes for lipids and lipases.

Novel ecological insights and functional roles during anaerobic digestion of saccharides unveiled by genome-centric metagenomics.

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.

Co-digestion of Laminaria digitata with cattle manure: A unimodel simulation study of both batch and continuous experiments.

This work investigated the thermophilic (55 °C) co-digestion performance both in batch and continuous mode operation. The biochemical methane potentials of L. digitata and cattle manure were 308 ±â€¯24 and 203 ±â€¯33 mL CH4/g VS, respectively. The optimum co-digestion feedstock ratio was found to be 80% macroalgae: 20% manure on a volatile solids basis, which produced 290 ±â€¯19 mL CH4/g VS under long-term and stable continuous operation at an organic loading rate of 2 g VS/L/d and hydraulic retention time of 15 days. Simulations of the batch and continuous experiments were, for the first time, carried out using an integrated anaerobic bioconversion model without structural modifications. Close fits between measured and simulated data provided mutual confirmation of experimental reliability and model robustness, and provided new perspectives for the use of the software tool.

Microbial profiling during anaerobic digestion of cheese whey in reactors operated at different conditions.

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.

Process performance and microbial community structure in thermophilic trickling biofilter reactors for biogas upgrading.

This study evaluated the process performance and determined the microbial community structure of two lab-scale thermophilic trickling biofilter reactors used for biological methanation of hydrogen and carbon-dioxide for a total period of 94 days. Stable and robust operation was achieved by means of a single-pass gas flow. The quality of the output gas (>97%) was comparable to the methane purity achieved by commercial biogas upgrading systems fulfilling the specifications to be used as substitute to natural gas. The reactors' methane productivity reached >1.7 LCH4/(LR·d) at hydrogen loading rate of 7.2 LH2/(LR·d). The spatial distribution of the microbial consortia localized in the liquid media and biofilm enabled us to gain a deeper understanding on how the microbiome is structured inside the trickling biofilter. Sequencing results revealed a significant predominance of Methanothermobacter sp. in the biofilm. Unknown members of the class Clostridia were highly abundant in biofilm and liquid media, while acetate utilising bacteria predominated in liquid samples.

Microbial activity response to hydrogen injection in thermophilic anaerobic digesters revealed by genome-centric metatranscriptomics.

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.

Spatial Distribution and Diverse Metabolic Functions of Lignocellulose-Degrading Uncultured Bacteria as Revealed by Genome-Centric Metagenomics.

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.

Hydrogen-Fueled Microbial Pathways in Biogas Upgrading Systems Revealed by Genome-Centric Metagenomics.

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.