Assessment of the in situ biomethanation potential of a deep aquifer used for natural gas storage.

The dihydrogen (H2) sector is undergoing development and will require massive storage solutions. To minimize costs, the conversion of underground geological storage sites, such as deep aquifers, used for natural gas storage into future underground hydrogen storage sites is the favored scenario. However, these sites contain microorganisms capable of consuming H2, mainly sulfate reducers and methanogens. Methanogenesis is, therefore expected but its intensity must be evaluated. Here, in a deep aquifer used for underground geological storage, 17 sites were sampled, with low sulfate concentrations ranging from 21.9 to 197.8 µM and a slow renewal of formation water. H2-selected communities mainly were composed of the families Methanobacteriaceae and Methanothermobacteriaceae and the genera Desulfovibrio, Thermodesulfovibrio, and Desulforamulus. Experiments were done under different conditions, and sulfate reduction, as well as methanogenesis, were demonstrated in the presence of a H2 or H2/CO2 (80/20) gas phase, with or without calcite/site rock. These metabolisms led to an increase in pH up to 10.2 under certain conditions (without CO2). The results suggest competition for CO2 between lithoautotrophs and carbonate mineral precipitation, which could limit microbial H2 consumption.

The Targeted Deletion of Genes Responsible for Expression of the Mth60 Fimbriae Leads to Loss of Cell-Cell Connections in Methanothermobacter thermautotrophicus ΔH.

This study is a continuation by the Environmental Biotechnology Group of the University of Tübingen in memoriam to Reinhard Wirth, who initiated the work on Mth60 fimbriae at the University of Regensburg. Growth in biofilms or biofilm-like structures is the prevailing lifestyle for most microbes in nature. The first crucial step to initiate biofilms is the adherence of microbes to biotic and abiotic surfaces. Therefore, it is crucial to elucidate the initial step of biofilm formation, which is generally established through cell-surface structures (i.e., cell appendages), such as fimbriae or pili, that adhere to biotic and abiotic surfaces. The Mth60 fimbriae of Methanothermobacter thermautotrophicus ΔH are one of only a few known archaeal cell appendages that do not assemble via the type IV pili assembly mechanism. Here, we report the constitutive expression of Mth60 fimbria-encoding genes from a shuttle-vector construct and the deletion of the Mth60 fimbria-encoding genes from the genomic DNA of M. thermautotrophicus ΔH. For this, we expanded our system for genetic modification of M. thermautotrophicus ΔH using an allelic-exchange method. While overexpression of the respective genes increased the number of Mth60 fimbriae, deletion of the Mth60 fimbria-encoding genes led to a loss of Mth60 fimbriae in planktonic cells of M. thermautotrophicus ΔH compared to the wild-type strain. This, either increased or decreased, number of Mth60 fimbriae correlated with a significant increase or decrease of biotic cell-cell connections in the respective M. thermautotrophicus ΔH strains compared to the wild-type strain. IMPORTANCE Methanothermobacter spp. have been studied for the biochemistry of hydrogenotrophic methanogenesis for many years. However, a detailed investigation of certain aspects, such as regulatory processes, was impossible due to the lack of genetic tools. Here, we amend our genetic toolbox for M. thermautotrophicus ΔH with an allelic exchange method. We report the deletion of genes that encode the Mth60 fimbriae. Our findings provide the first genetic evidence of whether the expression of these genes underlies regulation and reveal a role of the Mth60 fimbriae in the formation of cell-cell connections of M. thermautotrophicus ΔH.

Impact of carbon monoxide on performance and microbial community of extreme-thermophilic hydrogenotrophic methanation in horizontal rotary bioreactor.

A novel horizontal rotary bioreactor was developed for upgrading biogas from coke oven gas at extreme-thermophilic condition. The introduction of CO decreased the outlet methane content from 80% to 50% due to insufficient H2. This hindrance was overcome by increasing the proportion of incoming hydrogen, coupled with a prolonged gas retention time from 24 to 72 h, leading to a restoration of methane content to 91.6%. Notably, CO and CO2 exhibited a competitive relationship to hydrogen, which was determined by their contents. The substitution of Methanothermobacter for Methanobacterium as the dominant genus was observed at 70 °C, with relative abundance exceeding 98%. Incorporation of CO increased bacteria diversity and fostered a syntrophic relationship between the bacterial community and M. thermautotrophicus. This study provides both theoretical basis and practical support for biogas upgrading from coke oven gas using a biofilm reactor, thus aiding its future industrialization prospects.

The effect of 3-nitrooxypropanol, a potent methane inhibitor, on ruminal microbial gene expression profiles in dairy cows.

BACKGROUND: Enteric methane emissions from dairy cows are an environmental problem as well as a gross feed energy loss to the animal. Methane is generated in the rumen by methanogenic archaea from hydrogen (H2) + carbon dioxide and from H2 + methanol or methylamines. The methanogenic substrates are provided by non-methanogens during feed fermentation. Methane mitigation approaches have yielded variable results, partially due to an incomplete understanding of the contribution of hydrogenotrophic and methylotrophic archaea to methanogenesis. Research indicates that 3-nitrooxypropanol (3-NOP) reduces enteric methane formation in dairy cows by inhibiting methyl-coenzyme M reductase (MCR), the enzyme responsible for methane formation. The purpose of this study was to utilize metagenomic and metatranscriptomic approaches to investigate the effect of 3-NOP on the rumen microbiome and to determine the fate of H2 that accumulates less than expected under inhibited methanogenesis. RESULTS: The inhibitor 3-NOP was more inhibitory on Methanobrevibacter species than methanol-utilizing Methanosphaera and tended to reduce the gene expression of MCR. Under inhibited methanogenesis by 3-NOP, fluctuations in H2 concentrations were accompanied by changes in the expression of [FeFe] hydrogenases in H2-producing bacteria to regulate the amount of H2 production. No previously reported alternative H2 sinks increased under inhibited methanogenesis except for a significant increase in gene expression of enzymes involved in the butyrate pathway. CONCLUSION: By taking a metatranscriptomic approach, this study provides novel insights on the contribution of methylotrophic methanogens to total methanogenesis and regulation of H2 metabolism under normal and inhibited methanogenesis by 3-NOP in the rumen. Video Abstract.

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.

A Shuttle-Vector System Allows Heterologous Gene Expression in the Thermophilic Methanogen Methanothermobacter thermautotrophicus ΔH.

Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of molecular hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis). Yet, a genetic system for these model microbes was missing despite intensive work for four decades. Here, we report the successful implementation of genetic tools for Methanothermobacter thermautotrophicus ΔH. We developed shuttle vectors that replicated in Escherichia coli and M. thermautotrophicus ΔH. For M. thermautotrophicus ΔH, a thermostable neomycin resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The shuttle-vector DNA was transferred from E. coli into M. thermautotrophicus ΔH via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus ΔH, we demonstrated heterologous gene expression of a thermostable ß-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assay, we found significantly different ß-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus ΔH on formate as the sole growth substrate, while this was not possible for the empty-vector control. IMPORTANCE The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production but also for production of high-value platform chemicals in power-to-x processes.

Effects of pH on ex-situ biomethanation with hydrogenotrophic methanogens under thermophilic and extreme-thermophilic conditions.

Ex-situ biogas upgrading based on hydrogenotrophic methanogenic process has attracted much attention with the depletion of fossil fuels. Consumption of CO2 leads to the pH increase in the mixed cultures of biogas upgrading system. The hydrogenotrophic methanogens were enriched at pH 5.5-6.0, 7.0-7.5, and 8.5-9.0 and at 55°C and 70°C. The methane production activity and microbial community structure were evaluated. Semi-continuous experimental results showed that stable and similar methane production was obtained at pH 7.0-7.5 and 8.5-9.0. In addition, pH 8.5-9.0 presented higher maximum methane production rate compared to pH 7.0-7.5. pH below 6 obtained the longest lag phase time of about 17.4 h, more than twice the values at pH 7.0-7.5 (8.8 h) and pH 8.5-9.0 (6.9 h) at 55°C. The predominant methanogen was the genus Methanothermobacter, a hydrogenotrophic methanogen at higher temperatures. Methanobacterium became predominant at pH 8.5-9.0 and the abundance increased to 83.6% at 55°C. Coprothermobacter and Caldanaerobacter were identified as the core functional bacteria under alkaline condition and were likely involved in syntrophic acetate oxidation with hydrogenotrophic methanogens.

Biological CO2 fixation in up-flow reactors via exogenous H2 addition.

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.

Links between the rumen microbiota, methane emissions and feed efficiency of finishing steers offered dietary lipid and nitrate supplementation.

Ruminant methane production is a significant energy loss to the animal and major contributor to global greenhouse gas emissions. However, it also seems necessary for effective rumen function, so studies of anti-methanogenic treatments must also consider implications for feed efficiency. Between-animal variation in feed efficiency represents an alternative approach to reducing overall methane emissions intensity. Here we assess the effects of dietary additives designed to reduce methane emissions on the rumen microbiota, and explore relationships with feed efficiency within dietary treatment groups. Seventy-nine finishing steers were offered one of four diets (a forage/concentrate mixture supplemented with nitrate (NIT), lipid (MDDG) or a combination (COMB) compared to the control (CTL)). Rumen fluid samples were collected at the end of a 56 d feed efficiency measurement period. DNA was extracted, multiplexed 16s rRNA libraries sequenced (Illumina MiSeq) and taxonomic profiles were generated. The effect of dietary treatments and feed efficiency (within treatment groups) was conducted both overall (using non-metric multidimensional scaling (NMDS) and diversity indexes) and for individual taxa. Diet affected overall microbial populations but no overall difference in beta-diversity was observed. The relative abundance of Methanobacteriales (Methanobrevibacter and Methanosphaera) increased in MDDG relative to CTL, whilst VadinCA11 (Methanomassiliicoccales) was decreased. Trimethylamine precursors from rapeseed meal (only present in CTL) probably explain the differences in relative abundance of Methanomassiliicoccales. There were no differences in Shannon indexes between nominal low or high feed efficiency groups (expressed as feed conversion ratio or residual feed intake) within treatment groups. Relationships between the relative abundance of individual taxa and feed efficiency measures were observed, but were not consistent across dietary treatments.

Characteristics of physiology of and genomic mutations in aggregation-enhanced mutants of Methanothermobacter sp. CaT2.

The thermophilic hydrogenotrophic methanogen Methanothermobacter sp. CaT2 aggregates by itself. CaT2 is known to have a surface sugar layer and extracellular proteins that may be related to its aggregation. Aggregation-enhanced mutants, CHA001 and CHA002, were isolated after repeated cultivation for more than two years. When treated with proteinase K, CHA001 and CaT2 similarly exhibited a very low degree of aggregation and CHA002 exhibited less aggregation but still retained aggregation, suggesting protein-based aggregation via extracellular proteins in both CHA001 and CHA002, presumably via a putative membrane-bound and extracellularly protruding protein, MTCT_1020, identified previously. Genomic analysis revealed that CHA001 and CHA002 shared a missense mutation of MTCT_1348 and had distinct mutations. These results suggested that the MTCT_1348 mutation provides subsidiary support to the adhesive function of extracellular proteins and that there is an additional mutation(s) in CHA002 for the non-proteinous aggregation capability.

Top-Down Enrichment Guides in Formation of Synthetic Microbial Consortia for Biomass Degradation.

Consortium-based approaches are a promising avenue toward efficient bioprocessing. However, many complex microbial interactions dictate community dynamics and stability that must be replicated in synthetic systems. The rumen and/or hindguts of large mammalian herbivores harbor complex communities of biomass-degrading fungi and bacteria, as well as archaea and protozoa that work collectively to degrade lignocellulose, yet the microbial interactions responsible for stability, resilience, and activity of the community remain largely uncharacterized. In this work, we demonstrate a "top-down" enrichment-based methodology for selecting a minimal but effective lignocellulose-degrading community that produces methane-rich fermentation gas (biogas). The resulting enrichment consortium produced 0.75-1.9-fold more fermentation gas at 1.4-2.1 times the rate compared to a monoculture of fungi from the enrichment. Metagenomic sequencing of the top-down enriched consortium revealed genomes encoding for functional compartmentalization of the community, spread across an anaerobic fungus (Piromyces), a bacterium (Sphaerochaeta), and two methanogenic archaea (Methanosphaera and Methanocorpusculum). Guided by the composition of the top-down enrichment, several synthetic cocultures were formed from the "bottom-up" using previously isolated fungi, Neocallimastix californiae and Anaeromyces robustus paired with the methanogen Methanobacterium bryantii. While cross-feeding occurred in synthetic co-cultures, removal of fungal metabolites by methanogens did not increase the rate of gas production or the rate of substrate deconstruction by the synthetic community relative to fungal monocultures. Metabolomic characterization verified that syntrophy was established within synthetic co-cultures, which generated methane at similar concentrations compared to the enriched consortium but lacked the temporal stability (resilience) seen in the native system. Taken together, deciphering the membership and metabolic potential of an enriched gut consortium enables the design of methanogenic synthetic co-cultures. However, differences in the growth rate and stability of enriched versus synthetic consortia underscore the difficulties in mimicking naturally occurring syntrophy in synthetic systems.

Meta-omics based analyses of microbiome involved in biomethanation of rice straw in a thermophilic anaerobic bioreactor under optimized conditions.

Biomethanation of rice straw was performed at 55 °C without thermochemical pretreatment using cattle dung supplemented with Methanothermobacter thermautotrophicus strains. Methane yield of 323 ml g-1 VS obtained under optimized conditions such as particle size (1 mm), carbon to nitrogen ratio (15:1), substrate to inoculum ratio (1:1), organic loading rate (7.5% w/v) and hydraulic retention time (20 days), was one of the highest ever reported from rice straw. Metagenome analysis revealed several putative novel taxa among resident microbes. The genomes of Clostridium, Hungateiclostridium, Alkaliphilus, Anaerocolumna, Olsenella, Paenibacillus, Pseudoclostridium, Tepidanaerobacter and Turicibacter were recovered as metagenome assisted genomes. Clostridium spp. and M. thermautotrophicus were the dominant hydrolytic and methanogenic microbes, respectively. Syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis was found to be the major pathway for methane production. Efficient thermophilic biomethanation of rice straw without thermochemical pretreatment using cattle dung supplemented with M. thermautotrophicus is reported for the first time.

Methanogenic degradation of branched alkanes in enrichment cultures of production water from a high-temperature petroleum reservoir.

Branched alkanes are important constituents of crude oil and are usually regarded as resistant to microbial degradation, resulting in little knowledge of biochemical processes involved in anaerobic branched alkanes biodegradation. Here, we initiated an incubation study by amendment of iso-C9 (2-methyl, 3-methyl, and 4-methyloctane) as substrates for methanogenic degradation in production water from a high-temperature petroleum reservoir. Over an incubation period of 367 days, significant methanogenesis was observed in samples amended with these branched alkanes. The strong methanogenic activity only observed in iso-C9 amendments suggested the presence of microbial transformation from iso-alkanes into methane. GC-MS-based examination of the original production water identified an intermediate tentatively to be iso-C9-like alkylsuccinate, but was not detected in the enrichment cultures, combined with the successful amplification of assA functional gene in inoculating samples, revealing the ability of anaerobic biodegradation of iso-C9 via fumarate addition pathway. Microorganisms affiliated with members of the Firmicutes, Synergistetes, and methanogens of genus Methanothermobacter spp. were highly enriched in samples amended with iso-C9. The co-occurrence of known syntrophic acetate oxidizers Thermoacetogenium spp. and Methanothermobacter spp. (known hydrogenotrophic methanogens) indicates a potential syntrophic acetate oxidation associated with the methanogenic biodegradation of iso-C9. These results provide some useful information on the potential biodegradation of branched alkanes via methanogenesis and also suggest that branched alkanes are likely activated via fumarate addition in high-temperature petroleum reservoirs.

Development of methanogens within cathodic biofilm in the single-chamber microbial electrolysis cell.

The aim of this study was to investigate the development of cathodic biofilm and its effect on methane production in a single-chamber microbial electrolysis cell (MEC). The MEC with 1 g/L acetate was successfully operated within 31 cycles (∼2400 h). The maximum methane production rate and average current capture efficiency in the MEC reached 93 L/m3·d and 82%, respectively. Distinct stratification of Methanobacteriaceae within cathodic biofilm was observed after 9 cycles of operation. The relative abundance of Methanobacteriaceae in the microbial community increased from 45.3% (0-15 µm), 57.6% (15-30 µm), 66.9% (30-45 µm) to 77.2% (45-60 µm) within the cathodic biofilm. The methane production rates were positively correlated with the mcrA gene copy numbers in the cathodic biofilm. Our results should be useful to understand the mechanism of methane and hydrogen production in the MEC.

Functional annotation of operome from Methanothermobacter thermautotrophicus ΔH: An insight to metabolic gap filling.

Methanothermobacter thermautotrophicus ΔH (MTH) is a potential methanogen known to reduce CO2 with H2 for producing methane biofuel in thermophilic digesters. The genome of this organism contains ~50.5% conserved hypothetical proteins (HPs; operome) whose function is still not determined precisely. Here, we employed a combined bioinformatics approach to annotate a precise function to HPs and categorize them as enzymes, binding proteins, and transport proteins. Results of our study show that 315 (35.6%) HPs have exhibited well-defined functions contributing imperative roles in diverse cellular metabolism. Some of them are responsible for stress-response mechanisms and cell cycle, membrane transport, and regulatory processes. The genome-neighborhood analysis found five important gene clusters (dsr, ehb, kaiC, cmr, and gas) involving in the energetic metabolism and defense systems. MTH operome contains 223 enzymes with 15 metabolic subsystems, 15 cell cycle proteins, 17 transcriptional regulators and 33 binding proteins. Functional annotation of its operome is thus more fundamental to a profound understanding of the molecular and cellular machinery at systems-level.

Comparative proteomic analysis of Methanothermobacter thermautotrophicus reveals methane formation from H2 and CO2 under different temperature conditions.

The growth of all methanogens is limited to a specific temperature range. However, Methanothermobacter thermautotrophicus can be found in a variety of natural and artificial environments, the temperatures of which sometimes even exceed the temperature growth ranges of thermophiles. As a result, the extent to which methane production and survival are affected by temperature remains unclear. To investigate the mechanisms of methanogenesis that Archaea have evolved to cope with drastic temperature shifts, the responses of Methanothermobacter thermautotrophicus to temperature were investigated under a high temperature growth (71°C) and cold shock (4°C) using Isobaric tags for relative and absolute quantitation (iTRAQ). The results showed that methane formation is decreased and that protein folding and degradation are increased in both high- and low-temperature treatments. In addition, proteins predicted to be involved in processing environmental information processing and in cell membrane/wall/envelope biogenesis may play key roles in affecting methane formation and enhancing the response of M. thermautotrophicus to temperature stress. Analysis of the genomic locations of the genes corresponding to these temperature-dependent proteins predicted that 77 of the genes likely to form 32 gene clusters. Here, we assess the response of M. thermautotrophicus to different temperatures and provide a new level of understanding of methane formation and cellular putative adaptive responses.

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.

Comparative Genomic Analysis of Members of the Genera Methanosphaera and Methanobrevibacter Reveals Distinct Clades with Specific Potential Metabolic Functions.

Methanobrevibacter and Methanosphaera species represent some of the most prevalent methanogenic archaea in the gastrointestinal tract of animals and humans and play an important role in this environment. The aim of this study was to identify genomic features that are shared or specific for members of each genus with a special emphasis of the analysis on the assimilation of nitrogen and acetate and the utilization of methanol and ethanol for methanogenesis. Here, draft genome sequences of Methanobrevibacter thaueri strain DSM 11995T, Methanobrevibacter woesei strain DSM 11979T, and Methanosphaera cuniculi strain 4103T are reported and compared to those of 16 other Methanobrevibacter and Methanosphaera genomes, including genomes of the 13 currently available types of strains of the two genera. The comparative genome analyses indicate that among other genes, the absence of molybdopterin cofactor biosynthesis is conserved in Methanosphaera species but reveals also that the three species share a core set of more than 300 genes that distinguishes the genus Methanosphaera from the genus Methanobrevibacter. Multilocus sequence analysis shows that the genus Methanobrevibacter can be subdivided into clades, potentially new genera, which may display characteristic specific metabolic features. These features include not only the potential ability of nitrogen fixation and acetate assimilation in a clade comprised of Methanobrevibacter species from the termite gut and Methanobrevibacter arboriphilus strains but also the potential capability to utilize ethanol and methanol in a clade comprising Methanobrevibacter wolinii strain DSM 11976T, Mbb. sp. AbM4, and Mbb. boviskoreani strain DSM 25824T.

How [Fe]-Hydrogenase from Methanothermobacter is Protected Against Light and Oxidative Stress.

[Fe]-hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyltetrahydromethanopterin (methenyl-H4 MPT+ ) with H2 . Hmd contains the iron-guanylylpyridinol (FeGP) cofactor, which is sensitive to light and oxidative stress. A natural protection mechanism is reported for Hmd based on structural and biophysical data. Hmd from Methanothermobacter marburgensis (mHmd) was found in a hexameric state, where an expanded oligomerization loop is detached from the dimer core and intrudes into the active site of a neighboring dimer. An aspartic acid residue from the loop ligates to FeII of the FeGP cofactor and thus blocks the postulated H2 -binding site. In solution, this enzyme is in a hexamer-to-dimer equilibrium. Lower enzyme concentrations, and the presence of methenyl-H4 MPT+ , shift the equilibrium toward the active dimer side. At higher enzyme concentrations-as present in the cell-the enzyme is predominantly in the inactive hexameric state and is thereby protected against light and oxidative stress.

Temporal and spatial impact of Spartina alterniflora invasion on methanogens community in Chongming Island, China.

Methane production by methanogens in wetland is recognized as a significant contributor to global warming. Spartina alterniflora (S. alterniflora), which is an invasion plant in China's wetland, was reported to have enormous effects on methane production. But studies on shifts in the methanogen community in response to S. alterniflora invasion at temporal and spatial scales in the initial invasion years are rare. Sediments derived from the invasive species S. alterniflora and the native species Phragmites australis (P. australis) in pairwise sites and an invasion chronosequence patch (4 years) were analyzed to investigate the abundance and community structure of methanogens using quantitative real-time PCR (qPCR) and Denaturing gradient gel electrophoresis (DGGE) cloning of the methyl-coenzyme M reductase A (mcrA) gene. For the pairwise sites, the abundance of methanogens in S. alterniflora soils was lower than that of P. australis soils. For the chronosequence patch, the abundance and diversity of methanogens was highest in the soil subjected to two years invasion, in which we detected some rare groups including Methanocellales and Methanococcales. These results indicated a priming effect at the initial invasion stages of S. alterniflora for microorganisms in the soil, which was also supported by the diverse root exudates. The shifts of methanogen communities after S. alterniflora invasion were due to changes in pH, salinity and sulfate. The results indicate that root exudates from S. alterniflora have a priming effect on methanogens in the initial years after invasion, and the predominate methylotrophic groups (Methanosarcinales) may adapt to the availability of diverse substrates and reflects the potential for high methane production after invasion by S. alterniflora.