Genus Methanothermobacter Research Articles

Carbon monoxide conversion by anaerobic microbiome in a thermophilic trickle bed reactor

Biomethanation offers a promising route for the valorization of synthesis gas, yet significant challenges arise from the limited conversion of carbon monoxide (CO). This study investigated the adaptation of an anaerobic microbiome in a continuous trickle bed reactor (TBR) with CO as the sole carbon and energy source. We evaluated reactor performance and microbial community changes under different CO loading rates and gas retention times (GRT). Optimal performance was achieved at a CO loading rate of 5.16 Nm³ m⁻³ d⁻¹ and a GRT of 60.6 min, resulting in average production rates of 0.99 Nm³ m⁻³ d⁻¹ for CH₄ and 2.55 Nm⁻³ m⁻³ d⁻¹ for CO₂, with an 88 % CO conversion rate. Microbial analysis indicated that the community was dominated by the genus Methanothermobacter, known for its ability to utilize CO as a sole substrate, followed by a co-dominance of syntrophic acetate-oxidizing bacteria Syntrophaceticus. This syntrophic relationship between Methanothermobacter and Syntrophaceticus is expected to be crucial for the efficient CO conversion process. Additionally, the study proposes a two-reactor system for converting synthesis gas to grid-quality methane.

Optimization of the Ex Situ Biomethanation of Hydrogen and Carbon Dioxide in a Novel Meandering Plug Flow Reactor: Start-Up Phase and Flexible Operation.

With the increasing use of renewable energy resources for the power grid, the need for long-term storage technologies, such as power-to-gas systems, is growing. Biomethanation provides the opportunity to store energy in the form of the natural gas-equivalent biomethane. This study investigates a novel plug flow reactor that employs a helical static mixer for the biological methanation of hydrogen and carbon dioxide. In tests, the reactor achieved an average methane production rate of 2.5 LCH4LR∗d (methane production [LCH4] per liter of reactor volume [LR] per day [d]) with a maximum methane content of 94%. It demonstrated good flexibilization properties, as repeated 12 h downtimes did not negatively impact the process. The genera Methanothermobacter and Methanobacterium were predominant during the initial phase, along with volatile organic acid-producing, hydrogenotrophic, and proteolytic bacteria. The average ratio of volatile organic acid to total inorganic carbon increased to 0.52 ± 0.04, while the pH remained stable at an average of pH 8.1 ± 0.25 from day 32 to 98, spanning stable and flexible operation modes. This study contributes to the development of efficient flexible biological methanation systems for sustainable energy storage and management.

An integrated systems biology approach reveals differences in formate metabolism in the genus Methanothermobacter

Methanogenesis allows methanogenic archaea to generate cellular energy for their growth while producing methane. Thermophilic hydrogenotrophic species of the genus Methanothermobacter have been recognized as robust biocatalysts for a circular carbon economy and are already applied in power-to-gas technology with biomethanation, which is a platform to store renewable energy and utilize captured carbon dioxide. Here, we generated curated genome-scale metabolic reconstructions for three Methanothermobacter strains and investigated differences in the growth performance of these same strains in chemostat bioreactor experiments with hydrogen and carbon dioxide or formate as substrates. Using an integrated systems biology approach, we identified differences in formate anabolism between the strains and revealed that formate anabolism influences the diversion of carbon between biomass and methane. This finding, together with the omics datasets and the metabolic models we generated, can be implemented for biotechnological applications of Methanothermobacter in power-to-gas technology, and as a perspective, for value-added chemical production.

Phylogenetically and physiologically diverse methanogenic archaea inhabit the Indian hot spring environments.

Mesophilic and thermophilic methanogens belonging to the hydrogenotrophic, methylotrophic, and acetotrophic groups were isolated from Indian hot spring environments using BY and BCYT growth media. Following initial Hinf I-based PCR-RFLP screening, 70 methanogens were sequenced to ascertain their identity. These methanogens were phylogenetically and physiologically diverse and represented different taxa distributed across three physiological groups, i.e., hydrogenotrophs (53), methylotrophs (14) and acetotrophs (3). Overall, methanogens representing three families, five genera, and ten species, including two putative novel species, were recognized. The highest number and diversity of methanogens was observed at 40℃, dominated by Methanobacterium (10; 3 species), Methanosarcina (9; 3 species), Methanothermobacter (7; 2 species), Methanomethylovorans (5; 1 species) and Methanoculleus (3; 1 species). Both putative novel methanogen species were isolated at 40℃ and belonged to the genera Methanosarcina and Methanobacterium. At 55℃, limited diversity was observed, and resulted in the isolation of only two genera of methanogens, i.e., Methanothermobacter (28; 2 species) and Methanosarcina (4; 1 species). At 70℃, only members of the genus Methanothermobacter (5; 2 species) were isolated, whereas no methanogen could be cultured at 85℃. Ours is the first study that documents the extensive range of cultivable methanogenic archaea inhabiting hot springs across various geothermal provinces of India.

Biomethane recovery through co-digestion of cheese whey and glycerol in a two-stage anaerobic fluidized bed reactor: Effect of temperature and organic loading rate on methanogenesis

Anaerobic digestion for CH4 recovery in wastewater treatment has been carried out with different strategies to increase process efficiency, among which co-digestion and the two-stage process can be highlighted. In this context, this study aimed at evaluating the co-digestion of cheese whey and glycerol in a two-stage process using fluidized bed reactors, verifying the effect of increasing the organic loading rate (OLR) (2–20 g-COD.L−1.d−1) and temperature (thermophilic and mesophilic) in the second stage methanogenic reactor. The mesophilic methanogenic reactor (R-Meso) (mean temperature of 22 °C) was more tolerant to high OLR and its best performance was at 20 g-COD.L−1.d−1, resulting in methane yield (MY) and methane production (MPR) of 273 mL-CH4.g-COD−1 and 5.8 L-CH4.L−1.d−1 (with 67% of CH4), respectively. Through 16S rRNA gene massive sequencing analysis, a greater diversity of microorganisms was identified in R-Meso than in R-Thermo (second stage methanogenic reactor, 55 °C). Firmicutes was the phyla with higher relative abundance in R-Thermo, while in R-Meso the most abundant ones were Proteobacteria and Bacteroidetes. Regarding the Archaea domain, a predominance of hydrogenotrophic microorganisms could be observed, being the genera Methanothermobacter and Methanobacterium the most abundant in R-Thermo and R-Meso, respectively. The two-stage system composed with a thermophilic acidogenic reactor + R-Meso was more adequate for the co-digestion of cheese whey and glycerol than the single-stage process, promoting increases of up to 47% in the energetic yield (10.3 kJ.kg-COD−1) and 14% in organic matter removal (90.5%).

Development of Stable Mixed Microbiota for High Yield Power to Methane Conversion

The performance of a mixed microbial community was tested in lab-scale power-to-methane reactors at 55 °C. The main aim was to uncover the responses of the community to starvation and stoichiometric H2/CO2 supply as the sole substrate. Fed-batch reactors were inoculated with the fermentation effluent of a thermophilic biogas plant. Various volumes of pure H2/CO2 gas mixtures were injected into the headspace daily and the process parameters were followed. Gas volumes and composition were measured by gas-chromatography, the headspace was replaced with N2 prior to the daily H2/CO2 injection. Total DNA samples, collected at the beginning and end (day 71), were analyzed by metagenome sequencing. Low levels of H2 triggered immediate CH4 evolution utilizing CO2/HCO3− dissolved in the fermentation effluent. Biomethanation continued when H2/CO2 was supplied. On the contrary, biomethane formation was inhibited at higher initial H2 doses and concomitant acetate formation indicated homoacetogenesis. Biomethane production started upon daily delivery of stoichiometric H2/CO2. The fed-batch operational mode allowed high H2 injection and consumption rates albeit intermittent operation conditions. Methane was enriched up to 95% CH4 content and the H2 consumption rate attained a remarkable 1000 mL·L−1·d−1. The microbial community spontaneously selected the genus Methanothermobacter in the enriched cultures.

Response of cattle manure anaerobic digestion to zinc oxide nanoparticles: Methane production, microbial community, and functions

The increasing use of zinc oxide nanoparticles (ZnO NPs) as feed additives has raised huge environmental concerns in the anaerobic digestion (AD) of livestock wastes. In this study the 30-day effect of ZnO NPs on AD performance of cattle manure was investigated under optimal conditions (temperature=55 °C, initial pH=10, total solids contents=10%), which were obtained from response surface methodology. Results showed that ZnO NPs (5–100 mg/g TS) promoted the accumulation of soluble chemical oxygen demand, soluble protein and polysaccharide, but inhibited their following fermentation and methane production processes. This inhibition became stronger as ZnO NPs concentration increased, leading to the methane production declined by 84.55% (5 mg/ g TS), 92.39% (30 mg/g TS), and 93.72% (100 mg/g TS), respectively. High-throughput sequencing results demonstrated that the decrease in the abundances of functional bacteria from 72.11% to 11.24% (in families Ruminococcaceae and Lachnospiraceae), and of methanogens from 96.82% to < 1% (in genus Methanothermobacter) led to poor fermentation and methanogenesis, respectively. Predictive functional analysis indicated that ZnO NPs can enhance abundances of AD-friendly functional genes instead of possible genetic expression, resulting in the low methane production. Our findings highlight a deep understanding of the role of ZnO NPs in cattle manure AD and are critical for guiding feed additive application.

Improved robustness of ex-situ biological methanation for electro-fuel production through the addition of graphene

Ex-situ biomethanation (CO2 + 4H2 → CH4 + 2H2O) can simultaneously achieve renewable electricity storage and CO2 valorisation. However, fluctuations in variable renewable electricity may lead to intermittent hydrogen supply, which is shown to adversely affect microbial activity and performance of the biomethanation process. Carbonaceous materials may act as an abiotic additive to enhance microbial robustness and improve system performance. Nanomaterial graphene and pyrochar were compared to assess their effects on biomethanation systems with an intermittent supply of hydrogen. Results revealed that intermittent gas supply caused deterioration in the restart performance with only 66% of theoretical methane production obtained in the control compared with 84% under steady state conditions. The addition of graphene in biomethanation led to 78% of the theoretical methane production after repetitive intermittent supply; this improvement is postulated to be due to its high electrical conductivity and large specific surface (500 m2/g). In comparison, pyrochar amendment did not lead to a significant improvement in upgrading performance. Microbial analysis showed that the OTUs affiliated to bacteria withinin the order SHA-98 (42.9% in abundance) and archaea from the genus Methanothermobacter (99%) may result in the establishment of a new syntrophic relationship to improve the robustness of biomethanation process.

Evidence of thermophilic waste decomposition at a landfill exhibiting elevated temperature regions

There have been several reports of landfills exhibiting temperatures as high as 80 to 100 °C. This observation has motivated researchers to understand the causes of the elevated temperatures and to develop predictive models of landfill temperature. The objective of this research was to characterize the methanogenic activity of microbial communities that were derived from landfill samples excavated from a section of a landfill exhibiting gas well temperatures above 55 °C. Specific objectives were to: (1) determine the upper temperature limit for methane production; (2) evaluate the kinetics of methane generation when landfill-derived microcosms are incubated above and below their excavation temperature and derive a temperature inhibition function; and (3) evaluate microbial community shifts in response to temperature perturbations. Landfill microcosms were derived from 57 excavated landfill samples and incubated within ±2.5 °C of their excavation temperature between 42.5 °C and 87.5 °C. Results showed an optimum temperature for methane generation of ~57 °C and a 95% reduction in methane yield at ~72 °C. When select cultures were perturbed between 5 °C below and 15 °C above their in-situ temperature, both the rate and maximum methane production decreased as incubation temperature increased. Microbial community characterization using 16S rRNA amplicon sequencing suggests that thermophilic methanogenic activity can be attributed to methanogens of the genus Methanothermobacter. This study demonstrated that from a microbiological standpoint, landfills may maintain active methanogenic processes while experiencing temperatures in the thermophilic regime (<72 °C).

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 17.4h, more than twice the values at pH 7.0-7.5 (8.8h) and pH 8.5-9.0 (6.9h) 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.

Dominant and Active Methanogens in the Production Waters From a High-Temperature Petroleum Reservoir by DNA- and RNA-Based Analysis

Methane metabolism in deep subsurface petroleum reservoirs is of paramount interest in the global biogeochemical cycle of carbon. Methanogenesis in such habitats is driven mostly by methanogens in syntrophic association with bacteria. In the present study, methanogenic communities in production water samples from six wells of a high-temperature petroleum reservoir were analyzed based on both genomic 16S rDNA and metabolically active 16S rRNA. The PCR-amplified mcrA gene analysis showed that Methanosaeta (26%) and Methanomassiliicoccus (56%) were separately the dominant members in sample W2_71 and W9_18 at the DNA level. In comparison, the RNA-based analysis showed that Methanosaeta (63 ∼ 83%) followed by Methanolinea (16 ∼ 23%) were the most active methanogens, which were different from the communities of genomic DNA. While several lines of studies indicated that CO2-reducing methanogens of the genus Methanothermobacter was the most frequently detected phylotype in deep-subsurface petroleum reservoirs. Redundancy analysis (RDA) showed the possible correlation between active methanogens (Methanosaeta) and environmental factors (CO3 2-). The datasets indicated the importance of investigating methanogenic community by integrating DNA- and RNA-based approaches. These results provide new insight into active methanogens in high-temperature petroleum reservoirs, and promote a comprehensive understanding of methanogens as well as their potential methanogenic pathways in such environments.

An Unexpectedly Broad Thermal and Salinity-Tolerant Estuarine Methanogen Community.

Moderately thermophilic (Tmax, ~55 °C) methanogens are identified after extended enrichments from temperate, tropical and low-temperature environments. However, thermophilic methanogens with higher growth temperatures (Topt ≥ 60 °C) are only reported from high-temperature environments. A microcosm-based approach was used to measure the rate of methane production and methanogen community structure over a range of temperatures and salinities in sediment from a temperate estuary. We report short-term incubations (<48 h) revealing methanogens with optimal activity reaching 70 °C in a temperate estuary sediment (in situ temperature 4–5 °C). While 30 °C enrichments amended with acetate, H2 or methanol selected for corresponding mesophilic trophic groups, at 60 °C, only hydrogenotrophs (genus Methanothermobacter) were observed. Since these methanogens are not known to be active under in situ temperatures, we conclude constant dispersal from high temperature habitats. The likely provenance of the thermophilic methanogens was studied by enrichments covering a range of temperatures and salinities. These enrichments indicated that the estuarine sediment hosted methanogens encompassing the global activity envelope of most cultured species. We suggest that estuaries are fascinating sink and source environments for microbial function study.

Variations in CH4 and CO2 productions and emissions driven by pollution sources in municipal sewers: An assessment of the role of dissolved organic matter components and microbiota

Variations in methane (CH4) and carbon dioxide (CO2) emissions in municipal sewer driven by pollution sources are complex and multifaceted. It is important to investigate the role of dissolved organic matter (DOM) components and microbiota to better understand what and how those variations occurred. For this purpose, this study provides a systematic assessment based on short-term in-sewer conditioned cultivations, in conjunction with a field survey in four typical sewers in Shanghai Megacity. The results are as follows: (1) Sediment plays a main role in driving the sewer carbon emission behavior owing to its strong associations with the utilized substrates and predominant microbes that significantly promoted the gas fluxes (genera Bacteroidete_vadinHA17, Candidatus_competibacter, and Methanospirillum). (2) Aquatic DOM in overlying water is an indispensable factor in promoting total carbon emissions, yet the dominant microbes present there inversely correlated with gas fluxes (genera Methanothermobacter and Bacteroides). (3) The total fluxes of both CH4 and CO2 enhanced by pavement runoff were limited. Its high COD-CH4/CO2 conversion efficiencies can be ascribed to its dominant anthropogenic humic-like components and the emerged aquatic tyrosine-like components. (4) Domestic sewage can significantly enhance the total fluxes because of its high concentration of bioavailable DOM. However, these substrates, which were more suitable for supporting microbial growth, as well as the substrate competition caused by sulfate reduction and the nitrogen cycle (revealed by the dominant functional microbes genera Acinetobacter, Pseudomonas, Dechloromona, and Candidatus_competibacter and their correlations with indicators), seemed to be responsible for the low COD-CH4/CO2 conversion efficiencies of domestic sewage. (5) A field survey indicated the distinct features of carbon emissions of sewer sewage discharged from different catchments. An extreme hydraulic condition in a sewer in the absence of influent showed unexpectedly high levels of CO2, while a small amount of CH4 emissions.

Genome Analyses and Genome-Centered Metatranscriptomics of Methanothermobacter wolfeii Strain SIV6, Isolated from a Thermophilic Production-Scale Biogas Fermenter.

In the thermophilic biogas-producing microbial community, the genus Methanothermobacter was previously described to be frequently abundant. The aim of this study was to establish and analyze the genome sequence of the archaeal strain Methanothermobacter wolfeii SIV6 originating from a thermophilic industrial-scale biogas fermenter and compare it to related reference genomes. The circular chromosome has a size of 1,686,891 bases, featuring a GC content of 48.89%. Comparative analyses considering three completely sequenced Methanothermobacter strains revealed a core genome of 1494 coding sequences and 16 strain specific genes for M. wolfeii SIV6, which include glycosyltransferases and CRISPR/cas associated genes. Moreover, M. wolfeii SIV6 harbors all genes for the hydrogenotrophic methanogenesis pathway and genome-centered metatranscriptomics indicates the high metabolic activity of this strain, with 25.18% of all transcripts per million (TPM) belong to the hydrogenotrophic methanogenesis pathway and 18.02% of these TPM exclusively belonging to the mcr operon. This operon encodes the different subunits of the enzyme methyl-coenzyme M reductase (EC: 2.8.4.1), which catalyzes the final and rate-limiting step during methanogenesis. Finally, fragment recruitment of metagenomic reads from the thermophilic biogas fermenter on the SIV6 genome showed that the strain is abundant (1.2%) within the indigenous microbial community. Detailed analysis of the archaeal isolate M. wolfeii SIV6 indicates its role and function within the microbial community of the thermophilic biogas fermenter, towards a better understanding of the biogas production process and a microbial-based management of this complex process.

Mesophilic and thermophilic anaerobic digestion of soybean curd residue for methane production: Characterizing bacterial and methanogen communities and their correlations with organic loading rate and operating temperature

To find the optimal operation parameters and provide an explanation of methanogenic pathway for methane production in mesophilic (35 °C) and thermophilic (55 °C) anaerobic digestion (MAD, TAD) of soybean curd residue (SCR), MAD and MAD were contrastively investigated for 95 days. The maximum available OLR was identified as 3.3 gVS/L for both MAD and TAD. Compared to MAD, TAD exhibited a 20% higher average methane yield (0.591 L/gVS) and a 7.5% higher volatile solids removal efficiency (74.1 ± 10.4%). Bacterial phyla Bacteroidetes, Firmicutes and Proteobacteria dominated in MAD digesters while genus Defluviitoga was selectively enriched in TAD digesters due to higher temperature and organic loading pressure. Principal coordinates analysis of methanogen community showed that both temperature and OLR were crucial environmental variables shifting the taxonomic patterns of the methanogens. The enriched methanogen genus Methanothermobacter (93%) with a hydrogenotrophic methanogenic pathway had a close correlation with the TAD performance.

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 367days, 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.

An explanation of the methanogenic pathway for methane production in anaerobic digestion of nitrogen-rich materials under mesophilic and thermophilic conditions

The impact of temperature on the anaerobic digestion of chicken manure was investigated by studying the process performance and pathway for continuously-fed digesters under mesophilic and thermophilic conditions. The mesophilic digester obtained a 15% higher methane yield compared with the thermophilic digester. Mesophilic and thermophilic digester had free ammonia of 31 and 145 mg/L, respectively. The stable carbon isotope analysis indicated that 41% and 50% of acetate was converted to methane through the syntrophic acetate oxidation and hydrogenotrophic methanogenesis (SAO-HM) pathway under mesophilic and thermophilic conditions, respectively. The genus Pseudomonas represented 10% and 16% under mesophilic and thermophilic conditions, respectively. A high abundance of the methanogens genus Methanoculleus (94% of total methanogens) in mesophilic and the genus Methanothermobacter (96%) in thermophilic digesters indicated they were the main hydrogenotrophic partners in SAO. The present study therefore illustrated that methanogenic pathway shifting, induced by free ammonia, closely correlated to the process performance.

Biogas production from food waste via co-digestion and digestion- effects on performance and microbial ecology

In this work, performance and microbial structure of a digestion (food waste-only) and a co-digestion process (mixture of cow manure and food waste) were studied at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The highest methane yield (480 mL/g VS) was observed in the mesophilic digester (MDi) fed with food waste alone. The mesophilic co-digestion of food waste and manure (McoDi) yielded 26% more methane than the sum of individual digestions of manure and food waste. The main volatile fatty acid (VFA) in the mesophilic systems was acetate, averaging 93 and 172 mg/L for McoDi and MDi, respectively. Acetate (2150 mg/L) and propionate (833 mg/L) were the main VFAs in the thermophilic digester (TDi), while propionate (163 mg/L) was the major VFA in the thermophilic co-digester (TcoDi). The dominant bacteria in MDi was Chloroflexi (54%), while Firmicutes was dominant in McoDi (60%). For the mesophilic reactors, the dominant archaea was Methanosaeta in MDi, while Methanobacterium and Methanosaeta had similar abundance in McoDi. In the thermophilic systems, the dominant bacteria were Thermotogae, Firmicutes and Synergistetes in both digesters, however, the relative abundance of these phyla were different. For archaea, the genus Methanothermobacter were entirely dominant in both TDi and TcoDi.

Variability of the composition of the microbial community of the deep subsurface thermal aquifer in Western Siberia

The deep subsurface biosphere is one of the least studied ecosystems on Earth, containing communities of extremophilic microorganisms. The present work was aimed at molecular genetic characterization of microbial communities of underground thermal waters in Western Siberia, lying at depths of 2–3 km. Water samples were collected from the 5P oil-exploration well, drilled to a depth of 2.8 km near the village Chazhemto (Tomsk region). The water had a temperature of about 20°C, a neutral pH and a low redox potential (–304 mV). Underground aquifers have a complex structure and may contain both planktonic microorganisms and those immobilized on the surface of rocks in the form of biofilms, which may be washed out and detected in the water flowing out of the well. Community composition was analyzed by amplification and pyrosequencing of the 16S rRNA gene fragments in seven water samples taken at different times during 26 hours. Bacteria, which constituted about half of the community, were represented mainly by uncultured lineages of the phyla Firmicutes, Ignavibacteria, Chloroflexi, Bacteroidetes, and Proteobacteria. Archaea belonged mainly to known methanogens of the genera Methanothermobacter, Methanosaeta, and Methanomassiliicoccus. Analysis of the samples taken at different times revealed large variations in the content of most groups of bacteria, with a decrease in Firmicutes abundance accompanied by an increase in the shares of Ignavibacteria and Chloroflexi. The share of archaea of the genus Methanothermobacter varied slightly during the day, while significant variations were observed for the phylotypes assigned to Methanosaeta and Methanomassiliicoccus. Hydrogenotrophic archaea of the genus Methanothermobacter are probably a permanent component of the microbial community occurring in the planktonic state, while most of the identified groups of bacteria are present in biofilms or spatially localized parts of the underground water reservoir, the material of which accidentally enters the well.

High-pressure thermophilic electromethanogenic system producing methane at 5 MPa, 55°C

Toward applications of bio-electrochemical systems in industrial processes and extreme environments, electromethanogenesis under high-pressure conditions was examined. Stainless-steel single-chamber reactors specifically designed to examine bio-electrochemical reactions under pressurized conditions were inoculated with thermophilic microorganisms originated from an oilfield formation water. The reactors were incubated at 5MPa, 55°C in fed-batch operational mode with an applied voltage of 0.7V. In the first few fed-batch cycles, hydrogen was mainly produced. After the third cycle, however, the reactors produced only methane simultaneously with current generation. The methane-production rate of the reactors showed an applied-voltage dependence and increased from 34.9 to 168.4mmolm-2day-1 with an increase in the applied voltage from 0.4 to 0.9V. The efficiency of capturing electrons in the produced methane on average exceeded 70% with the applied voltage of 0.4V or higher. Cyclic voltammetry further confirmed abilities of the bioelectrodes to catalyze electrochemical reactions at 5MPa. Performance of the electromethanogenesis system was not altered under lower pressure conditions (1.2 and 2.5MPa). An exoelectrogenic bacterium affiliated with the genus Thermincola and a methanogen belonging to the genus Methanothermobacter were detected as the dominant species in the bioanode and biocathode microbiotas, respectively. Thus, our results indicated that electromethanogenesis systems could be developed and operated under highly-pressurized conditions, suggesting that applications of the bio-electrochemical system in high-pressure environments (including high-temperature subsurface reservoirs) can be technically feasible.