Hydrogenotrophic methanogens of the mammalian gut: Functionally similar, thermodynamically different-A modelling approach.

Methanogenic archaea occupy a functionally important niche in the gut microbial ecosystem of mammals. Our purpose was to quantitatively characterize the dynamics of methanogenesis by integrating microbiology, thermodynamics and mathematical modelling. For that, in vitro growth experiments were performed with pure cultures of key methanogens from the human and ruminant gut, namely Methanobrevibacter smithii, Methanobrevibacter ruminantium and Methanobacterium formicium. Microcalorimetric experiments were performed to quantify the methanogenesis heat flux. We constructed an energetic-based mathematical model of methanogenesis. Our model captured efficiently the dynamics of methanogenesis with average concordance correlation coefficients of 0.95 for CO2, 0.98 for H2 and 0.97 for CH4. Together, experimental data and model enabled us to quantify metabolism kinetics and energetic patterns that were specific and distinct for each species despite their use of analogous methane-producing pathways. Then, we tested in silico the interactions between these methanogens under an in vivo simulation scenario using a theoretical modelling exercise. In silico simulations suggest that the classical competitive exclusion principle is inapplicable to gut ecosystems and that kinetic information alone cannot explain gut ecological aspects such as microbial coexistence. We suggest that ecological models of gut ecosystems require the integration of microbial kinetics with nonlinear behaviours related to spatial and temporal variations taking place in mammalian guts. Our work provides novel information on the thermodynamics and dynamics of methanogens. This understanding will be useful to construct new gut models with enhanced prediction capabilities and could have practical applications for promoting gut health in mammals and mitigating ruminant methane emissions.

[NiFe]-hydrogenases are constitutively expressed in an enriched Methanobacterium sp. population during electromethanogenesis.

Electromethanogenesis is the bioreduction of carbon dioxide (CO2) to methane (CH4) utilizing an electrode as electron donor. Some studies have reported the active participation of Methanobacterium sp. in electron capturing, although no conclusive results are available. In this study, we aimed at determining short-time changes in the expression levels of [NiFe]-hydrogenases (Eha, Ehb and Mvh), heterodisulfide reductase (Hdr), coenzyme F420-reducing [NiFe]-hydrogenase (Frh), and hydrogenase maturation protein (HypD), according to the electron flow in independently connected carbon cloth cathodes poised at- 800 mV vs. standard hydrogen electrode (SHE). Amplicon massive sequencing of cathode biofilm confirmed the presence of an enriched Methanobacterium sp. population (>70% of sequence reads), which remained in an active state (78% of cDNA reads), tagging this archaeon as the main methane producer in the system. Quantitative RT-PCR determinations of ehaB, ehbL, mvhA, hdrA, frhA, and hypD genes resulted in only slight (up to 1.5 fold) changes for four out of six genes analyzed when cells were exposed to open (disconnected) or closed (connected) electric circuit events. The presented results suggested that suspected mechanisms for electron capturing were not regulated at the transcriptional level in Methanobacterium sp. for short time exposures of the cells to connected-disconnected circuits. Additional tests are needed in order to confirm proteins that participate in electron capturing in Methanobacterium sp.

Anaerobic oxidation of methane coupled with extracellular electron transfer to electrodes.

Anaerobic oxidation of methane (AOM) is an important process for understanding the global flux of methane and its relation to the global carbon cycle. Although AOM is known to be coupled to reductions of sulfate, nitrite, and nitrate, evidence that AOM is coupled with extracellular electron transfer (EET) to conductive solids is relatively insufficient. Here, we demonstrate EET-dependent AOM in a biofilm anode dominated by Geobacter spp. and Methanobacterium spp. using carbon-fiber electrodes as the terminal electron sink. The steady-state current density was kept at 11.0 ± 1.3 mA/m2 in a microbial electrochemical cell, and isotopic experiments supported AOM-EET to the anode. Fluorescence in situ hybridization images and metagenome results suggest that Methanobacterium spp. may work synergistically with Geobacter spp. to allow AOM, likely by employing intermediate (formate or H2)-dependent inter-species electron transport. Since metal oxides are widely present in sedimentary and terrestrial environments, an AOM-EET niche would have implications for minimizing the net global emissions of methane.

Effect of Nickel Levels on Hydrogen Partial Pressure and Methane Production in Methanogens.

Hydrogen (H2) consumption and methane (CH4) production in pure cultures of three different methanogens were investigated during cultivation with 0, 0.2 and 4.21 µM added nickel (Ni). The results showed that the level of dissolved Ni in the anaerobic growth medium did not notably affect CH4 production in the cytochrome-free methanogenic species Methanobacterium bryantii and Methanoculleus bourgensis MAB1, but affected CH4 formation rate in the cytochrome-containing Methanosarcina barkeri grown on H2 and CO2. Methanosarcina barkeri also had the highest amounts of Ni in its cells, indicating that more Ni is needed by cytochrome-containing than by cytochrome-free methanogenic species. The concentration of Ni affected threshold values of H2 partial pressure (pH2) for all three methanogen species studied, with M. bourgensis MAB1 reaching pH2 values as low as 0.1 Pa when Ni was available in amounts used in normal anaerobic growth medium. To our knowledge, this is the lowest pH2 threshold recorded to date in pure methanogen culture, which suggests that M.bourgensis MAB1 have a competitive advantage over other species through its ability to grow at low H2 concentrations. Our study has implications for research on the H2-driven deep subsurface biosphere and biogas reactor performance.

Biochar alleviates combined stress of ammonium and acids by firstly enriching Methanosaeta and then Methanosarcina.

This investigation evaluated the effectiveness of biochar of different particle sizes in alleviating ammonium (NH4(+)) inhibition (up to 7 g-N/L) during anaerobic digestion of 6 g/L glucose. Compared to the control treatment without biochar addition, treatments that included biochar particles 2-5 mm, 0.5-1 mm and 75-150 µm in size reduced the methanization lag phase by 23.9%, 23.8% and 5.9%, respectively, and increased the maximum methane production rate by 47.1%, 23.5% and 44.1%, respectively. These results confirmed that biochar accelerated the initiation of methanization during anaerobic digestion under double inhibition risk from both ammonium and acids. Furthermore, fine biochar significantly promoted the production of volatile fatty acids (VFAs). Comparative analysis on the archaeal and bacterial diversity at the early and later stages of digestion, and in the suspended, biochar loosely bound, and biochar tightly bound fractions suggested that, in suspended fractions, hydrogenotrophic Methanobacterium was actively resistant to ammonium. However, acetoclastic Methanosaeta can survive at VFAs concentrations up to 60-80 mmol-C/L by improved affinity to conductive biochar, resulting in the accelerated initiation of acetate degradation. Improved methanogenesis was followed by the colonization of the biochar tightly bound fractions by Methanosarcina. The selection of appropriate biochar particles sizes was important in facilitating the initial colonization of microbial cells.

Selective microbial electrosynthesis of methane by a pure culture of a marine lithoautotrophic archaeon.

Reduction of carbon dioxide to methane by microorganisms attached to electrodes is a promising process in terms of renewable energy storage strategies. However the efficient and specific electrosynthesis of methane by methanogenic archaea on cathodes needs fundamental investigations of the electron transfer mechanisms at the microbe-electrode interface without the addition of artificial electron mediators. Using well-defined electrochemical techniques directly coupled to gas chromatography and surface analysis by scanning electron microscopy, it is shown that a pure culture of the marine lithoautotrophic Methanobacterium-like archaeon strain IM1 is capable to utilize electrons from graphite cathodes for a highly selective production of methane, without hydrogen serving as a cathode-generated electron carrier. Microbial electrosynthesis of methane with cultures of strain IM1 is achieved at a set potential of -0.4V vs. SHE and is characterized by a coulomb efficiency of 80%, with rates reaching 350 nmol d(-1) cm(-2) after 23 days of incubation. Moreover, potential step measurements reveal a biologically catalyzed hydrogen production at potentials more positive than abiotic hydrogen evolution on graphite, indicating that an excessive supply of electrons to strain IM1 results in proton reduction rather than in a further increase of methane production.

Methane production in microbial reverse-electrodialysis methanogenesis cells (MRMCs) using thermolytic solutions.

The utilization of bioelectrochemical systems for methane production has attracted increasing attention, but producing methane in these systems requires additional voltage to overcome large cathode overpotentials. To eliminate the need for electrical grid energy, we constructed a microbial reverse-electrodialysis methanogenesis cell (MRMC) by placing a reverse electrodialysis (RED) stack between an anode with exoelectrogenic microorganisms and a methanogenic biocathode. In the MRMC, renewable salinity gradient energy was converted to electrical energy, thus providing the added potential needed for methane evolution from the cathode. The feasibility of the MRMC was examined using three different cathode materials (stainless steel mesh coated with platinum, SS/Pt; carbon cloth coated with carbon black, CC/CB; or a plain graphite fiber brush, GFB) and a thermolytic solution (ammonium bicarbonate) in the RED stack. A maximum methane yield of 0.60 ± 0.01 mol-CH4/mol-acetate was obtained using the SS/Pt biocathode, with a Coulombic recovery of 75 ± 2% and energy efficiency of 7.0 ± 0.3%. The CC/CB biocathode MRMC had a lower methane yield of 0.55 ± 0.02 mol-CH4/mol-acetate, which was twice that of the GFB biocathode MRMC. COD removals (89-91%) and Coulombic efficiencies (74-81%) were similar for all cathode materials. Linear sweep voltammetry and electrochemical impedance spectroscopy tests demonstrated that cathodic microorganisms enhanced electron transfer from the cathode compared to abiotic controls. These results show that the MRMC has significant potential for production of nearly pure methane using low-grade waste heat and a source of waste organic matter at the anode.

Methanobacterium lacus sp. nov., isolated from the profundal sediment of a freshwater meromictic lake.

An autotrophic, hydrogenotrophic methanogen, designated strain 17A1(T), was isolated from the profundal sediment of the meromictic Lake Pavin, France. The cells of the novel strain, which were non-motile, Gram-staining-negative rods that measured 2-15 µm in length and 0.2-0.4 µm in width, grew as filaments. Strain 17A1(T) grew in a mineral medium and its growth was stimulated by the addition of yeast extract, vitamins, acetate or rumen fluid. Penicillin, vancomycin and kanamycin reduced growth but did not completely inhibit it. Growth occurred at 14-41 °C (optimum 30 °C), at pH 5.0-8.5 (optimum pH 6.5) and with 0-0.4 M NaCl (optimum 0.1 M). The novel strain utilized H(2)/CO(2) and methanol/H(2) as substrates but not formate, acetate, methylamine/H(2), isobutanol or 2-propanol. Its genomic DNA G+C content was 37.0 mol%. In phylogenetic analyses based on 16S rRNA gene sequences, strain 17A1(T) appeared to be a member of the genus Methanobacterium, with Methanobacterium beijingense 8-2(T) (96.3% sequence similarity) identified as the most closely related established species. Based on phenotypic and phylogenetic data, strain 17A1(T) represents a novel species of methanogen within the genus Methanobacterium, for which the name Methanobacterium lacus sp. nov. is proposed. The type strain is 17A1(T) (=DSM 24406(T)=JCM 17760(T)).

[Microbial community in granules from an EGSB reactor operated at 20 degrees C].

The microbial communities in 6 granular sludge samples taken from a lab-scale EGSB reactor operated under different organic loading rates (OLR) at 20 degrees C were studied using PCR-DGGE and RTQ-PCR technique. The results indicate that as the OLR increased from 1.5 kg/(m3 x d) to 10.0 kg/(m3 x d), the microbial communities of archaea and eubacteria in the 6 sludge samples are not changed greatly. The archaea population is highly similar among the 6 samples. The Shannon indexes of archaea population are 5.51, 5.88, 5.47, 5.25, 5.32 and 5.11, respectively. Except the seed sludge, the eubacteria population is similar among the other 5 samples. The Shannon indexes of eubacteria population are 2.97, 5.07, 5.44, 6.38, 6.66 and 5.21, respectively. The dominant archaea in the 6 granular samples are Methanobacterium, Methanocorpusculum, Methanosaeta and Methanospirillum. As the OLR of the reactor increased and the operation time elapsed, Methanocorpusculum parvum and Methanospirillum hungatei, both are hydrotrophic methanogens, become the dominant archaea. The archaea content in the samples decreases slightly at the beginning, but increases later and finally reaches to a level obviously higher than that in the seed sludge.

Methane production by methanogens following an aerobic washing procedure: simplifying methods for manipulation.

The recent discovery of methane in the martian atmosphere is arguably one of the most important discoveries in the field of astrobiology. One possible source of this methane could be a microorganism analogous to those on Earth in the domain Archaea known as methanogens. Methanogens are described as obligately anaerobic, and methods developed to work with methanogens typically include anaerobic media and buffers, gassing manifolds, and possibly anaerobic chambers. To determine if the time, effort, and supplies required to maintain anaerobic conditions are necessary to maintain viability, we compared anaerobically washed cells with cells that were washed in the presence of atmospheric oxygen. Anaerobic tubes were opened, and cultures were poured into plastic centrifuge tubes, centrifuged, and suspended in fresh buffer, all in the presence of atmospheric oxygen. Washed cells from both aerobic and anaerobic procedures were inoculated into methanogenic growth media under anaerobic conditions and incubated at temperatures conducive to growth for each methanogenic strain tested. Methane production was measured at time intervals using a gas chromatograph. In three strains, significant differences were not seen between aerobically and anaerobically washed cells. In one strain, there was significantly less methane production observed following aerobic washing at some time points; however, substantial methane production occurred following both procedures. Thus, it appears that aerobic manipulations for relatively short periods of time with at least a few species of methanogens may not lead to loss of viability. With the discovery of methane in the martian atmosphere, it is likely that there will be an increase in astrobiology-related methanogen research. The research reported here should simplify the methodology.

Syntrophomonas cellicola sp. nov., a spore-forming syntrophic bacterium isolated from a distilled-spirit-fermenting cellar, and assignment of Syntrophospora bryantii to Syntrophomonas bryantii comb. nov.

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

Degradation of methanethiol by methylotrophic methanogenic archaea in a lab-scale upflow anaerobic sludge blanket reactor.

In a lab-scale upflow anaerobic sludge blanket reactor inoculated with granular sludge from a full-scale wastewater treatment plant treating paper mill wastewater, methanethiol (MT) was degraded at 30 degrees C to H2S, CO2, and CH4. At a hydraulic retention time of 9 h, a maximum influent concentration of 6 mM MT was applied, corresponding to a volumetric loading rate of 16.5 mmol liter-1 day-1. The archaeal community within the reactor was characterized by anaerobic culturing and denaturing gradient gel electrophoresis analysis, cloning, and sequencing of 16S rRNA genes and quantitative PCR. Initially, MT-fermenting methanogenic archaea related to members of the genus Methanolobus were enriched in the reactor. Later, they were outcompeted by Methanomethylovorans hollandica, which was detected in aggregates but not inside the granules that originated from the inoculum, the microbial composition of which remained fairly unchanged. Possibly other species within the Methanosarcinacaea also contributed to the fermentation of MT, but they were not enriched by serial dilution in liquid media. The archaeal community within the granules, which was dominated by Methanobacterium beijingense, did not change substantially during the reactor operation. Some of the species related to Methanomethylovorans hollandica were enriched by serial dilutions, but their growth rates were very low. Interestingly, the enrichments could be sustained only in the presence of MT and did not utilize any of the other typical substrates for methylotrophic methanogens, such as methanol, methyl amine, or dimethylsulfide.

Spectroscopic and kinetic studies of the reaction of bromopropanesulfonate with methyl-coenzyme M reductase.

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis in which coenzyme B and methyl-coenzyme M are converted to methane and the heterodisulfide, CoMS-SCoB. MCR also appears to initiate anaerobic methane oxidation (reverse methanogenesis). At the active site of MCR is coenzyme F430, a nickel tetrapyrrole. This paper describes the reaction of the active MCR(red1) state with the potent inhibitor, 3-bromopropanesulfonate (BPS; I50 = 50 nM) by UV-visible and EPR spectroscopy and by steady-state and rapid kinetics. BPS was shown to be an alternative substrate of MCR in an ionic reaction that is coenzyme B-independent and leads to debromination of BPS and formation of a distinct state ("MCR(PS)") with an EPR signal that was assigned to a Ni(III)-propylsulfonate species (Hinderberger, D., Piskorski, R. P., Goenrich, M., Thauer, R. K., Schweiger, A., Harmer, J., and Jaun, B. (2006) Angew. Chem. Int. Ed. Engl. 45, 3602-3607). A similar EPR signal was generated by reacting MCR(red1) with several halogenated sulfonate and carboxylate substrates. In rapid chemical quench experiments, the propylsulfonate ligand was identified by NMR spectroscopy and high performance liquid chromatography as propanesulfonic acid after protonolysis of the MCR(PS) complex. Propanesulfonate formation was also observed in steady-state reactions in the presence of Ti(III) citrate. Reaction of the alkylnickel intermediate with thiols regenerates the active MCR(red1) state and eliminates the propylsulfonate group, presumably as the thioether. MCR(PS) is catalytically competent in both the generation of propanesulfonate and reformation of MCR(red1). These results provide evidence for the intermediacy of an alkylnickel species in the final step in anaerobic methane oxidation and in the initial step of methanogenesis.

Survival of methanogens during desiccation: implications for life on Mars.

The relatively recent discoveries that liquid water likely existed on the surface of past Mars and that methane currently exists in the martian atmosphere have fueled the possibility of extant or extinct life on Mars. One possible explanation for the existence of the methane would be the presence of methanogens in the subsurface. Methanogens are microorganisms in the domain Archaea that can metabolize molecular hydrogen as an energy source and carbon dioxide as a carbon source and produce methane. One factor of importance is the arid nature of Mars, at least at the surface. If one is to assume that life exists below the surface, then based on the only example of life that we know, liquid water must be present. Realistically, however, that liquid water may be seasonal just as it is at some locations on our home planet. Here we report on research designed to determine how long certain species of methanogens can survive desiccation on a Mars soil simulant, JSC Mars-1. Methanogenic cells were grown on JSC Mars-1, transferred to a desiccator within a Coy anaerobic environmental chamber, and maintained there for varying time periods. Following removal from the desiccator and rehydration, gas chromatographic measurements of methane indicated survival for varying time periods. Methanosarcina barkeri survived desiccation for 10 days, while Methanobacterium formicicum and Methanothermobacter wolfeii were able to survive for 25 days.

Washing methanogenic cells with the liquid fraction from a Mars soil simulant and water mixture.

Certain methanogens have been shown to grow on a Mars soil simulant following a washing procedure using a carbonate buffer. In experiments where liquid fractions from the soil simulant and water mixtures were used in place of the buffer, two out of three of the species demonstrated significantly greater methane production compared to the buffer.

Syntrophomonas erecta subsp. sporosyntropha subsp. nov., a spore-forming bacterium that degrades short chain fatty acids in co-culture with methanogens.

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

Kinetic characterization of Methanobacterium bryantii M.o.H.

H2 is a key electron donor for many anaerobic microorganisms; thus, keen competition for H2 occurs among H2-utilizing microbial groups. Monod kinetic parameters provide essential information for kinetic analysis of competition for H2. In this study, we estimated Monod kinetic parameter values for a methanogen that consumes only H2 as its electron donor, Methanobacterium bryantii M.o.H. Utilization of a single electron donor is an advantage in this study, because complications from alternate metabolic pathways are avoided. Using a set of batch experiments designed to provide the best estimates of each parameter, we obtained these values: maximum specific growth rate (mumax) = 0.77/ day, maximum substrate consumption rate (qmax) = 2.36 mol-H2/gcells/day, true yield (Y) = 0.325 gcell/mol H2, fraction of donor electrons to synthesis (fs degrees) = 0.03 e-cell/e- donor, half-maximum-rate substrate concentration (Ks) = 18 000 nM = 18 microM H2, and endogenous decay rate (b) = 0.088/ day. This self-consistent set of parameters indicates that, when H2 is not limiting, M. bryantii M.o.H. is a slow grower (low mumax) compared to other H2-oxidizing methanogens and sulfate reducers, and this is mainly due to its low true Y, not a low qmax. The relatively high Ks and b values suggest that M. bryantii also may not be a strong competitor when H2 is limiting.

Membrane lipid patterns typify distinct anaerobic methanotrophic consortia.

The anaerobic oxidation of methane (AOM) is one of the major sinks of this substantial greenhouse gas in marine environments. Recent investigations have shown that diverse communities of anaerobic archaea and sulfate-reducing bacteria are involved in AOM. Most of the relevant archaea are assigned to two distinct phylogenetic clusters, ANME-1 and ANME-2. A suite of specific (13)C-depleted lipids demonstrating the presence of consortia mediating AOM in fossil and recent environments has been established. Here we report on substantial differences in the lipid composition of microbial consortia sampled from distinct compartments of AOM-driven carbonate reefs growing in the northwestern Black Sea. Communities in which the dominant archaea are from the ANME-1 cluster yield internally cyclized tetraether lipids typical of thermophiles. Those in which ANME-2 archaea are dominant yield sn-2-hydroxyarchaeol accompanied by crocetane and crocetenes. The bacterial lipids from these communities are also distinct even though the sulfate-reducing bacteria all belong to the Desulfosarcina/Desulfococcus group. Nonisoprenoidal glycerol diethers are predominantly associated with ANME-1-dominated communities. Communities with ANME-2 yield mainly conventional, ester-linked diglycerides. ANME-1 archaea and associated sulfate-reducing bacteria seem to be enabled to use low concentrations of methane and to grow within a broad range of temperatures. Our results offer a tool for the study of recent and especially of fossil methane environments.

Methanol utilization in defined mixed cultures of thermophilic anaerobes in the presence of sulfate.

We studied thermophilic sulfate reduction with methanol as electron donor in continuous cultures. Mixed cultures of selected microorganisms were used, representing different methanol degrading pathways followed by various trophic groups of microorganisms. Our results show that direct competition for methanol between a homoacetogen, Moorella thermoautotrophica, and a sulfate reducer, Desulfotomaculum kuznetsovii, is in favour of the sulfate reducer due to its affinity for methanol. Methanogenesis as a result of interspecies hydrogen transfer between D. kuznetsovii and a hydrogen-consuming methanogenic archaeon, Methanothermobacter thermoautotrophicus occurred only below 5 mM total sulfide. A similar result was obtained when M. thermoautotrophica was grown on methanol in the presence of Mb. thermoautotrophicus. Interestingly, D. kuznetsovii could coexist with a non-methanol-utilizing sulfate reducer (Thermodesulfovibrio species). Our data show that it is possible to maintain a dominant sulfate-reducing process with methanol as electron donor at 60 degrees C in mixed continuous cultures.