Methanogen Methanothermobacter Thermautotrophicus Research Articles

Cobalt(III)–EDTA]− reduction by thermophilic methanogen Methanothermobacter thermautotrophicus

Cobalt is a metal contaminant at high temperature radioactive waste disposal sites. Past studies have largely focused on mesophilic microorganisms to remediate cobalt, despite the presence of thermophilic microorganisms at such sites. In this study, Methanothermobacter thermautotrophicus, a thermophilic methanogen, was used to reduce Co(III) in the form of [Co(III)–EDTA]−. Bioreduction experiments were conducted in a growth medium with H2/CO2 as a growth substrate at initial Co(III) concentrations of 1, 2, 4, 7, and 10mM. At low Co(III) concentrations (<4mM), a complete reduction was observed within a week. Wet chemistry, X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) analyses were all consistent in revealing the reduction kinetics. However, at higher concentrations (7 and 10mM) the reduction extents only reached 69.8% and 48.5%, respectively, likely due to the toxic effect of Co(III) to the methanogen cells as evidenced by a decrease in total cellular protein at these Co(III) concentrations. Methanogenesis was inhibited by Co(III) bioreduction, possibly due to impaired cell growth and electron diversion from CO2 to Co(III). Overall, our results demonstrated the ability of M. thermautotrophicus to reduce Co(III) to Co(II) and its potential application for remediating 60Co contaminant at high temperature subsurface radioactive waste disposal sites.

Reduction of hexavalent chromium by the thermophilic methanogen Methanothermobacter thermautotrophicus

Despite significant progress on iron reduction by thermophilic microorganisms, studies on their ability to reduce toxic metals are still limited, despite their common co-existence in high temperature environments (up to 70°C). In this study, Methanothermobacter thermautotrophicus, an obligate thermophilic methanogen, was used to reduce hexavalent chromium. Experiments were conducted in a growth medium with H2/CO2 as substrate with various Cr6+ concentrations (0.2, 0.4, 1, 3, and 5mM) in the form of potassium dichromate (K2Cr2O7). Time-course measurements of aqueous Cr6+ concentrations using 1,5-diphenylcarbazide colorimetric method showed complete reduction of the 0.2 and 0.4mM Cr6+ solutions by this methanogen. However, much lower reduction extents of 43.6%, 13.0%, and 3.7% were observed at higher Cr6+ concentrations of 1, 3 and 5mM, respectively. These lower extents of bioreduction suggest a toxic effect of aqueous Cr6+ to cells at this concentration range. At these higher Cr6+ concentrations, methanogenesis was inhibited and cell growth was impaired as evidenced by decreased total cellular protein production and live/dead cell ratio. Likewise, Cr6+ bioreduction rates decreased with increased initial concentrations of Cr6+ from 13.3 to 1.9μMh−1. X-ray absorption near-edge structure (XANES) spectroscopy revealed a progressive reduction of soluble Cr6+ to insoluble Cr3+ precipitates, which was confirmed as amorphous chromium hydroxide by selected area electron diffraction pattern. However, a small fraction of reduced Cr occurred as aqueous Cr3+. Scanning and transmission electron microscope observations of M. thermautotrophicus cells after Cr6+ exposure suggest both extra- and intracellular chromium reduction mechanisms. Results of this study demonstrate the ability of M. thermautotrophicus cells to reduce toxic Cr6+ to less toxic Cr3+ and its potential application in metal bioremediation, especially at high temperature subsurface radioactive waste disposal sites, where the temperature may reach ∼70°C.

Hydrogen isotope systematics among H2–H2O–CH4 during the growth of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus strain ΔH

Stable hydrogen isotope systematics among H2, H2O, and CH4 during hydrogenotrophic methanogenesis were investigated by growing a thermophilic methanogen, Methanothermobacter thermautotrophicus strain ΔH, in batch cultures spiked with deuterium-labeled H2 and/or H2O. The hydrogen isotope ratio of the product, CH4, reflected not only the isotope ratio of the H2O in the medium but also that of the substrate, H2. The D/H ratios of the CH4 were highest during the early phase of growth, and the growth-phase-dependent changes were greatest in the deuterium-enriched H2 cultures. The hydrogen isotope systematics among H2, H2O, and CH4 during growth of the methanogen could be described with the following equations: δDCH4=a×δDH2O+b×δDH2-ca=0.71-0.55×b0.17⩽b⩽0.38c=1000×(a+b-1) The greatest effect of δDH2 on δDCH4 (b=0.38) was observed during the earliest phase of growth. In contrast to this study, the possible disappearance of the effect (b=0) has been suggested in a previous study (Valentine et al., 2004a) in which Methanothermobacter marburgensis was cultured and hydrogen isotope systematics during growth was monitored, as was the case in this study. The close phylogenetic relationship between M. thermautotrophicus and M. marburgensis, which likely have similar biochemical pathways, suggests a possibly broad range of the b value, 0–0.38. To explain the observed hydrogen isotope systematics, two cellular mechanisms were proposed. One is that the hydrogen atoms of both H2 and H2O are directly incorporated into the product, CH4. The other is that all four hydrogen atoms in the product, CH4, are derived from intracellular H2O, which consists of a mixture of medium-derived pristine H2O and isotopically distinct H2O derived from methanogenic H2 oxidation. Although we attempted to evaluate the feasibility of these mechanisms, both cellular mechanisms remain hypothetical. The hydrogen isotope systematics shown here contribute to put forward utility of δDCH4 value as a geochemical tracer of the origins of environmental CH4.

Redox-sensitive DNA binding by homodimeric Methanosarcina acetivorans MsvR is modulated by cysteine residues

BackgroundMethanoarchaea are among the strictest known anaerobes, yet they can survive exposure to oxygen. The mechanisms by which they sense and respond to oxidizing conditions are unknown. MsvR is a transcription regulatory protein unique to the methanoarchaea. Initially identified and characterized in the methanogen Methanothermobacter thermautotrophicus (Mth), MthMsvR displays differential DNA binding under either oxidizing or reducing conditions. Since MthMsvR regulates a potential oxidative stress operon in M. thermautotrophicus, it was hypothesized that the MsvR family of proteins were redox-sensitive transcription regulators.ResultsAn MsvR homologue from the methanogen Methanosarcina acetivorans, MaMsvR, was overexpressed and purified. The two MsvR proteins bound the same DNA sequence motif found upstream of all known MsvR encoding genes, but unlike MthMsvR, MaMsvR did not bind the promoters of select genes involved in the oxidative stress response. Unlike MthMsvR that bound DNA under both non-reducing and reducing conditions, MaMsvR bound DNA only under reducing conditions. MaMsvR appeared as a dimer in gel filtration chromatography analysis and site-directed mutagenesis suggested that conserved cysteine residues within the V4R domain were involved in conformational rearrangements that impact DNA binding.ConclusionsResults presented herein suggest that homodimeric MaMsvR acts as a transcriptional repressor by binding Ma PmsvR under non-reducing conditions. Changing redox conditions promote conformational changes that abrogate binding to Ma PmsvR which likely leads to de-repression.

Examination of metal corrosion byDesulfomicrobium thermophilum, Archaeoglobus fulgidus,andMethanothermobacter thermautotrophicus

Metal corrosion resulting from microbial processes constitutes a serious expense in many industrial applications. Oil pipelines are plagued by these events leading not only to economic losses but also severe environmental consequences. Therefore, our understanding of the microorganisms that contribute to metal corrosion is vital. Here, the roles of a methanogen and either a sulfate-reducing bacterium or archaeon in corrosion are addressed. The methanogen Methanothermobacter thermautotrophicus was used to assess the effect of methanogenesis in corrosion. The sulfate-reducing bacterium Desulfomicrobium thermophilum and the sulfate-reducing archaeon Archaeoglobus fulgidus were used to measure the effect of sulfate reduction on corrosion. To assess the combined effect of sulfate-reduction and methanogenesis on corrosion, M. thermautotrophicus was grown in combination with A. fulgidus or D. thermophilum. This allowed investigation of corrosion impact at different temperatures and salinities. All organisms contributed to corrosion, measured by metal weight loss over time, either independently or in conjunction with a partner organism. Corrosion resulting from strictly biotic processes was dominant at lower temperature and salinities. At higher salinities and temperatures, approximately half of the corrosion measured was because of abiotic reactions as indicated by experimental controls. This work demonstrates the role of these microbial metabolisms in metal corrosion in anaerobic environments.

Electrochemical control of redox potential affects methanogenesis of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus

To investigate the precise effect of the redox potential on the methanogenesis of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus by using an electrochemical redox controlling system without adding oxidizing or reducing agents. A bioelectrochemical system was applied to control the redox conditions in culture and to measure the methane-producing activity of M.thermautotrophicus at a constant potential from +0·2 to -0·8V (vs Ag/AgCl). Methane production and growth of M.thermautotrophicus were 1·6 and 3·5 times increased at -0·8V, compared with control experiments without electrolysis, respectively, while methanogenesis was suppressed between +0·2 and -0·2V. A clear relationship between an electrochemically regulated redox potential and methanogenesis was revealed.

Microbial reduction of Fe(III) in smectite minerals by thermophilic methanogen Methanothermobacter thermautotrophicus

Clay minerals and thermophilic methanogens can co-exist in hot anoxic environments, including the continental subsurface, geysers, terrestrial hot springs, and deep-sea hydrothermal vent systems. However, it is unclear whether thermophilic methanogens are able to reduce structural Fe(III) in clay minerals. In this study, the ability of a thermophilic methanogen Methanothermobacter thermautotrophicus to reduce structural Fe(III) in iron-rich and iron-poor smectites, (nontronite NAu-2 and Wyoming montmorillonite SWy-2) and the relationship between iron reduction and methanogenesis were investigated. M. thermautotrophicus reduced Fe(III) in nontronite NAu-2 and montmorillonite SWy-2 with H2/CO2 as substrate. The extent of bioreduction was 27% for nontronite and 13–15% for montmorillonite. Anthraquinone-2,6-disulfonate (AQDS) did not enhance the extent of bioreduction, but accelerated the rate. When methanogenesis was inhibited via addition of 2-bromoethane sulfonate (BES), the extent of bioreduction decreased to 16% for NAu-2 and 9% for SWy-2. These data suggest that Fe(III) bioreduction and methanogenesis were mutually beneficial. The likely mechanism was that Fe(III) bioreduction lowered the reduction potential of the system so that methanogenesis became favorable, and methanogenesis in turn stimulated the growth of the methanogen, which enhanced Fe(III) bioreduction. NAu-2 was partly dissolved and high charge smectite and biogenic silica formed as a result of bioreduction.

Acceleration of cellulose degradation and shift of product via methanogenic co-culture of a cellulolytic bacterium with a hydrogenotrophic methanogen

Although the effects of syntrophic relationships between bacteria and methanogens have been reported in some environments, those on cellulose decomposition using cellulolytic bacteria from methanogenic reactors have not yet been examined. The effects of syntrophic co-culture on the decomposition of a cellulosic material were investigated in a co-culture of Clostridium clariflavum strain CL-1 and the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus strain ΔH and a single-culture of strain CL-1 under thermophilic conditions. In this study, strain CL-1 was newly isolated as a cellulolytic bacterium from a thermophilic methanogenic reactor used for degrading garbage slurry. The degradation efficiency and cell density of strain CL-1 were 2.9- and 2.7-fold higher in the co-culture than in the single-culture after 60h of incubation, respectively. Acetate, lactate and ethanol were the primary products in both cultures, and the concentration of propionate was low. The content of acetate to total organic acids plus ethanol was 59.3% in the co-culture. However, the ratio decreased to 24.9% in the single-culture, although acetate was the primary product. Therefore, hydrogen scavenging by the hydrogenotrophic methanogen strain ΔH could shift the metabolic pathway to the acetate production pathway in the co-culture. Increases in the cell density and the consequent acceleration of cellulose degradation in the co-culture would be caused by increases in adenosine 5'-triphosphate (ATP) levels, as the acetate production pathway includes ATP generation. Syntrophic cellulose decomposition by the cellulolytic bacteria and hydrogenotrophic methanogens would be the dominant reaction in the thermophilic methanogenic reactor degrading cellulosic materials.

Methane production by Methanothermobacter thermautotrophicus to recover energy from carbon dioxide sequestered in geological reservoirs

To recover energy from carbon dioxide sequestered in geological reservoirs, the geochemical effects of acidic and substrate- and nutrient-limiting conditions on methane production by the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus were investigated in a simulated deep saline aquifer environment using formation water media retrieved from petroleum reservoirs.

Thermodesulfovibrio aggregans sp. nov. and Thermodesulfovibrio thiophilus sp. nov., anaerobic, thermophilic, sulfate-reducing bacteria isolated from thermophilic methanogenic sludge, and emended description of the genus Thermodesulfovibrio

Four obligately anaerobic, thermophilic, sulfate-reducing bacterial strains, designated TGE-P1(T), TDV(T), TGL-LS1 and TSL-P1, were isolated from thermophilic (operated at 55 degrees C) methanogenic sludges from waste and wastewater treatment. The optimum temperature for growth of all the strains was in the range 55-60 degrees C. The four strains grew by reduction of sulfate with a limited range of electron donors, such as hydrogen, formate, pyruvate and lactate. In co-culture with the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus DeltaH(T), strains TGE-P1(T), TGL-LS1 and TSL-P1 were able to utilize lactate syntrophically for growth. The DNA G+C contents of all the strains were in the range 34-35 mol%. The major cellular fatty acids of the strains were iso-C(17 : 0), iso-C(16 : 0), C(16 : 0) and anteiso-C(15 : 0). Phylogenetic analyses based on 16S rRNA gene sequences revealed that the strains belong to the Thermodesulfovibrio clade of the phylum 'Nitrospirae'. On the basis of their physiological, chemotaxonomic and genetic properties, strains TGL-LS1 (=JCM 13214) and TSL-P1 (=JCM 13215) were classified as strains of Thermodesulfovibrio islandicus. Two novel species of the genus Thermodesulfovibrio are proposed to accommodate the other two isolates: Thermodesulfovibrio aggregans sp. nov. (type strain TGE-P1(T) =JCM 13213(T) =DSM 17283(T)) and Thermodesulfovibrio thiophilus sp. nov. (type strain TDV(T) =JCM 13216(T) =DSM 17215(T)). To examine the ecological aspects of Thermodesulfovibrio-type cells in the sludge from which the strains were originally isolated, an oligonucleotide probe targeting 16S rRNA of all Thermodesulfovibrio species was designed and applied to thin sections of thermophilic sludge granules. Fluorescence in situ hybridization using the probe revealed rod- or vibrio-shaped cells as a significant population within the sludge, indicating their important role in the original ecosystem.

Tepidanaerobacter syntrophicus gen. nov., sp. nov., an anaerobic, moderately thermophilic, syntrophic alcohol- and lactate-degrading bacterium isolated from thermophilic digested sludges

Three anaerobic, moderately thermophilic, syntrophic primary alcohol- and lactate-degrading microbes, designated strains JL(T), JE and OL, were isolated from sludges of thermophilic (55 degrees C) digesters that decomposed either municipal solid wastes or sewage sludge. The strains were strictly anaerobic organisms. All three strains grew at 25-60 degrees C and pH 5.5-8.5 and optimum growth was observed at 45-50 degrees C and pH 6.0-7.0. The three organisms grew chemo-organotrophically on a number of carbohydrates in the presence of yeast extract. In co-culture with the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus, all strains could utilize ethanol, glycerol and lactate syntrophically for growth, although these compounds were not metabolized in pure culture without additional external electron acceptors. All strains could reduce thiosulphate. Quinones were not detected. The DNA G+C contents of strains JL(T), JE and OL were 38.0, 37.3 and 37.7 mol%, respectively. Major cellular fatty acids of the strains were iso-C(15 : 0), C(16 : 0) and unsaturated species of C(15 : 1). Phylogenetic analyses based on 16S rRNA gene sequences revealed that the strains belong to a deeply branched lineage of the phylum Firmicutes; the most closely related species was Thermovenabulum ferriorganovorum (16S rRNA gene sequence similarity of 88 %). The three strains were phylogenetically very closely related to each other (99-100 % 16S rRNA gene sequence similarity) and were physiologically and chemotaxonomically similar. These genetic and phenotypic properties suggest that the strains should be classified as representatives of a novel species and genus; the name Tepidanaerobacter syntrophicus gen. nov., sp. nov. is proposed. The type strain of Tepidanaerobacter syntrophicus is strain JL(T) (=JCM 12098(T)=NBRC 100060(T)=DSM 15584(T)).

Pelotomaculum thermopropionicum gen. nov., sp. nov., an anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium.

An anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium, strain SI(T), isolated previously from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) reactor, was characterized. The strain could grow fermentatively on pyruvate and fumarate in pure culture. The strain grew on propionate, ethanol, lactate, 1-butanol, 1-pentanol, 1,3-propanediol, 1-propanol and ethylene glycol in co-culture with the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus strain deltaH(T). The optimum temperature for growth was 55 degrees C and the pH optimum was 7.0. The G+C content of the DNA was 52.8 mol %. Strain SI(T) contained MK-7 and MK-7(H4) as the major quinones and contained iso-C15:0 as the major fatty acid. Based on 16S rDNA sequence analysis, strain SI(T) formed a novel lineage within the gram-positive, spore-forming, sulphate-reducing bacterial group Desulfotomaculum. However, the strain lacked the ability to conduct dissimilatory sulphate reduction. Instead, it could reduce fumarate to succinate with concomitant growth on several organic substances as electron donor. These phenotypic and genetic properties support the formation of a novel species of a new genus, for which the name Pelotomaculum thermopropionicum gen. nov., sp. nov. is proposed. The type strain is strain SI(T) (= DSM 13744T = JCM 10971T).