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PubMed2004

Reverse methanogenesis: testing the hypothesis with environmental genomics.

Microbial methane consumption in anoxic sediments significantly impacts the global environment by reducing the flux of greenhouse gases from ocean to atmosphere. Despite its significance, the biological mechanisms controlling anaerobic methane oxidation are not well characterized. One current model suggests that relatives of methane-producing Archaea developed the capacity to reverse methanogenesis and thereby to consume methane to produce cellular carbon and energy. We report here a test of the "reverse-methanogenesis" hypothesis by genomic analyses of methane-oxidizing Archaea from deep-sea sediments. Our results show that nearly all genes typically associated with methane production are present in one specific group of archaeal methanotrophs. These genome-based observations support previous hypotheses and provide an informed foundation for metabolic modeling of anaerobic methane oxidation.

Anaerobiosis↗
PubMed1978

Methanogenesis from acetate: enrichment studies.

An acetate enrichment culture was initiated by inoculating anaerobic sludge from a mesophilic methane digestor into a mineral salts medium with calcium acetate as the sole carbon and energy source. This enrichment was maintained indefinitely by weekly transfer into medium of the same composition. A study of this enrichment disclosed an unexpected age-dependent inhibition of methanogenesis by H2 and formate which apparently differed from the inhibition by chloroform and benzyl viologen. This age-dependent inhibition indicated that microbial interactions of the mixed enrichment population may play a regulatory role in methane formation. Futhermore, stimulation of methanogenesis in the acetate enrichment by addition of yeast extract showed a nutrient limitation which indicated that syntrophic interactions leading to formation of growth factors may also occur. A model is presented to illustrate the possible interrelationships between methanogenic and nonmethanogenic bacteria in their growth and formation of methane and carbon dioxide from acetate.

Acetates↗
PubMed1983

Effects of metals on methanogenesis, sulfate reduction, carbon dioxide evolution, and microbial biomass in anoxic salt marsh sediments.

The effects of several metals on microbial methane, carbon dioxide, and sulfide production and microbial ATP were examined in sediments from Spartina alterniflora communities. Anaerobically homogenized sediments were amended with 1,000 ppm (ratio of weight of metal to dry weight of sediment) of various metals. Time courses in controls were similar for CH(4), H(2)S, and CO(2), with short initial lags (0 to 4 h) followed by periods of constant gas production (1 to 2 days) and declining rates thereafter. Comparisons were made between control and experimental assays with respect to initial rates of production (after lag) and overall production. Methane evolution was inhibited both initially and overall by CH(3)HgCl, HgS, and NaAsO(2). A period of initial inhibition was followed by a period of overall stimulation with Hg, Pb, Ni, Cd, and Cu, all as chlorides, and with ZnSO(4), K(2)CrO(4), and K(2)Cr(2)O(7). Production of CO(2) was generally less affected by the addition of metals. Inhibition was noted with NaAsO(2), CH(3)HgCl, and Na(2)MoO(4). Minor stimulation of CO(2) production occurred over the long term with chlorides of Hg, Pb, and Fe. Sulfate reduction was inhibited in the short term by all metals tested and over the long term by all but FeCl(2) and NiCl(2). Microbial biomass was decreased by FeCl(2), K(2)Cr(2)O(7), ZnSO(4), CdCl(2), and CuCl(2) but remained generally unaffected by PbCl(2), HgCl(2), and NiCl(2). Although the majority of metals produced an immediate inhibition of methanogenesis, for several metals this was only a transient phenomenon followed by an overall stimulation. The initial suppression of methanogenesis may be relieved by precipitation, complexation, or transformation of the metal (possibly by methylation), with the subsequent stimulation resulting from a sustained inhibition of competing organisms (e.g., sulfate-reducing bacteria). For several environmentally significant metals, severe metal pollution may substantially alter the flow of carbon in sediments.

Journal Article↗
PubMed1985

Effect of gramicidin on methanogenesis by various methanogenic bacteria.

Methanogenesis by Methanobacterium thermoautotrophicum strains was extremely sensitive to gramicidin, total inhibition being observed at 0.2 mug/ml. In contrast, methane synthesis by Methanococcus voltae, Methanogenium marisnigri, Methanosarcina mazei, and Methanospirillum hungatei were resistant to the highest concentrations of gramicidin tested (40 mug/ml), although spheroplasts of Methanospirillum hungatei were extremely sensitive. Other species tested showed intermediate sensitivity to gramicidin, methanogenesis inhibition occurring at 4 to 20 mug/ml.

Journal Article↗
PubMed1987

Stable Carbon Isotope Fractionation by Methanosarcina barkeri during Methanogenesis from Acetate, Methanol, or Carbon Dioxide-Hydrogen.

Methanosarcina barkeri was cultured on methanol, H(2)-CO(2), and acetate, and the C/C ratios of the substrates and the methane produced from them were determined. The discrimination against C in methane relative to substrate decreased in the order methanol > CO(2) > acetate. The isotopic fractionation for methane derived from acetate was only one-third of that observed with methanol as the substrate. The data presented indicate that the last enzyme of methanogenesis, methylreductase, is not the primary site of isotopic discrimination during methanogenesis from methanol or CO(2). These results also support biogeochemical interpretations that gas produced in environments in which acetate is the primary methane precursor will have higher C/C ratios than those from environments where other substrates predominate.

Journal Article↗
PubMed1988

Methanogenesis from Methylated Amines in a Hypersaline Algal Mat.

Methane ebullition and high rates of methane production were observed in sediments of a hypersaline pond (180 per thousand) which contained sulfate in excess of 100 mM. The highest rates of methane production were observed in surface sediments associated with an algal mat dominated by a Spirulina sp. The mat contained a methylated amine, glycine betaine (GBT), at levels which accounted for up to 20% of the total mat nitrogen. GBT was apparently the source of trimethylamine (TMA), which was also present in the sediment at relatively high concentrations. Patterns of substrate metabolism by the methanogenic populations in sediment slurries suggested that TMA was a major methane precursor. Neither exogenous hydrogen nor acetate stimulated methanogenesis, while addition of a variety of amines including TMA, trimethylamine oxide, GBT, and choline resulted in substantial increases with yields of >70%. The temperature optimum for methanogenesis in this system was 45 to 55 degrees C, which coincided with the observed sediment temperature. Patterns and rates of methane production in this and other hypersaline algal mats may be determined by a complex interaction between salinity, the use of methylated amines for osmoregulation by algae, and the formation of TMA by fermentation.

Journal Article↗
PubMed1988

Use of a three-stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria.

A mixed culture of human fecal bacteria was grown for 120 days in a three-stage continuous culture system. To reproduce some of the nutritional and pH characteristics of the large gut, each vessel had a different operating volume (0.3, 0.5, and 0.8 liter) and pH (6.0, 6.5, and 7.0). A mixture of polysaccharides and proteins was used as carbon and nitrogen sources. Measurements of H2, CH4, S2-, sulfate reduction rates, sulfate-reducing bacteria (SRB), and volatile fatty acids were made throughout the experiment. After 48 days of running, porcine gastric mucin (5.8 g/day) was independently fed to vessel 1 of the multichamber system. The mucin was extensively degraded as evidenced by the stimulation of volatile fatty acid production. In the absence of mucin, sulfate-reducing activity was comparatively insignificant and methanogenesis was the major route for the disposal of electrons. The reverse occurred upon the addition of mucin; sulfate reduction predominated and methanogenesis was completely inhibited. This was attributed to release of sulfate from the mucin which enabled SRB to outcompete methanogenic bacteria for H2. SRB stimulated by mucin were acetate-utilizing Desulfobacter spp., lactate- and H2-utilizing Desulfovibrio spp., and propionate-utilizing Desulfobulbus spp. When the mucin pump was switched off, the multichamber system reverted to a state close to its original equilibrium. These data provide further evidence that sulfated polysaccharides such as mucin may be a source of sulfate for SRB in the human large gut.

Bacteria, Anaerobic↗
PubMed1989

Methanogenesis from ethanol by defined mixed continuous cultures.

Methanogenesis from ethanol by defined mixed continuous cultures was studied. Under sulfate-free conditions, a Desulfovibrio strain was used as the ethanol-degrading species producing acetic acid and hydrogen. In a two-membered mutualistic coculture, the hydrogen was converted to methane by a Methanobacterium sp. and pH was maintained at neutrality by the addition of alkali. Introduction of a third species, the acetate-utilizing Methanosarcina mazei, obviated the need for external pH control. Methanogenesis by the co-and triculture was studied at various dilution rates in the steady state. The mutualistic coculture performed like a composite single species, as predicted from the theory of mutualistic interactions. Coupling between the mutualistic coculture and the acetate-utilizing methanogen was less tight. Increasing the dilution rate destabilized the triculture; at low dilution rates, instability was soon recovered, but at higher dilution rates imbalance between the rates of production and removal of acetic acid led to a drop in pH. Flocs formed in the triculture. An annulus of the Methanobacterium sp. and Desulfovibrio sp. was retained around the Methanosarcina sp. by strands of material probably derived from the Methanosarcina sp.

Journal Article↗
PubMed2004

Effect of dilution rate on metabolic pathway shift between aceticlastic and nonaceticlastic methanogenesis in chemostat cultivation.

Acetate conversion pathways of methanogenic consortia in acetate-fed chemostats at dilution rates of 0.025 and 0.6 day(-1) were investigated by using (13)C-labeled acetates, followed by gas chromatography-mass spectrometry (GC-MS) analysis of the CH(4) and CO(2) produced. Nonaceticlastic syntrophic oxidation by acetate-oxidizing syntrophs and hydrogenotrophic methanogens was suggested to occupy a primary pathway (approximately 62 to 90%) in total methanogenesis at the low dilution rate. In contrast, aceticlastic cleavage of acetate by aceticlastic methanogens was suggested to occupy a primary pathway (approximately 95 to 99%) in total methanogenesis at the high dilution rate. Phylogenetic analyses of transcripts of the methyl coenzyme M reductase gene (mcrA) confirmed that a significant number of transcripts of the genera Methanoculleus (hydrogenotrophic methanogens) and Methanosarcina (aceticlastic methanogens) were present in the chemostats at the low and high dilution rates, respectively. The mcrA transcripts of the genus Methanosaeta (aceticlastic methanogens), which dominated the population in a previous study (T. Shigematsu, Y. Tang, H. Kawaguchi, K. Ninomiya, J. Kijima, T. Kobayashi, S. Morimura, and K. Kida, J. Biosci. Bioeng. 96:547-558, 2003), were poorly detected at both dilution rates due to the limited coverage of the primers used. These results demonstrated that the dilution rate could cause a shift in the primary pathway of acetate conversion to methane in acetate-fed chemostats.

Acetates↗
PubMed2005

Evidence for aceticlastic methanogenesis in the presence of sulfate in a gas condensate-contaminated aquifer.

The anaerobic metabolism of acetate was studied in sediments and groundwater from a gas condensate-contaminated aquifer in an aquifer where geochemical evidence implicated sulfate reduction and methanogenesis as the predominant terminal electron-accepting processes. Most-probable-number tubes containing acetate and microcosms containing either [2-(14)C]acetate or [U-(14)C]acetate produced higher quantities of CH(4) compared to CO(2) in the presence or absence of sulfate.(14)CH(4) accounted for 70 to 100% of the total labeled gas in the [(14)C]acetate microcosms regardless of whether sulfate was present or not. Denaturing gradient gel electrophoresis of the acetate enrichments both with and without sulfate using Archaea-specific primers showed identical predominant bands that had 99% sequence similarity to members of Methanosaetaceae. Clone libraries containing archaeal 16S rRNA gene sequences amplified from sediment from the contaminated portion of the aquifer showed that 180 of the 190 clones sequenced belonged to the Methanosaetaceae. The production of methane and the high frequency of sequences from the Methanosaetaceae in acetate enrichments with and without sulfate indicate that aceticlastic methanogenesis was the predominant fate of acetate at this site even though sulfate-reducing bacteria would be expected to consume acetate in the presence of sulfate.

Acetates↗
PubMed1985

Tetrahydromethanopterin-dependent methanogenesis from non-physiological C1 donors in Methanobacterium thermoautotrophicum.

Methanogenesis from the non-physiological C1 donors thioproline, thiazolidine, hexamethylenetetramine, formaldehyde (HCHO), and HOCH2-S-coenzyme M (CoM) was catalyzed by cell extracts of Methanobacterium thermoautotrophicum under a hydrogen atmosphere. Tetrahydromethanopterin (H4MPT) and HS-CoM were required in the reaction mixture. The non-physiological compounds were found to be in chemical equilibrium with HCHO, which has been shown to react spontaneously with H4MPT to form methylene-H4MPT, an intermediate of the methanogenic pathway at the formaldehyde level of oxidation. Highfield (360 MHZ) 1H and 13C nuclear magnetic resonance studies performed on the interaction between HCHO and HS-CoM showed that these compounds are in equilibrium with HOCH2-S-CoM and that the equilibrium is pH dependent. When methanogenesis from the non-physiological donors was followed under a nitrogen atmosphere, the C1 moiety from each compound underwent a disproportionation, forming methenyl-H4MPT+ and methane. The compounds tested served as substrates for the enzymatic synthesis of methenyl-H4MPT+.

Carbon↗
PubMed1998

Clustered genes encoding the methyltransferases of methanogenesis from monomethylamine.

Coenzyme M (CoM) is methylated during methanogenesis from monomethyamine in a reaction catalyzed by three proteins. Using monomethylamine, a 52-kDa polypeptide termed monomethylamine methyltransferase (MMAMT) methylates the corrinoid cofactor bound to a second polypeptide, monomethylamine corrinoid protein (MMCP). Methylated MMCP then serves as a substrate for MT2-A, which methylates CoM. The genes for these proteins are clustered on 6.8 kb of DNA in Methanosarcina barkeri MS. The gene encoding MMCP (mtmC) is located directly upstream of the gene encoding MMAMT (mtmB). The gene encoding MT2-A (mtbA) was found 1.1 kb upstream of mtmC, but no obvious open reading frame was found in the intergenic region between mtbA and mtmC. A single monocistronic transcript was found for mtbA that initiated 76 bp from the translational start. Separate transcripts of 2.4 and 4.7 kb were detected, both of which carried mtmCB. The larger transcript also encoded mtmP, which is homologous to the APC family of cationic amine permeases and may therefore encode a methylamine permease. A single transcriptional start site was found 447 bp upstream of the translational start of mtmC. MtmC possesses the corrinoid binding motif found in corrinoid proteins involved in dimethylsulfide- and methanol-dependent methanogenesis, as well as in methionine synthase. The open reading frame of mtmB was interrupted by a single in-frame, midframe, UAG codon which was also found in mtmB from M. barkeri NIH. A mechanism that circumvents UAG-directed termination of translation must operate during expression of mtmB in this methanogen.

Amino Acid Sequence↗
PubMed1987

Inhibition of methanogenesis in pure cultures by ammonia, fatty acids, and heavy metals, and protection against heavy metal toxicity by sewage sludge.

The effect of ammonium chloride, sodium butyrate, sodium propionate, and the heavy metals nickel, zinc, and copper on methanogenesis by pure cultures of Methanospirillum hungatei, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobacterium formicicum at pH 6.5 was studied. The latter three strains were resistant to greater than 60 g/L of the volatile fatty acids and to greater than 10 g/L of NH3 N. Methanospirillum hungatei was somewhat more sensitive with 50% inhibition of methanogenesis occurring at 4.2 g/L NH3 N, 27 g/L butyrate, and 41 g/L propionate. All strains were very sensitive to both copper (1-5 mg/L) and zinc (1-10 mg/L), but much more resistant to nickel. Zinc and copper concentrations 30 to 270 times higher were required to cause inhibition of Msp. hungatei incubated in sewage sludge compared with buffer, indicating a strong protective environment was afforded the methanogens against heavy metal toxicity in the sludge.

Ammonium Chloride↗
PubMed2004

A genomic timescale of prokaryote evolution: insights into the origin of methanogenesis, phototrophy, and the colonization of land.

BACKGROUND: The timescale of prokaryote evolution has been difficult to reconstruct because of a limited fossil record and complexities associated with molecular clocks and deep divergences. However, the relatively large number of genome sequences currently available has provided a better opportunity to control for potential biases such as horizontal gene transfer and rate differences among lineages. We assembled a data set of sequences from 32 proteins (approximately 7600 amino acids) common to 72 species and estimated phylogenetic relationships and divergence times with a local clock method. RESULTS: Our phylogenetic results support most of the currently recognized higher-level groupings of prokaryotes. Of particular interest is a well-supported group of three major lineages of eubacteria (Actinobacteria, Deinococcus, and Cyanobacteria) that we call Terrabacteria and associate with an early colonization of land. Divergence time estimates for the major groups of eubacteria are between 2.5-3.2 billion years ago (Ga) while those for archaebacteria are mostly between 3.1-4.1 Ga. The time estimates suggest a Hadean origin of life (prior to 4.1 Ga), an early origin of methanogenesis (3.8-4.1 Ga), an origin of anaerobic methanotrophy after 3.1 Ga, an origin of phototrophy prior to 3.2 Ga, an early colonization of land 2.8-3.1 Ga, and an origin of aerobic methanotrophy 2.5-2.8 Ga. CONCLUSIONS: Our early time estimates for methanogenesis support the consideration of methane, in addition to carbon dioxide, as a greenhouse gas responsible for the early warming of the Earths' surface. Our divergence times for the origin of anaerobic methanotrophy are compatible with highly depleted carbon isotopic values found in rocks dated 2.8-2.6 Ga. An early origin of phototrophy is consistent with the earliest bacterial mats and structures identified as stromatolites, but a 2.6 Ga origin of cyanobacteria suggests that those Archean structures, if biologically produced, were made by anoxygenic photosynthesizers. The resistance to desiccation of Terrabacteria and their elaboration of photoprotective compounds suggests that the common ancestor of this group inhabited land. If true, then oxygenic photosynthesis may owe its origin to terrestrial adaptations.

Bacteria↗
PubMed2006

Effects of ferric hydroxide on methanogenesis from lipids and long-chain fatty acids in anaerobic digestion.

The addition of ferric hydroxide to sludge from a municipal anaerobic digester stimulated the rate of methanogenesis from canola oil when the initial oil concentration was high (4600 mg/L; P < 0.002), but not when it was low (920 mg/L; P > 0.05). Similar trends were observed when oleic acid, a fatty acid that is a major component of canola oil triglycerides, was provided, but the effects were statistically significant only when the initial concentration of ferric hydroxide was also high (18 g/L; P = 0.015). Iron reduction occurred when ferric hydroxide was added to microcosms containing anaerobic digester sludge, but the extent of ferrous iron production was much less in acetate-amended microcosms than in those that were provided with canola oil or oleic acid. Methanogenesis and acetate consumption were completely inhibited when the initial acetate concentration was approximately 5000 mg/L, regardless of the initial ferric hydroxide concentration. The main effect of ferric hydroxide in this system appears to have been a result of stimulation of the rate of fatty acid oxidation.

Anaerobiosis↗
PubMed2001

A mechanistic model of whole-tract digestion and methanogenesis in the lactating dairy cow: model development, evaluation, and application.

Dietary intervention to reduce methane emissions from lactating dairy cattle is both environmentally and nutritionally desirable due to the importance of methane as a causative agent in global warming and as a significant loss of feed energy. Reliable prediction systems for methane production over a range of dietary inputs could be used to develop novel dietary regimes for the limitation of feed energy loss to methane. This investigation builds on previous attempts at modeling methanogenesis and involves the development of a dynamic mechanistic model of wholerumen function. The model incorporates modifications to certain ruminal fermentation parameters and the addition of a postruminal digestive element. Regression analysis showed good agreement between observed and predicted results for experimental data taken from the literature (r2 = 0.76, root mean square prediction error = 15.4%). Evaluation of model predictions for experimental observations from five calorimetry studies (67 observations) with lactating dairy cows at the Centre for Dairy Research, in Reading, U.K., shows an underprediction (2.1 MJ/d) of methane production (r2 = 0.46, root mean square prediction error = 12.4%). Application of the model to develop diets for minimizing methanogenesis indicated a need to limit the ratio of lipogenic to glucogenic VFA in the rumen and hindgut. This may be achieved by replacing soluble sugars in the concentrate with starch or substituting corn silage for grass silage. On a herd basis, the model predicted that increasing dietary energy intake per cow can minimize the annual loss of feed energy through methane production. The mechanistic model is a valuable tool for predicting methane emissions from dairy cows.

Animal Feed↗
PubMed2000

Thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor.

Sulfate reduction outcompeted methanogenesis at 65 degrees C and pH 7.5 in methanol and sulfate-fed expanded granular sludge bed reactors operated at hydraulic retention times (HRT) of 14 and 3.5 h, both under methanol-limiting and methanol-overloading conditions. After 100 and 50 days for the reactors operated at 14 and 3.5 h, respectively, sulfide production accounted for 80% of the methanol-COD consumed by the sludge. The specific methanogenic activity on methanol of the sludge from a reactor operated at HRTs of down to 3.5 h for a period of 4 months gradually decreased from 0. 83 gCOD. gVSS(-1). day(-1) at the start to a value of less than 0.05 gCOD. gVSS(-1). day(-1), showing that the relative number of methanogens decreased and eventually became very low. By contrast, the increase of the specific sulfidogenic activity of sludge from 0. 22 gCOD. gVSS(-1). day(-1) to a final value of 1.05 gCOD. gVSS(-1). day(-1) showed that sulfate reducing bacteria were enriched. Methanol degradation by a methanogenic culture obtained from a reactor by serial dilution of the sludge was inhibited in the presence of vancomycin, indicating that methanogenesis directly from methanol was not important. H(2)/CO(2) and formate, but not acetate, were degraded to methane in the presence of vancomycin. These results indicated that methanol degradation to methane occurs via the intermediates H(2)/CO(2) and formate. The high and low specific methanogenic activity of sludge on H(2)/CO(2) and formate, respectively, indicated that the former substrate probably acts as the main electron donor for the methanogens during methanol degradation. As sulfate reduction in the sludge was also strongly supported by hydrogen, competition between sulfate reducing bacteria and methanogens in the sludge seemed to be mainly for this substrate. Sulfate elimination rates of up to 15 gSO(4)(2-)/L per day were achieved in the reactors. Biomass retention limited the sulfate elimination rate.

Acetates↗
PubMed2003

Inhibition of methanogenesis by interaction of aluminium ion with co-factor, F-420, in Methanosarcina barkeri.

Methane emission was inhibited by aluminium ion in paddy fields. Addition of Al3+ (20 mM) to the culture medium containing cells of pure Methanosarcina barkeri, inhibited methanogenesis. Methanogenic co-factor, F-420, was isolated and purified from Methanosarcina barkeri MS. Spectrophotometric and spectrofluorometric analysis of interaction between co-factor, F-420, and Al3+ revealed that they formed a complex compound that might have blocked methanogenesis.

Aluminum Compounds↗