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PubMed2003

Hydrogenotrophic methanogenesis by moderately acid-tolerant methanogens of a methane-emitting acidic peat.

The emission of methane (1.3 mmol of CH(4) m(-2) day(-1)), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH(4) (0.4 to 1.7 micro mol g [dry wt] of soil(-1) day(-1)) under anoxic conditions. At in situ pH, supplemental H(2)-CO(2), ethanol, and 1-propanol all increased CH(4) production rates while formate, acetate, propionate, and butyrate inhibited the production of CH(4); methanol had no effect. H(2)-dependent acetogenesis occurred in H(2)-CO(2)-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H(2) that were subsequently consumed. The accumulation of H(2) was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H(2); these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H(2)-utilizing methanogens. A total of 10(9) anaerobes and 10(7) hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H(2)-CO(2)-supplemented, fatty acid-enriched defined medium. CH(4) production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH(4)-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H(2) that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H(2) is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.

Anaerobiosis↗

PubMed1990

Environmental factors influencing methanogenesis in a shallow anoxic aquifer: a field and laboratory study.

The environmental factors influencing methanogenesis in a shallow anoxic aquifer were probed in a combined field and laboratory study. Field data collected over a year revealed that 'in situ' rates of methane production were depressed in winter and elevated in summer. Over the same period, ground water pH values ranged from 6.0 to 7.8 while temperatures varied from 7-22 degrees C. 'In situ' methanogenesis was severely inhibited at temperatures less than 13 degrees C or by pH values less than 7. The influence of these factors on microbial methane formation from both endogenous and exogenous substrates were tested in aquifer slurries adjusted to pH 5-9 and incubated at temperatures ranging from 5-45 degrees C. Temperature optima for methane production from endogenous substrates varied as a function of pH, but the pH optimum was 8 at all temperatures. Optimal conditions for acetoclastic methanogenesis were found at pH 8 and 35 degrees C. An analysis of variance revealed that pH, temperature, and a pH-temperature interaction are all significant variables influencing aquifer methanogenesis. In addition transient sulfate accumulations were also found to limit methane production in some areas. A comparison of field and laboratory methane production patterns suggest that pH, temperature, and sulfate accumulations are important, but not the only environmental variables influencing the mineralization of organic matter in shallow aquifers.

Analysis of Variance↗

PubMed1984

The bioenergetics of methanogenesis.

The reduction of CO2 or any other methanogenic substrate to methane serves the same function as the reduction of oxygen, nitrate or sulfate to more reduced products. These exergonic reactions are coupled to the production of usable energy generated through a charge separation and a protonmotive-force-driven ATPase. For the understanding of how methanogens derive energy from C-1 unit reduction one must study the biochemistry of the chemical reactions involved and how these are coupled to the production of a charge separation and subsequent electron transport phosphorylation. Data on methanogenesis by a variety of organisms indicates ubiquitous use of CH3-S-CoM as the final electron acceptor in the production of methane through the methyl CoM reductase and of 5-deazaflavin as a primary source of reducing equivalents. Three known enzymes serve as catalysts in the production of reduced 5-deazaflavin: hydrogenase, formate dehydrogenase and CO dehydrogenase. All three are potential candidates for proton pumps. In the organisms that must oxidize some of their substrate to obtain electrons for the reduction of another portion of the substrate to methane (e.g., those using formate, methanol or acetate), the latter two enzymes may operate in the oxidizing direction. CO2 is the most frequent substrate for methanogenesis but is the only substrate that obligately requires the presence of H2 and hydrogenase. Growth on methanol requires a B12-containing methanol-CoM methyl transferase and does not necessarily need any other methanogenic enzymes besides the methyl-CoM reductase system when hydrogenase is present. When bacteria grow on methanol alone it is not yet clear if they get their reducing equivalents from a reversal of methanogenic enzymes, thus oxidizing methyl groups to CO2. An alternative (since these and acetate-catabolizing methanogens possess cytochrome b) is electron transport and possible proton pumping via a cytochrome-containing electron transport chain. Several of the actual components of the methanogenic pathway from CO2 have been characterized. Methanofuran is apparently the first carbon-carrying cofactor in the pathway, forming carboxy-methanofuran. Formyl-FAF or formyl-methanopterin (YFC, a very rapidly labelled compound during 14C pulse labeling) has been implicated as an obligate intermediate in methanogenesis, since methanopterin or FAF is an essential component of the carbon dioxide reducing factor in dialyzed extract methanogenesis. FAF also carries the carbon at the methylene and methyl oxidation levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetates↗

PubMedDate not supplied

Shotgun proteomics of Methanococcus jannaschii and insights into methanogenesis.

Methanococcus jannaschii is an autotrophic hyperthermophilic archaeon isolated from an oceanic hydrothermal vent. Its primary pathway for energy production is methanogenesis from H2 and CO2. High-throughput Multidimensional Protein Identification Technology based on microcapillary LC/LC/ MS/MS was used to investigate the proteome of M. jannaschii and the methanogenesis pathway in cells grown in complex medium with high H2 supply. A total of 963 proteins have been unambiguously identified. The identified proteins represent approximately 54% of the whole genome of M. jannaschii. About 44% of the identified proteins are either conserved hypothetical or hypothetical proteins. We identified 83-95% of the proteins predicted to be involved in amino acid biosynthesis, cellular processes, central intermediary metabolism, energy metabolism, protein synthesis, transcription, and purine, pyridine, nucleoside, and nucleotide synthesis. Over 40% of these proteins have better than 50% sequence coverage. Approximately 90% of the predicted methanogenesis proteins were detected. In contrast, only 27-37% of predicted hypothetical proteins, proteins involved in transport and binding, and proteins with regulatory functions were identified. High peptide number, spectrum count, and sequence coverage have been used as indicators of high expression levels and are in good agreement with codon bias analysis. Predicted intein peptides were detected in MJ1043 (DNA-directed RNA polymerase, subunit A"), MJ0542 (phosphoenolpyruvate synthase), MJ0782 (transcription initiation factor IIB), and MJ1422 (putative replication factor C subunit). New peptides created by protein splicing were detected in MJ0885 (DNA dependent DNA polymerase), MJ0542, and MJ0782. The methanogenesis pathway and the enzymes involved are also discussed.

Amino Acid Sequence↗

PubMed1996

In vitro study of the age-dependent caecal fermentation pattern and methanogenesis in young rabbits.

The caecal fermentation pattern, including methanogenesis, was studied in young rabbits using in vitro batch incubations. Six conventional litters of eight rabbits each were used. At the age of 22, 25, 28, 32, 36, 42 and 56 days, an animal was slaughtered from each litter and its caecal contents were used for in vitro batch incubations at 39 degrees C/24 h. The incubated samples were analysed for volatile fatty acids (VFA), methane, hydrogen, ammonia nitrogen (NH3-N) and lactic acid (LA). The net total in vitro VFA production did not differ clearly with age, although a significant decrease was observed on day 36, reflecting the reduced zootechnical performances probably related to an infection with Clostridium spiroforme that occurred in the same period. The molar proportions of butyrate and propionate formed a change in the opposite direction with age, starting with a sudden shift from propionate to butyrate at day 25. In vitro NH3-N production was suggestive of a progressive and significant decrease with age; in vitro LA production was always low. Methane production was almost absent from fermentation until 32 days of age, after which it suddenly shifted from 1.6 to 52.0 mumol/flask/day and increased further with age. A significant litter effect on methanogenesis was observed which suggested the existence of a genetic effect. The hydrogen production was quite low and decreased significantly from day 36 with increasing methanogenesis. The calculated hydrogen recoveries showed a gradual increase from day 32 and were positively correlated (r = 0.92) with methane production. In conclusion, it would seem that in young suckling rabbits, reductive acetogenesis is a major characteristic of caecal fermentation, to be replaced gradually and partially by methanogenesis with the increasing intake of solid feed.

Aging↗

PubMedDate not supplied

Use of some novel alternative electron sinks to inhibit ruminal methanogenesis.

Several compounds were evaluated in vitro as alternative electron sinks to ruminal methanogenesis. They were incubated with ruminal fluid, buffer mixture, and finely ground alfalfa hay for 24 h, at 0, 6, 12, and 18 mM initial concentrations. The propionate enhancer oxaloacetic acid, the butyrate enhancer beta-hydroxybutyrate, and the butyrate unsaturated analog 3-butenoic acid were ineffective in decreasing methanogenesis. Nevertheless, beta-hydroxybutyrate increased apparent fermentation of the alfalfa hay substrate from 58.0 to 63.4%, and 3-butenoic acid seemed to increase it from 62.0 to 73.7%. Almost all of added oxaloacetic acid disappeared during the incubation, while only between 30.3 and 53.4% of beta-hydroxybutyrate disappeared. The butyrate enhancers acetoacetate and crotonic acid, and the butyrate unsaturated analog 2-butynoic acid, decreased methanogenesis by a maximum of 18,9 and 9%, respectively. Crotonic acid at 18 mM initial concentration seemed to increase the substrate apparent fermentation from 57.0 to 68.2%. Between 78.6 and 100% of acetoacetate disappeared during the incubation. The propionate unsaturated analog propynoic acid, and the unsaturated ester ethyl 2-butynoate, decreased methanogenesis by a maximum of 76 and 79%, respectively. Less than 5% of propynoic acid disappeared. The substrate apparent fermentation was decreased by propynoic acid from 62.0 to 57.4%, and seemed to have been decreased by ethyl 2-butynoate from 62.0 to 29.3%. More accurate measurements of the disappearance of some of the compounds studied are needed to better understand how they are metabolized and how they affect fermentation.

3-Hydroxybutyric Acid↗

PubMed1994

Competition between reductive acetogenesis and methanogenesis in the pig large-intestinal flora.

Washed bacterial suspensions obtained from the pig hindgut were incubated under 13CO2 in a buffer containing NaH13CO3 and carbohydrates. Incorporation of 13C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H2 added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H2 markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H2 significantly increased the incorporation of 13CO2 into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H2 partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO2 and H2 (represented mostly by double labelling). A highly significant interaction between BES and H2 concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.

Acetates↗

PubMed1988

Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials. An explanation for the apparent uncoupler insensitivity of ATP synthesis.

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (delta mu H+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 mumol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 mumol min-1 (mg protein)-1, the intracellular ATP content was 20 nmol/mg protein and the delta mu H+ was 200 mV (inside negative). When K+ was added to the suspensions the measured delta mu H+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of delta mu H+ adjustment, it was found that lowering delta mu H+ from 200 mV ([K+]in/[K+]out = 1000) to 100 mV ([K+]in/[K+]out = 40) had no effect on the rate of methane formation and on the intracellular ATP content. At delta mu H+ values below 100 mV ([K+]in/[K+]out less than 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when delta mu H+ was adjusted to values lower 50 mV ([K+]in/[K+]out less than 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri. Protonophoric uncouplers like 3,5,3',4'-tetrachlorosalicylanilide (TCS) or 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF 6847) were found not to dissipate delta mu H+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.

Adenosine Triphosphate↗

PubMedDate not supplied

A possible role for bile acid in the control of methanogenesis and the accumulation of hydrogen gas in the human colon.

This study investigated a possible role for primary bile acid in the control of methanogenesis in the human colon. Production of hydrogen and methane was measured in anaerobic faecal cultures derived from faeces of six 'non-methanogenic' and three methanogenic healthy humans. Using a sensitive technique for gas measurement, methane was detected in all faecal cultures, including those from 'non-methanogenic' humans. Bile acid inhibited methanogenesis in a dose-response fashion in the in vitro 'non-methanogenic' and methanogenic faecal cultures. Inhibition was significant at bile acid concentrations > 0.05%. Methanogenesis correlated with methanogen (methanogenic bacteria) numbers. If this inhibition occurs in vivo, then it would explain much of the epidemiology of non-methanogenesis in humans. From an analysis of net hydrogen production by the faecal cultures, it is inferred that bile acid inhibits other hydrogen-consuming bacteria in addition to methanogens. These in vitro data suggest a major role for bile acid in the accumulation of hydrogen gas in the colon. Possible links between bile acid induced accumulation of gas and irritable bowel syndrome are discussed.

Bacteria↗

PubMed2000

Competition between methanogenesis and quinone respiration for ecologically important substrates in anaerobic consortia.

Anaerobic consortia obtained from a wide variety of environments were tested for oxidizing several ecologically significant substrates with the humic model compound, anthraquinone-2,6-disulfonate (AQDS), as terminal electron acceptor. All the substrates, including hydrogen, acetate, propionate, methanol and lactate, were completely or partially converted to methane when bicarbonate was the only electron acceptor available. Addition of AQDS (20 mM) to the cultures prevented methanogenesis in most cases and AQDS reduction became the preferred pathway. AQDS was shown to be toxic for methanogenesis and this effect played an important role in enabling quinone-respiring bacteria to outcompete methanogens. Furthermore, AQDS respiration is thermodynamically more favorable than methanogenesis. All the consortia evaluated were capable of oxidizing hydrogen linked to the reduction of AQDS. Most inocula tested were also able to oxidize acetate and lactate in the same way. When methanol was provided as an electron donor competition between methanogenesis and acetogenesis occurred. Acetate accumulated from the latter process was responsible for quinone respiration. These results suggest that quinone-respiring bacteria are ubiquitous and that quinones in humus may significantly contribute to carbon cycling process by serving as a terminal electron acceptor for the anaerobic microbial oxidation of a wide variety of ecologically important substrates.

Journal Article↗

PubMed1977

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.

Acetates↗

PubMed1979

Isolation and Characterization of a Thermophilic Strain of Methanosarcina Unable to Use H(2)-CO(2) for Methanogenesis.

A thermophilic strain of Methanosarcina, designated Methanosarcina strain TM-1, was isolated from a laboratory-scale 55 degrees C anaerobic sludge digestor by the Hungate roll-tube technique. Penicillin and d-cycloserine, inhibitors of peptidoglycan synthesis, were used as selective agents to eliminate contaminating non-methanogens. Methanosarcina strain TM-1 had a temperature optimum for methanogenesis near 50 degrees C and grew at 55 degrees C but not at 60 degrees C. Substrates used for methanogenesis and growth by Methanosarcina strain TM-1 were acetate (12-h doubling time), methanol (7- to 10-h doubling time), methanol-acetate mixtures (5-h doubling time), methylamine, and trimethylamine. When radioactively labeled acetate was the sole methanogenic substrate added to the growth medium, it was predominantly split to methane and carbon dioxide. When methanol was also present in the medium, the metabolism of acetate shifted to its oxidation and incorporation into cell material. Electrons derived from acetate oxidation apparently were used to reduce methanol. H(2)-CO(2) was not used for growth and methanogenesis by Methanosarcina strain TM-1. When presented with both H(2)-CO(2) and methanol, Methanosarcina strain TM-1 was capable of limited hydrogen metabolism during growth on methanol, but hydrogen metabolism ceased once the methanol was depleted. Methanosarcina strain TM-1 required a growth factor (or growth factors) present in the supernatant of anaerobic digestor sludge. Growth factor requirements and the inability to use H(2)-CO(2) are characteristics not found in other described Methanosarcina strains. The high numbers of Methanosarcina-like clumps in sludges from thermophilic digestors and the fast generation times reported here for Methanosarcina TM-1 indicate that Methanosarcina may play an important role in thermophilic methanogenesis.

Journal Article↗

PubMed1980

Populations of methane-producing bacteria and in vitro methanogenesis in salt marsh and estuarine sediments.

Most probable numbers (MPNs) of methanogens in various salt marsh and estuarine sediments were determined with an anaerobic, habitat-simulating culture medium with 80% H(2) plus 20% CO(2) as substrate. Average MPNs for the short Spartina (SS) marsh sediments of Sapelo Island, Ga., were maximal at the 5- to 7-cm depth (1.2 x 10/g of dry sediment). Populations decreased to approximately 880/g of dry sediment at the 34- to 36-cm depth. There was no significant difference between summer and winter populations. In tall Spartina (TS) marsh sediments, average populations were maximal (1.2 x 10/g of dry sediment) in the upper 0- to 2-cm zone; populations from the 5- to 36-cm zones were similar (average of 9 x 10/g of dry sediment). Methanogenic populations for TS sediments of James Island Creek marsh, Charleston, S.C., were similar (average of 3 x 10/g of dry sediment) for all depths tested (0 to 22 cm), which was comparable to the trend observed for TS sediments at Sapelo Island, Ga. Sediment grab samples collected along a transect of James Island Creek and its adjacent Spartina marsh had MPNs that were approximately 20 times greater for the region of Spartina growth (average of 10/g of dry sediment) compared with the channel (approximately 5 x 10 methanogens per g of dry sediment). A similar trend was found at Pawley's Island marsh, S.C., but populations were approximately one order of magnitude lower. In vitro rates of methanogenesis with SS sediments incubated under 80% H(2)-20% CO(2) showed that the 5- to 7-cm region exhibited maximal activity (51 nmol of CH(4) g h), which was greater than rates for sediments above and below this depth. SS sediment samples (5 to 7 cm) incubated under 100% N(2) and supplemented with formate exhibited rates of methanogenesis similar to those generated by samples under 80% H(2)-20% CO(2). Replacing the N(2) atmosphere with H(2) resulted in an eightfold decrease in the rate of methanogenesis. In vitro methanogenic activity by TS salt marsh sediments, incubated under 80% H(2)-20% CO(2), was similar for all depths tested (0 to 22 cm). TS sediment samples (0 to 7 cm) supplemented with formate and incubated under 100% N(2) had greater rates of methanogenesis compared with unsupplemented samples.

Journal Article↗

PubMed1984

Methanogenesis in a Thermophilic (58 degrees C) Anaerobic Digestor: Methanothrix sp. as an Important Aceticlastic Methanogen.

Aceticlastic methanogens and other microbial groups were enumerated in a 58 degrees C laboratory-scale (3 liter) anaerobic digestor which was fed air-classified municipal refuse, a lignocellulosic waste (loading rate = 1.8 to 2.7 g of volatile solids per liter per day; retention time = 10 days). Two weeks after start-up, Methanosarcina sp. was present in high numbers (10 to 10 CFU/ml) and autofluorescent Methanosarcina-like clumps were abundant in sludge examined by using epifluorescence microscopy. After about 4 months of digestor operation, numbers of Methanosarcina sp. dropped 2 to 3 orders of magnitude and large numbers (most probable number = 10 to 10/ml) of a thermophilic aceticlastic methanogen morphologically resembing Methanothrix sp. were found. Methanothrix sp. had apparently displaced Methanosarcina sp. as the dominant aceticlastic methanogen in the digestor. During the period when Methanothrix sp. was apparently dominant, acetate concentrations varied between 0.3 and 1.5 mumol/ml during the daily feeding cycle, and acetate was the precursor of 63 to 66% of the methane produced during peak digestor methanogenesis. The apparent K(m) value obtained for methanogenesis from acetate, 0.3 mumol/ml, indicated that the aceticlastic methanogens were nearly saturated for substrate during most of the digestor cycle. CO(2)-reducing methanogens were capable of methanogenesis at rates more than 12 times greater than those usually found in the digestor. Added propionate (4.5 mumol/ml) was metabolized slowly by the digestor populations and slightly inhibited methanogenesis. Added n-butyrate, isobutyrate, or n-valerate (4.5 mumol/ml each) were broken down within 24 h. Isobutyrate was oxidized to acetate, a novel reaction possibly involving isomerization to n-butyrate. The rapid growth rate and versatile metabolism of Methanosarcina sp. make it a likely organism to be involved in start-up, whereas the low K(m) value of Methanothrix sp. for acetate may cause it to be favored in stable digestors operated with long retention times.

Journal Article↗

PubMed1984

Selective inhibition by 2-bromoethanesulfonate of methanogenesis from acetate in a thermophilic anaerobic digestor.

The effects of 2-bromoethanesulfonate, an inhibitor of methanogenesis, on metabolism in sludge from a thermophilic (58 degrees C) anaerobic digestor were studied. It was found from short-term experiments that 1 mumol of 2-bromoethanesulfonate per ml completely inhibited methanogenesis from CH(3)COO, whereas 50 mumol/ml was required for complete inhibition of CO(2) reduction. When 1 mumol of 2-bromoethanesulfonate per ml was added to actively metabolizing sludge which was then incubated for 24 h. it caused a 60% reduction in methanogenesis and a corresponding increase in acetate accumulation; at 50 mumol/ml it caused complete inhibition of methanogenesis and accumulation of acetate. H(2), and ethanol.

Journal Article↗

PubMed1986

Relationship of formate to growth and methanogenesis by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is a methanogenic archaebacterium that can use either H2 or formate as its source of electrons for reduction of CO2 to methane. Growth and suspended-whole-cell experiments show that H2 plus CO2 methanogenesis was constitutive, while formate methanogenesis required adaptation time; selenium was necessary for formate utilization. Cells grown on formate had 20 to 100 times higher methanogenesis rates on formate than cells grown on H2-CO2 and transferred into formate medium. Enzyme assays with crude extracts and with F420 or methyl viologen as the electron acceptor revealed that hydrogenase was constitutive, while formate dehydrogenase was regulated. Cells grown on formate had 10 to 70 times higher formate dehydrogenase activity than cells grown on H2-CO2 with Se present in the medium; when no Se was added to H2-CO2 cultures, even lower activities were observed. Adaptation to and growth on formate were pH dependent, with an optimal pH for both about one pH unit above that optimal for H2-CO2 (pH 5.8 to 6.5). When cells were grown on H2-CO2 in the presence of formate, formate (greater than or equal to 50 mM) inhibited both growth and methanogenesis at pH 5.8 to 6.2, but not at pH greater than 6.6. Both acetate and propionate produced similar inhibition. Formate inhibition was also observed in Methanospirillum hungatei.

Acetates↗

PubMed1988

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs.

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO(2) and either ethanol or lactate but not H(2) gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO(2) reduction, with a formate production rate and methanogenic turnover constant of 660 muM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 muM) than digestor contents, and the free flora was the primary site for formate cleavage to CO(2) and H(2) (90 muM formate per h). Inhibition of methanogenesis by CHCl(3) resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H(2) gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H(2) transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.

Journal Article↗

PubMed1980

Microbiology of methanogenesis in thermal, volcanic environments.

Microbial methanogenesis was examined in thermal waters, muds, and decomposing algal-bacterial mats associated with volcanic activity in Yellowstone National Park. Radioactive tracer studies with [(14)C]glucose, acetate, or carbonate and enrichment culture techniques demonstrated that methanogenesis occurred at temperatures near 70 degrees C but below 80 degrees C and correlated with hydrogen production from either geothermal processes or microbial fermentation. Three Methanobacterium thermoautotrophicum strains (YT1, YTA, and YTC) isolated from diverse volcanic habitats differed from the neotype sewage strain DeltaH in deoxyribonucleic acid guanosine-plus-cytosine content and immunological properties. Microbial methanogenesis was characterized in more detail at a 65 degrees C site in the Octopus Spring algal-bacterial mat ecosystem. Here methanogenesis was active, was associated with anaerobic microbial decomposition of biomass, occurred concomitantly with detectable microbial hydrogen formation, and displayed a temperature activity optimum near 65 degrees C. Enumeration studies estimated more than 10(9) chemoorganotrophic hydrolytic bacteria and 10(6) chemolithotrophic methanogenic bacteria per g (dry weight) of algal-bacterial mat. Enumeration, enrichment, and isolation studies revealed that the microbial population was predominantly rod shaped and asporogenous. A prevalent chemoorganotrophic organism in the mat that was isolated from an end dilution tube was a taxonomically undescribed gram-negative obligate anaerobe (strain HTB2), whereas a prevalent chemolithotrophic methanogen isolated from an end dilution tube was identified as M. thermoautotrophicum (strain YTB). Taxonomically recognizable obligate anaerobes that were isolated from glucose and xylose enrichment cultures included Thermoanaerobium brockii strain HTB and Clostridium thermohydrosulfuricum strain 39E. The nutritional properties, growth temperature optima, growth rates, and fermentation products of thermophilic bacterial strains 39E, HTB2, and YTB were determined.

Anaerobiosis↗