Methanogenic pathways in Methanosphaera stadtmanae

Methanosphaera stadtmanae reduces methanol to CH4 in a similar way as Methanosarcina barkeri. Low activities of 5,10-methylenetetrahydromethanopterin dehydrogenase (MTDH) and reductase (MTR) were found. From studies on formaldehyde oxidation and reduction it was concluded that most likely the inability to reduce CO2 to CH4 was due to the lack of an active or the presence of an inactive CO2 reductase system and methyltetrahydromethanopterin (methyl-H4MPT): coenzyme M methyltransferase. Methanofuran was not detected, while the presence of a pterin, analogous to H4MPT, could be substantiated from its degradation products in boiled extracts.     

One-carbon metabolism in methanogenic bacteria: analysis of short-term fixation products of 14CO2 and 14CH3OH incorporated into whole cells.

Methanobacterium thermoautotrophicum, M. ruminantium, and Methanosarcina barkeri were labeled with 14CO2 (14CO2 + H14CO3- + 14CO32-) for from 2 to 45 s. Radioactivity was recovered in coenzyme M derivatives, alanine, aspartate, glutamate, and several unidentified compounds. The properties of one important structurally unidentified intermediate (yellow fluorescent compound) displayed UV absorbance maxima at pH 1 of 290 and 335 nm, no absorbance in the visible region, and a fluorescence maximum at 460 nm. Label did not appear in organic phosphates until after 1 min. 14CH3OH was converted by M. barkeri primarily into coenzyme M derivatives at 25 s. [2-14C]acetate was assimilated by M. thermoautotrophicum mainly into alanine and succinate during 2 to 240 s, but not into coenzyme M derivatives or yellow fluorescent compound. Cell-free extracts of M. thermoautotrophicum lacked ribulose 1,5-bisphosphate carboxylase activity. The data indicated the absence of the Calvin, serine, and hexulose phosphate paths of C1 assimilation in the methanogens examined and indicated that pyruvate was an early intermediate product of net CO2 fixation. The in vivo importance of coenzyme M derivatives in methanogenesis was demonstrated.     

Sodium ions and an energized membrane required by Methanosarcina barkeri for the oxidation of methanol to the level of formaldehyde.

Methanogenesis from methanol by cell suspensions of Methanosarcina barkeri was inhibited by the uncoupler tetrachlorosalicylanilide. This inhibition was reversed by the addition of formaldehyde. 14C labeling experiments revealed that methanol served exclusively as the electron acceptor, whereas formaldehyde was mainly oxidized to CO2 under these conditions. These data support the hypothesis (M. Blaut and G. Gottschalk, Eur. J. Biochem. 141: 217-222, 1984) that the first step in methanol oxidation depends on the proton motive force or a product thereof. Cell extracts of M. barkeri converted methanol and formaldehyde to methane under an H2 atmosphere. Under an N2 atmosphere, however, formaldehyde was disproportionated to CH4 and CO2, whereas methanol was metabolized to a very small extent only, irrespective of the presence of ATP. It was concluded that cell extracts of M. barkeri are not able to oxidize methanol. In further experiments, the sodium dependence of methanogenesis and ATP formation by whole cells was investigated. Methane formation from methanol alone and the corresponding increase in the intracellular ATP content were strictly dependent on Na+. If, in contrast, methanol was utilized together with H2, methane and ATP were synthesized in the absence of Na+. The same is true for the disproportionation of formaldehyde to methane and carbon dioxide. From these experiments, it is concluded that in M. barkeri, Na+ is involved not in the process of ATP synthesis but in the first step of methanol oxidation.     

Coupling of methyl coenzyme M reduction with carbon dioxide activation in extracts of Methanobacterium thermoautotrophicum

The stimulation of carbon dioxide reduction to methane by addition of 2-(methylthio)ethanesulfonate (CH3-S-CoM) to cell extracts of Methanobacterium thermoautotrophicum was investigated. Similar stimulation of CO2 reduction by CH3-S-CoM was found for cell extracts of Methanobacterium bryantii and Methanospirillum hungatei. The CH3-S-CoM requirement could be met by the methanogenic precursors formaldehyde, serine, or pyruvate, or by 2-(ethylthio)ethanesulfonate (CH3CH2-S-CoM), but not by other coenzyme M derivatives. Efficient reduction of CO2 to CH4 was favored by low concentrations of CH3-S-CoM and high concentrations of CO2. Sulfhydryl compounds were identified as effective inhibitors of CO2 reduction. Both an allosteric model and a free-radical model for the mechanism of CO2 activation and reduction are discussed.     

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.     

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri.

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.     

N-furfurylformamide as a pseudo-substrate for formylmethanofuran converting enzymes from methanogenic bacteria

Methanofuran (4-[N-(4,5,7-tricarboxyheptanoyl-gamma-L-glutamyl)-gamma-L- glutamyl)-p-(beta-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan is a coenzyme involved in methanogenesis. The N-formyl derivative is an intermediate in the reduction of CO2 to CH4 and the disproportionation of methanol to CO2 and CH4. Formylmethanofuran dehydrogenase and formylmethanofuran:tetrahydromethanopterin formyltransferase are the enzymes catalyzing its conversions. We report here that the two enzymes from Methanosarcina barkeri and the formyltransferase from Methanobacterium thermoautotrophicum can also use N-furfurylformamide as a pseudo-substrate albeit with higher apparent Km and lower apparent Vmax values. N-Methylformamide, formamide, and formate were not converted indicating that the furfurylamine moiety of methanofuran is the minimum structure required for the correct binding of the coenzyme.     

Carbon monoxide-dependent methyl coenzyme M methylreductase in acetotrophic Methosarcina spp

Cell extracts of acetate-grown Methanosarcina strain TM-1 and Methanosarcina acetivorans both contained CH3-S-CoM methylreductase activity. The methylreductase activity was supported by CO and H2 but not by formate as electron donors. The CO-dependent activity was equivalent to the H2-dependent activity in strain TM-1 and was fivefold higher than the H2-dependent activity of M. acetivorans. When strain TM-1 was cultured on methanol, the CO-dependent activity was reduced to 5% of the activity in acetate-grown cells. Methanobacterium formicicum grown on H2-CO2 contained no CO-dependent methylreductase activity. The CO-dependent methylreductase of strain TM-1 had a pH optimum of 5.5 and a temperature optimum of 60 degrees C. The activity was stimulated by the addition of MgCl2 and ATP. Both acetate-grown strain TM-1 and acetate-grown M. acetivorans contained CO dehydrogenase activities of 9.1 and 3.8 U/mg, respectively, when assayed with methyl viologen. The CO dehydrogenase of acetate-grown cells rapidly reduced FMN and FAD, but coenzyme F420 and NADP+ were poor electron acceptors. No formate dehydrogenase was detected in either organism when grown on acetate. The results suggest that a CO-dependent CH3-S-CoM methylreductase system is involved in the pathway of the conversion of acetate to methane and that free formate is not an intermediate in the pathway.     

Involvement of methyltransferase-activating protein and methyltransferase 2 isoenzyme II in methylamine:coenzyme M methyltransferase reactions in Methanosarcina barkeri Fusaro.

The enzyme systems involved in the methyl group transfer from methanol and from tri- and dimethylamine to 2-mercaptoethanesulfonic acid (coenzyme M) were resolved from cell extracts of Methanosarcina barkeri Fusaro grown on methanol and trimethylamine, respectively. Resolution was accomplished by ammonium sulfate fractionation, anion-exchange chromatography, and fast protein liquid chromatography. The methyl group transfer reactions from tri- and dimethylamine, as well as the monomethylamine:coenzyme M methyltransferase reaction, were strictly dependent on catalytic amounts of ATP and on a protein present in the 65% ammonium sulfate supernatant. The latter could be replaced by methyltransferase-activating protein isolated from methanol-grown cells of the organism. In addition, the tri- and dimethylamine:coenzyme M methyltransferase reactions required the presence of a methylcobalamin:coenzyme M methyltransferase (MT2), which is different from the analogous enzyme from methanol-grown M. barkeri. In this work, it is shown that the various methylamine:coenzyme M methyltransfer steps proceed in a fashion which is mechanistically similar to the methanol:coenzyme M methyl transfer, yet with the participation of specific corrinoid enzymes and a specific MT2 isoenzyme.     

Evidence that the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate is a product of the methylreductase reaction in Methanobacterium

When 7-mercaptoheptanoylthreonine phosphate (HS-HTP) was used as the sole source of electrons for reductive demethylation of 2-(methylthio)-ethanesulfonic acid (CH3-S-CoM) by cell extracts of Methanobacterium thermoautotrophicum strain delta H, the heterodisulfide of coenzyme M and HS-HTP (CoM-S-S-HTP) was quantitatively produced: HS-HTP + CH3-S-CoM----CH4 + CoM-S-S-HTP. CH4 and CoM-S-S-HTP were produced stoichiometrically in a ratio of 1:1. Coenzyme M (HS-CoM) inhibited HS-HTP driven methanogenesis indicating that CH3-S-CoM rather than HS-CoM was the substrate for CoM-S-S-HTP formation.     

Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.

To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation.     

Reductive activation of the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H.

When titanium(III) citrate was used as electron donor for the reduction of methyl coenzyme M by the methyl coenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H, component A1 was no longer required. The simpler system thus obtained required components A2, A3, and C as well as catalytic amounts of ATP, vitamin B12, and the disulfide of 7-mercaptoheptanoylthreonine phosphate in addition to titanium(III) citrate. This three component enzyme system also could produce CH4 when stoichiometric amounts of 7-mercaptoheptanoylthreonine phosphate were used as a source of electrons under an H2 atmosphere. When 7-mercaptoheptanoylthreonine phosphate or H2 was used alone no CH4 was produced, indicating a dual requirement for reducing equivalents: one to activate the methylreductase system and the other to reduce methyl coenzyme M. This is the first evidence that the activation of methyl coenzyme M methylreductase is a reductive process.     

Identification of methyl coenzyme M as an intermediate in methanogenesis from acetate in Methanosarcina spp

The transfer of the methyl group of acetate to coenzyme M (2-mercaptoethanesulfonic acid; HS-CoM) during the metabolism of acetate to methane was investigated in cultures of Methanosarcina strain TM-1. The organism metabolized CD3COO- to 83% CD3H and 17% CD2H2 and produced no CDH3 or CH4. The isotopic composition of coenzyme M in cells grown on CD3COO- was analyzed with a novel gas chromatography-mass spectrometry technique. The cells contained CD3-D-CoM and CD2H-S-CoM) in a proportion similar to that of CD3H to CD2H2. These results, in conjunction with a report (J.K. Nelson and J.G. Ferry, J. Bacteriol. 160:526-532, 1984) that extracts of acetate-grown strain TM-1 contain high levels of CH3-S-CoM methylreductase, indicate that CH3-S-CoM is an intermediate in the metabolism of acetate to methane in this organism.     

Genetic analysis of mch mutants in two Methanosarcina species demonstrates multiple roles for the methanopterin-dependent C-1 oxidation/reduction pathway and differences in H(2) metabolism between closely related species

A mutation in the mch gene, encoding the enzyme 5,10-methenyl tetrahydromethanopterin (H(4)MPT) cyclohydrolase, was constructed in vitro and recombined onto the chromosome of the methanogenic archaeon Methanosarcina barkeri. The resulting mutant does not grow in media using H(2)/CO(2), methanol, or acetate as carbon and energy sources, but does grow in media with methanol/H(2)/CO(2), demonstrating its ability to utilize H(2) as a source of electrons for reduction of methyl groups. Cell suspension experiments showed that methanogenesis from methanol or from H(2)/CO(2) is blocked in the mutant, explaining the lack of growth on these substrates. The corresponding mutation in Methanosarcina acetivorans C2A, which cannot grow on H(2)/CO(2), could not be made in wild-type strains, but could be made in strains carrying a second copy of mch, suggesting that M. acetivorans is incapable of methyl group reduction using H(2). M. acetivorans mch mutants could also be constructed in strains carrying the M. barkeri ech hydrogenase operon, suggesting that the block in the methyl reduction pathway is at the level of H(2) oxidation. Interestingly, the ech-dependent methyl reduction pathway of M. acetivorans involves an electron transport chain distinct from that used by M. barkeri, because M. barkeri ech mutants remain capable of H(2)-dependent methyl reduction.     

Acetate catabolism by Methanosarcina barkeri: evidence for involvement of carbon monoxide dehydrogenase, methyl coenzyme M, and methylreductase.

The pathway of acetate catabolism in Methanosarcina barkeri strain MS was studied by using a recently developed assay for methanogenesis from acetate by soluble enzymes in cell extracts. Extracts incubated with [2-14C]acetate, hydrogen, and ATP formed 14CH4 and [14C]methyl coenzyme M as products. The apparent Km for acetate conversion to methane was 5 mM. In the presence of excess acetate, both the rate and duration of methane production was dependent on ATP. Acetyl phosphate replaced the cell extract methanogenic requirement for both acetate and ATP (the Km for ATP was 2 mM). Low concentrations of bromoethanesulfonic acid and cyanide, inhibitors of methylreductase and carbon monoxide dehydrogenase, respectively, greatly reduced the rate of methanogenesis. Precipitation of CO dehydrogenase in cell extracts by antibodies raised to 95% purified enzyme inhibited both CO dehydrogenase and acetate-to-methane conversion activity. The data are consistent with a model of acetate catabolism in which methylreductase, methyl coenzyme M, CO dehydrogenase, and acetate-activating enzymes are components. These results are discussed in relation to acetate uptake and rate-limiting transformation mechanisms in methane formation.     

Characterization of a CO: heterodisulfide oxidoreductase system from acetate-grown Methanosarcina thermophila.

During the methanogenic fermentation of acetate by Methanosarcina thermophila, the CO dehydrogenase complex cleaves acetyl coenzyme A and oxidizes the carbonyl group (or CO) to CO2, followed by electron transfer to coenzyme M (CoM)-S-S-coenzyme B (CoB) and reduction of this heterodisulfide to HS-CoM and HS-CoB (A. P. Clements, R. H. White, and J. G. Ferry, Arch. Microbiol. 159:296-300, 1993). The majority of heterodisulfide reductase activity was present in the soluble protein fraction after French pressure cell lysis. A CO:CoM-S-S-CoB oxidoreductase system from acetate-grown cells was reconstituted with purified CO dehydrogenase enzyme complex, ferredoxin, membranes, and partially purified heterodisulfide reductase. Coenzyme F420 (F420) was not required, and CO:F420 oxidoreductase activity was not detected in cell extracts. The membranes contained cytochrome b that was reduced with CO and oxidized with CoM-S-S-CoB. The results suggest that a novel CoM-S-S-CoB reducing system operates during acetate conversion to CH4 and CO2. In this system, ferredoxin transfers electrons from the CO dehydrogenase complex to membrane-bound electron carriers, including cytochrome b, that are required for electron transfer to the heterodisulfide reductase. The cytochrome b was purified from solubilized membrane proteins in a complex with six other polypeptides. The cytochrome was not reduced when the complex was incubated with H2 or CO, and H2 uptake hydrogenase activity was not detected; however, the addition of CO dehydrogenase enzyme complex and ferredoxin enabled the CO-dependent reduction of cytochrome b.     

Characterization of the MCRred2 form of methyl-coenzyme M reductase: a pulse EPR and ENDOR study

Methyl-coenzyme M reductase (MCR), which catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (H-S-CoB) to CH(4) and CoM-S-S-CoB, contains the nickel porphinoid F430 as prosthetic group. The active enzyme exhibits the Ni(I)-derived axial EPR signal MCR(red1) both in the absence and presence of the substrates. When the enzyme is competitively inhibited by coenzyme M (HS-CoM) the MCR(red1) signal is partially converted into the rhombic EPR signal MCR(red2). To obtain deeper insight into the geometric and electronic structure of the red2 form, pulse EPR and ENDOR spectroscopy at X- and Q-band microwave frequencies was used. Hyperfine interactions of the four pyrrole nitrogens were determined from ENDOR and HYSCORE data, which revealed two sets of nitrogens with hyperfine couplings differing by about a factor of two. In addition, ENDOR data enabled observation of two nearly isotropic (1)H hyperfine interactions. Both the nitrogen and proton data indicate that the substrate analogue coenzyme M is axially coordinated to Ni(I) in the MCR(red2) state.     

Anaerobic metabolism of immediate methane precursors in Lake Mendota.

Lake Mendota sediments and the immediate overlying water column were studied to better understand the metabolism of the methanogenic precursors H2/CO2 and acetate in nature. The pool size of acetate (3.5 microns M) was very small, and the acetate turnover time (0.22h) was very rapid. The dissolved inorganic carbon pool was shown to be large (6.4 to 8.3 mM), and the turnover time was slow (111 H.). CO2 was shown to account for 41 +/- 5.5% of the methane produced in sediment. Acetate and H2/CO2 were simultaneously converted to CH4. The addition of H2 to sediments resulted in an increase specific activity of CH4 from H(14)CO3- and a decrease in specific activity of CH4 from [2-14C]acetate. Acetate addition resulted in a decrease in specific activity of CH4 from H(14)CO3-. The metabolism of H(14)CO3- or [2-14C]acetate to 14CH4 was not inhibited by addition of acetate or H2. After greater than 99% of added [2-14C]acetate had been turned over, 42% of the label was recovered as 14CH4 20% was recovered as 14CO2 and 38% was incorporated into sediment. Inhibitor studies of [2-14C]acetate metabolism in sediments demonstrated that CHCl3 completely inhibited CH4 formation, but not CO2 production. Air and nitrate addition inhibited CH4 formation and stimulated CO2 production, whereas fluoroacetate addition totally inhibited acetate metabolism. The oxidation of [2-14C]acetate to 14CO2 was shown to decrease with time when sediment was incubated before the addition of label, suggesting depletion of low levels of an endogenous sediment electron acceptor. Acetate metabolism varied seasonally and was related to the concentration of sulfate in the lake and interstitial water. Methanogenesis occurred in the sediment and in the water immediately overlying the sediment during period of lake stratification and several centimeters below the sediment-water interface during lake turnovers. These data indicate that methanogenesis in Lake Mendota sediments was limited by "immediate" methane precursor availability (i.e., acetate and H2), by competition for these substrates by nonmethanogens, and by seasonal variations which altered sediment and water chemistry.     

Anaerobic metabolism of immediate methane precursors in Lake Mendota.

Lake Mendota sediments and the immediate overlying water column were studied to better understand the metabolism of the methanogenic precursors H2/CO2 and acetate in nature. The pool size of acetate (3.5 microns M) was very small, and the acetate turnover time (0.22h) was very rapid. The dissolved inorganic carbon pool was shown to be large (6.4 to 8.3 mM), and the turnover time was slow (111 H.). CO2 was shown to account for 41 +/- 5.5% of the methane produced in sediment. Acetate and H2/CO2 were simultaneously converted to CH4. The addition of H2 to sediments resulted in an increase specific activity of CH4 from H(14)CO3- and a decrease in specific activity of CH4 from [2-14C]acetate. Acetate addition resulted in a decrease in specific activity of CH4 from H(14)CO3-. The metabolism of H(14)CO3- or [2-14C]acetate to 14CH4 was not inhibited by addition of acetate or H2. After greater than 99% of added [2-14C]acetate had been turned over, 42% of the label was recovered as 14CH4 20% was recovered as 14CO2 and 38% was incorporated into sediment. Inhibitor studies of [2-14C]acetate metabolism in sediments demonstrated that CHCl3 completely inhibited CH4 formation, but not CO2 production. Air and nitrate addition inhibited CH4 formation and stimulated CO2 production, whereas fluoroacetate addition totally inhibited acetate metabolism. The oxidation of [2-14C]acetate to 14CO2 was shown to decrease with time when sediment was incubated before the addition of label, suggesting depletion of low levels of an endogenous sediment electron acceptor. Acetate metabolism varied seasonally and was related to the concentration of sulfate in the lake and interstitial water. Methanogenesis occurred in the sediment and in the water immediately overlying the sediment during period of lake stratification and several centimeters below the sediment-water interface during lake turnovers. These data indicate that methanogenesis in Lake Mendota sediments was limited by "immediate" methane precursor availability (i.e., acetate and H2), by competition for these substrates by nonmethanogens, and by seasonal variations which altered sediment and water chemistry.     

Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS).

Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO2 and CH4 at a specific rate of 50 nmol min-1 mg-1. When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridium pasteurianum. The apparent Km for ferredoxin from M. barkeri was determined to be 2.5 M. A ferredoxin dependence was also found for the formation of CO2, H2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl-coenzyme M with H2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.