Reversal of 2-bromoethanesulfonate inhibition of methanogenesis in Methanosarcina sp.

2-Bromoethanesulfonate (BES) inhibition of methanogenesis from methanol by resting-cell suspensions or cell extracts of Methanosarcina was reversed by coenzyme M. BES inhibition of methylcoenzyme M methylreductase activity in cell-free extracts was reversed by methylcoenzyme M but not by coenzyme M. Methanol/coenzyme M methyltransferase activity was not inhibited by 10 microM BES. Inhibition of methylreductase by BES and 3-bromopropionate was competitive with methylcoenzyme M, but inhibition by 2-bromoethanol exhibited mixed kinetics. The Ki values for the inhibitors in cell-free extracts were similar to the concentrations which inhibited intact cells. BES-resistant mutants of strain 227 were apparently permeability mutants because in vitro assays showed that mutant and parent strain methylreductases were equally sensitive to BES.     

Hydrogen metabolism during methanogenesis from acetate by Methanosarcina barkeri

A tritium exchange assay and a sensitive gas chromatographic technique were used to demonstrate that hydrogenase was active and that hydrogen was produced by Methanosarcina barkeri strain MS grown on acetate. Both methane and hydrogen production rates were dependent on the concentration of acetate in the medium. H₂ was produced at 0.5–2% of the rate of CH₄ formation. Chloroform and potassium cyanide, inhibitors of methanogenesis from acetate, inhibited H₂ production but not hydrogenase activity. The addition of hydrogen gas to cell suspensions did not inhibit CH₄ or carbon dioxide production from the methyl group of acetate. H₂ production appears to be linked to several intracellular redox processes which follow the cleavage of acetate.     

Bioenergetics of methanogenesis from acetate by Methanosarcina barkeri.

Methane formation from acetate by resting cells of Methanosarcina barkeri was accompanied by an increase in the intracellular ATP content from 0.9 to 4.0 nmol/mg of protein. Correspondingly, the proton motive force increased to a steady-state level of -120 mV. The transmembrane pH gradient however, was reversed under these conditions and amounted to +20 mV. The addition of the protonophore 3,5,3',4'-tetrachlorosalicylanilide led to a drastic decrease in the proton motive force and in the intracellular ATP content and to an inhibition of methane formation. The ATPase inhibitor N,N'-dicyclohexylcarbodiimide stopped methanogenesis, and the intracellular ATP content decreased. The proton motive force decreased also under these conditions, indicating that the proton motive force could not be generated from acetate without ATP. The overall process of methane formation from acetate was dependent on the presence of sodium ions; upon addition of acetate to cell suspensions of M. barkeri, a transmembrane Na+ gradient in the range of 4:1 (Na+ out/Na+ in) was established. Possible sites of involvement of the Na+ gradient in the conversion of acetate to methane and carbon dioxide are discussed. Na+ is not involved in the CO dehydrogenase reaction.     

Growth and methanogenesis by Methanosarcina strain 227 on acetate and methanol.

Methanosarcina strain 227 exhibited exponential growth on sodium acetate in the absence of added H(2). Under these conditions, rates of methanogenesis were limited by concentrations of acetate below 0.05 M. One mole of methane was formed per mole of acetate consumed. Additional evidence from radioactive labeling studies indicated that sufficient energy for growth was obtained by the decarboxylation of acetate. Diauxic growth and sequential methanogenesis from methanol followed by acetate occurred in the presence of mixtures of methanol and acetate. Detailed studies showed that methanol-grown cells did not metabolize acetate in the presence of methanol, although acetate-grown cells did metabolize methanol and acetate simultaneously before shifting to methanol. Acetate catabolism appeared to be regulated in response to the presence of better metabolizable substrates such as methanol or H(2)-CO(2) by a mechanism resembling catabolite repression. Inhibition of methanogenesis from acetate by 2-bromoethanesulfonate, an analog of coenzyme M, was reversed by addition of coenzyme M. Labeling studies also showed that methanol may lie on the acetate pathway. These results suggested that methanogenesis from acetate, methanol, and H(2)-CO(2) may have some steps in common, as originally proposed by Barker. Studies with various inhibitors, together with molar growth yield data, suggest a role for electron transport mechanisms in energy metabolism during methanogenesis from methanol, acetate, and H(2)-CO(2).     

Effect of redox potential on methanogenesis by Methanosarcina barkeri

Concentrations of 0.5% O₂ immediately inhibited CH₄ production from methanol by Methanosarcina barkeri. Simultaneously, the redox potential of the medium increased to about +100 mV. However, the rates of CH₄ production were not significantly affected, when the redox potential of an anoxic medium was adjusted to values between -420 mV and +100 mV by addition of titanium (III) citrate, sodium dithionite, flavin adenine dinucleotide, or sodium ascorbate. When the redox potential was adjusted to values between -80 mV and +550 mV by means of mixtures of ferrocyanide and ferricyanide, CH₄ production was not inhibited until a redox potential of about +420 mV was reached. M. barkeri was able to reduce 0.5 mM ferricyanide solution at +430 mV within <30 min to a value of about +50 mV, and then to start CH₄ production. Higher ferricyanide concentrations were only partially reduced. The extent of reduction of ferricyanide was also dependent on the substrate concentration (methanol) and the density of the bacterial suspension. The results show that M. barkeri was able to generate to a certain extent by itself the redox environment which suited the production of CH₄. However, the bacteria probably have not enough reducing power to decrease the redox potential below the critical level of +50 mV, if O₂ is present at concentrations >0.005%.     

The effect of detergent on methanogenesis by Methanosarcina barkeri

Abstract both growth and methanogenesis of Methanosarcina barkeri are completely inhibited by sodium dodecylbenzene sulphonate at between 15 and 20 mg·1⁻¹. At lower concentrations growth of cultures was delayed, but no uncoupling of methanogenesis from growth was observed. Higher concentrations of detergent (50 mg·1⁻¹) produced marked alterations in the surface structures of organisms observed in scanning electron micrographs. Thus levels of a detergent common in anaerobic sewage treatment plants can inhibit methanogenesis, the terminal stage in the anaerobic digestion process.     

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.     

Nutrient control by the gas evolution in methanogenesis of methanol by Methanosarcina barkeri

A practical fed-batch culture, in which consumed amounts of methanol and other nutrients were supplied in response to a direct signal of the gas production of CH₄ and CO₂, was carried out for the cell production of methanol-utilizing Methanosarcina barkeri. In this fed-batch culture system equipped with level sensors to detect the gas production, a high cell concentration of 24.4 g/l was attained in 175-h cultivation maintaining the optimized nutrient concentrations of methanol, NH₄⁺, PO₄³⁻, Na⁺, Mg²⁺, Ca²⁺, Fe²⁺, Ni²⁺, Co²⁺ and cysteine (S source) throughout the culture.     

Mixotrophic growth of two thermophilic Methanosarcina strains, Methanosarcina thermophila TM-1 and Methanosarcina sp. SO-2P, on methanol and hydrogen/carbon dioxide

Two thermophilic strains, Methanosarcina thermophila TM-1 and Methanosarcina sp. SO-2P, were capable of mixotrophic growth on methanol and H₂/CO₂. Activated carbon was, however, found to be necessary to support good growth. Both strains used hydrogen and methanol simultaneously. When methanol was depleted, hydrogen utilization continued and methane was further produced with concurrent cell growth. UV epifluorescence microscopy revealed that aggregates of both strains exhibited a bright red fluorescence besides the usual blue-green fluorescence.     

Electron-transport-driven sodium extrusion during methanogenesis from formaldehyde and molecular hydrogen by Methanosarcina barkeri

Methanogenesis from formaldehyde or formaldehyde + H2, as carried out by Methanosarcina barkeri, was strictly dependent on sodium ions whereas methane formation from methanol + H2 or methanol + formaldehyde was Na+-independent. This indicates that the reduction of formaldehyde to the formal redox level of methanol exhibits a Na+ requirement. During methanogenesis from formaldehyde, a delta pNa in the range of -62 mV to -80 mV was generated by means of a primary, electron-transport-driven sodium pump. This could be concluded from the following results obtained on cell suspensions of M. barkeri. 1. The addition of proton conductors or inhibitors of the Na+/H+ antiporter had no effect on sodium extrusion. 2. During methanogenesis from formaldehyde + H2 a delta psi of -60 mV to -70 mV was generated even in the presence of proton conductors. 3. ATPase inhibitors, applied in the presence of proton conductors, had no effect on primary sodium extrusion or generation of a delta psi. Evidence for a Na+-translocating ATPase could not be obtained.     

Methyltetrahydromethanopterin as an intermediate in methanogenesis from acetate in Methanosarcina barkeri

Cell extracts (100,000×g) of acetate grown Methanosarcina barkeri (strain MS) catalyzed CH₄ and CO₂ formation from acetyl-CoA with specific activities of 50 nmol·min^-1·mg protein^-1. CH₄ formation was found to be dependent on tetrahydromethanopterin (H₄MPT) (apparent K _M=4 μM), coenzyme M (H-S-CoM), and 7-mercaptoheptanoylthreonine phosphate (H-S-HTP=component B) rather than on methanofuran (MFR) and coenzyme F₄₂₀ (F₄₂₀). Methyl-H₄MPT was identified as an intermediate. This compound accumulated when H-S-CoM and H-S-HTP were omitted from the assays. These and previous results indicate that methanogenesis from acetate proceeds via acetyl phosphate, acetyl-CoA, methyl-H₄MPT, and CH₃-S-CoM as intermediates. The disproportionation of formaldehyde to CO₂ and CH₄ was also studied. This reaction was shown to be dependent on H₄MPT, MFR, F₄₂₀, H-S-CoM, and H-S-HTP.     

Kinetic studies of acetate fermentation by Methanosarcina sp. MSTA-1

Summary The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp. MSTA-1. The optimum temperature for growth ranged around 55° C, and optimum pH was 6.5–7.5, giving a minimum generation time of 12.6–13.9 h (µ_max = 0.050–0.055 h^–1) and a maximum value of the specific acetate consumption rate (q _infs ^supps ) of 14–20 mmol/g cells per hour. Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature. Methanosarcina sp. MSTA-1 showed a low affinity for acetate substrate. Growth at 55° C and at constant pH 7 resulted in a K _m value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively. Offprint requests to: R. Moletta     

Involvement of a corrinoid enzyme in methanogenesis from acetate in Methanosarcina barkeri

Abstract Extracts of acetate-grown Methanosarcina barkeri strain Fusaro formed methane from acetate plus ATP and form acetyl phosphate under H₂. Coenzyme A (CoA) is stimulatory. Inhibitors of methanogenesis are cyanide, propyliodide and bromoethanesulfonic acid. In cofactor-free extracts methanogenic activity from acetate was restored by addition of ATP, CoA, coenzyme M and 7-mercaptoheptanoylthreonine phosphate.
An enzyme-bound corrinoid was found to be involved in methanogenesis from acetate.     

Inhibition of methanogenesis by ethylene and other unsaturated hydrocarbons

Abstract Ethylene (ethene) was found to inhibit methane formation in slurries from sewage sludge and sediment samples taken from freshwater and marine sources. Methane formation from sediment contents was inhibited by 50% at 0.07% ethylene concentration in the gas phase (approx. 5 μmol · 1⁻¹ in the aqueous phase) and by 94% at ≥0.05% ethylene in the gas phase (≥36 μ mol · 1⁻¹ in the aqueous phase). Sulphate reduction was not impaired. Methane formation from added acetate, hydrogen or methanol was inhibited by ≥98%, from lactate by about 90%. The inhibition was reversible, and methanogenic activity recuperated completely after ethylene removal. Cyclopentadiene and cycloheptatriene led to strong inhibition; benzene, toluene, isoprene, and 1-hexine to moderate inhibition of methanogenesis; several unsaturated linear hydrocarbons were without effect. Pure cultures of Methanospirillum hungatei, Methanothrix soehngenii , and Methanosarcina barkeri were all inhibited by 50% at 0.05–0.1% ethylene concentration in the gas phase (3.6–7.2 μmol · 1⁻¹ in the aqueous phase). Pure cultures of Acetobacterium woodii, Halobacterium halobium and Sulfolobus acidocaldarius were not significantly inhibited by either ethylene or acetylene. Ethylene is recommended as a selective inhibitor of methanogenesis for physiological and enrichment experiments with sediment and sludge samples.     

Inhibition of Methanosarcina barkeri strain 227 by cadmium

Abstract The effect of cadmium (Cd) on methane formation from methanol and/or H₂–CO₂ by Methanosarcina barkeri was examined in a defined growth medium and in a simplified buffer system containing 50 mM Tes with or without 2 mM dithiothreitol (DTT). No inhibition of methanogenesis by high concentrations of cadmium was observed in growth medium. Similarly, little inhibition of methanogenesis by whole cells in the Tes buffer system was observed in the presence of 430 μM Cd or 370 μM mercury (Hg) with 2 mM DTT. When the concentration of DTT was reduced to 0.4 mM, almost complete inhibition of methanogenesis from H₂–CO₂ and methanol by 600 μM Cd was observed. In the absence of DTT, 150 μM Cd inhibited methanogenesis from H₂–CO₂ completely and from methanol by 97%. Methanogenesis from H₂–CO₂ was more sensitive to Cd than that from methanol.     

5-Formyl-5,6,7,8-tetrahydromethanopterin is the intermediate in the process of methanogenesis in Methanosarcina barkeri.

Formylmethanofuran:tetrahydromethanopterin (H4MPT) formyltransferase and 5,10-methenyl-H4MPT cyclohydrolase purified from Methanosarcina barkeri catalyze a formyl group transfer and the hydrolysis of the methenyl function, respectively. The results from UV spectroscopy and HPLC analyses, and comparison with results obtained with the enzymes isolated from Methanobacterium thermoautotrophicum showed 5-formyl-H4MPT to be the product of the formyltransferase and cyclohydrolase reactions in M. barkeri. The findings disagree with an earlier report in which 10-formyl-H4MPT was identified as the product of the cyclohydrolase in the latter organism. In addition, it was observed that 10-formyl-H4MPT, which is non-enzymically formed from 5,10-methenyl-H4MPT at alkaline pH, becomes rapidly converted into the 5-formyl derivative. The latter finding explains why the nature of the formyl species previously had been improperly assigned.     

Methanogenesis from Choline by a Coculture of Desulfovibrio sp. and Methanosarcina barkeri

A sulfate-reducing vibrio was isolated from a methanogenic enrichment with choline as the sole added organic substrate. This organism was identified as a member of the genus Desulfovibrio and was designated Desulfovibrio strain G1. In a defined medium devoid of sulfate, a pure culture of Desulfovibrio strain G1 fermented choline to trimethylamine, acetate, and ethanol. In the presence of sulfate, more acetate and less ethanol were formed from choline than in the absence of sulfate. When grown in a medium containing sulfate, a coculture of Desulfovibrio strain G1 and Methanosarcina barkeri strain Fusaro degraded choline almost completely to methane, ammonia, and hydrogen sulfide and presumably to carbon dioxide. Methanogenesis occurred in two distinct phases separated by a lag of about 6 days. During the first phase of methanogenesis choline was completely converted to trimethylamine, acetate, hydrogen sulfide, and traces of ethanol by the desulfovibrio. M. barkeri fermented trimethylamine to methane, ammonia, and presumably carbon dioxide via dimethyl- and methylamine as intermediates. Simultaneously, about 60% of the acetate expected was metabolized. In the second phase of methanogenesis, the residual acetate was almost completely catabolized.