Activities of formylmethanofuran dehydrogenase,methylenetetrahydromethanopterin dehydrogenase,methylenetetrahydromethanopterin reductase,and heterodisulfide reductase in methanogenic bacteria

The activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin reductase, and heterodisulfide reductase were tested in cell extracts of 10 different methanogenic bacteria grown on H₂/CO₂ or on other methanogenic substrates. The four activities were found in all the organisms investigated: Methanobacterium thermoautotrophicum,M. wolfei, Methanobrevibacter arboriphilus, Methanosphaera stadtmanae, Methanosarcina barkeri (strains Fusaro and MS), Methanothrix soehngenii, Methanospirillum hungatei, Methanogenium organophilum, and Methanococcus voltae. Cell extracts of H₂/CO₂ grown M. barkeri and of methanol grown M. barkeri showed the same specific activities suggesting that the four enzymes are of equal importance in CO₂ reduction to methane and in methanol disproportionation to CO₂ and CH₄. In contrast, cell extracts of acetate grown M. barkeri exhibited much lower activities of formylmethanofuran dehydrogenase and methylenetetrahydromethanopterin dehydrogenase suggesting that these two enzymes are not involved in methanogenesis from acetate. In M. stadtmanae, which grows on H₂ and methanol, only heterodisulfide reductase was detected in activities sufficient to account for the in vivo methane formation rate. This finding is consistent with the view that the three other oxidoreductases are not required for methanol reduction to methane with H₂.     

Interspecific hydrogen transfer during methanol degradation by Sporomusa acidovorans and hydrogenophilic anaerobes

In the presence of active hydrogenophilic sulfate-reducing bacteria, the homoacetogenic bacterium Sporomusa acidovorans did not produce acetate during methanol degradation. H₂S and presumably CO₂ were the only end products. Since the sulfate-reducer did not degrade methnol or acetate, the sulfidogenesis from methanol was related to a complete interspecific hydrogen transfer between both species.In coculture with hydrogenophilic methanogenic bacteria (Methanobacterium formicicum, Methanospirillum hungatei), the interspecific hydrogen transfer with S. acidovorans was incomplete. Beside CH₄ and presumably CO₂, acetate was produced. The results suggested that H₂-production and H₂-consumption were involved during anaerobic methanol degradation by S. acidovorans and the hydrogenophilic anaerobes play an important role during methanol degradation by homoacetogenic bacteria in anoxic environments.     

Anaerobic degradation of benzoate to methane by a microbial consortium

A stabilized consortium of microbes which anaerobically degraded benzoate and produced CH₄ was established by inoculation of a benzoate-mineral salts medium with sewage sludge; the consortium was routinely subcultured anaerobically in this medium for 3 years. Acetate, formate, H₂ and CO₂ were identified as intermediates in the overall conversion of benzoate to CH₄ by the culture. Radioactivity was equally divided between the CH₄ and CO₂ from the degradation of uniformly ring-labeled [¹⁴C]benzoate. The methyl group of acetate was stoichiometrically converted to CH₄. Acetate, cyclohexanecarboxylate, 2-hydroxycyclohexanecarboxylate, o-hydroxybenzoic acid and pimelic acid were converted to CH₄ without a lag suggesting that benzoate was degraded by a reductive pathway. Addition of o-chlorobenzoate inhibited benzoate degradation but not acetate degradation or methane formation. Two methanogenic organisms were isolated from the mixed culture, neither organism was able to degrade benzoate, showing that the methanogenic bacteria served as terminal organisms of a metabolic food chain composed of several organisms. Removal of intermediates by the methanogenic bacteria provided thermodynamically favorable conditions for benzoate degradation.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H2 on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H₂ and CO₂ for methanogenesis, degraded lactate, with the production of acetate and CH₄. When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H₂-CO₂ and acetate for methanogenesis, lactate was stoichiometrically degraded to CH₄ and presumably to CO₂. During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH₄. Later, acetate was degraded to CH₄ and presumably to CO₂. In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H₂ was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H₂, but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH₄ from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H₂-CO₂ as the methanogenic substrate produced CH₄ from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H₂ produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Stimulation of CO2 reduction to methane by methylcoenzyme M in extracts Methanobacterium.

Addition of methyl-coenzyme M (CH₃SCH₂CH₂SO₃^?) to undialized, anaerobic, cell-extracts of $\text{Methanobacterium thermoautotrophicum}$ under an atmosphere of H₂ and CO₂ (80:20 v/v) stimulates 30-fold the rate of CO₂ reduction to methane. For each mol of CH₃SCH₂CH₂SO₃^? added 12 mol of methane is produced. This stimulation phenomenon requires magnesium ion, ATP, H₂, and CH₃SCH₂CH₂SO₃^?. Neither the reduced form of the cofactor, HSCH₂CH₂SO₃^?, nor the oxidized, disulfide form will replace the methylated coenzyme.     

Growth of Methanobacterium thermoautotrophicum on H2CO2: High CH4 productivities in continuous culture

The thermophilic methanogenic bacterium, Methanobacterium thermoautotrophicum, was grown on H₂CO₂. In continuous culture, high CH₄ productivities were obtained (288 litres litre⁻¹ day⁻¹) with 96% CH₄ in the effluent gas, i.e. the productivity was twice as high as that obtained previously by other authors, with pure or mixed cultures; the biomass was 3·6 g dry wt litre⁻¹.     

Methanogenesis from Sucrose by Defined Immobilized Consortia

A bacterial consortium capable of sucrose degradation primarily to CH₄ and CO₂ was constructed, with acetate as the key methanogenic precursor. In addition, the effect of agar immobilization on the activity of the consortium was determined. The primary fermentative organism, Escherichia coli, produced acetate, formate, H₂, and CO₂ (known substrates for methanogens), as well as ethanol and lactate, compounds that are not substrates for methanogens. Oxidation of the nonmethanogenic substrates, lactate and ethanol, to acetate was mediated by the addition of Acetobacterium woodii and Desulfovibrio vulgaris. The methanogenic stage was accomplished by the addition of the acetophilic methanogen Methanosarcina barkeri and the hydrogenophilic methanogen Methanobacterium formicicum. Results of studies with low substrate concentrations (0.05 to 0.2% [wt/vol]), a growth-limiting medium, and the five-component consortium indicated efficient conversion (40%) of sucrose carbon to CH₄. Significant decreases in yields of CH₄ and rates of CH₄ production were observed if any component of the consortium was omitted. Approximately 70% of the CH₄ generated occurred via acetate. Agar-immobilized cells of the consortium exhibited yields of CH₄ and rates of CH₄ production from sucrose similar to those of nonimmobilized cells. The rate of CH₄ production decreased by 25% when cysteine was omitted from reaction conditions and by 40% when the immobilized consortium was stored for 1 week at 4°C.     

Bioconversion of organic carbon to CH4 and CO2

The activities of populations in complex anaerobic microbial communities that perform complete bioconversion of organic matter to CH₄ and CO₂ are reviewed. Species of eubacteria produce acetate, H₂, and CO₂ from organic substrates, and methanogenic species of archaebacteria transform the acetate, H₂, and CO₂ to CH₄. The characteristics and activities of the methanogenic bacteria are described. The impact of the use of H₂ by methanogens on the fermentations that produce acetate, H₂, and CO₂ and the importance of syntrophy in complete bioconversion are discussed.     

Secondary alcohols as hydrogen donors for CO2-reduction by methanogens

Out of 22 methanogens Methanobacterium formicicum, Methanobacterium bryantii M.o.H., Methanogenium marisnigri, Methanomicrobium paynteri, Methanocorpusculum parvum and the new coccoid methanogenic isolates GKZPZ and SZSXXZ were found to grow at the expense of 2-propanol and 2-butanol + CO₂. 2-Propanol was oxidized to acetone and 2-butanol was converted to 2-butanone during CO₂-reduction to methane. Growth was poor compared to that on H₂/CO₂, and in the presence of both, 2-propanol and H₂, molecular hydrogen was the preferred reductant. Acetone, formed during oxidation of 2-propanol in the absence of hydrogen, was reduced again to 2-propanol, when the culture was supplied with H₂/CO₂. Ethanol, 1-propanol, 1-butanol, 2-pentanol and cyclohexanol could not serve as hydrogen donors for methanogenesis.     

Transient Production of Formate During Chemolithotrophic Growth of Anaerobic Microorganisms on Hydrogen

The homoacetogenic bacteria Acetobacterium woodii, A. carbinolicum, Sporomusa ovata, and Eubacterium limosum, the methanogenic archaeon Methanobacterium formicicum, and the sulfate-reducing bacterium Desulfotomaculum orientis all produced formate as an intermediate when they were growing chemolithoautotrophically with H₂ and CO₂ as sources of energy, electrons, and carbon. The sulfate-reducing bacterium Desulfovibrio vulgaris grew chemolithoheterotrophically with H₂ and CO₂ using acetate as carbon source, but also produced formate when growth was limited by sulfate. All these bacteria were also able to grow on formate as energy source. Formate accumulated transiently while H₂ was consumed. The maximum formate concentrations measured in cultures of A. woodii and A. carbinolicum were proportional to the initial H₂ partial pressure, giving a ratio of about 0.5 mM formate per 10 kPa H₂. The methanogen Methanobacterium bryantii, on the other hand, was unable to grow on formate and did not produce formate during chemolithoautotrophic growth on H₂. The results indicate that the ability to utilize formate, that is, to possess a formate dehydrogenase, was the precondition for the production of formate during chemolithotrophic growth on H₂. Received: 24 November 1998 / Accepted: 30 December 1998     

A Coupled Ecosystem-Climate Model for Predicting the Methane Concentration in the Archean Atmosphere

A simple coupled ecosystem-climate model is described that canpredict levels of atmospheric CH₄, CO₂, and H₂during the Late Archean, given observed constraints on Earth'ssurface temperature. We find that methanogenic bacteria shouldhave converted most of the available atmospheric H₂ intoCH₄, and that CH₄ may have been equal in importance to CO₂ as a greenhouse gas. Photolysis of this CH₄ may have produced a hydrocarbon smog layer that would have shielded the surface from solar UV radiation. Methanotrophic bacteria would have consumed some of the atmospheric CH₄,but they would have been incapable of reducing CH₄ to modern levels. The rise of O₂ around 2.3 Ga would have drastically reduced the atmospheric CH₄ concentrationand may thereby have triggered the Huronian glaciation.     

Purine and pyrimidine biosynthesis in methanogenic bacteria

Following long-term labeling with [1-¹³C]acetate, [2-¹³C]acetate, ¹³CO₂, H¹³COOH, or ¹³CH₃OH, NMR spectroscopy was used to determine the labeling patterns of the purified ribonucleosides of Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii. Major differences were observed among the methanogens studied, specifically at carbon positions 2 and 8 of the purines, positions at which one-carbon carriers are involved during synthesis. In Methanospirillum hungatei and Methanosarcina barkeri, the labcl at both positions came from carbon atom C-2 of acetate, as predicted from known eubacterial pathways, whereas in Methanococcus voltae and Methanobacterium bryantii both originated from CO₂. In Methanosphaera stadtmanae grown in the presence of formate, the C-2 of purines originated exclusively from formate and the C-8 was labeled by the C-2 of acetate. When grown in media devoid of formate, the C-2 of the purine ring originated mainly from the C-2 of acetate and in part from CH₃OH. In Methanobrevibacter smithii grown in the presence of formate, C-2 and C-8 of purines were derived from CO₂ and/or formate. The labeling patterns obtained for pyrimidines are consistent with the biosynthetic pathways common to eubacteria and eucaryotes.Abbreviations CODH Carbon monoxide dehydrogenase - FH₄ tetrahydrofolate - H₄MPT tetrahydromethanopterin Issued as NRCC Publication No. 37383     

Function of methanofuran,tetrahydromethanopterin, and coenzyme F420 in Archaeoglobus fulgidus

Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO₂ and H₂S. The organism contains coenzyme F₄₂₀, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F₄₂₀ oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO₂ via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C₁-intermediates otherwise only used by methanogenic bacteria.Non-standard abbreviations APS adenosine 5-phosphosulfate - BV benzyl viologen - DCPIP 2,6-dichlorophenolindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - H₄F tetrahydrofolate - H₄MPT tetrahydromethanopterin - CH₂ H₄MPT, methylene-H₄MPT - CH H₄MPT, methenyl-H₄MPT - Mes morpholinoethane sulfonic acid - MFR methanofuran - Mops morpholinopropane sulfonic acid - MV methyl viologen - Tricine N-tris(hydroxymethyl)-methylglycine - U mol product formed per min     

The pterin lumazine inhibits growth of methanogens and methane formation

The pterin compound lumazine [2, 4-(1H, 3H)-pteridinedione] inhibited the growth of several methanogenic archaea completely at a concentration of ≤ 0.6 mM and was bacteriocidal for Methanobacterium thermoautotrophicum strain Marburg. In contrast, growth of two non-methanogenic archaea, several eubacteria, and one eukaryote was not strongly affected at much higher concentrations. In washed-cell suspensions, methanogenesis from H₂ and CO₂ by Mb. thermoautotrophicum or from H₂ and methanol by Methanosarcina barkeri was inhibited by addition of lumazine. In cell-free extracts of Mb. thermoautotrophicum, H₂-driven methane production from CO₂ or CH₃-S-CoM was completely inhibited by 0.6 mM lumazine. The results suggest that the compound may be useful in probing the methanogenesis pathway or in selecting against methanogens. Received: 30 January 1996 / Accepted 15 May 1996     

Enzymes and coenzymes of the carbon monoxide dehydrogenase pathway for autotrophic CO2 fixation in Archaeoglobus lithotrophicus and the lack of carbon monoxide dehydrogenase in the heterotrophic A. profundus

Archaeoglobus lithotrophicus is a hyperthermophilic Archaeon that grows on H₂ and sulfate as energy sources and CO₂ as sole carbon source. The autotrophic sulfate reducer was shown to contain all the enzyme activities and coenzymes of the reductive carbon monoxide dehydrogenase pathway for autotrophic CO₂ fixation as operative in methanogenic Archaea. With the exception of carbon monoxide dehydrogenase these enzymes and coenzymes were also found in A. profundus. This organism grows lithotrophically on H₂ and sulfate, but differs from A. lithotrophicus in that it cannot grow autotrophically: A. profundus requires acetate and CO₂ for biosynthesis. The absence of carbon monoxide dehydrogenase in A. profundus is substantiated by the observation that this organism, in contrast to A. lithotrophicus, is not mini-methanogenic and contains only relatively low concentrations of corrinoids.Abbreviations F ₄₂₀ coenzyme F₄₂₀ - MFR methanofuran - CHO-MFR formylmethanofuran - H ₄MPT 5,6,7,8-tetrahydromethanopterin - CHO–H ₄MPT N⁵ formyl-H₄MPT - CHH₄MPT⁺N⁵ methenyl-H₄MPT - CH ₂=H₄MPT N⁵, N¹⁰ methylene-H₄MPT - CH ₃–H₄MPT N⁵ methyl-H₄MPT - H ₄F tetrahydrofolate - I U 1 mol/min - t _d doubling time     

An adenine nucleotide translocase in the procaryote Methanobacterium thermoautotrophicum

ATP synthesis, ATP hydrolysis and ADP uptake by membrane vesicles of Methanobacterium thermoautotrophicum are inhibited by inhibitors of mitochondrial $\text{ADP}\text{ATP}$ translocases. Atractyloside binds to one of the membrane proteins. These data demonstrate the presence of an eucaryotic type of $\text{ADP}\text{ATP}$ translocase in a procaryotic microorganism and stress the unique position of methanogenic bacteria in evolution.     

Valinomycin inhibited methane synthesis in Methanobacteriumthermoautotrophicum

$\text{Methanobacterium}\text{thermoautotrophicum}$ cells, incubated anaerobically under H₂ in 0.1 M KCl or 0.1 M NaCl, above pH 7.5, are interior acid with respect to the incubation medium. The pH gradient thus established can be discharged by either carbonyl cyanide m-chlorophenylhydrazone or valinomycin at high concentration (17μM). In these cells, which actively synthesize CH₄ from CO₂ and H₂, methanogenesis is strongly inhibited when the pH gradient is discharged.     

Processes involved in formation and emission of methane in rice paddies

The seasonal change of the rates of production and emission of methane were determined under in-situ conditions in an Italian rice paddy in 1985 and 1986. The contribution to total emission of CH₄ of plant-mediated transport, ebullition, and diffusion through the flooding water was quantified by cutting the plants and by trapping emerging gas bubbles with funnels. Both production and emission of CH₄ increased during the season and reached a maximum in August. However, the numbers of methanogenic bacteria did not change. As the rice plants grew and the contribution of plant-mediated CH₄ emission increased, the percentage of the produced CH₄ which was reoxidized and thus, was not emitted, also increased. At its maximum, about 300 ml CH₄ were produced per m² per hour. However, only about 6% were emitted and this was by about 96% via plant-mediated transport. Radiotracer experiments showed that CH, was produced from H₂/CO₂. (30–50%) and from acetate. The pool concentration of acetate was in the range of 6–10 mM. The turnover time of acetate was 12–16 h. Part of the acetate pool appeared to be not available for production of CH₄ or CO₂     

Methane Production from Formate by Syntrophic Association of Methanobacterium bryantii and Desulfovibrio vulgaris JJ

Coculture of a sulfate-reducing bacterium, when grown in the absence of added sulfate, with Methanobacterium bryantii, which uses only H₂ and CO₂ for methanogenesis, degraded formate to CH₄. A pure culture of Desulfovibrio vulgaris JJ was able to produce small amounts of H₂. Such a syntrophic relationship might provide an additional way to avoid formate accumulation in anaerobic environments.