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Showing papers on "Methanosarcina barkeri published in 1982"


Journal ArticleDOI
01 Mar 1982-Nature
TL;DR: This paper showed that methanol and trimethylamine are important substrates for methanogenic bacteria in salt marsh sediments and that these compounds may account for the bulk of methane produced therein.
Abstract: It has been generally believed that sulphate reduction precludes methane generation during diagenesis of anoxic sediments1,2. Because most biogenic methane formed in nature is thought to derive either from acetate cleavage or by hydrogen reduction of carbon dioxide3–6, the removal of these compounds by the energetically more efficient sulphate-reducing bacteria can impose a substrate limitation on methanogenic bacteria7–9. However, two known species of methanogens, Methanosarcina barkeri and Methanococcus mazei, can grow on and produce methane from methanol and methylated amines10–13. In addition, these compounds stimulate methane production by bacterial enrichments from the rumen11,14 and aquatic muds13,14. Methanol can enter anaerobic food webs through bacterial degradation of lignins15 or pectin16, and methylated amines can be produced either from decomposition of substances like choline, creatine and betaine13,14 or by bacterial reduction of trimethylamine oxide17, a common metabolite and excretory product of marine animals. However, the relative importance of methanol and methylated amines as precursors of methane in sediments has not been previously examined. We now report that methanol and trimethylamine are important substrates for methanogenic bacteria in salt marsh sediments and that these compounds may account for the bulk of methane produced therein. Furthermore, because these compounds do not stimulate sulphate reduction, methanogenesis and sulphate reduction can operate concurrently in sulphate-containing anoxic sediments.

327 citations


Journal ArticleDOI
TL;DR: Difference in substrate affinities can account for the inhibition of methanogenesis from acetate in sulfate-rich environments, where the acetate concentration is well below 1 mM.
Abstract: Methanosarcina barkeri and Desulfobacter postgatei are ubiquitous anaerobic bacteria which grow on acetate or acetate plus sulfate, respectively, as sole energy sources. Their apparent K s values for acetate were determined and found to be approximately 0.2 mM for the sulfate-reducing bacterium and 3 mM for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V max) the rate of acetate consumption by D. postgatei approached 15-fold the rate of M. barkeri at low acetate concentrations. The apparent inhibition of methanogenesis was of the same order as expected from the different K s value for acetate. Difference in substrate affinities can thus account for the inhibition of methanogenesis from acetate in sulfate-rich environments, where the acetate concentration is well below 1 mM.

327 citations


Journal ArticleDOI
TL;DR: Five strains of rod-shaped, Gram-negative, non-sporing, strictly anaerobic bacteria were isolated from limnic and marine mud samples with gallic acid or phloroglucinol as sole substrate, and the new isolates converted also syringic acid completely to acetate.
Abstract: Five strains of rod-shaped, Gram-negative, non-sporing, strictly anaerobic bacteria were isolated from limnic and marine mud samples with gallic acid or phloroglucinol as sole substrate. All strains grew in defined mineral media without any growth factors; marine isolates required salt concentrations higher than 1% for growth, two freshwater strains only thrived in freshwater medium. Gallic acid, pyrogallol, 2,4,6-trihydroxybenzoic acid, and phloroglucinol were the only substrates utilized and were fermented stoichiometrically to 3 mol acetate (and 1 mol CO2) per mol with a growth yield of 10g cell dry weight per mol of substrate. Neither sulfate, sulfur, nor nitrate were reduced. The DNA base ratio was 51.8% guanine plus cytosine. A marine isolate, Ma Gal 2, is described as type strain of a new genus and species, Pelobacter acidigallici gen. nov. sp. nov., in the family Bacteroidaceae. In coculture with Acetobacterium woodii, the new isolates converted also syringic acid completely to acetate. Cocultures with Methanosarcina barkeri converted the respective substrates completely to methane and carbon dioxide.

196 citations


Journal ArticleDOI
TL;DR: It is suggested that a significant intramolecular redox pathway is possible for the generation of CH(4) from acetate, that energy metabolism from acetates by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity.
Abstract: We examined the unitrophic metabolism of acetate and methanol individually and the mixotrophic utilization of these compounds by using detailed 14C-labeled tracer studies in a strain of Methanosarcina barkeri adapted to grow on acetate as the sole carbon and energy source. The substrate consumption rate and methane production rate were significantly lower on acetate alone than during the unitrophic or mixotrophic metabolism of methanol. Cell yields (in grams per mole of substrate) were identical during exponential growth on acetate and exponential growth on methanol. During unitrophic metabolism of acetate, the methyl moiety accounted for the majority of the CH4 produced, but 14% of the CO2 generated originated from the methyl moiety. This correlated with the concurrent reduction of equivalent amounts of the C-1 of acetate to CH4. 14CH4 was also produced from added 14CO2, although to a lesser extent than from reduction of the C-1 of acetate. During mixotrophic metabolism, methanol and acetate were catabolized simultaneously. The rates of 14CH4 and 14CO2 generation from [2-14C]acetate were logarithmic and higher in mixotrophic than in unitrophic cultures at substrate concentrations of 50 mM. A comparison of the oxidoreductase activities in cell extracts of the acetate-adapted strain grown on acetate and of strain MS grown on methanol or on H2 plus CO2 indicated that the pyruvate, α-ketoglutarate, and isocitrate dehydrogenase activities remained constant, whereas the CO dehydrogenase activity was significantly higher (5,000 nmol/min per mg of protein) in the acetate-adapted strain. These results suggested that a significant intramolecular redox pathway is possible for the generation of CH4 from acetate, that energy metabolism from acetate by M. barkeri is not catabolite repressed by methanol, and that the acetate-adapted strain is a metabolic mutant with derepressed CO dehydrogenase activity.

123 citations


Journal ArticleDOI
TL;DR: The effect of methanogens is interpreted as a shift in the flow of electrons away from the formation of electron sink products lactate and ethanol to methane via hydrogen, favoring an increase in acetate, which is in turn converted to methane and carbon dioxide by M. barkeri.
Abstract: The fermentation of cellulose by a rumen anaerobic fungus in the presence of Methanobrevibacter sp. strain RA1 and Methanosarcina barkeri strain 227 resulted in the formation of 2 mol each of methane and carbon dioxide per mol of hexose fermented. Coculture of the fungus with either Methanobrevibacter sp. or M. barkeri produced 0.6 and 1.3 mol of methane per mol of hexose, respectively. Acetate, formate, ethanol, hydrogen, and lactate, which are major end products of cellulose fermentation by the fungus alone, were either absent or present in very low quantities at the end of the triculture fermentation (

110 citations


Journal ArticleDOI
TL;DR: One-carbon metabolic transformations associated with cell carbon synthesis and methanogenesis were analyzed by long- and short-term ( 14)CH(3)OH or (14)CO(2) incorporation studies during growth and by cell suspensions and it was not possible to distinguish between acetate and acetyl coenzyme A as the immediate product of two-carbon synthesis with the methods employed.
Abstract: One-carbon metabolic transformations associated with cell carbon synthesis and methanogenesis were analyzed by long- and short-term 14CH3OH or 14CO2 incorporation studies during growth and by cell suspensions. 14CH3OH and 14CO2 were equivalently incorporated into the major cellular components (i.e., lipids, proteins, and nucleic acids) during growth on H2-CO2-methanol. 14CH3OH was selectively incorporated into the C-3 of alanine with decreased amounts fixed in the C-1 and C-2 positions, whereas 14CO2 was selectively incorporated into the C1 moiety with decreasing amounts assimilated into the C-2 and C-3 atoms. Notably, 14CH4 and [3-14C]alanine synthesized from 14CH3OH during growth shared a common specific activity distinct from that of CO2 or methanol. Cell suspensions synthesized acetate and alanine from 14CO2. The addition of iodopropane inhibited acetate synthesis but did not decrease the amount of 14CH3OH or 14CO2 fixed into one-carbon carriers (i.e., methyl coenzyme M or carboxydihydromethanopterin). Carboxydihydromethanopterin was only labeled from 14CH3OH in the absence of hydrogen. Cell extracts catalyzed the synthesis of acetate from 14CO (∼1 nmol/min per mg of protein) and an isotopic exchange between CO2 or CO and the C-1 of pyruvate. Acetate synthesis from 14CO was stimulated by methyl B12 but not by methyl tetrahydrofolate or methyl coenzyme M. Methyl coenzyme M and coenzyme M were inhibitory to acetate synthesis. Cell extracts contained high levels of phosphotransacetylase (>6 μmol/min per mg of protein) and acetate kinase (>0.14 μmol/min per mg of protein). It was not possible to distinguish between acetate and acetyl coenzyme A as the immediate product of two-carbon synthesis with the methods employed.

92 citations


Journal ArticleDOI
TL;DR: The numbers of pectinolytic anaerobes varied seasonally in both sediments, and were highest during the fall after sedimentation of algal blooms and/or leaf detritus, and the pH remaining neutral.
Abstract: SUMMARY: Anaerobic digestion of pectin by bacteria was examined in two freshwater lakes in Wisconsin and in defined laboratory cultures of species prevalent in the lake sediment. The turnover times for pectin biodegradation to methane in sediments incubated at in situ temperature were much longer (100 h in Lake Mendota and 185 h in Knaack Lake) than either that observed for glucose (12 h in Lake Mendota) or previously reported for acetate (0·22 h in Lake Mendota). The numbers of pectinolytic anaerobes varied seasonally in both sediments (102--105 and 103--105 ml-1 in Knaack Lake and Lake Mendota, respectively), and were highest during the fall after sedimentation of algal blooms and/or leaf detritus. Clostridium butyricum was identified as a prevalent pectinolytic anaerobe in both lakes. In mono-culture pectin fermentations, C. butyricum produced methanol, H2/CO2, acetate, ethanol and butyrate; growth stopped in the presence of excess energy source when the pH fell to 4·3. In co-culture pectin fermentations of C. butyricum/Methanosarcina barkeri, H2/CO2, methanol and acetate were detected as intermediary metabolites, and pectin was completely degraded to CH4 and CO2, the pH remaining neutral. 14C-radiotracer analysis substantiated the simultaneous conversion of H2/CO2, methanol and acetate to CH4 by M. barkeri as these metabolites were generated from pectin hydrolysis by C. butyricum.

83 citations


Journal ArticleDOI
TL;DR: In this paper, 5-hydroxybenzimidazolylcobamide is the only base detected in cobamide compounds from methanol-grown Methanosarcina barkeri, accounting for about 83 and 90% of the total corrinoids of whole cells and cell-free extracts, respectively.

77 citations


Journal ArticleDOI
TL;DR: The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture.
Abstract: The mechanism of ammonia assimilation in Methanosarcina barkeri and Methanobacterium thermoautotrophicum was documented by analysis of enzyme activities, 13NH3 incorporation studies, and comparison of growth and enzyme activity levels in continuous culture. Glutamate accounted for 65 and 52% of the total amino acids in the soluble pools of M. barkeri and M. thermoautotrophicum. Both organisms contained significant activities of glutamine synthetase, glutamate synthase, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. Hydrogen-reduced deazaflavin-factor 420 or flavin mononucleotide but not NAD, NADP, or ferredoxin was used as the electron donor for glutamate synthase in M. barkeri. Glutamate dehydrogenase activity was not detected in either organism, but alanine dehydrogenase activity was present in M. thermoautotrophicum. The in vivo activity of the glutamine synthetase was verified in M. thermoautotrophicum by analysis of 13NH3 incorporation into glutamine, glutamate, and alanine. Alanine dehydrogenase and glutamine synthetase activity varied in response to [NH4+] when M. thermoautotrophicum was cultured in a chemostat with cysteine as the sulfur source. Alanine dehydrogenase activity and growth yield (grams of cells/mole of methane) were highest when the organism was cultured with excess ammonia, whereas growth yield was lower and glutamine synthetase was maximal when ammonia was limiting.

74 citations


Journal ArticleDOI
TL;DR: The results support those of in vivo studies and provide conclusive evidence that archaebacterial Ribosomes despite being 70S ribosomes lack binding sites for many classical eubacterial ribosome inhibitors, but at the same time they possess sites for others, as well as for some inhibitors of 80Sribosomes.
Abstract: An in vitro polypeptide synthesis system was set up for three methanogenic bacteria, Methanococcus vannielii, Methanobacterium formicicum and Methanosarcina barkeri, and the effect of classical 70S and 80S protein synthesis inhibitors studied. The following results were obtained: (i) The activity of ribosomes from all three methanogens was unaffected by a number of 70S inhibitors such as tetracycline, chloramphenicol, streptomycin, tiamulin and, probably, erythromycin as well; (ii) However, the ribosomes were sensitive to thiostrepton, virginiamycin and, to varying degrees, to those aminoglycosides containing a 2-deoxystreptamine moiety. Among the aminoglycosides examined, streptomycin induced no translational misreading. The compounds containing 2-deoxystreptamine stimulated misreading, albeit only at high concentrations (neomycin being an exception); (iii) Ribosomes from all three organisms were insensitive to the 80S inhibitors cycloheximide and ricin, but those from Methanobacterium formicicum were highly sensitive to anisomycin and moderately sensitive to verrucarin. The results support those of in vivo studies and provide conclusive evidence that archaebacterial ribosomes despite being 70S ribosomes lack binding sites for many classical eubacterial ribosome inhibitors. At the same time they possess sites for others, as well as for some inhibitors of 80S ribosomes.

66 citations


Journal ArticleDOI
TL;DR: The complete amino acid sequence for a 3Fe:3S ferredoxin from the "archaebacterium" Methanosarcina barkeri (DSM 800) was determined by repetitive Edman degradation on the whole protein and peptides derived from trypsin, thermolysin, and Staphylococcus aureus protease digestion.

Journal ArticleDOI
TL;DR: The combined EPR and Mössbauer studies show that M. barkeri ferredoxin contains only [3Fe-3S] clusters, similar to Azotobacter vinelandii Fd and mitochondrial beef heart aconitase.
Abstract: Methanosarcina barkeri ferredoxin was purified and characterized by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy. The purification procedure included chromatographic steps on DEAE-cellulose and gel filtration. The isolated protein is unstable under aerobic conditions. The ferredoxin exhibits charge transfer bands at 283 nm and 405 nm with an absorption ratio A405/A283= 0.73. Its molecular weight has been estimated to be 20000–22000 by gel filtration chromatography. The native ferredoxin exhibits an intense EPR signal at g= 2.02 and only a very weak g= 1.94 signal develops upon reduction with dithionite. The Mossbauer spectra of the reduced protein are characteristic of a [3 Fe-3S] center. The combined EPR and Mossbauer studies show that M. barkeri ferredoxin contains only [3 Fe-3S] clusters, similar to Azotobacter vinelandii Fd [Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-Johnson, W. H. & Munck, M. (1980) J. Biol. Chem. 255, 1793–1796], Desulfovibrio gigas FdII [Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V. & Munck, E. (1980) J. Biol. Chem. 255, 3242–3244] and mitochondrial beef heart aconitase [Kent, T. A., Dreyer, J.-L., Kennedy, M. C., Huynh, B. H., Emptage, M. H., Beinert, H. & Munck, E. (1982) Proc. Natl Acad. Sci. USA, 79, 1096–1100].

Journal ArticleDOI
TL;DR: In this paper, the growth of Methanosarcina barkeri strain Fusaro on a mixture of trimethylamine and acetate was investigated and it was shown that only 2.5% of the methane produced in the first phase originated from acetate.
Abstract: During growth of Methanosarcina barkeri strain Fusaro on a mixture of trimethylamine and acetate, methane production and acetate consumption were biphasic. In the first phase trimethylamine (33 mmol x l-1) was depleted and some acetate (11–14 from 50 mmol x l-1) was metabolized simultaneously. In the second phase the remaining acetate was cleaved stoichiometrically into CH4 and CO2. Kinetic experiments with (2-14C)acetate revealed that only 2.5% of the methane produced in the first phase originated from acetate: 18% of the acetate metabolized was cleaved into CH4 and CO2, 23% of the acetate was oxidized, and 55% was assimilated. Methane produced from CD3−COOH in the first phase consisted of CD2H2 and CD3H in a ratio of 1:1.

Journal ArticleDOI
Thauer Rk1
TL;DR: It is concluded that acetate oxidation with sulphate in D. postgatei proceeds via the citric acid cycle with the synthesis of pyruvate from acetyl CoA and CO2 as an anaplerotic reaction.
Abstract: Acetate oxidation by sulphate was studied with Desulfobacter postgatei. Cell extracts of the organism were found to contain high activities of the following enzymes: citrate synthase, aconitase, isocitrate dehydrogenase, $\alpha$ -ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase and pyruvate synthase. It is concluded that acetate oxidation with sulphate in D. postgatei proceeds via the citric acid cycle with the synthesis of pyruvate from acetyl CoA and CO $\_2$ as an anaplerotic reaction. The apparent K $\_S$ for acetate oxidation by D. postgatei as determined in vivo was near 0.2 mM. The apparent K $\_S$ for acetate fermentation to methane and CO $\_2$ by Methanosarcina barkeri was 3 mM. The significantly lower K $_S$ for acetate of the sulphate reducer explains why methane formation from acetate in natural habitats is apparently inhibited by sulphate.

Journal ArticleDOI
TL;DR: A ferredoxin isolated from the methanogenic organism Methanosarcina barkeri functions as an electron carrier in the pyruvate dehydrogenase system and its possible role in a variety of electron transfer reactions is discussed.

Journal ArticleDOI
TL;DR: A newly isolated methanogenic archaebacterium, Methanothrix soehngenii (“acetate organism”) was characterized by oligonucleotide cataloguing of its 16S ribosomal RNA, using an improved method for sequenzing ribonuclease T1 resistant oligon nucleotides.

Journal ArticleDOI
16 Apr 1982-Science
TL;DR: Findings indicate that a B12-dependent system is operative in the biological formation of methane in addition to other systems that are B 12-independent.
Abstract: When Methanosarcina barkeri is grown on methanol as the sole carbon source, a B12-containing protein is synthesized by this organism. This B12 protein contains bound aquocobalamin, and when this cofactor is reduced and methylated with [14C]methyl iodide, the resultant [14C]methyl B12 protein is extremely active in the biosynthesis of 14C-labeled methane. These findings indicate that a B12-dependent system is operative in the biological formation of methane in addition to other systems that are B12-independent.

Journal ArticleDOI
TL;DR: A protein which catalyses the 6-electron reduction step from sulfite to sulfide was isolated from the methanogenic bacterium, Methanosarcina barkeri and its spectral characteristics are similar to the siroheme containing sulfite reductases.

Journal ArticleDOI
TL;DR: Results indicate that methylcobalamin-HS-CoM methyl-transferase is not involved in the biosynthesis of CH3-S- CoM or CH4 from CH3OH by M. barkeri.
Abstract: The participation of corrinoids in the biosynthesis of CH3-S-CoM and CH4 from CH3OH by extracts of heterotrophically and autotrophically grown cells of Methanosarcina barkeri was investigated. Alkyl-B12 derivatives severely inhibited the transfer of the Co-methyl moiety of CH3-B12 to HS-CoM, but hardly affected the formation of CH3-S-CoM and CH4 from CH3OH. 1-Iodoalkanes completely suppressed methanogenesis from CH3OH, but only slightly inhibited the biosynthesis of CH3-S-CoM. Additionally, [methyl-3H]methylcobalamin could not be detected in cell-free extracts following biosynthesis of [methyl-3H]CH3-S-CoM from HS-CoM and excess [methyl-3H]CH3OH. These results indicate that methylcobalamin-HS-CoM methyl-transferase is not involved in the biosynthesis of CH3-S-CoM or CH4 from CH3OH by M. barkeri.

Journal ArticleDOI
TL;DR: Cell-free extracts incubated with Mg-ATP, and then exhaustively dialysed, exhibit substantial methyltransferase activity in the absence of added ATP, which is inhibited by the addition of Mg2+ to the reaction mixture.
Abstract: The methanol-HS-CoM methyltransferase system in extracts of autotrophically cultivated Methanosarcina barkeri undergoes a time-dependent (hysteretic) activation by ATP. Cell-free extracts incubated with Mg-ATP, and then exhaustively dialysed, exhibit substantial methyltransferase activity in the absence of added ATP, which is inhibited by the addition of Mg2+ to the reaction mixture.

Journal ArticleDOI
TL;DR: 57Fe-Mössbauer Spectroscopy of methanol grown cells oxidized in the presence of air demonstrated only the state of Fe3+ whereas active cells under reducing conditions offered spectra of two additional iron sites in the Fe2+ state.
Abstract: Abstract Cells of the archaebacterium Methanosarcina barkeri were grown strictly anaerobic in defined media. 57Fe-Mössbauer Spectroscopy of methanol grown cells oxidized in the presence of air demonstrated only the state of Fe3+ whereas active cells under reducing conditions offered spectra of two additional iron sites in the Fe2+ state. Furthermore the 57Fe hyperfine interaction data at different temperatures gave evidence that cells cultivated with methanol or acetate as substrate contained ferredoxin like compounds.