Showing papers on "Methanosarcina barkeri published in 1986"

Characterization and purification of the membrane-bound ATPase of the archaebacterium Methanosarcina barkeri.

Abstract: Membrane-bound ATPase was found in membranes of the archaebacterium Methanosarcina barkeri. The ATPase activity required divalent cations, Mg2+ or Mn2+, and maximum activity was obtained at pH 5.2. The activity was specifically stimulated by HSO3- with a shift of optimal pH to 5.8, and N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. The enzyme could be solubilized from membranes by incubation in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. The solubilized ATPase was purified by DEAE-Sepharose and Sephacryl S-300 chromatography. The molecular weight of the purified enzyme was estimated to be 420,000 by gel filtration through Sephacryl S-300. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two classes of subunit, Mr 62,000 (alpha) and 49,000 (beta) associated in the molar ratio 1:1. These results suggest that the ATPase of M. barkeri is similar to the F0F1 type ATPase found in many eubacteria.

Coupling of carbon monoxide oxidation to CO2 and H2 with the phosphorylation of ADP in acetate‐grown Methanosarcina barkeri

Abstract: Cell suspensions of Methanosarcina barkeri, grown on acetate, catalyzed the conversion of carbon monoxide and H2O to CO2 and H2 in stoichiometric amounts when methane formation was inhibited by bromoethanesulfonate. The specific activity was 80-120 nmol min-1 mg protein-1 at 5% CO in the gas phase. CO oxidation was coupled with the phosphorylation of ADP as indicated by a rapid increase of the intracellular ATP level upon start of the reaction. At least 0.1 mol ATP was formed/mol CO consumed. The onset of CO oxidation was also accompanied by an increase of the proton motive force (delta p) from 100 mV to 150 mV (inside negative). Addition of the uncoupler tetrachlorosalicylanilide to CO-metabolizing cells led to a rapid decrease of the ATP level and of delta p, and to an increase of the CO oxidation rate up to 70%. In the presence of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide the phosphorylation of ADP was inhibited and CO oxidation slowed down, whereas delta p was almost unaffected. Inhibition of CO oxidation under these conditions was relieved by the addition of the protonophore tetrachlorosalicylanilide. The results indicate that in acetate-grown M. barkeri the free-energy change associated with the formation of CO2 and H2 from CO and H2O (delta G degrees = -20 kJ/mol) can be used to drive the phosphorylation of ADP and that the coupling proceeds via a chemiosmotic mechanism. A possible role of the carbon monoxide oxidation reaction as an energy-conserving site in acetate fermentation to CH4 and CO2 is discussed.

Microbial ecophysiology of whey biomethanation: characterization of bacterial trophic populations and prevalent species in continuous culture

Abstract: The organization and species composition of bacterial trophic groups associated with lactose biomethanation were investigated in a whey-processing chemostat by enumeration, isolation, and general characterization studies. The bacteria were spatially organized as free-living forms and as self-immobilized forms appearing in flocs. Three dominant bacterial trophic group populations were present (in most probable number per milliliter) whose species numbers varied with the substrate consumed: hydrolytic, 10; acetogenic, 10 to 10; and methanogenic, 10 to 10. The three prevalent species utilizing lactose were identified as Leuconostoc mesenteroides, Klebsiella oxytoca, and Clostridium butyricum. Clostridium propionicum and Desulfovibrio vulgaris were the dominant lactate-consuming, hydrogen-producing acetogenic bacteria, while D. vulgaris was the only significant ethanol-degrading species. Methanosarcina barkeri and Methanothrix soehngenii were identified as the dominant acetate-utilizing methanogens, and Methanobacterium formicicum was the prevalent hydrogen-utilizing methanogen. A microbial food chain is proposed for lactose biomethanation that comprises multiple species in three different groups, with the major hydrogen-producing acetogen being a sulfate-reducing species, D. vulgaris, which functioned in the absence of significant levels of environmental sulfate.

Effect of Sulfur-Containing Compounds on Growth of Methanosarcina barkeri in Defined Medium.

Abstract: Methanosarcina barkeri Fusaro (DSM 804) could grow on methanol in a mineral medium containing cysteine or thiosulfate as the sole sulfur source. Optimum growth occurred at cysteine concentrations of 1 to 2.8 mM and at thiosulfate concentrations of 2.5 to 5 mM. No inhibition of growth was observed even when these concentrations were doubled in the culture medium. Under the optimum cysteine and thiosulfate concentrations, the generation times of the organism were about 8 to 10 and 10 to 12 h, respectively, giving a cell yield of about 0.14 to 0.17 and 0.08 to 0.11 g (dry weight)/g of methanol consumed. The organism metabolized cysteine and thiosulfate during growth, giving rise to sulfide in the culture medium. H(2)S evolution from cysteine and thiosulfate was catalyzed by two enzymes, namely cysteine desulfhydrase and thiosulfate reductase, respectively, as revealed by enzyme assay in the crude cell-free extract of the organism.

Utilization of Methanol plus Hydrogen by Methanosarcina barkeri for Methanogenesis and Growth

Abstract: Methanosarcina barkeri grew on methanol plus H2. Both substrates were consumed in equimolar amounts. Growth was strictly dependent on the presence of acetate, which was required for the biosynthesis of cellular constituents. Only about 0.4% of the methane produced originated from acetate. By using deuterated methanol, it was demonstrated that methanogenesis from this compound under H2 did not occur via oxidation of methanol to CO2 and subsequent reduction but by direct reduction with H2. Growth yields with methanol plus H2 and with methanol alone were not significantly different: 2.8 g of cells per mol of methanol in mineral medium and 4.6 g of cells per mol of methanol in complex medium, respectively. Growth of M. barkeri on methanol plus H2 depended strictly on the presence of sodium ions in the medium. In the presence of 50 mM K+ the Ks for Na+ was 5 mM.

Fermentation of isoleucine and arginine by pure and syntrophic cultures of Clostridium sporogenes

Abstract: The fermentation of isoleucine, arginine and isoleucine + arginine by pure and syntrophic cultures of Clostridium sporogenes was investigated. Growth of C. sporogenes on isoleucine, if any, was poor, but some isoleucine was fermented to 2-methylbutyrate and hydrogen. In syntrophic cultures with Methanobacterium formicicum or Methanosarcina barkeri growth was better, and isoleucine was completely fermented, the hydrogen being used for methane production. Pure cultures of C. sporogenes grew on arginine and produced 5-aminovalerate, ornithine and acetate. The reducing equivalents for 5-aminovalerate production from intermediarily formed proline were provided by oxidative conversion of arginine to acetate and by oxidative metabolism of some amino acids present in the yeast extract. However, when isoleucine was available together with arginine in syntrophic cultures of C. sporogenes and M. formicicum, the reducing equivalents for arginine fermentation came mainly from the oxidation of isoleucine (Stickland reaction), and the hydrogen produced in excess served for the reduction of CO2 to methane.

Production of corrinoids including vitamin B-12 byMethanosarcina barkeri growing on methanol

Abstract: A preliminary attempt was made for producing vitamin B-12 byMethanosarcina barkeri strain Fusaro in a fed-batch culture with a methanol minimum medium. After 11 days, total methanol consumption, cell density and corrinoid concentration were 145 g/l, 8.5 g(dry cell weight)/l, and 135 mg/l (73% in supernatant) respectively. Electrophoretic separation revealed that 33% of the total corrinoids was B-12 Factor III (5-hydroxybenzimidazolylcobamide) and the remaining corrinoids were cobinamide (Factor B) and its derivatives.

Primary structure of the chromosomal protein HMb from the archaebacteria Methanosarcina barkeri

Abstract: The amino acid sequence of the protein HMb, a protein of 93 residues (Mr 10757) which represents the major acid-soluble component of the Methanosarcina barkeri nucleoprotein complex, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid, arginine and methionine residues. The protein HMb is mainly characterized by a high amount of charged residues (15% of acidic residues and 26.8% of basic residues) which are distributed all along the polypeptide chain. The amino acid sequence of the protein HMb is not homologous to any eubacterial, archaebacterial or eukaryotic chromosomal proteins known up to now.

Flocculation in methanogens, a comparative study of Methanosarcina barken strains Jülich and Fusaro

Abstract: Two strains of Methanosarcina barkeri grown on methanol as substrate were investigated for their ability to aggregate: the new, flocculent strain Julich formed stable flocs of several millimetres in diameter during rapid growth on a methanol-containing medium ("bread-crumb growth"). When observed with an electron microscope, the Julich strain showed a unique parenchymatic texture with thick-walled cells inside a floc, and coccoidlike cells on the periphery. In contrast to the Julich strain, the strain Fusaro grew on methanol in dispersed form, and generated macroscopically visible clumps only under poor growth conditions, induced, for example, by calcium deficiency. The formation of large cell aggregations of the Fusaro strain could also be induced during growth in the presence of 0.01% of the stain Calcofluor which is known to interact specifically with β-1,4 and β-1,3 glucan moieties. Sugar analyses revealed a different pattern for both strains: the exopolymer of the flocculent strain Julich contained h...

Methanogenesis from methanol in Methanosarcina barkeri studied using membrane inlet mass spectrometry

Abstract: A mass spectrometer with membrane inlet was used to study methanol metabolism by Methanosarcina barkeri strain MS. The addition of methanol to methanol grown culture samples in the mass spectrometer vessel stimulated methanogenesis and hydrogen production. The apparent Ks for methanol was determined as 0.5 mM and the Vmax as 8.14 mmol g (dry weight) h−1. The Vmax for methane production was fairly constant during growth of the culture on methanol implying that growth is tightly coupled to methanogenesis. The addition of methanol to culture samples in the mass spectrometer vessel stimulated methanogenesis with no lag which indicated that methanogenesis can be uncoupled from growth. Exposure of the culture sample in the mass spectrometer vessel to an atmosphere of 2 kPa oxygen for 80 min resulted in a decrease in the rate of methanogenesis from methanol but on returning the atmosphere to nitrogen the addition of further methanol stimulated methanogenesis. The effect of other inhibitors of methanogenesis (2-bromoethane sulphonate and monensin); Kj values 21.5 μM and 0.3 mM, respectively) were also studied.

Production of formic acid

Abstract: PURPOSE:To produce formic acid easily and economically by biochemical means, by reacting carbon monoxide with water in the presence of a cultured liquid, cultured cells or treated cells of microorganism belonging to Methanosarcina genus CONSTITUTION:A microbial strain belonging to Methanosarcina genus and capable of producing methane, eg Methanosarcina barkeri, etc, is cultured under anaerobic condition, and the cultured liquid, microbial cells separated from the cultured liquid, or treated microbial cells are collected The obtained cells or treated cells, etc, may be used in immobilized state Carbon monoxide is made to react with water under anaerobic condition in the presence of the microbial cells in a reaction liquid added with an electron acceptor such as methyl viologen to obtain formic acid The produced formic acid is separated and purified by conventional extraction process

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.