Showing papers on "Methanosarcina barkeri published in 1987"

Stable Carbon Isotope Fractionation by Methanosarcina barkeri during Methanogenesis from Acetate, Methanol, or Carbon Dioxide-Hydrogen.

Abstract: Methanosarcina barkeri was cultured on methanol, H2-CO2, and acetate, and the 13C/12C ratios of the substrates and the methane produced from them were determined. The discrimination against 13C in methane relative to substrate decreased in the order methanol > CO2 > acetate. The isotopic fractionation for methane derived from acetate was only one-third of that observed with methanol as the substrate. The data presented indicate that the last enzyme of methanogenesis, methylreductase, is not the primary site of isotopic discrimination during methanogenesis from methanol or CO2. These results also support biogeochemical interpretations that gas produced in environments in which acetate is the primary methane precursor will have higher 13C/12C ratios than those from environments where other substrates predominate.

Inhibition of methanogenesis in pure cultures by ammonia, fatty acids, and heavy metals, and protection against heavy metal toxicity by sewage sludge.

Abstract: The effect of ammonium chloride, sodium butyrate, sodium propionate, and the heavy metals nickel, zinc, and copper on methanogenesis by pure cultures of Methanospirillum hungatei, Methanosarcina ba...

Kinetic study and mathematical modeling of methanogenesis of acetate using pure cultures of methanogens

Abstract: Kinetics of methanogenesis from acetate was studied using pure cultures of Methanosarcina barkeri and Methanosarcina mazei. Methane formation was found to be associated with cell growth. Nearly equimolar methane was produced from acetate during the methanogenic growth, and about 1.94 g of cells were formed from each mole of acetate consumed. Cell growth can be estimated from methane production. Significant substrate inhibition was found when acetate concentration was higher than 0.12 M. Among the three methanogenic strains studied, M. mazei strain S6 had the highest specific growth rate at all acetate concentrations studied and was least sensitive to environmental factors investigated (e.g., acetate concentration). The maximum specific growth rate found for strain S6 was 0.022 hr(-1) at acetic acid concentration around 7 g/L. The other two strains studied were M. barkeri strain 227 and strain MS. Growth of M. barkeri was completely inhibited at sodium acetate concentrations higher than 0.24 M. The maximum specific growth rate found for strains 227 and MS was 0.019 and 0.021 h(-1) at acetic acid concentrations of 3.6 and 6.8 g/L, respectively. A kinetic model with substrate inhibition was developed and can be used to simulate the methane formation from M. mazei strain S6 grown on acetate at 35 degrees C, pH 7.

Production of extracellular vitamin B-12 compounds from methanol by Methanosarcina barkeri

Abstract: Production of vitamin B-12 compounds from methanol was carried out by Methanosarcina barkeri Fusaro, an anaerobic methanogen. The methanogen released about 40% to 70% of corrinoids irrespective of the culture medium used. The use of cysteine instead of Na2S as the sole sulphur source for cell growth led to an increase in the cobalt chloride concentration in the culture medium up to 16 times the normal (0.6 mg·l-1) without medium precipitation. This in turn resulted in an intracellular vitamin B-12 content of 5.6 mg·g dry cell-1, the rest being discharged into the culture supernatant; this was 87 mg·l-1, 73% of the total corrinoids after 20 repeated intermittently fed cultures and the final cell concentration was 5.8 g dry cell·l-1. Taking advantage of this, continuous production of extracellular vitamin B-12 compounds was attempted with a fixed-bed bioreactor (carrier: diatomaceous clay). At a steady state operation at space velocity of 9 to 11 day-1, the concentration of the discharged corrinoid was 6.8 to 7.9 mg·l-1, having a vitamin B-12 activity of about 4 mg·l-1. Total cell mass retained in the reactor was 39.6 g dry cell l-reactor-1. Identification of the corrinoids revealed that 19% of the total corrinoids was comprised of the vitamin B-12 Factor III (5-hydroxybenzimidazolyl cobamide) and the remainder were mainly the base-free vitamin B-12 Factor B (cobinamide and its derivatives).

Emended Description of Strain MST (DSM 800T), the Type Strain of Methanosarcina barkeri

Abstract: The isolation and characteristics of Methanosarcina barkeri MST(DSM 800T), accomplished in 1966 by Marvin P. Bryant, are described.

Proton-motive-force-driven formation of CO from CO2 and H2 in methanogenic bacteria

Abstract: Cell suspensions of methanogenic bacteria (Methanosarcina barkeri, Methanospirillum hungatei, Methanobrevibacter arboriphilus, and Methanobacterium thermoautotrophicum) were found to form CO from CO2 and H2 according to the reaction: CO2+ H2 CO + H2O; ΔG0=+20 kJ/mol. Up to 15000 ppm CO in the gas phase were reached which is significantly higher than the equilibrium concentration calculated from ΔG0 (95 ppm under the experimental conditions). This indicated that CO2 reduction with H2 to CO is energy-driven and indeed the cells only generated CO when forming CH4. The coupling of the two reactions was studied in more detail with acetate-grown cells of M. barkeri using methanol and H2 as methanogenic substrates. The effects of the protonophore tetrachlorosalicylanilide (TCS) and of the proton-translocating ATPase inhibitor N,N′-dicyclohexylcarbodiimide (cHxN)2C were determined. TCS completely inhibited CO formation from CO2 and H2 without affecting methanogenesis from CH3OH and H2. In the presence of the protonophore the proton motive force Δp and the intracellular ATP concentration were very low. (cHxN)2C, which partially inhibited methanogenesis from CH3OH and H2, had no effect on CO2 reduction to CO. In the presence of (cHxN)2C Δp was high and the intracellular ATP content was low. These findings suggest that the endergonic formation of CO from CO2 and H2 is coupled to the exergonic formation of CH4 from CH3OH and H2 via the proton motive force and not via ATP. CO formation was not stimulated by the addition of sodium ions.

Generation of a transmembrane gradient of Na+ in Methanosarcina barkeri.

Abstract: A transmembrane Na+ gradient was generated by Methanosarcina barkeri during methanogenesis. The intracellular Na+ concentration amounted to approximately one fifth of the extracellular one. A secondary Na+/H+ antiport system was shown to be responsible for Na+ extrusion. This system could be inhibited by amiloride. In the presence of amiloride the ΔpH across the cytoplasmic membrane increased and a transmembrane Na+ gradient could neither be generated nor maintained. The possible role of Na+ in the oxidation of methanol to the level of formaldehyde is discussed.

Effects of hydrogen pressure during growth and effects of pregrowth with hydrogen on acetate degradation by Methanosarcina species. [Methanosarcina barkeri; Methanosarcina mazei]

Abstract: Methanosarcina barkeri 227 and Methanosarcina mazei S-6 grew with acetate as the substrate; little effect of H/sub 2/ on the rate of aceticlastic growth was found in the presence of various H/sub 2/ pressures between 2 and 810 Pa. Physical (H/sub 2/ addition or flushing the headspace to remove H/sub 2/) and biological (H/sub 2/-producing or -utilizing bacteria in cocultures) methods were used for controlling H/sub 2/ pressure in Methanosarcina cultures growing on acetate. Added H/sub 2/ (ca. 100 Pa) was removed rapidly (a few hours) by M. barkeri and slowly (within a day) by M. mazei. When the H/sub 2/ produced by the aceticlastic methanogens was removed by coculturing with an H/sub 2/-using Desulfovibrio sp., the H/sub 2/ pressure was about 2.2 Pa. Under these conditions the stoichiometry of aceticlastic methanogenesis did not change. H/sub 2/-grown inocula of M. barkeri grew with acetate as the sole catabolic substrate if the inoculum culture was transferred during logarithmic growth to acetate-containing medium or if the transfer was accomplished with 1 or 2 days after exhaustion of H/sub 2/. H/sub 2/-grown cultures incubated for 4 or more days after exhaustion of H/sub 2/ were able to grow with H/sub 2/ but notmore » with acetate as the sole catabolic substrate. Addition of small quantities of H/sub 2/ to acetate-containing medium permitted these cultures to initiate growth on acetate.« less

Stereochemical course of methyl transfer from methanol to methyl coenzyme M in cell-free extracts of methanosarcina barkeri

Abstract: The transformation of the methyl group of methanol into methyl coenzyme M proceeds with net retention of methyl group configuration and without significant racemization. This is consistent with a proposed mechanism in which the methyl group is transferred from methanol first to the cobalt of the corrinoid enzyme MT/sub 1/ and then to the sulfur of coenzyme M. This resembles the transfer of the methyl group of methyltetrahydrofolate to homocysteine, catalyzed by the B/sub 12/-dependent methionine synthase from E. coli, which the authors have demonstrated also occurs with net retention of methyl group configuration. Both reactions pose the same question of how a relatively inert bond, the C-O bond of methyltetrahydrofolate in the case of methionine synthase, is cleaved in the transfer of a methyl group.

Effects of Hydrogen Pressure during Growth and Effects of Pregrowth with Hydrogen on Acetate Degradation by Methanosarcina Species.

Abstract: Methanosarcina barkeri 227 and Methanosarcina mazei S-6 grew with acetate as the substrate; we found little effect of H(2) on the rate of aceticlastic growth in the presence of various H(2) pressures between 2 and 810 Pa. We used physical (H(2) addition or flushing the headspace to remove H(2)) and biological (H(2)-producing or -utilizing bacteria in cocultures) methods for controlling H(2) pressure in Methanosarcina cultures growing on acetate. Added H(2) (ca. 100 Pa) was removed rapidly (a few hours) by M. barkeri and slowly (within a day) by M. mazei. When the H(2) produced by the aceticlastic methanogens was removed by coculturing with an H(2)-using Desulfovibrio sp., the H(2) pressure was about 2.2 Pa. Under these conditions the stoichiometry of aceticlastic methanogenesis did not change. H(2)-grown inocula of M. barkeri grew with acetate as the sole catabolic substrate if the inoculum culture was transferred during logarithmic growth to acetate-containing medium or if the transfer was accomplished within 1 or 2 days after exhaustion of H(2). H(2)-grown cultures incubated for 4 or more days after exhaustion of H(2) were able to grow with H(2) but not with acetate as the sole catabolic substrate. Addition of small quantities of H(2) to acetate-containing medium permitted these cultures to initiate growth on acetate.

Methanogenesis from Acetate by Methanosarcina barkeri: Catalysis of Acetate Formation from Methyl Iodide, CO2 , and H2 by the Enzyme System Involved

Abstract: Cell suspensions of Methanosarcina barkeri grown on acetate catalyze the formation of methane and CO2 from acetate as well as an isotopic exchange between the carboxyl group of acetate and CO2. Here we report that these cells also mediate the synthesis of acetate from methyl iodide, CO2, and reducing equivalents (H2 or CO), the methyl group of acetate being derived from methyl iodide and the carboxyl group from CO2. Methyl chloride and methyltosylate but not methanol can substitute for methyl iodide in this reaction. Acetate formation from methyl iodide, CO2, and reducing equivalents is coupled with the phosphorylation of ADP. Evidence is presented that methyl iodide is incorporated into the methyl group of acetate via a methyl corrinoid intermediate (deduced from inhibition experiments with propyl iodide) and that CO2 is assimilated into the carboxyl group via a C1 intermediate which does not exchange with free formate or free CO. The effects of protonophores, of the proton-translocating ATPase inhibitor N.N′-di- cyclohexylcarbodiimide, and of arsenate on acetate formation are interpreted to indicate that the reduction of CO2 to the oxidation level of the carboxyl group of acetate requires the presence of an electrochemical proton potential and that acetyl-CoA or acetyl-phosphate rather than free acetate is the immediate product of the condensation reaction. These results are discussed with respect to the mechanism of methanogenesis from acetate.

Analysis of hydrogen metabolism in Methanosarcina barkeri: regulation of hydrogenase and role of CO-dehydrogenase in H2 production

Abstract: Methanosarcina barkeri strain MS had significant levels of in vivo hydrogenase activity and both produced and consumed hydrogen when grown on CO or methanol as sole carbon and energy source. The activities of multiple hydrogenases were detected in extracts and cells of M. barkeri grown on CO or methanol. Uptake hydrogenase activity was 4 times higher and production hydrogenase was 3 times lower in methanol than in CO-grown cells. When 2-bromoethane sulfonate was added to M. barkeri growing on methanol, methane production was inhibited while hydrogen was produced but not consumed, indicating that hydrogen may be an intermediate in methane production from methanol. CO inhibited in vivo hydrogenase activity of M. barkeri cells suggesting that CO might inhibit uptake hydrogenase activity and not production hydrogenase activity. Purified CO-dehydrogenase (CODH) from M. barkeri produced 278.9 nmol hydrogen min−1· mg−1 protein using methyl viologen as electron carrier. Purified CODH did not display either detectable uptake hydrogenase activity or significant tritium exchange activity. Notably, 20 μM KCN inhibited both hydrogen producing and CO-oxidizing activity of purified CODH, but not uptake hydrogenase activity. Thus, we propose that in addition to its role in acetate synthesis and degradation, CODH produces hydrogen as a by-product of carbonyl group transformation.

Hydrogen metabolism during methanogenesis from acetate by Methanosarcina barkeri

Abstract: 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.

Spectroscopic and enzymatic evidence for membrane bound electron transport carriers and hydrogenase and their relation to cytochrome b function in methanosarcina barkeri

Abstract: Membranes prepared from Methanosarcina barkeri cultured on acetate were examined for electron carriers using electron paramagnetic resonance (EPR) and optical spectroscopy. EPR analysis of membrane suspensions demonstrated multiple iron-sulfur centers of the 4Fe-4S type, a hihg-spin heme-like species and possibly rebredoxin. Optical spectroscopy demonstrated that a b-type cytochrome was reduced by molecular hydrogen and oxidized by methyl coenzyme M. A membrane-bound hydrogenase activity (14 μM · min−1 (mg protein)−1) was detected. This suggests a putative role for cytochrome b and hydrogenase in electron transfer and methyl-group reduction during aceticlastic methanogenesis.

Microbial Ecophysiology of Whey Biomethanation: Comparison of Carbon Transformation Parameters, Species Composition, and Starter Culture Performance in Continuous Culture

Abstract: Changes in lactose concentration and feed rate altered bacterial growth and population levels in a whey-processing chemostat. The bacterial population and methane production levels increased in relation to increased lactose concentrations comparable to those in raw whey (6%) and converted over 96% of the substrate to methane, carbon dioxide, and cells. Sequential increases in the chemostat dilution rate demonstrated excellent biomethanation performance at retention times as low as 25 h. Retention times shorter than 25 h caused prevalent bacterial populations and methane production to decrease, and intermediary carbon metabolites accumulated in the following order: acetate, butyrate, propionate, lactate, ethanol, and lactose. Bacterial species dominated in the chemostat as a function of their enhanced substrate uptake and growth kinetic properties. The substrate uptake kinetic properties displayed by the mixed chemostat population were equivalent to those of individual species measured in pure culture, whereas the growth kinetic properties of species in mixed culture were better than those measured in pure culture. A designed starter culture consisting of Leuconostoc mesenteroides, Desulfovibrio vulgaris, Methanosarcina barkeri, and Methanobacterium formicicum displayed biomethanation performance, which was similar to that of a diverse adapted mixed-culture inoculum, in a continuous contact digestor system to which 10 g of dry whey per liter was added. Preserved starter cultures were developed and used as inocula for the start-up of a continuous anaerobic digestion process that was effective for biomethanation of raw whey at a retention time of 100 h.

Fermentation of alanine and glycine by pure and syntrophic cultures of Clostridium sporogenes

Abstract: Clostridium sporogenes was grown on alanine, glycine and alanine + glycine in pure culture and in syntrophic cultures with methanogens. Batch and continuous pure cultures of C. sporogenes grew only poorly on alanine as the sole amino acid, due to an elevated hydrogen partial pressure and converted alanine to acetate, CO2, H2 and presumably NH3. Growth on glycine was also poor and accompanied by the formation of acetate, CO2, some H2 and NH3. In the presence of alanine + glycine C. sporogenes performed an almost stoichiometric Stickland reaction in batch cultures. However, some hydrogen accumulated and little more CO2 was produced than could be accounted for by alanine utilization. Excellent growth of C. sporogenes was observed in a chemostat culture on alanine and glycine. Both amino acids were quantitatively fermented, as judged from the acetate production. When alanine was the sole substrate in syntrophic cultures of C. sporogenes and Methanobacterium formicicum it was almost quantitatively utilized and methane was produced by interspecies hydrogen transfer. More methane was generated, when a H2/CO2− and acetate-utilizing Methanosarcina barkeri was co-cultured with C. sporogenes . Compared to pure cultures, glycine fermentation in syntrophic cultures of C. sporogenes and M. formicicum was not improved, but less of it was reductively converted to acetate, in favour of oxidative metabolism with the formation of CO2 and methane. More glycine was fermented in cocultures of C. sporogenes and Ms. barkeri . In syntrophic cultures growing in the presence of alanine and glycine not more glycine was utilized than in cultures growing in the presence of glycine alone. Judging from the acetate production, a reductive metabolism was predominant.

Purification and properties of carbon monoxide dehydrogenase from Methanococcus vannielii.

Abstract: Carbon monoxide dehydrogenase was purified to homogeneity from Methanococcus vannielii grown with formate as the sole carbon source. The enzyme is composed of subunits with molecular weights of 89,000 and 21,000 in an alpha 2 beta 2 oligomeric structure. The native molecular weight of carbon monoxide dehydrogenase, determined by gel electrophoresis, is 220,000. The enzyme from M. vannielii contains 2 g-atoms of nickel per mol of enzyme. Except for its relatively high pH optimum of 10.5 and its slightly greater net positive charge, the enzyme from M. vannielii closely resembles carbon monoxide dehydrogenase isolated previously from acetate-grown Methanosarcina barkeri. Carbon monoxide dehydrogenase from M. vannielii constitutes 0.2% of the soluble protein of the cell. By comparison the enzyme comprises 5% of the soluble protein in acetate-grown cells of M. barkeri and approximately 1% in methanol-grown cells.

Spontaneous Disaggregation of Methanosarcina mazei S-6 and Its Use in the Development of Genetic Techniques for Methanosarcina spp.

Abstract: When monomethylamine was the growth substrate, spontaneous disaggregation of Methanosarcina mazei S-6 commenced at the mid-exponential phase and resulted in the formation of a suspension containing 108 to 109 free cells per ml. Free cells were osmotically fragile and amenable to extraction of DNA. Hypertonic media for the manipulation and regeneration of free cells into aggregates were developed, and plating efficiencies of 100% were achieved for M. mazei S-6 and LYC. Free cells of strain S-6 required MgCl2 (10 mM) for growth, whereas aggregates did not. Specific growth rates of strains S-6 and LYC were increased by MgCl2. Treatment with pronase caused sphere formation and removal of the protein wall of cells of strain S-6, but protoplasts could not be regenerated. The disaggregating enzyme produced by strain S-6 facilitated the preparation of suspensions of free cells of some strains of Methanosarcina barkeri. Although this provided a means of extracting high-molecular-weight DNA from M. barkeri, less than 0.1% of free cells were viable.

Oxidation of trimethylamine to the level of formaldehyde by Methanosarcina barkeri is dependent on the protonmotive force

Abstract: The conversion of trimethylamine to methane, carbon dioxide and ammonia as catalyzed by cell suspensions of Methanosarcina barkeri was coupled to the generation of a protonmotive force and to the synthesis of ATP. Methanogenesis as well as ATP formation and protonmotive force generation was abolished by the uncoupler tetrachloro-salicylanilide (TCS). Inhibition of methane formation was reversed by addition of formaldehyde, which was predominantly oxidized to carbon dioxide, whereas trimethylamine was predominantly reduced to methane and ammonia under these conditions. Cell extracts of M. barkeri were unable to convert trimethylamine to methane, carbon dioxide and ammonia independent from the presence or absence of ATP.

Effect of molecular hydrogen on acetate degradation by methanosarcina

Abstract: Acetate is the ultimate intermediate for over two-thirds of the methane produced by anaerobic digestors, and methanosarcinae are the predominant aceticlastic methanogens, especially in high-rate digestors. The remainder of the methane produced in anaerobic digestors (about one third) comes from the oXidation'of hydrogen and the concomitant reduction of carbon dioxide to form methane. Hydrogen concentrations control many important catabolic reactions in anaerobic digestion, especially those involved in hydrogen production and degradation. Because hydrogen concentration is normally very low (several tenths of a micromole) and its turnover is very rapid, the entire pool size is degraded many times per second, and so minute changes in the production or degradation rate of hydrogen may be instantaneously translated into fluctuations in its concentration. Also, hydrogen concentration may not be uniform in the digestor. It is likely that there are microenvironments in anaerobic digestors having higher and lower concentrations than the average since hydrogen production is not uniform, and particles may account for a major portion of the hydrogen production. These fluctuations and the presence of microenvironments may be important in the inhibition of acetate degradation by Methanosarcina barkeri.

Recent Developments on the Biochemistry of Methanogenesis from Acetate

Abstract: Athough acetate is the major precursor for methanogenesis in nature, biochemical studies on this pathway have lagged behind studies on the pathway of carbon dioxide reduction to methane. In 1955, it was demonstrated that the methyl group of acetate is metabolized intact to methane by mixed cultures [17]. Research was stimulated 23 years later when Mah et al. [12] showed that Methanosarcina barkeri utilizes acetate as a sole growth substrate and produces methane at rates comparable to that observed in nature. Methods for large-scale culture of these slow growing organisms have been improved by use of the pH auxostat which delivers the large amounts of acetic acid required for growth while maintaining a constant pH [21]. Methanogenesis from acetate in cell exracts [1, 9] is an important development. Coenzyme M was shown to be a methyl carrier in the pathway by demonstrating deuterated methyl coenzyme M in Methanosarcina thermophila cells utilizing deuterated methyl acetate for methanogenesis [11]. Recently, the involvement of coenzyme M was also demonstrated in Methanosarcina barkeri using 14C-methyl labelled acetate [6]. The terminal step in methanogenesis from acetate is the reductive demethylation of methyl coenzyme M [6, 14]; thus, a common mechanism exists for the terminal step in methanogenesis from all known substrates. However, the mechanism of acetate cleavage and the steps leading to methyl coenzyme M in acetotrophic methanogens are unknown.

Effect of Molecular Hydrogen on Acetate Degradation by Methanosarcina Barkeri 227

Abstract: Acetate is the ultimate intermediate for over two-thirds of the methane produced by anaerobic digestors, and methanosarcinae are the predominant aceticlastic methanogens, especially in high-rate digestors. The remainder of the methane produced in anaerobic digestors (about one third) comes from the oxidation of hydrogen and the concomitant reduction of carbon dioxide to form methane. Hydrogen concentrations control many important catabolic reactions in anaerobic digestion, especially those involved in hydrogen production and degradation. Because hydrogen concentration is normally very low (several tenths of a micromole) and its turnover is very rapid, the entire pool size is degraded many times per second, and so minute changes in the production or degradation rate of hydrogen may be instantaneously translated into fluctuations in its concentration. Also, hydrogen concentration may not be uniform in the digestor. It is likely that there are microenvironments in anaerobic digestors having higher and lower concentrations than the average since hydrogen production is not uniform, and particles may account for a major portion of the hydrogen production. These fluctuations and the presence of microenvironments may be important in the inhibition of acetate degradation by Methanosarcina barkeri.