Pseudo vitamin B12 or 5-hydroxybenzimidazolyl-cobamide are the corrinoids found in methanogenic bacteria

Thirteen species of methanogenic bacteria were analyzed for corrinoids. Pseudo vitamin B₁₂ (Co-[-(7-adenyl)]-cobamide) was the predominant cobamide of methanococcales and Methanoplanus. All other methanogens contained factor III (Co-[-(5-hydroxybenzimidazolyl)]-cobamide). Vitamin B₁₂ (Co-[-(5,6-dimethylbenzimidazolyl)]-cobamide) was not detected in any of these archaebacteria. Their cobamide content was 100 to 1400 nmol per gram cell dry weight, indicating that abundant cobamides are essential for methanogens.     

Polysaccharide reserve material in the acetotrophic methanogen,Methanosarcina thermophila strain TM-1: accumulation and mobilization

The ability of Methanosarcina thermophila strain TM-1 to store a reserve polysaccharide was studied using both biochemical methods and thin-section electron microscopy. When grown under conditions of excess carbon and energy (either methanol or acetate) and limiting nitrogen, M. thermophila accumulated a polysaccharide which could be hydrolyzed to glucose by the enzyme amyloglucosidase. This polysaccharide reached levels of 20 mg polysaccharide per g protein in nitrogen-limited cells, while cells limited for carbon, as well as cells in the exponential phase of growth, did not accumulate significant amounts of this polysaccharide. Thin-section electron micrographs of M. thermophila showed glycogen-like inclusion granules in nitrogen-limited cells but not in carbon-limited or exponential-phase cells. These granules were stained by a polysaccharide-specific staining procedure, the PATO stain. The polysaccharide was purified from cell extracts, the iodine-polysaccharide complex gave a maximum absorption at between 500 and 510 nm. The polysaccharide was mobilized within 21 h by cells starved for a carbon/energy source. N-Limited (polysaccharide-containing) acetategrown cells could shift to methanogenesis from methanol more quickly than did C-limited acetate-grown cells lacking polysaccharide, and ATP levels remained higher in N-limited cells. The results are consistant with the hypothesis that this polysaccharide can provide carbon and energy for metabolic shifts but other storage compounds, such as polyphosphate, may also play a similar role.     

Methanogenic diversity and activity in municipal solid waste landfill leachates

Archaeal microbial communities present in municipal solid waste landfill leachates were characterized using a 16S rDNA approach. Phylogenetic affiliations of 239 partial length 16S rDNA sequences were determined. Sequences belonging to the order Methanosarcinales were dominant in the clone library and 65% of the clones belonged to the strictly acetoclastic methanogenic family Methanosaetaceae. Sequences affiliated to the metabolically versatile family Methanosarcinaceae represented 18% of the retrieved sequences. Members of the hydrogenotrophic order Methanomicrobiales were also recovered in limited numbers, especially sequences affiliated to the genera Methanoculleus and Methanofollis. Eleven euryarchaeal and thirteen crenarchaeal sequences (i.e. 10%) were distantly related to any hitherto cultivated microorganisms, showing that archaeal diversity within the investigated samples was limited. Lab-scale incubations were performed with leachates mixed with several methanogenic precursors (acetate, hydrogen, formate, methanol, methylamine). Microbial populations were followed using group specific 16S rRNA targeted fluorescent oligonucleotidic probes. During the incubations with acetate, acetoclastic methanogenesis was rapidly induced and led to the dominance of archaea hybridizing with probe MS1414 which indicates their affiliation to the family Methanosarcinaceae. Hydrogen and formate addition induced an important acetate synthesis resulting from the onset of homoacetogenic metabolism. In these incubations, species belonging to the family Methanosarcinaceae (hybridizing with probe MS1414) and the order Methanomicrobiales (hybridizing with probe EURY496) were dominant. Homoacetogenesis was also recorded for incubations with methanol and methylamines. In the methanol experiment, acetoclastic methanogenesis took place and archaea hybridizing with probe MS821 (specific for Methanosarcina spp.) were observed to be the dominant population. These results confirm that acetoclastic methanogenesis performed by the members of the order Methanosarcinales is predominant over the hydrogenotrophic and methylotrophic pathways in landfill leachates.     

Long-term performance and microbial community analysis of a full-scale synthesis gas fed reactor treating sulfate- and zinc-rich wastewater

The performance of a full-scale (500 m³) sulfidogenic synthesis gas fed gas-lift reactor treating metal- and sulfate-rich wastewater was investigated over a period of 128 weeks. After startup, the reactor had a high methanogenic activity of 46 Nm³·h⁻¹. Lowering the carbon dioxide feed rate during the first 6 weeks gradually lowered the methane production rate. Between weeks 8 and 93, less than 1% of the hydrogen supplied was used for methanogenesis. Denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified 16S rRNA gene fragments showed that the archaeal community decreased in diversity but did not disappear completely. After the carbon dioxide feed rate increased in week 88, the methane production rate also increased, confirming that methane production was carbon dioxide limited. Even though lowering the carbon dioxide feed appeared to affect part of the sulfate-reducing community, it did not prevent achieving the desired rates of sulfate reduction. The average sulfate conversion rate was 181 kg∙h⁻¹ for the first 92 weeks. After 92 weeks, the sulfate input rate was increased and from week 94 to 128, the average weekly sulfate conversion rate was 295 kg·h⁻¹ (SD ± 87). Even higher sulfate conversion rates of up to 400 kg·h⁻¹ could be sustained for weeks 120–128. The long-term performance and stability together with the ability to control methanogenesis demonstrates that synthesis gas fed reactor can be used successfully at full scale to treat metal and sulfate-rich wastewater.     

Enzymology of one-carbon metabolism in methanogenic pathways

Methanoarchaea, the largest and most phylogenetically diverse group in the Archaea domain, have evolved energy-yielding pathways marked by one-carbon biochemistry featuring novel cofactors and enzymes. All of the pathways have in common the two-electron reduction of methyl-coenzyme M to methane catalyzed by methyl-coenzyme M reductase but deviate in the source of the methyl group transferred to coenzyme M. Most of the methane produced in nature derives from acetate in a pathway where the activated substrate is cleaved by CO dehydrogenase/acetyl-CoA synthase and the methyl group is transferred to coenzyme M via methyltetrahydromethanopterin or methyltetrahydrosarcinapterin. Electrons for reductive demethylation of the methyl-coenzyme M originate from oxidation of the carbonyl group of acetate to carbon dioxide by the synthase. In the other major pathway, formate or H2 is oxidized to provide electrons for reduction of carbon dioxide to the methyl level and reduction of methyl-coenzyme to methane. Methane is also produced from the methyl groups of methanol and methylamines. In these pathways specialized methyltransferases transfer the methyl groups to coenzyme M. Electrons for reduction of the methyl-coenzyme M are supplied by oxidation of the methyl groups to carbon dioxide by a reversal of the carbon dioxide reduction pathway. Recent progress on the enzymology of one-carbon reactions in these pathways has raised the level of understanding with regard to the physiology and molecular biology of methanogenesis. These advances have also provided a foundation for future studies on the structure/function of these novel enzymes and exploitation of the recently completed sequences for the genomes from the methanoarchaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii.     

Tungstate can substitute for molybdate in sustaining growth of Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to grow on media supplemented with tungstate rather than with molybdate. The Archaeon then synthesized a tungsten iron-sulfur isoenzyme of formylmethanofuran dehydrogenase. The isoenzyme was purified to apparent homogeneity and shown to be composed of four different subunits of apparent molecular masses 65 kDa, 53 kDa, 31 kDa, and 15 kDa and to contain per mol 0.4 mol tungsten, <0.05 mol molybdenum, 8 mol non-heme iron, 8 mol acid-labile sulfur and molybdopterin guanine dinucleotide. Its molecular and catalytic properties were significantly different from those of the molybdenum isoenzyme characterized previously. The two isoenzymes also differed in their metal specificity: the active molybdenum isoenzyme was only synthesized when molybdenum was available during growth whereas the active tungsten isoenzyme was also generated during growth of the cells on molybdate medium. Under the latter conditions the tungsten isoenzyme was synthesized containing molybdenum rather than tungsten.Abbreviations MFR methanofuran - CHO-MFR N-formylmethanofuran - MGD molybdopterin guanine dinucleotide - MAD molybdopterin adenine dinucleotide - MHD molybdopterin hypoxanthine dinucleotide - FPLC fast protein liquid chromatography - SDS/PAGE sodium dodecylsulfate/polyacrylamide gel electrophoresis - ICP-MS inductively coupled plasma mass spectrometry     

Biogenesis of methane in primate dental plaque

Dental plaque samples collected from monkeys (Macaca mulatta) were found to contain a large amount of dissolved methane gas (0.6 nmol CH4/mg wet wt plaque). Enrichment cultures inoculated with dental plaque obtained from Macaca fascicularis produced methane when the medium contained ethanol, methanol, lactate, acetate or a hydrogen + CO2 atmosphere. Methane formation in the enrichments was inhibited by oxidation of the culture medium, autoclaving or the addition of 2-bromoethane sulfonic acid (BES). The methane producing enrichments were observed to contain fluorescent cocci occurring singly and in short chains. It was concluded that methane formation in the monkey dental plaque was the result of the presence of methanogenic bacteria.     

Acetate metabolism inMethanosarcina barkeri

Methanosarcina barkeri was grown by acetate fermentation in complex medium (N₂ gas phase). The molar growth yield was 1.6–1.9 g cells/mol methane formed. Under these conditions 63–82% of the methane produced byMethanosarcina strains was derived from the methyl carbon of acetate, indicating that some methane was derived from other media components. Growth was not demonstrated in complex media lacking acetate or mineral acetate medium containing acetate but lacking H₂/CO₂, methanol, or trypticase and yeast extract. Acetate metabolism byM. barkeri strain MS was further exmined in mineral acetate medium containing H₂/CO₂ and/or methanol, but lacking cysteine. Under these conditions, more methane was derived from the methyl carbon of acetate than from the carboxyl carbon. Methanogenesis from the methyl group increased with increasing acetate concentration. The methyl carbon contributed up to 42% of the methane formed with H₂/CO₂ and up to 5% with methanol. Methanol stimulated the oxidation of the methyl group of acetate to CO₂. The average rates of methane formation from acetate were 1.3 nomol/min ·ml/culture (0.04mg² cell dry weight) in defined media (gas phase H₂/CO₂) and complex media (gas phase N₂). Acetate contributed up to 60% of cell carbon formed under the growth conditions examined. Similar quantities of cell carbon were derived from the methyl and carboxyl carbons of acetate, suggesting incorporation of this compound as a two-carbon unit. Incorporated acetate was not preferentially localized in lipid material, as 70% of the incorporated acetate was found in the wall and protein cell fractions. Acetate catabolism was stimulated by pregrowing of cultures in media containing acetate, while acetate anabolism was not influenced. The results are discussed in terms of the differences between the mechanisms of acetate catabolism and anabolism.Abbreviations CH₃-S-CoM methyl coenzyme M - TCA trichloroacetic acid - CoM coenzyme M (2-mercaptoethane sulfonic acid) - E^o standard potential change (pH 7) - F₄₂₀ Factor 420, a low redox electron carrier - G^o standard free energy change (pH 7) - kJ kilojoules (=0.24 kilocalories) - PBBW Weimer's phosphate-buffered basal medium - X unknown C₁ carrier     

Hydrogen as a substrate for methanogenesis and sulphate reduction in anaerobic saltmarsh sediment

Hydrogen gas stimulated sulphate reduction in a saltmarsh sediment and the importance of H₂ transferred from organotrophic bacteria to the sulphate-reducers is discussed. -fluorolactate inhibited sulphate reduction whether lactate, ethanol or hydrogen was being used as growth substrate. When added to sediment -fluorolactate inhibited sulphate reduction with a consequent increase in methane production.Addition of H₂ stimulated methanogenesis in sediment and this stimulation was greater if CO₂ was also present. Hydrogen availability was the primary limitation of methanogenesis but the low concentration of dissolved CO₂ in seawater may limit methane production even if H₂ is available.The removal of inhibition of methanogenesis by the use of fluorolactate to suppress sulphate reduction or by the provision of hydrogen indicates competitive inhibition of methanogens by sulphate reducers utilizing transferred hydrogen.Abbreviations HSRB hydrogen utilizing sulphate reducing bacteria - HDO hydrogen donating organism     

One carbon metabolism in methanogenic bacteria

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H₂ oxidation/CO₂ reduction. The optimum temperature for growth and methanogenesis was 37°C. H₂ oxidation proceeded via an F₄₂₀-dependent NADP⁺-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 mol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H₂/CO₂ and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH₄ formed, respectively. During mixotrophic growth on H₂/CO₂ plus methanol, most methane was derived from methanol rather than from CO₂. Similar activities of hydrogenase were observed in cell-free extracts from H₂/CO₂-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO₂, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO₂ and from an organic intermediate more reduced than CO₂. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.Abbreviations CAPS cycloaminopropane sulfonic acid - CH₃-SCoM methyl coenzyme M - DCPIP 2,6-dichlorophenolindophenol - DEAE diethylaminoethyl - dimethyl POPOP 1,4-bis-2-(4-mothyl-5-phenyloxazolyl)-benzene - DNA deoxyribonucleic acid - dpm dismtegrations per min - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - F₄₂₀ factor 420 - G+C guanosine plus cytosine - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - PBBW phosphate buffered basal Weimer - PMS phenazine methosulfate - PPO 2,5-diphenyloxazole - rRNA ribosomal ribonucleic acid - RuBP ribulose-1,5-bisphosphate - Tris tris-hydroxymethyl-aminomethane - max maximum specific growth rate