H2: heterodisulfide oxidoreductase,a second energy-conserving system in the methanogenic strain Gö1

Washed everted vesicles of the methanogenic bacterium strain Gö1 catalyzed an H₂-dependent reduction of the heterodisulfide of HS-CoM (2-mercaptoethanesulfonate) and HS-HTP (7-mercaptoheptanoylthreonine phosphate) (CoM-S-S-HTP). This process was independent of coenzyme F₄₂₀ and was coupled to proton translocation across the cytoplasmic membrane into the lumen of the everted vesicles. The maximal H⁺/CoM-S-S-HTP ratio was 2. The tranmembrane electrochemical gradient thereby generated was shown to induce ATP synthesis from ADP+P_i, exhibiting a stoichiometry of 1 ATP synthesized per 2 CoM-S-S-HTP reduced (H⁺/ATP=4). ATP formation was inhibited by the uncoupler 3,5-di-tert-butyl-4-hydroxy-benzylidene-malononitrile (SF 6847) and by the ATP synthase inhibitor N,N-dicyclohexylcarbodiimide (DCCD). This energy-conserving system showed a stringent coupling. The addition of HS-CoM and HS-HTP at 1 mM each decreased the heterodisulfide reductase activity to 50% of the control. Membranes from Methanolobus tindarius showed F₄₂₀H₂-dependent but no H₂-dependent heterodisulfide oxidoreductase activity. Neither of these activities was detectable in membranes of Methanococcus thermolithotrophicus.Abbreviations _H+ transmembrane electrochemical gradient of H⁺ - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - DCCD N,N-dicyclohexylcarbodiimide - SF 6847 3,5-di-ert-butyl-4-hydroxybenzylidenemalononitrile - Mb. Methanobacterium - Ml. Methanolobus - Mc. Methanococcus - MV methylviologen - BV benzylviologen - MTZ metronidazole     

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.     

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.     

高温油藏内源微生物及其提高采收率潜力研究

大港孔店油田油藏特征、流体和微生物性质分析结果表明,属于高温生态环境,地层水矿化度较低,氮、磷浓度低,而且缺乏电子受体,主要的有机物来源是油气.油田采用经过除油处理的油藏产出水回注方式开发,油层中存在的微生物类型主要是厌氧嗜热菌,包括发酵菌(102个/mL～105个/mL),产甲烷菌(103个/mL);好氧菌主要存在于注水井周围.硫酸盐还原菌(SRB)还原速率0.002 μg S2-/(L·d)～18.9 μg S2-/(L·d),产甲烷菌产甲烷速率0.012 μgCH4/(L·d)～16.2 μgCH4/(L·d).好氧菌能够氧化油形成生物质,部分氧化产物为挥发性脂肪酸和表面活性荆.产甲烷菌在油氧化菌液体培养基中产生CH4,CO2为好氧微生物和厌氧微生物的共同代谢产物.这些产物具有提高原油流动性的作用.用示踪剂研究了注入水渗流方向.通过综合分析,油藏微生物具有较大的潜力,基于激活油层茵的提高采收率方法在该油田是可行的.     

Hydrogen metabolism in a mildly acidic lake sediment (Knaack Lake)

Hydrogen metabolism was studied in anoxic Knaack Lake sediments by measuring the in situ concentrations of dissolved H₂, as well as the V_max, turnover rate constant, and K_m for H₂. The results show that the relatively low rate of H₂/CO₂-dependent methanogenesis is paralleled by a low turnover of the dissolved H₂ pool. H₂-dependent acetate formation did not appear to be of importance based on the discrepancy of the K_m for H₂ consumption between the sediment and the prevalent homoacetogenic microbial population. In this mildly acidic lake sediment, H₂ turnover apparently was limited by H₂ production from organic matter. During incubation of sediment under a gaseous headspace, H₂ escaped from the aqueous phase, and steady state concentrations of dissolved H₂ were significantly lower than under in situ conditions. H₂ concentrations increased upon addition of various organic substrates. H₂ turnover within the sediment appeared unrelated to the concentration of H₂ detected in the water column, especially in the epilimnetic water layers.     

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

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.     

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.     

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     

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