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PubMed2001

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding.

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.

Binding Sites↗
PubMed1976

Hydrogenase activity in nitrogen-fixing methane-oxidizing bacteria.

Hydrogenase activity in cells of the nitrogen-fixing methane-oxidizing bacterium strain 41 of the Methylosinus type increased markedly when growth was dependent upon the fixation of gaseous nitrogen. A direct relationship may exist between hydrogenase and nitrogenase in this bacterium. Acetylene reduction was supported by the presence of hydrogen gas.

Acetylene↗
PubMed1990

Elemental metals as electron sources for biological methane formation from CO2.

Several elemental metals were examined as potential electron donors for methanogenic bacteria, using both a single tube system where the metal was in direct contact with the cells, and a two-flask system, where metal and cells were not in direct contact, but had contact via the gas phase. With all organisms examined in the direct contact system, Fe degree, Al degree and Zn degree served as electron donors for methanogenesis; some organisms used Ni degree or Sn degree as low-level electron donors. Of the metals tested, methanogenesis from H2 + CO2 was inhibited by direct contact with Zn degree or Cu degree, but not by Fe degree or Al degree. Ni degree and Co degree were inhibitory to some methanogens, with Ni degree being particularly inhibitory to the thermophilic strains tested. With all organisms examined in the two-flask system, Fe degree and Zn degree served as good electron sources for both methanogenesis and growth; Co degree generated a very low level of methane and Cu degree did not work at all. In either system V degree, Ti degree or Cd degree did not serve as electron donors. The results suggest that some elemental metals (notably Fe degree, Al degree and Zn degree) produce gaseous H2 by cathodic depolarization which is then consumed by the methanogen, thus accelerating oxidation of the metal by its metabolic activity. All of these reactions are thermodynamically favorable; however, some other metals that are clearly favorable for such a reaction on thermodynamic grounds (Ti degree and V degree) are very stable and do not serve as electron donors.

Aluminum↗
PubMed1976

Anaerobic degradation of benzoate to methane by a microbial consortium.

A stabilized consortium of microbes which anaerobically degraded benzoate and produced CH4 was established by inoculation of a benzoate-mineral salts medium with sewage sludge; the consortium was routinely subcultured anaerobically in this medium for 3 years. Acetate, formate, H2 and CO2 were identified as intermediates in the overall conversion of benzoate to CH4 by the culture. Radioactivity was equally divided between the CH4 and CO2 from the degradation of uniformly ring-labeled [14C]benzoate. The methyl group of acetate was stoichiometrically converted to CH4. Acetate, cyclohexanecarboxylate, 2-hydroxycyclohexanecarboxylate, o-hydroxybenzoic acid and pimelic acid were converted to CH4 without a lag suggesting that benzoate was degraded by a reductive pathway. Addition of o-chlorobenzoate inhibited benzoate degradation but not acetate degradation or methane formation. Two methanogenic organisms were isolated from the mixed culture; neither organism was able to degrade benzoate, showing that the methanogenic bacteria served as terminal organisms of a metabolic food chain composed of several organisms. Removal of intermediates by the methanogenic bacteria provided thermodynamically favorable conditions for benzoate degradation.

Acetates↗
PubMed1978

The occurrence and identification of intracellular polyglucose storage granules in Methylococcus NCIB 11083 grown in chemostat culture on methane.

The accumulation of intracellular storage granules (0.03--0.5 micrometer) by Methylococcus NCIB 11083 when grown under conditions of ammonia limitation with methane as the sole source of carbon and energy was inversely proportional to the dilution rate. The isolated material was composed entirely of glucose residues and the infra-red spectrum exhibited characteristic absorption bands at 925 cm(-1), 845 cm(-1) and 745 +/- 4cm(-1), indicating the presence of alpha (1 leads to 4) glycosidic linkages. The polymer dissolved in hot water to give an opalescent solution that formed a violet iodine complex with an absorption maximum at 550nm, identical to that observed with reference amylopectin. The percentage of the polysaccharide released as maltose by the action of beta- and alpha-amylases was 55--64% and 80--90% respectively, values very similar to those obtained by the action of these enzymes on reference amylopectin and glycogen. Methylation analysis indicated that the average interior and exterior chain lengths of the polymer were 2.7 and 10.0 glucose units respectively and confirmed that the Methylococcus polyglucose is a branched polymer composed of units joined by 1 leads to 4 and 1 leads to 6 linkages. The number average molecular weight of the polymer is 2--4.5 x 10(5). The stored polymer was metabolised by the organism and its metabolism resulted in the synthesis of protein.

Ammonia↗
PubMedDate not supplied

Genetics of methane and methanol oxidation in gram-negative methylotrophic bacteria.

Within the past few years, considerable progress has been made in the understanding of the molecular genetics of methane and methanol oxidation. In order to summarize this progress and to illustrate the important genetic methods employed, this review will focus on several well-studied organisms. These organisms include the gram-negative faculative methylotrophs Methylobacterium extorquens, Methylobacterium organophilum and Paracoccus denitrificans. In addition, the obligate methanotrophs Methylococcus capsulatus and Methylosinus trichosporium are discussed. We have chosen not to discuss the genetics of methanol oxidation in the yeasts or in gram-positive bacteria. Likewise, the genetics of related topics (for example, methylamine oxidation and carbon assimilation pathways) are not reviewed here. Broad host range conjugatable plasmids have enabled researchers to complement mutations and clone genes from gram-negative methylotrophic bacteria. More recently, 'promoter probe' derivative plasmids have been used to elucidate aspects of gene regulation. Also, alternative gene-cloning techniques are proving useful in circumventing problems in the genetic studies of the obligate methanotrophs, the group of bacteria that is the most refractory to traditional methods.

Cloning, Molecular↗
PubMed1995

The effect of sulfate and nitrate on methane formation in a freshwater sediment.

A freshwater sediment from a ditch of a peat grassland near Zegveld (Province of Utrecht, The Netherlands) was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Methanogenesis started after a 10 days lagphase. After 35-40 days aceticlastic methanogens were sufficiently enriched to cause a net decrease of acetate. In the presence of sulfate methane formation was only slightly affected. The addition of nitrate led to an outcompetition of aceticlastic methanogens by nitrate reducers. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. Addition of inorganic electron acceptors resulted in an outcompetition of the syntrophic propionate and butyrate degrading consortia by the sulfate and nitrate reducers.

Acetates↗
PubMed1993

Relationship between methane production and breath hydrogen excretion in lactose-malabsorbing individuals.

Recent studies have shown reduced breath hydrogen (H2) excretion in methane (CH4)-producing healthy individuals following ingestion of lactulose. This questions the reliability of the breath hydrogen test (BHT) in CH4 excretors, but the relationship between CH4 and H2 excretion in other clinical applications of the BHT is not known. We reviewed BHT results in two groups of subjects: (1) 385 children tested for lactose malabsorption in a hospital setting, and (2) 109 lactose-malabsorbing patients tested with a home kit. The percentage of lactose malabsorbers in group 1 (51%) was the same regardless of CH4-producing status (P = 0.97). The BHT data from group 2 showed a positive correlation (r = 0.6, P < 0.000001) between the magnitude of the rise in CH4 and H2 concentrations, and the H2 excretion curves were significantly higher in the CH4-producing individuals. We conclude that attention to CH4-producing status is not necessary in the interpretation of the lactose BHT.

Adolescent↗
PubMed1990

Methane excretion and experimental colonic carcinogenesis.

To examine the association between methane (CH4) excretion and experimental colonic carcinogenesis, we measured CH4 excretion in rats treated with the colonic carcinogen azoxymethane (AOM, 7 mg/kg weekly for 10 weeks) and paired controls. CH4 excretion was not initially detected in either experimental or control groups, but all animals acquired positive CH4 excretion status by time of sacrifice (week 26). There was no difference between groups or among AOM-treated animals with and without tumors in the median time to onset of detectable CH4 excretion or in the amount of CH4 excreted. Our results fail to provide support for a link between CH4 excretion and experimental colonic dysplasia or adenocarcinoma.

Adenocarcinoma↗
PubMed1989

Methane plasma as a protective coating on intraocular lenses: an in vitro study.

A new methane plasma polymer was used to treat eight polymethylmetacrylate intraocular lenses (IOL). Another group of four lenses which had no treatment was used as control. The groups was compared in terms of the amount of endothelial cell damage caused after each of the twelve IOLs touched the central endothelium of 12 separate New Zealand rabbit corneas for 30 seconds in vitro. The amount of damage was estimated by means of vital staining with nitroblue tetrazolium and scanning electron microscopy. We found that the untreated (control) group of IOL produced significantly more endothelial cell damage in comparison with the treated groups. Further studies are needed to evaluate the clinical significance of our findings.

Animals↗
PubMed1996

Breath hydrogen and methane excretion produced by commercial beverages containing dietary fiber.

Soft drinks containing dietary fiber are popular in Japan. There seem to be two types, one containing polydextrose and the other, oligosaccharide. These beverages are claimed to be useful for constipation or obesity, but data are scanty. We examined four such fiber-containing beverages [Fibe-mini Otsuka Pharmaceuticals (Tokyo, Japan), Seni and Oligo Takeda Food Engineering (Osaka, Japan), Oligo CC (Calpis Food Engineering, Tokyo, Japan), and Sapitus 5289 Nakakita Pharmaceuticals (Nagoga, Japan)] for large intestine fermentability by measuring breath hydrogen (H2) and methane (CH4). Five healthy subjects (two men, three women, 22-48 years old) participated in the study. Breath H2 and CH4 were measured with a MicroLyzer (Quintron Instruments, Milwaukee, Wis.). Breath H2 increased within 2h of beverage consumption, but CH4 excretion was observed in only two subjects. Orocecal transit time was constant for all beverages. Total H2 plus CH4 excretion (AUC; area under the curve) after lactulose was 1294 +/- 250 ppm x min/g fiber. AUC for Oligo CC was significantly greater than that for Fibe-mini or Sapitus 5289 (P < 0.05). The AUCs of Fibe-mini, Seni and Oligo, Oligo CC, and Sapitus 5289 were 41%, 129%, 174%, and 40%, respectively, that of lactulose. It is concluded that commercial fiber-containing drinks produce H2, and CH4 in some people. Oligosaccharide produces more H2 and CH4 than polydextrose.

Adult↗
PubMed2004

Archaeal community structure and pathway of methane formation on rice roots.

The community structure of methanogenic Archaea on anoxically incubated rice roots was investigated by amplification, sequencing, and phylogenetic analysis of 16S rRNA and methyl-coenzyme M reductase (mcrA) genes. Both genes demonstrated the presence of Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae, Methanosaetaceae, and Rice cluster I, an uncultured methanogenic lineage. The pathway of CH4 formation was determined from the 13C-isotopic signatures of the produced CH4, CO2 and acetate. Conditions and duration of incubation clearly affected the methanogenic community structure and the pathway of CH4 formation. Methane was initially produced from reduction of CO2 exclusively, resulting in accumulation of millimolar concentrations of acetate. Simultaneously, the relative abundance of the acetoclastic methanogens (Methanosarcinaceae, Methanosaetaceae), as determined by T-RFLP analysis of 16S rRNA genes, was low during the initial phase of CH4 production. Later on, however, acetate was converted to CH4 so that about 40% of the produced CH4 originated from acetate. Most striking was the observed relative increase of a population of Methanosarcina spp. (but not of Methanosaeta spp.) briefly before acetate concentrations started to decrease. Both acetoclastic methanogenesis and Methanosarcina populations were suppressed by high phosphate concentrations, as observed under application of different buffer systems. Our results demonstrate the parallel change of microbial community structure and function in a complex environment, i.e., the increase of acetoclastic Methanosarcina spp. when high acetate concentrations become available.

Acetates↗
PubMed2000

Denitrification with methane as electron donor in oxygen-limited bioreactors.

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10(7) cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10(7) cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10(4) cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. "In vivo" 13C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification.

Anaerobiosis↗
PubMed1998

Degradation of cyanide in agroindustrial or industrial wastewater in an acidification reactor or in a single-step methane reactor by bacteria enriched from soil and peels of cassava.

During cassava starch production, large amounts of cyanoglycosides were released and hydrolysed by plant-borne enzymes, leading to cyanide concentrations in the wastewater as high as 200 mg/l. For anaerobic degradation of the cyanide during pre-acidification or single-step methane fermentation, anaerobic cultures were enriched from soil residues of cassava roots and sewage sludge. In a pre-acidification reactor this culture was able to remove up to 4 g potassium cyanide/l of wastewater at a hydraulic retention time (tHR) of 4 days, equivalent to a maximal cyanide space loading of 400 mg CN- 1(-1) day-1. The residual cyanide concentration was 0.2-0.5 mg/l. Concentrated cell suspensions of the mixed culture formed ammonia and formate in almost equimolar amounts from cyanide. Little formamide was generated by chemical decay. A concentration of up to 100 mmol ammonia/l had no inhibitory effect on cyanide degradation. The optimal pH for cyanide degradation was 6-7.5, the optimal temperature 25-37 degrees C. At a pH of 5 or lower, cyanide accumulated in the reactor and pre-acidification failed. The minimal tHR for continuous cyanide removal was 1.5 days. The enriched mixed culture was also able to degrade cyanide in purely mineralic wastewater from metal deburring, either in a pre-acidification reactor with a two-step process or in a one-step methanogenic reactor. It was necessary to supplement the wastewater with a carbon source (e.g. starch) to keep the population active enough to cope with any possible inhibiting effect of cyanide.

Aerobiosis↗
PubMed1996

Diversity of H2/CO2-utilizing acetogenic bacteria from feces of non-methane-producing humans.

The purpose of this work was to study H2/CO2-utilizing acetogenic population in the colons of non-methane-producing individuals harboring low numbers of methanogenic archaea. Among the 50 H2-consuming acetogenic strains isolated from four fecal samples and an in vitro semi-continuous culture enrichment, with H2/CO2 as sole energy source, 20 were chosen for further studies. All isolates were Gram-positive strict anaerobes. Different morphological types were identified, providing evidence of generic diversity. All acetogenic strains characterized used H2/CO2 to form acetate as the sole metabolite, following the stoichiometric equation of reductive acetogenesis. These bacteria were also able to use a variety of organic compounds for growth. The major end product of glucose fermentation was acetate, except for strains of cocci that mainly produced lactate. Yeast extract was not necessary, but was stimulatory for growth and acetogenesis from H2/CO2.

Acetates↗
PubMed2006

Vertical distribution and diversity of bacteria and archaea in sulfide and methane-rich cold seep sediments located at the base of the Florida Escarpment.

The bacterial and archaeal communities of the sediments at the base of the Florida Escarpment (Gulf of Mexico, USA) were investigated using molecular phylogenetic analysis. The total microbial community DNA of each of three vertical zones (top, middle and bottom) of a sediment core was extracted and the 16S rRNA genes were amplified by PCR, cloned and sequenced. Shannon-Weaver Diversity measures of bacteria were high in all three zones. For the archaea, diversity was generally low, but increased with depth. The archaeal clonal libraries were dominated by representatives of four groups of organisms involved in the anaerobic oxidation of methane (ANME groups). Phylogenetic analysis of bacteria suggests the dominance of epsilon-proteobacteria in the top zone, the epsilon-, delta- and gamma-proteobacteria in the middle zone and the delta-proteobacteria in the bottom zone of the core. Members of the Cytophaga-Flexibacter-Bacteroidetes group, the Chloroflexi/green non-sulfur bacteria, the Gram+ (Firmicutes), the Planctomyces, candidate division WS3 and Fusobacterium were also detected. Our data suggest that the community structure and diversity of microorganisms can shift greatly within small vertical distances, possibly in response to changes in the physical and chemical conditions.

Archaea↗
PubMed2005

Molecular evidence demonstrating the basidiomycetous fungus Cryptococcus curvatus is the dominant microbial eukaryote in sediment at the Kuroshima Knoll methane seep.

The Kuroshima Knoll, located in the southern Ryukyu Arc, is known to actively bubble with gas containing methane and hydrogen sulfide from numerous fissures in the large carbonate pavement. Although ecological studies regarding macrobenthos and bacteria from Kuroshima Knoll have been intensively conducted, the community structure and ecological importance of microbial eukaryotes (protists) have not yet been investigated. In the present study, we directly extracted DNA from sediment of the Kuroshima Knoll at a depth of 640 m and constructed genetic libraries of PCR-amplified eukaryotic small-subunit ribosomal DNA (SSU rDNA). Although the SSU rDNA sequences of several types of benthic foraminifers were retrieved from the surface of the sediment, all other sequences (just below the sediment surface to approximately 9 cm below sediment surface) were derived from the basidiomycetous yeast Cryptococcus curvatus. Furthermore, sequences of the internal transcribed spacer of rDNA (ITS-rDNA) retrieved from the same sediment were identical to that of C. curvatus originating from terrestrial habitats. The diversity of microbial eukaryotes in the Kuroshima Knoll sediment seems to be extremely low and significantly different from that of other marine environments previously reported.

Animals↗