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PubMed1986

Breath hydrogen and methane: poor indicators of apparent digestion of soy fiber.

To examine whether end-alveolar breath hydrogen and methane could be used as indicators of fiber digestion in humans, 16 male subjects were fed four fiber-free, complete liquid diets with 0, 30 g (heat processed), 30 g, and 60 g/day soy polysaccharide. Breath hydrogen was measured hourly and breath methane every 4 h on days 6 and 8 of each study period. Feces were collected, homogenized, dried, and analyzed for neutral detergent fiber (NDF). NDF in diets was determined and apparent NDF digestibility calculated. NDF from soy was extensively fermented, greater than 80%, on the fiber-containing diets. No significant relationship was found between breath-gas excretion and fiber digestion, although breath-gas values varied greatly. Breath hydrogen and methane were not significantly different when subjects consumed diets containing 0, 30, or 60 g soy polysaccharide.

Adult↗

PubMed1992

Guar, but not psyllium, increases breath methane and serum acetate concentrations in human subjects.

Guar and psyllium are fermented by human fecal bacteria in vitro. To see if they were fermented in vivo, eight subjects were studied over 3 separate days, in random order, while eating a polysaccharide-free diet. Twenty grams guar or psyllium, taken at breakfast, had no effect on breath hydrogen levels over 14 h. Mean breath methane and serum acetate concentrations after guar, 37 +/- 1 ppm and 93 +/- 6 mumol/L, respectively, were significantly greater than after control, 20 +/- 2 ppm (P less than 0.05) and 62 +/- 4 mumol/L (P less than 0.01), and psyllium, 20 +/- 2 ppm (P less than 0.05) and 78 +/- 6 mumol/L (P less than 0.05). Serum acetate increased after guar (area under curve 193 +/- 56 mumol.h/L; P less than 0.02) but decreased after psyllium and control. We conclude that guar is fermented in the human colon, producing rises in breath methane and serum acetate but not hydrogen. Although psyllium had no effect on hydrogen, methane, or acetate, this does not prove that it is not fermented.

Acetates↗

PubMed1981

Effect of Fluori-Methane spray on passive hip flexion.

The purpose of the study was to evaluate the influence of Fluori-Methane spray as a method of affecting passive range of motion measured at the right hip joint. Subjects were 30 normal volunteers randomly divided into an experimental group and a control group. A special table was constructed to position and stabilize each subject for monitoring the right lower extremity's resistance to side-lying straight leg raising. Specific right hip flexion goniometric measurements were compared and analyzed before and after application of Fluori-Methane spray to the soft tissue overlying the posterior part of the right thigh. The results of the study showed that the experimental group, which received application of Fluori-Methane spray and static passive stretch, did significantly (p less than .02) increase the range of passive hip flexion over that of the control group, which received only static passive stretch.

Adult↗

PubMed1975

Oxidation of carbon monoxide and methane by Pseudomonas methanica.

The oxidation of carbon monoxide and methane by suspensions and ultrasonic extracts of Pseudomonas methanica was studied. A continuous assay for the oxidation of CO to CO2 was devised, using O2 and CO2 electrodes in combination. Stoicheiometries of CO-dependent CO2 formation, O2 consumption and NADH oxidation, and the partial stoicheiometries of methane-dependent NADH oxidation, suggest the involvement of a mono-oxygenase in these oxidations. Evidence is presented suggesting methane and CO oxidation are catalysed by a single enzyme system, distinct, at least in part, from the NADH oxidase present in extracts. Ethanol was able to provide the reductant necessary for CO oxidation by cell suspensions, though the metabolism of ethanol by P. methanica was found unlikely to result in substrate-level formation of NADH; the means whereby alcohol oxidation could supply reductant for the mono-oxygenase are discussed.

Ammonium Chloride↗

PubMed2005

Effect of methanobactin on the activity and electron paramagnetic resonance spectra of the membrane-associated methane monooxygenase in Methylococcus capsulatus Bath.

Improvements in the purification of methanobactin (mb) from either Methylosinus trichosporium OB3b(T) or Methylococcus capsulatus Bath resulted in preparations that stimulated methane-oxidation activity in both whole-cell and cell-free fractions of Methylococcus capsulatus Bath expressing the membrane-associated methane monooxygenase (pMMO). By using washed membrane factions with pMMO activities in the 290 nmol propylene oxidized min(-1) (mg protein)(-1) range, activities approaching 400 nmol propylene oxidized min(-1) (mg protein)(-1) were commonly observed following addition of copper-containing mb (Cu-mb), which represented 50-75 % of the total whole-cell activity. The stimulation of methane-oxidation activity by Cu-mb was similar to or greater than that observed with equimolar concentrations of Cu(II), without the inhibitory effects observed with high copper concentrations. Stimulation of pMMO activity was not observed with copper-free mb, nor was it observed when the copper-to-mb ratio was <0.5 Cu atoms per mb. The electron paramagnetic resonance (EPR) spectra of mb differed depending on the copper-to-mb ratio. At copper-to-mb ratios of <0.4 Cu(II) per mb, Cu(II) addition to mb showed an initial coordination by both sulfur and nitrogen, followed by reduction to Cu(I) in <2 min. At Cu(II)-to-mb ratios between 0.4 and 0.9 Cu(II) per mb, the intensity of the Cu(II) signal in EPR spectra was more representative of the Cu(II) added and indicated more nitrogen coordination. The EPR spectral properties of mb and pMMO were also examined in the washed membrane fraction following the addition of Cu(II), mb and Cu-mb in the presence or absence of reductants (NADH or duroquinol) and substrates (CH4 and/or O2). The results indicated that Cu-mb increased electron flow to the pMMO, increased the free radical formed following the addition of O2 and decreased the residual free radical following the addition of O2 plus CH4. The increase in pMMO activity and EPR spectral changes to the pMMO following Cu-mb addition represent the first positive evidence of interactions between the pMMO and Cu-mb.

Cell Membrane↗

PubMed2006

Characterizing a stable methane-utilizing mixed culture used in the synthesis of a high-quality biopolymer in an open system.

AIMS: To characterize a methane-utilizing poly-beta-hydroxybutyrate (PHB)-producing microbial community. METHODS AND RESULTS: Three different approaches based on microbiology, analytical chemistry and molecular biology were used to determine the composition of the mixed culture. The dominant species, Methylocystis sp. GB25, represents more than 86% of the total biomass. Seven accompanying bacterial species are present in the mixed culture of which two are methylotrophic bacteria and five are utilizers of complex carbon sources. Both these groups were found to be present at the same ratio with respect to each other. Results of fatty acid analysis and PCR-DGGE fingerprints reflect the stability of the mixed-culture composition in the open system during multiple continuous growth and polymer formation processes throughout a period of 29 months. The consistently high quality of the accumulated polymer further corroborates this finding. CONCLUSION: The methane-utilizing mixed culture has the potential of self-regulation resulting in a stable composition even under non-aseptic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Avoiding the necessity of sterile conditions, as demonstrated in this paper, is an important step towards the development of a viable large-scale process for the production of PHB using cheap substrates like methane from natural or renewable sources. This is the first report characterizing a bacterial mixed culture being used for the biotechnological production of a high-value product in an open system.

Bacteria↗

PubMed1986

Methane formation from methyl-coenzyme M in a system containing methyl-coenzyme M reductase, component B and reduced cobalamin.

Purified methyl-CoM reductase of Methanobacterium thermoautotrophicum was found to catalyze the reduction of methyl-coenzyme M to methane at a specific rate of 100 nmol X min-1 X mg protein-1 in a system containing partially purified component B, cob(III)alamin (B-12a), and, as electron donors, dithiothreitol or SnCl2. Under these experimental conditions B-12a was reduced to cob(II)alamin (B-12r), which is known to disproportionate to cob(I)alamin (B-12s) and B-12a to a slight extent. Methane formation from methyl-CoM was inhibited by propyl iodide, methyl iodide, chloroform and carbon tetrachloride, which were shown to react with B-12s to the corresponding light-sensitive alkyl cobalamin. Inhibition by propyl iodide was less effective in light-exposed samples. From these findings it is concluded that in this assay system B-12s might serve as electron donor for the enzymatic reduction of methyl-CoM to methane.

Catalysis↗

PubMed1997

The particulate methane monooxygenase gene pmoA and its use as a functional gene probe for methanotrophs.

The particulate methane monooxygenase gene pmoA, encoding the 27 kDa polypeptide of the membrane-bound particulate methane monooxygenase, was amplified by PCR from DNA isolated from a blanket peat bog and from enrichment cultures established, from the same environment, using methane as sole carbon and energy source. The resulting 525 bp PCR products were cloned and a representative number of clones were sequenced. Phylogenetic analysis of the derived amino acid sequences of the pmoA clones retrieved directly from environmental DNA samples revealed that they form a distinct cluster within representative PmoA sequences from type II methanotrophs and may originate from a novel group of acidophilic methanotrophs. The study also demonstrated the utility of the pmoA gene as a phylogenetic marker for identifying methanotroph-specific DNA sequences in the environment.

DNA Probes↗

PubMed2001

Methane-consuming archaea revealed by directly coupled isotopic and phylogenetic analysis.

Microorganisms living in anoxic marine sediments consume more than 80% of the methane produced in the world's oceans. In addition to single-species aggregates, consortia of metabolically interdependent bacteria and archaea are found in methane-rich sediments. A combination of fluorescence in situ hybridization and secondary ion mass spectrometry shows that cells belonging to one specific archaeal group associated with the Methanosarcinales were all highly depleted in 13C (to values of -96 per thousand). This depletion indicates assimilation of isotopically light methane into specific archaeal cells. Additional microbial species apparently use other carbon sources, as indicated by significantly higher 13C/12C ratios in their cell carbon. Our results demonstrate the feasibility of simultaneous determination of the identity and the metabolic activity of naturally occurring microorganisms.

Anaerobiosis↗

PubMed2002

Microbial reefs in the Black Sea fueled by anaerobic oxidation of methane.

Massive microbial mats covering up to 4-meter-high carbonate buildups prosper at methane seeps in anoxic waters of the northwestern Black Sea shelf. Strong 13C depletions indicate an incorporation of methane carbon into carbonates, bulk biomass, and specific lipids. The mats mainly consist of densely aggregated archaea (phylogenetic ANME-1 cluster) and sulfate-reducing bacteria (Desulfosarcina/Desulfococcus group). If incubated in vitro, these mats perform anaerobic oxidation of methane coupled to sulfate reduction. Obviously, anaerobic microbial consortia can generate both carbonate precipitation and substantial biomass accumulation, which has implications for our understanding of carbon cycling during earlier periods of Earth's history.

Anaerobiosis↗

PubMed2004

Detection of methane in the atmosphere of Mars.

We report a detection of methane in the martian atmosphere by the Planetary Fourier Spectrometer onboard the Mars Express spacecraft. The global average methane mixing ratio is found to be 10 +/- 5 parts per billion by volume (ppbv). However, the mixing ratio varies between 0 and 30 ppbv over the planet. The source of methane could be either biogenic or nonbiogenic, including past or present subsurface microorganisms, hydrothermal activity, or cometary impacts.

Archaea↗

PubMed1997

Methane and trichloroethylene oxidation by an estuarine methanotroph, Methylobacter sp. strain BB5.1.

An estuarine methanotroph was isolated from sediment enrichments and designated Methylobacter sp. strain BB5.1. In cells grown on medium with added copper, oxidation of methane and trichloroethylene occurred with similar Ks values, but the Vmax for trichloroethylene oxidation was only 0.1% of the methane oxidation Vmax. Cells grown on low-copper medium did not oxidize trichloroethylene and showed a variable rate of methane oxidation.

Copper↗

PubMed1999

High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph.

Methanotrophic bacteria in an organic soil were enriched on gaseous mixing ratios of <275 parts per million of volume (ppmv) of methane (CH4). After 4 years of growth and periodic dilution (>10(20) times the initial soil inoculum), a mixed culture was obtained which displayed an apparent half-saturation constant [Km(app)] for CH4 of 56 to 186 nM (40 to 132 ppmv). This value was the same as that measured in the soil itself and about 1 order of magnitude lower than reported values for pure cultures of methane oxidizers. However, the Km(app) increased when the culture was transferred to higher mixing ratios of CH4 (1,000 ppmv, or 1%). Denaturing gradient gel electrophoresis of the enrichment grown on <275 ppmv of CH4 revealed a single gene product of pmoA, which codes for a subunit of particulate methane monooxygenase. This suggested that only one methanotroph species was present. This organism was isolated from a sample of the enrichment culture grown on 1% CH4 and phylogenetically positioned based on its 16S rRNA, pmoA, and mxaF gene sequences as a type II strain of the Methylocystis/Methylosinus group. A coculture of this strain with a Variovorax sp., when grown on <275 ppmv of CH4, had a Km(app) (129 to 188 nM) similar to that of the initial enrichment culture. The data suggest that the affinity of methanotrophic bacteria for CH4 varies with growth conditions and that the oxidation of atmospheric CH4 observed in this soil is carried out by type II methanotrophic bacteria which are similar to characterized species.

Culture Media↗

PubMed2000

Starvation alters the apparent half-saturation constant for methane in the type II methanotroph Methylocystis strain LR1.

When cells of a type II methanotrophic bacterium (Methylocystis strain LR1) were starved of methane, both the K(m(app)) and the V(max(app)) for methane decreased. The specific affinity (a(o)(s)) remained nearly constant. Therefore, the decreased K(m(app)) in starved cells was probably not an adjustment to better utilize low-methane concentrations.

Alphaproteobacteria↗

PubMed2002

Microbial communities from methane hydrate-bearing deep marine sediments in a forearc basin.

Microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35 degrees C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within the Euryarchaeota and Crenarchaeota domains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to the Bacteroidetes, Planctomycetes, Actinobacteria, Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments.

Anaerobiosis↗

PubMed2005

Abundance, activity, and community structure of pelagic methane-oxidizing bacteria in temperate lakes.

The abundance and activity of methane-oxidizing bacteria (MOB) in the water column were investigated in three lakes with different contents of nutrients and humic substances. The abundance of MOB was determined by analysis of group-specific phospholipid fatty acids from type I and type II MOB, and in situ activity was measured with a 14CH4 transformation method. The fatty acid analyses indicated that type I MOB most similar to species of Methylomonas, Methylomicrobium, and Methylosarcina made a substantial contribution (up to 41%) to the total bacterial biomass, whereas fatty acids from type II MOB generally had very low concentrations. The MOB biomass and oxidation activity were positively correlated and were highest in the hypo- and metalimnion during summer stratification, whereas under ice during winter, maxima occurred close to the sediments. The methanotroph biomass-specific oxidation rate (V) ranged from 0.001 to 2.77 mg CH4-C mg(-1) C day(-1) and was positively correlated with methane concentration, suggesting that methane supply largely determined the activity and biomass distribution of MOB. Our results demonstrate that type I MOB often are a large component of pelagic bacterial communities in temperate lakes. They represent a potentially important pathway for reentry of carbon and energy into pelagic food webs that would otherwise be lost as evasion of CH4.

Biomass↗

PubMed1997

Hydrogen regulation of growth, growth yields, and methane gene transcription in Methanobacterium thermoautotrophicum deltaH.

Changes in growth rate, methanogenesis, growth yield (Y(CH4)), and methane gene transcription have been correlated with changes in the supply of H2 to Methanobacterium thermoautotrophicum deltaH cells growing on H2 plus CO2 in fed-batch cultures. Under conditions of excess H2, biomass and methanogenesis increased exponentially and in parallel, resulting in cultures with a constant Y(CH4) and transcription of the mth and mrt genes that encode the H2-dependent N5,N10-methenyltetrahydromethanopterin (methenyl-H4MPT) reductase (MTH) and methyl coenzyme M reductase II (MRII), respectively. Reducing the H2 supply, by decreasing the percentage of H2 in the input gas mixture or by reducing the mixing speed of the fermentor impeller, decreased the growth rate and resulted in lower and constant rates of methanogenesis. Under such H2-limited growth conditions, cultures grew with a continuously increasing Y(CH4) and the mtd and mcr genes that encode the reduced coenzyme F420-dependent N5,N10-methenyl-H4MPT reductase (MTD) and methyl coenzyme M reductase I (MRI), respectively, were transcribed. Changes in the kinetics of growth, methanogenesis, and methane gene transcription directed by reducing the H2 supply could be reversed by restoring a high H2 supply. Methane production continued, but at a low and constant rate, and only mcr transcripts could be detected when the H2 supply was reduced to a level insufficient for growth. ftsA transcripts, which encode coenzyme F390 synthetase, were most abundant in cells growing with high H2 availability, consistent with coenzyme F390 synthesis signaling a high exogenous supply of reductant.

Bacteriological Techniques↗

PubMed1968

Hydrogen-oxidizing methane bacteria. I. Cultivation and methanogenesis.

A method for the mass culture of hydrogen-oxidizing methane bacteria has been developed; yields of 50 to 60 g (wet weight) of cells per 12 to 14 liters of culture medium were obtained. The methanogenic organism from the culture of Methanobacillus omelianskii was grown in a complex medium which was aerated with a gas mixture of hydrogen and carbon dioxide. Extracts prepared from hydrogen-grown cells formed methane from methyl cobalamin, 5-methyl tetrahydrofolate, serine, pyruvate, and carbon dioxide; these substrates have been shown to be precursors of methane in extracts of the ethyl alcohol-grown culture of M. omelianskii.

Bacteria↗