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Trace methane oxidation studied in several Euryarchaeota under diverse conditions.

We used (13)C-labeled methane to document the extent of trace methane oxidation by Archaeoglobus fulgidus, Archaeoglobus lithotrophicus, Archaeoglobus profundus, Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Methanosarcina acetivorans. The results indicate trace methane oxidation during growth varied among different species and among methanogen cultures grown on different substrates. The extent of trace methane oxidation by Mb. thermoautotrophicum (0.05 +/- 0.04%, +/- 2 standard deviations of the methane produced during growth) was less than that by M. barkeri (0.15 +/- 0.04%), grown under similar conditions with H(2) and CO(2). Methanosarcina acetivorans oxidized more methane during growth on trimethylamine (0.36 +/- 0.05%) than during growth on methanol (0.07 +/- 0.03%). This may indicate that, in M. acetivorans, either a methyltransferase related to growth on trimethylamine plays a role in methane oxidation, or that methanol is an intermediate of methane oxidation. Addition of possible electron acceptors (O(2), NO(3) (-), SO(4) (2-), SO(3) (2-)) or H(2) to the headspace did not substantially enhance or diminish methane oxidation in M. acetivorans cultures. Separate growth experiments with FAD and NAD(+) showed that inclusion of these electron carriers also did not enhance methane oxidation. Our results suggest trace methane oxidized during methanogenesis cannot be coupled to the reduction of these electron acceptors in pure cultures, and that the mechanism by which methane is oxidized in methanogens is independent of H(2) concentration. In contrast to the methanogens, species of the sulfate-reducing genus Archaeoglobus did not significantly oxidize methane during growth (oxidizing 0.003 +/- 0.01% of the methane provided to A. fulgidus, 0.002 +/- 0.009% to A. lithotrophicus and 0.003 +/- 0.02% to A. profundus). Lack of observable methane oxidation in the three Archaeoglobus species examined may indicate that methyl-coenzyme M reductase, which is not present in this genus, is required for the anaerobic oxidation of methane, consistent with the "reverse methanogenesis" hypothesis.

Carbon Dioxide↗

Field observations of methane concentrations and oxidation rates in the southeastern bering sea.

Measurements of methane oxidation rates were made in southeastern Bering Sea water samples with [C]methane. The rate at which CO(2) evolved from samples exposed to one methane concentration was defined as the relative methane oxidation rate. Rate determinations at three methane concentrations were used to estimate methane oxidation kinetics. The rate constant calculated from the kinetics and the observed methane concentration in the same water sample were used to calculate an in situ methane oxidation rate and the turnover time. First-order kinetics were observed in essentially all experiments in which methane oxidation kinetics were measured. Relative methane oxidation rates were greater in waters collected at inshore stations than at the offshore stations and were greater in bottom samples than in surface samples. In most water samples analyzed, there was essentially no radioactivity associated with the cells. The resulting respiration percentages were therefore very high with a mean of >98%. These data suggest that most of the methane was used by the microflora as an energy source and that very little of it was used in biosynthesis. The relative methane oxidation rates were not closely correlated with methane concentrations and did not appear to be linked to either oxygen or dissolved inorganic nitrogen concentrations. However, there was a significant correlation with relative microbial activity. Our data suggest that the methane oxidizers were associated with the general microbial heterotrophic community. Since these organisms did not appear to be using methane as a carbon source, it is unlikely that they have been isolated and identified as methane oxidizers in the past.

Journal Article↗

Carbohydrate malabsorption: quantification by methane and hydrogen breath tests.

BACKGROUND: Previous studies in small series of healthy adults have suggested that parallel measurement of hydrogen and methane resulting from gut fermentation may improve the precision of quantitative estimates of carbohydrate malabsorption. Systematic, controlled studies of the role of simultaneous hydrogen and methane measurements using end-expiratory breath test techniques are not available. METHODS: We studied seven healthy, adult methane and hydrogen producers and seven methane non-producers by means of end-expiratory breath test techniques. Breath gas concentrations and gastrointestinal symptoms were recorded at intervals for 12h after ingestion of 10, 20 and 30 g lactulose. RESULTS: In the seven methane producers the excretion pattern was highly variable; the integrated methane responses were disproportional and not reliably reproducible. However, quantitative estimates of carbohydrate malabsorption on the basis of individual areas under the methane and hydrogen excretion curves (AUCs) tended to improve in methane producers after ingestion of 20 g lactulose by simple addition of AUCs of methane to the AUCs of the hydrogen curves. Estimates were no more precise in methane producers than similar estimates in non-producers. Gastrointestinal symptoms increased significantly with increasing lactulose dose; correlation with total hydrogen and methane excretion was weak. CONCLUSIONS: Our study suggests that in methane producers, simple addition of methane and hydrogen excretion improves the precision of semiquantitative measurements of carbohydrate malabsorption. The status of methane production should, therefore, be known to interpret breath tests semiquantitatively. The weak correlation between hydrogen and methane excretion and gas-related abdominal complaints suggests that other factors than net production of these gases may be responsible for the symptoms.

Adult↗

Methane Oxidation by Nitrosococcus oceanus and Nitrosomonas europaea.

Chemolithotrophic ammonium-oxidizing and nitrite-oxidizing bacteria including Nitrosomonas europaea, Nitrosococcus oceanus, Nitrobacter sp., Nitiospina gracilis, and Nitrococcus mobilis were examined as to their ability to oxidize methane in the absence of ammonium or nitrite. All ammonium oxidizers tested had the ability to oxidize significant amounts of methane to CO(2) and incorporate various amounts into cellular components. None of the nitrite-oxidizing bacteria were capable of methane oxidation. The methane-oxidizing capabilities of Nitrosococcus oceanus and Nitrosomonas europaea were examined with respect to ammonium and methane concentrations, nitrogen source, and pH. The addition of ammonium stimulated both CO(2) production and cellular incorporation of methane-carbon by both organisms. Less than 0.1 mM CH(4) in solution inhibited the oxidation of ammonium by Nitrosococcus oceanus by 87%. Methane concentrations up to 1.0 mM had no inhibitory effects on ammonium oxidation by Nitrosomonas europaea. In the absence of NH(4)-N, Nitrosococcus oceanus achieved a maximum methane oxidation rate of 2.20 x 10 mumol of CH(4) h mg (dry weight) of cells, which remained constant as the methane concentration was increased. In the presence of NH(4)-N (10 ppm [10 mug/ml]), its maximum rate was 26.4 x 10 mumol of CH(4) h mg (dry weight) of cells at a methane concentration of 1.19 x 10 mM. Increasing the methane concentration above this level decreased CO(2) production, whereas cellular incorporation of methane-carbon continued to increase. Nitrosomonas europaea showed a linear response throughout the test range, with an activity of 196.0 x 10 mumol of CH(4) h mg (dry weight) of cells at a methane concentration of 1.38 x 10 mM. Both nitrite and nitrate stimulated the oxidation of methane. The pH range was similar to that for ammonium oxidation, but the points of maximum activity were at lower values for the oxidation of methane.

Journal Article↗

Methanol promotes atmospheric methane oxidation by methanotrophic cultures and soils.

Two methanotrophic bacteria, Methylobacter albus BG8 and Methylosinus trichosporium OB3b, oxidized atmospheric methane during batch growth on methanol. Methane consumption was rapidly and substantially diminished (95% over 9 days) when washed cell suspensions were incubated without methanol in the presence of atmospheric methane (1.7 ppm). Methanotrophic activity was stimulated after methanol (10 mM) but not methane (1,000 ppm) addition. M. albus BG8 grown in continuous culture for 80 days with methanol retained the ability to oxidize atmospheric methane and oxidized methane in a chemostat air supply. Methane oxidation during growth on methanol was not affected by methane deprivation. Differences in the kinetics of methane uptake (apparent K(m) and V(max)) were observed between batch- and chemostat-grown cultures. The V(max) and apparent K(m) values (means +/- standard errors) for methanol-limited chemostat cultures were 133 +/- 46 nmol of methane 10 cells h and 916 +/- 235 ppm of methane (1.2 muM), respectively. These values were significantly lower than those determined with batch-grown cultures (V(max) of 648 +/- 195 nmol of methane 10 cells h and apparent K(m) of 5,025 +/- 1,234 ppm of methane [6.3 muM]). Methane consumption by soils was stimulated by the addition of methanol. These results suggest that methanol or other nonmethane substrates may promote atmospheric methane oxidation in situ.

Journal Article↗

Stability of human methanogenic flora over 35 years and a review of insights obtained from breath methane measurements.

BACKGROUND & AIMS: Breath methane measurements reflect the in situ activity of the methanogenic colonic flora. Thirty-five years ago we found that 34% of healthy adult subjects were methane producers (breath methane level >1 ppm above atmospheric methane levels). The current study presents a new survey of breath methane levels designed to determine if the activity of the methanogenic flora has changed over the past 35 years. In addition, we review insights into the methanogenic flora that have resulted from breath methane measurements. METHODS: The end-alveolar breath methane concentrations of 212 healthy adults living in the Minneapolis area were determined via gas chromatography. The influence of sex, age, and bowel movement frequency on methane production was assessed. RESULTS: The findings that 36.4% of participants were methane producers, with a mean methane concentration in these producers of 16.6 ppm, are strikingly similar to the values of 33.6% and 15.2 ppm observed 35 years ago. Neither sex nor age showed a statistically significant relationship to methane production. There was a negative correlation between frequency of bowel movements and breath methane concentration in methane producers. CONCLUSIONS: The activity of the methanogenic flora of healthy adults remained remarkably stable over the past 35 years despite widespread antibiotic use and dietary changes. A literature review revealed that many associations have been shown between methane production and clinical states, but it remains to be determined if methanogens actively influence human physiology or are simply a marker of colonic function.

Adult↗

Kinetics of microbial landfill methane oxidation in biofilters.

A methane oxidizing biofilter system fitted to the passive venting system of a harbor sludge landfill in Germany was characterized with respect to the the methanotrophic population, methane oxidizing capacity, and reaction kinetics. Methanotrophic cell counts stabilized on a high level with 1.3 x 10(8) to 7.1 x 10(9) cells g dw(-1) about one year after first biofilter operation, and a maximum of 1.2 x 10(11) cells g dw(-1). Potential methane oxidizing activity varied between 5.3 and 10.7 microg h(-1) g dw(-1). Cell numbers correlated well with methane oxidation activities. Extrapolation of potential activities gave methane removal rates between 35 and 109 g CH4 h(-1) m(-3), calculated for 30 degrees C. Optimum temperature was 38 degrees C for freshly sampled biofilter material and 22 degrees C for a methanotrophic enrichment culture grown at 10 degrees C incubation temperature. Substrate kinetics revealed the presence of a low-affinity methane oxidizing community with a high Vmax of 1.78 micromol CH4 h(-1) g ww(-1) and a high K(M) of 15.1 microM. K(MO2) for methane oxidation was 58 microM. No substantial methane oxidizing activity was detected below 1.7-2.6 vol.-% O2 in the gaseous phase. Methane deprivation led to a decrease in methane oxidation activity within 5-9 weeks but could still be detected after 25 weeks of substrate deprivation and was fully restored within 3 weeks of continuous methane supply. Very high salt loads are leached from the novel biofilter material, expanded clay, yielding electric conductivity values of up to 15 mS cm(-1) in the leachate. Values > 6 mS cm(-1) were shown to depress methane consumption. Water retention characteristics of the material proved to be favourable for methane oxidizing systems with a gas permeable volume of 78% of bulk volume at field capacity water content. Correspondingly, no influence of water content on methane oxidation activity could be detected at water contents between 2.5 and 20 vol.-%.

Biodegradation, Environmental↗

Seasonal study of methane oxidation in lake washington.

The distribution of methane and methane-oxidizing bacteria in the water column of Lake Washington was determined monthly for 1 year. The methane profiles were relatively constant, with little stratification and low concentrations (0.05 to 0.5 muM). The number of methane-oxidizing bacteria detected by a filter-plating method was routinely <1/ml throughout the water column, and no incorporation or oxidation of methane was detected by radioisotopic labeling, even after methane was added. However, samples taken from the sediment-water interface contained as much as 3 muM methane and 50 CFU of methane-oxidizing bacteria per ml and showed significant rates of methane oxidation and incorporation. To define the region of maximum activity more precisely, vertical profiles of the sediment were examined. The concentration of methane increased with depth to a maximum of 150 to 325 muM at 2.5 cm, and significant rates of methane oxidation were found within the top 2.5 cm. The apparent K(m)s for methane and oxygen were determined for samples from the top 1.0 cm of the sediment and found to be ca. 10 and 20 muM, respectively. Projected values for methane oxidation rates suggested that maximum methane oxidation occurred in the top 0.5 cm of the sediment.

Journal Article↗

Distribution and rate of methane oxidation in sediments of the Florida everglades.

Rates of methane emission from intact cores were measured during anoxic dark and oxic light and dark incubations. Rates of methane oxidation were calculated on the basis of oxic incubations by using the anoxic emissions as an estimate of the maximum potential flux. This technique indicated that methane oxidation consumed up to 91% of the maximum potential flux in peat sediments but that oxidation was negligible in marl sediments. Oxygen microprofiles determined for intact cores were comparable to profiles measured in situ. Thus, the laboratory incubations appeared to provide a reasonable approximation of in situ activities. This was further supported by the agreement between measured methane fluxes and fluxes predicted on the basis of methane profiles determined by in situ sampling of pore water. Methane emissions from peat sediments, oxygen concentrations and penetration depths, and methane concentration profiles were all sensitive to light-dark shifts as determined by a combination of field and laboratory analyses. Methane emissions were lower and oxygen concentrations and penetration depths were higher under illuminated than under dark conditions; the profiles of methane concentration changed in correspondence to the changes in oxygen profiles, but the estimated flux of methane into the oxic zone changed negligibly. Sediment-free, root-associated methane oxidation showed a pattern similar to that for methane oxidation in the core analyses: no oxidation was detected for roots growing in marl sediment, even for roots of Cladium jamaicense, which had the highest activity for samples from peat sediments. The magnitude of the root-associated oxidation rates indicated that belowground plant surfaces may not markedly increase the total capacity for methane consumption. However, the data collectively support the notion that the distribution and activity of methane oxidation have a major impact on the magnitude of atmospheric fluxes from the Everglades.

Journal Article↗

Biogeochemistry and microbial ecology of methane oxidation in anoxic environments: a review.

Evidence supporting a key role for anaerobic methane oxidation in the global methane cycle is reviewed. Emphasis is on recent microbiological advances. The driving force for research on this process continues to be the fact that microbial communities intercept and consume methane from anoxic environments, methane that would otherwise enter the atmosphere. Anaerobic methane oxidation is biogeochemically important because methane is a potent greenhouse gas in the atmosphere and is abundant in anoxic environments. Geochemical evidence for this process has been observed in numerous marine sediments along the continental margins, in methane seeps and vents, around methane hydrate deposits, and in anoxic waters. The anaerobic oxidation of methane is performed by at least two phylogenetically distinct groups of archaea, the ANME-1 and ANME-2. These archaea are frequently observed as consortia with sulfate-reducing bacteria, and the metabolism of these consortia presumably involves a syntrophic association based on interspecies electron transfer. The archaeal member of a consortium apparently oxidizes methane and shuttles reduced compounds to the sulfate-reducing bacteria. Despite recent advances in understanding anaerobic methane oxidation, uncertainties still remain regarding the nature and necessity of the syntrophic association, the biochemical pathway of methane oxidation, and the interaction of the process with the local chemical and physical environment. This review will consider the microbial ecology and biogeochemistry of anaerobic methane oxidation with a special emphasis on the interactions between the responsible organisms and their environment.

Anaerobiosis↗

Pulmonary hydrogen and methane excretion following ingestion of an unabsorbable carbohydrate: a study of twins.

Pulmonary excretion of hydrogen and methane after administration of an unabsorbable disaccharide (lactulose) was determined in 228 adult Hungarian twins, 60 monozygous (MZ) and 54 dizygous (DZ) pairs. More than 98% of the subjects (224 of 228) excreted large amounts of hydrogen between 90 and 180 min after lactulose administration. Methane excretion in the fasting state was observed in 124 of 228 of the probands (54.4%), and 68 of 228 (29.8%) produced additional methane in response to lactulose ingestion. In contrast to hydrogen production, both methane excretion and production were significantly more frequent in females than in males. In the total group, and more distinctly in females, the correlation between peak hydrogen and methane concentrations was negative. Twin concordance of fasting methane excretion and lactulose-induced methane production was near 70% in both MZ and DZ pairs. Heritability estimates of methane excretion and production based on intrapair correlation and variance were smaller than unity, and intrapair correlation coefficients were larger in twin pairs living apart than in those living in the same household. Methane excretion is comparatively frequent in the Hungarian population, and a substantial proportion of fasting methane excreters (55%) produce additional methane from lactulose. The sex difference of methane excretion appears to be characteristic of European populations. The twin data disprove regular Mendelian inheritance of methane production and are suggestive of genetic effects in a multifactorial system.

Adult↗

Patterns of methane production in a Burmese (Myanmar) population.

While up to 50% of Western populations produce methane, this is less than that of rural black Africans and there is no information on methane production in populations from Asian developing countries. Females consistently produce methane more commonly than males, and methane production in children under the age of five years, except in Nigeria, is unusual. Breath methane was sampled in 1426 subjects from Myanmar ranging in age from 1 month to 88 years, with a mean age of 26.2 years. Half (49.8%) of the Myanmar population produced methane, this figure comprising 53% of females and 46% of males sampled. Methane production increases with age and reaches adult levels after 10 years of age. A high prevalence of methane production was found in children under 3 years of age (15.8%). Methane production was absent in 13 solely breast-fed children and increased as other food was introduced into the diet. There was an association of methane production within families and with smoking. The prevalence of methane production increased in male and female smokers, with 75% of smokers producing methane. Methane production was not associated with occupation, education, income, water source, latrine type, previous diarrhoea, antibiotic usage or socio-economic status.

Adolescent↗

Characterization of methanotrophic bacterial populations in soils showing atmospheric methane uptake.

The global methane cycle includes both terrestrial and atmospheric processes and may contribute to feedback regulation of the climate. Most oxic soils are a net sink for methane, and these soils consume approximately 20 to 60 Tg of methane per year. The soil sink for atmospheric methane is microbially mediated and sensitive to disturbance. A decrease in the capacity of this sink may have contributed to the approximately 1%. year(-1) increase in the atmospheric methane level in this century. The organisms responsible for methane uptake by soils (the atmospheric methane sink) are not known, and factors that influence the activity of these organisms are poorly understood. In this study the soil methane-oxidizing population was characterized by both labelling soil microbiota with (14)CH(4) and analyzing a total soil monooxygenase gene library. Comparative analyses of [(14)C]phospholipid ester-linked fatty acid profiles performed with representative methane-oxidizing bacteria revealed that the soil sink for atmospheric methane consists of an unknown group of methanotrophic bacteria that exhibit some similarity to type II methanotrophs. An analysis of monooxygenase gene libraries from the same soil samples indicated that an unknown group of bacteria belonging to the alpha subclass of the class Proteobacteria was present; these organisms were only distantly related to extant methane-oxidizing strains. Studies on factors that affect the activity, population dynamics, and contribution to global methane flux of "atmospheric methane oxidizers" should be greatly facilitated by use of biomarkers identified in this study.

Amino Acid Sequence↗

Characterization of C1-metabolizing prokaryotic communities in methane seep habitats at the Kuroshima Knoll, southern Ryukyu Arc, by analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA genes.

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (approximately 34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the delta-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 x 10(6) copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 x 10(6) copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in gamma-Proteobacteria and had a high copy number of mxaF (7.1 x 10(6) copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 x 10(2) copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C1 metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.

Alcohol Oxidoreductases↗