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Methane influences infrared technique anesthetic agent monitors.

OBJECTIVE: During closed-circuit anesthesia, anesthetic vapor analysis by infrared absorption at 3.3 microm can be influenced by the concentration of accumulated methane, resulting in inaccurate readings of anesthetic concentrations. The current study examined the influence of different known methane concentrations on the analysis of halothane or isoflurane concentrations by the infrared absorption technique. METHODS: Three different gas mixtures containing 100, 500 and 1000 ppm methane were given through an experimental sampling bar. Four infrared technique anesthetic agent monitors were examined: (1) the Ultima (Datex), (2) the Andros analyzer (Cato anesthesia machine, Driger), (3) the anesthetic gas monitor 1304 (Brüel & Kjaer) and (4) the mainstream analyzer Irina (Drager). All devices, except the Brüel & Kjaer anesthetic gas monitor, function at 3.3 microm wavelength. The Brüel & Kjaer apparatus functions at 10.3-13 microm wavelength. The readings were recorded with and without addition of halothane (or isoflurane) at a halothane (or an isoflurane) dedicated sensitivity after application of methane. RESULTS: At the two highest methane concentrations (500 and 1000 ppm) all studied devices except the Brüel & Kjaer anesthetic gas monitor 1304 displayed inaccurate anesthetic concentrations. This was more pronounced at halothane than at isoflurane sensitivity. Introduction of halothane (0.8%) or isoflurane (0.8%) vapor into the experimental sampling bar resulted in values that were additive to the falsely recorded ones. CONCLUSIONS: In closed circuit or low-flow anesthesia, in which methane can accumulate, infrared measuring techniques for potent inhalation anesthetics that do not use the 3.3 microm wavelength appear to be preferable.

Anesthesia, Closed-Circuit↗

Colonic hydrogen elimination and methane production in infants with and without infantile colic syndrome.

Our objective was to investigate the relationship between demographic factors, nutrition, stool gas production, and the existence of infantile colic (IC) syndrome. Hydrogen and methane production from stool specimens of infants with and without infantile colic was quantified at two separate time points, the age at presentation of colic (<12 weeks) and at >6 months of age. The relationship between demographic variables and IC was also studied. A total of 59 infants with ages ranging from 2 to 12 weeks were enrolled in the study. Of these, 30 infants developed symptoms of colic. No correlation was found between IC and birth weight, gestational age, sex, type of feeding, mean time of feeding, stool frequency, and consistency. There was also no correlation between IC and the parents' age or education or the infant's number of siblings. Analysis of the stool samples revealed that methane was produced at concentrations >2 ppm by 15.3% of the infants at age <3 months and by 46.4% of infants at age >6 months. The mean methane concentrations produced by stool increased with age (0.95 +/- 0.58 ppm at 3 months of age vs 1.29 +/- 0.65 ppm at 6 months of age. There was no difference in stool hydrogen concentration between infants with and without IC. In contrast, the mean methane level at 3 and 6 months of age was higher in infants without IC than with IC, but reached statistical significance only at 6 months of age (0.97 +/- 0.68 vs 0.93 +/- 0.46) (NS) at 3 months of age, and 1.56 +/- 0.55 vs 0.93 +/- 0.62 (P < 0.05) at 6 months of age respectively. Furthermore, infants that produced higher methane levels at 3 and 6 months of age had significantly (p < 0.05) less colic in the first months of life. In conclusions, methane production may play a role in the alleviation of IC. Future studies are needed to confirm our findings.

Colic↗

Methane-consuming archaebacteria in marine sediments.

Large amounts of methane are produced in marine sediments but are then consumed before contacting aerobic waters or the atmosphere. Although no organism that can consume methane anaerobically has ever been isolated, biogeochemical evidence indicates that the overall process involves a transfer of electrons from methane to sulphate and is probably mediated by several organisms, including a methanogen (operating in reverse) and a sulphate-reducer (using an unknown intermediate substrate). Here we describe studies of sediments related to a decomposing methane hydrate. These provide strong evidence that methane is being consumed by archaebacteria that are phylogenetically distinct from known methanogens. Specifically, lipid biomarkers that are commonly characteristic of archaea are so strongly depleted in carbon-13 that methane must be the carbon source, rather than the metabolic product, for the organisms that have produced them. Parallel gene surveys of small-subunit ribosomal RNA (16S rRNA) indicate the predominance of a new archael group which is peripherally related to the methanogenic orders Methanomicrobiales and Methanosarcinales.

Archaea↗

A marine microbial consortium apparently mediating anaerobic oxidation of methane.

A large fraction of globally produced methane is converted to CO2 by anaerobic oxidation in marine sediments. Strong geochemical evidence for net methane consumption in anoxic sediments is based on methane profiles, radiotracer experiments and stable carbon isotope data. But the elusive microorganisms mediating this reaction have not yet been isolated, and the pathway of anaerobic oxidation of methane is insufficiently understood. Recent data suggest that certain archaea reverse the process of methanogenesis by interaction with sulphate-reducing bacteria. Here we provide microscopic evidence for a structured consortium of archaea and sulphate-reducing bacteria, which we identified by fluorescence in situ hybridization using specific 16S rRNA-targeted oligonucleotide probes. In this example of a structured archaeal-bacterial symbiosis, the archaea grow in dense aggregates of about 100 cells and are surrounded by sulphate-reducing bacteria. These aggregates were abundant in gas-hydrate-rich sediments with extremely high rates of methane-based sulphate reduction, and apparently mediate anaerobic oxidation of methane.

Anaerobiosis↗

Methane synthesis without the addition of adenosine triphosphate by cell membranes isolated from Methanobacterium ruminantium.

The membrane fraction isolated from broken cells of Methanobacterium ruminantium actively synthesized methane from CO2 and H2 without the addition of ATP or other cofactors. This activity was lost unless strictly anaerobic conditions were maintained throughout the isolation and incubation procedures. 3H2, but not 3H2O, was readily incorporated into methane. This indicates that hydrogen atoms are used in the formation of methane without the prior equilibration of protons with the water phase. Methylenetetrahydrofolate was shown to be converted into methane, but less efficiently than CO2. The evidence indicates that tetrahydrofolate derivatives may not be of primary importance in the formation of methane from CO2 and H2. No requirement for ATP in methanogenesis could be demonstrated. However, chemical reagents that can increase proton conductance in membranes and therby abolish the membrane electrical potential were also effective inhibitors of methanogenesis. It was postulated that, although the reduction of CO2 to methane by bacterial membranes may require energy derived from a transmembrane potential, this does not appear to be coupled to the intermediary synthesis of ATP.

Adenine Nucleotides↗

Methane-induced haemolysis of human erythrocytes.

Human erythrocytes were exposed to high concentrations of methane and nitrogen through the application of elevated partial pressures of these gas molecules. Cell leakage (haemolysis) was measured for cells exposed to these gases under a wide range of experimental conditions. Application of methane produces haemolysis at pressures far below the hydrostatic pressures known to disrupt membrane or protein structure. The effects of changes in buffer, temperature, diffusion rate and detergents were studied. Methane acts co-operatively with detergents to produce haemolysis at much lower detergent concentration than is required in the absence of methane or in the presence of nitrogen. At sufficiently high concentrations of methane, all cells are haemolysed. Increased temperature enhances the effect. Methane produces 50% haemolysis at a concentration of about 0.33 M compared with about 7.5 M methanol required for the same degree of haemolysis.

Buffers↗

Evidence for large methane releases to the atmosphere from deep-sea gas-hydrate dissociation during the last glacial episode.

Past atmospheric methane-concentration oscillations recorded in polar ice cores vary together with rapid global climatic changes during the last glacial episode. In the "clathrate gun hypothesis," massive releases of deep-sea methane from marine gas-hydrate dissociation led to these well known, global, abrupt warmings in the past. If evidence for such releases in the water column exists, however, the mechanism and eventual transfer to the atmosphere has not yet been documented clearly. Here we describe a high-resolution marine-sediment record of stable carbon isotopic changes from the Papua Gulf, off Papua New Guinea, which exhibits two extremely depleted excursions (down to -9 per thousand ) at approximately 39,000 and approximately 55,000 years. Morphological, isotopic, and trace metal evidence dismisses authigenic calcite as the main source of depleted carbon. Massive methane release associated with deep-sea gas-hydrate dissociation is the most likely cause for such large depletions of delta(13)C. The absence of a delta(13)C gradient in the water column during these events implies that the methane rose through the entire water column, reaching the sea-air interface and thus the atmosphere. Foraminiferal delta(18)O composition suggests that the rise of the methane in the water column created an upwelling flow. These inferred emission events suggest that during the last glacial episode, this process was likely widespread, including tropical regions. Thus, the release of methane from the ocean floor into the atmosphere cannot be dismissed as a strong positive feedback in climate dynamics processes.

Air↗

An unconventional pathway for reduction of CO2 to methane in CO-grown Methanosarcina acetivorans revealed by proteomics.

Methanosarcina acetivorans produces acetate, formate, and methane when cultured with CO as the growth substrate [Rother M, Metcalf WW (2004) Proc Natl Acad Sci USA 101:], which suggests novel features of CO metabolism. Here we present a genome-wide proteomic approach to identify and quantify proteins differentially abundant in response to growth on CO versus methanol or acetate. The results indicate that oxidation of CO to CO2 supplies electrons for reduction of CO2 to a methyl group by steps and enzymes of the pathway for CO2 reduction determined for other methane-producing species. However, proteomic and quantitative RT-PCR results suggest that reduction of the methyl group to methane involves novel methyltransferases and a coenzyme F420H2:heterodisulfide oxidoreductase system that generates a proton gradient for ATP synthesis not previously described for pathways reducing CO2 to methane. Biochemical assays support a role for the oxidoreductase, and transcriptional mapping identified an unusual operon structure encoding the oxidoreductase. The proteomic results further indicate that acetate is synthesized from the methyl group and CO by a reversal of initial steps in the pathway for conversion of acetate to methane that yields ATP by substrate level phosphorylation. The results indicate that M. acetivorans utilizes a pathway distinct from all known CO2 reduction pathways for methane formation that reflects an adaptation to the marine environment. Finally, the pathway supports the basis for a recently proposed primitive CO-dependent energy-conservation cycle that drove and directed the early evolution of life on Earth.

Acetates↗

Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments.

No microorganism capable of anaerobic growth on methane as the sole carbon source has yet been cultivated. Consequently, information about these microbes has been inferred from geochemical and microbiological observations of field samples. Stable isotope analysis of lipid biomarkers and rRNA gene surveys have implicated specific microbes in the anaerobic oxidation of methane (AOM). Here we use combined fluorescent in situ hybridization and secondary ion mass spectrometry analyses, to identify anaerobic methanotrophs in marine methane-seep sediments. The results provide direct evidence for the involvement of at least two distinct archaeal groups (ANME-1 and ANME-2) in AOM at methane seeps. Although both archaeal groups often occurred in direct physical association with bacteria, they also were observed as monospecific aggregations and as single cells. The ANME-1 archaeal group more frequently existed in monospecific aggregations or as single filaments, apparently without a bacterial partner. Bacteria associated with both archaeal groups included, but were not limited to, close relatives of Desulfosarcina species. Isotopic analyses suggest that monospecific archaeal cells and cell aggregates were active in anaerobic methanotrophy, as were multispecies consortia. In total, the data indicate that the microbial species and biotic interactions mediating anaerobic methanotrophy are diverse and complex. The data also clearly show that highly structured ANME-2/Desulfosarcina consortia are not the sole entities responsible for AOM at marine methane seeps. Other microbial groups, including ANME-1 archaea, are capable of anaerobic methane consumption either as single cells, in monospecific aggregates, or in multispecies consortia.

Archaea↗

Methane production by methanogens following an aerobic washing procedure: simplifying methods for manipulation.

The recent discovery of methane in the martian atmosphere is arguably one of the most important discoveries in the field of astrobiology. One possible source of this methane could be a microorganism analogous to those on Earth in the domain Archaea known as methanogens. Methanogens are described as obligately anaerobic, and methods developed to work with methanogens typically include anaerobic media and buffers, gassing manifolds, and possibly anaerobic chambers. To determine if the time, effort, and supplies required to maintain anaerobic conditions are necessary to maintain viability, we compared anaerobically washed cells with cells that were washed in the presence of atmospheric oxygen. Anaerobic tubes were opened, and cultures were poured into plastic centrifuge tubes, centrifuged, and suspended in fresh buffer, all in the presence of atmospheric oxygen. Washed cells from both aerobic and anaerobic procedures were inoculated into methanogenic growth media under anaerobic conditions and incubated at temperatures conducive to growth for each methanogenic strain tested. Methane production was measured at time intervals using a gas chromatograph. In three strains, significant differences were not seen between aerobically and anaerobically washed cells. In one strain, there was significantly less methane production observed following aerobic washing at some time points; however, substantial methane production occurred following both procedures. Thus, it appears that aerobic manipulations for relatively short periods of time with at least a few species of methanogens may not lead to loss of viability. With the discovery of methane in the martian atmosphere, it is likely that there will be an increase in astrobiology-related methanogen research. The research reported here should simplify the methodology.

Aerobiosis↗

Correlation of hydrogen and methane production to rice carbohydrate malabsorption in Burmese (Myanmar) children.

Rice carbohydrate malabsorption is common in Burmese village children and adults and may contribute to diminished growth. Its diagnosis depends on a rice breath hydrogen test, which has limitations. Almost 20% of Burmese children under age 5 produce methane, compared with less than 7% of children in Africa and Hong Kong. If an increased carbohydrate load in the colon due to rice malabsorption provides increased substrate for methanogenic bacteria in the left colon, higher fasting breath methane concentrations might be a simpler method of diagnosing rice malabsorption. We tested breath hydrogen and methane over a 4-h period and did anthropometric measurements in 142 subjects, 79 children, and 63 adults. Seventy percent of children were rice-malabsorbers. Methane production occurred in 20% of children under 5 years of age and increased to 60% of adults. There is an association of rice malabsorption with reduced length. There was not correlation between rice malabsorption and breath methane, and the concentration of breath methane does not, therefore, indicate rice absorption status and cannot replace rice breath hydrogen tests.

Adult↗

Breath methane associated with slow colonic transit time in children with chronic constipation.

OBJECTIVE: This study analyzed the relationship between methane production and colonic transit time in children with chronic constipation. METHODOLOGY: Forty children, from 3 to 13 years of age, suffering from chronic constipation were included. Methane production was defined when the breath methane concentration was greater than 3 ppm. The total and segmental colonic transit times were measured with radio-opaque markers. RESULTS: Soiling was present in 34 (85.0%) of 40 patients with constipation. Methane production was present in 25 of 34 (73.5%) patients with constipation and soiling and only in 1 (16.7%) of 6 with constipation but without soiling (P = 0.014). The medians of total colonic transit time were 80.5 and 61.0 hours, respectively (P = 0.04), in methane and nonmethane producers. Segmental colonic transit times were 17.5 and 10.5 hours, respectively (P = 0.580), in right colon, 29.5 and 10.5 hours (P = 0.001), respectively, in left colon, and 31.5 and 27.0 hours (P = 0.202), respectively, in the rectosigmoid. By the sixth week of treatment, the reduction in the total colonic transit time was greater in patients who had become nonmethane producers. CONCLUSION: The presence of breath methane in children with chronic constipation may suggest the possibility of prolonged colonic transit time.

Adolescent↗

Coupling of ATP synthesis and methane formation from methanol and molecular hydrogen in Methanosarcina barkeri.

The addition of methanol to a cell suspension of Methanosarcina barkeri resulted in an increase of the intracellular ATP concentration from 1 nmol/mg to 10 nmol/mg protein and in the formation of a proton-motive force delta p of -130 mV. delta p consisted of more than 90% of the membrane potential delta psi. These values were similar under N2 and under H2. The addition of the uncoupler tetrachlorosalicylanilide to the above system under N2 led to a drastic decrease of both, the ATP concentration and the delta p and to a stop of methanogenesis. With methanol and H2, however, methane formation continued, although the effect of the uncoupler on the ATP pool and on delta p was a under N2. The proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide caused a rapid exhaustion of the ATP pool and a discontinuation of methane synthesis, whereas delta p was unaffected. Inhibition of methane formation under these conditions could be relieved by the addition of the uncoupler tetrachlorosalicylanilide. These results demonstrate that methane formation according to the equation CH3OH + H2----H2----CH4 + H2O was coupled to ATP synthesis by a chemiosmotic mechanism and was under the control of delta psi: Methane formation only proceeded if the delta psi generated was used for ATP synthesis or if an uncoupler was present. Under N2, methane formation according to the equation 4CH3OH ----CO2 + 3CH4 + 2H2O was abolished by an uncoupler, because one step in the oxidation of methanol to 1 CO2 apparently depended on an energized state of the membrane.

Adenosine Triphosphate↗

Environmental regulation of the anaerobic oxidation of methane: a comparison of ANME-I and ANME-II communities.

The anaerobic oxidation of methane (AOM) is one of the major sinks for methane on earth and is known to be mediated by at least two phylogenetically different groups of anaerobic methanotrophic Archaea (ANME-I and ANME-II). We present the first comparative in vitro study of the environmental regulation and physiology of these two methane-oxidizing communities, which occur naturally enriched in the anoxic Black Sea (ANME-I) and at Hydrate Ridge (ANME-II). Both types of methanotrophic communities are associated with sulfate-reducing-bacteria (SRB) and oxidize methane anaerobically in a 1:1 ratio to sulfate reduction (SR). They responded sensitively to elevated methane partial pressures with increased substrate turnover. The ANME-II-dominated community showed significantly higher cell-specific AOM rates. Besides sulfate, no other electron acceptor was used for AOM. The processes of AOM and SR could not be uncoupled by feeding the SRB with electron donors such as acetate, formate or molecular hydrogen. AOM was completely inhibited by the addition of bromoethanesulfonate in both communities, indicating the participation of methanogenic enzymes in the process. Temperature influenced the intensity of AOM, with ANME-II being more adapted to cold temperatures than ANME-I. The variation of other environmental parameters, such as sulfate concentration, pH and salinity, did not influence the activity of both communities. In conclusion, the ecological niches of methanotrophic Archaea seem to be mainly defined by the availability of methane and sulfate, but it remains open which additional factors lead to the dominance of ANME-I or -II in the environment.

Anaerobiosis↗

Abundance, distribution and potential activity of methane oxidizing bacteria in permafrost soils from the Lena Delta, Siberia.

The methane oxidation potential of active layer profiles of permafrost soils from the Lena Delta, Siberia, was studied with regard to its respond to temperature, and abundance and distribution of type I and type II methanotrophs. Our results indicate vertical shifts within the optimal methane oxidation temperature and within the distribution of type I and type II methanotrophs. In the upper active layer, maximum methane oxidation potentials were detected at 21 degrees C. Deep active layer zones that are constantly exposed to temperatures below 2 degrees C showed a maximum potential to oxidize methane at 4 degrees C. Our results indicate a dominance of psychrophilic methanotrophs close to the permafrost table. Type I methanotrophs dominated throughout the active layer profiles but their number strongly fluctuated with depth. In contrast, type II methanotrophs were constantly abundant through the whole active layer and displaced type I methanotrophs close to the permafrost table. No correlation between in situ temperatures and the distribution of type I and type II methanotrophs was found. However, the distribution of type I and type II methanotrophs correlated significantly with in situ methane concentrations. Beside vertical fluctuations, the abundance of methane oxidizers also fluctuated according to different geomorphic units. Similar methanotroph cell counts were detected in samples of a flood plain and a polygon rim, whereas cell counts in samples of a polygon centre were up to 100 times lower.

Arctic Regions↗

Diversity and abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea.

The Black Sea, with its highly sulfidic water column, is the largest anoxic basin in the world. Within its sediments, the mineralization of organic matter occurs essentially through sulfate reduction and methanogenesis. In this study, the sulfate-reducing community was investigated in order to understand how these microorganisms are distributed relative to the chemical zonation: in the upper sulfate zone, at the sulfate-methane transition zone, and deeply within the methane zone. Total bacteria were quantified by real-time PCR of 16S rRNA genes whereas sulfate-reducing microorganisms (SRM) were quantified by targeting their metabolic key gene, the dissimilatory (bi)sulfite reductase (dsrA). Sulfate-reducing microorganisms were predominant in the sulfate zone but occurred also in the methane zone, relative proportion was maximal around the sulfate-methane transition, c. 30%, and equally high in the sulfate and methane zones, 5-10%. The dsrAB clone library from the sulfate-methane transition zone, showed mostly sequences affiliated with the Desulfobacteraceae. While, the dsrAB clone libraries from the upper, sulfate-rich zone and the deep, sulfate-poor zone were dominated by similar, novel deeply branching sequences which might represent Gram-positive spore-forming sulfate- and/or sulfite-reducing microorganisms. We thus hypothesize that terminal carbon mineralization in surface sediments of the Black Sea is largely due to the sulfate reduction activity of previously hidden SRM. Although these novel SRM were also abundant in sulfate-poor, methanogenic areas of the Black Sea sediment, their activities and possibly very versatile metabolic capabilities remain subject of further study.

Colony Count, Microbial↗

Cultivation of methanotrophic bacteria in opposing gradients of methane and oxygen.

In sediments, methane-oxidizing bacteria live in opposing gradients of methane and oxygen. In such a gradient system, the fluxes of methane and oxygen are controlled by diffusion and consumption rates, and the rate-limiting substrate is maintained at a minimum concentration at the layer of consumption. Opposing gradients of methane and oxygen were mimicked in a specific cultivation set-up in which growth of methanotrophic bacteria occurred as a sharp band at either c. 5 or 20 mm below the air-exposed end. Two new strains of methanotrophic bacteria were isolated with this system. One isolate, strain LC 1, belonged to the Methylomonas genus (type I methantroph) and contained soluble methane mono-oxygenase. Another isolate, strain LC 2, was related to the Methylobacter group (type I methantroph), as determined by 16S rRNA gene and pmoA sequence similarities. However, the partial pmoA sequence was only 86% related to cultured Methylobacter species. This strain accumulated significant amounts of formaldehyde in conventional cultivation with methane and oxygen, which may explain why it is preferentially enriched in a gradient cultivation system.

Bacteriological Techniques↗

Oxidation of fugitive methane in ground water linked to bacterial sulfate reduction.

When fugitive methane migrates upward along boreholes of oil and gas wells, it may migrate into shallow ground water or pass through overlying soil to the atmosphere. Prior to this study, there was little information on the fate of fugitive methane that migrates into ground water. In a field study near Lloydminster, Alberta, Canada, we found hydrogeochemical evidence that fugitive methane from an oil well migrated into a shallow aquifer but has been attenuated by dissimilatory bacterial sulfate reduction at low temperature ( approximately 5 degrees C) under anaerobic conditions. Evidence includes spatial and temporal trends in concentrations of methane and sulfate in ground water and associated trends in concentrations of bicarbonate and sulfide. Within 10 m of the oil well, sulfate concentrations were low, and sulfate was enriched in both 34S and 18O. Sulfate concentrations had a strong positive correlation with delta13C values of bicarbonate, and sulfide was depleted in 34S compared to sulfate. These data indicate that bacterial sulfate reduction occurred near the production well. Near the oil well, elevated concentrations of bicarbonate were observed, and the bicarbonate was depleted in 13C. Modeling indicates that the main source of this excess 13C-depleted bicarbonate is oxidized methane. In concert with the sulfate concentration and isotope data, these results support an interpretation that in situ bacterial oxidation of methane has occurred, linked to bacterial sulfate reduction. Bacterial sulfate reduction may play a major role in bioattenuation of fugitive natural gas in ground water in western Canada.

Fossil Fuels↗