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The carbon assimilation pathways of Methylococcus capsulatus, Pseudomonas methanica and Methylosinus trichosporium (OB3B) during growth on methane.

d-arabino-3-Hexulose 6-phosphate was prepared by condensation of formaldehyde with ribulose 5-phosphate in the presence of 3-hexulose phosphate synthase from methane-grown Methylococcus capsulatus. The 3-hexulose phosphate was unstable in solutions of pH greater than 3, giving a mixture of products in which, after dephosphorylation, allulose and fructose were detected. A complete conversion of d-ribulose 5-phosphate and formaldehyde into d-fructose 6-phosphate was demonstrated in the presence of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase (prepared from methane-grown M. capsulatus). d-Allulose 6-phosphate was prepared from d-allose by way of d-allose 6-phosphate. No evidence was found for its metabolism by extracts of M. capsulatus, thus eliminating it as an intermediate in the carbon assimilation process of this organism. A survey was made of the enzymes involved in the regeneration of pentose phosphate during C(1) assimilation via a modified pentose phosphate cycle. On the basis of the presence of the necessary enzymes, two alternative routes for cleavage of fructose 6-phosphate are suggested, one route involves fructose diphosphate aldolase and the other 6-phospho-2-keto-3-deoxygluconate aldolase. A detailed formulation of the complete ribulose monophosphate cycle of formaldehyde fixation is presented. The energy requirements for carbon assimilation by this cycle are compared with those for the serine pathway and the ribulose diphosphate cycle of carbon dioxide fixation. A cyclic scheme for oxidation of formaldehyde via 6-phosphogluconate is suggested.

Aldehyde-Lyases↗

Some properties of a soluble methane mono-oxygenase from Methylococcus capsulatus strain Bath.

Soluble extracts of Methylococcus capsulatus (Bath), obtained by centrifugation of crude extracts at 160000g for 1h, catalyse the NAD(P)H- and O2-dependent disappearance of bromomethane, and also the formation of methanol from methane. Soluble methane mono-oxygenase is not inhibited by chelating agents or by most electron-transport inhibitors, and is a multicomponent enzyme.

Electron Transport↗

Purification and properties of the methane mono-oxygenase enzyme system from Methylosinus trichosporium OB3b.

1. A three-component enzyme system that catalyses the oxidation of methane to methanol has been highly purified from Methylosinus trichosporium. 2. The components are (i) a soluble CO-binding cytochrome c, (ii) a copper-containing protein and (iii) a small protein; the mol. wts. are 13 000, 47 000 and 9400 respectively. The cytochrome component cannot be replaced by similar cytochrome purified from Pseudomonas extorquens or by horse heart cytochrome c. 3. The stoicheiometry suggests a mono-oxygenase mechanism and the specific activity with methane as substrate is 6 micronmol/min per mg of protein. 4. Other substrates rapidly oxidized are ethane, n-propane, n-butane and CO. Dimethyl ether is not a substrate. 5. The purified enzyme system utilizes ascorbate or, in the presence of partially purified M. trichosporium methanol dehydrogenase, methanol as electron donor but not NADH or NADPH. 6. Activity is highly sensitive to low concentrations of a variety of chelating agents, cyanide, 2-mercaptoethanol and dithiothreitol. 7. Activity is highly pH-dependent (optimum 6.9-7.0) and no component of the enzyme is stable to freezing. 8. The soluble CO-binding cytochrome c shows oxidase acitivity and the relationship between this and the oxygenase activity is discussed.

Ascorbate Oxidase↗

Metabolism of dietary fibre components in man assessed by breath hydrogen and methane.

1. Breath hydrogen and methane were measured in eight normal individuals after acute and separate administration of different chemical components of dietary fibre. 2. Hemicellulose, raffinose and lactulose increased H2 production, while cellulose pectin and lignin did not. Methane production was found to be individual and unaffected by any of the substances. Differences in physical properties of the same chemical appear to have no influences on H2 and CH4 production.

Adult↗

Determination of environmental factors influencing methane oxidation in a sandy landfill cover soil.

It is advantageous to use coarse soils as landfill cover because they allow better aeration of the biologically active zone. In this study, therefore, patterns of methane oxidation were investigated under various environmental conditions including soil moisture content, temperature, and the addition of NH4+ in a sandy landfill cover soil. The kinetics of CH4 oxidation was also studied at different moisture contents and temperatures. Soil moisture content of 10% (wt/wt) resulted in the maximum CH4 oxidation rate (19.2-22.4 nmol gsoil DW(-1) min(-1)). A Vmax value was not significantly different when the moisture content was more than 10%, but a Km value increased from 5.23 to 75.24 microM as the moisture content increased. The ratio of Vmax to Km was the highest at 10% moisture content. The CH4 oxidation rate increased as the incubation temperature increased, and Q10 values and optimum temperature were determined to be 2.57-2.69 and 30 degrees C, respectively. Both Vmax and Km values decreased at the temperatures below and above 30 degrees C. The addition of various levels of NH4+ resulted in increased or decreased CH4 oxidation rates, however, the initiation of appreciable CH4 oxidation was delayed with increasing amounts of NH4+ application in all samples tested. Among the environmental variables tested, moisture content control seems to be the most important and an efficient means of managing methane oxidation when sandy soils are used in landfill cover.

Bacteria, Anaerobic↗

Kinetic studies on the CuO/gamma-Al2O3 sorbent regeneration by methane--topochemical approach.

CuO impregnated gamma-Al2O3 support provides convenient adsorption and regeneration characteristics. These features give the potential to regard this chemisorption process as more effective, feasible and widely accepted than lime and limestone scrubbing in the removal of SO2 from the outlet gases. Thermogravimetric tests on the regeneration of the CuO/gamma-Al2O3 exhausted sorbent (first stage of the regeneration, CuSO4-->Cu0) with methane were conducted. Commercially available 1/8" gamma-Al2O3 spheres, loaded with 5.8 wt % of Cu were investigated. The tests concerning the isothermal regeneration kinetics were performed at temperatures: T = 700, 727 and 750 K, while a 20 cm3 min(-1) flow of pure methane was used. To select the mechanism of the regeneration reaction, as well as to evaluate their kinetic parameters of the rate equations, the TGA data were compared with the series of theoretical topochemical kinetic models as advocated by Gardner, Hancock and Sharp's approach. The kinetic mechanism of the regeneration reaction in the initial stage of the process may be originally interpreted as the combination of both boundary-surface-controlled reaction (contracting sphere) and the first-order kinetics models. Various diffusion limited kinetic equations have proved useful for the description of the final stage of the process. Both proposed models (two kinetic and four diffusion ones) fit fairly well to the experimental data (in their limited validity ranges) obtained in all three temperature values studied.

Adsorption↗

Natural carbon isotopes used to study methane consumption and production in soil.

Changes in the isotopic composition of carbon can be used to reveal simultaneous occurrence of methane production and oxidation in soil. The method is conducted in laboratory jar experiments as well as in the field by using flux chambers. Simultaneous occurrence of production and oxidation of methane was suggested.

Carbon Isotopes↗

Methane emissions from domestic waste management facilities in Jordan--applicability of IPCC methodology.

In this paper, methane emissions from municipal wastewater treatment plants and municipal solid waste (MSW) landfills in Jordan for 1994 have been estimated using the methodology developed by the Intergovernmental Panel on Climate Change (IPCC). For this purpose, the 14 domestic wastewater treatment plants in the country were surveyed. Generation rates and characterization of MSW components as well as dumping and landfilling practices were surveyed in order to estimate 1994 CH4 emissions from these sites. Locally available waste statistics were used in cases where those of the IPCC guidelines were not representative of Jordan's statistics. Methane emissions from domestic wastewater in Jordan were estimated at 4.66 gigagrams (Gg). Total 1994 CH4 emissions from MSW management facilities in Jordan are estimated at 371.76 Gg--351.12 Gg (94.45%) from sanitary landfills, 19.83 Gg (5.33%) from MSW open dumps, and 0.81 Gg (0.22%) from raw sewage-water dumping ponds. Uncertainties associated with these estimations are presented.

Greenhouse Effect↗

Methane production in goats given diets based on lucerne hay and barley.

An analysis of data from 102 energy balance trials carried out with Granadina goats, 32 with castrated males and 70 with lactating females, was made with the aim of establishing relationships between methane production and some nutritive attributes of the diet. The diets were based on pelleted lucerne (Medicago sativa) hay and barley and differed widely in the amounts and proportions of their ingredients. Methane production was measured by open-circuit respirometry. The between-animal variation in CH4 production was found to be +/- 8%. In absolute terms, CH4 production increased on increasing the intake of energy. The CH4 loss was 6.56 or 9.75 kJ/100 kJ of gross energy or digestible energy intake, respectively. The prediction of the CH4 production was best described from knowledge of the amounts of the "in vitro" digestible fractions (g/d) of both the neutral-detergent solubles (DNDS) and the neutral-detergent fibre (DNDF), according to the equation CH4 = 2.24 + 0.0299 DNDS + 0.0889 DNDF. The regression was highly significant and the residual standard deviation +/- 6.63 or +/- 20% of the mean. It was also found that on increasing the feeding level by one multiple of maintenance there was a reduction in CH4 losses of 1.51 kJ or 2.19 kJ/100 kJ of gross energy or digestible energy intake, respectively. The CH4 production (kJ/100 kJ of gross energy intake) was also related to the apparent digestibility of energy (D, %) determined at the level of nutrition close to maintenance. The equation was CH4 = -2.58 + 0.151 D. The regression was highly significant statistically.

Animal Feed↗

Estimation of methane emission from rice cultivation in Korea.

This study focused on estimating a methane (CH4) inventory and developing mitigation options in South Korea, and was performed jointly in an integrated national research program on CH4 from rice fields conducted by three National Agricultural Research Institutes, under the Rural Development Administration during 1993-1997. Methane emissions were measured by a closed chamber method, in rice plots at three locations (Suwon, Iksan, and Milyang) with the single rice cropping system. All experimental data from 5 years of study were summarized and used for calculating nation-wide CH4 emissions. Temperature, soil type, cultural practices, water management, organic matter management, and cultivar selection significantly affected the fluctuations of CH4 emissions. The two most promising mitigation options for reducing emissions were altered water management, in particular mid-season aeration by short-term drainage, and improved organic matter management, by promotion of aerobic degradation through composing or soil incorporation. Annual total CH4 emission in Korea changed from 410Gg in 1990 to 339Gg in 2000, due mainly to a decrease in the cultivated area of paddy rice. If we convert annual CH4 emission to Global Warming Potential as CO2 equivalent, it amounts to 7.1 M CO2 t yr(-1) of greenhouse gas emitted to the atmosphere in 2000 from the rice fields in Korea, which is just 5.3% of the annual CO2 emission from the industry and energy sector. More importantly, the balance between CO2 uptake by photosynthesis and CO2 emission is positive (a net sink), so that rice culture actually has net benefits for the global atmospheric carbon issue. Further reductions in emission amounts, by following recommendations in this article, could make these benefits even greater.

Agriculture↗

Monodeuterated methane in the outer solar system. IV. Its detection and abundance on Neptune.

We have detected the 3 nu 2 band of CH3D in the spectrum of Neptune near 1.6 micrometers recorded at a spectral resolution of 4 cm-1 with the Cassegrain Fourier Transform Spectrometer at the 3.6 m Canada-France-Hawaii Telescope (CFHT) on Mauna Kea. Our analysis of this spectrum, using spectral synthesis techniques, yielded a CH3D/CH4 ratio of 6(+6)(-4) x 10(-4), which corresponds to a global D/H ratio for Neptune of 1.2(+1.2)(-0.8) x 10(-4), if CH3D is in isotopic fractionation equilibrium with HD. This value is about an order of magnitude larger than an earlier estimate by Orton et al. based on deconvolution measurements of unresolved molecular emission in the 8-10 micrometers region. Comparison of this new determination with previous studies of CH3D in the outer solar system shows that, as in the case of Uranus, the D/H on Neptune is strongly enhanced over that found on Jupiter and Saturn and is comparable to the D/H in methane on Titan and in terrestrial methane and water.

Astronomical Phenomena↗

An investigation of 11C-methane, 13N-nitrous oxide and 11C-acetylene as regional cerebral blood flow agents.

Radioisotopes of xenon or krypton are generally used as the inert diffusible indicators in regional cerebral blood flow (rCBF) studies. These gases have disadvantages when introduced into the body inhalation because of their low solubility in body tissues. The use of 11C-methane, 13N-nitrous oxide and 11C-acetylene as inhalation rCBF agents has been investigated. These gases span a wide range of solubilities: methane is four times less soluble than xenon in body tissues while nitrous oxide and acetylene are respectively 3 and 6 times more soluble. Cerebral blood flow measurements were performed on normal volunteers using the three labelled gases as well as 133Xe. All tracers were administered by inhalation and the cerebral clearance of the gases was monitored by coincidence detection. Uptake in the body was found to be dependent on solubility. The computed blood flow values found using the various indicators were in excellent agreement and were within the normal cerebral blood flow range. The high solubilities of acetylene and nitrous oxide result in a more efficient transfer of tracer from lung to brain than the conventionally used agents. The positron emission is also advantageous for tomographic rCBF measurements.

Acetylene↗

Recognition of fossil prokaryotes in Cretaceous methane seep carbonates: relevance to astrobiology.

Recovery of prokaryotic body fossils from methane seep carbonates such as those of the Cretaceous Tepee Buttes of Colorado serves as a model for sampling in future astrobiological missions. The fossils, found primarily at the interface between paragenetic fabrics, suggest a sharp physicochemical gradient. Evidence of these microbial fossils occurs at a variety of scales. In the field, microbialite is found as meter-scale thrombolitic zones and centimeterscale stromatolitic crusts lining voids inferred to be the sites of ancient methane seepage. Petrographic fabrics suggestive of microbialite include indistinct peloids (0.1-1 mm in diameter) and crusts of authigenic micrite. Primary evidence obtained from scanning electron microscopy coupled with energy-dispersive x-ray spectroscopy analysis comprises pinnate bacteria (0.3 microm in diameter and 1-1.5 microm long), sheaths (2-4 microm in diameter), coccoids (0.5-1 microm in diameter, up to 40 per cluster), and the presence of framboidal pyrite (6-8 microm in diameter). These results are in agreement with studies of other ancient and modern seeps and suggest a morphological conservatism of microbial form that can be incorporated into studies of extraterrestrial environments where it is presumed that reduced gases drive the metabolic activity of prokaryote-like organisms. Target areas that could serve as conduits for reduced gas seeps include tectonic or impact-driven faulting, zones of cryosphere melting, or other disruptions in crustal coherence. Ancient seeps, preserved as localized anomalous evaporite deposits in the sedimentary cover, could be detected by remote sensing.

Carbonates↗

Death due to a methane gas explosion in a tunnel on urban reclaimed land.

Studies of four male victims who were killed in an accidental tunnel gas explosion on urban reclaimed land are described. The studies were judicial autopsy examinations to determine the precise causes of death. Two men died of carbon monoxide intoxication, one died of massive brain damage, and the fourth died of drowning. The concentrations of methane in several organs were much lower than the lethal level, whereas those in adipose tissue were relatively high. These findings indicated that a low concentration of methane was almost always present in the atmosphere at the construction site. Recently, coal mine accidents have been decreasing in Japan. However, there is still a possibility of underground explosions or gas leaks in confined spaces other than coal mines. To determine the precise cause of death in such cases, careful autopsies and other examinations should be performed using methods similar to those used in coal mine accidents.

Adult↗

Respiratory excretion of hydrogen and methane in Italian subjects after ingestion of lactose and milk.

Lactose malabsorption, by the breath hydrogen test, and lactose intolerance (presence of symptoms) were studied in twenty healthy Italian subjects after intake of 12.5, 25 and 50 g lactose, whole milk and low-lactose milk. A rise in respiratory concentration of hydrogen (greater than 20 ppm) (malabsorption) was found in fifteen subjects after 50 g lactose, in thirteen after 25 g and in seven after 12.5 g. Symptoms generally occurred in subjects presenting a rise in respiratory hydrogen excretion, but such a rise was often observed without symptoms. Thirteen subjects presented symptoms after 50 g lactose, but only three after 25 g and one after 12.5 g. Whole milk (500 ml) gave a lower incidence of lactose malabsorption than 25 g lactose (7/20 versus 13/20, P less than 0.05) and more subjects developed symptoms (7/20 versus 3/20, NS). Low-lactose milk produced no malabsorbers and one intolerant. Breath methane was detected constantly in seven subjects and in three on some of the days of observation. Respiratory methane excretion generally appeared to be unrelated to lactose ingestion.

Adult↗

Properties of the methane mono-oxygenase from extracts of Methylosinus trichosporium OB3b and evidence for its similarity to the enzyme from Methylococcus capsulatus (Bath).

1. The methane mono-oxygenase from Methylosinus trichosporium OB3b was soluble. The only suitable electron donor was NAD(P)H, neither sodium L-ascorbate nor electrons derived from the oxidation of methanol could substitute for NAD(P)H. Evidence is presented for the existence of an NAD+-linked formaldehyde dehydrogenase. 2. Mono-oxygenase activity was not inhibited by a range of potential inhibitors including potassium cyanide, amytal, carbon monoxide or various metal-chelating agents, although 8-hydroxyquinoline and ethyne were effective in this respect. 3. Although the enzyme preparations were unstable on storage, the crude extract could be resolved into two components by ion-exchange chromatography. Activity could be restored to one of the components on addition of purified components from Methylococcus capsulatus (Bath). 4. Cross-reactivity of mono-oxygenase components and other similarities between the enzymes from M. trichosporium and M. capsulatus are discussed. The properties of the M. trichosporium methane mono-oxygenase reported here are contrasted with the properties of the same enzyme reported by others.

Aldehyde Oxidoreductases↗

ATP synthesis coupled to methane formation from methyl-CoM and H2 catalyzed by vesicles of the methanogenic bacterial strain Gö1.

Methanogenesis from methyl-CoM and H2, as catalyzed by inside-out vesicle preparations of the methanogenenic bacterium strain Gö1, was associated with ATP synthesis. That this ATP synthesis proceeded via an uncoupler-sensitive transmembrane proton gradient was concluded from the following results: 1. Various inhibitors that affected methane formation (e.g. 2-bromomethanesulfonate) also prevented ATP synthesis. 2. The protonophore 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile, in combination with the K+ ionophore valinomycin, inhibited ATP synthesis completely without affecting methanogenesis. 3. The ATP synthase inhibitor diethylstilbestrol inhibited ATP synthesis. 4. Addition of the detergent sulfobetaine inhibited both methane formation and ATP synthesis; the former but not the latter could be restored by adding titanium(III) citrate as electron donor. In addition it was shown that ATP synthesis could also be driven by transmembrane proton gradients artificially imposed on the vesicles. Furthermore net methanogenesis-dependent ATP formation was shown by measuring [32P]phosphate incorporation.

ATP Synthetase Complexes↗

Abundance and activity of uncultured methanotrophic bacteria involved in the consumption of atmospheric methane in two forest soils.

The activity and abundance of methanotrophic bacteria were measured in an acidic and a neutral forest soil. The soils exhibited high uptake rates (>30 microg CH4 m(-2) h(-1)) of atmospheric CH4 at all measurement times throughout the vegetation period. The abundances of various phylogenetic groups of methanotrophs, including some uncultured putative ones, were measured using real-time polymerase chain reaction assays. Each assay specifically targeted the pmoA gene or mmoX gene of a particular group of methanotrophs, or the amoA gene of ammonia-oxidizing bacteria. As yet uncultured methanotrophs of a group previously named 'forest soil cluster' or 'USC alpha' were numerically dominant in the acidic soil, while cultured but taxonomically uncharacterized methanotrophs of a group 'Cluster I' were dominant in the neutral soil. Each group was detected in numbers equivalent to about 10(6) pmoA gene copies per gram dry weight of soil and comprised >90% of the detectable methanotrophic bacteria in the respective soil. As the numbers of ammonia-oxidizing bacteria were similar but not higher, they could not have accounted for the observed CH4 uptake rates due to their low cell-specific CH4 oxidation activity. Based on CH4 flux and bacterial abundance data, estimated cell-specific CH4 oxidation rates of the detected methanotrophic bacteria were 540-800 x 10(-18) mol cell(-1) h(-1), which is high compared with literature values of cultured methanotrophic bacteria. These estimated cell-specific CH4 oxidation rates are sufficiently high to allow not only maintenance but even growth on atmospheric CH4 alone. Transcripts of mRNA of the pmoA gene were detected in the acidic soil, demonstrating that USC alpha methanotrophs expressed pmoA under ambient methane mixing ratios. On the other hand, pmoA transcripts of Cluster I or of other methanotrophic groups were not detectable. Our study suggests that USC alpha and Cluster I methanotrophs are adapted to the low concentration of methane in forest soils by possessing high cell-specific CH4 oxidation activities.

DNA, Bacterial↗