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Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.

Base Sequence↗

Enzymatic reactions of methionine sulfoximine. Conversion to the corresponding alpha-imino and alpha-keto acids and to alpha-ketobutyrate and methane sulfinimide.

L-Methionine sulfoximine is a substrate of L-amino acid oxidase (Crotalus adamanteus), glutamine transaminase, and gamma-cystathionase. In the reaction catalyzed by L-amino acid oxidase, methionine sulfoximine is converted to aplph-imino-gamma-methylsulfoximinylbutyrate, which undergoes rapid gamma elimination yielding methane sulfinimide and 2-imino-3-butenoic acid. Methane sulfinimide is converted to methane sulfonamide, methane sulfinic acid, and methane sulfonic acid; 2-imino-3-butenoic acid is hydrolyzed to vinylglyoxylate, which polymerizes to an insoluble product. When the reaction is carried out in the presence of semicarbazide, the imine formed initially is quantitatively trapped as alpha-keto-gamma-methylsulfoximinylbutyrate semicarbazone, from which the free alpha-keto acid may be obtained. When the reaction is carried out in the presence of a mercaptan (RSH), a gamma exchange reaction occurs leading to formation of a new alpha-keto acid substituted in the gamma position by an SR-group; thus, alpha-keto-gamma-(beta-hydroxyethiol)butyric acid (S-(hydroxyethyl)-2-keto-4-mercaptobutyric acid) was obtained when L-methionine sulfoximine was oxidized in the presence of 2-mercaptoethanol, and enzymatic transamination of this alpha-keto acid with L-glutamine gave the new amino acid, L-omega-hydroxyethionine. The reaction of L-methionine sulfoximine with gamma-cystathionase yields 1 mol each of alpha-ketobutyrate and methane sulfinimide; the latter is hydrolyzed almost exclusively to methane sulfinic acid. Transamination of L-methionine sulfoximine yields the corresponding alpha-keto acid (alpha-keto-gamma-methylsulfoximinylbutyrate), which is stable. Some of these reactions may occur in vivo, and thus contribute to the toxicity of L-methionine sulfoximine.

Amino Acid Oxidoreductases↗

Trichloroethene degradation in a two-step system by methylosinus trichosporium OB3b. Optimization of system performance: use of formate and methane.

The breakdown of dissolved TCE in a two-step bioremediation system is described. In the first reactor, the organism Methylosinus trichosporium OB3b is grown; in the second reactor, consisting of three 17-L column reactors in series, the cells degrade TCE. A special design allowed both for the addition of air (uG,s = 0.01-0. 04 mm s-1) in the conversion reactor to prevent oxygen limitation while minimizing stripping of TCE, and for the use of methane as exogenous electron donor. In two-step systems presented thus far, only formate was used (excess, 20 mM). We found formate additions could be reduced by 75% (15 degrees C), whereas small amounts of methane (0.02-0.04 mol CH4/g cells) could replace formate and led to equally optimal results. Example calculations show that up to 90% reduction in operating cost of chemicals can be obtained by using methane instead of formate. A model was developed to describe each of the conditions studied: excess formate and optimal methane addition, suboptimal formate addition and suboptimal methane addition. Using parameters obtained from independent batch experiments, the model gives a very good description of the overall TCE conversion in the two-step system. The system presented is flexible (oxygen/methane addition) and can easily be scaled up for field application. The model provides a tool for the design of an effective and low-cost treatment system based on methane addition in the conversion reactor.

Biodegradation, Environmental↗

Selective desorption of carbon dioxide from sewage sludge for in situ methane enrichment--part I: pilot-plant experiments.

In situ methane enrichment in anaerobic digestion of sewage sludge has been investigated by experiments and by modeling. In this first part, the experimental work on the desorption of carbon dioxide and methane from sewage sludge is reported. The bubble column, had a diameter of 0.3 m and a variable height up to 1.8 m. At operation the dispersion height in the column was between 1 and 1.3 m. Outdoor air was used. The column was placed close to a full-scale sewage sludge digester, at a municipal wastewater treatment plant. The digester was operated at mesophilic conditions with a hydraulic retention time of about 20 days. The bubble column was operated to steady-state, at which carbon dioxide concentration and alkalinity were determined on the liquid side, and the concentration of carbon dioxide and methane on the gas side. Thirty-eight experiments were performed at various liquid and gas flow rates. The experimental results show that the desorption rates achieved for carbon dioxide ranges from 0.07 to 0.25 m(3) CO(2)/m(3) sludge per day, which is comparable to the rate of generation by the anaerobic digestion. With increasing liquid flow rate and decreasing gas flow rate the amount of methane desorbed per amount of carbon dioxide desorbed increases. The lowest methane loss achieved is approximately 2% of the estimated methane production in the digestion process.

Anaerobiosis↗

On-line estimation of dissolved methane concentration during methanotrophic fermentations.

Knowledge of the aqueous phase methane concentration is critical to understanding and controlling process kinetics in methanotrophic bioreactors. Unfortunately since no dissolved methane probe is commercially available, this data must be obtained off-line by the time-consuming gas-liquid partition method. In this study we demonstrate how knowledge of the reactor's k(L)a for oxygen combined with gas phase methane analysis can be used to continuously estimate the aqueous phase concentration of dissolved methane. The on-line estimation was verified in two reactor systems with greatly different values of k (L)a. In both systems the measured and calculated dissolved methane concentrations were in good agreement although dissolved methane was underestimated in both cases. The utility of this methodology was demonstrated by revealing a possible metabolic bottleneck in the model system.

Automation↗

A process-based mathematical model on methane production with emission indices for control.

In this paper, a process-based mathematical model is developed for the production of methane through biodegradation. It is a three-dimensional model given by ordinary differential equations. The results of the analysis of the model are interpreted through three emission indices, which are introduced for the first time. The estimation of either one or all of them can interpret the feasibility of the equilibrium and the long-term emission tendency of methane. The vulnerability of the methane production process with respect to soil temperature effects in methanogenic phase has been discussed and a feasible condition within a specified temperature range has defined for the nonvulnerability of the methane production process and also it has shown that under the same condition, zero-emission process of methane will be nonvulnerable with respect to the soil temperature effects in methanogenic phase. Lastly, condition for zero emission of methane is also obtained and it is interpreted through the emission indices.

Biomass↗

Effects of nitro compounds and feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid.

Short-chain volatile fatty acids (VFA) and methane are the products from a wide variety of microorganisms living in the gastrointestinal tract. The objective of this study was to examine effects of feedstuff and select nitro compounds on VFA and methane production during in vitro incubation of laying hen cecal contents and rumen fluid from cattle and sheep. In the first experiment, one of the three nitro compound was added to incubations containing cecal contents from laying hens supplemented with either alfalfa (AF) or layer feed (LF). Both feed material influenced VFA production and acetic acid was the primary component. Incubations with nitro ethanol and 2-nitropropanol (NP) had significantly (P<0.05) higher propionate concentrations than incubations with added nitroethane (NE). The results further indicated that incubations containing LF produced significantly (P<0.05) more butyrate than incubations with added AF. Addition of NP and LF to incubations of avian cecal flora may promote Gram-positive, saccharolytic, VFA-producing bacteria, especially Clostridium spp. which is the predominant group in ceca. Similar to VFA production, both feed materials fostered methane production in the incubations although methane was lower (P<0.05) in incubations with added nitro compound, particularly NE. In experiments 3-8, NE was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either AF or LF. Unlike cecal contents, LF significantly (P<0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that NE impedes methane production, especially in incubations of chicken cecal contents.

Animal Feed↗

Effect of oral nitroethane and 2-nitropropanol administration on methane-producing activity and volatile fatty acid production in the ovine rumen.

Strategies are sought to reduce economic and environmental costs associated with ruminant methane emissions. The effect of oral nitroethane or 2-nitropropanol administration on ruminal methane-producing activity and volatile fatty acid production was evaluated in mature ewes. Daily administration of 24 and 72 mg nitroethane/kg body weight reduced (P<0.05) methane-producing activity by as much as 45% and 69% respectively, when compared to control animals given no nitroethane. A daily dose of 120 mg 2-nitropropanol/kg body weight was needed to reduce (P<0.05) methane-producing activity by 37% from that of untreated control animals. Reductions in methane-producing activity may have been diminished by the last day (day 5) of treatment, presumably due to ruminal adaptation. Oral administration of nitroethane or 2-nitropropanol had little or no effect on accumulations or molar proportions of volatile fatty acids in ruminal contents collected from the sheep. These results demonstrate that nitroethane was superior to 2-nitropropanol as a methane inhibitor and that both nitrocompounds reduced ruminal methanogenesis in vivo without redirecting the flow of reductant generated during fermentation to propionate and butyrate.

Administration, Oral↗

Iron-mediated reduction rates and pathways of halogenated methanes with nanoscale Pd/Fe: analysis of linear free energy relationship.

The influence of halogen substitution in chlorinated methanes and brominated methanes on their abiotic reductive dehalogenation with nanoscale Pd/Fe bimetallic particles was investigated in this study. The 0.2% and 1.0% Pd/Fe particles, with particle sizes of less than 100nm, were synthesized in the laboratory. Reduction of the halogenated methanes with the Pd/Fe particles followed pseudo-first-order kinetics. The Pd/Fe bimetallic particles demonstrated at least an order of magnitude higher in reactivity compared to the unpalladized Fe particles of similar particle size. Comparing the 0.2% Pd/Fe and the 1.0% Pd/Fe particles, the latter exhibited higher reduction rates of various halogenated methanes. On the other hand, the reduction rates of chlorinated methanes were consistently lower than those of their brominated counterparts. The compounds with higher number of halogen substitutions were more readily reduced than the lightly halogenated ones. Coupled hydrogenolysis-elimination processes were the important mechanism for complete dehalogenation of the chlorinated and brominated methanes. From the kinetic examination of their transformation rates and mechanistic insight into the associated pathways, an attempt to analyze linear free energy relationship between the observed kinetics and the associated thermodynamic constants was performed. The observed reaction rate constants were analyzed for their correlations with one-electron potential (E(1)), two-electron potential (E(2)), bond dissociation energy (BDE) and the lowest unoccupied molecular orbital (LUMO) energy (E(LUMO)). LUMO energy appeared to be the best descriptor for the kinetic prediction, while E(1) and E(2) might be more suitable for mechanistic analysis.

Environmental Pollutants↗

Investigations of methane emissions from rice cultivation in Indian context.

The increasing demand of the growing population requires enhancement in the production of rice. This has a direct bearing on the global environment since the rice cultivation is one of the major contributors to the methane emissions. As the rice cultivation is intensified with the current practices and technologies, the methane fluxes from paddy fields will substantially rise. Improved high yielding rice varieties together with efficient cultivation techniques will certainly contribute to the curtailment of the methane emission fluxes. In this paper, the system dynamic approach is used for estimating the methane emissions from rice fields in India till the year 2020. Mitigation options studied for curtailing the methane emissions include rice production management, use of low methane emitting varieties of rice, water management and fertilizer amendment. The model is validated quantitatively and sensitivity tests are carried out to examine the robustness of the model.

Agriculture↗

Methane fermentation of Japanese cedar wood pretreated with a white rot fungus, Ceriporiopsis subvermispora.

Methane fermentation of Japanese cedar wood was carried out after pretreatment with four strains of white rot fungi, Ceriporiopsis subvermispora ATCC 90467, CZ-3, CBS 347.63 and Pleurocybella porrigens K-2855. These fungi were cultivated on wood chip media with and without wheat bran for 4-8 weeks. The pretreated wood chip was fermented anaerobically with sludge from a sewage treatment plant. Pretreatments with C. subvermispora ATCC 90467, CZ-3 and CBS 347.63 in the presence of wheat bran for 8 weeks decreased 74-76% of beta-O-4 aryl ether linkages in the lignin to accelerate production of methane. After fungal treatments with C. subvermispora ATCC 90467 and subsequent 30-days methane fermentation, the methane yield reached 35 and 25% of the theoretical yield based on the holocellulose contents of the decayed and original wood, respectively. In contrast, treatment with the three strains of C. subvermispora without wheat bran cleaved 15-26% of the linkage and produced 6-9% of methane. There were no significant accelerating effects in wood chips treated with P. porrigens which has a lower ability to decompose the lignin. Thus, it was found that C. subvermispora, with a high ability to decompose aryl ether bonds of lignin, promoted methane fermentation of softwood in the presence of wheat bran.

Basidiomycota↗

Spatial distribution of methane over Lake Baikal surface.

The results of application of a high sensitivity methane laser detector to investigations of the methane concentration in the atmosphere over Baikal lake are presented as well as methane flows from the water into the atmosphere. The measurements were conducted at a stationary station and aboard the research vessel "Vereschagin" during two summer expeditions in 2003 and 2004. Mean background concentration was equal to (2.00+/-0.16) ppm in August 2003 and (1.90+/-0.07) ppm in June 2004. The areas of methane emission through the water's surface are found to be distinctly localized and to have a characteristic size of about 150-300 m in diameter. The methane concentration in the centers of these areas can reach approximately 27 ppm. Methane flows into the atmosphere in some Baikal regions were measured as well.

Atmosphere↗

On the volatile inventory of Titan from isotopic abundances in nitrogen and methane.

We analyze recently published nitrogen and hydrogen isotopic data to constrain the initial volatile abundances on Saturn's giant moon Titan. The nitrogen data are interpreted in terms of a model of non-thermal escape processes that lead to enhancement in the heavier isotope. We show that these data do not, in fact, strongly constrain the abundance of nitrogen present in Titan's early atmosphere, and that a wide range of initial atmospheric masses (all larger than the present value) can yield the measured enhancement. The enrichment in deuterated methane is now much better determined than it was when Pinto et al. (1986. Nature 319, 388-390) first proposed a photochemical mechanism to preferentially retain the deuterium. We develop a simple linear theory to provide a more reliable estimate of the relative dissociation rates of normal and deuterated methane. We utilize the improved data and models to compute initial methane reservoirs consistent with the observed enhancement. The result of this analysis agrees with an independent estimate for the initial methane abundance based solely on the present-day rate of photolysis and an assumption of steady state. This consistency in reservoir size is necessary but not sufficient to infer that methane photolysis has proceeded steadily over the age of the solar system to produce large quantities of less volatile organics. Our analysis indicates an epoch of early atmospheric escape of nitrogen, followed by a later addition of methane by outgassing from the interior. The results also suggest that Titan's volatile inventory came in part or largely from a circum-Saturnian disk of material more reducing than the surrounding solar nebula. Many of the ambiguities inherent in the present analysis can be resolved through Cassini-Huygens data and a program of laboratory studies on isotopic and molecular exchange processes. The value of, and interest in, the Cassini-Huygens data can be greatly enhanced if such a program were undertaken prior to the prime phase of the mission.

Atmosphere↗

Methane and carbon dioxide emissions from Shan-Chu-Ku landfill site in northern Taiwan.

To investigate the methane and carbon dioxide emissions from landfill, samples were taken of material up to 5 years old from Shan-Chu-Ku landfill located in the northern part of Taiwan. Atmospheric concentrations of carbon dioxide, methane and nitrous oxide ranged from 310 to 530, 2.64 to 20.16 and 0.358 to 1.516 ppmv with the measurement of gas-type open-path Fourier transform infra-red (FTIR) spectroscopy during February 1998 to March 2000, respectively. Average methane emission rate was 13.17, 65.27 and 0.99 mgm(-2)h(-1) measured by the gas chromatography chamber method in 1-2, 2-3 and 5 year-old landfill, respectively. Similarly, average carbon dioxide emission rate was 93.70, 314.60 and 48.46 mgm(-2)h(-1), respectively. About 2-3 year-old landfill had the highest methane and carbon dioxide emission rates among the tested areas, while 5 year-old landfill was the least. Methane emission rate at night in most tested locations was higher than that in the daytime. Total amount of methane and carbon dioxide emission from this landfill was around 171 and 828 ton in 1999, respectively.

Carbon Dioxide↗

Estimation of methane and nitrous oxide emission from animal production sector in Taiwan during 1990-2000.

To investigate the greenhouse gases emissions from the feeding and waste management of livestock and poultry, methane and nitrous oxide emissions were estimated from the local measurement and IPCC guidelines during 1990-2000 in Taiwan. Hog is the major livestock and is followed by goat and cattle, while chicken is the major poultry and is followed by duck and geese. Methane emission from enteric fermentation of livestock was 30.9 Gg in 1990, increased to 39.3 Gg in 1996, and then decreased gradually to 34.9 Gg in 2000. Methane emission from the waste management was 48.5 Gg in 1990, reached the peak value of 60.7 Gg in 1996, and then declined to 43.3 Gg in 2000. In the case of poultry, annual methane emission from enteric fermentation and waste management was 30.6-44.1 ton, and 8.7-13.2 Gg, respectively. Nitrous oxide emission from waste management of livestock was 0.78 ton in 1990, increased to 0.86 ton in 1996, and then decreased to 0.65 ton in 2000. Nitrous oxide emission from waste management of poultry was higher than that of livestock with 1.11 ton in 1990, 1.68 ton in 1999, and 1.65 ton in 2000. There is an urgent need to reduce methane emission from enteric fermentation and recover methane from anaerobic waste treatment for energy in livestock and poultry feeding in Taiwan.

Air Pollutants↗

Methane emission from lakes.

A physico-chemical model to describe methane and carbon dioxide emission from lakes was developed. The model describes the emission by diffusion, bubbles and plants. The intensity of the fluxes can be calculated either for total or particular emission in dependence on gas bubbles composition. It was found, that diffusional (Q) and bubble (J) fluxes depend on the methane molar ratio (X) in bubbles as follows: [formula: see text]. The model allows to estimate the role of the methane oxidation by atmospheric oxygen in the total methane flux. It was shown, that the methane oxidation does not influence much methane fluxes to the atmosphere for most of the experimentally observed situations.

Algorithms↗

New inhibitors of methane production by rumen micro-organisms. Experiments with animals and other practical possibilities.

1. Two inhibitors of rumen methane production were given to sheep and their effects were measured using an in vitro technique. 2. The substances used were trichloroethyl pivalate (TCE-P) and trichloroethyl adipate (TCE-A). In one experiment with two sheep TCE-P was injected into the rumen and in another experiment with two sheep the same inhibitor was mixed with the food just before feeding. In another experiment, TCE-A was given to two sheep at two dose levels, and in an experiment with four sheep two levels of inhibitor and two levels of diet were used. 3. In general the inhibition of methane production was greater with samples of rumen contents taken after feeding than with those taken before feeding. When sheep were given 120--300 mg TCE-P/d the inhibition of methane production ranged from 21 to 81%. When sheep given maintenance rations were given 125-300 mg TCE-A/d there was 28--90% inhibition of methane production. When sheep were given twice maintenance rations 150 mg TCE-A/d gave no inhibition and 300 mg TCE-A/d gave very low inhibition of methane production (about 16%). 4. In most experiments there was a significant increase in the propionic acid:acetic acid concentration ratio in the rumen when methane production was inhibited. 5. The possible practical use of inhibitors is discussed.

Adipates↗

Kinetics and activation thermodynamics of methane monooxygenase compound Q formation and reaction with substrates.

The transient kinetics of formation and decay of the reaction cycle intermediates of the Methylosinus trichosporium OB3b methane monooxygenase (MMO) catalytic cycle are studied as a function of temperature and substrate type and deuteration. Kinetic evidence is presented for the existence of three intermediates termed compounds O, P, and P forming after the addition of O(2) to diferrous MMO hydroxylase (H(r)) and before the formation of the reactive intermediate compound Q. The Arrhenius plots for these reactions are linear and independent of substrate concentration and type, showing that substrate does not participate directly in the oxygen activation phase of the catalytic cycle. Analysis of the transient kinetic data revealed only small changes relative to the weak optical spectrum of H(r) for any of these intermediates. In contrast, large changes in the 430 nm spectral region are associated with the formation of Q. The decay reaction of Q exhibits an apparent first-order concentration dependence for all substrates tested, and the observed rate constant depends on the substrate type. The kinetics of the decay reaction of Q yield a nonlinear Arrhenius plot when methane is the substrate, and the rates in both segments of the plot increase linearly with methane concentration. Together these observations suggest that at least two reactions with a methane concentration dependence, and perhaps two methane molecules, are involved in the decay process. When CD(4) is used as the substrate, a large isotope effect and a linear Arrhenius plot are observed. Analogous plots for all other MMO substrates tested (e.g., ethane) are linear, and no isotope effect for deuterated analogues is observed. This demonstrates that a step other than C-H bond breaking is rate limiting for alternative MMO substrates. A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of methane by Q exceeds that of many other hydrocarbons with weaker C-H bonds.

Catalysis↗