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PubMed1991

Unexpected Errors in Gas Chromatographic Analysis of Methane Production by Thermophilic Bacteria.

Unexpected errors in methane measurement by gas chromatography occurred when samples at thermophilic temperatures were analyzed. With a standard curve prepared at room temperature (25 degrees C), stoppered bottles incubated and sampled at 37 to 85 degrees C showed more methane upon analysis than bottles incubated at 25 degrees C: values at 50, 63, and 85 degrees C were 109, 126, and 125%, respectively, of the 25 degrees C value. All variation between 4 and 50 degrees C can be explained by the temperature difference between culture bottle and sampling syringe, and the variation of methane concentration can be predicted by the gas law. Between 50 and 63 degrees C, there was a more dramatic rise than predicted by theory. These variations are important to consider if thermophilic methane production is to be measured accurately. Methods to avoid errors are discussed.

Journal Article↗

PubMed1993

Methane consumption in temperate and subarctic forest soils: rates, vertical zonation, and responses to water and nitrogen.

Rates of methane consumption were measured in subarctic coniferous and temperate mixed-hardwood forest soils, using static chambers and intact soil cores. Rates at both sites were generally between 1 and 3 mg of CH(4) m day and decreased with increasing soil water contents above 20%. Addition of ammonium (1 mumol g of soil) strongly inhibited methane oxidation in the subarctic soils; a lesser inhibition was observed for temperate forest samples. The response to nitrogen additions occurred within a few hours and was probably due to physiological changes in the active methane-consuming populations. Methane consumption in soils from both sites was stratified vertically, with a pronounced subsurface maximum. This maximum was coincident with low levels of both nitrate and ammonium in the mixed-hardwood forest soil.

Journal Article↗

PubMed1999

Purification and characterization of the soluble methane monooxygenase of the type II methanotrophic bacterium Methylocystis sp. strain WI 14.

Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 microM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an alpha(2)beta(2)gamma(2) structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg(2+) ions (with a total loss of enzyme activity at 0.01 mM Hg(2+)) and Cu(2+), Zn(2+), and Ni(2+) ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath).

Alphaproteobacteria↗

PubMed2006

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell and tumor growth through PPARgamma-dependent and PPARgamma-independent pathways.

1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes containing p-trifluoromethyl, t-butyl, and phenyl [1,1-bis(3'-indolyl)-1-(p-phenyl)methane (DIM-C-pPhC(6)H(5))] substituents induce peroxisome proliferator-activated receptor gamma (PPARgamma)-mediated transactivation in SW480 colon cancer cells. These PPARgamma-active compounds also inhibit cell proliferation and modulate some cell cycle proteins. At concentrations from 2.5 to 7.5 micromol/L, the PPARgamma agonists induce caveolin-1 and phosphorylation of Akt and cotreatment with the PPARgamma antagonist GW9662 inhibited the induction response. In contrast, higher concentrations (10 micromol/L) of 1,1-bis(3'-indolyl)-1-(p-substituted phenyl)methanes containing 1,1-bis(3'-indolyl)-1-(p-trifluoromethyl)methane and DIM-C-pPhC(6)H(5) induce apoptosis, which is PPARgamma independent. This was accompanied by loss of caveolin-1 induction but induction of proapoptotic nonsteroidal anti-inflammatory drug activated gene-1. In athymic nude mice bearing SW480 cell xenografts, DIM-C-pPhC(6)H(5) inhibits tumor growth at doses of 20 and 40 mg/kg/d and immunohistochemical staining of the tumors showed induction of apoptosis and nonsteroidal anti-inflammatory drug activated gene-1 expression. Thus, the indole-derived PPARgamma-active compounds induce both receptor-dependent and receptor-independent responses in SW480 cells, which are separable over a narrow range of concentrations. This dual mechanism of action enhances their antiproliferative and anticancer activities.

Animals↗

PubMed1991

Redox properties of the hydroxylase component of methane monooxygenase from Methylococcus capsulatus (Bath). Effects of protein B, reductase, and substrate.

The reduction potentials of the hydroxylase component of the soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been investigated through potentiometric titrations. The potentials were determined by EPR spectroscopic quantitation of the mixed valent hydroxylase as a function of added sodium dithionite in the presence of appropriate mediators. The reduction of the oxidized Fe(III).Fe(III) form to the mixed valent Fe(II).Fe(III) form occurs at 48 mV versus NHE while the potential for the formation of the fully reduced Fe(II).Fe(II) species from the mixed valent form was determined to be -135 mV. Addition of the substrate propylene to the hydroxylase did not have a major effect on the reduction potentials. Introduction of the protein B and the reductase components, however, completely inhibited reduction of the hydroxylase at potentials as far negative as -200 mV. Addition of propylene to all three methane monooxygenase components greatly facilitated hydroxylase reduction. Under these conditions, the fully reduced form of the protein was obtained at potentials of greater than 150 mV. This high redox potential indicates that the oxidized form of the protein is highly reactive, as required for methane oxidation. The present results reveal aspects of how both protein B and substrate can regulate electron transfer into and out of the hydroxylase component of methane monooxygenase.

Dithionite↗

PubMed1989

Substrate specificity of soluble methane monooxygenase. Mechanistic implications.

Following the example set by studies of the mechanistic aspects of the substrate specificity of various cytochrome P-450 enzymes, we have undertaken a parallel investigation of the soluble methane monooxygenase from Methylococcus capsulatus (Bath). Soluble methane monooxygenase is a multicomponent enzyme with a broad substrate specificity. Using substrates previously tested with cytochrome P-450 enzymes and using purified enzyme preparations, this work indicates that soluble methane monooxygenase has a similar oxidative reaction mechanism to cytochrome P-450 enzymes. The evidence suggests that soluble methane monooxygenase oxidizes substrates via a nonconcerted reaction mechanism (hydrogen abstraction preceding hydroxylation) with radical or carbocation intermediates. Aromatic hydroxylation proceeds by epoxidation followed by an NIH shift.

Alkanes↗

PubMed1994

Methane utilizing bacteria and their biotechnological applications.

Methanotroph microorganisms oxidize methane in four steps, producing methanol, formaldehyde, formate intermediers and eventually degrade methane to carbon dioxide and water. It is possible to separate the pathway into four steps in the cell free extract or after partial purification of the various enzymes. The key enzyme is a metalloenzyme, methane monooxygenase (MMO) which catalyses the oxidation of methane to methanol. MMO is also capable of biodegrading exceptionally harmful and stable chlorinated hydrocarbons. Produced by various industrial activities, most chlorinated hydrocarbons are toxic, potential and/or proven carcinogens and their decomposition challenges water treatment technologies.

Biotechnology↗

PubMed2026

Ruminosignatures associated with methane emissions and feed efficiency across geographies and cattle breeds.

The cattle rumen microbiota represents a complex and dynamic ecosystem whose organization and relationship to host phenotypes are important for food security and environmental sustainability. We analyzed rumen microbiota profiles from 2496 cattle representing five breeds and production systems across five countries, identifying microbial co-abundance groups termed Ruminosignatures. We detected 14 distinct Ruminosignatures, including 2 observed across all populations dominated by Prevotella and UBA2810. Additional Ruminosignatures showed breed- and diet-specific patterns and collectively explained 96%-99% of variance in rumen microbial composition. Integrative cross-country analysis confirmed 10 out of 14 Ruminosignatures identified in cohort-specific analyses. Several Ruminosignatures were associated with methane emissions and feed efficiency traits and were partially under host genetic control, with heritability estimates ranging from 0.09 to 0.58. Structural equation modeling revealed consistent negative genetic and phenotypic correlations between the UBA2810-dominated Ruminosignature (RS_UBA2) and methane emissions across cohorts (rg = -0.40 to -0.65), with structural coefficients concordant in sign across all populations, supporting the expected direction of phenotypic response to selection on RS_UBA2. Meta-analysis confirmed positive associations of RS_UBA2 with average daily gain and negative associations with methane-related traits and feed conversion ratio. Functional genome-based predictions suggested RS_UBA2 may reduce methanogenesis through alternative hydrogen utilization pathways competing with methanogenic archaea. Production system type influenced both Ruminosignature occurrence and relationships with host phenotypes, emphasizing the relevance of context-specific strategies for microbiome modulation. Our findings highlight the potential of the Ruminosignatures framework for microbiome-informed breeding programs aimed at improving feed efficiency while reducing the environmental impact of cattle production.

Animals↗

PubMed1980

Ethane oxidation by methane-oxidizing bacteria.

The relationship between the rates of methane and ethane oxidation by washed suspensions of methane-oxidizing bacteria has been investigated. Considerable differences between bacterial strains were observed. Two closely related Methylomonas strains which differed in their oxidizing capacity were further investigated. The low ethane oxidation rate of one strain could be strongly stimulated by the addition of oxidizable co-substrates and the presence of ethane stimulated formate oxidation. The other strain had a much higher ethane oxidation rate and stimulation by co-substrates was negligible. Differences between the levels of dissimilative enzymes in cell-free extracts could not be detected. Attempts to produce extracts with methane mono-oxygenase activity failed. When cells were made permeable with chitosan the results suggested that strains with a low ethane oxidizing capacity obtain the required reductant for the moo-oxygenase from endogenous respiration. In strains with a high ethane oxidation rate, the reductant appears to be derived from oxidation of ethanol or acetaldehyde.

Chitin↗

PubMed1980

Methane formation from fructose by syntrophic associations of Acetobacterium woodii and different strains of methanogens.

When Acetobacterium woodii was co-cultured in continuous or in stationary culture with Methanobacterium strain AZ, fructose instead of being converted to 3 mol of acetate was converted to 2 mol of acetate and 1 mol each of carbon dioxide and methane, showing that interspecies hydrogen transfer occurred. In continuous culture the organisms formed a close physical association in clumps; the doubling time for each organism was 6 h at 33 degrees C. Methane mainly was derived from carbon positions 3 and 4 of the sugar, but other carbons also yielded methane; this was shown to be due to carbon dioxide-acetate exchange reactions by A. woodii in a manner similar to that carried out by Clostridium thermoaceticum. Four other methanogens, Methanobacterium M.o.H. and M.o.H. G, Methanobacterium formicicum, and Methanosarcina barkeri (not acetate-adapted) also produced similar results, when co-cultured with A. woodii.

Acetobacter↗

PubMed1994

Optimization and maintenance of soluble methane monooxygenase activity in Methylosinus trichosporium OB3b.

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity in Methylosinus trichosporium OB3b. Methylosinus trichosporium OB3b attained peak sMMO activity (275-300 nmol of naphthol formed h-1 mg of protein-1 at 25 degrees C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (mu max 0.08 h-1) and growth yields (0.4-0.5 g cells/g CH4) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45-50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B12 (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25-75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.

Biotransformation↗

PubMed2001

Detection and localization of two hydrogenases in Methylococcus capsulatus (Bath) and their potential role in methane metabolism.

Methylococcus capsulatus (Bath) was shown to contain two distinct hydrogenases, a soluble hydrogenase and a membrane-bound hydrogenase. This is the first report of a membrane-bound hydrogenase in methanotrophs. Both enzymes were expressed apparently constitutively under normal growth conditions. The soluble hydrogenase was capable of reducing NAD(+) with molecular hydrogen. The activities of both soluble and particulate methane monooxygenases could be driven by molecular hydrogen. This confirmed that molecular hydrogen could be used as a source of reducing power for methane oxidation. Hydrogen-driven methane monooxygenase activities tolerated elevated temperatures and moderate oxygen concentrations. The significance of these findings for biotechnological applications of methanotrophs is discussed.

Hydrogen↗

PubMed2002

Assessment of metabolic properties and kinetic parameters of methanogenic sludge by on-line methane production rate measurements.

This report presents a new approach to studying the metabolic and kinetic properties of anaerobic sludge from single batch experiments. The two main features of the method are that the methane production is measured on-line with a relatively cheap system, and that the methane production data can be plotted as rate vs time curves. The case studies of specific methanogenic activity, biodegradability and toxicity tests here presented show that very accurate kinetic data can be obtained. The method is specifically useful in experiments in which strong changes in methane production occur, and it is proposed as a powerful tool to study methanogenic systems. Furthermore, the method is simple and could be implemented by industry in the routine analysis of sludge.

Anaerobiosis↗

PubMed2000

Dechlorination of polychlorinated methanes by a sequential methanogenic-denitrifying bioreactor system.

A two-stage bioreactor has been developed to link dechlorination of halogenated methane compounds to the anaerobic processes of methanogenesis and denitrification. A digester methanogenic consortium was shown to dechlorinate chloroform (CF) and carbon tetrachloride (CT) to dichloromethane (DCM), and DCM was then mineralized by an acclimated denitrifying biological activated carbon consortium. Combining these two processes, a sequential methanogenic-denitrifying bioreactor (SMDB) system that completely degraded polychlorinated methanes including CT, CF, and DCM was developed. More than 95% of the added CT and CF was dechlorinated in the methanogenic bioreactor with methanol as the primary substrate, and the resultant DCM was biodegraded in the denitrifying bioreactor with nitrate as the electron acceptor. In the denitrifying bioreactor, the residual CF was completely removed, and the DCM removal efficiency was more than 95%. This novel bioreactor system eliminates the need for aeration and so avoids the air contamination associated with aerobic biotreatment of volatile chlorinated pollutants. This SMDB system provides an alternative to conventional biotreatment of wastewaters and other matrices contaminated with polychlorinated methanes and is, to our knowledge, the first report on such a sequential anoxic system.

Bacteria↗

PubMed2001

Natural and anthropogenic methane emission from coastal wetlands of South India.

For the first time, the methane emissions from diverse coastal wetlands of South India have been measured. Annual emission rates varied widely, ranging from 3.10 mg/m2/hr (Bay of Bengal) to 21.56 mg/m2/hr (Adyar River), based on nature of the perturbance to each of the ecosystems studied. Distinct seasonality in methane emission was noticed in an unpolluted ecosystem (mangrove: 7.38 mg/m2/hr) and over a twofold increase was evident in the ecosystem that was disturbed by human activities (21.56 mg/m2/hr). The wide ranges in estimate suggest that methanogenesis occurs by both natural and anthropogenic activities in these coastal wetlands. Several physical and chemical factors such as salinity, sulfate, oxygen, and organic matter content influenced methanogenesis to a large degree in each of these ecosystems in addition to individual responses to human-induced stress. For example, there was a clear negative correlation between oxygen availability (0.99), sulfate (0.98), and salinity (0.98) with CH4 emission in the Adyar river ecosystem. Although similar results were obtained for the other wetland ecosystems, CH4 emission was largely influenced by tidal fluctuations, resulting in a concomitant increase in methanogenesis with high sulfate concentrations. This study demonstrates that coastal wetlands are potentially significant sources of atmospheric methane and could be a greater source if anthropogenic perturbations continue at the current rate.

Ecosystem↗

PubMed2005

Degradation of organic pollutants by methane grown microbial consortia.

Microbial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge ("AAE-A2"), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent Km and Vmax for DBP degradation by AAE-A2 at 20 degrees C was 3.1 +/- 1.2 mg l(-1) and 8.7 +/- 1.1 mg DBP (g protein x h)(-1), respectively. AAE-A2 also showed fast degradation of BTX (230 +/- 30 nmol benzene (mg protein x h)(-1) at 20 degrees C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinus trichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83-92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas, Flavobacterium, Janthinobacterium and Rubivivax).

Bacteria↗

PubMed1983

Biomass measurement of methane forming bacteria in environmental samples.

Methane-forming bacteria contain unusual phytanylglycerol ether phospholipids which can be extracted from the bacteria in sediments and assayed quantitatively by high performance liquid chromatography (HPLC). In this procedure the lipids were extracted, the phospholipids recovered, hydrolyzed, purified by thin layer chromatography, derivatized and assayed by HPLC. Ether lipids were recovered quantitatively from Methanobacterium thermoautotrophicum and sediments at levels as low as 8 x 10(-14) moles. In freshwater and marine sediments the flux of methane to the atmosphere and the methane levels in the pore water reflects the recovery of the phytanyl glycerol ether lipid 'signature'. The proportion of the ether phospholipid to the total recoverable phospholipid was highest in anaerobic digester sewage sludge and deeper subsurface freshwater sediment horizons.

Biomass↗

PubMed1995

Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related.

Genes encoding particulate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides (pmoA and amoA) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (alpha- and gamma-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (beta- and gamma-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the gamma-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.

Amino Acid Sequence↗