The soluble methane monooxygenase gene cluster of the trichloroethylene-degrading methanotroph Methylocystis sp. strain M.

In methanotrophic bacteria, methane is oxidized to methanol by the enzyme methane monooxygenase (MMO). The soluble MMO enzyme complex from Methylocystis sp. strain M also oxidizes a wide range of aliphatic and aromatic compounds, including trichloroethylene. In this study, heterologous DNA probes from the type II methanotroph Methylosinus trichosporium OB3b were used to isolate souble MMO (sMMO) genes from the type II methanotroph Methylocystis sp. strain M. sMMO genes from strain M are clustered on the chromosome and show a high degree of identity with the corresponding genes from Methylosinus trichosporium OB3b. Sequencing and phylogenetic analysis of the 16S rRNA gene from Methylocystis sp. strain M have confirmed that it is most closely related to the type II methanotroph Methylocystis parvus OBBP, which, unlike Methylocystis sp. strain M, does not possess an sMMO. A similar phylogenetic analysis using the pmoA gene, which encodes the 27-kDa polypeptide of the particulate MMO, also places Methylocystis sp. strain M firmly in the genus Methylocystis. This is the first report of isolation and characterization of methane oxidation genes from methanotrophs of the genus Methylocystis.     

Methylosinus trichosporium OB3b Mutants Having Constitutive Expression of Soluble Methane Monooxygenase in the Presence of High Levels of Copper

The methanotrophic bacterium Methylosinus trichosporium OB3b is unusually active in degrading recalcitrant haloalkanes such as trichloroethylene (TCE). The first and rate-limiting step in the degradation of TCE is catalyzed by a soluble methane monooxygenase (sMMO). This enzyme is not expressed when the cells are grown in the presence of copper at concentrations typically found in polluted groundwater. Under these conditions, M. trichosporium OB3b expresses a particulate form of the enzyme (pMMO), which has a narrow substrate specificity and does not degrade TCE at any significant rate. We have isolated M. trichosporium OB3b mutants that are deficient in pMMO and express sMMO constitutively in the presence of elevated concentrations of copper. One mutant (PP358) exhibited a TCE degradation rate which was almost twice as high as that of the wild-type strain grown under optimal conditions (without copper). All of the mutants lost the ability to express pMMO activity and to form stacked intracellular membranes characteristic of wild-type cells expressing pMMO.     

Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity

Methylosinus trichosporium OB3b biosynthesizes a broad specificity soluble methane monooxygenase that rapidly oxidizes trichloroethylene (TCE). The selective expression of the soluble methane monooxygenase was followed in vivo by a rapid colorimetric assay. Naphthalene was oxidized by purified soluble methane monooxygenase or by cells grown in copper-deficient media to a mixture of 1-naphthol and 2-naphthol. The naphthols were detected by reaction with tetrazotized o-dianisidine to form purple diazo dyes with large molar absorptivities. The rate of color formation with the rapid assay correlated with the velocity of TCE oxidation that was determined by gas chromatography. Both assays were used to optimize conditions for TCE oxidation by M. trichosporium OB3b and to test several methanotrophic bacteria for the ability to oxidize TCE and naphthalene.Abbreviations A₆₀₀ absorbance due to cell density measured at 600 nm - HPLC high pressure liquid chromatography - NADH reduced nicotinamide adenine dinucleotide - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - sMMO soluble methane monooxygenase - TCE trichloroethylene     

Detection of methanotrophs with highly divergent pmoA genes from Arctic soils

Tundra soil samples from the Canadian Arctic community, Kuujjuaq, were analyzed for the presence of the soluble (sMMO) and particulate (pMMO) methane monooxygenase genes. Total genomic DNA extracted from these soils was used as template for PCR using sMMO- and pMMO-specific primers, mmoX1-mmoX2 and A189-A682, respectively. pMMO and sMMO genes were detected in the Kuujjuaq soil samples. Isolation of sMMO-possessing methanotrophic microorganisms from the three soils, as determined by the colony naphthalene oxidation assay, was carried out using direct plating (5 degrees C) and methane enrichment studies (5 degrees C and 25 degrees C). Direct plating did not yield sMMO-possessing methanotrophic bacteria, whereas methane enrichments yielded isolates possessing and expressing sMMO activity. Analysis of derived amino acid sequences of pmoA genes and partial 16S rRNA genes obtained by PCR, using DNA isolated directly from this environment and from isolates, revealed the presence of highly divergent PmoA/AmoA sequences and 16S rRNA sequences that cluster closely with but are distinct from the genes from the genera Methylosinus and Methylocystis.     

Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas methanica 68-1

A methanotroph (strain 68-1), originally isolated from a trichloroethylene (TCE)-contaminated aquifer, was identified as the type I methanotroph Methylomonas methanica on the basis of intracytoplasmic membrane ultrastructure, phospholipid fatty acid profile, and 16S rRNA signature probe hybridization. Strain 68-1 was found to oxidize naphthalene and TCE via a soluble methane monooxygenase (sMMO) and thus becomes the first type I methanotroph known to be able to produce this enzyme. The specific whole-cell sMMO activity of 68-1, as measured by the naphthalene oxidation assay and by TCE biodegradation, was comparatively higher than sMMO activity levels in Methylosinus trichosporium OB3b grown in the same copper-free conditions. The maximal naphthalene oxidation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 551 ± 27 and 321 ± 16 nmol h^-1 mg of protein ^-1, respectively. The maximal TCE degradation rates of Methylomonas methanica 68-1 and Methylosinus trichosporium OB3b were 2,325 ± 260 and 995 ± 160 nmol h^-1 mg of protein^-1, respectively. The substrate affinity of 68-1 sMMO to naphthalene (K_m, 70 ± 4 μM) and TCE (K_m, 225 ± 13 μM), however, was comparatively lower than that of the sMMO of OB3b, which had affinities of 40 ± 3 and 126 ± 8 μM, respectively. Genomic DNA slot and Southern blot analyses with an sMMO gene probe from Methylosinus trichosporium OB3b showed that the sMMO genes of 68-1 have little genetic homology to those of OB3b. This result may indicate the evolutionary diversification of the sMMOs.     

Evaluation of Toxic Effects of Aeration and Trichloroethylene Oxidation on Methanotrophic Bacteria Grown with Different Nitrogen Sources

In this study we evaluated specific and nonspecific toxic effects of aeration and trichloroethylene (TCE) oxidation on methanotrophic bacteria grown with different nitrogen sources (nitrate, ammonia, and molecular nitrogen). The specific toxic effects, exerted directly on soluble methane monooxygenase (sMMO), were evaluated by comparing changes in methane uptake rates and naphthalene oxidation rates following aeration and/or TCE oxidation. Nonspecific toxic effects, defined as general cellular damage, were examined by using a combination of epifluorescent cellular stains to measure viable cell numbers based on respiratory activity and measuring formate oxidation activities following aeration and TCE transformation. Our results suggest that aeration damages predominantly sMMO rather than other general cellular components, whereas TCE oxidation exerts a broad range of toxic effects that damage both specific and nonspecific cellular functions. TCE oxidation caused sMMO-catalyzed activity and respiratory activity to decrease linearly with the amount of substrate degraded. Severe TCE oxidation toxicity resulted in total cessation of the methane, naphthalene, and formate oxidation activities and a 95% decrease in the respiratory activity of methanotrophs. The failure of cells to recover even after 7 days of incubation with methane suggests that cellular recovery following severe TCE product toxicity is not always possible. Our evidence suggests that generation of greater amounts of sMMO per cell due to nitrogen fixation may be responsible for enhanced TCE oxidation activities of nitrogen-fixing methanotrophs rather than enzymatic protection mechanisms associated with the nitrogenase enzymes.     

Field Evidence for Intrinsic Aerobic Chlorinated Ethene Cometabolism by Methanotrophs Expressing Soluble Methane Monooxygenase

Idaho National Laboratory's Test Area North is the site of a trichloroethene (TCE) plume resulting from waste injections. Previous investigations revealed that TCE was being attenuated relative to two codisposed internal tracers, tritium and tetrachloroethene, with a half-life of 9 to 21 years. Biological attenuation mechanisms were investigated using a novel suite of assays, including enzyme activity probes designed for the soluble methane monooxygenase (sMMO) enzyme. Samples were analyzed for chlorinated solvents, tritium, redox parameters, primary substrates, degradation products, bacterial community methanotrophic potential, and bacterial DNA. The enzyme probe assays, methanotrophic enrichments and isolations, and DNA analysis documented the presence and activity of indigenous methanotrophs expressing the sMMO enzyme. Three-dimensional groundwater data showed plume-wide aerobic conditions, with low levels of methane and detections of carbon monoxide, a by-product of TCE cometabolism. The TCE half-life attributed to aerobic cometabolism is 13 years relative to tritium, based on the tracer-corrected method. Similarly, a half-life of 8 years was estimated for cis-dichloroethene (DCE). Although these rates are slower than most anaerobic degradation processes, they can be significant for large plumes. This investigation is believed to be the first documentation of intrinsic aerobic TCE and DCE cometabolism in an aquifer by indigenous methanotrophs.     

Transformation yields of chlorinated ethenes by a methanotrophic mixed culture expressing particulate methane monooxygenase.

Transformation yields for the aerobic cometabolic degradation of five chlorinated ethenes were determined by using a methanotrophic mixed culture expressing particulate methane monooxygenase (pMMO). Transformation yields (expressed as moles of chlorinated ethene degraded per mole of methane consumed) were 0.57, 0.25, 0.058, 0.0019, and 0.00022 for trans-1,2-dichloroethylene (t-DCE), vinyl chloride (VC), cis-1,2-dichloroethylene (c-DCE), trichloroethylene (TCE), and 1,1-dichloroethylene (1,1-DCE), respectively. Degradation of t-DCE and VC was observed only in the presence of formate or methane, sources of reducing energy necessary for cometabolism. The t-DCE and VC transformation yields represented 35 and 15%, respectively, of the theoretical maximum yields, based on reducing-energy availability from methane dissimilation to carbon dioxide, exclusive of all other processes that require reducing energy. The yields for t-DCE and VC were 20 times greater than the yields reported by others for cells expressing soluble methane monooxygenase (sMMO). Transformation yields for c-DCE, TCE, and 1,1-DCE were similar to or less than those for cultures expressing sMMO. Although methanotrophic biotreatment systems have typically been designed to incorporate cultures expressing sMMO, these results suggest that pMMO expression may be highly advantageous for degradation of t-DCE or VC. It may also be much easier to maintain pMMO expression in treatment systems, because pMMO is expressed by all methanotrophs whereas sMMO is expressed only by type II methanotrophs under copper-limited conditions.     

Trichloroethylene and chloroform degradation by a recombinant pseudomonad expressing soluble methane monooxygenase from Methylosinus trichosporium OB3b.

Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b can degrade many halogenated aliphatic compounds that are found in contaminated soil and groundwater. This enzyme oxidizes the most frequently detected pollutant, trichloroethylene (TCE), at least 50 times faster than other enzymes. However, slow growth of the strain, strong competition between TCE and methane for sMMO, and repression of the smmo locus by low concentrations of copper ions limit the use of this bacterium. To overcome these obstacles, the 5.5-kb smmo locus of M. trichosporium OB3b was cloned into a wide-host-range vector (to form pSMMO20), and this plasmid was electroporated into five Pseudomonas strains. The best TCE degradation results were obtained with Pseudomonas putida F1/pSMMO20. The plasmid was maintained stably, and all five of the sMMO proteins (alpha, beta, and gamma hydroxylase proteins, reductase, and component B) were observed clearly by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. TCE degradation rates were quantified for P. putida F1/pSMMO20 with a gas chromatograph (Vmax = 5 nmol per min per mg of protein), and the recombinant strain mineralized 55% of the TCE (10 microM) as indicated by measuring chloride ion concentrations with a chloride ion-specific electrode. The maximum TCE degradation rate obtained with the recombinant strain was lower than that of M. trichosporium OB3b but greater than other TCE-degrading recombinants and most well-studied pseudomonads. In addition, this recombinant strain mineralizes chloroform (a specific substrate for sMMO), grows much faster than M. trichosporium OB3b, and degrades TCE without competitive inhibition from the growth substrate.     

Measurement and modeling of multiple substrate oxidation by methanotrophs at 20 degrees C

Earlier experiments have shown that when Methylosinus trichosporium OB3b was grown at 30 degrees C, greater growth and degradation of chlorinated ethenes was observed under particulate methane monooxygenase (pMMO)-expressing conditions than sMMO-expressing conditions. The effect of temperature on the growth and ability of methanotrophs to degrade chlorinated ethenes, however, has not been examined, particularly temperatures more representative of groundwater systems. Thus, experiments were performed at 20 degrees C to examine the effect of mixtures of trichloroethylene, trans-dichloroethylene and vinyl chloride in the presence of methane on the growth and ability of Methylosinus trichosporium OB3b cells to degrade these pollutants. Although the maximal rates of chlorinated ethane degradation were greater by M. trichosporium OB3b expressing sMMO as compared with the same cell expressing pMMO, the growth and ability of sMMO-expressing cells to degrade these cosubstrates was substantially inhibited in their presence as compared with the same cell expressing pMMO. The Delta model developed earlier was found to be useful for predicting the effect of chlorinated ethenes on the growth and ability of M. trichosporium OB3b to degrade these compounds at a growth temperature of 20 degrees C. Finally, it was also discovered that at 20 degrees C, cells expressing pMMO exhibited faster turnover of methane than sMMO-expressing cells, unlike that found earlier at 30 degrees C, suggesting that temperature may exert selective pressure on methanotrophic communities to express sMMO or pMMO.     

Molecular analysis of the methane monooxygenase (MMO) gene cluster of Methylosinus trichosporium OB3b

The oxidation of methane to methanol in methanotrophic bacteria is catalysed by the enzyme methane monooxygenase (MM0). This multicomponent enzyme catalyses a range of oxidations including that of aliphatic and aromatic compounds and therefore has potential for commercial exploitation. This study details the molecular characterization of the soluble MMO (sMMO) genes from the Type II methanotroph Methylosinus trichosporium OB3b. The structural genes encoding the alpha, beta and gamma subunits of sMMO protein A and the structural gene encoding component B have been isolated and sequenced. These genes have been expressed and their products identified using an in vitro system. A comparative analysis of sMMO predicted sequences of M. trichosporium OB3b and the taxonomically related M. capsulatus (Bath) is also presented.     

Applications of a colorimetric plate assay for soluble methane monooxygenase activity.

A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in the development of a colored complex between 1-naphthol, which is formed when sMMO reacts with naphthalene, and o-dianisidine (tetrazotized). Methanotrophic colonies expressing sMMO turned deep purple when exposed successively to naphthalene and o-dianisidine. The method was evaluated within the contexts of two potential applications. The first was for the enumeration of Methylosinus trichosporium OB3b in a methane-amended, unsaturated soil column dedicated to vinyl chloride treatment. The second application was for the isolation and enumeration of sMMO-bearing methanotrophs from sanitary landfill soils. The technique was effective in both applications.     

Natural Attenuation of Trichloroethylene in Rhizosphere Soils at the Savannah River Site

Extensive trichloroethylene (TCE) groundwater contamination has resulted from discharges to a former seepage basin in the A/M Area at the Department of Energy's Savannah River Site. The direction of groundwater flow has been determined and a seep line where the contaminated groundwater is estimated to emerge as surface water has been identified in a region of the Southern Sector of the A/M Area. This study was undertaken to estimate the potential of four rhizosphere soils along the seep line to naturally attenuate TCE. Microcosms were setup to evaluate both biotic and abiotic attenuation of TCE. Results demonstrated that sorption to soil was the dominant mechanism during the first week of incubation, with as much as 90% of the TCE removed from the aqueous phase. Linear partitioning coefficients (K_d) ranged from 0.83 to 7.4?mL/g, while organic carbon partition coefficients (K_oc) ranged from 72 to 180?mL/gC. Diffu-sional losses from the microcosms appeared to be a dominant fate mechanism during the remainder of the experiment, as indicated by results from the water controls. A limited amount of TCE biodegradation was observed, and attempts to stimulate TCE biodegradation by either methanotrophic or methanogenic activity through amendments with methane, oxygen, and methanol were unsuccessful. The appearance of cis-1,2-dichloroethylene (c-DCE), and trans-1,2-dichloroethylene (t-DCE) confirmed the potential for anaerobic reductive dechlorination. However, these daughter products represented less than 5% of the initial TCE added. The sorption results indicate that natural attenuation may represent a viable remediation option for the TCE plume as it passes through the rhizosphere.     

Applications of a colorimetric plate assay for soluble methane monooxygenase activity.

A straightforward method is described for screening methanotrophic colonies for soluble methane monooxygenase (sMMO) activity on solid media. Such activity results in the development of a colored complex between 1-naphthol, which is formed when sMMO reacts with naphthalene, and o-dianisidine (tetrazotized). Methanotrophic colonies expressing sMMO turned deep purple when exposed successively to naphthalene and o-dianisidine. The method was evaluated within the contexts of two potential applications. The first was for the enumeration of Methylosinus trichosporium OB3b in a methane-amended, unsaturated soil column dedicated to vinyl chloride treatment. The second application was for the isolation and enumeration of sMMO-bearing methanotrophs from sanitary landfill soils. The technique was effective in both applications.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus 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 inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°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 (_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.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

TCE degradation in a methanotrophic attached-film bioreactor

Trichloroethene was degraded in expanded-bed bioreactors operated with mixed-culture methanotrophic attached films. Biomass concentrations of 8 to 75 g volatile solids (VS) per liter static bed (L(sb)) were observed. Batch TCE degradation rates at 35 degrees C followed the Michaelis-Menten model, and a maximum TCE degradation rate (q(max)) of 10.6 mg TCE/gVS . day and a half velocity coefficient (K(S)) of 2.8 mg TCE/L were predicted. Continuous-flow kinetics also followed the Michaelis-Menten model, but other parameters may be limiting, such as dissolved copper and dissolved methane-q(max) and K(S) were 2.9 mg TCE/gVS . day and 1.5 mg TCE/L, respectively, at low copper concentrations (0.003 to 0.006 mg Cu/L). The maximum rates decreased substantially with small increases in dissolved copper. Methane consumption during continuous-flow operation varied from 23 to 1200 g CH(4)/g TCE degraded. Increasing the influent dissolved methane concentration from 0.01 mg/L to 5.4 mg/L reduced the TCE degradation rate by nearly an order of magnitude at 21 degrees C. Exposure of biofilms to 1.4 mg/L tetrachloroethene (PCE) at 35 degrees C resulted in the loss of methane utilization ability. Tests with methanotrophs grown on granular activated carbon indicated that lower effluent TCE concentrations could be obtained. The low efficiencies of TCE removal and low degradation rates obtained at 35 degrees C suggest that additional improvements will be necessary to make methanotrophic TCE treatment attractive. (c) 1993 John Wiley & Sons, Inc.     

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.     

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase.

Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.