Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.     

Effects of toxicity, aeration, and reductant supply on trichloroethylene transformation by a mixed methanotrophic culture.

The trichloroethylene (TCE) transformation rate and capacity of a mixed methanotrophic culture at room temperature were measured to determine the effects of time without methane (resting), use of an alternative energy source (formate), aeration, and toxicity of TCE and its transformation products. The initial specific TCE transformation rate of resting cells was 0.6 mg of TCE per mg of cells per day, and they had a finite TCE transformation capacity of 0.036 mg of TCE per mg of cells. Formate addition resulted in increased initial specific TCE transformation rates (2.1 mg/mg of cells per day) and elevated transformation capacity (0.073 mg of TCE per mg of cells). Significant declines in methane conversion rates following exposure to TCE were observed for both resting and formate-fed cells, suggesting toxic effects caused by TCE or its transformation products. TCE transformation and methane consumption rates of resting cells decreased with time much more rapidly when cells were shaken and aerated than when they remained dormant, suggesting that the transformation ability of methanotrophs is best preserved by storage under anoxic conditions.     

Influence of endogenous and exogenous electron donors and trichloroethylene oxidation toxicity on trichloroethylene oxidation by methanotrophic cultures from a groundwater aquifer

Trichloroethylene (TCE)-transforming aquifer methanotrophs were evaluated for the influence of TCE oxidation toxicity and the effect of reductant availability on TCE transformation rates during methane starvation. TCE oxidation at relatively low (6 mg liter-1) TCE concentrations significantly reduced subsequent methane utilization in mixed and pure cultures tested and reduced the number of viable cells in the pure culture Methylomonas sp. strain MM2 by an order of magnitude. Perchloroethylene, tested at the same concentration, had no effect on the cultures. Neither the TCE itself nor the aqueous intermediates were responsible for the toxic effect, and it is suggested that TCE oxidation toxicity may have resulted from reactive intermediates that attacked cellular macromolecules. During starvation, all methanotrophs tested exhibited a decline in TCE transformation rates, and this decline followed exponential decay. Formate, provided as an exogenous electron donor, increased TCE transformation rates in Methylomonas sp. strain MM2, but not in mixed culture MM1 or unidentified isolate, CSC-1. Mixed culture MM2 did not transform TCE after 15 h of starvation, but mixed cultures MM1 and MM3 did. The methanotrophs in mixed cultures MM1 and MM3, and the unidentified isolate CSC-1 that was isolated from mixed culture MM1 contained lipid inclusions, whereas the methanotrophs of mixed culture MM2 and Methylomonas sp. strain MM2 did not. It is proposed that lipid storage granules serve as an endogenous source of electrons for TCE oxidation during methane starvation.     

Influence of endogenous and exogenous electron donors and trichloroethylene oxidation toxicity on trichloroethylene oxidation by methanotrophic cultures from a groundwater aquifer.

Trichloroethylene (TCE)-transforming aquifer methanotrophs were evaluated for the influence of TCE oxidation toxicity and the effect of reductant availability on TCE transformation rates during methane starvation. TCE oxidation at relatively low (6 mg liter-1) TCE concentrations significantly reduced subsequent methane utilization in mixed and pure cultures tested and reduced the number of viable cells in the pure culture Methylomonas sp. strain MM2 by an order of magnitude. Perchloroethylene, tested at the same concentration, had no effect on the cultures. Neither the TCE itself nor the aqueous intermediates were responsible for the toxic effect, and it is suggested that TCE oxidation toxicity may have resulted from reactive intermediates that attacked cellular macromolecules. During starvation, all methanotrophs tested exhibited a decline in TCE transformation rates, and this decline followed exponential decay. Formate, provided as an exogenous electron donor, increased TCE transformation rates in Methylomonas sp. strain MM2, but not in mixed culture MM1 or unidentified isolate, CSC-1. Mixed culture MM2 did not transform TCE after 15 h of starvation, but mixed cultures MM1 and MM3 did. The methanotrophs in mixed cultures MM1 and MM3, and the unidentified isolate CSC-1 that was isolated from mixed culture MM1 contained lipid inclusions, whereas the methanotrophs of mixed culture MM2 and Methylomonas sp. strain MM2 did not. It is proposed that lipid storage granules serve as an endogenous source of electrons for TCE oxidation during methane starvation.     

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     

Effects of aeration and organic loading rates on degradation of trichloroethylene in a methanogenic-methanotrophic coupled reactor

The effects of four aeration and four organic loading (OLR) rates on trichloroethylene (TCE) degradation in methanogenic-methanotrophic coupled reactors were studied using ethanol as the carbon source for the methanogens. Microcosm and PCR studies demonstrated that methanotrophs capable of mineralizing TCE and methanogens were present in the biomass throughout the study. The gene for the particulate form of methane monooxygenase (pMMO) was detected by PCR, but not that for the soluble form (sMMO). TCE mineralization by methanotrophs was therefore due primarily to pMMO activity. Low TCE concentrations were measured in effluent and off-gas samples in all cases. Volatilization losses were 0-5%. Dichloroethylene (DCE) was also observed, but vinyl chloride and ethylene were never detected. Changes in the aeration rate had no effect on TCE removal, but did influence DCE degradation. Reductive dechlorination of TCE to DCE was favored at low and no-aeration conditions, and DCE accumulation occurred due to slow DCE degradation. Low DCE levels were observed at the higher aeration rates, which indicated that conditions in these reactors were amenable to the aerobic co-metabolism of TCE and DCE. The OLR did have an effect on TCE removal. TCE and DCE removal were negatively affected when the OLR was increased. An OLR of 0.3 g COD l(rx)(-1)day(-1) or lower with an aeration rate of 3 l(O2 )l(rx)(-1)day(-1) and higher is the recommended operating condition of a coupled reactor for removal of TCE.     

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.     

Characterization of the methanotrophic bacterial community present in a trichloroethylene-contaminated subsurface groundwater site.

Groundwater, contaminated with trichloroethylene (TCE) and tetrachloroethylene (PCE), was collected from 13 monitoring wells at Area M on the U.S. Department of Energy Savannah River Site near Aiken, S.C. Filtered groundwater samples were enriched with methane, leading to the isolation of 25 methanotrophic isolates. The phospholipid fatty acid profiles of all the isolates were dominated by 18:1 omega 8c (60 to 80%), a signature lipid for group II methanotrophs. Subsequent phenotypic testing showed that most of the strains were members of the genus Methylosinus and one isolate was a member of the genus Methylocystis. Most of the methanotroph isolates exhibited soluble methane monooxygenase (sMMO) activity. This was presumptively indicated by the naphthalene oxidation assay and confirmed by hybridization with a gene probe encoding the mmoB gene and by cell extract assays. TCE was degraded at various rates by most of the sMMO-producing isolates, whereas PCE was not degraded. Savannah River Area M and other groundwaters, pristine and polluted, were found to support sMMO activity when supplemented with nutrients and then inoculated with Methylosinus trichosporium OB3b. The maximal sMMO-specific activity obtained in the various groundwaters ranged from 41 to 67% compared with maximal rates obtained in copper-free nitrate mineral salts media. This study partially supports the hypothesis that stimulation of indigenous methanotrophic communities can be efficacious for removal of chlorinated aliphatic hydrocarbons from subsurface sites and that the removal can be mediated by sMMO.     

Trichloroethylene oxidation by the membrane-associated methane monooxygenase in type I,type II and type X methanotrophs

Trichloroethylene (TCE) oxidation was examined in 9 different methanotrophs grown under conditions favoring expression of the membrane associated methane monooxygenase. Depending on the strain, TCE oxidation rates varied from 1 to 677 pmol/min/mg cell protein. Levels of TCE in the reaction mixture were reduced to below 40 nmolar in some strains. Cells incubated in the presence of acetylene, a selective methane monooxygenase inhibitor, did not oxidize TCE.Cultures actively oxidizing TCE were monitored for the presence of the soluble methane monooxygenase (sMMO) and membrane associated enzyme (pMMO). Transmission electron micrographs revealed the cultures always contained the internal membrane systems characteristic of cells expressing the pMMO. Naphthalene oxidation by whole cells, or by the cell free, soluble or membrane fractions was never observed. SDS denaturing gels of the membrane fraction showed the polypeptides associated with the pMMO. Cells exposed to ¹⁴C-acetylene showed one labeled band at 26 kDa, and this protein was observed in the membrane fraction. In the one strain examined by EPR spectroscopy, the membrane fraction of TCE oxidizing cells showed the copper complexes characteristic of the pMMO. Lastly, most of the strains tested showed no hybridization to sMMO gene probes. These findings show that the pMMO is capable of TCE oxidation; although the rates are lower than those observed for the sMMO.     

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.     

Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells

The rate and capacity for chloroform (CF) and trichloroethylene (TCE) transformation by a mixed methanotrophic culture of resting cells (no exogenous energy source) and formate-fed cells were measured. As reported previously for TCE, formate addition resulted in an increased CF transformation rate (0.35 day-1 for resting cells and 1.5 day-1 for formate-fed cells) and transformation capacity (0.0065 mg of CF per mg of cells for resting cells and 0.015 mg of CF per mg of cells for formate-fed cells), suggesting that depletion of energy stores affects transformation behavior. The observed finite transformation capacity, even with an exogenous energy source, suggests that toxicity was also a factor. CF transformation capacity was significantly lower than that for TCE, suggesting a greater toxicity from CF transformation. The toxicity of CF, TCE, and their transformation products to whole cells was evaluated by comparing the formate oxidation activity of acetylene-treated cells to that of non-acetylene-treated cells with and without prior exposure to CF or TCE. Acetylene arrests the activity of methane monooxygenase in CF and TCE oxidation without halting cell activity toward formate. Significantly diminished formate oxidation by cells exposed to either CR or TCE without acetylene compared with that with acetylene suggests that the solvents themselves were not toxic under the experimental conditions but their transformation products were. The concurrent transformation of CF and TCE by resting cells was measured, and results were compared with predictions from a competitive-inhibition cometabolic transformation model. The reasonable fit between model predictions and experimental observations was supportive of model assumptions.     

Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells.

The rate and capacity for chloroform (CF) and trichloroethylene (TCE) transformation by a mixed methanotrophic culture of resting cells (no exogenous energy source) and formate-fed cells were measured. As reported previously for TCE, formate addition resulted in an increased CF transformation rate (0.35 day-1 for resting cells and 1.5 day-1 for formate-fed cells) and transformation capacity (0.0065 mg of CF per mg of cells for resting cells and 0.015 mg of CF per mg of cells for formate-fed cells), suggesting that depletion of energy stores affects transformation behavior. The observed finite transformation capacity, even with an exogenous energy source, suggests that toxicity was also a factor. CF transformation capacity was significantly lower than that for TCE, suggesting a greater toxicity from CF transformation. The toxicity of CF, TCE, and their transformation products to whole cells was evaluated by comparing the formate oxidation activity of acetylene-treated cells to that of non-acetylene-treated cells with and without prior exposure to CF or TCE. Acetylene arrests the activity of methane monooxygenase in CF and TCE oxidation without halting cell activity toward formate. Significantly diminished formate oxidation by cells exposed to either CR or TCE without acetylene compared with that with acetylene suggests that the solvents themselves were not toxic under the experimental conditions but their transformation products were. The concurrent transformation of CF and TCE by resting cells was measured, and results were compared with predictions from a competitive-inhibition cometabolic transformation model. The reasonable fit between model predictions and experimental observations was supportive of model assumptions.     

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.     

Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase

The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.     

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.     

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.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide.

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

Inhibition of trichloroethylene oxidation by the transformation intermediate carbon monoxide

Inhibition of trichloroethylene (TCE) oxidation by the transformation intermediate carbon monoxide (CO) was evaluated with the aquifer methanotroph Methylomonas sp. strain MM2. CO was a TCE transformation intermediate. During TCE oxidation, approximately 9 mol% of the TCE was transformed to CO. CO was oxidized by Methylomonas sp. strain MM2, and when formate was provided as an electron donor, the CO oxidation rate doubled. The rate of CO oxidation without formate was 4.6 liter mg (dry weight)-1 day-1, and the rate with formate was 10.2 liter mg (dry weight)-1 day-1. CO inhibited TCE oxidation, both by exerting a demand for reductant and through competitive inhibition. The Ki for CO inhibition of TCE oxidation, 4.2 microM, was much less than the Ki for methane inhibition of TCE oxidation, 116 microM. CO also inhibited methane oxidation, and the degree of inhibition increased with increasing CO concentration. When CO was present, formate amendment was necessary for methane oxidation to occur and both substrates were simultaneously oxidized. CO at a concentration greater than that used in the inhibition studies was not toxic to Methylomonas sp. strain MM2.     

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