Comparison of TCE Transformation Abilities of Methane- and Propane-Utilizing Microorganisms

Subsurface microorganisms from McClellan Air Force Base (AFB) were grown in batch aquifer microcosms on methane, propane, and butane to evaluate the potential for aerobic trichloroethylene (TCE) cometabolism. Microorganisms stimulated on all three substrates indicated the existence of a subsurface microbial community capable of utilizing alkanes as growth substrates. Initial growth substrate utilization lag periods of 2 weeks for methane and 3 weeks for propane and butane were observed. Methane- and propane-utilizers were active toward TCE cometabolism, whereas butane-utilizers showed no ability to transform TCE. Gradually increasing TCE concentrations were effectively transformed with uniform additions of methane and propane for up to 1 year. TCE was transformed most rapidly during active methane utilization, and continued at a slower rate for approximately 1 week after methane was consumed. Propane microcosms maintained first-order TCE transformation for up to 4 weeks after propane was consumed. The microbial communities remained active toward primary substrate utilization as the TCE concentration was gradually increased. Both methane- and propane-utilizers showed positive correlations between TCE transformation rates and primary substrate utilization rates. Observed maximum TCE transformation yields were 0.068 g TCE/g methane and 0.048 g TCE/g propane. The methane-utilizers also transformed chloroform (CF) but not 1,1,1-trichloroethane (1,1,1-TCA). Propane-utilizers transformed both CF and 1,1,1-TCA, indicating they were better suited for cometabolizing chlorinated aliphatic hydrocarbon mixtures in the McClellan AFB subsurface.     

Laboratory evaluation of a two-stage treatment system for TCE cometabolism by a methane-oxidizing mixed culture

The objective of this research was to evaluate several factors affecting the performance of a two-stage treatment system employing methane-oxidizing bacteria for trichloroethylene (TCE) biodegradation. The system consists of a completely mixed growth reactor and a plug-flow transformation reactor in which the TCE is cometabolized. Laboratory studies were conducted with continuous growth reactors and batch experiments simulating transformation reactor conditions. Performance was characterized in terms of TCE transformation capacity (T(C), g TCE/g cells), transformation yield (T(Y), g TCE/g CH(4)), and the rate coefficient ratio k(TCE)/K(S,TCE) (L/mg-d). The growth reactor variables studied were solids retention time (SRT) and nutrient nitrogen (N) concentration. Formate and methane were evaluated as potential transformation reactor amendments. Comparison of cultures from 2- and 8-day SRT (nitrogen-limited) growth reactors indicated that there was no significant effect of growth reactor SRT or nitrogen availability on T(C) or T(Y), but N-limited conditions yielded higher k(TCE)/K(S,TCE). The TCE cometabolic activity of the 8-day SRT, N-limited growth reactor culture varied significantly during a 7-year period of operation. The T(C) and T(Y) of the resting cells increased gradually to levels a factor of 2 higher than the initial values. The reasons for this increase are unknown. Formate addition to the transformation reactor gave higher T(C) and T(Y) for 2-day SRT growth reactor conditions and significantly lower T(C), T(Y), and k(TCE)/K(S,TCE) for 8-day SRT N-limited conditions. Methane addition to the transformation reactor inhibited TCE cometabolism at low TCE concentrations and enhanced TCE cometabolism at high TCE concentrations, indicating that the TCE cometabolism in the presence of methane does not follow simple competitive inhibition kinetics. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 650-659, 1997.     

Effect of carbon starvation on toluene degradation activity by toluene monooxygenase-expressing bacteria

Subsurface bacteria commonly exist in a starvation state with only periodic exposure to utilizable sources of carbon and energy. In this study, the effect of carbon starvation on aerobic toluene degradation was quantitatively evaluated with a selection of bacteria representing all the known toluene oxygenase enzyme pathways. For all the investigated strains, the rate of toluene biodegradation decreased exponentially with starvation time. First-order deactivation rate constants for TMO-expressing bacteria were approximately an order of magnitude greater than those for other oxygenase-expressing bacteria. When growth conditions (the type of growth substrate and the type and concentration of toluene oxygenase inducer) were varied in the cultures prior to the deactivation experiments, the rate of deactivation was not significantly affected, suggesting that the rate of deactivation is independent of previous substrate/inducer conditions. Because TMO-expressing bacteria are known to efficiently detoxify TCE in subsurface environments, these findings have significant implications for in situ TCE bioremediation, specifically for environments experiencing variable growth-substrate exposure conditions.     

Experimental evaluation of a model for cometabolism: Prediction of simultaneous degradation of trichloroethylene and methane by a methanotrophic mixed culture

A model for cometabolism is verified experimentally for a defined methanotrophic mixed culture. The model includes the effects of cell growth, endogenous cell decay, product toxicity, and competitive inhibition with the assumption that cometabolic transformation rates are enhanced by reducing power obtained from oxidation of growth substrates. A theoretical transformation yield is used to quantify the enhancement resulting from growth substrate oxidation. A systematic method for evaluating model parameters independently is described. The applicability of the model is evaluated by comparing experimental data for methanotrophic cometabolism of TCE with model predictions from independently measured model parameters. Propagation of errors is used to quantify errors in parameter estimates and in the final prediction. The model successfully predicts TCE transformation and methane utilization for a wide range of concentrations of TCE (0.5 to 9 mg/L) and methane (0.05 to 6 mg/L). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 492-501, 1997.     

Trichloroethylene degradation by butane-oxidizing bacteria causes a spectrum of toxic effects

The physiological consequences of trichloroethylene (TCE) transformation by three butane oxidizers were examined. Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 utilize distinctly different butane monooxygenases (BMOs) to initiate degradation of the recalcitrant TCE molecule. Although the primary toxic event resulting from TCE cometabolism by these three strains was loss of BMO activity, species differences were observed. P. butanovora and Nocardioides sp. CF8 maintained only 4% residual BMO activity following exposure to 165 μM TCE for 90 min and 180 min, respectively. In contrast, M. vaccae maintained 34% residual activity even after exposure to 165 μM TCE for 300 min. Culture viability was reduced 83% in P. butanovora, but was unaffected in the other two species. Transformation of 530 nmol of TCE by P. butanovora (1.0 mg total protein) did not affect the viability of BMO-deficient P. butanovora cells, whereas transformation of 482 nmol of TCE by toluene-grown Burkholderia cepacia G4 caused 87% of BMO-deficient P. butanovora cells to lose viability. Together, these results contrast with those previously reported for other bacteria carrying out TCE cometabolism and demonstrate the range of cellular toxicities associated with TCE cometabolism.     

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.     

Effect of trichloroethylene (TCE) and toluene concentrations on TCE and toluene biodegradation and the population density of TCE and toluene degraders in soil.

Toluene is one of several cosubstrates able to support the cometabolism of trichloroethylene (TCE) by soil microbial communities. Indigenous microbial populations in soil degraded TCE in the presence, but not the absence, of toluene after a 60- to 80-h lag period. Initial populations of toluene and TCE degraders ranged from 0.2 x 10(3) to 4 x 10(3) cells per g of soil and increased by more than 4 orders of magnitude after the addition of 20 micrograms of toluene and 1 microgram of TCE per ml of soil solution. The numbers of TCE and toluene degraders and the percent removal of TCE increased with an increase in initial toluene concentration. As the initial TCE concentration was increased from 1 to 20 micrograms/ml, the numbers of toluene and TCE degraders and the rate of toluene degradation decreased, and no TCE degradation occurred. No toluene or TCE degradation occurred at a TCE concentration of 50 micrograms/ml.     

Correlation of TCE cometabolism with growth characteristics on aromatic substrates in toluene-degrading bacteria

We have constructed a bacterial library consisting of 97 strains of toluene-degrading bacteria from soil and activated sludge samples for examining their physiological properties in terms of cometabolism of TCE. Large variation of TCE degradation ability was observed in Gram-positive and Gram-negative strains, as well as diverse patterns of availability of aromatics as a growth substrate. No clear correlation was observed between the number of available substrates for growth and TCE degradation ability. However, the growth on some of the aromatics showed positive or negative correlations with TCE degradation ability. Kendall correlation constants (tau) for the growth on cumene, m-xylene, p-xylene, and m-cresol with TCE degradation ability were statistically significant (P < 0.001): their values were −0.44, −0.31, −0.26, 0.37, respectively. Among 12 of aromatics, only m-cresol showed positive correlation with TCE degradation ability. These findings would be useful for enrichment and isolation of the microbes that have TCE-cometabolism ability, which is a selective disadvantage through the toxicity or competitive inhibition against growth substrates.     

Inhibition, Inactivation, and Recovery of Ammonia-Oxidizing Activity in Cometabolism of Trichloroethylene by Nitrosomonas europaea

The kinetics of the cometabolism of trichloroethylene (TCE) by the ammonia-oxidizing soil bacterium Nitrosomonas europaea in short-term (<10-min) incubations were investigated. Three individual effects of TCE cometabolism on this bacterium were characterized. First, we observed that TCE is a potent competitive inhibitor of ammonia oxidation by N. europaea. The K(infi) value for TCE (30 (mu)M) is similar to the K(infm) for ammonia (40 (mu)M). Second, we examined the toxicity associated with TCE cometabolism by N. europaea. Stationary-phase cells of N. europaea oxidized approximately 60 nmol of TCE per mg of protein before ammonia-oxidizing activity was completely inactivated by reactive intermediates generated during TCE oxidation. At the TCE concentrations used in these experiments, ammonia did not provide significant protection against inactivation. Third, we have determined the ability of cells to recover ammonia-oxidizing activity after exposure to TCE. Cells recovering from TCE inactivation were compared with cells recovering from the specific inactivation of ammonia-oxidizing activity by light. The recovery kinetics were indistinguishable when 40% or less of the activity was inactivated. However, at increased levels of inactivation, TCE-inactivated cells did not recover as rapidly as light-inactivated cells. The kinetics of recovery appear to be dependent on both the extent of inactivation of ammonia-oxidizing activity and the degree of specificity of the inactivating treatment.     

Kinetic analysis of a tod-lux bacterial reporter for toluene degradation and trichloroethylene cometabolism

Kinetics of toluene and trichloroethylene (TCE) degradation and bioluminescence from the bioreporter Pseudomonas putida B2 and TVA8 were investigated utilizing batch and continuous culture, respectively. Degradation was modeled using a Michaelis-Menten expression for the competition of two substrates for a single enzyme system, and bioluminescence was modeled assuming a luciferase enzyme saturational dependence on toluene as the inducer and growth substrate. During the batch experiments, bioluminescence increased at approximately 90 namp/min for initial toluene concentrations of 10 to 50 mg/L, but more slowly at higher toluene concentrations, suggesting maximum promoter induction at below 10 mg/L and toxic effects above 50 mg/L toluene. TCE degradation did not occur until toluene depletion, presumably due to competition between toluene and TCE for the toluene dioxygenase enzyme. During continuous culture, bioluminescence transiently increased, then gradually decreased in response to increasing step changes in toluene feed concentration. Bioluminescence in the CSTR appeared to be limited by growth substrate and/or inducer.     

Effect of Chemical Oxidation on Subsurface Microbiology and Trichloroethene (TCE) Biodegradation

Research was conducted to determine the effect of chemical oxidation on subsurface microbiology and cometabolic biodegradation capacity in a trichloroethene (TCE)/perchloroethene (PCE)-contaminated aquifer previously treated with Fenton's reagent. Groundwater pH declined from 5 to 2.4 immediately after the treatment, and subsequently rose to a range of 3.4 to 4.0 after 17 months. Limited microbial growth and TCE degradation were detected in the treated zone (pH 3.37 and TCE 5 to 21 mg/L) with carbon addition (i.e., methane and phenol). Methane addition resulted in the enrichment of yeast and fungi in microcosms at low pH. In contrast, methane addition to groundwater from the control well (pH 4.9 and TCE ca. 0.7 mg/L) stimulated methanotrophic growth, indicated by methane consumption, fluorescent antibody analysis, phospholipid-based markers, and rDNA probes. TCE degradation was measured in the control microcosms, but only when phenol was added. Although higher TCE concentrations in the treated zone might have inhibited TCE cometabolism, these results also indicate that low groundwater pH resulting from the chemical oxidation process (pH 3.37 versus 4.9) inhibited TCE degradation. Methanotrophic growth and TCE biodegradation may be possible as pH increases both in the treated zone and at the leading edge of plume, as long as the local soil is able to buffer the groundwater pH. Moreover, the Fenton's reagent process could be designed to operate at a higher pH (e.g., ≥ 4.5) and/or lower hydrogen peroxide concentration to minimize detrimental effects, providing an optimal environment to couple advanced oxidation processes with bioremediation technologies.     

Biodegradation of tri- and perchloroethylene in sewage waters and soils by a microbial consortium of compost and phototrophic bacteria

Compost and phototrophic bacteria are found to be able to degrade trichloroethylene (TCE) and perchloroethylene (PCE). With a TCE dose increase by more than 10 mg/kg, the TCE degradation decreases due to the toxic effect of this pollutant on microbial consortium activity. The addition of compost combined with a liquid culture of phototrophic bacteria (PTB) is experimentally proved to effectively decrease the TCE content in soil and water.     

Factors Limiting Aliphatic Chlorocarbon Degradation by Nitrosomonas europaea: Cometabolic Inactivation of Ammonia Monooxygenase and Substrate Specificity

The soil nitrifying bacterium Nitrosomonas europaea is capable of degrading trichloroethylene (TCE) and other halogenated hydrocarbons. TCE cometabolism by N. europaea resulted in an irreversible loss of TCE biodegradative capacity, ammonia-oxidizing activity, and ammonia-dependent O(2) uptake by the cells. Inactivation was not observed in the presence of allylthiourea, a specific inhibitor of the enzyme ammonia monooxygenase, or under anaerobic conditions, indicating that the TCE-mediated inactivation required ammonia monooxygenase activity. When N. europaea cells were incubated with [C]TCE under conditions which allowed turnover of ammonia monooxygenase, a number of cellular proteins were covalently labeled with C. Treatment of cells with allylthiourea or acetylene prior to incubation with [C]TCE prevented incorporation of C into proteins. The ammonia-oxidizing activity of cells inactivated in the presence of TCE could be recovered through a process requiring de novo protein synthesis. In addition to TCE, a series of chlorinated methanes, ethanes, and other ethylenes were screened as substrates for ammonia monooxygenase and for their ability to inactivate the ammonia-oxidizing system of N. europaea. The chlorocarbons could be divided into three classes depending on their biodegradability and inactivating potential: (i) compounds which were not biodegradable by N. europaea and which had no toxic effect on the cells; (ii) compounds which were cooxidized by N. europaea and had little or no toxic effect on the cells; and (iii) compounds which were cooxidized and produced a turnover-dependent inactivation of ammonia oxidation by N. europaea.     

Trichloroethylene cometabolic degradation by Rhodococcus sp. L4 induced with plant essential oils

Cometabolic degradation of TCE by toluene-degrading bacteria has the potential for being a cost-effective bioremediation technology. However, the application of toluene may pose environmental problems. In this study, several plant essential oils and their components were examined as alternative inducer for TCE cometabolic degradation in a toluene-degrading bacterium, Rhodococcus sp. L4. Using the initial TCE concentration of 80 muM, lemon and lemongrass oil-grown cells were capable of 20 +/- 6% and 27 +/- 8% TCE degradation, which were lower than that of toluene-grown cells (57 +/- 5%). The ability of TCE degradation increased to 36 +/- 6% when the bacterium was induced with cumin oil. The induction of TCE-degrading enzymes was suggested to be due to the presence of citral, cumin aldehyde, cumene, and limonene in these essential oils. In particular, the efficiency of cumin aldehyde and cumene as inducers for TCE cometabolic degradation was similar to toluene. TCE transformation capacities (T (c)) for these induced cells were between 9.4 and 15.1 mug of TCE mg cells(-1), which were similar to the known toluene, phenol, propane or ammonia degraders. Since these plant essential oils are abundant and considered non-toxic to humans, they may be applied to stimulate TCE degradation in the environment.     

The kinetics of cometabolism

Experimental observations indicate that the rates of cometabolic transformation are linked to the consumption of growth substrate during growth and to the consumption of cell mass and/or energy substrate in the absence of growth substrate. Three previously proposed models (models 1 through 3) describing the kinetics of cometabolism by resting cells are compared, and the interrelationships and underlying assumptions for these models are explored. Models 1 to 3 are shown to converge at high concentrations of the nongrowth substrate. An expression describing nongrowth substrate transformation in the presence of growth substrate is proposed, and this expression is integrated with an expression for cell growth to give a single unstructured model (model 4) that encompasses models 1 to 3 and describes cometabolism by both resting and growing cells. Model 4 couples transformation of nongrowth substrate to consumption of growth substrate and biomass, and predicts that cometabolism will result, and decreased specific growth rates for a cometabolizing population. Competitive inhibition can also be incorporated in the model. Experimental aspects of model calibration and verification are discussed. The need for models that distinguish between the exhaustion of cell activity and cell death is emphasized. (c) 1993 Wiley & Sons, Inc.     

Cometabolism of methyl tert-butyl ether (MTBE) with alkanes

The release of methyl tert-butyl ether (MTBE) to the environment, mainly from damaged gasoline underground storage tanks or distribution systems spills, has provoked extended groundwater pollution. Biological treatments are, in general, a good alternative for bioremediation of polluted sites; however, MTBE elimination from environment has constituted a challenge because of its chemical structure and physicochemical properties. The combination of a stable ether link and the branched moiety hinder biodegradation. Initial studies found MTBE to be highly recalcitrant but, in the last decade, reports of its biodegradation have been published first under aerobic conditions and just recently under anaerobic conditions. Microbial MTBE degradation is characterized by bacteria having low growth rates (0.35 day⁻¹) and biomass yields (average value 0.24 g biomass/g MTBE). Alternatively, cometabolism (defined as the transformation of a non-growth substrate in the obligate presence of a growth substrate), has been considered since it uncouples biodegradation of the contaminant from growth, reducing the long adaptation and propagation period. This period has been reported to be of several months in systems where it is degraded as sole carbon source. Cometabolic degradation rates are between 0.3 and 61 nmol/min/mg protein (in the same range of direct aerobic metabolism). However, a major concern in MTBE cometabolism is that the accumulation of tert-butyl alcohol (TBA) may, under certain cases, result in an incomplete site cleanup. This paper reviews in detail the implicated enzymes and field treatments for the cometabolism of MTBE degradation with alkanes as growth substrates.     

Cometabolic Degradation of Trichloroethene by Rhodococcus sp. Strain L4 Immobilized on Plant Materials Rich in Essential Oils

The cometabolic degradation of trichloroethene (TCE) by Rhodococcus sp. L4 was limited by the loss of enzyme activity during TCE transformation. This problem was overcome by repeated addition of inducing substrates, such as cumene, limonene, or cumin aldehyde, to the cells. Alternatively, Rhodococcus sp. L4 was immobilized on plant materials which contain those inducers in their essential oils. Cumin seeds were the most suitable immobilizing material, and the immobilized cells tolerated up to 68 μM TCE and degraded TCE continuously. The activity of immobilized cells, which had been inactivated partially during TCE degradation, could be reactivated by incubation in mineral salts medium without TCE. These findings demonstrate that immobilization of Rhodococcus sp. L4 on plant materials rich in essential oils is a promising method for efficient cometabolic degradation of TCE.Various bacteria have been reported to degrade trichloroethene (TCE) aerobically via cometabolic degradation with broad-substrate-specificity enzymes (2). However, TCE cometabolic degradation is considered an unsustainable process due to cytotoxicity, inhibition, or inactivation of TCE-degrading enzymes. These phenomena have been observed in studies using both whole cells and purified enzymes, including soluble methane monooxygenases from Methylosinus trichosporium OB3b (9) and Nitrosomonas europaea (13), toluene 2-monooxygenase from Burkholderia cepacia G4 (19, 27), toluene dioxygenase (TDO) from Pseudomonas putida F1 (15, 18), and butane-oxidizing bacteria, i.e., Pseudomonas butanovora, Mycobacterium vaccae, and Nocardioides sp. CF8 (11). Nevertheless, the addition of an inducer or growth substrate can maintain TCE cometabolic degradation. For example, the TCE-degrading activity of P. putida F1 toluene dioxygenase was restored after adding benzene, cumene, or toluene to displace TCE and its reactive intermediates from the enzyme active site (18). Arp et al. (2) suggested that the rate of enzyme maintenance and recovery depended on the extent of inactivation and the balance of TCE and inducer/growth substrate concentrations.Plant essential oils and their components, such as citral, limonene, cumene, and cumin aldehyde, have been found to induce TCE degradation in Rhodococcus sp. L4 (24). However, the removal of TCE by this bacterium was effective only for a short period. The impacts of TCE on Rhodococcus spp. and their enzymes have not been studied in detail, even though many bacteria of this genus exhibited high TCE-degrading activities (i.e., Rhodococcus erythropolis JE 77, R. erythropolis BD2, Rhodococcus sp. Sm-1, and Rhodococcus sp. Wrink) (5, 6, 7, 16). This study therefore investigated the changes in TCE-degrading activity of Rhodococcus sp. L4 cells and TDO during exposure to TCE. Two enzyme maintenance approaches were evaluated, namely, repeated addition of essential oil components to the system and immobilization of the bacterial cells on plant material rich in essential oils. Immobilized microorganisms are generally capable of degrading pollutants at a higher initial concentration and for a longer period than those of free cells (21, 23), possibly because the microbial cells are protected from environmental stress and toxic compounds (3). In this study, the plant materials were used to provide a solid surface for bacterial attachment and a continuous source of essential oils for inducing TCE-degrading enzymes. Our results show that the repeated addition of limonene, cumene, or cumin aldehyde enhances TCE degradation and that bacteria immobilized on cumin seeds are able to maintain their TCE-degrading activity.     

Understanding the diversity in catabolic potential of microorganisms for the development of bioremediation strategies

Molecular ecological approaches have detected diverse microorganisms that occur in response to pollution and bioremediation; however, most of these organisms have not been isolated, and their physiological traits are poorly understood. One important objective in current bioremediation studies would therefore be an assessment of the physiology and functions of the diverse microbial population at a polluted site. Among the parameters relating to the diversity of the microbial catabolic potential, e.g., substrate specificity, inducer specificity, number of catabolic routes and kinetics of catabolic enzymes, our studies have focused on the kinetic diversity of phenol-degrading bacteria. In one example, a kinetic analysis allowed functionally important phenol-degrading bacteria to be identified in activated sludge; this information could be used to improve the performance of phenol-degrading activated sludge. In an analysis of phenol-degrading bacteria present in trichloroethylene (TCE)-contaminated aquifer soil, the kinetic data could be linked to group-specific monitoring of their phenol-hydroxylase genes. The results have suggested that one group of phenol-degrading bacteria can effectively contribute to TCE bioremediation, while other groups work poorly. Based on this information, we have succeeded in developing a high-performance TCE-degrading bioreactor. We suggest that a careful analysis of the diversity of microbial catabolic potential, particularly of the kinetic traits, may facilitate the development of new bioremediation strategies. This revised version was published online in August 2006 with corrections to the Cover Date.     

Numerical modeling and uncertainties in rate coefficients for methane utilization and TCE cometabolism by a methane-oxidizing mixed culture

The rates of methane utilization and trichloroethylene (TCE) cometabolism by a methanotrophic mixed culture were characterized in batch and pseudo-steady-state studies. Procedures for determination of the rate coefficients and their uncertainties by fitting a numerical model to experimental data are described. The model consisted of a system of differential equations for the rates of Monod kinetics, cell growth on methane and inactivation due to TCE transformation product toxicity, gas/liquid mass transfer of methane and TCE, and the rate of passive losses of TCE. The maximum specific rate of methane utilization (k(CH(4) )) was determined by fitting the numerical model to batch experimental data, with the initial concentration of active methane-oxidizing cells (X(0) (a)) also used as a model fitting parameter. The best estimate of k(CH(4) ) was 2.2 g CH(4)/g cells-d with excess copper available, with a single-parameter 95% confidence interval of 2.0-2.4 mg/mg-d. The joint 95% confidence region for k(CH(4) ) and X(0) (a) is presented graphically. The half-velocity coefficient (K(S,CH(4) )) was 0.07 mg CH(4)/L with excess copper available and 0.47 mg CH(4)/L under copper limitation, with 95% confidence intervals of 0.02-0.11 and 0.35-0.59 mg/L, respectively. Unique values of the TCE rate coefficients k(TCE) and K(S,TCE) could not be determined because they were found to be highly correlated in the model fitting analysis. However, the ratio k(TCE)/K(S,TCE) and the TCE transformation capacity (T(C)) were well defined, with values of 0.35 L/mg-day and 0.21 g TCE/g active cells, respectively, for cells transforming TCE in the absence of methane or supplemental formate. The single-parameter 95% confidence intervals for k(TCE)/K(S,TCE) and T(C) were 0.27-0.43 L/mg-d and 0.18-0.24 g TCE/g active cells, respectively. The joint 95% confidence regions for k(TCE)/K(S,TCE) and T(C) are presented graphically. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 320-331, 1997.