INTRODUCTION

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Like many other chlorinated hydrocarbons, trichloroethylene (TCE) has become an important environmental pollutant because of its toxic properties and widespread occurrence in groundwater. TCE is the most commonly reported volatile organic pollutant of groundwater in the United States (20). While there are no reports of bacterial growth on TCE as a sole carbon and energy source, cometabolic oxidation of TCE by nonspecific catabolic oxygenases has been described for several types of microorganisms (3) and TCE is perhaps the best-studied compound subject to aerobic cometabolism.

The application of bacteria for the aerobic bioremediation of TCE has been proposed and investigated for a wide variety of microorganisms. The most critical factors in consideration for such studies are the specific activity of the cells for TCE and the possible formation of toxic intermediates. For example, in wild-type Pseudomonas putida and P. putida F1, in which TCE oxidation is mediated by toluene dioxygenase, observed inhibition of growth has been attributed to covalent modification of cellular molecules through reactive products of TCE degradation (9, 23). For the wild type and F1, the rate of TCE removal declines rapidly in batch cultures when TCE is supplied at initial concentrations greater than 10 or 80 µM, respectively. Furthermore, it has been shown that growth substrates added to induce the catabolic genes involved in oxidation of TCE can be competitive inhibitors of TCE conversion (4).

Most studies on the kinetics of aerobic TCE degradation have been done with methane- and toluene-utilizing mixed and pure cultures (5, 9, 12, 14, 16-18, 22). Limited data on the kinetics of aerobic TCE degradation by phenol-induced monooxygenases and on the possible toxic effects of TCE oxidation metabolites are available.

Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134 is able to degrade TCE by an inducible, chromosomally encoded phenol hydroxylase (7, 11). The isolation of the chromosomally encoded phenol hydroxylase genes, through complementation of a mutant deficient in phenol degradation with a JMP134 genomic cosmid library, has been reported previously (10). The subcloning of restriction fragments from this cosmid resulted in a recombinant plasmid (pYK3021) conferring phenol hydroxylase activity and TCE degradation in the absence of phenol induction. Preliminary studies using this construct have shown a high capacity for TCE removal in the absence of aromatic induction with limited sensitivity to TCE-mediated toxicity (11).

The purpose of the work presented here was to determine the whole-cell kinetics of TCE degradation by suspended batch cultures of R. eutropha containing the recombinant plasmid pYK3021. K_s and V_max were estimated for comparison to previously published values for these parameters. Our objective was to establish the feasibility of stable constitutive expression for application to the remediation of TCE contamination. Constitutive expression allows TCE degradation to occur in the absence of induction by the normal aromatic substrate. This obviates the biochemical problems related to competitive inhibition at the active site and regulatory problems which would prohibit the introduction of aromatic inducers during in situ remediation. The degree of TCE degradation was determined during growth on a variety of noninducing carbon sources in an effort to maximize TCE removal.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, media, growth conditions, and chemicals. R. eutropha AEK301 was derived from R. eutropha JMP134. The plasmid pJP4 was cured to create strain AEO106 (8). AEO106 was subjected to rifampin selection to produce the Rif^r strain AEK101 (11). Random Tn5 mutagenesis was used to obtain strain AEK301, deficient in phenol metabolism (10). The recombinant plasmid pYK3021 contains an 8.6-kb XhoI-BamHI fragment in the pMMB67EH vector, confers TCE degradation in the absence of phenol induction, and confers resistance to carbenicillin when placed in AEK301 (10). Cultures of R. eutropha AEK301 with and without pYK3021 were maintained at 30°C on minimal salts medium (MMO) (21) supplemented with 20 mM sodium citrate or on tryptone-yeast extract-glucose medium (TNB) (19). Where indicated, MMO was supplemented with benzoate (2.5 mM), citrate (20 mM), sodium citrate (20 mM), gluconate (20 mM), lactate (20 mM), malate (20 mM), or 0.3% Casamino Acids. Concentrated stock solutions of each carbon source were prepared and adjusted to a pH of 7.0 with NaOH where necessary. When required, 50 µg of carbenicillin, 150 µg of rifampin, or 100 µg of kanamycin per ml was added to the growth medium. Yeast extract, tryptone, and agar were purchased from Difco. Other medium additives, bovine serum albumin, and chromatography-quality n-pentane were all purchased from Sigma. Chromatography-quality TCE and 1,2-dibromoethane (EDB) were purchased from Aldrich. Teflon-butyl septa and reactor vials were purchased from Fisher Scientific.

Analytical methods. TCE was measured by gas chromatography (GC) analysis with a Hewlett-Packard model 5890 gas chromatograph equipped with a 25-m cross-linked methyl silicone gum capillary column (Hewlett-Packard) and an electron capture detection system. Peak integrations were obtained with a Hewlett-Packard model 3390A integrator. The following operating conditions were used: injector temperature, 150°C; detector temperature, 250°C; column temperature, 40 to 100°C at 20°C/min; and helium carrier gas flow, 6 ml/min. Under these conditions TCE and EDB (internal standard) in pentane extracts had retention times of 2.2 and 2.9 min, respectively.

Standard TCE degradation kinetics assay. AEK301/pYK3021 was grown in MMO containing 10 mM sodium citrate and appropriate levels of kanamycin and carbenicillin at 30°C with shaking at 180 rpm to mid-log phase at an optical density of 0.6 to 0.8 at 425 nm. Cells were harvested by centrifugation at 8,000 × g for 10 min. Cell pellets were then suspended in fresh MMO containing 10 mM sodium citrate at an optical density of 1.0 at 425 nm. The cultures were then returned to 30°C and shaking at 180 rpm. After 1 h, 2-ml samples were dispensed into 20-ml glass vials, and the vials were crimp-sealed with Teflon-butyl septa. The appropriate volume of an 8 mM TCE stock was added to each vial by injection through the septum with a gastight syringe (Hamilton, Reno, Nev.). The vials were inverted to minimize TCE loss and returned to 30°C and shaking at 180 rpm. All assays were performed in triplicate.

No-headspace assay. Cultures were incubated and prepared as described above. Following the 1-h preincubation, approximately 2 ml of each cell suspension and a glass bead (3-mm diameter) were placed in a 2-ml vial crimp-sealed with a Teflon-butyl septum and with no trapped air. The glass beads were added to facilitate thorough mixing of the contents. The appropriate volume of an 8 mM TCE stock was added by injection through the septum of each vial with a gastight syringe, and the vials were incubated at 30°C with constant mixing. At 5-min intervals, the reactions were stopped by the transfer of 0.5-ml aliquots of TCE-cell suspensions into other sealed vials containing 0.5 ml of n-pentane and 1 ppm of EDB. These vials were placed at room temperature for extraction as described above, and 1 µl of the organic phase was analyzed by GC for TCE concentrations.

Protein determinations. Cell suspensions were solubilized by adding 0.2 volume of 5 M NaOH and heating at 85°C for 10 min. Following the addition of 0.2 volume of 4 M HCl, the total protein concentrations were determined by the Lowry assay (13). Bovine serum albumin which had been treated with NaOH, heat, and HCl in parallel was used as a standard in these assays.

Calculations and equations. The doubling time of batch cultures, in hours, was determined during the logarithmic phase of growth by measuring the optical density of the culture at 425 nm at two separate time points. Standard logarithmic calculation of doubling time was then applied.

	RESULTS AND DISCUSSION

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Effects of substrates on TCE removal by AEK301/pYK3021. While TCE degradation by AEK301/pYK3021 occurred in the absence of phenol induction, the apparent rate of TCE cometabolism varied depending on the carbon and energy source provided (10, 11). To further characterize this observation and enhance rates of TCE removal by AEK301/pYK3021, the degree of TCE oxidation was examined with a variety of noninducing carbon sources. AEK301/pYK3021 was grown to mid-log phase in MMO supplemented with different single substrates or in TNB, an enriched medium, and subjected to a standard TCE degradation assay at an initial concentration of 40 µM TCE. After 2 h the reactions were stopped by the addition of pentane, and the concentrations of remaining TCE were determined. While measurable amounts of TCE were removed from all reaction mixtures within 2 h, the degree of removal varied among substrates. MMO containing citrate, sodium citrate, or gluconate provided the highest TCE removal rates. MMO containing malate provided relatively poor TCE removal, as did TNB (Table 1). The doubling time for AEK301/pYK3021 in MMO supplemented with these different substrates or in TNB ranged from 1.2 h in TNB to 2.6 h in MMO supplemented with citrate (Table 1). MMO containing citrate, sodium citrate, or gluconate had the slowest doubling times (2.6, 2.5, and 2.4 h, respectively) yet provided the greatest TCE removal of the carbon sources tested. Conversely, TNB had the fastest doubling time (1.2 h) while providing relatively poor TCE removal. No significant effect on doubling time was observed in the presence of 40 µM TCE (Table 1). Catabolite repression of phenol hydroxylase catabolic genes has been demonstrated, for example, in P. putida H (15) and R. eutropha (1). In P. putida H, repression is mediated by inhibition of a phenol hydroxylase-specific transcriptional activator and subsequent reduction in transcription of the phenol hydroxylase catabolic genes (15). Repression was observed with the addition of glucose, succinate, lactate, or acetate and was least affected by the addition of pyruvate or citrate (15). In our test system, the addition of citrate or sodium citrate appeared to be the least repressive. No significant difference between the addition of 10 mM sodium citrate or that of 20 mM sodium citrate was noted (data not shown). Therefore, 10 mM sodium citrate was selected as the carbon source for all subsequent TCE degradation assays.

No-headspace assay. Although TCE is highly volatile, for convenience it is often reported as an aqueous concentration in closed systems containing both air and water phases. This could be considered misleading when reporting kinetic data. To determine the effects, if any, of TCE volatility and phase partitioning on the measured kinetic parameters, a no-headspace assay was developed for comparison. Degradation of TCE by AEK301/pYK3021 is an aerobic process and is dependent on available oxygen. In such a closed system with no headspace to replenish the consumed dissolved oxygen, it is likely that oxygen would become a limiting factor. Accordingly, the degradation of TCE in a no-headspace assay was examined at a low initial concentration of TCE. Rate measurements were made immediately following the induction lag and were found to be consistent for several sampling intervals. After this short period of stable measurements, the rates in the no-headspace assay tended to diminish significantly. This probably indicates the eventual development of limiting oxygen levels.

Time course of TCE degradation by AEK301/pYK3021. In previous studies of AEK301/pYK3021, TCE degradation was monitored over a period of many hours or even days (10, 11). For the purpose of determining the whole-cell kinetics of TCE degradation by AEK301/pYK3021, TCE degradation was instead monitored for 3 h at 15-min intervals at two different initial concentrations of TCE (16 and 80 µM). The negative control (AEK301 alone) was unable to degrade detectable amounts of TCE. For each initial TCE concentration tested, an initial lag period of approximately 40 min was observed prior to the onset of TCE degradation by AEK301/pYK3021 (Fig. 1). Following the initial lag, the rate of TCE degradation by AEK301/pYK3021 at an initial concentration of 80 µM TCE was sustained and remained essentially constant for a period of almost 2 h, until TCE was no longer detectable.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Degradation of TCE by AEK301/pYK3021 at an initial concentration of 16 µM TCE () and AEK301/pYK3021 at an initial concentration of 80 µM TCE (). The negative control, AEK301 alone (), was used at an initial concentration of 80 µM TCE. Cultures were grown in MMO supplemented with 10 mM sodium citrate to mid-log phase, harvested by centrifugation, and suspended in fresh medium to an optical density of 1.0 at 425 nm. After 1 h at 30°C, 2-ml samples of each strain were then distributed into vials and the vials were sealed. Reactions were initiated by the injection of TCE through the septum. Samples were collected in duplicate every 15 min for a total of 3 h. Each data point represents the average of two or more samples, and error bars are provided where visible.

Whole-cell kinetics of TCE degradation by AEK301/pYK3021. To accurately measure initial rates of TCE degradation by whole cells of AEK301/pYK3021, 5-min assays (beginning 50 min following the addition of TCE to the reactor vials) were conducted at various concentrations of TCE. For seven different initial TCE concentrations ranging from 16 to 1,600 µM, TCE degradation rates and total protein concentrations were measured. These results, summarized in Table 3, represent the averages for at least three different samples. The velocity or rate of degradation for each concentration was determined, and these values were plotted as a function of the initial TCE concentration (Fig. 2). The rate of TCE degradation by AEK301/pYK3021 was essentially linear from 16 to 800 µM TCE. The rate observed at 1,600 µM TCE was similar to that observed at 800 µM TCE, indicating that the saturation or inhibition of the catabolic enzymes occurred somewhere between these two concentrations.

View larger version (17K): [in this window] [in a new window]   FIG. 2.   Whole-cell kinetics of TCE degradation by AEK301/pYK3021. The initial rates of TCE degradation were determined as described in Materials and Methods. Rates are plotted as a function of initial TCE concentration.