Oxidation of Trichloroethylene, 1,1-Dichloroethylene, and Chloroform by Toluene/o-Xylene Monooxygenase from Pseudomonas stutzeri OX1

Growth conditions. E. coli JM109(pBZ1260) was grown at 37°C overnight in Luria-Bertani (LB) medium (10) containing ampicillin (150 μg/ml; Sigma Chemical Co., St. Louis, Mo.) and then subcultured in fresh medium at an optical density of 0.1 at 600 nm. When the culture attained an optical density of 0.8 to 1.0 (note that a blue, insoluble pigment was clearly seen), 1 mM IPTG (Fisher Scientific, Fairlawn, N.J.) was added, and the cultures were grown for an additional 2 h. Cells were harvested by centrifugation at 2,000 × g for 15 min at 4°C (model J2-21 centrifuge; Beckman; Palo Alto, Calif.). The cells were washed twice with modified minimal salts (M9) medium (10) which lacked chloride (MgSO₄ [0.24 g/l] and CaSO₄[0.17 g/l] replaced MgCl₂ and CaCl₂) and diluted to an optical density of 1.0 at 600 nm.

Chemicals.TCE, chloroform, dichloromethane, and phenol were purchased from Fisher Scientific (Tustin, Calif.), and 1,1-DCE,cis-1,2-DCE, and trans-1,2-DCE were purchased from Supelco, Inc. (Bellefonte, Pa.). The chlorinated phenols 2,4,5- and 2,4,6-trichlorophenol, and 2,3,4,5,6-pentachlorophenol were purchased from Sigma Chemical Co. 2,3,5,6-Tetrachlorophenol was purchased from AccuStandard, Inc. (New Haven, Conn.), and 2,4-dichlorophenol was purchased from Eastman Kodak Co. (Rochester, N.Y.). All materials used were of the highest purity available and were used without further purification.

Extents of degradation and initial degradation rates.To determine the extents of degradation of the chlorinated aliphatics and the initial degradation rates of TCE, chloroform, and 1,1-DCE, these compounds were added at an initial concentration of 10 μM (assuming all substrates to be in the liquid phase) from a 0.1 M stock solution in N,N-dimethylformamide (Fisher Scientific). Ten milliliters of IPTG-induced cells in M9 medium containing no chloride was added to 60-ml glass vials to yield the actual liquid concentrations shown in Table 1 (calculated with Henry’s law constants of 0.4, 1.39, and 0.11 for TCE [6], 1,1-DCE [13], and chloroform [13], respectively). The vials were shaken at room temperature at 200 rpm on an IKA-Vibrax-VXR shaker (IKA-Works, Inc., Cincinnati, Ohio). The concentrations of the chlorinated aliphatics were determined by gas chromatography after 24 h (extent of degradation performed in duplicate) and at 0, 5, 10, and 15 min after substrate addition (initial degradation rate) by injecting a 10-μl headspace sample by using a 50-μl gas-tight syringe (Hamilton, Reno, Nev.) into a 5890 Series II gas chromatograph (Hewlett-Packard Co., Palo Alto, Calif.) equipped with an electron capture detector and fitted with a 0.1% AT-1000 on an 80/100 Graphpac packed column (Alltech Associates, Inc., Deerfield, Ill.). The samples were analyzed isocratically with the column and injector at 180°C and the detector at 210°C. To determine the initial degradation rates, two independent experiments were used for TCE, 1,1-DCE, and chloroform, and each of these experiments had duplicate or triplicate vials. Both abiotic (no cells plus chlorinated aliphatic) and biotic (live JM109 plus chlorinated aliphatic) controls were used.

To determine the extents of degradation of phenol and the chlorinated phenols, these compounds were added at 10 μM initial concentrations (assuming all substrates to be in the liquid phase) to 25-ml liquid cultures containing IPTG-induced cells in 250-ml Erlenmeyer flasks. 2,4-Dichlorophenol stock was prepared in ethanol, whereas the other phenol stocks were prepared in water. After 4, 8, and 12 h, phenol and 2,4-dichlorophenol were assayed by a colorimetric assay (11), and the disappearance of the other phenols was monitored by measuring the absorption at 310 nm for 2,4,5- and 2,4,6-trichlorophenol, 306 nm for 2,3,5,6-tetrachlorophenol, and 318 nm for 2,3,4,5,6-pentachlorophenol. For each assay, cells were removed by centrifugation prior to monitoring the absorbance change.

Plasmid stability.After 2 h of induction with IPTG, segregational plasmid stability of pBZ1260 in E. coli JM109 was measured at the time of the addition of the substrates TCE, 1,1-DCE, or chloroform and 15 min later (to cover the period of the initial degradation experiments). Appropriate dilutions of the culture were plated on LB plates and LB plates containing ampicillin (150 μg/ml). The colonies were enumerated after incubating the plates at 37°C overnight.

Chloride analysis and total protein.Chloride released by the degradation of TCE, 1,1-DCE, and chloroform was measured in duplicate by a colorimetric assay after 24 h (2). After cells were removed from a 1-ml sample by centrifugation, 200 μl of 0.25 M ferric ammonium sulfate in 9 N HNO₃ and 200 μl of saturated mercuric thiocyanate in ethanol were added and incubated for 10 min, and then the absorbance at 460 nm was measured. Total protein of cells at an absorbance of 1.0 (at 600 nm) was determined with a Sigma Diagnostics Protein Assay Kit (catalog no. P 5656).