INTRODUCTION

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1,2-Dichloroethane (DCA) is a synthetic chemical which has no known natural sources. DCA is mainly used as an intermediate in the synthesis of vinyl chloride, but it is also used in the production of chlorinated solvents such as trichloroethene, tetrachloroethene, and 1,1,1-trichloroethane. Based on the low sorption coefficient (log K_oc = 1.28) and the good solubility in water, leaching of DCA to groundwater is expected when DCA is released to the environment (23). Abiotic degradation of DCA does occur, however, very slowly. Under conditions similar to those in groundwater (15°C, pH 7, and in the presence of 1 mM total sulfide) the half-life of DCA is 23 years (3).

Aerobic biodegradation of DCA was first demonstrated in 1983 by Stucki et al. (16). The isolated Pseudomonas sp. strain DE2, is able to grow on DCA as the sole source of carbon and energy with a growth rate of 0.08 h¹ at 30°C. A strain studied in more detail is Xanthobacter autotrophicus GJ10 (11, 12). This strain was isolated from a mixture of activated sludge and contaminated soil. The first step in DCA metabolism in X. autotrophicus GJ10 is a hydrolytic dehalogenation to 2-chloroethanol, which is subsequently converted to chloroacetaldehyde by an alcohol dehydrogenase (12). Chloroacetaldehyde is further dehydrogenated to form chloroacetic acid. The latter compound is presumably dehalogenated by chloroacetate dehalogenase to glycolic acid, which can enter the central metabolic pathways (12).

X. autotrophicus GJ10 has been used in several studies aimed at biological treatment of synthetic waste waters containing DCA (5-7). In most of these studies, however, DCA concentrations were in the millimolar range whereas, for practical application, DCA in groundwater has to be removed at concentrations in the micromolar range. However, the kinetic parameters determined for suspended cells of strain GJ10 (20) indicate that the affinity of this strain for DCA is too low for the efficient removal of DCA at these low concentrations. In an attempt to isolate microorganisms with a higher affinity for DCA, van den Wijngaard et al. (19) isolated the bacterial strain Ancylobacter aquaticus AD25. They reported a K_s value of 24 µM for this strain compared to 260 µM for strain GJ10. Interestingly, strain AD25 expressed the same dehalogenase as strain GJ10, but at much higher levels, which explains the higher apparent affinity of strain AD25 for DCA (19, 20). However, A. aquaticus AD25, as well as X. autotrophicus GJ10 and Pseudomonas sp. strain DE2, require vitamins for optimal growth (16, 21). Therefore, we set out to isolate DCA-degrading bacteria with a high affinity for DCA but without the dependency on vitamins for optimal growth. In this report we describe the initial characterization of a new isolate, Pseudomonas sp. strain DCA1, which has a high affinity for DCA and utilizes a novel pathway for DCA degradation.

	MATERIALS AND METHODS

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Isolation of bacteria. Enrichments in batch cultures were set up in closed bottles containing mineral salts medium (10), with mixtures of soil and water from various sources as inocula. Incubations were carried out with 1 mM DCA at different temperatures (21 and 30°C).

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Analytical methods. Concentrations of DCA were determined by analyzing 100-µl headspace samples on a Chrompack CP9000 gas chromatograph equipped with a CP-Sil 8CB column (Chrompack B.V., Middleburg, The Netherlands). The oven temperature was kept at 100°C. All experiments were performed at 25°C unless stated otherwise. The partition coefficient for DCA between the gas phase and the liquid phase at this temperature is 0.05 (1). Concentrations of CO₂ were measured by injecting 100-µl gas phase samples into a Hewlett-Packard 6890 gas chromatograph containing a Chrompack Poraplot Q column. Qualitative determination of the chiral composition of the formed 1,2-epoxypropane was performed by headspace analysis on a Carlo Erba Strumentazione (series 4200) gas chromatograph with a -cyclodextrin 225 column (Supelco, Zwijndrecht, The Netherlands) at 50°C. Samples were heated to 60°C and 500-µl headspace was injected into the column. Retention times were 8.9 and 9.3 min for the R- and S-enantiomers, respectively.

Batch cultures. Growth experiments in batch cultures were performed in 250-ml serum bottles containing 50 ml of mineral salts medium (10). In the case of volatile compounds, such as DCA, Boston bottles with Teflon caps (Mininert; Phase Separations, Waddinxveen, The Netherlands) were used in order to prevent substrate loss by evaporation. In the case of DCA as the carbon and energy source, the substrate concentration was 1 mM unless stated otherwise. Growth rates were assessed by measuring CO₂ production during the exponential phase.

Continuous culture of strain DCA1. Strain DCA1 was grown continuously in a 2-liter fermenter containing 1 liter of mineral salts medium (10). The pH was maintained at 7.0 by the addition of sterile 2 N NaOH. The dilution rate was 0.05 h¹, and the temperature was 25°C. Air was bubbled through a column containing pure DCA at a rate of 5 ml/min. This air stream was diluted by mixing with a second stream of air (2,000 ml/min), resulting in an ingoing DCA concentration in the gas phase of 1 mg/liter which was bubbled through the liquid phase. The biomass density was monitored by determining the optical density at 660 nm (OD₆₆₀). Under steady-state conditions, the OD₆₆₀ was approximately 0.5.

Preparation of cell extracts. Cells were centrifuged for 10 min at 16,000 × g and then washed in an equal volume of 50 mM Tris-HCl buffer (pH 7.5). The pellet was resuspended in a 100-times smaller volume, and the cells were disrupted by sonication for 1 min on ice followed by centrifugation (20 min at 39,000 × g). The supernatant was used immediately for the determination of enzyme activities. The protein content was determined by using BCA Protein Assay Reagent (Pierce, Ill.).

Enzyme assays. DCA monooxygenase activity in cell extracts of strain DCA1 was determined in 25-ml glass vials (Supelco, Zwijndrecht, The Netherlands) closed with Teflon valves (Mininert). Cell extract was diluted in 50 mM Tris-HCl (pH 7.5) to a final volume of 2 ml. NADH or NADPH was added to a final concentration of 2 mM. DCA was added at an initial concentration of 100 µM unless stated otherwise, and DCA degradation was followed by headspace analysis as described above. For anaerobic incubations, 35-ml serum bottles with rubber septa were used. Bottles were flushed with nitrogen for 10 min before the DCA was added. A control incubation showed that no significant loss of DCA occurred during the time scale of the experiments. Monooxygenase activity in washed whole cells was determined with resuspended cells in a total volume of 2 ml of mineral salts medium. Monooxygenase activities in cell extracts as well as in whole cells were determined at 25°C.

Effect of growth substrate on enzyme activities in strain DCA1. To determine the effect of different growth substrates on enzyme activities, cells of strain DCA1 were grown at 21°C in stirred batch cultures by using 5-liter Erlenmeyer flasks containing 1 liter of mineral salts medium (10). The concentration of phosphate buffer was 2.5-times higher than in the standard mineral salts medium, and growth substrates were added at a concentration of 5 mM. Flasks were inoculated with cells that were pregrown on the same substrate. Cells were harvested in the exponential phase. One part of the cell suspension was washed in mineral salts medium (10) and used immediately for the determination of monooxygenase activity in whole cells. The other part of the cell suspension was washed and concentrated in 50 mM Tris-HCl buffer (pH 7.5) and stored at 20°C until the preparation of cell extracts, and the determination of enzyme activities was performed as described above.

Competition experiments. Competitive inhibition of the monooxygenase was measured with DCA-grown cells of strain DCA1 harvested from the fermenter. Cells were diluted in mineral salts medium (10) to a final OD₆₆₀ of 0.13. Incubations were performed in 135-ml serum bottles with an initial DCA concentration of 250 µM. Different amounts of propene were added to the bottles. Based on the partition coefficient of 0.12 between liquid and air phases at 25°C (14), propene concentrations in the liquid phase were 0, 8, and 40 µM. Bottles were shaken vigorously at 25°C, and DCA degradation was measured as described above.

	RESULTS

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Isolation of DCA-degrading bacteria. In order to isolate bacteria with a high affinity for DCA, a fermenter was inoculated with a biofilm sample, kindly provided by G. Stucki, that originated from a rotating biological contactor treating groundwater polluted with DCA as the sole contaminant (17). Within 3 weeks, a DCA-degrading mixed culture was obtained. This mixed culture did not grow very well on mineral salts medium agar plates with DCA vapor present as carbon and energy source; therefore, serial dilutions in liquid cultures were used to obtain a pure culture. The obtained bacterial culture gave one colony type on rich (yeast-glucose) medium agar plates. The new isolate was identified by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) as a bacterium belonging to the genus Pseudomonas. The partial sequence of the 16S ribosomal DNA showed 99.4% similarity with P. fluorescens and 99.1% similarity with P. viridiflava. However, important characteristics such as fluorescence and hydrolysis of gelatin, which are typical for these subspecies, were all negative for the new isolate. The new isolate was designated Pseudomonas sp. strain DCA1.

Temperature. In preliminary experiments with strain DCA1 it was observed that, when precultured at 25°C, inocula were not always viable at higher temperatures. Therefore, the optimum temperature for growth of strain DCA1 on DCA was determined. As can be seen in Fig. 1, the temperature has a pronounced effect on the growth rate of strain DCA1. The highest maximum growth rate of 0.14 h¹ was at 25°C. At 30°C, the maximum growth rate was twofold lower. No growth of strain DCA1 occurred at 35°C.

View larger version (11K): [in this window] [in a new window]   FIG. 1.   Influence of temperature on maximal specific growth rates of Pseudomonas sp. strain DCA1 growing on 1,2-dichloroethane (1 mM).

Growth kinetics. We could not determine the K_m value for DCA with washed whole cells of strain DCA1 since this value was below the detection limit of approximately 0.5 µM. Therefore, a comparative growth experiment between Pseudomonas sp. strain DCA1 and X. autotrophicus GJ10 was performed to study the kinetic differences between these two strains at low substrate concentrations. Substrate depletion curves in closed batch cultures were determined for both strains while they were growing on DCA at an initial concentration of 5 mM. In Fig. 2, the last part of the DCA depletion curves is shown. It is evident that the DCA degradation rate of strain GJ10 decreased dramatically at lower concentrations. Strain DCA1, however, did not show any decrease in the DCA depletion rate, even when the substrate concentration became very low, indicating a much higher affinity for DCA.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   DCA depletion curves of growing cells of Pseudomonas sp. strain DCA1 () and X. autotrophicus GJ10 ().

First step in DCA degradation by Pseudomonas sp. strain DCA1. In the absence of a cofactor, no DCA degradation was observed in cell extracts of DCA-grown cells of strain DCA1 (Fig. 3). This suggests that a hydrolytic DCA dehalogenase, as is involved in the DCA degradation pathway of X. autotrophicus GJ10, is apparently not present in strain DCA1. Alternatively, the first step in DCA degradation could be an oxidation reaction by a monooxygenase (24). In that case, oxygen and a source of reducing power (NADH or NADPH) would be required for DCA degradation in cell extracts. Degradation of DCA in cell extract did indeed occur when NADH was added to the reaction mixture (Fig. 3). An oxygen dependency of DCA degradation was first observed with whole cells of strain DCA1 (results not shown). In the absence of oxygen, no DCA degradation occurred. However, after the addition of air to the incubation mixture, DCA degradation was restored. Similarly, DCA was only degraded after the addition of air when nitrogen-flushed cell extract was used (Fig. 3). These results indicate that the first step in DCA degradation by Pseudomonas sp. strain DCA1 requires molecular oxygen and NAD(P)H, suggesting that DCA is oxidized by a monooxygenase.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Oxygen and NADH dependency of DCA degradation in a cell extract of Pseudomonas sp. strain DCA1. Symbols: , aerobic incubation in the presence of NADH; , nitrogen-flushed cell extract in the presence of NADH; , aerobic incubation without NADH. Arrow indicates the replacement of 3 ml of headspace by air.

Competitive inhibition of DCA degradation. Alkane monooxygenases generally have a broad substrate specificity and are often capable of performing both epoxidation and hydroxylation reactions (8). Oxidation of alkenes by cells expressing alkane monooxygenase activity often results in the accumulation of the corresponding epoxides. To determine whether the DCA-oxidizing enzyme would act similarly, DCA-grown cells of strain DCA1 were incubated with propene. This resulted in the formation of 1,2-epoxypropane, confirming the presence of monooxygenase activity in whole cells. Analysis of headspace samples of the reaction mixture on a chiral column revealed that both enantiomers of 1,2-epoxypropane were formed.

View larger version (13K): [in this window] [in a new window]   FIG. 4.   Effect of different propene concentrations on DCA degradation rates of whole cells of Pseudomonas sp. strain DCA1. Symbols: , no propene; , 8 µM propene; , 40 µM propene.

Influence of protein content on monooxygenase activity. Many monooxygenases are multicomponent enzymes. This can often be illustrated by determining the enzyme activity as a function of the protein content in the assay. Figure 5 shows that the relation between protein content and DCA monooxygenase activity is nonlinear, resulting in lower specific activities at protein concentrations below approximately 0.4 mg/ml.

View larger version (11K): [in this window] [in a new window]   FIG. 5.   Relationship between protein content in the reaction mixture and monooxygenase activity.

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Enzymes involved in DCA degradation pathway. To further study the DCA degradation pathway, Pseudomonas sp. strain DCA1 was tested to see if it could grow on the chlorinated intermediates in the proposed DCA degradation pathway shown in Fig. 6. Strain DCA1 could grow on chloroacetic acid, but no growth was observed on chloroacetaldehyde. This is probably due to the toxicity of this compound, since growth on DCA was inhibited in the presence of 1 mM chloroacetaldehyde. Strain DCA1 could also grow on 2-chloroethanol. The expression of DCA monooxygenase, chloroacetaldehyde dehydrogenase, and chloroacetic acid dehalogenase was determined in cells of strain DCA1, grown on DCA, chloroacetic acid, and ethanol as a control (Table 1). DCA monooxygenase activities were determined in washed whole cells instead of cell extracts, since DCA monooxygenase is a rather unstable enzyme. DCA monooxygenase activity was found in DCA-grown cells, whereas negligible activities were found in cells grown on chloroacetic acid or ethanol. Chloroacetaldehyde dehydrogenase activity was found in cells grown on all of the tested substrates, with the lowest activity in cells grown on chloroacetic acid. Cells grown on DCA and chloroacetic acid expressed high chloroacetic acid dehalogenase activities, whereas no dehalogenase activity was present in ethanol-grown cells of strain DCA1.

View larger version (27K): [in this window] [in a new window]   FIG. 6.   Different pathways of DCA degradation by Pseudomonas sp. strain DCA1 (proposed), X. autotrophicus GJ10, and A. aquaticus AD25.

	DISCUSSION

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To obtain new DCA-degrading microorganisms, we set up batch enrichment cultures with 1 mM DCA at different temperatures with various inocula. However, no DCA degradation was observed in any of these cultures. Apparently, DCA-degrading bacteria are not widespread in the environment.

Stucki and Thüer described the treatment of groundwater that contained DCA as the sole contaminant (17). The groundwater treatment plant consisted of activated carbon filters and a rotating biological contactor (RBC) and was inoculated with X. autotrophicus GJ10 and Pseudomonas sp. strain DE2. Initially, the RBC had little effect on DCA removal. However, during the fourth year of operation, DCA elimination by the RBC increased continuously. During the fifth year, most of the DCA was removed by the RBC, resulting in effluent concentrations of DCA which were usually much lower than 10 µM. The initially black surface of the RBC had by then changed to a yellow color, suggesting the presence of X. autotrophicus GJ10 as the predominant DCA degrader. Reisolation of DCA-degrading microorganisms confirmed the presence of Xanthobacter sp. with an identical gene encoding for the DCA dehalogenase (17). The average DCA concentration in the effluent, however, was much lower than could be expected, based on the kinetic parameters determined for suspended cells of X. autotrophicus GJ10 (20). We therefore anticipated that another DCA-degrading microorganism with a high affinity for DCA must be present in the RBC biofilm. To isolate this strain, a sample of the RBC biofilm was used to inoculate a fermentor. Enrichment in a continuous culture under DCA limitation was used to select the microorganism with the highest affinity for DCA (lowest K_s value). The isolated Pseudomonas sp. strain DCA1 did indeed exhibit a much higher affinity for DCA than strain GJ10, as was shown by the DCA depletion curves determined in closed cultures (Fig. 2). Considering the high affinity of strain DCA1 and also the relatively high growth rate with DCA as a carbon source, it is somewhat surprising that strain GJ10 was still present in the RBC biofilm. However, besides these kinetic differences, other factors such as decay rates and biofilm-forming capabilities, will affect the performance of the microorganisms in the biofilm.

The first DCA-utilizing pseudomonad described in the literature was Pseudomonas sp. strain DE2 (16), which was also used to inoculate the RBC (17). The physiological similarities between strains DE2 and DCA1 are striking; however, there are some distinctions. Strain DE2 does not form colonies on nutrient agar or other solidified media (16). Strain DCA1 does not grow on mineral salt agar plates either, but it does form small colonies on rich-medium agar plates. The growth rates of both strains at 30°C are comparable. Strain DE2 is reported to be dependent on vitamins, whereas we found that strain DCA1 does not require any additional organic nutrients for optimal growth. Further studies, e.g., 16S sequence analysis, are required to establish whether these two strains are intrinsically different.

In contrast to the hydrolytic dehalogenation of DCA in X. autotrophicus GJ10 and A. aquaticus AD25 (12, 19), an oxidation reaction seems to be the first step in DCA degradation in both Pseudomonas strains DE2 and DCA1. No DCA degradation was observed in the cell extracts of strain DE2, and a hydroxylation of DCA, yielding the unstable intermediate 1,2-dichloroethanol, was suggested as the first step in DCA metabolism (16). We also did not observe degradation of DCA in cell extracts of strain DCA1 unless NAD(P)H was added to the incubation mixture. In the presence of NAD(P)H, DCA degradation in cell extract was oxygen dependent, suggesting the involvement of a monooxygenase (Fig. 3). Oxygen-dependent dehalogenation of long-chain (C₅ to C₁₂) ,-dichloroalkanes was recently observed in Pseudomonas sp. strain 273 (22), and hexadecane-grown cells of Rhodococcus erythropolis Y2 showed oxygenase-type dehalogenation activity towards C₄ to C₁₈ -chloroalkanes (2). Conversion of DCA by a monooxygenase was suggested by Yokota et al. (24). They described the conversion of DCA by resting cells of two different methanotrophic strains.

Additional proof that a monooxygenase activity was responsible for DCA degradation in our strain was provided by conversion of propene to 1,2-epoxypropane and the results of competition experiments (Fig. 4). Propene competitively inhibited DCA degradation in DCA-grown cells of strain DCA1, suggesting that both substrates compete for the same active site. In general, alkane monooxygenases yield racemic mixtures of epoxides (18). Therefore, the observed conversion of propene to both enantiomers of 1,2-epoxypropane was expected. Interestingly, at a DCA concentration of 250 µM, the presence of 8 µM propene already inhibited DCA degradation by the monooxygenase for more than 50%, indicating an even higher affinity of the monooxygenase for propene than for DCA.

The nonlinear relationship between the protein content in the enzyme assay and the specific activity of DCA monooxygenase (Fig. 5) could indicate that DCA monooxygenase is a multicomponent enzyme, which is not uncommon for monooxygenases (9). Significant levels of DCA monooxygenase activity were only present in DCA-grown cells of strain DCA1 (Table 1). Apparently, DCA monooxygenase is an inducible enzyme, and DCA itself or chloroacetaldehyde could be the inducer.

The next enzymatic step in DCA degradation is probably the dehydrogenation of chloroacetaldehyde. Chloroacetaldehyde dehydrogenases are reported to have broad substrate specificities and are also induced during growth with ethanol (13, 15). Chloroacetaldehyde dehydrogenase activities in cell extracts of strain DCA1 were rather low, probably due to nonoptimal assay conditions. The activity in cell extracts was higher in DCA- or ethanol-grown cells of strain DCA1 than in cells grown on chloroacetic acid (Table 1).

The product of chloroacetaldehyde dehydrogenase is chloroacetic acid, which subsequently has to be dehalogenated. Dehalogenation of chloroacetic acid by halidohydrolases was observed in all DCA-utilizing bacteria described in the literature (12, 16, 19). Chloroacetic acid dehalogenase in strain DCA1 is obviously an inducible enzyme, since no activity was found in ethanol-grown cells. Dehalogenation of chloroacetic acid will most likely result in the formation of glycolic acid (15).

The complete proposed DCA degradation pathway of Pseudomonas sp. strain DCA1 is shown in Fig. 6. Only the initial attack of the DCA molecule seems to be different from the DCA degradation pathway in X. autotrophicus GJ10 and Ancylobacter aquaticus AD25 (12, 19).

The isolation of a DCA-degrading bacterial strain with a high affinity for DCA offers promising opportunities for the efficient biological removal of this compound from groundwater.