1,1-Dichloroethene as a Predominant Intermediate of Microbial

Origin and Propagation of an Incorrect Chemical Degradation Pathway in the Literature: cis-1,2-Dichloroethylene as a Daughter Product of 1,1,1-Trichlo...
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Environ. Sci. Technol. 2006, 40, 1830-1836

1,1-Dichloroethene as a Predominant Intermediate of Microbial Trichloroethene Reduction JINGJING ZHANG, ANDREW P. JOSLYN,† AND PEI C. CHIU* Department of Civil and Environmental Engineering, University of Delaware, Newark, Delaware 19716

A microbial culture derived from a landfill site in Dover, DE consistently reduced trichloroethene (TCE) to ethene through 1,1-dichloroethene (DCE) as a dominant intermediate in the presence of ampicillin. A constant 1,1-DCE-to-cisDCE ratio of 2.4 ( 0.3 was observed for more than two years, while trans-DCE was never detected. Without ampicillin, however, TCE was reduced to ethene almost exclusively through cis-DCE, suggesting that the culture contained at least two TCE-dechlorinating populations. Two subcultures, which were established using 1,1-DCE or vinyl chloride as an electron acceptor, exhibited the same 1,1-DCE-to-cisDCE ratio when TCE was introduced. PCR amplification of 16S rRNA gene followed by sequencing and DGGE analysis indicate that these (sub)cultures contained a Dehalococcoides population(s). TCE dechlorination assays with crude cell extract showed a DCE distribution pattern similar to that with whole cells. The enzyme involved in 1,1-DCE formation was likely a cobalt corrinoid enzyme, as suggested by the inhibitory effect of CH3I and photoreversibility of the inhibition. This study provides a possible biological mechanism for the occurrence of 1,1-DCE in TCE-contaminated sites.

Introduction Trichloroethene (TCE) is a chlorinated solvent commonly used in various industrial applications. As a result of improper handling and disposal in the past, TCE has become one of the most prevalent groundwater contaminants (1, 2). Microbial reductive dechlorination is arguably the most important process to degrade TCE in affected environments and has been used to remediate TCE-contaminated aquifers (3). Under anaerobic conditions, TCE can be dechlorinated sequentially by microorganisms to dichloroethenes (DCEs), vinyl chloride (VC), and ethene. While three DCE isomers, cis-1,2-, trans-1,2-, and 1,1-DCE, may be formed from TCE, most TCE-dechlorinating cultures appear to produce cisDCE as a predominant intermediate or end product (4-11). Several anaerobic isolates have been shown to respire TCE, including Desulfitobacterium species (4, 5), Desulfuromonas species (6, 7), Sulfurospirillum multivorans (8, 9), and Dehalococcoides species (10, 11). Most of these dehalogenators convert TCE to predominantly cis-DCE, and a cobalt corrinoid (i.e., a vitamin B12 derivative) is often suggested to be involved in the dechlorination reaction (4, 8, 12-14). This * Corresponding author phone: (302) 831-3104; fax: (302) 8313640; e-mail: [email protected]. † Currently with Golder Associates Inc., 1951 Old Cuthbert Road, Suite 301, Cherry Hill, NJ 08034. 1830

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is consistent with the observation that cis-DCE is the dominant isomer formed in abiotic TCE reduction catalyzed by vitamin B12 with Ti(III) citrate or dithiothreitol as a reductant (15-17). Only a few exceptions to this regio-selectivity have been reported. trans-DCE was found by Christiansen and coworkers (18) to be the predominant DCE isomer during tetrachloroethene (PCE) reduction in an upflow anaerobic sludge blanket reactor. The trans-DCE-to-cis-DCE ratio was between 1.2 and 2.2, and only traces of 1,1-DCE were found (18). Lo¨ffler et al. (19) observed formation of trans-DCE and cis-DCE from PCE at a ratio of 2.45 by a sediment-free, nonmethanogenic culture that was originally enriched on 1,2-dichloropropane. Two other mixed cultures, also reported by Lo¨ffler and co-workers (20), reduced PCE to trans-DCE and cis-DCE at a ratio of 3.0 ( 0.5. The organisms involved in the preferential production of trans-DCE in these mixed cultures were not identified. Griffin et al. reported that transDCE production was favored in PCE and TCE dechlorination: trans- and cis-DCE were generated at a ratio of 3.0 ( 0.5 in six mixed cultures and Dehalococcoides was the putative population responsible for the preferential production of trans-DCE (21). A pure culture of Dehalococcoides strain CBDB1 (22) was shown to reduce PCE to predominantly transDCE after a prolonged incubation. More recently, transformation of PCE to trans-DCE and cis-DCE at a ratio between 1.2 and 1.7 by a pure culture of the polychlorinated biphenyldechlorinating bacterium DF-1 was reported by Miller et al. (23). The 16S rRNA gene sequence of DF-1 was 89% identical to that of D. ethenogenes strain 195. DCEs were the end products of TCE reduction in all the above cultures and no dechlorination beyond DCEs was observed. 1,1-DCE was reported as the only TCE transformation product by a methanol-enriched anaerobic culture (24). However, the authors could not exclude formation of cisand trans-DCE since the 1,1-DCE concentration measured was low (0.14 µM) and the detection limit (0.25 µM) for the other two DCEs was about twice as high as that for 1,1-DCE. Eekert et al. (25) investigated the reduction of chlorinated ethenes by nonacclimated methanogenic sludge granules and observed production of 1,1-DCE instead of cis-DCE. However, the authors could not exclude the possibility of chemical reduction by hydrogen sulfide. 1,1-DCE was formed and suggested to be a potentially significant intermediate in TCE dechlorination by D. ethenogenes strain 195, although much greater amounts of cis-DCE were observed with this organism (26). 1,1-DCE is a common industrial solvent and an EPA priority pollutant that is frequently detected in hazardous waste sites. 1,1-DCE was present at at least 636 (38%) of the 1674 National Priority List sites, approximately half of cisand trans-DCE-contaminated sites combined (27). Presence of 1,1-DCE at waste sites has been explained by source contamination and abiotic transformation (i.e., dehydrochlorination) of 1,1,1-trichloroethane (TCA), a common cocontaminant of TCE (3, 28-30). 1,1-DCE is also a product of abiotic reduction of PCE and TCE by hydrogen sulfide (31) and biological reduction of 1,1,1,2-tetrachloroethane (32). To our knowledge, no conclusive evidence is available to date for the preferential production of 1,1-DCE from biological TCE dechlorination. In this paper we present evidence for the formation of 1,1-DCE as a predominant intermediate product of TCE in an enrichment culture that completely dechlorinated TCE to ethene. 10.1021/es051829m CCC: $33.50

 2006 American Chemical Society Published on Web 02/07/2006

Materials and Methods Chemicals and Culture. TCE (>99.5%), 1,1-DCE (99%), cisDCE (97%), and VC (99.5%) were obtained from SigmaAldrich (Milwaukee, WI). Ethene (>99.5%) and hydrogen (>99.99%) were acquired from Scotty Specialty Gases (Plumsteadsville, PA). Gas samples were handled using gastight syringes (VICI, Baton Rouge, LA). All other chemicals used in this study for culture medium and dechlorination assays were obtained from Sigma-Aldrich. Groundwater collected from Landfill 13 at Dover Air Force Base (Dover, DE) was used as the inoculum to establish the enrichment culture in this study. Enrichments were established using a liquid medium containing ammonium acetate (0.3 g/L), magnesium acetate (0.21 g/L), calcium acetate (0.11 g/L), phosphate buffer (0.05 M, pH 6.8), yeast extract (0.1 g/L), sodium lactate (1.0 g/L), sodium propionate (0.5 g/L), sodium butyrate (0.56 g/L), sodium sulfate (0.0284 g/L), a mineral solution (6.7 mL/L), and a vitamin solution (0.1 mL/L) (33). The stock enrichment culture (hereafter referred to as “TCE-fed culture”) was maintained in 250-mL amber bottles each capped with a Mininert valve (Precision, Baton Rouge, LA) and sealed with low-permeability vinyl tape (3M, St. Paul, MN). Each bottle contained 125 mL of culture medium and 125 mL of N2 headspace and received 2 µL of pure TCE at each transfer, which was carried out once a month in an anaerobic glovebag (I2R, Cheltenham, PA) under N2. The bottles were incubated at room temperature in an inverted position. Complete dechlorination of TCE to ethene was sustained over repeated transfers (8%, v/v) into fresh sterile medium for more than 3 years. Two subcultures were derived from the TCE-fed culture by feeding 1,1-DCE or VC instead of TCE (hereafter referred to as 1,1-DCE-fed culture and VCfed culture, respectively). These subcultures were transferred monthly to fresh medium amended with 2-4 µL of neat 1,1DCE or 250-500 µL of VC. The resulting aqueous concentrations were 0.1-0.2 mM for 1,1-DCE and 0.04-0.08 mM for VC, based on published Henry’s constants for these chlorinated ethenes (34). TCE Dechlorination Assays. Dechlorination of TCE by the TCE-fed culture was carried out in both the absence and presence of ampicillin (at 25, 50, and 500 mg/L). Ampicillin interferes with cell wall synthesis in most bacteria but not Dehalococcoides species (10, 11, 35, 36), which lack peptidoglycan. Five mL of H2 was added as an electron donor to some of the ampicillin-amended bottles during the assay. TCE dechlorination assays were also performed using the 1,1-DCE-fed and VC-fed cultures (both without ampicillin) to obtain the TCE dechlorination patterns of these subcultures. The typical inoculum size was 4 vol %. In addition, different inoculum sizes were tested using 1,1-DCE-fed culture (100%) and 15%, 0.6%, and 0.15% of the 1,1-DCE-fed culture in fresh medium. Dechlorination assays were conducted in a benchtop anaerobic glovebag using 250-mL amber bottles containing 125 mL of solution, as described above. Neat TCE (2-3 µL; corresponding to an aqueous concentration of 0.13-0.2 mM) was added to each bottle to initiate an assay. Bottles were incubated at room temperature in an inverted position. Medium with and without inoculum was autoclaved and used as control. Cell Extracts and Assays. Cell extracts were obtained from TCE-fed culture with ampicillin, 1,1-DCE-fed culture, and VC-fed culture. After g80% of the fed chlorinated ethene was consumed, cells were harvested by centrifuging 250 mL of culture at 8000g for 30 min and then re-suspended in 15 mL of 30 mM HEPES buffer (pH 8.0). One protease inhibitor cocktail tablet (Roche, Mannhein, Germany) was added to each 15 mL suspension to inhibit serine and cysteine proteases during extraction. The resulting suspension was then subjected to French press treatment at 6.8 MPa three times. The lysate was centrifuged at 5600g for 30 min at 4 °C

and the crude cell extract was used for the assays. Assays were performed in 63-mL amber bottles under an atmosphere of N2 and H2 (95/5, v/v) in an anaerobic box (Coy, MI). Each bottle was filled with 31.5 mL of anaerobic assay solution containing 2 mM titanium(III) citrate, 1 mM methyl viologen, 140 mM HEPES buffer (pH 8.0), and 3 mL of cell extract (approximately 110 µg protein/mL). Methyl viologen reduced by titanium(III) citrate served as an electron donor to reduce enzymes in cell extract (37). Reactions were initiated by introducing TCE (0.9 µmol/bottle). Bottles were shaken on an orbital shaker at 100 rpm at room temperature. The effect of cyanide on TCE dechlorination pattern was assessed through addition of potassium cyanide to the assay solution 10 min prior to introduction of TCE. Cyanide is a strong σ base that forms strong field complexes with 3d transition metals and has been used to probe the potential involvement of transition metal ions in TCE dechlorination (38). Different concentrations of cyanide (0, 5, 10, and 20 mM) were examined. Methyl iodide was used as an inhibitor to probe the involvement of Co(I) corrinoids in TCE dechlorination. Co(I) corrinoids can be inactivated by methyl iodide through SN2-like oxidative substitution to form a Co(III)CH3 adduct, which is catalytically inactive. This inhibition can be reversed by illumination due to the photolabile nature of the Co(III)-C bond (39). The inhibition experiments were performed under the same conditions described above except that cell extract was pretreated as follows. Cell extract was incubated with 3 mM titanium(III) citrate in the presence or absence of 3 µM methyl iodide for 30 min in 20-mL foilwrapped clear glass vials. Foil was then removed and bottles containing methyl iodide-treated or nontreated cell extract were placed on ice and subjected to illumination using a 300-watt slide projector lamp for 5 min prior to TCE dechlorination assay. The procedures used for the inhibition and photoreversibility assays were validated by substituting vitamin B12 or cell extract from the TCE-fed culture (which produced predominantly cis-DCE, Figure 1a) for the cell extracts from the three 1,1-DCE-producing cultures: TCEfed culture amended with ampicillin, 1,1-DCE-fed culture, and VC-fed culture. Methyl viologen was omitted for assays with vitamin B12. Chemical Analysis. At different times, 100 µL gas samples were withdrawn from reactor headspace using a gastight syringe and injected into an Agilent 6890 gas chromatograph (GC) (Wilmington, DE) equipped with a flame ionization detector (FID) and a 30-m GS-GasPro capillary column (J&W, Folson, CA). The GC temperature program was 40 °C for 2 min, 25 °C/min to 115 °C, 10 °C/min to 200 °C, and 200 °C for 1 min. The analytes had the following retention times: ethene, 0.77 min; VC, 5.67 min; 1,1-DCE, 7.77 min; transDCE, 8.60 min; cis-DCE, 9.68 min; and TCE, 10.67 min. Quantification was based on external calibration curves. 16S rRNA Gene Analysis. To obtain DNA for polymerase chain reaction (PCR), cells were collected by centrifuging (at 8000g for 30 min) 125 mL of the 1,1-DCE-fed or VC-fed culture. Most of the supernatant was discarded and the leftover (approximately 3 mL) was vortexed. The suspension was then transferred to a 1.5-mL micro-tube and centrifuged at 5600g for 2 min and the supernatant was discarded. This step was repeated to remove all liquid. A 100-µL portion of aqueous solution of Tween 20 (0.1% v/v, Bio-Rad, Hercules, CA) was added to the cell pellet and the content was vortexed to promote cell lysis. The mixture was then incubated at 100 °C for 10 min, vortexed for re-suspension, and centrifuged at 5600g for 1 min. InstaGene matrix (80 µL) was added to remove cell lysis products during vortexing. The mixture was then incubated at 56 °C for 20 min, vortexed, incubated again at 100 °C for 8 min, and vortexed again. The mixture was then centrifuged at 5600g for 2 min and the supernatant containing DNA was stored at -20 °C until PCR. VOL. 40, NO. 6, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Denaturing gradient gel electrophoresis (DGGE) was performed using a Bio-Rad DCode System. Twenty µL of PCR products and 5 µL of 6× gel loading dye (6% bromophenol blue, 6% xylene cyanol, 100% glycerol, and DI water) were loaded onto an 8% (w/v) polyacrylamide gel in 1× Tris-acetate-EDTA running buffer. The linear 60-90% denaturing gradient was created using urea and formamide (7 M urea and 40% formamide being 100%). Electrophoresis was performed at 60 °C and 75 V for 12 h. After electrophoresis, the gel was stained in an aqueous ethidium bromide solution (0.5 µg/mL) for 30 min. The gel was then visualized using a UV trans-illuminator and pictures were taken with a Polaroid camera (Cambridge, MA). Bands in the gel were excised with a sterile blade and the DNA was extracted using a Qiagen gel extraction kit. The DNA was then sequenced using an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA) at the University of Delaware’s Center for Agricultural Biotechnology. The same PCR primers were used for twoway sequencing. The sequences obtained (Figure S1) were compared to those of the known Dehalococcoides species using BLAST 2.2 from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov). DNA was also extracted and PCR amplification was performed for the TCE-fed stock culture to determine whether other known dehalogenators were present in the culture. Primers targeting the following five species were used: Dehalobacter, Desulfitobacterium frappieri PCP-1, Desulfomonile tiedjei, Desulfitobacterium dehalogenans, and Desulfuromonas (41-44). The primers and PCR methods used were as described elsewhere (41-44).

Results and Discussion

FIGURE 1. Reductive dechlorination of TCE by the TCE-fed enrichment culture (a) in the absence of ampicillin, (b) in the presence of 50 mg/L ampicillin, and (c) three transfers (without ampicillin) after initial ampicillin treatment. Six symbols are shown: TCE (open diamonds), 1,1-DCE (solid triangles), cis-DCE (solid circles), VC (open squares), ethene (asterisks with dashed line), and total C2 mass (plus signs). Data are averages of duplicate samples from one of at least six independent experiments, all of which showed similar results. The presence of Dehalococcoides 16S rRNA was tested through direct PCR by using one pair of Dehalococcoidestargeted primers, Fp DHC 774 (5′GGG AGT ATC GAC CCT CTC3′) and Rp DHC 1212 (5′GGA TTA GCT CCA GTT CAC ACT G3′) (40). The PCR reaction mixture contained 5 µL of 10× Taq buffer, 1 µL of deoxynucleotide triphosphate mix (10 mM each), 0.5 µL of Taq polymerase (5 U/µL), 10 µL of Q solution (PCR Core Kit, Qiagen, Valencia, CA), and 1 µL of extracted DNA. Concentration of the primer pair was 0.4 µM. Sterilized distilled water was added to make up a total volume of 50 µL. Negative controls consisted of all components except template DNA. Amplification was performed in a Bio-Rad iCycler. The temperature program for Fp DHC 774/Rp DHC 1212 was (40) 1 cycle of 95 °C for 2 min, 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. A 5-µL portion of each PCR product was visualized on a 1% ethidium bromide-stained agarose gel using a UV transilluminator (VWR, Baltimore, MD). 1832

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TCE Dechlorination by Enrichment Cultures. The TCE-fed culture consistently dechlorinated TCE to ethene. A typical TCE dechlorination profile is shown in Figure 1a. cis-DCE was the predominant DCE isomer and accounted for >99% of the total DCEs at any time. In contrast to the rapid conversion of TCE to cis-DCE, dechlorination of cis-DCE to VC was slow and seemed to commence after a short lag. Interestingly, in the presence of ampicillin (from 25 to 500 mg/L) 1,1-DCE became the dominant DCE isomer, accounting for up to 70% of the total DCEs. A typical TCE dechlorination result in the presence of 50 mg/L of ampicillin is shown in Figure 1b. After a lag of about 3 days, TCE was reduced to ethene through 1,1-DCE, cis-DCE, and VC. The ratio of 1,1-DCE to cis-DCE was 2.4 ( 0.3 (mean ( standard deviation) before significant formation of VC. trans-DCE was never detected. Reduction of TCE was much slower with ampicillin, suggesting that the dehalogenator(s) involved in TCE-to-cis-DCE dehalogenation in Figure 1a was adversely affected by ampicillin, either directly or indirectly through inhibition of other organisms that this dehalogenator(s) depended on. In fact, the observed shift from cis-DCE to 1,1-DCE suggests that the organism(s) responsible for TCE reduction in the presence of ampicillin was a different dehalogenator(s). In the absence of ampicillin, this dehalogenator(s) was probably out-competed and its activity (and thus 1,1-DCE formation) might have been masked by that of the cis-DCE-producing dehalogenator(s). Increasing the concentration of ampicillin up to 500 mg/L did not affect the 1,1-DCE-to-cis-DCE ratio, but it increased the lag time to 60 days. This long lag time was reduced to 8 days when 5 mL of H2 was added. A possible reason for the prolonged lag time might be that ampicillin inhibited organisms that produced H2, which the 1,1-DCE-producing TCE dehalogenator(s) presumably utilized as an electron donor. The ampicillin-treated, TCE-fed culture could be transferred for 2-4 feeding cycles in the absence of ampicillin while maintaining the predominant 1,1-DCE formation pattern. Beyond that, however, the major TCE dechlorination

product would shift back to cis-DCE. An example of such shift is shown in Figure 1c, where a crossover from a 1,1DCE-dominated regime to a cis-DCE-dominated one was observed. Such crossover was always accompanied by lower and more variable 1,1-DCE-to-cis-DCE ratios during TCE dechlorination. No TCE dechlorination or DCE production was observed with sterilized cultures. The results support the earlier supposition that at least two TCE-dechlorinating populations existed that degraded TCE through different pathways and that distribution of the DCE isomers would depend on the relative activities of these populations. Furthermore, the results also indicate that the 1,1-DCEproducing dehalogenator(s) could not compete well with the cis-DCE producer(s) for TCE when there was no ampicillin present to inhibit the latter. The fact that 1,1-DCE production was not significantly affected by the peptidoglycan inhibitor ampicillin suggests that ampicillin might help to select for the population that produced 1,1-DCE by inhibiting the other TCE dehalogenator(s) that generated exclusively cis-DCE. However, attempts to establish a stable 1,1-DCE-producing, TCE-fed culture that could be transferred indefinitely without ampicillin were unsuccessful by using different electron donors (H2 and organic acids), pH buffers (phosphate and HEPES), and electron acceptors (with and without sulfate). In all cases the cis-DCE-producing activity eventually dominated after a few transfers without ampicillin. As noted earlier, following rapid reduction of TCE to cisDCE in Figure 1a, there appeared to be a short lag before cis-DCE reduction to VC began, suggesting that TCE and cis-DCE might be transformed by different organisms. In contrast, when cis-DCE production was inhibited with ampicillin and 1,1-DCE was the main product of TCE, there did not appear to be a lag (Figure 1b), which suggests that dechlorination of TCE to ethene in Figure 1b might be carried out by the same population. This observation led to the attempt to enrich the 1,1-DCE-producing population by using lesser chlorinated ethenes as electron acceptors. Two subcultures were established through serial transfers of the ampicillin-treated, TCE-fed culture into ampicillinfree medium amended with 1,1-DCE or VC. The TCE dechlorination pattern in each subculture was examined periodically by introducing TCE to sterile medium inoculated with one of the subcultures. Dechlorination of TCE was regioselective and the 1,1-DCE-to-cis-DCE ratio was similar in the two subcultures: 2.5 ( 0.4 for the 1,1-DCE-fed culture and 2.7 ( 0.4 for the VC-fed culture. These ratios have been constant to date over 14 transfers for the 1,1-DCE-fed culture and 5 transfers for the VC-fed culture. Results of TCE dechlorination by the 1,1-DCE-fed culture at different dilutions are shown in Figure 2. With increasing amount of inoculum, TCE dechlorination rate increased and the initial lag time shortened (Figure 2a). Despite the different dechlorination rates and lag times, 1,1-DCE was always the dominant intermediate and the product selectivity (i.e., 1,1to-cis ratio) was constant regardless of the inoculum size (Figure 2b and c). These results indicate that 1,1-DCE can selectively enrich the population(s) that produced 1,1-DCE as a predominant intermediate. The observations that TCE product distribution was essentially the same for the cultures receiving TCE plus ampicillin and 1,1-DCE or VC without ampicillin, and that 1,1-DCE predominance was preserved in these cultures over repeated transfers, suggest 1,1-DCE, VC, and TCE could all serve as electron acceptors to support growth of the 1,1DCE-producing organism(s). In addition, comparing Figure 2b and c, the longer lag time as a result of smaller inoculum size also suggests that TCE supported growth of the 1,1DCE-producing organism in the first two weeks shown in Figure 2c, since no DCEs or VC was utilized (i.e., no VC or

FIGURE 2. (a) TCE dechlorination by the 1,1-DCE-fed culture (100%, closed squares) and by 15% (open diamonds), 0.6% (open triangles), and 0.15% (open circles) of this culture in fresh medium. TCE reduction and product distributions for the (b) 15% and (c) 0.15% inocula are shown. The six symbols in (b) and (c) are TCE (open diamonds), 1,1-DCE (solid triangles), cis-DCE (solid circles), VC (open squares), ethene (asterisks with dashed line), and total C2 mass (plus signs). Data are averages of duplicate samples from one of two or more independent experiments, all of which showed similar results. ethene was formed) to support growth of dehalogenators during this lag period. The ability of the enrichment cultures described here to reduce TCE through mostly 1,1-DCE distinguishes them from all other dechlorinating cultures reported to date, which produce predominantly cis-DCE (4-11) or trans-DCE (1823). The few cultures recently reported to produce primarily trans-DCE exhibit a trans-DCE-to-cis-DCE ratio in the range of 1.2-3.0 (18-23), comparable to the 1,1-DCE-to-cis-DCE ratio observed in this study. However, in contrast to these trans-DCE-producing cultures, which cannot dechlorinate beyond DCEs, the 1,1-DCE-producing culture dechlorinated TCE completely to ethene. TCE Dechlorination by Cell Extract. Cell extracts from the TCE-fed culture receiving ampicillin, 1,1-DCE-fed culture, and VC-fed culture could all catalyze the reductive dechlorination of TCE in vitro in the presence of reduced methyl VOL. 40, NO. 6, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. TCE dechlorination with cell extract from the 1,1-DCEfed culture (a) in the absence of CH3I, and (b) treated with CH3I and subjected to illumination. The six symbols are TCE (open diamonds), 1,1-DCE (solid triangles), cis-DCE (solid circles), VC (open squares), ethene (asterisks with dashed line), and total C2 mass (plus signs). Data are averages of duplicate samples from one of two or more independent experiments, all of which showed similar results. viologen. No TCE dechlorination was observed without reduced methyl viologen. TCE was reduced to predominantly 1,1-DCE with cell extract from any of the three cultures. Result of a typical TCE dechlorination assay with cell extract from the 1,1-DCE-fed culture is shown in Figure 3a. The 1,1-DCEto-cis-DCE ratio was 2.1 ( 0.2, slightly lower than that observed with whole cells. The same ratio was observed for cell extract from the VC-fed culture. The DCEs were further reduced to VC, but reduction of VC to ethene was slow. In additional assays, crude cell extract was heated for 45 min at 95 °C prior to the assay. All cell extracts lost their ability to catalyze TCE dechlorination after heating (data not shown), suggesting that the observed dechlorination was mediated by a heat-sensitive enzyme(s). Cyanide was found to decrease the catalytic activity of the cell extract: the amount of TCE transformed in 70 min was lowered by 53%, 70%, and 93% relative to control in the presence of 5, 10, and 20 mM of potassium cyanide, respectively. However, the ratio of 1,1-DCE to cis-DCE (2.1 ( 0.2) remained unchanged. This suggests that a transition metal might be involved in the formation of 1,1-DCE from TCE. Methyl iodide almost completely inhibited TCE dechlorination by cell extract. TCE dechlorination activity was partially restored by illumination of CH3I-inhibited samples (Figure 3b). The reactivated (illuminated) cell extract showed a TCE dechlorination activity between 31 and 54% of that of the positive control containing noninhibited cell extract (i.e., no CH3I). Dechlorination with light-reactivated cell extract exhibited the same predominance of 1,1-DCE as in the positive control with CH3I-free cell extract. The inhibitory effect of CH3I and the photoreversible nature of the inhibition suggest that a cobalt corrinoid was involved in the formation of 1,1-DCE from TCE. To confirm the validity of the procedure used, the CH3I and photoreversibility assays were repeated with vitamin B12 or cell extract from the original TCE-fed culture (which 1834

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dechlorinated TCE through cis-DCE in the absence of ampicillin) replacing cell extract from the 1,1-DCE-fed culture. In both cases, inhibition by CH3I and photoreactivation were observed and cis-DCE was the predominant DCE isomer (data not shown). This result not only validates the assay procedure but also suggests that dechlorination of TCE by the cis-DCE-producing organism(s) in Figure 1a was also mediated by a corrinoid cofactor. The involvement of a cobalt corrinoid is consistent with the cyanide inhibition, since cyanide can bind strongly to Co(III) (which has an empty dz2* antibonding orbital to accommodate the σ base) and thus hinder its reduction to the active Co(I) state. Gantzer and Wackett reported that cis-DCE was the primary dichloroethene isomer (>90% of the total DCEs) in the reductive transformation of TCE catalyzed by the metallocofactors vitamin B12, hematin, and cofactor F430 (15). In a number of studies with a purified enzyme (8) or cell extract (4, 12, 13), the dehalogenase enzymes were shown (8) or suggested (4, 12, 13) to contain a corrinoid cofactor through light-reversible inhibition by alkyl halides. Consistent with the regio-selectivity observed in cofactor-mediated TCE dechlorination, cis-DCE was the dominant DCE isomer in these in vitro studies. As suggested by Glod et al. (17) and Nonnenberg et al. (45), if TCE reduction is initiated by a one-electron transfer from a Co(I) corrinoid followed by elimination of a chloride ion to form an ethenyl radical, the cis selectivity may be due to the higher stability of the cis1,2-dichloroethen-1-yl radical than other radicals such as the 1,1-dichloro-vinyl radical, the precursor of 1,1-DCE. While this mechanism is consistent with TCE reduction by cob(I)alamin (17), it cannot explain the preferential 1,1-DCE formation in this study. In fact, the instability of 1,1-dichlorovinyl radical argues that dechlorination of TCE to 1,1-DCE by this culture probably occurred via formation of an alkylcorrinoid adduct (46, 47) rather than one-electron transfer. The result that replacing the physiological reductant with Ti(III)-reduced methyl viologen did not significantly change the 1,1-DCE-to-cis-DCE ratio suggests the regio-selectivity was controlled by the dehalogenase enzyme rather than the reductant. In addition, formation of different dominant DCE isomers by the cell extract and by vitamin B12 further suggests that the selectivity was controlled by the apoprotein, although difference between vitamin B12 and the corrinoid cofactor involved might also play a role. Nucleic Acid Characterization. PCR products of the expected size with primers targeting the Dehalococcoides 16S rRNA gene were obtained with DNA from both 1,1-DCE-fed and VC-fed cultures. DGGE analysis produced only one band from each of these PCR products, suggesting each culture contained either only one Dehalococcoides species or multiple Dehalococcoides populations with an identical sequence over the range covered by the primers. This was confirmed through sequencing of the PCR products, the result of which shows that the Dehalococcoides species in these cultures was 99% identical to D. ethenogenes strain 195 (AF004928) over a range of 440 bp (Figure S1) and belonged to the Cornell or Victoria subgroup (40). Several observations suggest that a Dehalococcoides species might be responsible for the production of 1,1-DCE from TCE. First, the same Dehalococcoides 16S rRNA gene sequence was recovered from both 1,1-DCE-fed and VC-fed culture that produced 1,1-DCE from TCE. Second, dechlorination of TCE to primarily 1,1-DCE was not adversely affected by ampicillin, the resistance to which is a characteristic of the known Dehalococcoides isolates (10, 11, 35, 36). Third, as described earlier, H2 appeared to be the direct electron donor to support 1,1-DCE production in the presence of ampicillin in our cultures. H2 is the only electron donor the known Dehalococcoides isolates can utilize (10, 11, 35, 36).

There are four isolates in the Dehalococcoides group to date (10, 11, 35, 36): Dehalococcoides ethenogenes strain 195, Dehalococcoides sp. strain CBDB1, Dehalococcoides sp. strain BAV1, and Dehalococcoides sp. strain FL2. These organisms are close relatives phylogenetically and are affiliated with the “Dehalococcoides” subphylum within the phylum Chloroflexi (green non-sulfur bacteria) (36). They can obtain energy through reduction of different types of chlorinated compounds including chlorinated ethenes and chlorinated aromatic compounds (10, 11, 35, 36, 48). The substrate ranges of these isolates can vary quite dramatically. For example, strains 195 and FL2 can respire TCE and DCE isomers but dechlorinate VC only cometabolically (10, 11, 26, 49). In contrast, strain BAV1 can respire VC and DCEs but its reduction of TCE does not yield energy. In addition, these isolates dechlorinate TCE through different pathways and yield different end products. Strain CBDB1 converts TCE to primarily trans-DCE as an end product, whereas strains 195 and FL2 appear to produce cis-DCE as the dominant intermediate, although accumulation of the DCEs was limited and transient (11, 22, 26). Strains 195 and CBDB1 also differ in their dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin (48, 50), which was converted to 1,2,4-trichlorodibenzo-pdioxin and 1,3-dichlorodibenzo-p-dioxin by strain 195 but to 2,3-dichlorodibenzo-p-dioxin and 2-monochlorodibenzop-dioxin by strain CBDB1. These observations and the results of this study indicate that these organisms, while phylogenetically close, differ widely in terms of substrate range, transformation pathway, and products. No PCR product was obtained from the original TCE-fed, ampicillin-free culture using primers targeting the following five species: Dehalobacter, Desulfitobacterium frappieri PCP1, Desulfomonile tiedjei, Desulfitobacterium dehalogenans, Desulfuromonas. However, because no positive controls were included in these analyses, the possibility that one or more of these organisms were present in the culture cannot be ruled out. The identity of the dehalogenator that reduced TCE to cis-DCE (Figure 1a) remains unknown. This study demonstrates that TCE can be dechlorinated microbially to 1,1-DCE as a major intermediate instead of the usual cis-DCE. Presence of 1,1-DCE at contaminated sites is often attributed to chemical hydrolysis of 1,1,1-TCA, a common co-contaminant of TCE (3, 28-30). Our findings provide an alternative, biological mechanism for the occurrence of 1,1-DCE in TCE-contaminated sites. Our results also illustrate that competition for TCE can exist between different dehalogenating populations. Competition among different dehalogenators, which may control the fate of chlorinated ethenes at field sites, has received limited attention and needs to be better understood.

Acknowledgments We thank Thomas Hanson of the Graduate College of Marine Studies and the Delaware Biotechnology Institute for reviewing the manuscript, Ulhas Naik of the Department of Biological Sciences and Jeff Rockwood for their assistance in the cell extract assays, and Timothy McHale of U.S. Air Force Research Laboratory for collecting groundwater samples. This material is based upon work supported by the National Science Foundation under Grant No. 9984669.

Supporting Information Available Sequence comparison between the amplicon from our culture and the 16S rRNA gene sequence from Dehalococcoides ethenogenes strain 195. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review September 15, 2005. Revised manuscript received January 5, 2006. Accepted January 12, 2006. ES051829M