Environ. Sci. Technol. 2005, 39, 2631-2635
Reductive Dechlorination of Tetrachloroethene to trans-Dichloroethene and cis-Dichloroethene by PCB-Dechlorinating Bacterium DF-1 GREGORY S. MILLER,† CHARLES E. MILLIKEN,† KEVIN R. SOWERS,‡ AND H A R O L D D . M A Y * ,† Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Ave., 201 BSB, Charleston, South Carolina 29425-2230, and Center of Marine Biotechnology, University of Maryland, Biotechnology Institute of Maryland, Suite 236, Columbus Center, 701 E Pratt St, Baltimore, Maryland 21202
Polychlorinated biphenyls (PCBs) and chlorinated ethenes (CEs) are known to pollute sediment, soil, and groundwater. The anaerobic dechlorination of these compounds is an integral part of their biodegradation in polluted environments. We report for the first time the dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) by bacterium DF-1. This PCB and chlorobenzene dechlorinating bacterium dechlorinated PCE to TCE, which was then converted into trans-1,2-dichloroethene (trans-DCE) and cis1,2-dichloroethene (cis-DCE). The ratio of trans-DCE to cisDCE produced by the culture had a range of 1.2-1.7. Bacterium DF-1 has been enriched in co-culture with a desulfovibrio-like microorganism. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the 16S rRNA genes of the co-culture demonstrated that DF-1 was enriched during the dechlorination of PCE, PCB, and chlorobenzene. DF-1 was not detected in the absence of PCE dechlorination and the desulfovibrio-like organism, isolated in pure culture, did not dechlorinate PCE. This is the first identification of a microorganism capable of producing high amounts of trans-DCE from PCE and indicates that microorganisms such as DF-1 are a possible biological source of trans-DCE in the environment.
Introduction Polychlorinated biphenyls (PCBs) and chlorinated ethenes (CEs), for example, tetrachloroethene (PCE) and trichloroethene (TCE), are common pollutants of aquatic sediments and groundwater. Microbial transformation alters the toxicity of these compounds, and complete microbial dechlorination can remove the threat from the environment. The more extensively chlorinated species of both of these classes of compounds resist aerobic degradation, but they are susceptible to anaerobic dechlorination. The latter process can result in the complete dechlorination and detoxification of * Corresponding author phone: (843)792-7140; fax: (843)792-2464; e-mail:
[email protected]. † Medical University of South Carolina. ‡ University of Maryland. 10.1021/es048849t CCC: $30.25 Published on Web 03/11/2005
2005 American Chemical Society
PCE and some PCBs or render the compounds sufficiently dechlorinated for subsequent aerobic biodegradation (1, 2, 3). PCR-denaturing gradient gel electrophoresis (PCRDGGE) of PCB dechlorinating cultures and selective enrichment for PCB dechlorination were used to make the first identification of PCB dechlorinating microorganisms, bacteria DF-1 (4) and o-17 (5). Both microorganisms exist in co-culture with a different sulfate-reducing vibrio. These desulfovibrio-like organisms do not dechlorinate PCBs when in pure culture. However, DF-1 and o-17 are enriched during PCB dechlorination, and dechlorination does not occur when these bacteria are undetected by DGGE. Similar investigations have proven that bacterium DF-1 catalyzes chlorobenzene dechlorination (6). The PCB dechlorinating bacteria DF-1 and o-17 are most similar phylogenetically (89% similarity) to Dehalococcoides spp. on the basis of 16S rRNA gene sequence, yet these organisms belong to a distinctly different genus (unpublished results). Since many Dehalococcoides spp. dechlorinate CEs (7, 8, 9), it was considered possible that the PCB dechlorinating bacteria may do likewise. Bacterium DF-1 was used to test this hypothesis. Since the organism is one of two members of a co-culture, PCR-DGGE was again used to examine the growth of DF-1 and its companion desulfovibrio-like bacterium in response to the dechlorination of PCE. The results of this study indicated that DF-1 reductively dechlorinates CEs. This is the first identification of a microorganism that dechlorinates PCE and TCE more so to trans-DCE than to cis-DCE.
Materials and Methods Chemicals. Chlorobenzenes, CEs (PCE, TCE, 1,1-DCE, transDCE, cis-DCE, vinyl chloride), ethene, and ethane, were purchased from Sigma-Aldrich (Milwaukee, WI). All PCBs were purchased from Accustandard (New Haven, CT). Culture Conditions. A culture containing bacterium DF-1 was grown under anaerobic conditions as described previously (4) in estuarine-chloride (E-Cl) medium with the addition of 10 mM sodium formate. The E-Cl was prepared as described (10) except 0.5 mM titanium(III) nitrilotriacetic acid was substituted for cysteine-HCl. The titanium(III) nitrilotriacetic acid was prepared according to the methods of Zehnder and Wuhrmann (11) with sodium nitrilotriacetate substituted for sodium citrate. Solvent-resistant 0.2 µ Nalgene nylon membrane filters (Nalge Company, Rochester, NY) were used to sterilize PCE and other CEs, which were directly added to the culture medium with a Gastight 10-µL glass syringe (model 1701, Hamilton Co., Reno, NV); 173 µM 2,3,4,5PCB or pentachlorobenzene was added in acetone. The cultures were grown in 160-mL serum bottles containing 50-100 mL E-Cl and sealed with 20-mm Teflon-coated butyl stoppers (West Co., Lionville, PA) or blue butyl rubber stoppers (Bellco, Vineland, NJ) or were grown in 1-L cultures in a bioreactor fabricated from a 2.0-L spinner flask (Bellco), which allowed for the direct addition of low concentrations of neat PCE to the culture. The vessel consisted of a closed glass top with two ports (20-mm diameter) that could accommodate the same Teflon-coated stoppers used with the serum bottles. The total volume of the modified vessel was 2.3 L. Aluminum crimp seals (Wheaton) were used to secure the stoppers to the bottles and custom vessels. PCE was periodically replenished by direct addition to the culture medium while the culture vessel was flushed with 80% N220% CO2. The 2.3-L vessels and 160-mL serum bottles were sealed immediately after the addition of PCE. Cultures were incubated statically in the dark at 30 °C. To repeatedly VOL. 39, NO. 8, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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replenish the PCE, the cultures were flushed with N2/CO2 (130 mL per min) after each headspace sampling until the CEs were no longer detected (10 min for 160-mL serum bottles and 15 min for the 2.3-L vessels). PCE was replenished and a new sterile Teflon-coated stopper was used to seal the container. Analysis of Chlorinated Organic Compounds. PCBs were analyzed with a HP6890 GC/ECD according to the methods described by Berkaw et al. (10) and Wu et al. (6). CEs and ethene were measured with an HP6890 GC/FID equipped with an HP-1MS capillary column (5 m × 0.53 mm × 2.65 um film thickness). The following oven temperature program was used to separate the CEs: initial hold at 35 °C for 2.2 min, increase to 55 °C at a rate of 10 °C per min, final hold at 55 °C for 1.8 min. Baseline separation was achieved with all of the CEs and ethene. Standard curves for each of the CEs were determined by adding the compounds individually to 2.3-L vessels containing 1 L of sterile E-Cl medium. The standards were allowed to equilibrate overnight and headspace gas samples were assayed in triplicate by GC-FID to determine gaseous standard concentrations. Aqueous values were determined using Gossett’s published Henry’s Law constants for each CE and ethene (12). The minimum detection limits for each CE were PCE, 1.6 µmoles (per 1-L medium in 2.3-L vessel); TCE, 2.7; 1,1-DCE, 6.3; cDCE, 9.6; tDCE, 2.7; VC, 5.6; and ethene, 6.5. Samples from live cultures and standard abiotic samples were drawn from the headspace of the vessels using a Gastight 50-µL glass syringe (model 1705, Hamilton Co., Reno, NV) equipped with a sample lock. Since stoppers had to be replaced in live cultures after analysis, the residual CEs had to be removed by gas flushing after each sampling. To determine the efficacy of the gas flushing, abiotic controls (sterile, killed-cell) with each CE were flushed for 15 min, sealed with a new Teflon-coated stopper, and allowed to equilibrate overnight. GC-FID analysis on the day following the flushing of the headspace gas revealed that the residual CE was below the minimum detection limits. Analysis of 16S rDNA. Genomic DNA was extracted from 1.5 mL of culture using Instagene Matrix (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. PCR, DGGE, and sequencing analysis were performed as described by Cutter et al. (5) using PCR primers 1055-1070 forward and 1406-1392 reverse, which are Bacteria domain specific and universal, respectively (13). Amplified rDNA restriction analyses (ARDRA) and comparative sequence analyses were conducted as described in Pulliam-Holoman et al. (14). 16S rRNA clone libraries were generated with 1.5 kb universal primers described by Edwards et al. (15) and 940 bp archaeal primers described by Lane et al. (16) and screened by restriction fragment length polymorphism analysis after digesting with restriction endonucleases HaeII and HhaI. Digestion products were discriminated by gel electrophoresis on a 3% Trevigel at 25 V for 3 h at 0 °C. Plasmids and PCR products were used as templates for dye terminator cycle sequencing using Big Dye 3.1 kit (Applied Biosystems) and were run in an ABI 3100 (Applied Biosystems).
Results Dechlorination of Chlorinated Ethenes. To accurately determine the CE dechlorination by bacterium DF-1, several issues pertaining to potential toxicity and abiotic loss of the CEs had to be addressed. Initially adding 0.5-1.0 mM of PCE to the culture proved unsuccessful, that is, no dechlorination was observed. However, TCE, trans-DCE, and cis-DCE were detected in the headspace of DF-1 cultures within one week when low amounts (
