Environ. Sci. Technol. 2004, 38, 4300-4303
Anaerobic Microbial Reductive Dechlorination of Tetrachloroethene to Predominately trans-1,2-Dichloroethene BENJAMIN M. GRIFFIN,† JAMES M. TIEDJE,† AND FRANK E. LO ¨ F F L E R * ,‡ Center for Microbial Ecology, Department of Microbiology and Molecular Genetics, and Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824-1325, and School of Civil and Environmental Engineering and School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0512
While most sites and all characterized PCE and TCE dechlorinating anaerobic bacteria produce cis-DCE as the major DCE isomer, significant amounts of trans-DCE are found in the environment. We have obtained microcosms from some sites and enrichment cultures that produce more trans-DCE than cis-DCE. These cultures reductively dechlorinated PCE and TCE to trans-DCE and cis-DCE simultaneously and in a ratio of 3((0.5):1 that was stable through serial transfers with a variety of electron donors and occurred in both methanogenic and nonmethanogenic enrichments. Two sediment-free, nonmethanogenic enrichment cultures produced trans-DCE at rates of up to 2.5 µmol L-1 day-1. Dehalococcoides populations were detected in both trans-DCE producing cultures by their 16S rRNA gene sequences, and trans-DCE was produced in the presence of ampicillin. Because trans-DCE can be the major product from PCE and TCE microbial dechlorination, high fractions of trans-DCE at chloroethene-contaminated sites are not necessarily from source contamination.
Introduction Chlorinated ethenes are significant groundwater contaminants and are present in aquifers as parent compounds (PCE and TCE) and daughter products (DCEs and VC). Of the current or former U.S. Environmental Protection Agency National Priority List Sites, the Agency for Toxic Substances and Disease Registry reports tetrachloroethene (perchloroethylene, PCE) at 54% of the sites; trichloroethene (TCE) at 60%; 1,1-dichloroethene (1,1-DCE) at 36%; trans-1,2-dichloroethene (trans-DCE) at 39%; cis-1,2-dichloroethene (cisDCE) at 10%; and vinyl chloride (VC) at 37% (1). Under anaerobic conditions, chloroethenes are subject to reductive dechlorination (hydrogenolysis) resulting in the stepwise conversion of PCE to TCE, DCE isomers, VC, and ethene. Several anaerobic bacterial populations have been isolated that use PCE or TCE as respiratory electron acceptors for growth through a process termed (de)chlororespiration (2-5). These isolates belong to several genera including * Corresponding author phone: (404)894-0279; fax: (404)894-8266; e-mail:
[email protected]. † Michigan State University. ‡ Georgia Institute of Technology. 4300
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 16, 2004
Dehalobacter (6, 7), Desulfuromonas (8, 9), Desulfitobacterium (10-13), Dehalococcoides (4, 14), Clostridium (15), Enterobacter (16), and Sulfurospirillum (formerly Dehalospirillum) (17-18). Except for a few Desulfitobacterium strains that only convert PCE to TCE and a few Dehalococcoides isolates that dechlorinate PCE or TCE to VC and ethene (4, 14, 19, 20), all other isolates dechlorinate PCE to predominately cis-DCE as the end product. Chloroethene reductive dehalogenases have been characterized from several organisms including Sulfurospirillum multivorans (21), Desulfitobacterium sp. strain PCE-S (22), Desulfitobacterium sp. strain Y51 (23), Dehalococcoides ethenogenes (24), and Dehalobacter restrictus (25). The reductively dechlorinating enzyme systems contain corrinoid cofactors in addition to iron-sulfur clusters, and in Dehalobacter restrictus, dechlorination is suggested to occur through a radical mechanism (25). A recent computational study examining the stability and transformation rates of radical intermediates explains the favored cis-DCE production by B12 catalyzed reactions (26). Interestingly, trans-DCE is often found at chloroethenecontaminated sites (1), and its presence has been explained by source contamination or generation through abiotic mechanisms acting on polychlorinated ethenes. No biotic mechanisms that produce predominantly trans-DCE from PCE or TCE are known. A comprehensive understanding of the mechanisms that contribute to trans-DCE occurrence is relevant for natural attenuation monitoring, point-source tracking, and the choice of the most promising remediation strategies. This study describes microcosms and enrichment cultures that produce trans-DCE and cis-DCE in a ratio of 3:1 from PCE and TCE.
Materials and Methods Chemicals. The following analytical grade chlorinated compounds were used in this study: PCE, TCE, trans-DCE, cisDCE, all of which were obtained from Supelco (Bellefonte, PA), and VC, which was obtained from Fluka Chemical Corp. (Ronkonkoma, NY). Other chemicals used in the study were obtained from Sigma-Aldrich (Milwaukee, WI). Inoculum Sources, Microcosm Preparation, and Growth Conditions. Microcosms that produced a mixture of transDCE and cis-DCE were derived from sediment and soil materials collected from the Tahquamenon River and the Pine River, both located in Michigan, the Perfume River near Hue´ in Vietnam, and a swampy area in Chitwan National Park, Nepal, as described (27, 28). In addition, PCE dechlorinating mircososms were established from a soil and sediment slurry consisting of over 15 agricultural soils, forest soils, and river sediment samples collected in Michigan. The slurry was maintained under nitrate reducing conditions to reduce readily bioavailable electron donors by 10 repeated additions of 0.5 mM nitrate. Following complete removal of nitrate and nitrite, approximately 10 g of slurry was added to 90 mL of basal salts medium reduced with cysteine and sulfide (0.2 mM each) (3) in 160 mL serum bottles. All microcosms were amended with lactate (5 mM) as a source of reducing equivalents and PCE (20 µmol). Three microcosms from each site were autoclaved for 60 min at 121 °C on three consecutive days and served as sterile controls. Chloroethenes were analyzed in headspace samples performed following inoculation and periodically thereafter. All cultures were monitored for chloroethene transformation (by gas chromatography) and electron donor consumption (by high performance liquid chromatography) as described previously, and substrates were replenished as needed 10.1021/es035439g CCC: $27.50
2004 American Chemical Society Published on Web 06/25/2004
TABLE 1. Summary of Microcosms and Enrichment Cultures that Produced Mixtures of trans-DCE and cis-DCE source
enriched with
transfersa
sediment-free
methane production
trans/cis DCEb
Tahquamenon River, MI Perfume River, Vietnam Red Cedar River, MI Pine River, MI Chitwan National Park, Nepal mixed MI soil and sediment inocula
PCE PCE 1,2-Dc PCE PCE PCE
>30 >25 12 6 1 1
yes yes yes yes no no
no no no yes yes yes
3.1 2.9 2.5 3.4 3 3.5
a Transfers were 1% (vol/vol) into fresh minimal medium. b Ratio is the average of at least five measurements (SD
