Characterization of Microbial Communities in TCE-Contaminated Seep Zone Sediments. (2004)

Hundreds of sites across the United States contain trichloroethene (TCE) contamination, including the Department of Energy's Savannah River Site (SRS) in Aiken, South Carolina. Previous studies have indicated that microorganisms are capable of efficiently degrading TCE to nonhazardous end products. In this project, molecular and growth based methods were used for microbial characterization of a TCE impacted seepzone where TCE degradation is naturally occurring. The results from this work provide clear evidence that the SRB may play a significant role in TCE degradation along the Twin Lakes seepline. INTRODUCTION Trichloroethene (TCE) was a common degreasing agent in dry-cleaning and machining industries, but the health hazards associated with this compound have largely restricted its use. At many locations TCE waste materials were simply dumped or buried in unlined pits or basins, and over time pollutants have seeped into subsurface groundwater supplies threatening downstream environments and public exposure (Hendrickson et al., 2002, Smidt, 2000). Flowing groundwater not only serves to transport TCE over large areas within the subsurface environment, but many contaminant plumes resurface through seepzones into streams, rivers, or wetland areas (Hendrickson et al., 2002). Hundreds of sites within the DOE complex have TCE contaminated groundwater, including the Savannah River Site in Aiken SC. A TCE plume originating from the C-Area Burning Rubble Pit (CBRP) on the Savannah River Site is now beginning to resurface at the Twin Lakes seep zone, a tributary of the Savannah River. Bioremediation is an attractive, passive strategy for effective removal of TCE polluted sites. The success of bioremediation relies on the presence and activities of microorganisms that are capable of degrading TCE by catalyzing sequential dechlorination reactions, thereby transforming trichloroethene (TCE) —» dichloroethene (DCE) -» vinyl chloride (VC) -» ethene (Hendrickson et al., 2002; Holliger et al., 1999; Smidt et al., 2000). Dehalococcoides ethogenes is the only known microorganism that can perform this entire process to completion. Other microbes, like the sulfate-reducing bacteria (SRB), can degrade TCE but incompletely to DCE or VC. Along the CBRP seepzone at the Savannah River Site TCE degradation is occurring naturally but this activity is inconsistent from site to site. TCE degradation is occurring at some sites but not others, and where degradation is occurring, the process stops at DCE or VC. The microbiology of the CBRP seepzone has not been studied in detail. The goals of this study were to 1) determine why TCE degradation activity differs along the seepzone, 2) determine the role of SRB in TCE degradation, and 3) explore the use of DNA fingerprinting tools for rapid identification of bacteria capable of TCE degradation at different sites along the seepzone. To accomplish these goals classic microbiological growth based methods and DNA molecular based methods were used to examine the microbiology in CBRP seepzone sediments directly to explain TCE degradation. MATERIALS AND METHODS Field Site Description and Sample Collection. Sediment samples were collected in June 2003 along the flow path of a subsurface TCE plume originating at the C-Area Burning Rubble Pit (CBRP). Six groundwater sampling wells marked sediment collection sites in study. Furthest from the seepzone were sites 21 and 41. The saturated zone at these sites occurred at 6 8' depth and TCE degradation products had not been detected at these sites. The saturated zone at the intermediate sites 42 and 44 was at 5 6' depth and TCE degradation occurred at these sites but stopped at cis-1,2dichloroethene (cDCE). Along the low-lying wetlands seepzone fringe, sites 48 and 50 were inundated and TCE degradation products cDCE and vinyl chloride (VC) had been detected at these locations. Sediment samples from these sites were collected within the 3 5 ' depth range. Sediment samples were collected using a hand auger. Upon collection, sediments were packed in ziplock bags and stored on ice during transport to the laboratory. Two grams of sediment per site were reserved for direct inoculation into SRB growth medium, and all remaining sediment was frozen at -80°C to await molecular analysis. SRB Growth Medium. Postgate's Medium B with lactate as the sole carbon and energy source was used to select for the growth of SRB from field sediments (Flemming and Ingvorsen, 1998). One gram of sediment (2 replicates per site) was directly inoculated into 10ml (final volume) of Postgate's Medium B in an anaerobic chamber having an atmosphere of 95%N2 and 5% FL. Enrichment tubes were incubated in the dark at room temperature. Active sulfate reduction in Postgate's Medium B is evidenced by the production of FeS2 that appears as a black precipitate in the growth medium. Growth of S0 4 reducing enrichments on TCE. After 6 consecutive transfers on Postgate's Medium B, all positive sulfate reducing enrichment cultures were examined for their ability to degrade TCE in the absence of sulfate. Enrichment cultures were subcultured into a modified Postgate's Medium B with TCE (20ul of TCE saturated dH20, 2ppm) as the sole terminal electron acceptor. After 5 days incubation at room temperature in the dark, headspace gases were analyzed by gas chromatography mass spectrometry (GC-MS) for analysis of TCE degradation products. Genomic DNA extractions from sulfate reducing enrichment cultures. The protocol for DNA extraction used the Promega DNA Purification kit as recommended by the manufacturer (Promega, Madison, WI). Cell biomass from 3ml of each enrichment culture was collected by centrifugation at 10,000xg for 2min. Cell pellets were then resuspended in 480uL of 50mM EDTA and 60uL of lysozyme (lOmg/mL), and incubated at 37°C for 60 minutes. Cell lysis was performed using 600uL of Nuclei Lysis Solution and incubated at 80°C for 5 min. Cell lysates were treated with RNase-A (37°C for