Field Demonstration of Successful Bioaugmentation To Achieve Dechlorination of Tetrachloroethene To Ethene DAVID W. MAJOR,* MICHAYE L. MCMASTER, AND EVAN E. COX GeoSyntec Consultants, Inc., 130 Research Lane, Suite 2, Guelph, Ontario, N1G 5G3 Canada ELIZABETH A. EDWARDS AND SANDRA M. DWORATZEK Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Ontario, M5S 3E5 Canada EDWIN R. HENDRICKSON, MARK G. STARR, JO ANN PAYNE, AND LOIS W. BUONAMICI E.I. DuPont de Nem ours & Com pany, Inc., Central Research and Developm ent, P.O. Box 6101, Glasgow 300, Newark, Delaware 19714-6101 A laboratory microcosm study and a pilot scale field test were conducted to evaluate biostimulation and bioaugmen- tation to dechlorinate tetrachloroethene (PCE) to ethene at Kelly Air Force Base. The site groundwater contained about 1 mg/L of PCE and lower amounts of trichloroethene (TCE) and cis- 1,2-dichloroethene (cDCE). Laboratory microcosms inoculated with soil and groundwater from the site exhibited partial dechlorination of TCEto cDCEwhen amended with lactate or methanol. Following the addition of a dechlorinating enrichment culture, KB-1, the chlorinated ethenes in the microcosms were completely converted to ethene. The KB-1 culture is a natural dechlorinating microbial consortium that contains phyloge- netic relatives of Dehalococcoides ethenogenes. The ability of KB-1 to stimulate biodegradation of chlorinated ethenes in situ was explored using a closed loop recirculation cell with a pore volume of approximately 64 000 L. The pilot test area (PTA) groundwater was first amended with methanol and acetate to establish reducing conditions. Under these conditions, dechlorination of PCE to cDCE was observed. Thirteen liters of the KB-1 culture were then injected into the subsurface. Within 200 days, the concentrations of PCE, TCE, and cis-1,2-DCE within the PTA were all below 5 μg/L, and ethene production accounted for the observed mass loss. The maximum rates of dechlorination estimated from field data were rapid (half- lives of a few hours). Throughout the pilot test period, groundwater samples were assayed for the presence of Dehalococcoides using both a Dehalococcoides-specific PCR assay and 16S rDNA sequence information. The sequences detected in the PTA after bioaugmentation were specific to the Dehalococcoides species in the KB-1 culture. These sequences were observed to progressively increase in abundance and spread downgradient within the PTA. These results confirm that organisms in the KB-1 culture populated the PTA aquifer and contributed to the stimulation of dechlorination beyond cDCE to ethene. Introduction Chlorinated ethenes such as tetrachloroethene (PCE) and trichloroethene (TCE) are some of the most pervasive groundwater contaminants. Biodegradation is a promising remedial alternative in many cases. The principle biodeg- radation mechanism for chlorinated ethenes is reductive dechlorination, which involves the sequential replacement ofchlorine atomson the alkene molecule byhydrogen atoms (1-3).Some microorganisms,termed dehalorespiringmicro- organisms, including Dehalospirillium multivorans (4), De- halobacter restrictus (5), and Dehalococcoides ethenogenes (1), use chlorinated ethenes as their terminal electron acceptors in metabolism and gain energy from reductive dechlorination (6-8). Hydrogen produced by fermentation is often the electron donor. Of these microorganisms, D. ethenogenes is the only one able to completely dechlorinate chlorinated ethenes to ethene but does not appear to be present at all sites. At some sites, dechlorination of PCE and TCE stalls at cis- 1,2-dichloroethene (cDCE) resulting in an accumulation ofpartiallydechlorinated products.In a recent survey of 24 chlorinated ethene-contaminated sites, Deha- lococcoides spp. (Dhc) were not detected at “partially dechlorinating” sites (9). Severalstable enrichment culturesthat contain organisms phylogeneticallycloselyrelated to D.ethenogenes are capable of mediating complete dechlorination of TCE to ethene. Examplesinclude the CornellCulture (1),the Victoria Culture (9), and the Pinellas culture (10-13). Afield demonstration by the Remediation Technologies Development Forum (RTDF) at Dover Air Force Base in Delaware demonstrated that dechlorination of cDCE to ethene occurred after the pilot test area (PTA) was bioaugmented with the Pinellas culture, a culture enriched from soil and groundwater samples from a U.S. Department of Energy site in Largo, FL (11, 12).After complete dechlorination ofTCEto ethene had occurred, samples taken from the Dover PTAwere shown to contain, via 16S rDNA analysis, at least three different dechlorinatingspeciessimilarto D. restrictus, D.multivorans, and D. ethenogenes (9, 10, 13). Furthermore, the 16S rDNA sequences obtained from the Dover pilot site were found to have signature sequences that were the same as those found in the Pinellas bioaugmentation culture. These findings suggest, although did not prove, that the inoculum abetted the dechlorination and that some of the culture had indeed survived and populated the pilot subsurface (10, 13).To date, allcultures that dechlorinate chlorinated ethenes complete- ly to ethene have been found to contain a phylogenetically close relative of Dehalococcoides (1, 9, 14). While Dehalo- coccoides may not be the only group of organisms capable ofcomplete dechlorination (othershave yet to be discovered), they have been shown to be an important indicator for dechlorination beyond cDCE (9, 14). In this study, we report the results of a second successful pilot scale bioaugmentation demonstration.The goalsofthis studywere to correlate the dechlorination to ethene with the presence of dechlorinating organisms, in particular Deha- lococcoides, whether they were indigenous to the Kelly Air *Correspondingauthor phone: (519)822-2230;fax: (519)822-3151; e-mail: dmajor@geosyntec.com. Environ. Sci. Technol. 2002, 36, 5106-5116 5106 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36, NO. 23, 2002 10.1021/es0255711 CCC: $22.00  2002 American Chemical Society Published on Web 10/26/2002