Further Biogeochemical Characterization of a Trichloroethene-Contaminated Fractured Dolomite Aquifer: Electron Source and Microbial Communities Involved in Reductive Dechlorination A. M. HOHNSTOCK-ASHE, † S. M. PLUMMER, ‡ R. M. YAGER, | P. BAVEYE, § AND E. L. MADSEN* ,† Departm ent of Microbiology, Wing Hall Cornell University, Ithaca, New York 14853, Department of Crop and Soil Sciences, Bradfield Hall, Cornell University, Ithaca, New York 14853, and USGS, Ithaca, New York 14850 A recent article presented geochemical and microbial evidence establishing metabolic adaptation to and in-situ reductive dechlorination of trichloroethene (TCE) in a fractured dolomite aquifer. This study was designed to further explore site conditions and microbial populations and to explain previously reported enhancement of reductive dechlorination by the addition of pulverized dolomite to laboratory microcosms.A survey of groundwater geochemical parameters (chlorinated ethenes, ethene, H 2 , CH 4 , DIC, DOC, and δ 13 C values for CH 4 , DIC, and DOC) indicated that in situ reductive dechlorination was ongoing and that an unidentified pool of organic carbon was contributing, likely via microbial respiration, to the large and relatively light on- site DIC pool. Petroleum hydrocarbons associated with the dolomite rock were analyzed by GC/MS and featured a characteristically low δ 13 C value. Straight chain hydrocarbons were extracted from the dolomite previously found to stimulate reductive dechlorination; these were particularly depleted in hexadecane (HD). Thus, we hypothesized that HD and related hydrocarbons might be anaerobically respired and serve both as the source of on- site DIC and support reductive dechlorination of TCE. Microcosms amended with pulverized dolomite demonstrated reductive dechlorination, whereas a combusted dolomite amendment did not. HD-amended microcosms were also inactive. Therefore, the stimulatory factor in the pulverized dolomite was heat labile, but that component was not HD. Amplified Ribosomal DNA Restriction Analysis (ARDRA) of the microbial populations in well waters indicated that a relatively low diversity, sulfur-transforming community outside the plume was shifted toward a high diversity community including Dehalococcoides ethenogenes-type microorganisms inside the zone of contamination. These observations illustrate biogeochemical intricacies of in situ reductive dechlorination reactions. Introduction The industrial solvent trichloroethylene (TCE) is among the most ubiquitous of groundwater contaminants (1). Under anaerobic conditions, both TCE and the related solvent tetrachloroethylene (PCE)can undergo a varietyofreactions (2, 3) that include sequential microbial reductive dechlori- nation to lesser chlorinated ethenes, dichloroethylene (cis- DCE), and vinyl chloride (VC) and to nontoxic ethene (4, 5). These chemical transformations can be carried out cometa- bolicallybysulfate-reducing,methanogenic,and acetogenic microorganisms that produce reactive transition metal coenzymes (4, 6). More importantly, PCE and TCE can be used as physiologicalelectron acceptors for dehalorespiring bacteria (7, 8).Because dehalorespiration leadsto the growth of microorganisms, this process is robust and provides a foundation for potential intrinsic bioremediation of sites contaminated with chlorinated solvents (2, 3, 9, 10). Dehalorespiration has become the focus ofconsiderable scientific inquiry (11-13). Many pure cultures capable of dehalorespiration have been isolated and include micro- organisms from the phylogenetically diverse genera De- sulfitobacterium , Desulfurom onas, Dehalospirillum , De- halobacter, and Dehalococcoides (14-20). A single repre- sentative of the latter genus, Dehalococcoides ethenogenes 195, can completely dechlorinate PCE to ethene (4, 8). Understanding the microbial ecology of these organisms, particularly D.ethenogenes in contaminated sites,contributes to accruing fundamental knowledge of bioremediation technology (21). Reductive dechlorination reactions can only occur in geochemical settings conducive to key electron donor/ acceptor reactions. In such sites, tangible geochemical and microbiological“footprints”ofdehalorespiration are evident (2). Clear evidence for complete reductive dechlorination of TCEin situ in a fractured dolomite aquifer near Niagara Falls, NYwas obtained in a previous study (22). Yager et al. (22) reported the presence of cis-DCE and VC on site, although neither of these compounds was among the pollutants released. Ethene, the product of complete reductive dechlo- rination of TCE, was also detected. In addition, Yager et al. (22)demonstrated that microorganisms native to site waters inside,butnotoutside,thecontaminantplumewereadapted to catalyze reductive dechlorination reactions.Furthermore, stimulation of reductive dechlorination activity was dem- onstrated in laboratorymicrocosmscontainingcontaminated groundwater amended with samples ofthe geologic aquifer matrix(pulverized fractured dolomite). Yet questions about the in situ source ofelectronsdrivingreductive dechlorination and the mechanism ofstimulation (surface area effect versus nutrients versus carbon source) remain unanswered. The objectivesofthe present investigation were to advance case-specific biogeochemical knowledge of reductive dechlo- rination processes by (i) expanding the study site’s geochem- ical database; (ii) investigating the in-situ source of carbon that might support microbial reductive dechlorination reac- tions, (iii) attempting to explain the previously reported stimulation of reductive dechlorination by the pulverized dolomite,and (iv)analyzingthe composition ofthe microbial community both inside and outside of the contaminant plume using 16S rDNA procedures. Methods Study Site and Sampling. The study site is adjacent to a manufacturing facility 8 km east of Niagara Falls, NY. *Correspondingauthor phone: (607)255-3086;fax: (607)255-3904; e-mail: elm3@cornell.edu. † Department of Microbiology, Cornell University. ‡ Present address: Colorado School of Mines, Golden, CO. | USGS. § Department of Crop and Soil Sciences, Cornell University. Environ. Sci. Technol. 2001, 35, 4449-4456 10.1021/es0110067 CCC: $20.00  2001 American Chemical Society VOL. 35, NO. 22, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4449 Published on Web 10/06/2001