Scale-up and Characterization of an Enrichment Culture for Bioaugmentation of the P-Area Chlorinated Ethene Plume at the Savannah River Site

Bioaugmentation has become an increasingly popular remediation strategy for groundwater sites contaminated with chlorinated solvents. When biostimulation is not an option due to the lack of necessary microorganisms required for dechlorination of the contaminants, bioaugmentation is an attractive option for remediation. The P-Area groundwater plumes at the Savannah River Site (SRS) are just such a case. The P-Area site is contaminated with tetrachloroethene (PCE), trichloroethene (TCE), and cis-dichloroethene (cDCE), and no dechlorination past cDCE is occurring. A similar site, the C-Area site, is near one of the P-Area site\u27s source zones but displays complete reduction of TCE to ethene. An enrichment culture was developed from the C-Area wetland for possible use as an indigenous bioaugmentation culture for the P-Area site. The culture underwent characterization in terms of potential terminal electron acceptors, pathogenicity, susceptibility to 1,1,1-trichloroethane (1,1,1-TCA), potential use of emulsified vegetable oil as an electron donor, and response to oxygen exposure and a range of pH levels. The ability of the SRS culture, which was enriched on PCE and TCE, to use other halogenated alkenes and alkanes as terminal electron acceptors was investigated. The SRS culture is capable of utilizing PCE, TCE, cDCE, trans-dichloroethene, 1,1-dichloroethene, vinyl chloride (VC), 1,2-dichloroethane, 1,2-dibromoethane, and vinyl bromide as electron acceptors. Additionally, the culture\u27s ability to dechlorinate several chlorinated benzenes was investigated. The SRS culture can dechlorinate hexachlorobenzene, pentachlorobenzene, 1,2,4,5-tetrachlorobenzene, and 1,2,4-trichlorobenzene; however, further testing is required to determine if these electron acceptors are used metabolically as with the halogenated alkenes and alkanes. The culture cannot dechlorinate the dichlorobenzene isomers or chlorobenzene. The SRS culture was tested for potential pathogenicity, which would hinder its regulatory approval as a bioaugmentation culture. Initially, the culture\u27s ability to grow on a rich substrate (trypticase soy broth) at the temperature of the human body was tested. The culture grew aerobically at 37¼C, and further analysis using commercial coliform and E. coli testing kits revealed that the SRS culture does contain coliforms. However, E. coli is not present in the culture. Further molecular testing is being conducted at the Savannah River National Laboratory (SRNL) to determine if other pathogenic species might be present in the culture. As 1,1,1-TCA has been shown to be inhibitory to many dechlorinating cultures, the SRS culture\u27s susceptibility to this known inhibitor was evaluated. The culture\u27s ability to completely dechlorinate TCE to ethene was inhibited by 300 µM 1,1,1-TCA; a mixture of VC and ethene were produced as end products. Lower concentrations of 1,1,1-TCA (0.7 and 3.6 µM) did not inhibit TCE conversion to ethene. Additionally, the SRS culture was not capable of dechlorinating 1,1,1-TCA. The SRS culture was enriched on lactate as an electron donor, however, the use of emulsified oil substrate (EOS¨) as an electron donor was investigated as it is a longer lasting electron donor. A microcosm evaluation suggested that EOS¨ is a better electron donor than lactate. Reductive dechlorination of PCE and TCE occurred faster and with less accumulation of daughter products in treatments amended with EOS¨ than in those amended with lactate. The SRS culture was tested for its vulnerability to oxygen exposure, as it is an anaerobic culture, and exposure to oxygen could be detrimental to the success of the culture in the field. Quiescent exposure to air (21% oxygen in headspace) for 24 hours slowed the dechlorination of PCE and TCE. However, the culture was able to overcome the aerobic conditions and completely dechlorinate PCE and TCE to ethene. The low redox conditions provided by the media in which the culture is maintained allowed for anaerobic conditions in the bottles to be reestablished. Given low redox conditions in contaminated groundwater, the SRS culture should be able to sustain brief oxygen exposure and retain its reductive dechlorinating ability. Related to oxygen exposure, the SRS culture\u27s susceptibility to extreme pH levels was investigated. The SRS culture is maintained in buffered minimal media within the pH range of 6.5-7.5. The culture was exposed to a range of pH levels (5.5, 6.0, 6.5, 7.0, and 8.5) as well as a treatment in which pH was allowed to decrease from neutral, as a result of HCl release. At pH 6.0, the dechlorinating activity of the SRS culture was slowed, and cDCE and VC accumulation was higher than at pH 7.0. At pH 5.5, reductive dechlorination stopped at cDCE, with no production of VC or ethene. When the pH was allowed to decrease from neutral, the culture exhibited a decrease in ethene production and accumulation of VC as the pH dropped below 6.0. The culture was most strongly inhibited at pH 8.5; some PCE was dechlorinated, but no TCE was consumed. Little to no cDCE or VC was produced. Given these results, it will be necessary for groundwater pH to be adequately buffered at a pH of 6.5 or higher for successful bioaugmentation with the SRS culture