Date of Award

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Environmental Engineering and Science

Advisor

Lee, Cindy M

Committee Member

Kurtz, Jr. , Harry D

Committee Member

Elzerman , Alan

Abstract

To date, all attempts to isolate PCB dechlorinators using traditional isolation techniques have failed. In addition, the study of PCB dechlorinating organisms has been hampered because researchers were unable to grow the organisms in sediment-free, defined media. Three microbial species capable of reductive dechlorination of polychlorinated biphenyls have been identified. These three species are from two phylogenetically distinct branches, Dehalococcoides (Bedard et al., 2007) and the DF-1/o-17 group (Wu et al., 2002a; Cutter et al., 2001). The three cultures from which polychlorinated biphenyl dechlorinators have been identified used similar strategies to 'wean' the microbes from the sediments. In this research, a sediment-free culture capable of dechlorinating 2,2',3,3',4,6'-hexachlorobiphenyl (PCB 132) and 2,2',3,4',6-pentachlorobiphenyl (PCB 91) was developed from the sediments of Lake Hartwell, South Carolina. The culture is designated the LH culture. The microorganisms in the LH culture responsible for dechlorinating the PCBs were identified as two Dehalococcoides strains.
Quantitative real time polymerase chain reaction showed that the number of Dehalococcoides present in the PCB amended treatment increased as PCB 132 was dechlorinated, while the numbers remained constant in the treatment without PCBs added. This indicated that PCBs were required for growth of Dehalococcoides. Quantitative real time polymerase chain reaction using reductive dehalogenase specific primers and probes for reductive dehalogenase genes showed that the ardA gene was present, but the tceA, bvcA, and vcrA genes were not. There were approximately half as many copies of the ardA gene as there were Dehalococcoides 16S ribosomal ribonucleic acid copies, indicating the presence of two organisms, one of which contained the ardA gene, while the other did not. Dehalococcoides specific primers were used to amplify and clone nearly complete 16S rRNA sequences from the LH second enrichment culture. The sequences were nearly identical. The LH 16S rRNA sequence most closely matched all known Dehalococcoides strains (>98%).
The LH culture was tested to determine if it would dechlorinate chlorinated benzenes and/or chlorinated ethenes. These two classes of compounds were chosen because they are known to be dechlorinated by various Dehalococcoides strains. The LH culture failed to dechlorinate any of the chlorinated compounds tested. This suggests that the two Dehalococcoides strains in the LH culture are novel strains; all of the previously identified Dehalococcoides strains dechlorinate one or more of the chlorinated compounds tested.
Previous research has shown that different electron donors can affect the rate of polychlorinated biphenyl dechlorination (Pulliam Holoman et al., 1998; Wu et al., 2000). Lactate, acetate, propionate, and hydrogen were provided as electron donors to determine their effects on the LH culture. Polychlorinated biphenyl dechlorination occurred with each electron donor. However, dechlorination was the greatest with hydrogen as the electron donor. This differs from two of the known polychlorinated biphenyl dechlorinators, o-17 and DF-1. It is possible that the LH dechlorinators utilized the hydrogen available in each of the treatments, while other organisms in the mixed culture utilized the lactate, propionate, and acetate.
Three microbial inhibitors, 2-bromoethanesulfonate, molybdate, and vancomycin, were tested to determine their effects on the LH culture. By selecting compounds that specifically inhibit certain types of microorganisms their role in the mixed culture can be inferred. 2-Bromoethanesulfonate inhibits methanogens. Molybdate inhibits sulfate reducing bacteria. Vancomycin inhibits the gram positive bacteria. BES and molybdate completely inhibited dechlorination in the LH culture but vancomycin did not, suggesting that methanogens and sulfate reducing bacteria provide growth factors to the dechlorinators.
A series of experiments was conducted to determine if the addition of a culture known to contain Dehalococcoides that chlororespire chlorinated ethenes could enhance polychlorinated biphenyl dechlorination in Lake Hartwell. Under the experimental conditions the commercial culture did not enhance dechlorination of the polychlorinated biphenyls and appears to be unable to dechlorinate PCB 132. The commercial culture used in these experiments contains strains BAV1, FL2, and CBDB1 (Duhamel et al., 2004). Considering that the commercial culture used contains three strains of Dehalococcoides, two of which have been shown to be present in a PCB dechlorinating culture, it is surprising that activity was not seen in these experiments.

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