Date of Award

5-2012

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Environmental Engineering and Science

Advisor

Freedman, David L

Committee Member

Kurtz Jr., Harry D

Committee Member

Lee, Cindy

Abstract

In a previous study using microcosms from a site contaminated with γ- hexachlorocyclohexane (γ-HCH), rapid anaerobic biodegradation of γ-HCH to benzene and chlorobenzene was observed. Enrichment cultures were developed and subsequently transferred to an anaerobic sulfate-free HEPES buffered medium without loss of γ-HCH dechlorination, thus suggesting γ-HCH undergoes anaerobic biodegradation via chlororespiration. The overall objective of this thesis was to further characterize the enrichment culture.
To do so, one specific objective was to determine the effect of vancomycin on γ-HCH in an enrichment culture grown in a sulfate-free HEPES buffered medium using hydrogen as the electron donor. This objective would help to gain insight into the type of microbe responsible for the dechlorination of γ-HCH to benzene and chlorobenzene. The other specific objective was to measure the yield of the HEPES-buffered enrichment culture by monitoring the increase in 16S rRNA gene copies for Bacteria versus hydrogen consumption and dechlorination of γ-HCH. An increase in biomass in treatments with γ-HCH and no observed increase in biomass in treatments without γ-HCH would establish that the growth of the culture was directly linked to the anaerobic dechlorination of γ-HCH.
The addition of 100 mg/L of vancomycin had a strong effect on γ-HCH dechlorination. In treatments containing vancomycin the total benzene and chlorobenzene formed ranged from 0.15-1.47 μmol/bottle as opposed to the treatments not containing vancomycin in which total benzene and chlorobenzene formed ranged from 25.0-31.0 μmol/bottle over the 125 day incubation period. The results with vancomycin further demonstrate that the microbe responsible for γ-HCH reduction is likely to be non-spore forming and gram positive.
Due to the formation of organic acids, including propionate and formate, as well as no clear distinction of total growth between treatments with and without γ-HCH reduction, it was not possible to determine a cell yield using a universal Bacteria primer. However, in the treatments that were inoculated and hydrogen was consumed there was a positive correlation between gene copies per mL and hydrogen consumed. It is recommended that additional research be completed to determine the specific microbe responsible for the dechlorination of γ-HCH and then a more specific primer be used to measure growth by qPCR.

Share

COinS