Anaerobic Biodegradation of Chlorinated Benzenes and Hexachlorocyclohexane by Mixed Microbial Cultures Derived from Contaminated Field Sites

Environmental contamination with toxic organohalides is of worldwide concern. Chlorinated ethenes, benzenes, and pesticides are organohalides frequently found in contaminated soil and groundwater. Bioaugmentation with mixed microbial cultures derived from contaminated sites has been successfully applied to remediate groundwater contaminated with chlorinated ethenes and ethanes, in which the halogenated compounds are electron acceptors in microbial respiration. On the other hand, in situ bioremediation of soil, sediment, and groundwater contaminated with chlorinated benzenes and the pesticide γ-hexachlorocyclohexane (lindane) has yet to be comprehensively explored. Hexachlorocyclohexane has been reported to dechlorinate into a mixture of chlorinated benzenes, so the bioremediation of these two types of organohalides is highly interrelated. In this thesis, the organohalide bioremediation potential of previously established and novel anaerobic mixed cultures was investigated using analytical and molecular omics techniques. In the commercial chlorinated ethene-to-ethene dechlorinating culture KB-1, cobalamin (vitamin B12), an essential corrinoid cofactor for the reductive dehalogenation of vinyl chloride, was determined to be supplied to organohalide-respiring Dehalococcoides by a growing Acetobacterium population. After switching the electron acceptor from trichloroethene to 1,2,4-trichlorobenzene, native KB-1 Dehalobacter populations that respire chlorinated benzenes were identified along with a KB-1 reductive dehalogenase enzyme that dechlorinates 1,2,4-trichlorobenzene to 1,3- and 1,4-dichlorobenzene. In addition, the metagenome-assembled genomes of three distinct KB-1 Dehalobacter spp. were shown to contain more than 70 reductive dehalogenase genes (rdh) which considerably increases the number of known KB-1 rdh genes and will allow for the examination of evolutionary relationships in Dehalobacter genomes. A Dehalobacter-containing culture (referred to as GT), enriched from soils and sediments contaminated with hexachlorocyclohexane, was shown to dechlorinate lindane to monochlorobenzene and benzene, and was found to contain a Dehalobacter population that use γ-hexachlorocyclohexane as terminal electron acceptor. A GT culture was combined with a KB-1 subculture that dechlorinates monochlorobenzene to benzene, and a benzene-degrading culture, to achieve complete biotransformation of lindane to non-toxic end products. Using metagenomics and proteomics data, a novel reductive dehalogenase enzyme, HchA, was for the first time identified. Altogether, this body of work demonstrates that the mixed cultures KB-1 and GT have bioremediation potential against the chlorinated benzenes and hexachlorocyclohexane, respectively.Ph.D