Characterization of the Microbial Community Composition and Benzene Activation Mechanisms in Anaerobic Benzene-Degrading Enrichment Cultures

Benzene is a carcinogenic petroleum hydrocarbon that is prevalent and particularly persistent in anoxic aquifers. It can be biodegraded under anaerobic conditions; however, this process is relatively slow compared to aerobic conditions, and the microorganisms and the pathways involved therein remain poorly understood. The objectives of this thesis were to accelerate the microbially-mediated benzene degradation, to characterize benzene-metabolizing microbial communities, and to identify the biochemical mechanisms involved in anaerobic benzene activation. The growth rate and substrate range of the laboratory enrichment cultures were investigated. Conclusive evidence was also obtained to support that a Deltaproteobacterium sp. (ORM2) and a Peptococcaceae sp. are the microorganisms directly involved in benzene activation. The Deltaproteobacterium ORM2 was observed to dominate under methanogenic conditions while Peptococcacceaea sp. dominated only under nitrate-reducing conditions. Syntrophic partners in these cultures were also identified. Organisms belonging to Parcubacteria were identified in all analyzed cultures and observed to grow up to 15% of the total bacteria. This is the first time a member of this clade has been observed to grow in the laboratory. In nitrate-reducing cultures, transcripts related to a putative anaerobic benzene carboxylase (abcDA) were specific to benzene, supporting carboxylation as the benzene activation mechanism in these cultures. The benzene-activating genes and mechanism in the methanogenic cultures remain unknown. Methods to extract protein from the cultures for biochemical assays were developed, and lysis using a French pressure cell press yielded crude extracts with the highest biochemical activity. Assays were developed to test various enzymatic steps, including benzoyl-CoA ligation, benzene carboxylation and carbonylation. Benzoyl-CoA ligase activity was detected in nitrate-reducing, but not in methanogenic cultures, consistent with the absence of this gene in ORM2 genome. Further assay development is required for detecting benzene transformation; however, these first experiments have provided some guidance for the next steps in functional analysis.Ph.D