Developing Protocols to Facilitate the Enrichment and Characterization of Hydrocarbon-degrading Anaerobic Microbial Communities.

摘要

This thesis investigates the use of density centrifugation with Percoll to separate and enrich the organisms involved in the first step of toluene degradation within a methanogenic toluene degrading consortium. Protocol development resulted in the enrichment of bacteria and archaea in separate layers. However the separation of Eub-1 (an organism suspected to be responsible for the first step in toluene degradation), and bssA (a gene encoding the benzylsuccinate synthase enzyme) using previously developed qPCR primers could not be established. Cloning and sequencing of the toluene degrading consortia were conducted and phylogenetic analysis showed a change in community composition from what had previously been observed, suggesting why previously established primers were not effective. In parallel with these studies, microcosms using soil obtained from a petroleum hydrocarbon contaminated area in North Carolina were constructed. These microcosms showed benzene degradation in all but one sample over the 444 day period.