Doctor of Philosophy (PhD)


Civil and Environmental Engineering

Document Type



In support of an effort to determine the feasibility of using an in-situ bioremediation strategy for cleanup of groundwater in an area containing chlorinated solvents present as a dense non-aqueous phase liquid (DNAPL), the bacterial population in the groundwater at a Superfund site located near Baton Rouge, Louisiana was characterized. More than 3¡Á107 cells/mL in the groundwater were observed via microscopy. Universal bacterial and ¡°Dehalococcoides¡±-specific 16S rRNA gene libraries were constructed and analyzed. Universal clones grouped into 18 operational taxonomic units (OTUs), as defined by sequence similarity ¡Ý97.0%, which included several as yet undescribed organisms. Multiple unique sequences closely related to ¡°Dehalococcoides ethenogenes¡± were detected. Sequences of 168 anaerobically grown isolates grouped into 18 OTUs, of which only three were represented in the clone library. Collectively, these results revealed that large numbers of novel microorganisms are present in groundwater within the DNAPL source zone, including bacteria closely related to known dechlorinators, fermentors, and hydrogen producers. This suggests that the population contains the bacterial components necessary to carry out reductive dechlorination. To further characterize the functional role that they may play in chlorinated solvent biotransformation, representatives from nine isolate OTUs and nine Clostridium type strains were tested to determine their ability to fermentatively produce hydrogen in the presence of 1,2-dichloroethane (DCA), 1,1,2-trichloroethane (TCA), and tetrachloroethene (PCE). All of the strains produced hydrogen in the presence of at least 7.4 mM DCA, 2.4 mM TCA, and 0.31 mM PCE. These results demonstrated that many Clostridium species are tolerant of chlorinated solvents and can produce hydrogen even in the presence of high concentrations of this class of contaminants. This suggests that Clostridum species may functionally support chlorinated solvent bioremediation through intra-species hydrogen transfer. Four isolates were characterized using a polyphasic approach to establish their taxonomic status. Three of the strains were phylogenetically and phenotypically identical. Phenotypic and chemotaxonomic properties differentiated these isolates from their closest phylogenetic relative. Phylogenetic analysis of the fourth strain revealed it was divergent from other Clostridium species. Based on these results, two new Clostridium species are proposed with names of Clostridium hydrogeniformans sp. nov. and Clostridium cavendishii sp. nov.



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Committee Chair

William Moe