Identification of Novel Biomarkers from Supportive Microorganisms in TCE-dechlorinating Microbial Communities

Chlorinated ethenes are toxic and carcinogenic compounds that have contaminated a large quantity of groundwater in the U.S. and other developed countries. In order to protect public health, in situ bioremediation via dehalorespiration by Dehalococcoides bacteria is a promising solution. The overall goal of this research is to understand the ecological relationship between Dehalococcoides mccartyi (Dhc) and supportive microorganisms in a community and identify novel biomarkers for monitoring TCE dechlorination activities during bioremediation. To accomplish these goals, traditional molecular and high throughput microarray techniques, as well as newly established analytical approaches were applied./nThe first objective of this research was to characterize four Dhc-containing microbial communities enriched from contaminated groundwater under different cobalamin stress and methanogenic conditions. Microarray analyses targeting four Dhc genomes revealed a commonly shared core Dhc genome most similar to strain 195. Physiological characterization revealed that inhibited methanogensis optimized the dechlorination performance. Experimental evidence demonstrated the presence of Bacterial species providing corrinoids to Dhc. The dominance of closely related Pelosinus spp., Dendrosporobacter spp. and Sporotalea spp. and the significant effects of cobalamin addition and methanogenic inhibition on the distribution of Clostridium spp. in the communities suggest that species in these genera are potential corrinoid providers to Dhc./nIn order to further target corrinoid production in enrichments without exogenous cobalamin, a detection method was established that successfully differentiates various corrinoid and lower ligand forms. Corrinoid and lower ligand profiles of different Dhc-containing enrichments indicate that cobalamin was the major corrinoid utilized by Dhc. Further evidence demonstrated that in enrichments without exogenous cobalamin, p-cresolylcobamide was produced, likely by Pelosinus spp., and then modified by Dhc into cobalamin in the presence of dimethylbenzimidazole (DMB) to support dechlorination reactions./nDifferential gene expression between enrichments with and without exogenous cobalamin was investigated using microarrays targeting four sequenced Dhc genomes. Results suggest that cobT and btuF genes, the Nuo and Hym operons and the tryptophan operon could serve as biomarkers indicating cobalamin stress and corrinoid salvaging. Temporal global gene expression of the enrichment without exogenous cobalamin corroborates the importance of hydrogenases at an early stage of dechlorination and the close relation between TceA reductive dehalogenase and Hup hydrogenase./nBased on the knowledge obtained in the previous studies, defined consortia were constructed by growing Dhc strain 195 with potential supportive microorganisms: Desulfovibrio vulgaris Hildenborough, Methanobacterium congolense, and Pelosinus fermentans strain R7. Physiological, transcriptomic and proteomic analyses of strain 195 containing consortia provide strong evidence supporting the previous hypotheses that Desulfovibrio spp. play important roles in hydrogen transfer, Pelosinus spp. provide corrinoid forms to Dhc but not DMB, while methanogens do not contribute to Dhc cell growth or biological corrinoid generation in the presence of the other two species./nThe significance of this research is that supportive microorganisms and related chemical and molecular biomarkers have been identified. And they may be useful for optimizing bioremediation processes by obtaining accurate feedback information from cells that are indicative of Dhc physiology. Knowledge developed in this research will aid practitioners to better design, monitor and optimize future in situ bioremediation systems.

Advisor: Alvarez-Cohen, Lisa