Use of Compound-specific Isotope Analysis to Investigate Enzymatic Reaction Mechanisms

To maximize the efficiency of biodegradation in groundwater at contaminated sites, it is imperative to understand biodegradation reaction mechanisms. Compound-specific isotope analysis (CSIA) is a powerful approach that provides novel insights into biodegradation reaction mechanisms, enzyme kinetics and bioremediation potential and efficiency. This thesis aims to extend the discipline of CSIA in several new directions including its use as a means for probing reaction mechanisms, and for examining enzyme kinetics and biodegradation efficiency for halogenated alkanes. This thesis expands the application of CSIA to a new class of compounds, chlorofluorocarbons (CFCs). Significant carbon isotope fractionation was observed during biotransformation of CFC- 113 and CFC-11, and experimental findings applied to investigation of a contaminated field site. Carbon isotope fractionation of CFC-11 in groundwater suggested up to 86% transformation was occurring. Science and public attention remain focused on CFCs, due to recently reported unexplained source inputs to the atmosphere, and the potential for CFC biotransformation in surface and groundwaters remains unclear. Further biotransformation experiments integrated chlorine isotope with carbon isotope analysis for 1,1,1-trichloroethane (1,1,1- TCA) and 1,1-dichloroethane (1,1-DCA). Carbon isotope effects for 1,1,1-TCA and 1,1-DCA biotransformation are suppressed while ΛCl/C are consistent, suggesting a non-fractionating masking effect is present in 1,1,1-TCA and 1,1-DCA biotransformation. In contrast CF biotransformation by the enriched culture SC05 produced highly suppressed carbon and chlorine isotope effects, resulting in significantly different ΛCl/C values. These results are inconsistent with a non-fractionating masking effect and indicate differences in reaction mechanisms or an isotopically fractionating masking effect. The observational data and insights into underlying controls on isotopic fractionation observed for the experiments and field applications herein emphasize the need to understand mechanistic and kinetic controls on carbon and chlorine isotope effects. Studying RDases using CSIA has implications for understanding the role of reductive dehalogenation in the global halogen cycle that influences environmental contamination, atmospheric chemistry and, in turn, climate.

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