Multi-Isotope Analysis as a Natural Reaction Probe of Biodegradation Mechanisms of 1,2- Dichloroethane

1,2-Dichloroethane (1,2-DCA), a chlorinated aliphatic hydrocarbon, is an EPA priority pollutant and a widespread groundwater contaminant. Stable isotope fractionation during biodegradation of 1,2-DCA occurs due to differences in the reaction rates of heavy versus light atoms present at a reacting bond in the 1,2-DCA molecule. In general, light isotopic bonds react more quickly, producing a relative enrichment in the heavy isotope in the remaining contaminant pool. Compound specific isotope analysis has the potential to demonstrate the occurrence and extent of biodegradation at chlorinated solvent contaminated groundwater sites. In this study, stable carbon isotope fractionation was used as a novel reaction probe to provide information about the mechanism of 1,2-DCA biodegradation. Isotopic fractionation was measured during 1,2-DCA degradation by a microbial culture capable of degrading 1,2-DCA under O2-reducing and NO3-reducing conditions. The microbial culture produced isotopic enrichment values that are not only large and reproducible, but are the same whether O2 or NO3 was used as an electron acceptor. The mean isotopic enrichment value of -25.8 permil measured for the microbial culture during 1,2-DCA degradation under both O2 and NO3- reducing conditions can be converted into a kinetic isotope effect (KIE) value to relate the observed isotopic fractionation to the mechanism of degradation. This KIE value (1.05) is consistent with degradation via a hydrolysis (SN2) reaction under both electron-accepting conditions. Isotope analysis was able to provide a first line of evidence for the reaction mechanism of 1,2-DCA biodegradation by the microbial culture. Using a multi-isotope approach incorporating both carbon and hydrogen isotopic data, compound specific isotope analysis also has the potential to determine degradation mechanisms for 1,2-DCA under aerobic conditions where 1,2-DCA is known to be degraded by two distinct enzymatic pathways. Biodegradation of 1,2-DCA via a hydrolysis (SN2) reaction produces a large carbon isotope enrichment (isotopic enrichment factor = -29.2 permil) while oxidation of 1,2-DCA via a monooxygenase enzyme produces a small carbon isotope enrichment (isotopic enrichment factor = -3.9 permil). Conversely, because a hydrogen bond is not broken in the hydrolysis (SN2) reaction, only a small secondary hydrogen isotope enrichment is expected, while a large hydrogen isotope enrichment is expected during oxidation of 1,2-DCA via a monooxygenase enzyme. The complementary information of both carbon and hydrogen isotopic data may be instrumental in identifying the mechanism of biodegradation in the subsurface, and illustrates the potential of compound specific isotope analysis as a natural reaction probe to provide insight into the enzymatic mechanism of subsurface contaminant degradation.