Effect of Metabolic Constraints on the Observable Isotope Fractionation Associated with Aerobic Biodegradation of Hexachlorocyclohexanes

Biodegradation of the highly persistent hexachlorocyclohexanes is difficult to assess in contaminated soils and sediments because this process occurs only slowly over timescales of years to decades. Recent instrumental advances for compound-specific isotope analysis (CSIA) now make it possible to monitor such processes based on the isotope fractionation of multiple elements as changes of 13C/12C, 37Cl/35Cl, and 2H/1H ratios in the residual HCH contamination. However, metabolic constraints from the expression of enzymes that can compete with the primary metabolic reactions leading to HCH biodegradation and thus alter the observable contaminant isotope fractionation through co metabolic side reactions have largely been overlooked. Here, we developed activity-based assays to assess the competitive behaviour of mixtures of lindane dehydrochlorinase LinA and haloalkane dehydrochlorinase LinB which catalyze the dehydrochlorination and hydrolytic dechlorination of several HCH isomers. Using X-HCH as model contaminant that can be transformed by both enzymes in mixtures of different LinA2/LinB activity, we observed preferential formation of products from hydrolytic dechlorination. This observation suggests that LinB was more reactive than predicted from the nominal enzyme activities. The C and H isotope fractionation of X-HCH in LinA2/LinB mixtures can be rationalized by a combination of isotope enrichment factors from independent dehydrochlorination and hydrolytic dechlorination reactions where LinA2 contributed more to H isotope fractionation than LinB, thus contrasting the assessment of competitive enzyme activity. Our study shows that metabolic constraints associated with the expression of multiple enzymes can potentially compromise inferences of extent and pathways of contaminant biodegradation from CSIA.