Compound-Specific Carbon, Nitrogen, and Hydrogen Isotope Analysis to Characterize Aerobic Biodegradation of 2,3-Dichloroaniline by a Mixed Enrichment Culture

Compound-specific isotope analysis (CSIA) is an established tool to track the in situ transformation of organic chemicals at contaminated sites. In this work, we evaluated the potential of multi-element CSIA to assess biodegradation of 2,3-dichloroaniline (2,3-DCA), which is a major industrial feedstock. Using controlled laboratory experiments, we determined, for the first time, negligible carbon (<0.5 parts per thousand) and hydrogen (<10 parts per thousand) isotope fractionation and a significant inverse nitrogen isotope fractionation (>10 parts per thousand) during aerobic 2,3-DCA biodegradation by a mixed enrichment culture. The tentative identification of a glutamate conjugate of 2,3-DCA as a reaction intermediate indicates that the initial multistep enzymatic reaction may be rate-limiting. The formation of the glutamate adduct would increase the bond energy at the N atom, thus likely explaining the observed inverse N isotope fractionation. The corresponding nitrogen enrichment factor was +6.8 +/- 0.6 parts per thousand. This value was applied to investigate the in situ 2,3-DCA biodegradation at a contaminated site where the carbon and nitrogen isotope signatures from field samples suggested similar aerobic processes by native microorganisms. Under the assumption of the applicability of the Rayleigh model in a pilot wetland treating contaminated groundwater, the extent of biodegradation was estimated to be up to 80-90%. This study proposes multi-element CSIA as a novel application to study 2,3-DCA fate in groundwater and surface water and provides insights into biodegradation pathways.