Dual Carbon-Chlorine Isotope Analysis Indicates Distinct Anaerobic Dichloromethane Degradation Pathways in Two Members of Peptococcaceae

Abstract

Dichloromethane (DCM) is a probable human carcinogen and frequent groundwater contaminant and contributes to stratospheric ozone layer depletion. DCM is degraded by aerobes harboring glutathione-dependent DCM dehalogenases; however, DCM contamination occurs in oxygen-deprived environments, and much less is known about anaerobic DCM metabolism. Some members of the Peptococcaceae family convert DCM to environmentally benign products including acetate, formate, hydrogen (H₂), and inorganic chloride under strictly anoxic conditions. The current study applied stable carbon and chlorine isotope fractionation measurements to the axenic culture Dehalobacterium formicoaceticum and to the consortium RM comprising DCM degrader Candidatus Dichloromethanomonas elyunquensis. Degradation-associated carbon and chlorine isotope enrichment factors (ε_C and ε_Cl) of -42.4 ± 0.7‰ and -5.3 ± 0.1‰, respectively, were measured in D. formicoaceticum cultures. A similar ε_Cl of -5.2 ± 0.1‰, but a substantially lower ε_C of -18.3 ± 0.2‰, were determined for Ca. Dichloromethanomonas elyunquensis. The ε_C and ε_Cl values resulted in distinctly different dual element C-Cl isotope correlations (Λ^C/Cl = Δδ¹³C/Δδ³⁷Cl) of 7.89 ± 0.12 and 3.40 ± 0.03 for D. formicoaceticum and Ca. Dichloromethanomonas elyunquensis, respectively. The distinct Λ^C/Cl values obtained for the two cultures imply mechanistically distinct C-Cl bond cleavage reactions, suggesting that members of Peptococcaceae employ different pathways to metabolize DCM. These findings emphasize the utility of dual carbon-chlorine isotope analysis to pinpoint DCM degradation mechanisms and to provide an additional line of evidence that detoxification is occurring at DCM-contaminated sites.