Mechanistic insight into co-metabolic dechlorination of hexachloro-1,3-butadiene in Dehalococcoides

Hexachloro-1,3-butadiene (HCBD) as one of emerging persistent organic pollutants (POPs) poses potential risk to human health and ecosystems. Organohalide-respiring bacteria (OHRB)-mediated reductive dehalogenation represents a promising strategy to remediate HCBD-contaminated sites. Nonetheless, information on the HCBD-dechlorinating OHRB and their dechlorination pathways remain unknown. In this study, both in vivo and in vitro experiments, as well as quantum chemical calculation, were employed to successfully identify and characterize the reductive dechlorination of HCBD by Dehalococcoides. Results showed that some Dehalococcoides extensively dechlorinated HCBD to (E)-1,2,3-tri-CBD via (E)-1,1,2,3,4-penta-CBD and (Z,E)-1,2,3,4-tetra-CBD in a co-metabolic way. Both qPCR and 16S rRNA gene amplicon sequencing analyses suggested that the HCBD-dechlorinating Dehalococcoides coupled their cell growth with dechlorination of perchloroethene (PCE), rather than HCBD. The in vivo and in vitro ATPase assays indicated ≥78.89% decrease in ATPase activity upon HCBD addition, which suggested HCBD inhibition on ATPase-mediated energy harvest and provided rationality on the Dehalococcoides-mediated co-metabolic dechlorination of HCBD. Interestingly, dehalogenation screening of organohalides with the HCBD-dechlorinating enrichment cultures showed that debromination of bromodichloromethane (BDCM) was active in the in vitro RDase assays but non-active in the in vivo experiments. Further in vitro assays of hydrogenase activity suggested that significant inhibition of BDCM on the hydrogenase activity could block electron derivation from H₂ for consequent reduction of organohalides in the in vivo experiments. Therefore, our results provided unprecedented insight into metabolic, co-metabolic and RDase-active-only dehalogenation of varied organohalides by specific OHRB, which could guide future screening of OHRB for remediation of sites contaminated by HCBD and other POPs.