High-Throughput Screening of Microbial Reductive Dechlorination of Polychlorinated Biphenyls: Patterns in Reactivity and Pathways

Polychlorinated biphenyls (PCBs) are pervasive pollutants that pose risks to ecosystems and human health. Microbial reductive dehalogenation plays crucial roles in attenuating PCBs, but comprehensive insights into PCB dechlorination pathways, reactivity, and governing factors are limited by the vast number of congeners and costly experimental approaches. We address this challenge by establishing a high-throughput <i>in vitro</i> assay approach of reductive dehalogenation (HINVARD), which increases dechlorination test throughput by 30-fold and enhances reagents and cell utilization efficiency by over 10-fold compared to conventional assay methods. Using HINVARD, we screened 61 PCB congeners across 9 enrichment cultures and 3 <i>Dehalococcoides</i> isolates, identifying active dechlorination of 31-44 congeners. Results showed that PCB congener properties (chlorine substitution patterns, steric hindrance, and solubility) primarily determine the dechlorination potential, leading to consistent reactivity trends across cultures. In contrast, different organohalide-respiring bacteria catalyzed distinct dechlorination pathways, preferentially removing <i>para</i>- or <i>meta</i>-chlorines. Structural modeling of reductive dehalogenases revealed unique binding orientations governing substrate specificity, offering molecular insights into these pathways. This study provides a high-efficiency strategy for investigating microbial reductive dehalogenation, yielding the first comprehensive understanding of PCB dechlorination patterns and mechanisms. These findings guide the design of tailored microbial consortia for effective PCB bioremediation.