Multi-omics reveals thiosulfate-driven sulfur-carbon coupling and cross-feeding that enhance anaerobic dechlorination of chlorinated organophosphate esters in sewage sludge.

Sulfur cycling is closely linked to methane metabolism, nutrient removal, and iron cycling, yet its role in microbial dechlorination remains poorly understood, particularly in sludge anaerobic fermentation systems. Here we developed a thiosulfate-assisted biostimulation strategy and used tris(2-chloroethyl) phosphate (TCEP), a representative chlorinated organophosphate ester to investigate how sulfur cycling affects anaerobic dechlorination. Results showed that 1000 mg/L thiosulfate supplementation increased TCEP degradation from 26.7% to 59.6% within 20 days. Transformation product analysis indicated that TCEP underwent concurrent reductive and hydrolytic transformations, yielding TEP via reductive dechlorination, and BCEP, MCEP, and BCEP-OH through stepwise hydrolysis. The estimated contributions of hydrolysis and reductive dechlorination shifted from 58.5% and 28.4% in the control to 29.7% and 46.3% in S1000, respectively, indicating that thiosulfate preferentially promoted reductive dechlorination. Model substrate assays showed that thiosulfate enhanced acetate production (+44.3%) and reducing equivalent levels (NADH, +24.6%; FADH2, +29.4%), aligning with enhanced flavin-based electron bifurcation and energy generation. Metagenomic analyses revealed thiosulfate enrichment of sulfur-metabolizing (e.g., MAG180), fermentative (e.g., MAG1), and Dehalococcoides-related lineage (e.g., MAG190), forming a potential cross-feeding organohalide-respiring consortium. Proteomic analyses further revealed a protein network involving sulfur metabolism (e.g., soxY, cysD, and cysH), acetate generation (e.g., por), electron transfer (e.g., fixAB), and dehalogenation-related proteins (e.g., 2-haloacid dehalogenase). This work provides a mechanistic basis for sulfur-assisted biostimulation of chlorinated organic pollutant removal in complex anaerobic systems.