Sulfur-driven hidden carbon flux enables reductive dechlorinating in sulfur-metabolism biofilm

The co-occurrence of nitrate and chlorinated phenols in industrial wastewater presents a significant challenge for simultaneous removal under carbon-limited conditions. This study evaluates a sulfur-metabolism biofilm reactor for the concurrent removal of nitrate and 2,4,6-trichlorophenol (2,4,6-TCP) without external organic carbon as electron source. A 180-day continuous-flow operation demonstrated efficient nitrate reduction (>90%) and effective 2,4,6-TCP dichlorination (∼80%) to 2,4-dichlorophenol (2,4-DCP) and 4-chlorophenol (4-CP). Batch experiments revealed that increasing nitrate concentrations enhanced 2,4,6-TCP dechlorination rates. 16S rRNA gene sequencing and qPCR indicated the enrichment of sulfur-oxidizing bacteria (e.g., Thiobacillus) and potential dechlorinators (e.g., Chloroflexota), along with elevated levels of dehalogenation gene (cprA). Metagenomic analysis suggested that sulfur oxidizers mediate CO₂ fixation and downstream central carbon metabolism. This process may generate low-molecular-weight metabolites such as formate and butyrate, which could serve as electron donors for potential dechlorinators (Chloroflexota). Follow-up nitrate-resupplemented batch tests confirmed that formate and butyrate accumulation was nitrate-dependent and declined upon 2,4,6-TCP addition, indicating their consumption during reductive dechlorination. These findings revealed a syntrophic linkage between autotrophic denitrifiers and organohalide-respiring bacteria, thereby facilitating simultaneous nitrogen elimination and transformation of recalcitrant organics under carbon-limited conditions. This study highlights a promising strategy for sustainable treatment of mixed contaminants via sulfur-metabolism biofilm.