Deciphering nitrogen and sulfur metabolic pathways in thiosulfate-driven denitrification coupled with Anammox for enhanced nitrogen removal

The integration of thiosulfate-driven denitrification with anaerobic ammonium oxidation (Anammox) presents a sustainable approach for nitrogen removal in wastewater treatment. However, the microbial interactions and underlying mechanisms within this system remain unclear. Here, a thiosulfate-driven denitrification coupled with Anammox (TDDA) system was successfully established, achieving a nitrogen removal rate of 190.5 mg N/L/d, with near-complete nitrogen (99.3 %) and thiosulfate (100 %) removal. Batch tests showed that nitrate enhanced thiosulfate oxidation and that anammox had higher competitiveness for nitrite utilization than thiosulfate-driven denitrification, with the combined system showing improved nitrogen removal. This interaction effectively enhanced overall nitrogen removal efficiency in the TDDA system. Metagenomic and metatranscriptomic analyses revealed that branched thiosulfate oxidation was the dominant thiosulfate oxidation pathway, with elemental sulfur as an intermediate. Simultaneously, Candidatus Jettenia caeni and Candidatus Brocadia sapporoensis performed nitric oxide-dependent Anammox and hydroxylamine-dependent Anammox, respectively. Overall, this study elucidated the microbial interactions and metabolic pathways involved thiosulfate-mediated nitrogen-sulfur biotransformation. The findings provided valuable insights for optimizing TDDA process and advancing sustainable, low-carbon wastewater treatment technologies.