Mechanistic insights into sulfur-driven denitrifying phosphorus removal: Roles of zero-valent sulfur and Thiothrix in...

Denitrifying sulfur conversion-associated enhanced biological phosphorus removal (DS-EBPR) has emerged as a promising strategy for integrated nutrient removal; however, the sulfur-driven mechanisms and functional microorganisms governing phosphorus removal remain poorly understood. In this study, a DS-EBPR system was established and operated long-term to elucidate the coupling mechanisms among carbon, sulfur, nitrogen, and phosphorus transformations. Stable and efficient simultaneous removal of multiple nutrients was achieved, demonstrating robust process performance. Sulfur speciation analyses identified zero-valent sulfur (ZVS) as the dominant sulfur intermediate, dynamically coupled with anaerobic phosphate release and subsequent anoxic denitrification and phosphate uptake. XPS and TEM-EDS further verified intracellular elemental sulfur storage in the anaerobic phase and its depletion during the anoxic phase concurrent with polyphosphate accumulation. Genome-resolved multi-omics revealed that intracellular ZVS was generated from sulfide oxidation by sulfur-oxidizing bacteria, among which Thiothrix exhibited particularly high transcriptional activity, consistent with the absence of detectable sulfide during the anaerobic phase. Thiothrix was identified as the primary sulfur-driven denitrifying phosphorus-removing microorganism, supported by the active expression of genes involved in sulfur oxidation, nitrate reduction, and polyphosphate metabolism. Electron- and energy-equivalent accounting further indicated that intracellular ZVS oxidation constituted a major internal source of reducing power during the anoxic phase, supporting denitrification and providing energy for anoxic phosphate uptake. These results demonstrate that sulfur-metabolizing microorganisms utilize intracellular ZVS as an electron and energy carrier to drive denitrification and phosphorus removal, providing a mechanistic basis for energy-efficient treatment of low-carbon wastewater.