Immobilized anthraquinone-2-sulfonate enhances hydroxylamine-induced partial nitrification-endogenous denitrification and phosphorus removal: Electron-transfer and multi-omics mechanisms.

Hydroxylamine-driven partial nitrification lowers oxygen and carbon demand, but the resulting nitrite accumulation and free nitrous acid (FNA) stress constrain endogenous denitrification (EnD) and denitrifying phosphorus removal (DPR). Here, immobilized anthraquinone-2-sulfonate (AQS) was used to overcome this electron-transfer bottleneck in three SPNEDPR biofilm reactors containing unmodified carriers or AQS-modified carriers at 0.29 and 0.51 mmol/L. At an influent C/N ratio of 3.05 ± 0.11, 0.51 mmol/L AQS achieved stable removal efficiencies of 98.99 ± 1.28% for total inorganic nitrogen (TIN), 94.96 ± 2.42% for total phosphorus (TP), and 86.19 ± 2.13% for chemical oxygen demand (COD). Relative to the control, AQS increased electron transfer system activity by 93.16 ± 7.36% and enhanced ammonia oxidation, nitrogen removal, and phosphate uptake rates by 86.89%, 108.11%, and 186.21%, respectively. The mediator also accelerated polyhydroxyalkanoate consumption and glycogen cycling, thereby converting intracellular carbon reserves into reducing equivalents. Multi-omics analyses revealed a distinctive mechanism: AQS primarily activated existing metabolic potential at the transcriptional level rather than broadly increasing functional-gene abundance. Upregulation of Complexes Ⅱ/Ⅲ, ubiquinone biosynthesis, ATP synthase, and carbon-nitrogen-phosphorus cycling genes strengthened electron delivery to denitrifying reductases and proton motive force-driven ATP regeneration. Nitrosomonas europaea supplied nitrite, whereas Candidatus Accumulibacter clade ⅡF and Dechloromonas phosphprivorans drove nitrite-based DPR within a functionally redundant EnD-DPR consortium. These coupled responses reduced nitrite accumulation, alleviated FNA inhibition, and sustained simultaneous nitrogen and phosphorus conversion in aerobic biofilm microzones and the subsequent anoxic stage. Overall, immobilized AQS enhanced SPNEDPR through coordinated intracellular carbon mobilization, electron-transfer acceleration, bioenergetic regeneration, and microbial cooperation.