Multi-omics reveals mechanism of hydroxylamine-enhanced ultimate nitrogen removal in pilot-scale anaerobic/aerobic/anoxic system

Hydroxylamine (HA) dosing is an effective strategy for promoting partial nitrification (PN); however, its impact on endogenous denitrification remains underexplored. In this study, long-term continuous HA dosing (1.4 mg/L) was introduced for over 110 days in a pilot-scale anaerobic/aerobic/anoxic (AOA) system treating municipal wastewater (66.7-75 m3/d). The HA dosing significantly increased the nitrite accumulation ratio to 67.6 f 5.0 % (p<0.001) and reduced the effluent total inorganic nitrogen concentration from 6.2 f 2.0 to 2.4 f 1.1 mg/L (p<0.001), achieving a nitrogen removal efficiency of 87.4 f 4.5 % (p<0.001) at a hydraulic retention time of 8 h. During the HA dosing, aerobic nitrogen removal contribution increased from 2.4 f 3.4 % to 25.8 f 8.1 % (p<0.001), and the anoxic nitrogen removal rate improved from 1.63 f 0.11 to 2.35 f 0.13 mg N/(L & sdot;h) (p<0.001). Enhanced nitrogen removal was not only achieved through the rapid establishment of PN but also driven by the long-term impact of HA dosing on microbial community dynamics. Multi-omics analyses revealed that HA disrupted the polyphosphate (poly-P) cycle, evidenced by enhanced transcription of ppx (poly-P degradation) and suppressed ppk (poly-P synthesis), thereby reducing energy availability for phosphateaccumulating organisms (PAOs) and shifting the carbon source competition toward glycogen-accumulating organisms (GAOs), with Ca. Competibacter abundance increased from 0.16 % to 1.13 % (p < 0.001). The economic analysis demonstrated that HA reduced sludge production by 11.2 % and saved operating costs by 31.4-42.8 % compared to conventional carbon sources. These findings highlight the potential of HA dosing to achieve sustainable and highly efficient wastewater treatment.