Simulated Sidestream Nitrite Transfer Enables In Situ Anammox Biostimulation for Sustained NOB Suppression in Mainstream PN/A Systems

Mainstream partial nitritation–anammox (PN/A) is often hindered by incomplete suppression of nitrite-oxidizing bacteria (NOB). This study investigated a strategy to reinforce NOB control through controlled nitrite (NO 2 – –N) supplementation simulating sidestream NO 2 – –N transfer, with the aim of stimulating in situ anammox activity and regulate NO 2 – –N partitioning within biofilms. A laboratory-scale PN/A biofilm reactor was continuously operated for 300 days with periodic addition of NO 2 – –N. The ratio of NO 3 – –N production to NH 4 + –N removal (ΔNO 3 – –N/ΔNH 4 + –N) of the system declined from 0.49 ± 0.11 to 0.22 ± 0.03, while nitrogen removal efficiency (NRE) increased from 36.2 ± 11.3% to approximately 70%. Activity assays demonstrated that NO 2 – –N supplementation strengthened anammox competitiveness and shifted the NO 2 – –N utilization toward the anammox pathway, resulting in NOB suppression. Community analysis revealed a decline in Nitrospira abundance from 3.62% at day 60 to approximately 0.3% by days 250–300, accompanied by persistently low nxrAB gene abundance. Model-based results indicated that intermittent NO 2 – –N supplementation (approximately 1–5 days per month) can maintain anammox functional dominance, indicating that continuous NO 2 – –N input is unnecessary. Collectively, these findings demonstrate that chemically driven in situ biostimulation can regulate microbial competition without external biomass transfer, providing mechanistic insight and operational guidance for stabilizing mainstream PN/A systems.