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). Herein, this study investigated a practical strategy to reinforce NOB control through simulated sidestream nitrite (NO2-N) transfer via controlled NO2-N supplementation, aiming to stimulate in situ anammox activity and regulate NO2-N partitioning within the biofilm. A laboratory-scale PN/A biofilm reactor was continuously operated for 300 days with controlled NO2-N addition. The ratio of NO3-N production to NH4-N removal (Delta NO3-N/Delta NH4-N) of system declined from 0.49+/-0.11 to 0.22+/-0.03, while nitrogen removal efficiency (NRE) increased from 36.15+/-11.33% to approximately 70%. Activity assays demonstrated that NO2-N supplementation strengthened anammox competitiveness and shifted NO2-N utilization toward the anammox pathway, resulting in effective NOB suppression. Community analysis further revealed a pronounced 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 analysis further indicated that intermittent NO2-N supplementation (approximately 1-5 days per month) is sufficient to sustain anammox functional dominance, indicating that continuous input is unnecessary. Collectively, these findings establish a chemically driven in situ biostimulation strategy that enables sustained NOB control without external biomass transfer, providing mechanistic insight and operational guidance for stabilizing mainstream PN/A systems.