Excessive biomass retention decouples ammonia-oxidizer abundance and activity in sidestream partial nitritation

Partial nitritation (PN) underpins two-stage anaerobic ammonium oxidation-based nitrogen removal, but biomass management in sidestream PN reactors remains largely empirical. This study tested three PN sequencing batch reactors maintained at contrasting biomass-retention states (2, 4, and 6 g L^-1 MLSS) under stepwise loading. Among the tested conditions, the intermediate-biomass reactor showed the most favorable performance, shortening recovery periods by 40.0-67.0% and reaching the highest apparent stable nitrogen loading rate of 2.30 g N L^-1 d^-1. Metagenomic analysis linked this response to more deterministic community assembly and stronger species-level dominance of Nitrosomonas stercoris, whereas the low- and high-biomass states showed greater stochasticity and enrichment of heterotrophic or stress-tolerant taxa. Metatranscriptomics further showed that the intermediate-biomass state maintained the strongest ammonia oxidizing bacteria (AOB)-centred transcriptional configuration, together with peak expression of amoABC and hao, and higher AMO and HAO levels. This state was also associated with higher expression of Calvin-Benson-Bassham cycle and electron transport genes, indicating stronger coupling among ammonia oxidation, energy metabolism, and autotrophic carbon fixation. By contrast, the high-biomass reactor retained nitrifier-related potential, but this potential was not converted into proportional transcriptional or functional output. It showed weakened AOB-centered dominance and a broader reduction in chemolithoautotrophic functional expression. These findings reveal abundance-activity decoupling under excessive biomass retention and show that PN control should move beyond biomass concentration alone. A more informative operational perspective is the loading-to-biomass state, which helps indicate whether retained biomass is translated into active nitritation function.