Nitrite Stress Shapes Ecological Shifts from Generalists to Specialists in Denitrifying Phosphorus Removal Wastewater Treatment Systems

Abstract Denitrifying phosphorus removal (DPR) systems in wastewater treatment often face challenges from nitrite accumulation, which acts as both an electron acceptor and a metabolic inhibitor. The stability of these systems depends on the microbial community’s composition and adaptability, which can be disrupted under nitrite stress. Here, we distinguish generalist taxa with broad niches from specialist taxa with narrow but critical functions to understand how nitrite stress drives ecological transitions that impact nutrient removal. We analyzed DPR microbiomes under gradient nitrite stress (10, 20, and 25 mg/L) over 120 operational cycles, using metagenomics. At low nitrite (10 mg/L), phosphorus (TP: 92.67 ± 5.72%) removal remained stable, but high nitrite (25 mg/L) impaired phosphorus removal (67.21 ± 13.86%) after 60 cycles. Increasing the nitrite loading markedly suppressed DPR-associated carbon and phosphorus transformations. Under high nitrite stress (25 mg/L), phosphorus release and uptake declined by 3.32-fold and 6.12-fold, respectively. Glycogen consumption and accumulation reduced by 2.18-fold and 2.38-fold, respectively. PHB consumption exhibited a 1.44-fold decrease. Nitrite stress reduced community diversity but increased the niche breadth. Ecological niche analysis identified 16 generalists and 164 specialists, with high nitrite increasing specialist abundance by 30.77% while generalists declined by 25.77%. Evolutionary modeling suggested a shift from generalists to specialists under nitrite stress (tg → s = 11.28). Notably, Thauera aminoaromatica, carrying complete nitrogen and phosphorus pathways, increased significantly under high nitrite stress. These results reveal how nitrite stress drives microbial community transitions and associated functional shifts in DPR systems, providing mechanistic insights into guide strategies for maintaining nutrient removal performance.