Mitigating nitrite stress and restoring functional redundancy in anammox reactor via acetate-driven DNRA-anammox...

Frequent fluctuations in nitrite concentrations and unstable control of partial nitritation often lead to excessive NO₂^- accumulation, resulting in performance deterioration in anammox-based systems. To address this challenge, an anammox reactor was operated for 180 days to investigate the inhibitory effects of elevated NO₂^-/NH₄⁺ratios on anammox activity and to evaluate the effectiveness of external carbon supplementation in promoting dissimilatory nitrate reduction to ammonium (DNRA)-related pathways that contribute to NO₂^- reduction. Increasing NO₂^-/NH₄⁺ratio from 1.3 to 3.0 decreased the nitrogen removal efficiency from 96.7% to 26.6%, reduced the relative abundance of anammox bacteria (Ca. Kuenenia and Ca. Jettenia) from 41.5% to 7.0% and promoted Nitrospira to 7.7%. In contrast, acetate supplementation at a C/N ratio of 0.2 suppressed Nitrospira to 0.2% and enhanced the abundance of anammox and DNRA-performing bacteria (e.g., Fimbriimonadaceae, Mycobacterium, Anaerolineales, Caldilineaceae, and Ignavibacteriaceae) to 31.2% and 15.7%, respectively. Metagenome-assembled genome analysis confirmed the enrichment of functional genes associated with anammox (hzsABC and hdh) and DNRA metabolism (nirBD and nrfAH), corresponding to the recovery of nitrogen removal efficiency to 82.3%. Quantitative microbial network analysis further revealed that functional redundancy index declined from 0.56 to 0.42 under nitrite stress but recovered to 0.53 following acetate supplementation, indicating the restoration of a functionally buffered microbial community. Overall, these results demonstrate that low-level acetate supplementation (C/N = 0.2) effectively stimulated DNRA-mediated NO₂^- reduction to NH₄⁺ by DNRA-performing bacteria, thereby supporting anammox activity and providing an energy-efficient strategy to mitigate NO₂^- accumulation and stabilize nitrogen removal in anammox-based systems.