Nitrogen loading fluctuations impact microbial community assembly and functional redundancy in anammox reactors

Nitrogen loading rate (NLR) fluctuations are common in full-scale anammox systems and can compromise process stability, yet the ecological mechanisms underlying system resilience under disturbed conditions remain insufficiently understood. This study investigated how different intensities of NLR disturbances influence microbial community assembly processes and functional redundancy, and how these ecological responses shape nitrogen removal performance. Two anammox sequencing batch reactors were operated for 180 days under either stable (R1) or fluctuating (R2) NLR conditions. Moderate NLR fluctuations (1.4-fold, Phase A) enhanced nitrogen removal efficiency (up to 99.7%) and increased the relative abundance of anammox bacteria to 26.4%, whereas severe fluctuations (2.0-fold, Phase B) caused deterioration in nitrogen removal efficiency (to 72.2%) and a decline in anammox bacteria abundance (7.1%). Metagenome-assembled genome analysis revealed pathway-level reorganization of nitrogen metabolism under fluctuating conditions, with increases in anammox-associated genes (hzsABC and hdh/hao-like) and the DNRA gene (nrfAH) during Phase A, followed by partial declines in Phase B. Neutral community modeling showed that stochastic processes dominated microbial assembly under moderate fluctuations (R² = 0.77), promoting coexistence and community adaptability, while deterministic selection prevailed under severe fluctuations (R² = 0.56). Functional redundancy exhibited a similar non-linear response, increasing under moderate disturbance (0.73) and declining sharply under severe disturbance (0.48), indicating reduced buffering capacity. These findings provide quantitative insight into the mechanistic link between loading disturbances and ecosystem resilience, offering a foundation for developing operational strategies that enhance the robustness of anammox-based nitrogen removal systems.