Functional imbalance between Thauera and anammox governs PD/A pathway stability under contrasting reactor modes

Partial denitrification coupled with anammox (PD/A) is a promising energy-efficient alternative for mainstream nitrogen removal, yet its stability under different reactor modes remains unclear. This study systematically compared sequencing batch (R_S) and continuous-flow (R_C) PD/A systems using performance monitoring, activity assays, and metagenomic analyses. R_C maintained superior long-term stability, achieving >80% nitrogen removal with anammox contributing above 90%, whereas R_S remained less stable (∼65% NRE, ∼75% anammox contribution) and relied more on complete denitrification. Activity assays revealed that PD activity in R_S was 5.03 times that of anammox, favoring complete denitrification. In contrast, R_C maintained balanced activities, with PD activity being 0.89 times that of anammox, supporting cooperative PD/A interactions. Metagenomic analysis further revealed that Thauera-associated Nir/Nor/Nos genes were markedly enriched in R_S, indicating a stronger potential for complete denitrification. These findings demonstrate that reactor mode regulates PD/A interactions through distinct stabilization mechanisms, with continuous-flow operation enhancing microbial cooperation and system resilience. This study provides mechanistic insights and practical guidance for the stable implementation of mainstream PD/A processes.