Innovatively Unlocking Anammox Driven by Nitrite Accumulation in a nrfA-Deficient Shewanella oneidensis MR-1...

With the advancement of industrialization and urbanization, nitrogen pollution has become increasingly severe, and accelerating the nitrogen cycle is of great significance for nitrogen removal in wastewater treatment. To enhance the nitrogen (N) removal efficiency, we constructed a novel coupled system integrating anammox with Shewanella oneidensis MR-1. We specifically focused on the functional differences between its wild-type and a nrfA-deficient mutant (ΔnrfA). We systematically evaluated the N removal performance, microbial community structure, and nitrogen/carbon metabolic functional genes under different inoculation ratios. Although the wild-type strain could supply nitrite (NO₂ ^--N) for anammox via dissimilatory nitrate reduction to ammonium (DNRA), its excessive inoculation triggered substrate competition with denitrification, weakening the anammox dominance. Conversely, the mutant strain can cause the accumulation of NO₂ ^--N by blocking the DNRA pathway, thereby forming a stable synergistic interaction with anammox bacteria. The coupled system achieved a maximum total N removal efficiency of 97% with the fastest reaction kinetics at an optimal anammox-to-mutant volume ratio of 5:1. Metagenomic analysis corroborated these findings, revealing significant enrichment of key anammox functional genes (hzs and hdh) under this optimized condition. In summary, strategically coupling anammox with an electron-transfer-capable, DNRA-deficient mutant ensures a sustained NO₂ ^--N supply. This genetic manipulation strategy offers a novel, efficient, and stable paradigm for advancing anaerobic nitrogen removal in nitrate-laden wastewater treatment.