Dual Anammox-Based Nitrogen Removal Pathways Enhance a A2/O System Treating Low-COD/N Municipal Wastewater

Conventional biological nitrogen removal in wastewater treatment plants requires substantial energy for aeration and considerable organic carbon for denitrification, with limited efficiency in pre-denitrification systems. Therefore, in the globally widespread anaerobic/anoxic/oxic (A2/O) process for municipal wastewater, implementing anammox-based pathways in both anoxic and aerobic zones is crucial for further improving nitrogen removal efficiency, especially under low COD/N conditions. This study investigated the feasibility of establishing dual anammox-based nitrogen removal pathways in a A2/O system treating real municipal wastewater with a low COD/N ratio (~3). Building upon a previously established partial denitrification/anammox (PD/A) process in the anoxic zone, we introduced nitrifying biofilm carriers into the aerobic zone to develop complementary partial nitritation/anammox (PNA). Through comprehensive performance monitoring, batch tests, stable isotope tracing, qPCR and metagenomic analysis, we successfully demonstrated the establishment of aerobic PNA within 80 days, with anammox abundance achieving 1.63%. Total nitrogen removal increased from 70.9% to 80.4%, reducing effluent TN from 12.0 to 8.2 mg-N/L without additional carbon supplementation. Overall anammox contribution to nitrogen removal rose from 35.2% to 46.4%. The rapid enrichment of anammox in the aerobic zone resulted from three synergistic factors: (1) a stable anammox inoculum from the anoxic biofilm, (2) inherent oxygen tolerance of the dominant Candidatus Brocadia species, and (3) protective microenvironments within the biofilm structure. This dual-pathway approach significantly enhances nitrogen removal without additional infrastructure, offering a cost-effective solution for existing A2/O systems worldwide to meet increasingly stringent discharge standards, particularly for environmentally sensitive water bodies.