Enhanced synergistic interaction between AOA and anammox bacteria: a novel mechanism for stability under nitrite...

Fluctuating ammonia concentrations in wastewater often exceed the adaptive capacity of ammonia-oxidizing archaea (AOA), thereby limiting the efficacy of partial nitritation/anammox (PN/A) systems that rely on AOA-driven nitritation. Although nitrite addition may enhance process stability, the underlying microbial mechanisms remain unclear. This study investigated the response of an AOA/anammox bacteria enriched counter-current biological aerated filter under low intermittent aeration to nitrite shock loading. At an NO₂^--N concentration of 21.95 ± 1.36 mg/L and NH₄⁺/NO₂^- ratio of 0.44 ± 0.027, the system achieved high nitrogen removal efficiencies: 99.10 ± 0.72 % for NH₄⁺-N and 83.89 ± 0.35 % for total nitrogen. AOA were identified as the dominant nitrifiers via dual-inhibition, phylogenetic, and functional gene analyses. Metagenomics revealed upregulation of key functional genes, including AOA-associated nirK, anammox-related hzs/hzo/hdh, and denitrification genes nir/nap/nor/nos, enhancing AOA-anammox synergy. Copiotrophic AOA (93.49 % of AOA community, primarily Nitrososphaera and Nitrosocosmicus) and Candidatus Brocadia (35.95 % of bacteria) dominated the system. Genomic binning further revealed horizontal transfer of nap/nor genes between AOA and denitrifiers, reinforcing metabolic adaptability under selective conditions. Metabolic profiling indicated that AOA competitiveness under nutrient stress was reinforced by genes involved in nitrogen assimilation, urea utilization, polysaccharide synthesis and energy metabolism, thereby strengthening their partnership with anammox bacteria. These findings demonstrate that nitrite supplementation can reinforce AOA-based PN/A systems under variable ammonia loads, supporting energy-efficient mainstream wastewater treatment.