Novel incomplete nitritation-regulated multi-pathway anammox process for advanced nitrogen removal from real...

Mainstream anammox continues to face fundamental operational bottlenecks, due to unstable nitrite supply and the limited competitive strength of autotrophic anammox bacteria in municipal wastewater. In this study, a novel strategy based on incomplete nitritation was established to initiate and regulate the multi-pathway anammox process. Short-term hydroxylamine induction (5 mg/L) rapidly established coexistence of NH₄⁺, NO₂^-, and NO₃^-, sustaining nitrite supply via nitritation (PN) and denitritation (PD). Advanced nitrogen removal (NRE = 93.3%) was achieved at extremely low C/N (2.5) during real municipal wastewater treatment. The activity and abundance of anammox bacteria increased markedly (6.2 → 16.7 mg NH₄⁺-N/gMLVSS/d; 5.6 × 10⁷→9.0 × 10⁸ gene copies/g VSS). Genomic and transcriptomic analyses revealed enriched Candidatus Brocadia (0.02%→0.18%) enhanced nitrogen removal while suppressing nitrite competition from nitrite oxidizing bacteria and denitrifiers, thereby strengthening both PN and PD pathways. Functional gene ratios demonstrated strong coordination between substrate supply pathways, with AOB/NOB increasing from 1.3 to 6.2 and (narG + napA)/(nirS + nirK) rising from 0.2 ± 0.1 to 1.0 ± 0.1. Importantly, incomplete rather than full nitrite oxidizing bacteria suppression was identified as a sustainable control point, ensuring robustness. Overall, multi-pathway anammox regulated by incomplete nitritation offers superior efficiency and stability compared to single-path systems, providing a promising strategy for mainstream anammox applications.