Enhancing nitrogen removal performance under Anammox subjected to nitrite limitation: The impact of Fe2O3 dosing modes and underlying mechanisms

As an energy-saving and sustainable method for nitrogen removal, the stable acquisition of NO₂⁻-N is a bottleneck for the application of Anammox. To address this limitation, this study investigated Fe₂O₃ as a potential electron acceptor for NH₄⁺-N oxidation, examining its addition modes and underlying mechanisms for nitrogen removal. Short-term pulse dosing of Fe₂O₃ (10–250 mg/L) sustained nitrogen removal, whereas absence or excess (500 mg/L) impaired efficiency due to NO₂⁻-N limitation and microbial inhibition by Fe₂O₃. Long-term incremental Fe₂O₃ addition enhanced NH₄⁺-N and total nitrogen removal efficiencies by facilitating Fe(III) reduction coupled to anaerobic ammonium oxidation (Feammox) and NO_x⁻-dependent Fe(II) oxidation (NDFO) processes. Fe₂O₃ addition improved the activity of electron transfer system, with intracellular Fe(II)-mediated key electron transfer and energy metabolism processes. The abundance of functional genes associated with gene expression regulation and transmembrane transport was increased by Fe₂O₃ addition, enhancing the transcriptional activity of genes involved in nitrogen transformation and carbon metabolism. Furthermore, Fe₂O₃ promoted the secretion of extracellular polymeric substances and microbial lysis, supporting the growth of heterotrophic bacteria. RNA stable isotope tracing identified autotrophic Candidatus Kuenenia and heterotrophic Pseudomonas as key microorganisms in NH₄⁺-N removal. Concurrently, autotrophic and heterotrophic denitrifiers contributed to a progressive decrease in effluent NO₃⁻-N. This study provides an in-depth understanding of the impact of Fe₂O₃ on nitrogen removal and offers an effective strategy to solve performance decline associated with NO₂⁻-N limitations for Anammox.