Metagenomic and Micro-CT insights into Fe(III)-modulated extracellular polymeric substances driving anammox granulation and enhancement of nitrogen removal.

Multivalent cations are commonly employed to accelerate sludge aggregation and granulation, yet they often compromise intragranular mass transfer and diminish microbial activity. Here, the effect of Fe(III) dosing on granule formation and anammox-driven nitrogen removal over a 110-day continuous operation was investigated. Fe(III) supplementation enhanced interactions with extracellular polymeric substances (EPS), transforming flocculent biomass into highly porous granules and yielding a 67.9 % increase in specific anammox activity (SAA). During the first 30 days of Fe(III) dosing, the Zeta potential shifted from -12.0 to -9.3 mV and EPS content surged from 32.8 mg/g VSS to 49.0 mg/g VSS, accelerating primary floc aggregation. Subsequently, the protein-to-polysaccharide ratio of EPS escalated from 1.7 to 5.3, reflecting a transition from a carbohydrate-rich, hydrophilic gel to a protein-dominated, hydrophobic network that facilitated granulation. Micro-computed tomography revealed that smaller (1.5 mm) granules exhibited dense architectures, whereas larger (2.5 mm) granules developed internal cavities as a result of mass transfer limitations, approaching structural fragmentation. Metagenomic analysis showed that Fe(III) upregulated pathways for hydrophobic amino acids (e.g., isoleucine, leucine, valine) synthesis and downregulated polysaccharide biosynthesis, thereby increasing EPS hydrophobicity and promoting granulation. The observed SAA enhancement was linked to Fe(III)-dependent activation of iron-containing enzymes (e.g., hzs ABC), cytochrome c maturation, and Fe-S cluster assembly. Concurrently, the relative abundance of Ca. Kuenenia rose from 14.1 % during aggregation to 18.6 % after sludge granulation. This work elucidates the mechanism of Fe(III)-EPS-mediated granulation and offers a sustainable strategy to reconcile structural integrity with metabolic efficiency in metal-driven anammox wastewater treatment processes.