Underestimated role of modulating sludge structural evolution in hematite-enhanced anammox systems beyond electron...

Anaerobic ammonium oxidation (anammox) represents a promising sustainable nitrogen removal technology, yet the slow growth of anammox bacteria results in suboptimal reaction rates and operational stability, while nitrate accumulation further diminishes total nitrogen removal efficiency. In this study, 1.0 g L^-1 hematite was introduced into a bioreactor as a redox-active mineral to overcome these limitations. Hematite amendment enhanced electron transfer capacity, promoted sludge structural evolution, and established Fe-N coupling for diverse nitrogen removal pathways, ultimately improving total nitrogen removal efficiency by 13.4 %. Hematite addition stimulated microbial activity, as evidenced by a 103 % increase in adenosine triphosphate and a 31 % elevation in electron transport system activity, thereby improving electron transfer capacity. Sludge structural evolution was improved, with D₅₀ increasing from 535 ± 18 µm to 1333 ± 96 µm, extracellular polymeric substance-protein content reaching 40.3 ± 6.8 mg g^-1 VSS, and microbial viability under shock loading improving from 52.8 % to 58.1 %. Fe-N coupling was confirmed by intensified N₂ production attributable to Feammox and NDFO processes, though their quantitative contribution remained minor (<0.1 % of total N₂ production), upregulation of the rnfG gene (+136.9 %), and the enrichment of Candidatus Brocadia and iron-reducing bacteria. Partial least squares structural equation modeling (PLS-SEM) analysis suggested that sludge structural evolution accounted for 41.69 % of the observed performance enhancement. The direct contribution of electron transfer capacity was comparatively modest (22.95 %), with its primary influence mediated through an indirect effect on sludge structural evolution (35.36 %). These findings highlight that sludge structural evolution represents a previously underappreciated yet critical pathway in mineral-enhanced anammox systems.