Iron as an Electron Reservoir in Two-Zone Biofilters for Robust Nitrogen Removal

Biological nitrogen removal in on-site wastewater treatment is often constrained by unbalanced electron acceptors and donors. This study evaluated iron-rich basalt as an auxiliary electron reservoir for nitrogen transformation in a dual-zone biofilter. Three biofilters with separate anaerobic and microaerobic zones were packed with basalt, iron-coated perlite, or perlite and operated under an influent with varying alkalinity and carbon-to-nitrogen (C/N) ratios. In all bioreactors, NH4+–N removal increased progressively from Stage I to IV, with high removal efficiencies (>80%) under elevated alkalinity (Stage III) and sustained performance (>70%) at a C/N ratio of 10:1 (Stage IV). Across all stages, basalt-packed biofilters demonstrated superior NH4+–N removal and the highest overall nitrogen loss (>60%) compared with iron-coated perlite and perlite systems. To elucidate nitrogen removal mechanisms, batch activity assays and microbial community analyses were conducted. The assays indicated that iron-mediated pathways, including Fe3+-mediated ammonium oxidation (Feammox) and nitrate-dependent Fe2+ oxidation (NDFO), played central roles in nitrogen transformation in iron-containing media. Correspondingly, iron-reducing bacteria (e.g., Ferruginibacter) were enriched in anaerobic zones, while iron-oxidizing bacteria (e.g., Rhodanobacter) dominated microaerobic zones in biofilters packed with basalt- and iron-coated perlite. X-ray diffraction analyses further confirmed the phase composition changes in iron on the surfaces of basalt and iron-coated perlite. This study provides a proof-of-concept for leveraging iron-cycling biofilters to enhance ammonia removal in on-site wastewater treatment systems, offering a promising strategy for sustainable nutrient management in on-site applications.