Nitrogen removal driven by zonal supply of thiosulfate and pyrite in an up-flow blanket filter (UBF) reactor

Despite its effectiveness in nitrogen removal, the anammox process faces challenges in practical applications due to NO₃^--N accumulation, which can lead to effluent breaches of discharge standards. This study developed a single-stage up-flow blanket filter (UBF) reactor featuring three axially stratified functional layers with multi-point supplementation of thiosulfate and pyrite. During stable operation, the high-intensity anammox layer (L1) contributed to 82.3 % of total nitrogen (TN) removal and generated alkalinity to support subsequent sulfur autotrophic denitrification (SAD). The SAD-coupled anammox layer (L2) effectively removed accumulated NO₃^--N, reducing TN from 42.8 mg/L to 25.2 mg/L. The advanced nitrogen removal layer (L3) further polished the effluent to a TN concentration of 4.3 ± 2.8 mg/L and a pH of 7.1 ± 0.1, achieving advanced nitrogen removal and pH self-regulation. Anammox bacteria exhibited an inhibition-recovery response to thiosulfate addition, while pyrite served a triple role as a filter medium, metabolic accelerator, and electron donor. Metagenomic analysis revealed that thiosulfate supplementation selectively enrichedThiobacillusandPFJX01in each functional layer, with denitrification gene abundance increasing along the reactor's height. Notably, functional and associated microorganisms showed layer-specific gene expression and co-metabolism patterns. Even within the same genus, differential gene regulation in response to local microenvironments drove functional divergence at the genetic level. In summary, this work innovatively establishes spatially segregated functional layers within a single UBF reactor, offering a novel low-carbon strategy for advanced nitrogen removal and facilitating the resource utilization of tailings.