Experiments were conducted using simulated secondary effluent from wastewater treatment plants. A composite filler was prepared by mixing sulfur and pyrite (FeS₂) as electron donors for microorganisms to evaluate the nitrate removal efficiency under different filler compositions. Three iron-carbon-based composite sulfur autotrophic denitrification fillers were synthesized with varying pyrite-to-sulfur mass ratios (2:1, 1:1, and 1:2, labeled as FS1, FS2, and FS3, respectively). A synergistic autotrophic-heterotrophic denitrification biofilter system was established to compare the operational conditions and nitrogen removal performance among mixed autotrophic-heterotrophic, heterotrophic, and autotrophic denitrification processes. The results showed that the FS2 system (FeS₂:S = 1:1) achieved the shortest start-up time (7 days), 33% and 50% faster than FS1 (FeS₂:S = 2:1) and FS3 (FeS₂:S = 1:2), respectively. During the start-up phase, FS2 exhibited the highest NO₃⁻-N removal rate (1.82 mg/(L·h)), significantly outperforming FS1 (1.25 mg/(L·h)) and FS3 (0.98 mg/(L·h)). The optimal HRT for pyrite-based systems was 24 h, with FS2 achieving the highest NO₃⁻-N removal efficiency (94.2%), followed by FS3 (84.2%) and the pure sulfur system (80%). When HRT was reduced to 12 h, FS2 maintained a removal efficiency of 81.3%, while FS1 and FS3 declined to 78.5% and 65.2%, respectively. Within pH 6.5–8. 0, FS2 demonstrated stable NO₃⁻-N removal (95%–98%) with minimal effluent pH fluctuation (±0.3 from initial pH 7.0), whereas FS1 and FS3 exhibited larger pH variations (±1.2 and ±1.8, respectively), indicating inferior buffering capacity. The FS2 filler (FeS₂:S = 1:1) exhibited superior denitrification efficiency, rapid system start-up, and robust pH stability, making it a promising candidate for enhancing nitrogen removal in secondary effluent treatment
