Boosting Nitrogen Removal through Fe0-Coupled Sulfur Autotrophic Denitrification: Mediated by Enhanced Sulfur Disproportionation and Electron Transfer

• Fe 0 -coupled SAD system enabled efficient N removal and less sulfate generation. • Fe 0 sustained neutral-alkaline pH for facilitated sulfur bioavailability. • Encapsulation and dispersion of Fe 0 by sulfur helped to maintain its reactivity. • SRT of sulfur and iron substrates was extended with in-situ FeS deposition. • Fe 0 boosted the overall electron flux towards denitrifying. Sulfur autotrophic denitrification (SAD) has gained increasing attention in advanced nitrogen removal from secondary effluent, yet its application is constrained by excessive sulfate emission, acidity accumulation, low sulfur bioavailability and limited solid retention time (SRT). To address these limitations, a Fe 0 -coupled SAD system was proposed based on novel sulfur-iron composite fillers (SICF), in which iron scraps were encapsulated into sulfur matrix. Under HRT of 1h, SICF 10% (SICF with 10 wt.% iron scraps) achieved the highest denitrification rate of 490.3 ± 24.5 mg/(L·d), outperforming the sole SAD system by 36.9%, and effluent sulfate concentration was reduced to 5.3 ± 0.2 mg·mg⁻¹-N. Alkalinity from Fe° corrosion sustained a favorable pH range (7.1 ± 0.1–8.2 ± 0.1) for sulfur disproportionation and polysulfide (S n 2- ) generation, leading to enhanced sulfur bioavailability for denitrifiers. Besides, protons from SAD effectively mitigated Fe 0 passivation, ensuring sustained electron supply of Fe 0 that accounted for 17.6%–84.3% to overall denitrification. Then, FeS was generated in-situ through reaction of Fe 2+ /Fe 3+ (from Fe 0 oxidation) with HS - (from sulfur disproportionation), effectively extending SRT of sulfur-iron substrates and mitigating reduced iron and sulfur donors overflow. Fe 0 supplementation promoted enrichment of sulfur/iron autotrophic denitrifier ( Thiobacillus ) and sulfur-disproportionating bacteria ( Dissulfurimicrobium ). Metagenomic analysis revealed enhanced expression of genes related to denitrification ( napAB and nosZ ), sulfur disproportionation ( sdo, asrA/B and psrA ), iron oxidation/transport ( KorA/B, feoB and afuC ), and electron transfer chain, Complex I ( NQ01 and e1.6.99.1 ), Complex III ( qcrA/B/C ), cytochrome c ( ccdA and CYC ), MtrABC porin-cytochrome complex ( MtrABC ), and quinones ( pqqB ), establishing an efficient sulfur/iron/nitrogen metabolism network coupled with active electron transfer.