ZVI-modified carriers boost S0-partial autotrophic denitrification-anammox for effective nitrogen removal under nitrite-deficient conditions

The ZVI-modified carrier enhanced TNRE to 93.9% under nitrite-deficient conditions. • ZVI promoted biofilm formation, EPS secretion, and heme c synthesis in AnAOB. • ZVI induced nirK downregulation and napA/napB upregulation for nitrite accumulation. • Anammox contributed >80% of N 2 production and Ca. Kuenenia dominated at 20.87%. • S 0 -PAD-Anammox with the ZVI carrier reduced chemical costs by 13.9–32.4% vs. heterotrophic. S 0 -driven partial autotrophic denitrification couple with anaerobic ammonium oxidation (S 0 -PAD-Anammox) is an emerging nitrogen removal process that reduces the strict NO 2 − -N requirement of anammox. However, both sulfur-oxidizing bacteria (SOB) and anammox bacteria (AnAOB) typically exhibit slow growth and low bioactivity, limiting the synergistic nitrogen removal performance. Zero-valent iron (ZVI) can facilitate heme synthesis and enhance electron transfer in anammox, while also serving as a cofactor for key enzymes in sulfur oxidation. Nevertheless, direct ZVI dosing poses operational challenges, including particle aggregation, surface passivation, and difficulties in dosage control. In this study, ZVI15@HDPE carriers (15 wt% ZVI loading) were prepared via melt-blending and used in an integrated floating-film activated sludge (IFFAS) system. The ZVI-modified carrier significantly enhanced biofilm formation, extracellular polymeric substance (EPS) secretion, and heme c synthesis, as well as promoted the enrichment and metabolic activity of AnAOB and SOB. Under influent conditions of 75 mg/L NH 4 + -N, 25 mg/L NO 2 − -N, and 75 mg/L NO 3 − -N, the total nitrogen removal efficiency (TNRE) reached 93.9 ± 0.2%, substantially higher than the control reactor filled with high-density polyethylene (HDPE) carriers (72.3 ± 0.2%). 15 N isotope analysis confirmed that anammox or S 0 -PAD-anammox contributed over 80% of total N 2 production, while S 0 -AD accounted for 12.2–18.4%. The dominant functional genera Candidatus_Kuenenia (20.87%) and Thiobacillus (5.98%) were selectively colonized on ZVI15@HDPE carriers. Metagenomic sequencing revealed that ZVI induced lower abundance of nirK gene and higher abundance of napA / napB genes, promoting preferential NO 3 − -N reduction to NO 2 − -N and enabling stable S 0 -PAD coupled with anammox. This study provides a viable and cost-effective approach for efficient nitrogen removal under nitrite-deficient conditions. Download: Download high-res image (201KB) Download: Download full-size image