High-efficiency simultaneous ammonia and nitrate removal in iron-sulfur coupled system under carbon limitation: Multi-omics insights into metabolic regulation and microbial interactions.

Iron‑sulfur coupling is a promising process for efficient nitrate removal, but its potential for simultaneous ammonia removal remains poorly recognized. This study established a biofilter (ISBF) using sponge iron and elemental sulfur as mixed fillers. Over 228 days of operation, ISBF achieved excellent nitrate (98.1%) and ammonia (89.7%) removal within 2 h. In-situ batch tests and ¹⁵N isotope tracing revealed synergistic nitrogen removal via autotrophic denitrification (56.6%), Feammox (9.1%), and Anammox (30.9%). X-ray diffraction confirmed FeOOH formation within biofilm, providing highly available substrates for iron metabolism. The analysis of microbial and functional genes revealed that Ca. Brocadia was enriched (3.57% and 26.67% at genomic and transcriptional levels, respectively) with high expression of hzsA (cDNA/DNA: 0.73-0.88). The bottom region of ISBF drove multi-pathway nitrogen removal, and middle/upper zones promoted complete denitrification and sulfate reduction, thereby improving nitrogen loss and reducing sulfate pollution. Batch tests and multi-omics analyses suggested that Ca. Brocadia possessed the potential for dual Feammox-Anammox metabolism, thereby facilitating its enrichment and maintenance of activity under NO₂⁻-deficient startup conditions. The Thiobacillus-dominated denitrification consortia exhibited a high narG and low nirKS expression pattern, implying a robust capacity for NO₂⁻ accumulation and supporting efficient nitrogen removal through anammox metabolism. Additionally, the increased abundance of genes involved in the electron transfer process suggested that ISBF could efficiently regulate multi-pathway synergistic nitrogen removal and ensure functional robustness. Therefore, this study provides novel insights for achieving simultaneous nitrate and ammonia removal under carbon limitation.