Fe²⁺ primarily regulates nitrate nitrogen removal from groundwater in sulfur-driven autotrophic denitrification...

Nitrate-nitrogen (NO₃^--N) contamination of groundwater poses a serious threat to human health and ecosystems. Sulfur autotrophic denitrification (SADN) has garnered significant interest owing to its cost-effectiveness and operational efficiency. Through batch experimentation, this research evaluated Fe²⁺ concentration-dependent effects on microbial community organization and denitrification performance in SADN systems. The experimental data revealed that high Fe²⁺ concentrations (≥4 mM) significantly inhibited the NO₃^--N removal in SADN systems during the initial phase (day 6), achieving only 71.9% removal efficiency, which was significantly lower than treatment with Fe²⁺ ≤2 mM (90% removal). However, by day 12, even with 5 mM Fe²⁺, the system achieved >95% NO₃^--N removal. Fe²⁺ addition inhibited the growth of microorganisms and downregulated denitrification-related genes (narG, nirS, nirK, norB, and nosZ) and sulfur-oxidizing genes (dsrA, soxB), which impaired NO₂^--N degradation, N₂O transformation, and sulfur oxidation. In the system supplemented with 5 mM Fe²⁺, 0.38 mM NO₂^--N remained undegraded on day 12, and the N₂O accumulation peaked at 265 μM on day 10. Conversely, iron oxidation gene mtrA exhibited upregulated expression with the increase of Fe²⁺ concentrations. The microbial community analysis indicated the coexistence of SADN, iron-autotrophic denitrification, and denitrification synergism in this system. Integrated analysis confirmed that NO₂^--N reduction was the primary factor limiting the nitrogen removal rate in the Fe²⁺-regulated SADN system. These findings offer a theoretical basis for the remediation of nitrate pollution in Fe²⁺-containing groundwater.