Coexistence of Fe2+ and Mn2+ inhibits nitrate removal in sulfur autotrophic denitrification systems

Sulfur autotrophic denitrification (SAD) is commonly utilized for nitrate (NO₃ ^--N) removal from groundwater because of its efficiency, minimal sludge production, cost-effectiveness, and carbon source independence. However, elevated Fe²⁺ and Mn²⁺ concentrations in groundwater may influence its efficiency. The purpose of this work was to explore the effects of coexisting Fe²⁺ and Mn²⁺ at varying 5 mM ratios on SAD efficiency and its underlying mechanisms. The results showed that adding 5 mM Fe²⁺ and Mn²⁺ at different ratios inhibited NO₃ ^--N removal, reducing efficiency from 92.73% (without Fe²⁺/Mn²⁺) to 60.96% (Fe²⁺: Mn²⁺ = 9:1) by Day 6. All the systems with coexisting Fe²⁺ and Mn²⁺ accumulated NO₂ ^--N and N₂O. The generation of SO₄ ^2- by the system gradually diminished, the Fe²⁺ removal rate gradually decreased, and the Mn²⁺ removal rate gradually increased as Fe²⁺ and Mn²⁺ concentrations increased and decreased, respectively. The coexistence of Fe²⁺ and Mn²⁺ reduced pH, decreased the relative abundance of <i>Thiobacillus</i>, and downregulated the expression of key denitrification (<i>nirS</i>, <i>norB</i>, <i>nosZ</i>) and sulfur oxidation (<i>dsrA</i>, <i>soxB</i>) genes, thereby compromising the denitrification efficiency of the SAD system. The rate-limiting reactions for system denitrogenation with Fe²⁺ and Mn²⁺ coexistence included NO reduction and N₂O reduction. Furthermore, the key driving factors were the <i>nosZ</i>/<i>narG</i>, <i>nosZ</i>/<i>nirK</i>, <i>norB</i>/<i>nirK</i>, <i>dsrA</i>/<i>16S rRNA</i>, <i>soxB</i>/<i>nirK</i>, and <i>soxB</i>/<i>nirK</i> gene ratios. The findings of this study provide theoretical support for employing SAD technology to remove NO₃ ^--N from water with elevated levels of coexisting Fe²⁺ and Mn²⁺.