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

## TL;DR

This study shows that the presence of Fe2+ and Mn2+ in groundwater reduces the efficiency of sulfur-based nitrate removal systems.

## Contribution

The study reveals the mechanisms by which Fe2+ and Mn2+ coexistence inhibits sulfur autotrophic denitrification.

## Key findings

- Coexisting Fe2+ and Mn2+ reduced nitrate removal efficiency from 92.73% to 60.96%.
- The presence of Fe2+ and Mn2+ led to accumulation of NO2−-N and N2O.
- Key denitrification and sulfur oxidation genes were downregulated in the presence of Fe2+ and Mn2+.

## Abstract

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 SO42− 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 Thiobacillus, and downregulated the expression of key denitrification (nirS, norB, nosZ) and sulfur oxidation (dsrA, soxB) 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 nosZ/narG, nosZ/nirK, norB/nirK, dsrA/16S rRNA, soxB/nirK, and soxB/nirK 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+.

Bar chart and flow diagram illustrating nitrate nitrogen (NO₃⁻-N) removal rates with different Fe²⁺ to Mn²⁺ ratios and associated mechanisms on Day 6. The bar chart shows removal rates between 92.73% and 60.96% across ratios from 0 to 9:1. The flow diagram details the conversion pathways of nitrogen species using various enzymes, such as narG, nirS, and norB, within the SADN system, alongside sulfur transformations involving dsrA and soxB. Arrows indicate processes influenced by pH and *Thiobacillus*.