Chemolithotrophic Biological Nitrogen Fixation Fueled by Antimonite Oxidation May Be Widespread in Sb-Contaminated Habitats

Nitrogen (N) deficiency in mining-contaminated habitats usually hinders plant growth and thus hampers tailing revegetation. Biological N fixation (BNF) is an essential biogeochemical process that contributes to the initial accumulation of N in oligotrophic mining-contaminated regions. Previous studies reported that chemolithotrophic rather than heterotrophic diazotrophs frequently dominated in the mining-contaminated regions. Chemolithotrophic diazotrophs may utilize elements abundant in such habitats (e.g., sulfur (S), arsenic (As), and antimony (Sb)) as electron donors to fix N2. BNF fueled by the oxidation of S and As has been detected in previous studies. However, BNF fueled by Sb(III) oxidation (Sb-dependent BNF) has never been reported. The current study observed the presence of Sb-dependent BNF in slurries inoculated from Sb-contaminated habitats across the South China Sb belt, suggesting that Sb-dependent BNF may be widespread in this region. DNA-stable isotope probing identified bacteria associated with Rhodocyclaceae and Rhizobiaceae as putative microorganisms responsible for Sb-dependent BNF. Furthermore, metagenomic-binning demonstrated that Rhodocyclaceae and Rhizobiaceae contained essential genes involved in Sb(III) oxidation, N2 fixation, and carbon fixation, suggesting their genetic potential for Sb-dependent BNF. In addition, meta-analysis indicated that these bacteria are widespread among Sb-contaminated habitats with different niche preferences: Rhodocyclaceae was enriched in river sediments and tailings, while Rhizobiaceae was enriched only in soils. This study may broaden our fundamental understanding of N fixation in Sb-mining regions.