A novel chemolithotrophic biological nitrogen fixation process fueled by antimonite oxidation in antimony contaminated tailings and soils

Nitrogen (N) deficiency in mining regions usually impairs the soil ecosystems and thus hinders the bioremediation of contamination introduced by mining activities. Biological N fixation (BNF) is an essential biogeochemical process required for the initial accumulation of N in oligotrophic habitats. Our previous studies reported that chemolithotrophic diazotrophs rather than heterotrophic diazotrophs dominate in mining-contaminated region. It is proposed that diazotrophs may utilize reduced sulfur (S), arsenic (As), and antimony (Sb), which are abundant in mining-contaminated regions, as electron donors to fix N2. BNF fueled by the oxidation of S and As has been detected in previous studies. It is hypothesized that microorganisms may use the abundant Sb in Sb-contaminated soil to fuel nitrogen fixation. However, BNF fueled by Sb(III) oxidation (Sb-dependent BNF) has never been reported previously. The current study observed Sb-dependent BNF in cultures inoculated from Sb-contaminated tailing soils, upland soils, and rice paddy soils, suggesting that Sb-dependent BNF is probably widespread in Sb-contaminated environments. In addition, microorganisms responsible for Sb-dependent BNF were identified by DNA-stable isotope probing. Members of Rhodocyclaceae and Rhizobiaceae were identified as putative microorganisms responsible for Sb-dependent BNF. Furthermore, metagenomic-binning suggested that Rhodocyclaceae and Rhizobiaceae contained essential genes involved in Sb(III) oxidation, N2 fixation, and carbon fixation, confirming their metabolic potentials for Sb-dependent BNF. The detection of the novel Sb-dependent BNF and the illustration of its microbial mechanisms may broaden the fundamental understanding of chemolithotrophic BNF processes, which can be harnessed to improve remediation strategies in Sb-contaminated areas.