Bacteria responsible for nitrate-dependent antimonite oxidation in antimony-contaminated paddy soil revealed by the combination of DNA-SIP and metagenomics

Antimonite (Sb(III)) oxidation (SbO) can decrease the toxicity of antimony (Sb) and its uptake into rice, thus serving an ecological role in bioremediation of Sb contamination in rice paddies and decreasing the translocation and accumulation of Sb in rice. Nitrate as the electron acceptor can be coupled to SbO in anoxic environments, which, however, has not been reported in paddy soils. Here we investigate the potential for nitrate-dependent SbO in Sb-contaminated rice paddies and identify nitrate-dependent Sb(III)-oxidizing bacteria (SbOB) using stable isotope probing (SIP) coupled with amplicon and shotgun metagenomic sequencing. Anaerobic SbO was exclusively observed in the paddy soil amended with both Sb(III) and NO 3 − , whereas no apparent SbO was detected in the soil amended with Sb(III) only. The increasing abundance of the arsenite oxidase gene ( aioA ) suggests that nitrate-dependent SbO was catalysed by microorganisms harbouring the aioA gene. After 60-day DNA-SIP incubation, an obvious shift in the relative abundance of aioA gene to heavy DNA fractions occurred only in the treatment amended with 13 C–NaHCO 3 , Sb(III) and NO 3 − , suggesting the incorporation of 13 C by nitrate-dependent SbOB. Accordingly, a number of putative nitrate-dependent SbOB were identified in the paddy soil, including Azoarcus , Azospira and Chelativorans . Metagenomic analysis further revealed that they contained aioA genes and genes involved in denitrification and carbon fixation, supporting their capability for nitrate-dependent SbO. These observations suggested the occurrence of nitrate-dependent SbO in paddy soils. A number of putative nitrate-dependent SbOB ( i.e. , Azoarcus , Azospira and Chelativorans ) were reported here, which expands our current knowledge regarding the diversity of nitrate-dependent SbOB. In addition, this study provides a proof of concept using DNA-SIP to identify nitrate-dependent SbOB. • Nitrate-dependent Sb(III) oxidation can take place in paddy soil. • Nitrate-dependent Sb(III) oxidation was mediated by the microbiota in paddy soil. • The aioA gene may be responsible for nitrate-dependent Sb(III) oxidation. • Azoarcus , Azospira and Chelativorans were identified as putative nitrate-dependent Sb(III) oxidizers.