amplicon and shotgun metagenomic sequencing of nitrate-dependent Sb(III) oxidizers

Sb(III) oxidation can decrease the toxicity of Sb and its uptake into plants (e.g., rice), thus serving an ecological role in bioremediation of Sb contamination. In some anoxic environments such as rice paddies, Sb(III) can be oxidized coupled with nitrate as the electron acceptor. Here we investigate the potential for nitrate-dependent Sb(III) oxidization in Sb contaminated rice paddies and identify nitrate-dependent Sb(III)-oxidizing bacteria (SbO-bacteria) with stable isotope probing (SIP) combined with amplicon and shotgun metagenomic sequencing. Anaerobic Sb(III) oxidation was exclusively observed in the paddy soil slurry treatment amended with both Sb(III) and NO3-, whereas no apparent Sb(III) oxidation was observed with Sb(III) only. The increase in the abundance of the arsenite oxidase (aioA) gene suggests that nitrate-dependent Sb(III) oxidation (SbO) was catalysed by microorganisms harbouring the aioA gene. After 60-day of DNA-SIP incubation, obvious shift in the abundance of the aioA gene to heavy fractions only in the treatment amended with 13C-NaHCO3, Sb(III) and nitrate suggested the incorporation of 13C by nitrate-dependent SbO-bacteria. Accordingly, DNA-SIP identified a number of putative nitrate-dependent SbO-bacteria in the paddy soil, including Azoarcus, Azospira and Chelativorans. Metagenomic analysis further revealed that they contained aioA gene and genes for denitrification and carbon fixation, supporting their capability for Nitrate-dependent SbO. Collectively, these observations suggested the occurrence of nitrate-dependent SbO in paddy soils. A number of putative nitrate-dependent SbO-bacteria never correlated with Sb(III) oxidation were reported here, which expands our current knowledge regarding the diversity of nitrate-dependent SbO-bacteria. In addition, this study provides a proof of concept using DNA-SIP to identify nitrate-dependent SbO-bacteria although a long incubation period is required and a combination of metagenomics is suggested.