Antimony (Sb) isotopic fractionation is frequently used as a proxy for biogeochemical processes in nature. However, to date, little is known about Sb isotope fractionation in biologically driven reactions. In this study, Pseudomonas sp. J1 was selected for Sb isotope fractionation experiments with varying initial Sb concentration gradients (50-200 μM) at pH 7.2 and 30 °C. Compared to the initial Sb(III) reservoir (δ123Sb = 0.03 ± 0.01 ∼ 0.06 ± 0.01‰), lighter isotopes were preferentially oxidized to Sb(V). Relatively constant isotope enrichment factors (ε) of -0.62 ± 0.06 and -0.58 ± 0.02‰ were observed for the initial Sb concentrations ranging between 50 and 200 μM during the first 22 days. Therefore, the Sb concentration has a limited influence on Sb isotope fractionation during Sb(III) oxidation that can be described by a kinetically dominated Rayleigh fractionation model. Due to the decrease in the Sb-oxidation rate by Pseudomonas sp. J1, observed for the initial Sb concentration of 200 μM, Sb isotope fractionation shifted toward isotopic equilibrium after 22 days, with slightly heavy Sb(V) after 68 days. These findings provide the prospect of using Sb isotopes as an environmental tracer in the Sb biogeochemical cycle.

Microbial oxidation of environmental antimonite (Sb(III)) to antimonate (Sb(V)) is an antimony (Sb) detoxification mechanism. Ensifer adhaerens ST2, a bacterial isolate from a Sb-contaminated paddy soil, oxidizes Sb(III) to Sb(V) under oxic conditions by an unknown mechanism. Genomic analysis of ST2 reveals a gene of unknown function in an arsenic resistance (ars) operon that we term arsO. The transcription level of arsO was significantly upregulated by the addition of Sb(III). ArsO is predicted to be a flavoprotein monooxygenase but shows low sequence similarity to other flavoprotein monooxygenases. Expression of arsO in the arsenic-hypersensitive Escherichia coli strain AW3110Δars conferred increased resistance to Sb(III) but not arsenite (As(III)) or methylarsenite (MAs(III)). Purified ArsO catalyzes Sb(III) oxidation to Sb(V) with NADPH or NADH as the electron donor but does not oxidize As(III) or MAs(III). The purified enzyme contains flavin adenine dinucleotide (FAD) at a ratio of 0.62 mol of FAD/mol protein, and enzymatic activity was increased by addition of FAD. Bioinformatic analyses show that arsO genes are widely distributed in metagenomes from different environments and are particularly abundant in environments affected by human activities. This study demonstrates that ArsO is an environmental Sb(III) oxidase that plays a significant role in the detoxification of Sb(III).

Resistant bacteria are potential natural materials for the bioremediation of soil metalloid pollution. A strain isolated from farmland soil chronically exposed to Sb was identified as K. aerogenes X with high antimonite [Sb(III)] tolerance and oxidation ability. The resistance mechanism of K. aerogenes X and its extracellular polymeric substances (EPS), antioxidant enzymes, and oxidation characteristics in Sb(III) stress were investigated in this study by stress incubation experiments and FTIR. The biotoxicity of Sb was limited by the binding of the organic compounds in EPS, and the anionic functional groups (e.g., amino, carboxyl and hydroxyl groups, etc.) present in the cell envelope were the components primarily responsible for the metalloid-binding capability of K. aerogenes X. The K. aerogenes X can oxidize Sb(III), and its metabolites induce changes in reactive oxygen species (ROS), catalase (CAT), total superoxide dismutase (SOD) and glutathione s-transferase (GSH-S) activity, indicating that the resistance mechanisms of K. aerogenes X are mediated by oxidative stress, EPS restriction and cell damage. Oxidation of Sb(III) is driven by interactions in intracellular oxidation, cell electron transport, extracellular metabolism including proteins and low molecular weight components (LMWs). LMWs (molecular weight <3 kDa) are the main driving factor of Sb(III) oxidation. In addition, Sb resistance genes arsA, arsB, arsC, arsD and acr3 and potential oxidation gene arsH were identified in K. aerogenes X. Owing to its natural origin, high tolerance and oxidation ability, K. aerogenes X could serve as a potential bioremediation material for the mitigation of Sb(III) in contaminated areas.

Antimony (Sb), the analog of arsenic (As), is a toxic metalloid that poses risks to the environment and human health. Antimonite (Sb(III)) oxidation can decrease Sb toxicity, which contributes to the bioremediation of Sb contamination. Bacteria can oxidize Sb(III), but the current knowledge regarding Sb(III)-oxidizing bacteria (SbOB) is limited to pure culture studies, thus underestimating the diversity of SbOB. In this study, Sb(III)-oxidizing microcosms were set up using Sb-contaminated rice paddies as inocula. Sb(III) oxidation driven by microorganisms was observed in the microcosms. The increasing copies and transcription of the arsenate-oxidizing gene, aioA, in the microcosms during biotic Sb(III) oxidation indicated that microorganisms mediated Sb(III) oxidation via the aioA genes. Furthermore, a novel combination of DNA-SIP and shotgun metagenomic was applied to identify the SbOB and predict their metabolic potential. Several putative SbOB were identified, including Paracoccus, Rhizobium, Achromobacter and Hydrogenophaga. Furthermore, the metagenomic analysis indicated that all of these putative SbOB contained aioA genes, confirming their roles in Sb(III) oxidation. These results suggested the concept of proof of combining DNA-SIP and shotgun metagenomics directly. In addition, the identification of the novel putative SbOB expands the current knowledge regarding the diversity of SbOB.