Characterization of Nitrate-Dependent As(III)-Oxidizing Communities in Arsenic-Contaminated Soil and Investigation of Their Metabolic Potentials by the Combination of DNA-Stable Isotope Probing and Metagenomics

Abstract

Arsenite (As(III)) oxidation has important environmental implications by decreasing both the mobility and toxicity of As in the environment. Microbe-mediated nitrate-dependent As(III) oxidation (NDAO) may be an important process for As(III) oxidation in anoxic environments. Our current knowledge of nitrate-dependent As(III)-oxidizing bacteria (NDAB), however, is largely based on isolates, and thus, the diversity of NDAB may be underestimated. In this study, DNA-stable isotope probing (SIP) with ¹³C-labeled NaHCO₃ as the sole carbon source, amplicon sequencing, and shotgun metagenomics were combined to identify NDAB and investigate their NDAO metabolism. As(III) oxidation was observed in the treatment amended with nitrate, while no obvious As(III) oxidation was observed without nitrate addition. The increase in the gene copies of <i>aioA</i> in the nitrate-amended treatment suggested that As(III) oxidation was mediated by microorganisms containing the <i>aioA</i> genes. Furthermore, diverse putative NDAB were identified in the As-contaminated soil cultures, such as <i>Azoarcus</i>, <i>Rhodanobacter</i>, <i>Pseudomonas</i>, and Burkholderiales-related bacteria. Metagenomic analysis further indicated that most of these putative NDAB contained genes for As(III) oxidation and nitrate reduction, confirming their roles in NDAO. The identification of novel putative NDAB expands current knowledge regarding the diversity of NDAB. The current study also suggests the proof of concept of using DNA-SIP to identify the slow-growing NDAB.