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In this study, the Cr(VI)-resistant bacterium LYB-23 was isolated from waste chromium residues in Sichuan and identified as Bacillus velezensis. The chromium tolerance of LYB-23 was improved by domestication (continuously increasing the chromium concentration in the medium from 60 to 200 mg/L). Subsequently, we screened a mutant strain, B. velezensis SA-23, using atmospheric pressure room temperature plasma (ARTP) mutagenesis for 90 s based on the domesticated strain. The minimum inhibitory concentration of B. velezensis on chromium increased from 80 mg/L for the original strain to 200 mg/L for the domesticated strain and to 400 mg/L for the mutant strain. The Cr(VI) reduction experiment results show that SA-23 can completely reduce 100 mg/L Cr(VI) within 24 h, and the reduction rate of 200 mg/L Cr(VI) reached 78.45% within 72 h. Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) analysis showed that ARTP mutagenesis did not change the type of functional groups on the surface of bacteria after reductive adsorption of Cr(VI) and the crystalline state of Cr(III); however, mutagenesis changed the biological morphology of bacteria and greatly enhanced the role of phosphate groups in the process of adsorption and reduction of Cr(VI). This experiment for the first time used the combined domestication and ARTP mutagenesis to effectively improve the tolerance of bacteria to Cr(VI) and their reduction adsorption efficiency, which proved to be an efficient method for the breeding of environmental remediation engineering bacteria and for promoting the application of microbial remediation. [Display omitted] • The MIC of LYB-23 for Cr(VI) increased from 80 to 200 mg/L after domestication. • A mutant strain SA-23 was screened with MIC of 400 mg/L to Cr(VI). • The reduction rate of 200 mg/L Cr(VI) by SA-23 reached 78.45% within 72 h. • Mutagenesis significantly enhanced the function of phosphate groups of SA-23. • This study established a new breeding method of environmental remediation bacteria. [ABSTRACT FROM AUTHOR]