Reduction of Hexavalent Chromium [Cr(VI)] by Heavy Metal Tolerant Bacterium Alkalihalobacillus clausii CRA1 and Its Toxicity Assessment Through Flow Cytometry.

Abstract

A chromate-resistant bacterial strain was isolated from tannery effluent; based on morphological, biochemical, and 16S rRNA gene sequencing, it was identified as Alkalihalobacillus clausii and designated A. clausii CRA1. It was found to be halophilic, alkaliphilic, and resistant to multiple heavy metals like Cr(VI), Cd(II), As(II), Pb(II), Ni(II), Hg(II), Cu(II), Zn(II), and Fe(II). The strain was found to reduce 72% of chromate in 6days in Cr(VI) spiked Luria Bertani medium with unaffected bacterial growth at an initial C(VI) concentration of 50mg L-1. Chromate reductase activity of culture supernatant (cultivated in LB broth) and cell lysate of the bacterium was found to be 23 and 43U, where 1U is µmol of Cr(VI) reduced/min/mg protein. Flow cytometry studies revealed that no significant effect of Cr(VI) on cell viability was observed till 12h of exposure at 100, 200, 400mg L-1 concentrations, indicated by non-significant cell death (propidium iodide positive cells). However, at 800 and 1000mg L-1 Cr(VI) concentration, toxicity (cell death) was observed after 12h of exposure. FACs studies also indicated that exposure to Cr(VI) increases cell size and cell granularity, which was also confirmed in SEM and TEM images of Cr(VI) treated cells. The presence of Cr(III) species in EDX spectra of Cr(VI) treated cells confirms that reduction of Cr(VI) to Cr(III) is the primary mechanism of Cr(VI) removal by the bacterium. Therefore, the bacterium A. clausii has potential for application in chromate removal from industrial waste effluents.