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

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 6 days in Cr(VI) spiked Luria Bertani medium with unaffected bacterial growth at an initial C(VI) concentration of 50 mg 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 12 h of exposure at 100, 200, 400 mg L-1 concentrations, indicated by non-significant cell death (propidium iodide positive cells). However, at 800 and 1000 mg L-1 Cr(VI) concentration, toxicity (cell death) was observed after 12 h 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.

Evaluation of the cytotoxicity and interaction of lead with lead resistant bacterium Acinetobacter junii Pb1.

This study explores the potential of lead resistant bacterium Acinetobacter junii Pb1 for adsorption/accumulation of lead using various techniques. In the present work, growth of A. junii Pb1 was investigated in the presence of a range of Pb(II) concentrations (0, 100, 250, 500, and 1000 mg l-1). Lead was found to have no toxic effect on the growth of A. junii Pb1 at 100 and 250 mg l-1 concentrations. However, further increase in Pb(II) concentration (500 mg l-1) showed increase in lag phase, though growth remained unaffected and significant growth inhibition was observed when concentration was increased to 1000 mg l-1. Same was confirmed by the observations of flow cytometry. Further, the effect of Pb(II) on A. junii Pb1 was evaluated by using fluorescence microscopy, spectrofluorimetry, and flow cytometry. The spectrofluorimetry and fluorescence microscopy results revealed the accumulation of Pb(II) inside the bacterial cells as evident by green fluorescence due to lead binding fluorescent probe, Leadmium Green AM dye. Flow cytometry observations indicate an increase in cell size and granularity of exposure to lead. Thus, present work provides a new understanding of Pb(II) tolerance in A. junii Pb1 and its potential use in remediation of lead from contaminated soil.