Cr(VI) reduction by Enterobacter sp. DU17 isolated from the tannery waste dump site and characterization of the bacterium and the Cr(VI) reductase

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Highlights

  • Newly isolate Enterobacter sp. DU17 reduced Cr(VI) extracellularly.

  • Complete reduction of 100 mg L−1 Cr(VI) noticed within 16 h in presence of glucose.

  • Maximum homology of Cr(VI) reducing gene was established with flavoprotein.

  • Strain DU17 showed low multiple antibiotic resistance (MAR) index.

Abstract

Out of nineteen bacteria screened from the tannery waste dump site, the most effective isolate, strain DU17 was selected for Cr(VI) reduction process among the non-pathogenic once. Based on 16S rRNA gene sequence analysis, the bacterium was identified as Enterobacter sp. DU17. Its amplified Cr(VI) reductase gene showed maximum homology with flavoprotein of Enterobacter cloacae. Enterobacter sp. DU17 reduced Cr(VI) maximally at 37 °C and pH 7.0. Various co-metals, electron (e) donors and inhibitors were tested to study their effect on Cr(VI) reduction. In presence (0.2% each) of glucose and fructose, Enterobacter sp. DU17 reduced Cr(VI) completely after 16 and 20 h, respectively. Since the concentration of total Cr was invariable after remediation as detected through AAS analysis, this experiment disclosed that responsible operation was associated with extracellular Cr(VI) reduction process rather than uptake mechanism. Multiple antibiotic resistance index of 0.08 for this bacterium was very low as compared to standard risk assessment value of 0.20. With high Cr(VI) reducing capability, non-pathogenicity and antibiotic sensitivity, Enterobacter sp. DU17 is found to be very efficient in removing Cr(VI) toxicity from the environment.

Introduction

Due to anthropogenic activities, elevated concentration of chromium (Cr) in environment has become a major concern for the scientists worldwide. Cr(VI) is considered to be highly toxic and classified as a Group ‘A’ human carcinogen because of its mutagenic, carcinogenic and teratogenic nature (Kathiravan et al., 2011, Mishra et al., 2012). Cr(VI) exists in oxoanionic form in solutions and shows structural similarity with phosphate/sulphate anions; hence enters the cells through membrane using phosphate/sulphate anionic transporters (O’Brien et al., 2003, Ramırez-Dıaz et al., 2008). Inside the cell, cysteine, ascorbate and glutathione like molecules and glutathione reductase and lipoyl dehydrogenase like metabolic enzymes vicariously stimulate reduction of Cr(VI) into Cr(III) via highly active intermediates of Cr(V) and Cr(IV) (O’Brien et al., 2003, Cheung and Gu, 2007, Eswaramoorthy et al., 2012). During this reaction, one of its intermediates, Cr(V) gets reconverted into Cr(VI) and releases electrons (e) to substantially generate reactive oxygen species (ROS). The ROS is the main cause of oxidative damage of cell constituents like DNA, protein and lipid (Leonard et al., 2003, Cheung and Gu, 2007).

Certain plants and microbial isolates of various groups like bacteria, yeast, fungi, algae and protozoa have wide range of adaptability against Cr(VI) (Cervantes et al., 2001, Polti et al., 2011). Their mechanisms of resistance include enzymatic reduction (chromosome encoded), Cr(VI) efflux system (plasmid encoded), biosorption, bioacculmulation, SOS response, enzymatic DNA repair system, etc. (Ramirez-Diaz et al., 2008). Among them, some of the resistance mechanisms are being emphasized in an effective way to remove metal toxicity using bioremediation approaches.

To date, a variety of Cr(VI) resistant bacteria have been screened to clean up the Cr(VI) toxicity (Colin et al., 2012). Much studies have been carried out on enzyme based reaction, which brings about ‘safe’ reduction of Cr(VI) by mitigating the redox cycling (Eswaramoorthy et al., 2012). Moreover, the transformed product Cr(III) is found to be nontoxic on account of its insolubility at biological pH and less permeability to biological membrane (Poljsak et al., 2010). Various aspects are being considered to achieve maximum level of Cr(VI) reduction but biosafety of those microbial agents in terms of its antibiotic resistivity and infectious nature is not much studied, which may generate considerable medical problems and therapeutic hurdles. Those variants may attract by their high Cr(VI) removal capability but their biosafety, in terms of reducing health (by infectious diseases) in human being and animals is of great concern (Allen et al., 2010, Garcia-Armisen et al., 2011).

Therefore, in the present study, it was considered necessary to screen highly Cr(VI) reducing bacterial strain that would also be non-pathogenic. Bacterial Cr(VI) reducing capability was characterized under various factors to examine its efficacy for the bioremediation purpose.

Section snippets

Sample collection and isolation of Cr(VI) resistant bacteria

The soil sample along with healthy plant roots (of Malvaceae family) were collected from the rhizosphere of tannery waste dump site located at Jajmau, Kanpur, India and maintained at 4 °C until used for microbial analysis. Soil pH was slightly basic (i.e. pH 7.59). Furthermore, the soil sample was digested with HNO3 and HCl (3:1) followed by dilution with double distilled water to detect the heavy metals using atomic absorption spectroscopy (AAS) (AA-6300 Shimadzu, Japan). The concentrations of

Screening and selection of bacteria

A total 19 bacterial strains (DU1-DU19) resistant to Cr(VI) were isolated from tannery waste dump site. Different strains showed different MIC of Cr(VI) ranging from 100 to 1100 mg L−1. Cr(VI) reduction in LB broth was carried out in all screened variants. Only 10 strains reduced Cr(VI) and other 9 strains showed negative result for the same. Previous studies also reported that some bacteria resistant to Cr(VI) were unable to reduce Cr(VI), suggesting that Cr(VI) tolerance did not relate to its

Conclusions

Most of the earlier reports on Cr(VI) bioreduction have been focused only for its optimization, whereas variants biosafety is highly ignored. Considering this, Enterobacter sp. DU17 was isolated from the Cr(VI) contaminated tannery waste dump site and selected on the basis of its non-pathogenicity, for the first time. Resistivity against different co-metal ions also makes bacterium suitable for bioremediation in multiple metal contaminated sites. Enterobacter sp. DU17 reduced Cr(VI) completely

Acknowledgements

This work was supported by R & D grant of University of Delhi (DU) and fellowship to ZR by CSIR, New Delhi. The authors are also highly thankful to Mr. Siva P.K. Chetri and colleges of Lab no. 28 of Department of Botany, DU for technical helps.

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