Hexavalent chromium reduction by chromate-resistant haloalkaliphilic Halomonas sp. M-Cr newly isolated from tannery effluent

Abstract

The current study aimed to isolate and characterize a chromate-resistant bacterium from tannery effluent, able to reduce Cr(VI) aerobically at high pH and salinity. Environmental contamination by hexavalent chromium, Cr(VI), presents a serious public health problem. Enrichment led to the isolation of 12 bacteria displaying different degrees of chromate reduction. Phenotypic characterization and phylogenetic analysis based on 16S rDNA sequence comparison indicated that the most potent strain belonged to the genus Halomonas. The new strain designated as Halomonas sp. M-Cr was able to reduce 82% of 50 mg L^-1 Cr(VI) in 48 h, concomitant with discolouring of yellow colour of the medium and formation of white insoluble precipitate of Cr(III). It exhibited growth up to 3500 mg L^-1 Cr(VI), 20% NaCl and showed strong Cr(VI) reduction under alkaline condition, pH 10. Scanning electron microscopy revealed precipitation of chromium hydroxide on bacterial cell surfaces, which showed characteristic peak of chromium in energy-dispersive X-ray analysis. Plackett-Burman design was used to evaluate the influence of related parameters for enhancing Cr(VI) reduction. Glucose, yeast extract and KH₂PO ₄ were confirmed as significant variables in the medium. Data suggest Halomonas sp. M-Cr as a promising candidate for bioremediation of Cr(VI) contaminated effluents particularly in saline and alkaline environments. Up to our knowledge, this is the first report on isolation of haloalkaliphilic Halomonas sp. from tannery effluent.

Keywords: Cr(VI) reduction; Halomonas sp. M-Cr; Plackett–Burman design; bioremediation; haloalkaliphilic; scanning electron microscopy.

Figure 1.

Phylogenetic tree based on 16S rDNA gene sequence, and reference sequences extracted from the GenBank Database, showing the phylogenetic relationship of Halomonas sp. M-Cr within representative species of the genus Halomonas. Numbers in bracket represents GenBank accession numbers.

Figure 2.

Growth and Cr(VI) reduction efficiency of Halomonas sp. M-Cr grown in LB broth, pH 10, in the absence and presence of 50 mg L⁻¹ Cr(VI) and incubated at 30 °C under shaking at 120 rpm.

Figure 3.

Chromate reduction by Halomonas sp. M-Cr. Cells were grown in alkaline LB medium (pH 10) amended with 50 mg L⁻¹ Cr(VI), and incubated at 30 °C with shaking at 120 rpm. Complete Cr(VI) reduction was achieved within 120 h and white-precipitate was visible at the bottom of the flask (A). Cell-free control was used to monitor any abiotic reduction of Cr(VI) (B).

Figure 4.

Effect of pH on growth and chromate reduction by Halomonas sp. M-Cr after incubation for 48 h with 50 mg L⁻¹ Cr(VI), NaCl 0.5%, and agitation of 120 rpm, 30 °C.

Figure 5.

Effect of different concentrations of NaCl on growth and chromate reduction by Halomonas sp. M-Cr growing in LB medium of pH 10 with initial Cr(VI) concentration of 50 mg L⁻¹ Cr(VI) after incubation for 48 h at 30 °C.

Figure 6.

SEM micrographs of Halomonas sp. M-Cr cells grown in: (A) LB medium without Cr(VI) (control); (B) LB medium amended with 50 mg L⁻¹ Cr(VI) for 24 h; (C) Cr(III) precipitates found as discrete particles bound to the cell surface (arrows); (D) Amorphous Cr(III) hydroxide precipitates not attached to cells are also present (arrows), the largest precipitates were slightly rounded.

Figure 7.

EDX spectrum analysis of amorphous precipitates that surrounded Halomonas sp. M-Cr cell surfaces during Cr(VI) reduction (A). An EDX spectrum from the dense particles generated a large Cr peak, indicating that it is most likely an amorphous Cr(III) hydroxide. Inset: SEM images of Halomonas sp. M-Cr cells and precipitates. The whole area was analysed with EDX.

Figure 8.

Positive and negative influence of different variables on Cr(VI) reduction by Halomonas sp. M-Cr based on the result of Plackett–Burman design.