Hexavalent chromium reduction and 16S rDNA identification of bacteria isolated from a Cr (VI) contaminated site

摘要

A Gram-positive, hexavalent chromium [chromate: Cr (VI)]-resistant & reducing bacterium, isolated from sukind chromite mines, jajpur, India, was identified as a Brevibacterium casei(Gene Bank Accession Number: EU781952) by gene sequence homology. The strain(designated as APD15) could tolerate chromium up to a maximum concentration of 500 ppm, at optimum temperature and pH 300C and 7 for maximum chromium reduction. Agar supplemented with 100g Cr (VI)/ml as K2Cr2O7 and 0.5% (w/v) dextrose used as a carbon source. The results of the study indicated removal of more than 94% chromium (VI) by Brevibacterium caseidetermined by diphenylcarbazide colorimetric assay. Introduction Hexavlent Chromium is widely use in various industrial and viable processes, including mining, electroplating, leather tanning, petroleum refining, textiles inorganic chemicals and pulp production, and many other metal finishing industries (Wang and Xiao 1995) & is considered as a serious environmental pollutant. Chromium exists in the environment in several diverse forms such as trivalent [Cr (III)] and hexavalent [Cr (VI)], (Fendorf 1995) of which hexavalent chromium is a so-called carcinogen and a potential soil, surface water and ground water contaminant. Whereas it’s reduced trivalent form (Cr3 + ) is much less toxic, insoluble and a vital nutrient for humans. Cr (III) occurs naturally in the environment and is an essential nutrient required by the human body (Mishra & Das 2007). In India, Sukinda mines in Jajpur district of Orissa state witnesses a vast mining and mineral processing waste that are continuously discharged into open fields and are gradually becoming a source of Cr toxicity for human life, environment and animals and hence pose a serious threat to the inhabitants of this region. About 26 lakhs people residing on the banks of Brahmini River have fallen prey to water contamination due to Chromite mines discharged water which has been highlighted by the Blacksmith Institute. Hence there is an urgent need to reduce Cr (VI) contamination in this region. Recently, bioremediation of Cr (VI) has gained considerable consideration (Wang et al. 1989; Yamamoto et al. 1993; Middleton et al. 2003). Some microbial species can utilize Cr (VI) as a terminal electron acceptor in their respiratory process and transform Cr (VI) to less toxic Cr (III) compounds (Lovley and Phillips 1994; Shen et al. 1996). A number of these microorganisms, particularly bacteria, can reduce Cr and therefore detoxify it (Fuji et al. 1990). The present study describes a microbiological treatment for industrial effluent that may be suitable for processing Cr-contaminated waste. This study proposes a remediation route for detoxification of Cr (VI) using an indigenous microorganism. Materials and Methods Bacterial strains and growth conditionsFour strains used in this study were originally isolated by Mr. A.P. Das sukind chromite mines, jajpur, India. Bacterial strains, resistant to Cr (VI), were isolated from the soil using the serial dilution technique in PYE medium (Peptone, Yeast extract). Agar supplemented with 100g Cr (VI)/ml as K2Cr2O7 and 0.5% (wt/vol) dextrose served as carbon source. The pH was maintained at 7±0.2 by using HCl or NaOH. The isolates are tested for their chromate tolerance at different concentrations (12.5, 25, 50, 75, 100?l/ml) of hexavalent chromium supplemented as K2Cr2O7. Significant growth of the specific bacterial species in the presence of 100 mg Cr (VI)/l in PYE medium during two-day incubation at 30°C, were considered as Cr (VI) resistant. A single strain was capable of growing at this condition & was selected for further experiments.Cr (VI) analysisChromate-reducing activity was estimated as the decrease in chromate concentration in supernatant with time using the Cr(VI)-specific colorimetric reagent 1,5-diphenylcarbazide (DPC), prepared in acetone/H2SO4 to minimize deterioration (Urone 1955) as follows: DPC (0.025 g) was dissolved in 9.67 ml acetone