Hexavalent chromium reduction and 16S rDNA identification of bacteria isolated from a Cr (VI) contaminated site
A Das, S Mishra
16s rdna, brevibacterium casei, detoxification, hexavalent chromium
A Das, S Mishra. Hexavalent chromium reduction and 16S rDNA identification of bacteria isolated from a Cr (VI) contaminated site. The Internet Journal of Microbiology. 2008 Volume 7 Number 1.
A Gram-positive, hexavalent chromium [chromate: Cr (VI)]-resistant & reducing bacterium, isolated from sukind chromite mines, jajpur, India, was identified as a
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 conditions
Four 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, 100l/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) analysis
Chromate-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 (AR) and 330 μl of 3 M H2SO4 was added. The reaction mixture was set up in an Eppendorf tube containing the following: 200 μl sample or standard sodium chromate solution, 400 μl 20 mM MOPS-NaOH buffer pH 7.0, 33 μl 3 M H2SO4, 40 μl 0.25% (w/v) DPC, and 327 μl distilled water. Spectrophotometric measurements were made immediately at 540 nm.
Identification of selected strains
Gram staining of bacterial strain was carried out using established methods (Collins and Patricia 1984). For PCR amplification, a small amount of a bacterial colony was resuspended in 100 μl of sterile deionised water (SDW), mixed and lysed at 70°C (10 min). Crude lysate (0.2 μl) was added to 19.8 μl SDW and used as a PCR template. Universal bacterial 16S rDNA gene primers pA (5′- AGAGTTTGATCCTGGCTCAG-3′) and pH′ (5′-AAGGAGGTGATCCAGCCGCA- 3′) were used to amplify the ~1.5 kb 16S rDNA gene fragment (Edwards et al. 1989). Sequence data was aligned and analyzed for finding the closest homology for the microbe.
Preliminary screening for Cr (VI)-reducing activity
Strain was grown aerobically in PYE at 30°C overnight. For anaerobic Cr(VI) reduction tests, 45 ml of PYE in 50 ml serum bottles was degassed with O2-free N2 (10–20 min). Each bottle was inoculated using a syringe with 10% (v/v) of the starter culture and incubated statically at 30°C. Potassium dichromate (100 μM) was added after 2 h. Samples (1 ml) were withdrawn periodically and the bacterial density was determined as OD600 prior to harvest by centrifugation at room temperature. The cell pellet was suspended in 1 ml of isotonic saline [0.85% (w/v) NaCl] for protein assay and the supernatant was assayed for residual Cr (VI).
Effect of initial Cr (VI) concentration and initial cell density on Cr (VI) reduction by strain APD15
Strain APD15 (deposited in the National Center for Biotechnology Information
Results and Discussion
Cr (VI)-reducing activity of isolates
The Cr (VI)-reducing activity of 4 isolates was investigated as shown in Table 1. Strains APD1, APD8, APD15, and APD39 were selected for further study, with a maxi-mum removal of 94% of the Cr (VI) (APD15). These bacteria were Gram-positive, non spore-forming rods. On PYE agar, colonies were 1–3 mm in diameter, circular, low convex with an entire margin, opaque, and moist.
16S RNA identification of selected strains
Based on nucleotides homology and phylogenetic analysis the Microbe (Sample: APD15) was detected to be
Chromate reduction activity by Brevibacterium casei (APD15)
The inoculum of the bacterial strains cultured overnight was used for this experiment. Culture flasks (150 ml) with a final volume of 100ml supplemented with (10-50mg/L) of Cr (VI) were inoculated with 2ml of inoculums for 24 hour. The growth kinetics of bacteria is characterized as initial lag phase, second exponential phase, stationary phase and death phase. In this experiment it is observed that lag phase is increasing with increased initial Cr (VI) concentration [Fig 2 & 3]. It is basically due to inhibitory effect of higher chromium concentration on the growth of the organism. Each organism has a specific resistance at a specified growth condition. As the initial age of the inoculum was fixed at 24 hours the acclimatization period at varying chromium concentration will not remain same. Hence the following behavior is observed. The chromium-resistant bacteria isolate exhibited reduced bioaccumulation when cells were in stationary phase. At higher concentrations the growth of the bacteria is inhibited due to fixed amount of inoculum for all the different concentration of Cr (VI) considered in the experiment.
Bacterial chromate reduction has been reported under aerobic (Ishibashi et al. 1990; Cooke et al. 1995; Wang and Xiao 1995), anaerobic (Romanenko and Koren‘kov 1977; Lebedeva and Lyalikova 1979), or both (Llovera et al. 1993; Shen and Wang 1993) conditions. In this study, a newly isolated Cr (VI)- reducing bacterium was identified as a