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, 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.

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.

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.

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culture and incubated statically at 30°C. Potassium dichromate (100 μM) was added after 2 h. Samples (1 ml) were withdrawn periodically and the bacterial density was determined as OD600 prior to harvest by centrifugation at room temperature.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).

Strain APD15 (deposited in the National Center for Biotechnology Information (NCBI) Genbank; accession no. EU781952) was precultured aerobically as described above. For aerobic Cr (VI) reduction tests, 45 ml of PYE in 250 ml flasks was inoculated with 10% (v/v) of the primary inoculum and incubated with shaking at 30°C with parallel anaerobic tests as described above but with various concentrations of sodium chromate added immediately after incubation. Growth, and residual chromate in the sample supernatants, was determined as above. To determine the effect of initial cell density, cells were precultured aerobically (30°C, 24 h) in PYE (40 ml). Cells were harvested by centrifugation at 4°C (15 min), and kept on ice until use (usually within 1 h). Anaerobic Cr(VI) reduction tests were performed as described above with initial cell densities of 2.7×106, 5.3×107, 1.4×108, 1.0×109, and 2.4×109 cells/ml. potassium dichromate was added to 100 μM and the cells were incubated at 30°C. Residual chromate in the sample supernatants was determined as described above.

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.

Figure 1

Table 1: Loss of hexavalent chromium [Cr (VI)]

Based on nucleotides homology and phylogenetic analysis the Microbe (Sample: APD15) was detected to be Brevibacterium casei (GenBank Accession Number: EU781952).The 16S rDNA nucleotide sequences from NCBI gen bank were compared with known sequences in the EMBL database using ClustalW2 to identify the most similar sequence.

Figure 2

Fig 1: phylogenetic tree of with similar sequence from NCBI genbank

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.

Figure 3

Fig-2: Cr (VI) degradation kinetics varying chromium concentration

Figure 4

Fig-3: Biomass growth at varying chromium concentration