Hexavalent chromium [Cr(VI)], a widespread pollutant, is released into the environment from industrial processes and exhibits mutagenic and carcinogenic effects on biological systems [8, 10]. Biotransformation of Cr(VI) to relatively nontoxic trivalent chromium [Cr(III)] by chromium-resistant bacteria (CRB) [1, 5, 7, 12] has offered an economical as well as eco-friendly option for chromate detoxification and bioremediation [11]. Microbial reduction of Cr(VI) occurs either directly (enzymatically) or indirectly through production of hydrogen sulfide that precipitates Cr(VI) as chromium sulfide (Cr2S3) [8]. Enzymatic reduction of chromate by CRB proceeds aerobically, anaerobically, or both. In aerobic organisms, chromate reductase activity is mostly localized in the cytosolic fraction of the cell [4] while under anaerobic reduction; chromate is reduced in the membrane [18]. Reduction of hexavalent chromate is a redox reaction and requires supply of electrons. The bacterial enzymes responsible for direct reduction of chromate have not been well characterized to date. Studies using partially purified chromate reductase from Pseudomonas ambigua G-1 [15] and P. putida MK1 [13] have indicated Cr(VI) reduction using NADH or NAD(P)H as a source of electrons. In Bacillus sp. ES 29, chromate reducing activity was localized in the soluble fraction of cell-free extract (CFE), which utilizes NADH as the sole electron donor [1].

Isolation of chromium-resistant bacteria from naturally occurring chromium percolated serpentine outcrops of Andaman, India has been reported, and their potential to reduce chromate during growth was assessed. Whole cell suspension of Bacillus sphaericus AND 303, the most potent strain, reduced chromate using glucose as an effective electron donor [12]. The present study was envisaged to elucidate the in vitro reduction of Cr(VI) by CFEs of B. sphaericus AND 303.

Materials and Methods

Microorganism and culture condition

Bacillus sphaericus AND 303 (MTCC 6512), a chromate-resistant and reducing bacterium [12] isolated in this laboratory from serpentine soil of Andaman, India, was used throughout this study. The bacterium was grown in Peptone Yeast Extract Glucose medium amended with 1.0 mM Cr(VI) (as K2CrO4) at 30°C for 24 h under continuous shaking (120 rpm).

Preparation of cell-free extracts

CFEs of B. sphaericus AND 303 were prepared following the modified procedure of Wang and Xiao [17]. Cells from overnight-grown culture were harvested, washed, and sonicated in phosphate buffer (pH 7.0) at 5% of the original culture volume using an ultrasonic probe (150 kHz) for 20 min. The sonicate was centrifuged (6000 g, 4°C, 10 min) and the supernatant was filtered (0.22 μm) to produce the crude CFE. The CFE was subsequently centrifuged (4°C) at 12,000 g (10 min), 32,000 g (20 min), and 1,50,000 g (40 min) to yield the supernatants S12, S32, and S150 and the corresponding pellets P12, P32, and P150, respectively. Chromate reduction experiments were performed using freshly prepared CFEs.

Chromate reductase assay

Chromate reductase activity of CFE was assayed following the procedure of Camargo et al. [1]. The reaction mixture (1.0 mL) contained 200 μM K2CrO4 in 0.6 mL of 0.2 M phosphate buffer (pH 7.0). An aliquot of 0.4 mL of CFE was added as the enzyme to initiate the reaction. Reduction of Cr(VI) was measured by estimating the decrease in Cr(VI) in the reaction mixture after 30 min of incubation at 30°C. One unit of Cr(VI) reductase activity was defined as the amount of enzyme that converts 1.0 μ M Cr(VI) per min at 30°C. Cr(VI) was quantified colorimetrically using 1, 5-diphenylcarbazide as the complexing reagent [13, 16]. Protein was estimated by the folin-phenol method of Lowry et al. [9] using bovine serum albumin as the standard.

Results

The cell-free extracts (CFEs) of B. sphaericus AND 303 fractionated into soluble (S12, S32, and S150) and insoluble (P12, P32, and P150) fractions were tested for Cr(VI) reduction. All soluble fractions reduced Cr(VI) readily under aerobic condition without the presence of any additional electron donors. However, reductase activity was negligible to almost nil in suspended pellet fractions. The chromate reduction efficiency of CFEs obtained from cells pre-grown in the absence or presence of 1.0 mM Cr(VI) was almost equal irrespective of their growth conditions (Table 1). The soluble S150 fractions from both sources were most active and reduced 56.2% and 57% of Cr(VI), respectively, in 30 min. However, fraction S32 was not inferior to S150. The active soluble fractions, when treated at 15 p.s.i. for 15 min, failed to show any chromate reduction. A soluble component of the cell and not the membrane fraction of B. sphaericus AND 303, therefore, appeared to be responsible for Cr(VI) reduction under aerobic condition. Characterization of chromate reductase of CFE was based on soluble fraction S150 only, prepared from cells pre-grown in Cr(VI)-free medium.

Table 1 Localization of chromate reductase activity in cell fractions of Bacillus sphaericus AND 303
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Time course of Cr(VI) reduction by the soluble fraction S150 at an initial Cr(VI) concentration of 200 μM shows that the extent of reduction was rapid during the first 10 min of incubation and continued till 30 min when more than 55% of Cr(VI) was reduced (Fig. 1). In subsequent experiments Cr(VI) reduction was, therefore, measured after 30 min of incubation. A gradual increase in Cr(VI) reduction was obtained with increasing concentration of S150 and the optimum concentration was found to be 0.4 mL of the fraction, which was equivalent to 4.56 mg protein/mL.

Fig. 1
figure 1

Chromate reduction by cell-free extract (S150) of Bacillus sphaericus AND 303 as a function of time. [The assay mixture (1.0 mL) contained 200 μM Cr(VI) in 0.2 M phosphate buffer (pH 7.0) and incubated at 30°C.]

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The effect of initial concentration of chromate on reductase activity of CFE was determined at a concentration range of 0 – 500 μM of Cr(VI). The specific chromate reduction activity increased rapidly with increase in initial Cr(VI) level up to 300 μM, beyond which the activity slowed but continued until 500 μM Cr(VI) (Fig. 2A). The kinetics of Cr(VI) reductase activity fit well with the linearized Lineweaver-Burk plot (Fig. 2B), and the Km and Vmax values thus obtained from the Lineweaver-Burk plot were 158.12 μM Cr(VI) and 1.432 μmol Cr(VI)/mg protein/min, respectively.

Fig. 2
figure 2

Effect of initial Cr(VI) concentration on chromate reduction (A) and linearized Lineweaver-Burk plot for Cr(VI) reduction by cell-free extract (S150) (B). [The assay mixture (1.0 mL) contained increasing concentration of Cr(VI) in 0.2 M phosphate buffer (pH 7.0), 0.4 mL of CFE; incubation 30°C for 30 min.]

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The effect of pH on Cr(VI) reduction by crude CFE (S150) was determined at a pH range of 4.0 – 8.0 using citrate phosphate (4.0 to 6.5), sodium phosphate (6.0 to 7.5), and Tris-HCl (7.0 to 8.0) buffers of overlapping pH. The Cr(VI) reductase activity was maximum at pH 6.0, and it declined sharply on either side of the pH scale. Similarly, the optimum temperature for Cr(VI) reduction by the soluble fraction S150 of B. sphaericus AND 303 was 30°C. However, Cr(VI) reductase activity of S150 was found to be heat labile, and 80% of the enzyme activity was lost when S150 was treated at 70°C for 15 min.

An increase in Cr(VI) reductase activity of CFE (S150) was recorded with addition of NADH (0.1 μM), glucose, and yeast extract (1.0 g/L each) as electron donors, and the activity was further increased with increasing concentration of the respective electron donors. An increase of 28% activity over control was recorded when NADH concentration was raised by 10-fold. However, the presence of additional metal cations, Ni(II), Cu(II), and Cd(II) inhibited Cr(VI) reduction activity (Table 2). Cu(II) and Cd(II) in particular caused nearly 70% inhibition at 100 μM. Co(II) was apparently noninhibitory, causing about 7% loss of activity at 100 μM.

Discussion

Bacillus sphaericus AND 303 has been reported to reduce chromate under aerobic condition [12]. The present findings with soluble fractions (S12, S32, and S150) of CFE of B. sphaericus AND 303 have clearly indicated the localization of Cr(VI) reductase activity solely in the soluble component of the cell (Table 1). Furthermore, the reductase activity of Bacillus AND 303 has been found to be a constitutive one, which is in conformity with the earlier studies on aerobic chromate reduction by constitutive soluble reductases [2, 17] from chromium-resistant Pseudomonas putida MK1 [13] and Bacillus ES 29 [1].

The optimal reductase activity of the crude soluble fraction (S150) was obtained at an enzyme concentration equivalent to 4.56 mg protein/mL and at 300 μM Cr(VI) (Figure 2A). On the other hand, temperature and pH optima of chromate reductase in in vitro condition coincide closely with those of optimal growth conditions of the organism [12]. However, differences in such parameters were recorded with E. coli [14], P. putida MK1 and P. putida PRS2000 [13]. The crude enzyme in the present study appeared to be heat labile with a significant loss (80%) of activity at 70°C. CFE of P. putida MK1 was reported to retain reductase activity even at 80°C [13]. Comparison of enzyme kinetics revealed that the Km value obtained with S150 fraction (Fig. 2B) of Bacillus AND 303 was nearly equal to that of Bacillus subtilis 168t+ [6], but the maximal velocity of Cr(VI) reduction by AND 303 was recorded to be highest among other chromate reducing Bacilli [1, 3].

The soluble fraction (S150) of CFE of Bacillus AND 303 was capable of reducing chromate in the absence of any added electron donor, which supported the concept that endogenous electron reserves can act as electron donors during Cr(VI) reduction [14]. The enzyme activity of AND 303 was enhanced further in the presence of additional NADH, glucose, or yeast extract. Similar enhancement of Cr(VI) reduction by external NADH has also been demonstrated in E. coli ATCC 33456 [14]. However, Cr(VI) reductase activity of Pseudomonas aeruginosa and P. putida was NADH or NAD(P)H-dependent [5, 13]. Inhibitory effects of metal ions on Cr(VI) reduction by intact cells [12] and CFEs [13] have been reported. Similarly, Cr(VI) reduction by CFE (S150) of B. sphaericus AND 303 was inhibited by 100 μM Ni(II), Cd(II), and Cu(II), whereas Co(II) at a similar concentration failed to show any significant inhibition (Table 2). On the contrary, chromate reductase activity of CFE of Bacillus ES 29 was stimulated by 1.0 mM Cu(II) [1].

Table 2 Effect of metal cations on chromate reductase activity by CFE (S150) of Bacillus sphaericus AND 303.
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