Removal efficiency of Cr6+ by indigenous Pichia sp. isolated from textile factory effluent.

Resistance of the indigenous strains P. jadinii M9 and P. anomala M10, to high Cr(6+) concentrations and their ability to reduce chromium in culture medium was studied. The isolates were able to tolerate chromium concentrations up to 104 μg mL(-1). Growth and reduction of Cr(6+) were dependent on incubation temperature, agitation, Cr(6+) concentration, and pH. Thus, in both studied strains the chromium removal was increased at 30 °C with agitation. The optimum pH was different, with values of pH 3.0 and pH 7.0 in the case of P. anomala M10 and pH 7.0 using P. jadinii M9. Chromate reduction occurred both in intact cells (grown in culture medium) as well as in cell-free extracts. Chromate reductase activity could be related to cytosolic or membrane-associated proteins. The presence of a chromate reductase activity points out a possible role of an enzyme in Cr(6+) reduction.

1. Introduction

Heavy metals found in wastewaters are harmful to the environment and their effects on biological systems are very severe. Chromium is one of the most widely used metals in industry, such as steel production, alloy preparation, wood preservation, leather tanning, metal corrosion inhibition, paints pigments, metal plating, tanning, and other industrial applications [1]. Chromium exists in several oxidation states from Cr2+ to Cr6+. In nature, trivalent and hexavalent forms are the dominant oxidation states. The toxicity of chromium is dependent on its oxidation state, Cr3+ is rather benign and easily adsorbed in soils and waters; whereas Cr6+, which is the toxic form, is not readily adsorbed and is soluble [2]. Thus, Cr6+, a carcinogenic element, is highly toxic to all forms of life but Cr3+, an essential micronutrient for many higher organisms, is relatively insoluble in water and 100 times less toxic than Cr6+ [3]. Chromium hexavalent toxicity is believed to be caused by the negatively charged chromate oxyanion, which can be easily transported into microbial cells. Once inside the cells, the oxyanion is believed to undergo immediate reduction reactions leading to the formation of various reactive intermediates, which are harmful to the cell organelles, proteins, and nucleic acids [4].

For that reason, it is important to develop an innovative, low cost, and ecofriendly method for the toxic heavy metal removal from the wastewater, instead of the conventional physical-chemical ones [1, 5]. Several microorganisms have the exceptional ability to adapt to and colonize the noxious metal-polluted environments. These microorganisms have developed the capabilities to protect themselves from heavy metal toxicity by various mechanisms such as adsorption, uptake, methylation, oxidation, and reduction.

Yeasts are known for playing an important role in the removal of toxic heavy metals [4, 6, 7]. Furthermore, the occurrence of indigenous Cr6+ reducing eukaryotic microorganisms, including those not related with Cr6+ contamination, has emerged as an important nonconventional yeasts-based bioremediation method with significant biological relevance and biotechnological applications.

Microbial Cr6+tolerance and Cr6+reduction are independent events. However, for the Cr6+-reduction cells must tolerate Cr6+, otherwise the cell growth is inhibited. Some authors argue that the microbial reduction of Cr6+ can be considered as an additional mechanism of resistance to chromate, which is usually not encoded in plasmids [8]. The enzymatic biospeciation of Cr6+ to Cr3+ with eukaryotic microorganisms was reported in Candida maltose [9], C. utilis [10], fungi Hypocrea tawa [11], and Aspergillus [12]. But it was not possible to continue with the purification and characterization of the protein involved, therefore available information is scarce. In this context, the study of specific chromate reductases is meaningful to understand the cellular mechanisms in future bioremediation processes.

The present study deals with the ability of P. jadinii M9 and P. anomala M10 to grow and remove chromium in batch cultures and using cell-free extracts. The effects of different factors on Cr6+ removal, including pH, temperature, agitation, and initial Cr6+ concentration were also considered and optimum removal parameters were established.

2. Materials and Methods

2.1. Yeast Strains and Culture Conditions

Chromate-resistant yeasts Pichia jadinii M9 and Pichia anomala M10, previously isolated from textile factory effluents (Tucumán, Argentina) were used [13]. For the inocula, the yeast strains were grown in 500 mL-Erlenmeyer flasks containing 100 mL of Czapek malta medium using methodology described by Fernández et al. [13].

Chromium removal experiments were performed using YNB' medium amended with Cr6+ and inoculated with a constant biomass. YNB' medium was chosen based on previous assays that confirmed lower interferences of this medium during Cr-bioremediation and Cr6+-quantification by 1,5-diphenylcarbazide (DPC) [14]. YNB' composition (in g L−1) was 10 × yeast nitrogen base (YNB w/o amino acids and ammonium sulfate; Difco), 10% (v v−1); sucrose, 50; ammonium sulfate, 0.6; pH 5.0. All the experimental sets were performed on a rotary shaker (250 rev min−1) at 25°C in 250 mL Erlenmeyer flasks containing 50 mL of culture medium, unless otherwise stated.

The Cr6+ (as K2Cr2O7 or K2CrO4) stock solution (5,200 μg mL−1) was prepared in bidistilled water and filter-sterilized (0.2 μm-cellulose acetate membrane filter; Sartorius).

2.2. Effect of Cr6+ on Yeasts Growth

Chromate resistance test and growth curves were determined in YNB' medium supplemented with the desired Cr6+ concentration and without chromium (control). Growth was monitored at specific time intervals by biomass dry weight (BDW). Samples from culture were spun down at 10,000× g for 10 min. The distilled water suspended pellet was filtered through a 0.45 μm cellulose acetate membrane filter (Sartorius) and dried at 85°C until constant weight to determine BDW in g L−1 [13]. For determination of Cr6+ concentration, a miniaturized protocol was developed as follows: to 50 μL of sample supernatant, 50 μL of 0.2 N H2SO4 were added and the volume was made up to 2 mL with distilled water. After mixing with 40 μL of 5 mg DPC mL−1 acetone, the mixture was allowed to stand for 10 min and spectrophotometric determinations were performed at 540 nm (Beckman DU640) against a reagent blank. Cr6+ concentrations were quantified by the use of an external K2Cr2O7 standard with a 7-point calibration curve [14].

2.3. Factors Affecting Cr6+ Removal

To characterize the Cr6+-reduction efficiency by strains M9 and M10, the effects of temperature (10, 20, 25, 30°C), initial pH (3.0, 5.0, 7.0, 9.0), agitation (0, 150, 250 rev min−1), and initial Cr6+ concentration (26–104 μg mL−1) were investigated. Cr6+ reduction was studied in aerobic batch cultures. The following set of standard conditions was chosen as the starting point: 52 μg mL−1 of initial Cr6+ concentration, pH 5.0, 25°C and 250 rev min−1. Samples were withdrawn at defined times and analyzed for disappearance of Cr6+ as described above. In order to monitor any abiotic Cr6+ reduction, cell-free control experiments were carried out for each assayed condition.

2.4. Preparation of Cell-Free Extract and Enzymatic Determinations

To prepare the crude cell-free extract, the yeast cultures were grown in 200 mL YNB' medium for 48 h at 25°C with 52 μg mL−1 Cr6+ and without chromium (control). Cells were harvested by centrifugation at 10,000 ×g for 10 min. Pellets were washed twice with 50 mM phosphate-citrate buffer (pH 5.0) and suspended in the same buffer with protease inhibitor cocktail (SET1; Calbiochem) plus a volume of sterilized glass beads. Cells were disrupted by sonication for 5 min in cold environment conidtions (5 cycles: 59 seg on, 30 seg off; Sonics Vibra Cell VCX 130). The homogenate was centrifuged at 10,000 ×g for 10 min at 4°C to remove cell walls and unbroken cells. The supernatant filtered through a 0.2 μm cellulose acetate membrane filter was used as a crude extract and called cell-free extract (CFE). Decrease of chromate concentration by CFE was assayed after 30 min at 30°C using 50 μL of sample preparation in 0.25 mL reaction mixtures containing (to a final concentration): 50 mM phosphate-citrate buffer (pH 5.0), 26 μg mL−1 K2CrO4, 1 mM NADH; these concentrations were saturating and noninhibitory under these conditions. The reaction was started by addition of chromate to the reaction mixture. Hexavalent chromium was spectrophotometrically quantified, as previously described. Protein was determined using Bicinchoninic Acid Kit (BCA, Sigma), with BSA as standard.

3. Results and Discussion

3.1. Effect of Initial Cr6+ Concentration on Cells Growth

Cr6+ resistance of P. jadinii M9 and P. anomala M10 was evaluated by growth response of the strains under different concentrations of Cr6+. Growth curves of yeast isolates with or without Cr6+ were plotted (Figures 1(a), 1(b)). The cells grew well in the medium with a range of initial Cr6+ concentration of 26–104 μg mL−1. However, the growth curves of P. jadinii M9 and P. anomala M10 in the medium containing Cr6+ did not follow the same growth pattern as the control, indicating a possible toxic effect of Cr6+ on the cells. It was obvious that the growth of cells was heavily influenced by Cr6+ at a concentration of 104 μg mL−1 (biomass concentration drop a 63% and 56% for P. jadinii M9 and P. anomala M10, resp.), but it did not suppressed the cells growth. The experiments conducted with Cr6+ concentrations of 26, 52, 78 μg mL−1 had only slight effects on the growth (Figures 1(a), 1(b)). The P. jadinii M9 and P. anomala M10 strains completely reduced all Cr6+ concentrations tested; thus, overall efficiency of Cr6+ reduction (100%) was not affected by initial Cr6+ concentration. The highest concentration of Cr6+ (104 μg mL−1) that allowed growth and was completely reduced by P. jadinii M9 and P. anomala M10 was much higher than concentrations commonly found to be reduced by bacteria [15], yeasts [9], and filamentous fungi [16]. However, it is important to consider that the microbial chromate-resistance and chromate-reduction parameters are correlated with medium composition and cell density [13]. The real toxicity of Cr6+ could be masked or underestimated due to complexation of Cr6+ with organic components. The minimal medium used in our study eliminated/minimized the possible complexation of Cr6+ with media components and allowed the assessment of the toxicity of Cr6+ more accurately.

Figure 1

Growth curves of P. jadinii M9 (a) and P. anomala M10 (b) at varying Cr6+ concentrations as K2Cr2O7.

In both strains, it was observed that, although residual Cr6+ concentration decreased as incubation progressed, total chromium in solution remained virtually constant (data not showed, Fernández et al., unpublished) and chromium did not accumulate in the cell, which indicates that P. jadinii M9 and P. anomala M10 were able to reduce chromium to forms of lower valency. Taking into consideration that the more stable forms of chromium are the trivalent and hexavalent ones [17], it seems most likely that the M9 and M10 strains were capable of transforming the highly toxic and soluble hexavalent chromium to the less toxic and mobile trivalent form.

Hexavalent chromium reduction potential of P. jadinii M9 and P. anomala M10 was assessed with two kinds of Cr6+ salts, K2CrO4 (chromate), and K2Cr2O7 (dichromate). Cr6+ (at initial concentration of 52 μg mL−1) was reduced up to 100% by both strains within 48 h (Figure 2). Importantly, Cr6+ occurs in aquatic environment either as CrO4 2− or Cr2O7 2− [18] and the strains used in this study were able to reduce both forms of hexavalent chromium.

Figure 2

Cr6+-removal yield by P. jadinii M9 and P. anomala M10 exposed to different forms of Cr6+ (chromate: CrO4 −2 and dichromate: Cr2O7 −2) at 52 μg mL−1 initial Cr6+ concentration during 48 h.

3.2. Factors Affecting Cr6+ Reduction

The effect of initial Cr6+ concentration on Cr6+ reduction was investigated over a range of 26–104 μg mL−1 under aerobic conditions. As shown in Table 1, Cr6+ reduction occurred even at the highest concentration of 104 μg mL−1, and the time taken for total reduction of Cr6+ increased with increasing concentration of Cr6+. Complete Cr6+ reduction was observed at 96 and 72 h, for P. jadinii M9 and P. anomala M10, respectively. Megharaj et al. [19] also observed that the time required for total Cr6+ reduction increased with increasing initial Cr6+ concentration. The Pseudomonad strain CRB5 showed complete reduction of 20 μg mL−1 of chromate after 120 h [18], whilst B. sphaericus AND303 failed to completely reduce 10 μg mL−1 of Cr6+ [20].

Table 1

The effect of factors on Cr6+ removal, including pH, temperature, agitation, and initial Cr6+ concentration.

Parameters	Time for complete Cr6+ removal (h)
	Strains
Initial Cr6+ concentration (μg mL−1)	P. jadinii M9	P. anomala M10
26	12	8
52	48	24
78	48	48
104	96	72
Temperature (°C)
10	+120	72
20	48	24
25	48	24
30	24	8
Agitation (rev min−1)
0	48	72
150	48	24
250	48	24
pH
3	+120	8
5	48	24
7	12	12
9	+120	48

Reference +120: complete Cr6+ removal was not achieved after 120 h culture.

Initial culture medium pH was considered as a relevant factor for growth and Cr6+ removal by strains M9 and M10. The time required for complete removal of Cr6+ in every experimental set is listed in Table 1. The optimum pH for the strain P. jadinii M9 was pH 7.0. In the case of P. anomala M10, the optimum pH for Cr6+ reduction was pH 3.0. Nonetheless, strain M10 was also capable of reducing Cr6+ in the range of 3.0–9.0 with an appreciable efficiency at neutral pH. Some authors have reported that reduction of chromium in various fungal strains, such as Rhizopus nigricans [21], R. arrhizus [22], and Mucor hiemalis [23] occurred at pH 2.0-3.0. It is known that a drop in pH causes the protonation of the adsorbent surface, inducing a strong attraction of negatively charged Cr6+-ions. Accordingly, biosorption increased with increasing acidity of the solution. The opposite would occur with increasing pH, inducing changes in the adsorbent surface, thereby preventing the Cr6+-ion biosorption. On the other side, Farrell and Ranallo [24] noted that in enzymatic Cr6+ reduction, changes in pH affect the degree of enzyme ionization, with protein conformation and enzyme activity modifications. This would explain why the acidity is not absolutely critical for a better Cr6+ removal. Related, P. anomala M10 showed two optimum pH values. The lowest (pH 3.0) could be related to stimulation of the biosorption phenomena, while pH 7.0 could be linked to improved enzymatic Cr6+ reduction. No measurable changes in Cr6+ concentrations were detected after 120 h of incubation in cell-free controls at the different pH values assayed. These results suggest that Cr6+ removal by medium components was not significant in these experiments and also indicate that Cr6+ reduction observed in the Cr6+ removal experiments conducted with cells was not due to the pH changes that occurred as result of metabolic activity of the growing cells.

Temperature was also an important factor on microbial Cr6+ removal. Chromate removal, by strains P. jadinii M9 and P. anomala M10 was evaluated under four different temperatures: 10, 20, 25, and 30°C for 120 h. These strains reduced Cr6+ in the culture medium more rapidly with an increment in temperature, with an optimum value of 30°C, as shown in Table 1. Generally, an increase in temperature increases the Cr6+-removal rate and reduces the contact time required for metal-removal, which is due to a direct increase in the rate of redox reaction [25]. Similarly, the optimum temperature for Cr6+ reduction by Bacillus sp. [26] and Pseudomonad strain CRB5 was 30°C [27].

The results of shaken versus stationary cultures are presented in Table 1. Generally, Cr6+ removal was enhanced by shaking the cultures, but strains P. jadinii M9 and P. anomala M10 could achieve a complete removal (100%) of the metal, both at stationary and shaken states. The aeration and the cell/metal contact are directly related to the removal of it. However, the alternative to remediate Cr6+ without agitation is particularly important for in situ bioremediation applications and may represent a valuable advantage from the economic point of view.

3.3. Chromate Reduction by Cell-Free Extract (CFE)

Yeast cells recovered from cultures grown in the presence of 52 μg mL−1 of Cr6+ and without Cr6+ (control) were tested for chromate reductase activity. The concentration of protein obtained in CFE from cultures with Cr6+ was two times higher than the control ones (Figure 3(a)). The chromate reductase specific activity in the CFE of P. jadinii M9 was higher in cultures with Cr6+, which could be interpreted as an induction by the metal present in the culture medium. In the case of P. anomala M10, there were no significant differences in chromate reductase specific activity between the different CFEs (Figure 3(b)). Das and Chandra [28] studied a strain of Streptomyces sp. M3 and noticed an increase in the chromate reductase activity when working in cultures with Cr6+. These same authors found that enzyme-expression was constitutive. Chromate reductase enzymes with constitutive expression were also discovered in Bacillus species [29, 30]. In the case of constitutive expression, it could be possible that the activity was not specific for this metal and, therefore, normally expressed in cells. It could also take place by induction of some other components of the culture medium with or without Cr6+. Kwak et al. [31] reported the presence of chromate reductase activity in V. harveyi, which also had nitroreductase activity. In P. denitrificans, the iron reductase (Ferb) also showed chromate reductase activity [32].

Figure 3

Total proteins (a) and chromate reductase-specific activity (b) in cell-free extract of P. jadinii M9 and P. anomala M10 grown with or without Cr6+. The reaction was started by addition of chromate, and the mixture was incubated at 30°C for 30 min.

It is important to point out that the specific chromate reductase activity in the cells from cultures with Cr6+ could be masked by an increase in the concentration of other proteins not related with the metal reduction. That could be happening in the case of P. anomala M10 (Figure 3(b)). This protein could be part of a protective mechanism in response to the stress suffered in the presence of Cr6+. However, to date most of the proteins that undergo changes in presence of Cr6+ have not yet been identified, and therefore, its particular function could not be determined.

These data indicate that the chromate reductase activity present in CFE of P. jadinii M9 and P. anomala M10 could be related with cytosolic or associated membrane proteins, which in this respect resembles the activity found in chromate-resistant bacteria [30], and Candida maltosa RR1 [9].

4. Conclusions

Environmental isolates P. jadinii M9 and P. anomala M10 can be exploited for bioremediation of hexavalent chromium, since they are chromate-resistant yeasts and possess the capability to reduce the toxic hexavalent form to its nontoxic trivalent form. The results obtained may provide useful information for the removal of chromate under a wide range of environmental conditions. Systematic studies are needed to determine the real nature of activities so far called as chromate reductases. A future communication will deal with the chromate reductase activities characterization. This information will greatly facilitate the use of the involved proteins to enhance the chromate remediation potential of P. jadinii M9 and P. anomala M10.