Localization of Cr(VI) removal activity in Candida tropicalis strain and the study of its Cr(VI) removal capacity in soil.
Localization of Cr(VI) removal activity in Candida tropicalis strain and the study of its Cr(VI) removal capacity in soil.
Candida tropicalis strain HE650140 showed a remarkable capacity to completely reduce 50 mg l−1 of Cr(VI) in 48 h under aerobic conditions; however, a small change in total content of chromium in the culture liquid was detected, which can be explained by the formation of Cr(III). Fractionation of the cells showed that chromium removal activity was present in both the cytoplasm and membrane. The bioaugmentation of Cr(VI)-contaminated soil microcosms by live and dead biomass showed that yeast inoculation diminishes phytoavailable chromium from soils, improving different growth parameters of clover.
The Cr(VI) removal activity was found in both cytoplasmic and membrane fractions. Both live and dead biomass of C. tropicalis were capable to reduce Cr(VI) in the soil and limit the toxicity of this metal to clover seedlings.
This study is one of the few documents that present the ability of dead yeast to limit phytoavailability of Cr(VI) from soil. This is of great significance in bioremediation of Cr(VI)-contaminated soil.
Chromium (Cr) toxicity is one of the major causes of environmental pollution. This metal is introduced into natural waters through various industrial activities, such as steel production, electroplating, leather tanning, textile industries, wood preservation, anodizing of aluminium, water cooling and chromate preparation (Garg et al. 2007). Chromium exists in nature as stable hexavalent and trivalent forms (Sen and Ghosh 2010). Hexavalent chromium compounds are approximately 1000-fold more cytotoxic and mutagenic than trivalent chromium (Biedermann and Landolph 1990). Cr(VI) is highly soluble and is a very strong oxidizing agent, which can easily penetrate into the living cells causing cell or tissue damage and is capable of inducing carcinogenic and mutagenic activities (Cervantes et al. 2001), while Cr(III) shows poor solubility and is easily adsorbed on mineral surfaces (Polti et al. 2007).
Conventional methods for the treatment of chromate wastewater include chemical reduction followed by precipitation under alkaline conditions, ion exchange and adsorption (Smith and Ghiassi 2006). These methods present high cost, low efficiency and the toxic sludge generation, which would require complex disposal operations (Sahin and Zturk 2005). Biological treatments, which utilize the potential of micro-organisms to transform or to adsorb heavy metals (Silver and Phung 1996), arouse great interest in Cr(VI) remediation of contaminated sites because it is an economical and environmental friendly way (Chai et al. 2009).
Soil bioremediation constitutes a special challenge because of its heterogeneity and also because well-adapted micro-organisms are needed to bioremediate this particular environment (Tabak et al. 2005). Hence, it is essential to research on the application of micro-organisms to experimentally chromium-polluted soil microcosms.
Yeasts, which can adapt and grow under various extreme conditions of pH, temperature and nutrient availability as well as at high pollutant concentrations (Anand et al. 2006; Gonen and Aksu 2008), might be applied in the management of chromium-containing wastes. Furthermore, yeast cells retain their ability to accumulate a broad range of heavy metals to varying degrees under a wide range of external conditions (Villegas et al. 2005). Several species of Candida were found to be able to efficiently remove heavy metals under a wide range of external conditions (Yin et al. 2008). In these yeasts, the general mechanism of chromate resistance is related to its adsorption (Pepi and Baldi 1992). However, some yeasts such as Candida utilis (Muter et al. 2001) and Candida maltosa (Ramirez-Ramirez et al. 2004) showed the ability to partially reduce Cr(VI) to Cr(III) and a good efficiency to accumulate chromium in the biomass. However, few studies have examined the potential of yeasts for bioremediation processes in soil and to diminish phytoavailability of chromium from soils.
In this study, we describe the mechanism of Cr(VI) removal by a Candida tropicalis strain isolated from chromium-contaminated site, and this strain exhibits high resistance level and has a very efficient removal capacity for Cr(VI). Bioaugmentation of soil microcosms experimentally polluted with chromium using live and dead biomass of C. tropicalis to evaluate the ability of this strain to effectively diminish bioavailable chromium from soils was also studied. Clover plants grown in this bioaugmented soil microcosm were used as bioindicators, to address the success of the bioremediation process.
Strain of Candida tropicalis HE650140 used in the present work has been isolated from chromium-contaminated site located in Oued Sebou, Fez (Morocco) (Bahafid et al. 2013). The yeast was able to tolerate high concentrations of Cr(VI) in YPG solid medium and also exhibits multiple metal (Ni, Zn, Hg, Pb, Co, Cu and Hg) tolerance. Stock culture of the strain was maintained on YPG solid medium (2% glucose, 2% peptone, 1% yeast extract and 2% agar) and subcultured at monthly intervals. The liquid culture medium was prepared by mixing 2% glucose, 0·2% peptone and 0·2% yeast extract. The growth temperature for HE650140 was 30°C.
A stock solution of chromium was prepared by dissolving potassium dichromate (K2Cr2O7) in distilled water and diluted to get the desired concentration.
For testing the ability of yeast to remove Cr(VI), concentration range of 25–100 mg l−1 of a sterile solution of chromate (K2Cr2O7) was added to the yeast culture with cell concentration of 2 g l−1 (an early exponential phase of the growth) and incubated with aeration at 30°C under continuous shaking (150 rev min−1). Uninoculated controls were included for each experiment and incubated under identical conditions. Aliquots (200 μl) were taken at regular time intervals, and the difference in initial and residual Cr(VI) levels was noted as Cr(VI) reduced.
The sum of chromium species remaining in the extracellular liquid, as well as the total chromium uptaken/adsorbed by the cells after their mineralization, was also determined. Yeast cells cultured in the presence of 25 and 50 mg l−1 of Cr(VI) for 48 h were harvested by centrifugation at 6000 g for 20 min at 4°C. The supernatant was collected and then sterilized by filtration through a nitrocellulose filter of 0·45 μm, while the pellet was washed and mineralized by the hot nitric acid (Guillen-Jiménez et al. 2008).
Cr(VI) analysis in the medium was carried out using hexavalent chromium-specific colorimetric reagent S-diphenyl carbazide (DPC), and spectrophotometric measurements were taken immediately at 540 nm (Urvashi et al. 2007). The concentration of total chromium in the extracellular liquid was also determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES).
To prepare various fractions of cells, the yeast culture grown overnight in 100 ml YPG was harvested by centrifugation at 6000 g for 20 min at 4°C. The supernatant was collected and then sterilized by filtration through a nitrocellulose filter of 0·45 μm, while cells (pellet) were washed twice with 20 ml of 10 mmol l−1 Tris-HCl buffer pH 7·2 and were suspended in 5 ml of Tris-HCl 25 mmol (pH 7·2). These cell suspensions were placed on ice (−20°C) and lysed using an ultrasonic probe (Sonics Vibra Cell 500, Sonics & Materials, Newtown, CT, USA) for 15 min at a temperature of 0°C. The sonicated cells thus obtained were then centrifuged at 12 000 g for 40 min at 4°C. The membrane fraction of the sonicated cells was suspended in the same volume of Tris-HCl (pH 7·2).
Supernatant fraction, cytosolic fraction and membrane fraction thus obtained were then tested for their ability to reduce Cr(VI) at a concentration of 10 mg l−1. The reaction mixture was incubated for 12 h at 30°C. After centrifugation, the residual concentration of Cr(VI) was determined using DPC method. All fractions that were heated at 100°C for 30 min acted as controls.
Neutral soil samples were taken from an uncontaminated site far from any industrial activity. The soil was kept for 36 h at room temperature to allow water to equilibrate in the soil (Jézéquel et al. 2005). After drying, the soil was sieved to recover the fraction below 2 mm. For the soil microcosm (SM) assay, Petri dishes were filled with 35 g of soil and steam-sterilized (three successive sterilizations 24 h apart, at 121°C for 1 h each). Solution of Cr(VI) was added to the soil up to a final concentration of 40 mg kg−1, and the soil humidity was adjusted to 100% with the culture of yeast isolate (sterilized SMs were inoculated with C. tropicalis pregrown in YPG to a final concentration of 2 g l−1). SMs not inoculated were used as control. All assays were performed in triplicate.
The dishes were incubated at 30°C, and a soil sample was taken after 1–8 days. The Cr(VI) soil content was determined by DPC method after alkaline digestion of the soil (Centre d'expertise en analyse environnementale du Québec (CEAEQ) 2008).
To evaluate the effect of bioremediation of Cr(VI) on germination and growth of clover (Trifolium fragiferum), clover seeds used were surface-disinfected and germinated in pots filled with 125 g of soil contaminated artificially with Cr(VI) solution at different concentrations (10–60 mg kg−1 soil). The SMs so prepared were inoculated with 25 ml of live and dead C. tropicalis cultures at 2 g l−1 (3·5–4 107 UFC ml−1). 30 clover seeds were subsequently transplanted into different pots. SMs not inoculated were used as control. The plants were grown for a month in a climate chamber at 30°C during daytime and 20°C during the night with 14 h of light alternating with 10 h of darkness. The pots were watered every day with deionized water as needed. After a month, the number of germinated seeds was counted and the seedlings were harvested, and the length of shoots and roots was measured.
The cell concentration was estimated by colony-forming units (CFU) and dry cell measurements. Absorbance from overnight grown cells was measured at 600 nm using a spectrophotometer. From this culture, serial dilutions were made, and 0·1 ml was plated onto YPG solid medium. After 24 h of incubation, colonies were counted and CFU ml−1 was calculated. To determine the dry weight of cells, sample of cells cultured with known cell concentrations was centrifuged at 6000 g for 10 min, and the cell pellet was washed with distilled water and dried at 100°C. Corresponding absorbance at 600 nm was converted to dry weight of cells and the correspondence with UFC ml−1 was made.
All the treatments were conducted in triplicates, and the data presented in the tables are mean ± SEM (standard error of mean) of three replicates. The data were analysed statistically by analysis of variance (anova), and Fisher's LSD test was performed to determine significant differences and to compare the differences between treatments. Statistical analyses were carried out using R (version 2.15.2).
Chromium-reducing capability of the yeast isolate was checked by adding 25 mg l−1 of Cr(VI) into the culture medium.
Figure 1 shows that residual Cr(VI) concentration decreased as incubation progressed, until no measurable Cr(VI) concentration could be detected in the culture medium after 48 h of incubation, while the calculation of the sum of the total chromium in the culture liquid and in the mineralized cells was almost close to the initial level of Cr(VI) (Table 1).
|25 mg l−1||50 mg l−1|
|Initial Cr(VI) in the medium||24·57 ± 1·04||48·13 ± 1·01|
|Total chromium in the medium||23·20 ± 0·64||32·2 ± 2·03|
|Cr(VI) remaining in the medium||0·36 ± 0·09||9·56 ± 1·14|
|Estimated Cr(III) in the medium||22·84||32·64|
It was also observed that the cellular concentration of Cr(VI) in this yeast increased with increasing concentration of Cr(VI) treatment. The concentrations of chromium accumulated by Candida tropicalis cells were 3·58 and 4·82 mg g−1 in the presence of 25 and 50 mg g−1 of Cr(VI), respectively.
Cr(III) concentration in the final solution may be calculated as the difference between the total chromium and soluble Cr(VI) in the medium (Table 1).
The effect of initial Cr(VI) concentration on microbial Cr(VI) removal and on the rate of Cr(VI) removal was investigated over a concentration range of 25–100 mg l−1. As seen in Fig. 1, when the initial Cr(VI) ion concentration increased from 25 to 100 mg l−1, the percentage removal of metal ions decreased. Reduction was complete within 48 h when the initial Cr(VI) concentration ranged between 25 and 50 mg l−1, while it was slower and required longer incubation time with initial concentrations of 100 mg l−1.
After cell disruption, evolution pattern of Cr(VI) concentration in extracellular, intracellular and membrane fractions obtained from C. tropicalis culture is shown in Fig. 2.
Significant difference was observed between treatments with different fractions (P < 0·0001). The highest removing activities (100% reduction) were found in both intracellular and membrane fractions, whereas a negligible activity was detected in the supernatant fractions. Interestingly, almost 100% of chromium reduction was also detected in membrane fraction that was heated at 100°C for 30 min, indicating that this fraction did involve in the adsorption process. This could be a good characteristic for this strain. However, the heat-killed control of cell-free extracts of the strain failed to reduce chromate, suggesting the presence of soluble enzymatic mechanism in the cytoplasmic fraction (crude cell-free extracts) of the strain, which was heat labile at 100°C.
The reduction of Cr(VI) by C. tropicalis was studied in a sterile microcosm soil artificially contaminated with 40 mg Cr(VI) per kg of soil. Chromate removal in soil microcosm was studied after 8 days. As shown in Table 2, there was very less removal of chromate (5%) in control soil after 8 days of incubation, while in the soil inoculated with the dead and live biomass of yeast isolate, concentration of Cr(VI) decreases significantly (up to 58·7 and 72·25% of reduction, respectively).
|Residual Cr(VI) in soil (mg kg−1)||Cr(VI) removal in soil (%)|
|Control||37·51 ± 0·78a||06·22 ± 1·95|
|Dead biomass||16·52 ± 0·85b||58·70 ± 2·12|
|Live biomass||11·10 ± 0·40c||72·25 ± 1·00|
Seed cultures of clover were used as bioindicators to confirm the effective decrease of bioavailable Cr(VI) in the SM bioaugmented with dead and live culture of C. tropicalis. The plants were seeded on SM containing different concentrations of potassium chromate (10–60 mg kg−1 of the soil) and inoculated with yeast. After 4 weeks, the growth of the plants on SM was evaluated by assessing germination, root and leaf length and total biomass of clover seedling (Table 3), and analysis of variance (anova) was carried out.
|Chromium treatment (mg kg−1)||Seed germination (%)||Seed germination inhibition (%)|
|Cr(VI) (control)||Cr(VI) + yeast||Cr(VI) (control)||Cr(VI) + yeast|
|10||30·00 ± 0·0||58·88 ± 1·1||70·00||41·11|
|30||10·00 ± 1·9||43·33 ± 0·0||90·00||56·66|
|20||25·55 ± 1·1||55·55 ± 1·1||74·44||44·44|
|60||05·55 ± 1·1||30·00 ± 0·0||94·44||70·00|
|Control (distilled water)||100·0||00·00|
From the statistical analysis, it has been inferred that different growth parameters of clover seedlings vary significantly (P < 0·0001) among the different treatments.
Results showed that different growth parameters were affected significantly under Cr(VI) stress. In the case of the soil microcosm without yeast (control), about 70, 74, 90 and 94% of reduction in seed germination was observed at 10, 20, 30 and 60 mg kg−1 of Cr(VI), respectively.
Interestingly, higher seedling germination and significant increase in different growth parameters (P < 0·0001) in soil microcosm, inoculated with dead and live cultures of yeast, were observed as compared with uninoculated control (Tables 3 and 4), almost 41, 44, 56 and 70% of reduction in seed germination was detected at 10, 20, 30 and 60 mg kg−1 of Cr(VI), respectively.
|Cr(VI) treatment (mg kg−1)||Leaf length (cm)||Root length (cm)||Fresh weight (g)|
|Cr(VI) (Control)||Cr(VI) + yeast||Cr(VI) (Control)||Cr(VI) + yeast||Cr(VI) (Control)||Cr(VI) + yeast|
|10||4·43 ± 0·23b||5·70 ± 0·25a||12·03 ± 0·14d||14·23 ± 0·18b||0·72 ± 0·01c||3·25 ± 0·06a|
|30||0·71 ± 0·01c||4·36 ± 0·23b||1·24 ± 0·09e||13·24 ± 0·22c||0·13 ± 0·01d||2·60 ± 0·05b|
|Control (distilled water)||5·78 ± 0·14a||16·43 ± 0·23a||3·26 ± 0·02a|
|20||1·50 ± 0·05d||4·81 ± 0·15b||2·22 ± 0·13c||13·55 ± 0·41b||0·25 ± 0·05c||3·12 ± 0·01a|
|60||0·62 ± 0·03e||3·46 ± 0·24c||0·38 ± 0·01d||13·18 ± 0·33b||0·06 ± 0·00c||0·96 ± 0·12b|
|Control (distilled water)||5·78 ± 0·14a||16·43 ± 0·23a||3·26 ± 0·02a|
Cr(VI)-polluted environments pose serious health and ecological risk. There is a continuous search for naturally occurring microbes that have better Cr(VI) transformation capabilities over a wider range of growth condition.
During the present investigation, Candida tropicalis was found to have excellent ability to remove Cr(VI). Complete reduction of 25 mg l−1 of Cr(VI) by C. tropicalis was observed after 48 h. The concentration removed in this study is near to that removed by Candida sp. FGSFEP and by Trichoderma viride in another study (Morales-Barrera and Cristiani-Urbina 2006) and is higher than the concentrations commonly reduced by fungi such as C. maltosa (Ramirez-Ramirez et al. 2004), Aspergillus sp. and Penicillium sp. (Acevedo-Aguilar et al. 2006) in other studies. However, it should be mentioned that the microbial Cr(VI) resistance and Cr(VI) removal are dependent on culture medium composition and cell density (Mergeay 1995; Wang 2000).
The reduction rate was found to be affected by the initial Cr(VI) concentration in the medium. The effects of different concentrations of chromium showed that the rate of chromium reduction decreased with increasing chromium concentration. The lesser chromium removal in C. tropicalis produced by higher initial Cr(VI) concentration may be due to the mutagenicity and toxicity of chromium for culture metabolism (Wang and Shen 1997). Our results are in agreement with those obtained by other authors. Sukumar (2010) reported that in the case of Rhizopus Oryzae, the percentage removal of chromium decreased with higher initial concentrations; however, this reduction increased with an increase in incubation time for all the range of initial concentrations studied. Jeyasingh and Philip (2005) also reported that the microbes are able to reduce/remediate Cr(VI) even at higher concentrations though it takes a long time.
In addition, the ability of this strain to accumulate/adsorb and/or reduce Cr(VI) on Cr(III) was studied. It was observed during the Cr(VI) removal experiments that although the residual Cr(VI) concentration decreased, the total chromium in solution remained almost constant. An amount of chromium was also detected in mineralized cells, which suppose the adsorption/accumulation of the highly toxic and soluble hexavalent chromium and its transformation to the less toxic and less mobile trivalent form by this yeast. Cr(VI) can be removed from the aqueous solution by yeast through two mechanisms, chemical reduction and enzymatic reduction (Wang and Shen 1995; Ramirez-Diaz et al. 2007). In chemical reduction, Cr(VI) is reduced to Cr(III) by contact with the electron-donor groups of the biomass surface or by some reducing agents in the cytoplasm such as the following: ascorbic acid (Goodgame and Joy 1988), glutathione (GSH) (Cupo and Wetterhahn 1985), cysteine (Kitagawa et al. 1988), hydrogen peroxide (H2O2) (Kawanishi et al. 1986) or riboflavin (Sugiyama et al. 1989) and by the action of flavoenzymes (Banks and Cooke 1986) namely glutathione reductase (GR) (Koutras et al. 1964). However, the enzymatic reduction of Cr (VI) is typically associated with soluble proteins requiring NAD(P)H as an electron donor. This reduction may be associated with the membrane, cytoplasmic and/or periplasmic fraction (Desjardin et al. 2002).
Localization of the Cr(VI)-reducing activity in C. tropicalis clearly indicated the presence of the activity in both intracellular and membrane fractions. The nonreduction of heat-killed control of cell-free extracts suggests that this yeast may have the enzymic capabilities for converting Cr(VI) to Cr(III). However, the reduction observed in the membrane fraction heated suggests that a plausible mechanism of chromium adsorption and/or chemical reduction could be present in this fraction. These results may indicate that this strain could remove chromium by employing both cytoplasmic and membrane activities, suggesting that after initial surface binding, a reduction of Cr(VI) to Cr(III) can be achieved by adjacent electron-donor groups. If there are a small number of electron-donor groups in the biomass or protons in the aqueous phase, the chromium bound on the biomass can enter into the cytosol, and the intracellular fraction of this yeast removes chromium mainly through its reduction to less toxic Cr(III) compound via enzymatic interaction. Similar result was reported using T. clypeatus and Vogococcus sp. (Sujoy et al. 2009; Mistry et al. 2010).
Cr(VI) removal ability of this strain could also be used to perform bioremediation process of polluted soils. Thus, bioremediation activity of yeast was further evaluated in soil microcosm containing 40 mg kg−1 of Cr(VI). The result of this study indicated that significant removal of Cr(VI) (P < 0.0001) was observed in soil inoculated with C. tropicalis (58% with dead biomass and 72% with live biomass), while un-inoculated soil had not shown much removal of chromium (5%). According to our results, removal of chromate in the soil inoculated with dead and live culture would be due to adsorption and/or reduction of Cr(VI) to Cr(III), respectively, by this yeast. Similar results were found by Srivastava and Thakur (2006) testing different Cr(VI) concentrations (200 and 400 mg kg−1) on soil inoculated with A. niger (FK1), which indicated that lower concentration of chromate (250 ppm) was reduced (75%) at 15 days compared with the control (5%), and chromium was removed by bioaccumulation in the mycelium of the fungus. Molokwane et al. (2008) also demonstrated that microcosm inoculated with Cr(VI)-reducing bacteria (R5) achieved significant removal of Cr(VI).
To evaluate the toxicity of chromate on seed germination, bioassay test was performed in soil microcosm artificially contaminated by Cr(VI) at different concentrations (10–60 mg kg−1). The result of the study indicated that the increase in concentration of chromate decreases the seed germination and different growth parameters of seedling. Comparable phenomenon was observed by Srivastava and Thakur (2006) who reported that seed germination, seedling length and seed vigour index of wheat seeds decreased as the chromate concentration increased. This reduction can be explicated by the Cr(VI) toxicity, which could be due to a depressive effect of Cr(VI) on the activity of amylases and on the subsequent transport of sugars to the embryo axes (Zeid 2001), or inhibition of root cell division/root elongation and subsequent inability of the roots to absorb water from the medium (Barcelo et al. 1986).
The reduction in the availability of Cr(VI) to plants by C. tropicalis was assessed using clover as bioindicator. As compared to uninoculated soil, significant increase in seed germination and growth of seedlings was detected in the presence of higher concentration of chromium together with the yeast. From this result, it is appearing that addition of C. tropicalis biomass induced better growth and was advantageous for combating toxicological effects and stimulating growth in clover seedlings by reducing the availability and toxicity of Cr(VI) to plants. These data confirmed the efficiency of the bioremediation process of C. tropicalis. Our results are in agreement with the finding of several authors. Faisal and Hasnain (2005) demonstrated that inoculation of Ochrobactrum intermedium caused a decrease in chromate uptake into seedlings as compared to their respective noninoculated control. Srivastava and Thakur (2006) also reported that the soil microcosm inoculated with A. niger increased the seed germination and seedling length under chromate stress. Based on this report and on our results, we can suggest that the yeast strains promoted clover growth mainly by adsorption of Cr(VI) on cells of C. tropicalis and/or reducing this toxic metal to less bioavailable Cr(III) by this yeast.
In summary, C. tropicalis yeast strain, isolated from effluents of tanneries and able to tolerate high concentrations of Cr(VI), was found capable of reducing high concentration of Cr(VI) in liquid medium in short time and was able to significantly diminish the bioavailable Cr(VI) on soil at both active and inactive states. This is very interesting in the sense that its introduction into soil will be successful and not harmful for the environment. This study is among a few works that report the potential of dead yeasts for bioremediation processes in polluted soil. In this sense, we propose C. tropicalis as a potential tool to perform bioremediation process of Cr(VI)-polluted soils and effluents in higher scale. Further research is being conducted in this direction.
The authors gratefully acknowledge the financial and scientific support of Microbial Biotechnology Laboratory, Faculty of Sciences and the City of Innovation, SMBA University, Fez, Morocco.