Ecotoxicology and Environmental Safety 106 (2014) 46–53

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Cadmium-induced oxidative stress tolerance in cadmium resistant Aspergillus foetidus: its possible role in cadmium bioremediation Shatarupa Chakraborty a,1, Abhishek Mukherjee a,n,1, Anisur Rahman Khuda-Bukhsh b, Tapan Kumar Das a a b

Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, India Department of Zoology, University of Kalyani, Kalyani 741235, India

art ic l e i nf o

a b s t r a c t

Article history: Received 6 August 2013 Received in revised form 19 March 2014 Accepted 4 April 2014

Toxic effects of cadmium (Cd) were examined on a cadmium-resistant strain of Aspergillus foetidus isolated from wastewater. The Cd removal potential was analyzed. The results indicated that the strain could tolerate up to 25 mM and 63 mM Cd in liquid and solid Czapek-Dox media, respectively. It efficiently removed Cd from liquid growth media and industrial wastewater by mycelial biosorption. The strain produced oxalic acid for the purpose of Cd bioleaching as confirmed by the presence of cadmium oxalate crystals on the mycelial surface. Intracellular proline contents and the antioxidative enzyme activities increased up to a certain level to detoxify the overproduced free radicals. These data indicate that the strain has inherent mechanisms to grow in Cd contaminated environment, tolerate high Cd doses and high Cd uptake potential which are pre-requisite for acting as a suitable candidate for Cd bioremediation. & 2014 Elsevier Inc. All rights reserved.

Keywords: Aspergillus foetidus Bioremediation Oxidative stress Antioxidative enzymes Lipid peroxidation Thiol

1. Introduction Cadmium (Cd) is one of the most deleterious trace heavy metals to plants and animals (Dong et al., 2007). It is used in rechargeable batteries, electronic equipments, bearing alloys, pigments for ceramic glazes, paints and plastics (Adamis et al., 2003). Cd is also present in phosphate fertilizers (Perez and Anderson, 2009). Cd has been accepted as a category 1 (human) carcinogen by the International Agency for Research on Cancer (Hossain and Huq, 2002). The environmental Cd pollution occurs due to its continuous release from the industrial and agricultural sources (Jarup and Akesson, 2009). Cd easily translocates from plant roots to above ground tissues (Zhou and Qiu, 2005) and interferes with physiological processes (Maksymiec et al., 2007; Li et al., 2008). Cd enters the food chain through plants and therefore induces its adverse effects on human and other organisms. The mechanism of Cd toxicity is of prime interest and it is important to see how the toxic effects are counterbalanced by the living cells. The thiol compounds, including reduced glutathione, phytochelatins (PCs), and metallothioneins, are essential components

n

Corresponding author. E-mail address: [email protected] (A. Mukherjee). 1 Those authors contributed equally.

http://dx.doi.org/10.1016/j.ecoenv.2014.04.007 0147-6513/& 2014 Elsevier Inc. All rights reserved.

of Cd detoxification pathways in various organisms (Hall, 2002; Brunetti et al., 2011). Cd induces oxidative stress by forming reactive oxygen species (ROS) in the living cells (Schutzendubel et al., 2001). The interaction of Cd with the antioxidative systems has been studied in several plants and animals (Vitoria et al., 2001; Fornazier et al., 2002). Cd2 þ inhibits nitrate reductase activity in Aspergillus niger (Aiken et al., 2003). The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) have been reported to increase as a result of excess amount of ROS formation induced by the Cd2 þ toxicity (Guelfi et al., 2003). Cd2 þ induces an increase in SOD activity in A. niger B 77 strain; however CAT activity decreases significantly with the increased Cd2 þ stress (Todorova et al., 2008). Cd-induced lipid peroxidation has also been reported (Howlett and Avery, 1997; Tao et al., 2007). Lipid peroxidation occurs via peroxidation of unsaturated fatty acids. Free radical damage to phospholipids is an important factor in developing toxic conditions. The free radical scavengers and antioxidants are shown to be useful in protection against Cd toxicity (Sarkar et al., 1998; Ognjanovic et al., 2003). Many plants and algae reduce heavy metal toxicity by the production of proline. Increased proline level in response to Cd toxicity in plants has been reported previously (Balestrasse et al., 2005; Dinakar et al., 2008). Proline may reduce hazardous effects of ROS by acting as an inhibitor of lipid peroxidation (Mehta and Gaur, 1999), a hydroxyl radical scavenger (Smirnoff and Cumbes, 1989) and a singlet oxygen scavenger (Alia and Matysik, 2001).

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Traditional methods like chemical precipitation, filtration, electrochemical treatments, reverse osmosis, ion exchange, adsorption etc. are expensive and inadequate for the removal of heavy metals from water. In this regard, microorganisms may be considered as a biological tool for metal processing as they can concentrate, remove and recover heavy metals from contaminated aquatic environments (Riggle and Kumamoto, 2000). In recent years, the filamentous fungi are gaining more importance as they are capable of removing heavy metals by biosorption as well as by intracellular uptake (Kapoor and Viraraghavan, 1995). Some Cd-resistant organisms have been studied and are of considerable values in the remediation of soils and aquatic systems heavily contaminated with Cd (Zhu et al., 1999). Several Aspergillus species have also been found to be efficient in bioleaching of Cd and other heavy metals (Aung and Ting, 2005; Santhiya and Ting, 2006). A. niger biomass pretreated by boiling in NaOH solution exhibits high Cd removal capacity (Kapoor et al., 1999). A. niger has been successfully used to remove Cd from oil field water (Barros Jnior et al., 2003). Dried, non-living and granulated biomass of Aspergillus fumigatus can remove Cd2 þ from solutions efficiently (Rama Rao et al., 2005). Aspergillus clavatus has been reported to immobilize high amount of Cd2 þ from aqueous solution (Cernansky et al., 2008). Aspergillus foetidus, the strain under the current report, can reduce chromium(VI) to chromium(III) by complexation of chromium(VI) with the organic compounds released by the fungi due to their metabolic activity and can take up chromium(VI) from solution (Prasenjit and Sumathi, 2005). Multimetal tolerant A. foetidus has been found to be effective in the bioleaching of nickel laterite ores (Le et al., 2006). In this work, we studied the mechanism of the Cd toxicity in a Cd-tolerant strain of A. foetidus. We also assessed the Cd tolerance mechanism of the strain by analyzing certain cellular responses evoked in respect of certain enzymes and biomolecules to counter the toxicity. The strain was used to quantify the Cd biosorption capacity from the liquid growth media and from experimental wastewater samples. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopic (EDS) studies were performed to characterize the biomass in order to determine the possible Cd binding mechanisms.

2. Materials and methods 2.1. Samples The sewage sediment sample was collected from the water treatment center, Kalyani, India. The sample was diluted serially with a sterile 145 mM NaCl solution and thoroughly shaken (10 times). Czapek Dox (CD) media were used for all the growth experiments. The CD media contained (per liter): NaNO3 (2.0 g), MgSO4 (0.5 g), KCl (0.5 g), FeSO4 (0.01 g), ZnSO4 (0.01 g), glucose (40.0 g). KH2PO4 was used as a phosphate source. The properly diluted (100 times) sample was used to spread onto solid Czapek Dox (CD) plates consisting of 4.95 ml CD media with 50 ml of 1 M cadmium chloride (CdCl2) solution to reach a final concentration of CdCl2 to 10 mM.

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supplemented with CdCl2 and allowed to grow at 32 1C for 96 h. The best grown colony was preserved in CDA slants. The slant cultures were routinely sub-cultured every one month prior to the experimental use; 8-day old spores were used as inoculums. 2.4. Preparation of fungal biomass The liquid CD broth with added streptomycin, (pH 5.0) was used for the growth of the fungus. Conical flasks containing CD media were inoculated with the spores (1010 conidia L  1) of the strain and shaken at 175 rpm at 32 1C. Ten different Cd concentrations including 10 mM, 50 mM, 100 mM, 500 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM and 25 mM were added separately to the growth media. For the enzymatic and biomolecule studies, the biomass grown at four different Cd doses (5, 10, 15 and 20 mM) along with a control (no Cd in growth media) were used. To study the effect of chloride ions (Cl  ), the fungus was grown at different Cl  doses (5, 10, 15, 20 mM). NaCl was used as the source of Cl  ions. The biomass was harvested after 96 h, filtered and washed with de-ionized water and kept at –20 1C for the biochemical analyses. 100 mg of the fungal biomass was introduced into wastewater samples collected from River Damodar near Asansol Industrial area, West Bengal and electroplating industry effluent at Kolkata, West Bengal, India. The biomass was allowed to stand for 96 h in the wastewater samples at room temperature with occasional shaking. 2.5. Estimation of Cd by atomic absorption spectroscopy The fungal mycelia, the spent media and the wastewater samples were analyzed by atomic absorption spectrometer (AAS) for Cd estimation. Approximately 300 mg of dried mycelia and 2 ml of spent media or wastewater sample were digested in 4 ml of concentrated HNO3 and 1 ml of 30 percent H2O2 in closed PTFE vessels in a digestion block at 90 1C for 3 h. The digest was diluted with MilliQ water up to a volume of 25 ml (Maihara et al., 2008). A Perkin Elmer AAnalyst 400 atomic absorption spectrometer with Zeeman background correction at 228.8 nm for Cd was used. 2.6. SEM and EDS analysis The samples were prepared for SEM according to Gharieb et al. (1995). The samples were analyzed by a field emission scanning electron microscope Jeol JSM 6700F with an accelerating voltage of 20 kV. 2.7. Changes in pH of the spent media The initial pH of the CD broth was maintained at 5.0 after autoclaving, followed by the addition of Cd for different treatment groups. The media were inoculated with the fungal spores and the biomass was harvested after a 96 h growth period. The pH of the spent media was measured for each treatment group. 2.8. Assay of total thiol (–SH) contents For total thiol assay, a modified Ellman (1959) method was followed. The fungal mycelia were ground with alumina and extracted with 50 mM phosphate buffer (pH 7.0) with and without 50 mM ethylenediaminetetraacetic acid (EDTA). The homogenate was centrifuged at 2000g for 20 min at 4 1C. The supernatant was mixed with phosphate buffer (pH 7.0) and distilled water (3:2:5). 20 ml of 10 mM 5,50 -dithiobis-(2-nitrobenzoic acid) (DTNB) solution was added to 3 ml of the reaction mixture, shaken well and absorbance was recorded at 412 nm. The thiol contents were calculated using the molar extinction coefficient value of 13600 M  1 cm  1 for DTNB at 412 nm. 2.9. Assay of CAT, GR and peroxidase activities

2.2. Isolation of microorganisms The isolation and enumeration of microorganisms were carried out in solid CD media, as described earlier (Raper and Thom, 1949). The pH of the media was maintained at 5.0. The media were solidified with 2 percent agar as solid CD medium (CDA). Streptomycin was added to the media to arrest bacterial growth. The sewage sample was plated on CDA and the plates were incubated at 32 1C for 96 h. The best grown fungal colony with black conidia was primarily identified as a high Cd tolerant strain and was preserved in the CDA slants containing 10 mM CdCl2. 2.3. Preparation of pure culture and its maintenance The conidia of the preserved strain were taken in sterile water and shaken vigorously. The properly diluted conidial suspension was spread onto CDA

Fresh mycelia were freeze-dried with liquid nitrogen, ground with alumina and extracted with an ice-cold 50 mM phosphate buffer (pH 7.0) containing 1 percent polyvinylpyrrolidone. The homogenate was centrifuged at 15,000g for 20 min at 4 1C. The supernatant was used for the assay of enzyme activities and the extent of lipid peroxidation. The CAT activity was measured according to Chance and Maehly (1955). One unit CAT activity was defined as the absorbance change of 0.01 unit per min. The GR activity was determined spectrophotometrically according to Carlberg and Mannervik (1985). One enzyme unit was defined as the oxidation of 1 μmol NADPH per min. The activity of peroxidase towards syringaldazine (SPX) was assayed according to Imberty et al. (1985). The peroxidase activity towards guaiacol (GPX) was assayed according to Maehly and Chance (1954). One unit peroxidase activity was defined as the oxidation of 1 μmol substrate per min.

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2.10. Determination of intracellular H2O2 content and the extent of lipid peroxidation The H2O2 content was measured according to the modified method of Velikova et al. (2000). The mycelia were digested and extracted with 0.1 percent trichloroacetic acid (w/v) at 4 1C. The homogenate was centrifuged at 12,000g for 15 min at 4 1C. 750 ml of the supernatant was added with 750 ml of phosphate buffer (pH 7.0) and 1.5 ml KI (1 M). The absorbance was read at 390 nm. The content of malondialdehyde (MDA), a final product of lipid peroxidation, was determined using the method described by Dhindsa et al. (1981).

observed for the 10 mM than for the lower Cd treatment groups (Table 2A). This may be due to more biomass formation and hence more metal uptake for the lower treatment groups than for the 10 mM treatment group. The pre-grown biomass of the test strain absorbed significant amount of Cd from the experimental water samples of river and electroplating industry (Table 2B).

2.11. Proline assay and protein measurement

3.4. Cadmium removal

The proline assay was done by the method of Chinard (1952). Fresh mycelia were ground with alumina and extracted with 3 percent sulfosalicylic acid. The homogenate was centrifuged at 2000g for 20 min. The supernatant was mixed with glacial acetic acid and acid-ninhydrin reagent (1:1:1). The mixture was heated to 100 1C for 45 min and subsequently placed in an ice bath. Toluene was added to the mixture, shaken vigorously and allowed to stay for 15 min for complete phase separation. Upper toluene layer was separated and kept at room temperature for 10 min and the red color intensity was read at 520 nm against toluene blank. The proline concentration was determined from a standard curve and calculated on a fresh weight basis (mM proline/gFW). The method of Lowry et al. (1951) was used to measure the protein contents in the above experiments.

The Cd content in the spent medium was taken as a measure of the ability of the strain to remove Cd(II) from the liquid media. The results showed that the fungal biomass could take in 79 percent of Cd from the liquid media at 100 mM Cd concentration (Table 2A). Significant removal efficacy was observed even for an unusually high Cd treatment, i.e., near 70 percent Cd removal for the 5 mM and 10 mM treatment groups respectively. The Cd removal decreased with a further increase in Cd concentration and the least (18 percent) was found for the 25 mM treatment group. For the 10 mM and 50 mM Cd treatment groups, no Cd was found in the spent media. Hence it may be presumed that the test strain showed a 100 percent Cd removal efficacy for those treatments groups. The test strain was able to remove large amount of Cd from the experimental water samples and worked efficiently even at wide ranges of Cd concentration in the experimental water samples (0.29–3.67 mg L  1).

2.12. Statistical analysis Each experiment was repeated three times. The statistical analysis was done by the one-way Analysis of Variance (ANOVA) to compare the means of two or more treatment groups followed by post-Hoc multiple comparisons by Tukey method. The difference was considered as significant when p o 0.05.

3. Results 3.5. Changes in pH of spent media 3.1. Strain identification The selected strain was identified from the Institute of Microbial Technology (IMTECH), Chandigarh and deposited as A. foetidus MTCC 8876. 3.2. Growth studies The effect of added Cd on the strain was determined by measuring the dry weight of fungal mycelia grown in liquid CD broth and the colony diameter in CDA plates supplemented with varied Cd concentrations. The mycelial pictures (Supplementary material Fig. 1) revealed that the growth of the strain gradually decreased with the increase in Cd concentration. The strain could tolerate up to 63 mM Cd in solid CD media. The spore formation capability decreased gradually with the increased Cd stress (Supplementary material Fig. 1). The hyphae propagation of the strain gradually decreased with the increase in Cd stress. No hyphae propagation was observed beyond 30 mM Cd treatment. In liquid CD media, a gradual decrease in the mycelial growth was observed with the increase in Cd stress (Table 1). The growth was severely stunted (90 percent) at 25 mM Cd concentration in respect to control and no growth was observed with a further increase in Cd concentration. Cl  showed no significant changes in growth of the strain in comparison to the control group. 3.3. Cadmium biosorption The total Cd content in the fungal biomass was measured as an indicator of the Cd biosorption as well as adsorption by the fungal mycelia. The results showed that the strain could take up the maximum amount of Cd at 10 mM after which the total Cd contents in the fungal mycelia gradually decreased (Table 2A). However, when the spent media were assayed for Cd, more Cd was

For the control group, the pH of the spent media was found to decrease from 5.00 to 3.74 70.12 (Table 2A). With an increase in Cd dosage, the pH of the spent media gradually declined, the decrease being mild at low Cd doses. A drastic decrease in the pH was observed when the Cd concentration was increased from 5 mM to 10 mM. For the 25 mM treatment group, the pH was reduced from 5.00 to 1.62 70.08.

3.6. SEM and EDS analysis The scanning electron micrographs of the strain grown in presence of Cd showed a number of crystals on the mycelial surface. The crystals were found to be concentrated to the mycelial surface (Fig. 1a) by the fibre-like threads of the fungal mycelia. As the harvested biomass was washed thoroughly and repeatedly before the sample preparation for SEM, it is likely that the crystals found must be strongly bound to the mycelial surface. The EDS analysis (Fig. 1b) showed that the crystals contained Cd. The ratio of cadmium (Cd), carbon (C) and oxygen (O) within the crystals as calculated from EDS data was found to be 1:2:4. Thus they might be cadmium oxalate (CdC2O4) crystals.

3.7. Intracellular protein content The intracellular protein contents of the strain initially increased at 5 mM Cd in comparison to control, the increase was 23 percent. However the protein contents decreased with a further increase in Cd concentrations and attained the minimum at 20 mM Cd concentration where a 50 percent reduction was observed in comparison to control (Table 1). Groups grown in the presence of Cl  showed no deviation in the intracellular protein contents from the control values.

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Fig. 1. Scanning electron micrographs of A. foetidus mycelia grown at 10 mM Cd in liquid Czapek Dox medium. (a) Bound cadmium crystals on mycelial surface. (b) EDS analysis of crystal confirming the presence of cadmium. CdC2O4 crystal is marked with circle. Table 1 Effects of Cd on growth, intracellular protein contents, intracellular proline contents; H2O2 generation and lipid peroxidation induced by Cd toxicity. Results are expressed as Mean 7 SE. Cd (II) concentrations (mM)

Dry weight of mycelia (gm)

Intracellular protein content (mg/gFW)

Intracellular H2O2 content (mg/gFW)

Intracellular MDA content (mg/gFW)

Intracellular proline content (mg/gFW)

0 5 10 15 20 25

2.81 70.13 2.21 70.16 1.98 70.27 1.00 70.12n 0.73 70.06 0.28 70.01

556 710 684 78nn 626 710nn 400 711nnn 279 78nnn –

0.737 0.02 0.93 7 0.02n 1.137 0.06n 2.09 7 0.06nnn 2.727 0.08nnn –

2.52 7 0.16 3.157 0.04nn 3.65 7 0.06nn 4.30 7 0.10nn 4.82 7 0.02nn –

26.4 7 1.0 33.5 7 0.7nnn 43.5 7 0.9nnn 50.4 7 0.9nnn 57.3 7 1.0nnn –

nnn

p o 0.001. p o0.01. po 0.05.

nn n

Table 2 (A) Cd absorbed by fungal mycelia, Cd left in spent media after 96 h growth, percentage of Cd removal by the strain and changes in pH of the spent media after fungal growth. Results are expressed as Mean7 SE. Cd (II) concentration in growth media

Cd (II) in Cd (II) spent media absorbed by mycelia (mg/g) (mg L  1)

% Cd (II) removal

0 10 mM 50 mM 100 mM 500 mM 1 mM 5 mM 10 mM 15 mM 20 mM 25 mM

N/A 0.08 7 .01 0.4 7 0.01 0.727 0.02 3.79 7 0.13 7.42 7 0.28 44.68 73.26 73.46 7 10.75 68.377 3.61 53.447 2.72 47.82 7 2.21

N/A 100 100 78.7 71.4 77.5 71.2 75.1 71.3 69.9 72.1 67.2 73.1 39.2 76.2 34.4 73.1 17.9 73.6nnn

N/A 0 0 2.34 7 0.16 12.6 7 0.7 27.5 7 2 168 7 12 3677 34n 10217 103nnn 14707 70nnn 2309 7110nnn

pH of the spent media

3.9. Thiol leakage 3.747 0.12 3.687 0.09n 3.617 0.05n 3.55 7 0.03n 3.53 7 0.17n 3.447 0.02n 3.247 0.03n 2.65 7 0.09nn 2.34 7 0.05n 2.117 0.01n 1.62 7 0.08nnn

nnn

p o 0.001. p o0.01. po 0.05.

nn n

buffer, the thiol content was found to be higher as compared with those where there was no EDTA in the extraction buffer (Fig. 2). No significant change in data was observed for the Cl  positive control groups.

3.8. Intracellular thiol content The intracellular thiol contents increased gradually with an increase in Cd stress. In groups treated with EDTA in the extraction

There was a drastic increase in the thiol leakage at a 5 mM Cd concentration in comparison to control, followed by a mild but steady increase with a further increase in Cd concentration (Fig. 2). Here again, the values for thiol contents were found to be higher when EDTA was used in the extraction buffer in comparison to that where there was no EDTA. Cl  treatment did not alter the thiol leakage values of the test strain as compared to control. 3.10. Intracellular H2O2 content and extent of lipid peroxidation Cd induced the formation of H2O2 within the cells of the strain as evident from Table 1. Initially, there was a mild induction at lower (5 and 10 mM) Cd concentrations and a drastic increase in intracellular H2O2 level was followed thereafter with higher Cd stress (15 and 20 mM). The values were 2.9 and 3.7 times greater than control, at the 15 and 20 mM Cd concentrations, respectively (Table 1).

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Table 2 (B) The initial Cd concentrations and pH of the experimental water samples and Cd removal by the mycelia of A. foetidus. Results are expressed as Mean 7 SE. Sample

Initial Cd concentration (mg L  1) pH

River water Electroplating industry effluent

0.29 7 0.01 3.677 0.27

Cd absorbed by mycelia (mg/g) Cd left in water sample (mg L  1) Cd removal (percent)

7.39 7 0.16 2.63 7 0.38 6.277 0.19 30.9 7 4.58

0 0

75

Enzyme activity (U/mg protein/min)

Thiol contents (µM/gFW mycelia)

350

100 100

300 250 200 150 100 50

60

45

30

15

0 0 0

5

10

15

Fig. 2. Effect of Cd on intracellular thiol content of the strain and thiol leakage in spent media by the strain during growth. Intracellular thiol without EDTA with EDTA. Thiol content in spent media without EDTA with EDTA. Values are Means 7 SE.

The MDA content of a cell is taken as a reliable indicator for the free radical formation (Velikova et al., 2000). An increase in the MDA contents was found with an increase in Cd concentration. MDA contents were 1.7 and 1.9 times greater than control, at the 15 and 20 mM Cd concentrations, respectively (Table 1). The Cl  treated groups showed no change in intracellular H2O2 and MDA contents when compared with the control group.

5

10

15

20

Cd concentrations (mM)

20

Cd concentrations (mM)

0

Fig. 3. Effect of Cd on enzyme activities of the strain. CAT activity GR activity GPX activity SPX activity. Values are Means 7SE. nnnp o0.001, nnp o 0.01, n p o0.05.

3.12. Intracellular proline content The production of proline was increased with an increase in the Cd stress. The proline contents increased by 65, 91 and 117 percent at 10, 15 and 20 mM Cd concentrations, respectively, with respect to control (Table 1). No significant changes in proline contents of the Cl  treated groups were observed in comparison to control.

4. Discussion 3.11. Activity of antioxidant enzymes The CAT activity initially increased with Cd stress and reached the maximum at a 5 mM Cd concentration where the increase was 2.37 times compared to control. This increase in CAT activity suggests that the Cd treatment induced antioxidative response within the strain. However, the CAT activity decreased with a further increase in Cd stress (Fig. 3) to attain the minimum at the 20 mM Cd concentration where a 1.5 time decrease was observed in comparison to control. Cl  treated groups showed no change in CAT activity when compared to the control group. With the increase in Cd stress, the GR activity sharply increased (Fig. 3). In comparison to control, the increase was nearly 2, 3 and 7 times at the 5, 10 and 20 mM Cd concentrations, respectively. Cl  treated groups showed no change in GR activity when compared to the control group. The GPX activity increased at 5 mM Cd in comparison to control. However, the GPX activity then decreased gradually with a further increase in Cd stress (Fig. 3). The SPX activities increased gradually with increased Cd stress and the maximum activity was observed at the 20 mM Cd concentration where the value was 2.3 times greater than control (Fig. 3). Treatment of Cl  produced no effect on peroxidase activities of the test strain compared to the values of the control group.

The growth inhibition by Cd was reported earlier in A. nidulans (Guelfi et al., 2003). A. fumigatus isolated from industrial waste could tolerate up to 1 g L  1 Cd however with a decrease in growth by 88.2 percent (Al-Garni et al., 2009). Aspergillus flavus and A. niger could grow in a medium containing 250 mg L  1 Cd, although a decrease of growth by more than 94 percent was observed (Al-Garni et al., 2009). The long term toxic effects of heavy metals on the growth of A. foetidus were reported (Ge et al., 2011). In the present study, the test strain could effectively grow and tolerate up to 25 mM Cd (2.8 g L  1) in liquid growth media. Presumably because of its prior adaptation in heavy metal contaminated environment, the test strain could grow under such a high concentration of Cd and take in high amounts of Cd which could be a destructive concentration for other life forms. This is in agreement with the earlier reports that microorganisms isolated from the heavy metal contaminated environments demonstrate the adaptive ability to sustain themselves in such environments (Cernansky et al., 2007). Generally, Cd concentrations in the ground water, river, wastewater, water bodies and river basins in the vicinity of Cd-releasing industries vary from 0.75 to 1300 mg L  1 (Srikanth et al., 1993; Rattan et al., 2005; Adomako et al., 2008; Beg and Ali, 2008). In the present study, the results showed that the fungus could grow in low as well as high Cd concentrations and the biomass produced, in turn, could absorb a significant amount of Cd. The decrease in Cd removal potential of

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the strain at high Cd doses may be due to (i) the low uptake of Cd by the strain and (ii) the decrease in biomass formation, at higher Cd concentrations. The pre-grown biomass of the test strain could remove Cd from experimental water samples also. Thus this strain appears to have a potential of field application for Cd removal from ground water, river, pond etc as it can act as a chelator of Cd in low as well as high Cd concentrations. Also the strain is capable to grow and biosorb Cd simultaneously from highly contaminated Cd media and this makes the strain even more suitable than other hypertolerant strains of Aspergillus to be used as a Cd chelator. The SEM and EDS data showed that the strain was capable of converting Cd(II) into insoluble cadmium oxalate crystals and to retain them strongly adhered to its mycelial surface. This conversion of the dissolved Cd to an insoluble compound makes the strain suitable for its subsequent growth and the Cd removal from the contaminated sites. This can prove to be a good strategy for an easy, effective and a low cost method for Cd bioremediation. Moreover, the formation of cadmium oxalate crystals may indicate the ability of the strain to chelate Cd by synthesizing organic acids as was earlier reported for A. niger (Sayer et al., 1999) and Beauveria caledonica (Fomina et al., 2005). Oxalic acid is a wellknown chelating agent and is able to mobilize metals effectively at neutral and even basic pH ranges (Fomina et al., 2005). Hence, chelation of Cd by the produced oxalate may be a reason for the unusually high Cd tolerance of the test strain. A drastic decrease in pH of the spent media may be correlated with oxalic acid production by the strain. The use of such strains in the bioleaching of heavy metals may reduce the cost of commercial heavy metal decontamination and decrease any environmental impacts resulting from metal contamination (Mulligan et al., 2004). In this way, the treated water may even be made suitable for sanitation and other household purposes, provided that the pH of the water is adjusted to near normality before use. Initial increase in the intracellular protein contents of the strain exposed to low Cd concentrations may be due to the induced protein synthesis involved in the chelation or detoxification of Cd. However, a decrease in protein contents at higher Cd concentrations may indicate the intolerance of the strain to such unusually high Cd concentrations. In the present study, the intracellular thiol contents of the strain gradually increased with the increase in Cd stress. Fungi synthesize increased amounts of metallothionein (MT) and phytochelatins (PC) to bind heavy metal ions as a part of cellular resistance to prevent heavy metal toxicity (Pal and Das, 2005). The increase in the intracellular thiol levels upon Cd exposure has been reported for several organisms (Pal and Das, 2005; Courbot et al., 2004; Jaeckel et al., 2005; Miersch and Grancharov, 2008). In the present study, when EDTA was used in the extraction buffer, the thiol contents were found to be higher than that in the absence of EDTA. Probably, EDTA chelated Cd(II) ions to leave the thiolate groups free to react with DTNB. Hence it may be said that thiol–Cd complexes were formed within the strain. For the control group, no such difference was observed in the values for thiol contents. The difference was found for the Cd-treated groups only. These data strongly support the phenomenon of complex formation among intracellular thiols and Cd only; this conjugation may lead to detoxification as previously reported for A. niger (Mukherjee et al., 2010). Hence Cd chelation by the intracellular thiols may be considered as an inherent mechanism of the strain to minimize the effects of intracellular Cd2 þ ions. Glutathione (GSH) plays an important role in ROS scavenging and the increased consumption of glutathione for PC production in response to Cd stress has been observed (Zenk, 1996; Mehra and Tripathi, 1999). The increase in thiol contents during Cd stress may be the strategy of the strain to produce more GSH to minimize the toxic effects of ROS formed due to Cd toxicity in the fungal cells.

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The significant leakage of the intracellular thiols in the spent media was observed during the growth experiments. The thiol leakage increased with an increase in Cd concentrations. Here again, when EDTA was used in the extraction buffer, the thiol contents were found to be higher than that without EDTA. Those results were in accordance with the fact that thiol–Cd complexes were formed within the strain and the leakage of thiol–Cd complexes from the cells would indicate that there was considerable effort made by the strain to minimize the Cd toxicity by removing those complexes outside the cell. A similar type of cellular detoxification against arsenic has been previously reported for A. niger (Mukherjee et al., 2010). Another reason behind such leakage may be the structural deformation of the fungal cell membrane as a result of membrane lipid peroxidation induced by ROS generated due to Cd stress. The use of lipid peroxidation as a molecular marker of toxicity with the cellular environment has been made in a variety of living organisms (Howlett and Avery, 1997; Pathak et al., 2006; Zhang et al., 2007). An increase in MDA levels indicates the occurrence of membrane damage due to the peroxidation of polyunsaturated fatty acids, resulting in the generation of ROS and subsequent oxidative stress (Montillet et al., 2005). Cd induced lipid peroxidation in the strain, the extent of which increased with the increase in Cd stress and thus indicated that Cd caused an extensive damage by peroxidation of the membrane lipids. Cd induced the production of H2O2 within the strain. However, the induction of H2O2 production was not significant at the lower Cd doses, which may be due to the action of the antioxidant enzymes within the cells. At higher Cd doses, very high amounts of H2O2 were generated within the cells and could not be detoxified by the cellular antioxidative defense system. Catalases protect cells against the damage caused by H2O2 and ROS to the cellular components (Imlay and Linn, 1988). For the test strain, the CAT activity increased at a 5 mM Cd dosage. This would indicate that H2O2 formed by Cd exposure was metabolized by CAT, since the activity of CAT is directly regulated by H2O2 concentration (Polidoros and Scandalios, 1999). A decrease in CAT activity with a further increase in Cd concentration would possibly imply that the strain could no longer protect itself from the overwhelmingly increased ROS generation that crossed the tolerance limit of the strain. Glutathione reductase reduces glutathione disulfide (GSSG) to sulfhydryl glutathione (GSH) (Yan et al., 2011) to maintain a high GSH/GSSG ratio as the cellular redox state (Willmore and Storey, 2007). For the test strain, the GR activity increased continuously with an increase in Cd treatment. This result explains the phenomenon of enhanced Cd chelation capacity by thiols, as an increased GR activity results in the reduction of GSSG to regenerate more GSH, which, in turn, chelates Cd as a part of the detoxification mechanism. Higher activities of peroxidases, in response to heavy metal stress, have been found in several species (Jouili and Ferjani, 2003; Malekzadeh et al., 2007; Singh et al., 2007; Haluskova et al., 2009). For the test strain, the GPX activity increased initially during a lower Cd treatment. This may reflect the cellular strategy to tolerate the Cd-induced toxicity. The SPX activity showed a gradual increase during the Cd stress. This increase in peroxidase activities may result in the breakage of excess H2O2 formed during the Cd stress, as a part of the cellular antioxidative defense mechanism of the strain. Proline can function as an osmolyte radical scavenger, electron sink, stabilizer of cell wall components and macromolecules (Matysik et al., 2002). High contents of proline are a typical feature routinely found in high metal tolerant populations. This tempts one to assume a functional role of proline in increased metal tolerance (Sharma and Dietz, 2006). Proline may act as an antioxidant to

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scavange ROS (Chen and Dickman, 2005). In the present study, a gradual increase in the intracellular proline contents during the Cd stress is indicative of a significant role that proline may play as a stabilizer and ROS scavenger, thus imparting a high Cd tolerance in the strain. The groups treated with Cl  ions did not show any significant alterations in the levels of intracellular biomolecules and the activities of antioxidant enzymes. Hence the Cl  ions generated during the treatment with high CdCl2 doses did not affect the metabolism and the enzymatic reactions within the cells of the test strain.

5. Conclusion The strain tolerated toxic effects of Cd by modifying its enzymatic machinery and synthesizing additional amounts of thiols and proline. The strain could maintain its cellular functions under Cd stress, suggesting that it possesses some inherent cellular mechanisms to counteract Cd toxicity due to its previous adaptation to a heavy metal contaminated environment. The strain efficiently removed Cd completely from experimental wastewater samples. After biomass separated and autoclaved for 15 min, no viable spores of fungus could be traced in the left over water. Hence, this is relatively non-toxic to living organisms for use. Hence the treated water may be made suitable for sanitation and other household uses.

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