Ecotoxicology and Environmental Safety 106 (2014) 46–53

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

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
Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, India
b
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: Toxic effects of cadmium (Cd) were examined on a cadmium-resistant strain of Aspergillus foetidus
Received 6 August 2013 isolated from wastewater. The Cd removal potential was analyzed. The results indicated that the strain
Received in revised form could tolerate up to 25 mM and 63 mM Cd in liquid and solid Czapek-Dox media, respectively. It
19 March 2014
efficiently removed Cd from liquid growth media and industrial wastewater by mycelial biosorption. The
Accepted 4 April 2014
Available online 14 May 2014
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
Keywords: activities increased up to a certain level to detoxify the overproduced free radicals. These data indicate
Aspergillus foetidus that the strain has inherent mechanisms to grow in Cd contaminated environment, tolerate high Cd
Bioremediation
doses and high Cd uptake potential which are pre-requisite for acting as a suitable candidate for Cd
Oxidative stress
bioremediation.
Antioxidative enzymes
Lipid peroxidation & 2014 Elsevier Inc. All rights reserved.
Thiol

1. Introduction of Cd detoxification pathways in various organisms (Hall, 2002;

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 cera-
mic 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 Interna-
tional 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 physio-
logical 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 of ROS by acting as an inhibitor of lipid peroxidation (Mehta and
Corresponding author.
E-mail address: abhi_biochem81@yahoo.in (A. Mukherjee).
1
Those authors contributed equally.
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 peroxida-
tion 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
Gaur, 1999), a hydroxyl radical scavenger (Smirnoff and Cumbes,
1989) and a singlet oxygen scavenger (Alia and Matysik, 2001).

http://dx.doi.org/10.1016/j.ecoenv.2014.04.007
0147-6513/& 2014 Elsevier Inc. All rights reserved.
 S. Chakraborty et al. / Ecotoxicology and Environmental Safety 106 (2014) 46–53
Traditional methods like chemical precipitation, filtration, elec-
trochemical 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 bioleach-
ing 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 chro-
mium(VI) with the organic compounds released by the fungi due to
their metabolic activity and can take up chromium(VI) from solu-
tion (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 per-
formed 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.
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
47
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. Approxi-
mately 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
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.
48 S. Chakraborty et al. / Ecotoxicology and Environmental Safety 106 (2014) 46–53
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 trichlor-
oacetic 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).
2.11. Proline assay and protein measurement
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.
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
3.1. Strain identification
The selected strain was identified from the Institute of Micro-
bial 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 (Sup-
plementary 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
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).
3.4. Cadmium removal
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).
3.5. Changes in pH of spent media
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 con-
centration where a 50 percent reduction was observed in com-
parison to control (Table 1). Groups grown in the presence of
Cl  showed no deviation in the intracellular protein contents from
the control values.
 S. Chakraborty et al. / Ecotoxicology and Environmental Safety 106 (2014) 46–53 49

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 Dry weight of Intracellular protein Intracellular H2O2 Intracellular MDA Intracellular proline
(mM) mycelia (gm) content (mg/gFW) content (mg/gFW) content (mg/gFW) content (mg/gFW)

0 2.81 70.13 556 710 0.737 0.02 2.52 7 0.16 26.4 7 1.0
5 2.21 70.16 684 78nn 0.93 7 0.02n 3.157 0.04nn 33.5 7 0.7nnn
10 1.98 70.27 626 710nn 1.137 0.06n 3.65 7 0.06nn 43.5 7 0.9nnn
15 1.00 70.12n 400 711nnn 2.09 7 0.06nnn 4.30 7 0.10nn 50.4 7 0.9nnn
20 0.73 70.06 279 78nnn 2.727 0.08nnn 4.82 7 0.02nn 57.3 7 1.0nnn
25 0.28 70.01 – – – –

nnn
p o 0.001.
nn
p o0.01.
n
po 0.05.

Table 2 buffer, the thiol content was found to be higher as compared with
(A) Cd absorbed by fungal mycelia, Cd left in spent media after 96 h growth,
those where there was no EDTA in the extraction buffer (Fig. 2).
percentage of Cd removal by the strain and changes in pH of the spent media after
fungal growth. Results are expressed as Mean7 SE. No significant change in data was observed for the Cl  positive
control groups.
Cd (II) Cd (II) Cd (II) in % Cd (II) pH of the
concentration absorbed by spent media removal spent media
in growth mycelia (mg/g) (mg L  1) 3.9. Thiol leakage
media
There was a drastic increase in the thiol leakage at a 5 mM Cd
0 N/A N/A N/A 3.747 0.12
10 mM 0.08 7 .01 0 100 3.687 0.09n concentration in comparison to control, followed by a mild but
50 mM 0.4 7 0.01 0 100 3.617 0.05n steady increase with a further increase in Cd concentration (Fig. 2).
100 mM 0.727 0.02 2.34 7 0.16 78.7 71.4 3.55 7 0.03n Here again, the values for thiol contents were found to be
500 mM 3.79 7 0.13 12.6 7 0.7 77.5 71.2 3.53 7 0.17n higher when EDTA was used in the extraction buffer in compar-
1 mM 7.42 7 0.28 27.5 7 2 75.1 71.3 3.447 0.02n
5 mM 44.68 73.26 168 7 12 69.9 72.1 3.247 0.03n
ison to that where there was no EDTA.
10 mM 73.46 7 10.75 3677 34n 67.2 73.1 2.65 7 0.09nn Cl  treatment did not alter the thiol leakage values of the test
15 mM 68.377 3.61 10217 103nnn 39.2 76.2 2.34 7 0.05n strain as compared to control.
20 mM 53.447 2.72 14707 70nnn 34.4 73.1 2.117 0.01n
25 mM 47.82 7 2.21 2309 7110nnn 17.9 73.6nnn 1.62 7 0.08nnn
3.10. Intracellular H2O2 content and extent of lipid peroxidation
nnn
p o 0.001.
nn
p o0.01.
n Cd induced the formation of H2O2 within the cells of the strain
po 0.05.
as evident from Table 1. Initially, there was a mild induction at
lower (5 and 10 mM) Cd concentrations and a drastic increase in
3.8. Intracellular thiol content intracellular H2O2 level was followed thereafter with higher Cd
stress (15 and 20 mM). The values were 2.9 and 3.7 times greater
The intracellular thiol contents increased gradually with an than control, at the 15 and 20 mM Cd concentrations, respectively
increase in Cd stress. In groups treated with EDTA in the extraction (Table 1).
50 S. Chakraborty et al. / Ecotoxicology and Environmental Safety 106 (2014) 46–53
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 0.29 7 0.01
Electroplating industry 3.677 0.27
effluent
350
300
Thiol contents (µM/gFW mycelia)
250
200
150
100
50
0
0 5 10
Cd concentrations (mM)
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.
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 com-
pared 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.
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 0 100
6.277 0.19 30.9 7 4.58 0 100
75
Enzyme activity (U/mg protein/min)
60
45
30
15
0
0 5 10 15 20
Cd concentrations (mM)
15 20
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
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
 S. Chakraborty et al. / Ecotoxicology and Environmental Safety 106 (2014) 46–53
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 conver-
sion 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 well-
known 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 result-
ing 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 concentra-
tions 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 phy-
tochelatins (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.
51
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 consider-
able 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 peroxida-
tion 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 pheno-
menon 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
52 S. Chakraborty et al. / Ecotoxicology and Environmental Safety 106 (2014) 46–53
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.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ecoenv.2014.04.007.
References
Adamis, P.D.B., Panek, A.D., Leite, S.G.F., Eleutherio, E.C.A., 2003. Factors involved
with cadmium absorption by a wild-type strain of Saccharomyces cerevisiae.
Braz. J. Microbiol. 34, 55–60.
Adomako, D., Nyarko, B.J.B., Dampare, S.B., Serfor-Armah, Y., Osae, S., Fianko, J.R.,
Akaho, E.H.K., 2008. Determination of toxic elements in waters and sediments
from River Subin in the Ashanti Region of Ghana. Environ. Monit. Assess. 141,
165–175.
Aiken, A.M., Peyton, B.M., Apel, W.A., Petersen, J.N., 2003. Heavy metal-induced
inhibition of Aspergillus niger nitrate reductase: applications for rapid con-
taminant detection in aqueous samples. Anal. Chim. Acta 480, 131–142.
Al-Garni, S.M., Ghanem, K.M., Bahobail, A.S., 2009. Biosorption characteristics of
Aspergillus fumigatus in removal of cadmium from an aqueous solution. Afr. J.
Biotechnol. 8, 4163–4172.
Alia, Mohanty, P., Matysik, J., 2001. Effect of proline on the production of singlet
oxygen. Amino Acids 21, 195–200.
Aung, K.M., Ting, Y.P., 2005. Bioleaching of spent fluid catalytic cracking catalyst
using Aspergillus niger. J. Biotechnol. 116, 159–170.
Balestrasse, K.B., Gallego, S.M., Benavides, M.P., Tomaro, M.L., 2005. Polyamines and
proline are affected by cadmium stress in nodules and roots of soyabean plants.
Plant Soil 270, 343–353.
Barros Jnior, L.M., Macedo, G.R., Duarte, M.M.L., Silva, E.P., Lobato, A.K.C.L., 2003.
Biosorption of cadmium using the fungus Aspergillus niger. Braz. J. Chem. Eng.
20, 229–239.
Beg, K.R., Ali, S., 2008. Chemical contaminants and toxicity of Ganga river sediments
from up and down stream area of Kanpur. Am. J. Environ. Sci. 4, 362–366.
Brunetti, P., Zanella, L., Proia, A., De Paolis, A., Falaska, G., Altamura, M.M., di Toppi,
L.S., Costantino, P., Cardarelli, M., 2011. Cadmium tolerance and phytochelatin
content of Arabidopsis seedlings over-expressing the phytochelatin synthase
gene AtPCS1. J. Exp. Bot. 62, 5509–5519.
Carlberg, I., Mannervik, B., 1985. Glutathione reductase assay. In: Colowick, S.P.,
Kaplan, N.O. (Eds.), Methods Enzymol, vol. 113. Academic Press, Orlando, FL,
pp. 484–495.
Cernansky, S., Urik, M., Sevc, J., Khun, M., 2007. Biosorption and biovolatilization of
arsenic by heat resistant fungi. Environ. Sci. Pollut. Res. 14, 31–35.
Cernansky, S., Urik, M., Sevc, J., Littera, P., Hiller, E., 2008. Biosorption of arsenic and
cadmium from aqueous solutions. Afr. J. Biotechnol. 6, 1932–1934.
Chance, B., Maehly, A.C., 1955. Assay of catalases and peroxidases. In: Colowick, S.P.,
Kaplan, N.O. (Eds.), Methods Enzymol, vol. 2. Academic Press, New York,
pp. 764–775.
Chen, C., Dickman, M.B., 2005. Proline suppresses apoptosis in the fungal pathogen
Colletotrichum trifolii. Proc. Natl. Acad. Sci. USA 102, 3459–3464.
Chinard, F.P., 1952. Photometric estimation of proline and ornithine. J. Biol. Chem.
199, 91–95.
Courbot, M., Diez, L., Ruotolo, R., Chalot, M., Leroy, P., 2004. Cadmium-responsive
thiols in the ectomycorrhizal fungus Paxillus involutus. Appl. Environ. Microbiol.
70, 7413–7417.
Dhindsa, R.S., Plumb-Dhindsa, P., Thorpe, T.A., 1981. Leaf senescence: correlated
with increased levels of membrane permeability and lipid peroxidation, and
decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93–101.
Dinakar, N., Nagajyothi, P.C., Suresh, S., Udaykiran, Y., Damodharam, T., 2008.
Phytotoxicity of cadmium on protein, proline and antioxidant enzyme activities
in growing Arachis hypogaea L. seedlings. J. Environ. Sci. 20, 199–206.
Dong, J., Mao, W.H., Zhang, G.P., Yu, F.B., Cai, Y., 2007. Root excretion and plant
tolerance to cadmium toxicity – a review. Plant Soil Environ. 53, 193–200.
Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77.
Fornazier, R.F., Ferreira, R.R., Vitoria, A.P., Molina, S.M.G., Lea, P.J., Azevedo, R.A.,
2002. Effects of cadmium on antioxidant enzyme activities in sugar cane. Biol.
Plant. 45, 91–97.
Fomina, M., Hillier, S., Charnock, J.M., Melville, K., Alexander, I.J., Gadd, G.M., 2005.
Role of oxalic acid overexcretion in transformations of toxic metal minerals by
Beauveria caledonica. Appl. Environ. Microbiol. 71, 371–381.
Ge, W., Zamri, D., Mineyama, H., Valix, M., 2011. Bioacuumulation of heavy metals
on adapted Aspergillus foetidus. Adsorption 17, 901–910.
Gharieb, M.M., Wilkinson, S.C., Gadd, G.M., 1995. Reduction of selenium oxyanions
by unicellular, polymorphic and filamentous fungi: cellular location of reduced
selenium and implications for tolerance. J. Ind. Microbiol. 14, 300–311.
Guelfi, A., Azevedo, R.A., Lea, P.J., Molina, S.M.G., 2003. Growth inhibition of the
filamentous fungus Aspergillus nidulans by cadmium: an antioxidant enzyme
approach. J. Gen. Appl. Microbiol 49, 63–73.
Hall, J.L., 2002. Cellular mechanisms for heavy metal detoxification and tolerance. J.
Exp. Bot. 53, 1–11.
Haluskova, L., Valentovikova, K., Huttova, J., Mistrik, I., Tamas, L., 2009. Effect of
abiotic stresses on glutathione peroxidase and glutathione S-transferase. Plant
Physiol. Biochem. 47, 1069–1074.
Hossain, Z., Huq, F., 2002. Studies on the interaction between Cd2 þ ions and
nucleobases and nucleotides. J. Inorg. Biochem. 90, 97–105.
Howlett, N.G., Avery, S.V., 1997. Induction of lipid peroxidation during heavy metal
stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid
unsaturation. Appl. Environ. Microbiol. 63, 2971–2976.
Imberty, A., Goldberg, R., Catessoen, A.M., 1985. Isolation and characterization of
Populus isoperoxidases involved in the last step of lignin formation. Planta 164,
221–226.
Imlay, J.A., Linn, S., 1988. DNA damage and oxygen radical toxicity. Science 240,
1302–1309.
Jaeckel, P., Krauss, G., Menge, S., Schierhorn, A., Rucknagel, P., Krauss, G.J., 2005.
Cadmium induces a novel metallothionein and phytochelatin 2 in an aquatic
fungus. Biochem. Biophys. Res. Commun. 333, 150–155.
Jarup, L., Akesson, A., 2009. Current status of cadmium as an environmental health
problem. Toxicol. Appl. Pharmacol. 238, 201–208.
Jouili, H., Ferjani, E.E., 2003. Changes in antioxidant and lignifying enzyme activities
in sunflower roots (Helianthus annuus L.) stressed with copper excess. C. R. Biol.
326, 639–644.
Kapoor, A., Viraraghavan, T., 1995. Fungal biosorption – an alternative treatment option
for heavy metal bearing wastewaters: a review. Bioresour. Technol. 53, 195–206.
Kapoor, A., Viraraghavan, T., Cullimore, D.R., 1999. Removal of heavy metals using
the fungus Aspergillus niger. Bioresour. Technol. 70, 95–104.
Le, L., Tang, J., Ryan, D., Valix, M., 2006. Bioleaching nickel laterite ores using multi-
metal tolerant Aspergillus foetidus organism. Miner. Eng. 19, 1259–1265.
Li, M., Zhang, L.J., Tao, L., Li, W., 2008. Ecophysiological responses of Jussiaea rapens
to cadmium exposure. Aquat. Bot. 88, 347–352.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement
with Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Maehly, A.C., Chance, B., 1954. The assay of catalase and peroxidase. In: Glick, D.
(Ed.), Methods of Biochemical Analysis, vol 1. Interscience Publishers Inc, New
York, pp. 357–425.
Maihara, V.A., Moura, P.L., Catharino, M.G., Castro, L.P., Figueira, R.C.L., 2008. Arsenic
and cadmium content in edible mushrooms from São Paulo, Brazil determined
by INAA and GF AAS. J. Radioanal. Nucl. Chem. 278, 395–397.
Maksymiec, W., Wojcik, M., Krupa, Z., 2007. Variation in oxidative stress and
photochemical activity in Arabidopsis thaliana leaves subjected to cadmium and
excess copper in the presence or absence of jasmonate and ascorbate. Chemo-
sphere 66, 421–427.
Malekzadeh, P., Khara, J., Farshian, S., 2007. Copper toxicity influence on antioxidant
enzyme activity in tomato plants and role of arbuscular mycorrhizal fungus Glomus
etunicatum in the tolerance of toxicity. Pak. J. Biol. Sci. 10, 2008–2013.
Matysik, J., AliaBhalu, B., Mohanty, P., 2002. Molecular mechanisms of quenching of
reactive oxygen species by proline under stress in plants. Curr. Sci. 82, 525–532.
Mehra, R.K., Tripathi, R.D., 1999. Phytochelatins and metal tolerance. In: Agrawal, S.
B., Agrawal, M. (Eds.), Environmental Pollution and Plant Responses. CRC Press,
Lewis Publisher, Boca Raton, FL, pp. 367–382.
Mehta, S.K., Gaur, J.P., 1999. Heavy metal-induced proline accumulation and its role
in ameliorating metal toxicity in Chlorella vulgaris. New Phytol. 143, 253–259.
Miersch, J., Grancharov, K., 2008. Cadmium and heat response of the fungus
Heliscus lugdunensis isolated from highly polluted and unpolluted areas. Amino
Acids34, 271–277.
 S. Chakraborty et al. / Ecotoxicology and Environmental Safety 106 (2014) 46–53
Montillet, J.L., Chamnongpol, S., Rusterucci, C., Dat, J., Van de Cotte, B., Agnel, J.P.,
Battesti, C., Inze, D., Van Breusegem, F., Triantaphylides, C., 2005. Fatty acid
hydroperoxides and H2O2 in the execution of hypersensitive cell death in
tobacco leaves. Plant Physiol. 138, 1516–1526.
Mukherjee, A., Das, D., Mondal, S.K., Biswas, R., Das, T.K., Boujedaini, N., Khuda-
Bukhsh, A.R., 2010. Tolerance of arsenate-induced stress in Aspergillus niger,
a possible candidate for bioremediation. Ecotoxicol. Environ. Saf. 73, 172–182.
Mulligan, C.N., KamLi, M., Gibbs, B.F., 2004. Bioleaching of heavy metals from a low-
grade mining ore using Aspergillus niger. J. Hazard. Mater. 110, 77–84.
Ognjanovic, B.I., Pavlovic, S.Z., Maletic, S.D., Zikic, R.V., Stajn, A.S., Radojicic, R.M.,
Saicic, Z.S., Patrovic, V.M., 2003. Protective influence of vitamin E on antiox-
idant defense system in the blood of rats treated with cadmium. Physiol. Res.
52, 563–570.
Pal, S.K., Das, T.K., 2005. Biochemical characterization of N-methyl N(-nitro-N-
nitrosoguanidine-induced cadmium resistant mutants of Aspergillus niger.
J. Biosci. 30, 639–646.
Pathak, S., Das, J.K., Biswas, S.J., Khuda-Bukhsh, A.R., 2006. Protective potentials of a
potentized homeopathic drug, Lycopodium-30, in ameliorating azo dye
induced hepatocarcinogenesis in mice. Mol. Cell. Biochem. 258, 121–131.
Perez, A.L., Anderson, K.A., 2009. DGT estimates cadmium accumulation in wheat
and potato from phosphate fertilizer applications. Sci. Total. Environ. 407,
5096–5103.
Polidoros, A.N., Scandalios, J.G., 1999. Role of hydrogen peroxide and different
classes of antioxidants in the regulation of catalase and glutathione S-
transferase gene expression in maize (Zea mays L.). Physiol. Plant. 106, 112–120.
Prasenjit, B., Sumathi, S., 2005. Uptake of chromium by Aspergillus foetidus. J. Mater.
Cycles Waste Manag. 7, 88–92.
Rama Rao, K., Rashmi, K., Lavanya Latha, J.N., Maruthi Mohan, P., 2005. Bioremedia-
tion of toxic metal ions using biomass of Aspergillus fumigatus from fermenta-
tive waste. Indian J. Biotechnol. 4, 139–143.
Raper, K.B., Thom, C., 1949. A Manual of the Penicillia. The Williams & Wilkins Co.,
Baltimore, Maryland.
Rattan, R.K., Datta, S.P., Chhonkar, P.K., Suribabu, K., Singh, A.K., 2005. Long-term
impact of irrigation with sewage effluents on heavy metal contents in soil,
crops and groundwater-a case study. Agric. Ecosyst. Environ. 109, 310–322.
Riggle, P.J., Kumamoto, C.A., 2000. Role of a Candida albicans P1-type ATPase in
resistance to copper and silver ion toxicity. J. Bacteriol. 182, 4899–4905.
Santhiya, D., Ting, Y.P., 2006. Use of adapted Aspergillus niger in the bioleaching of
spent refinery processing catalyst. J. Biotechnol. 121, 62–74.
Sarkar, S., Yadav, P., Bhatnagar, D., 1998. Lipid peroxidative damage on cadmium
exposure and alterations in antioxidant system in rat erythrocytes: a study with
relation to time. Biometals 11, 153–157.
Sayer, J.A., Cotter-Howells, J.D., Watson, C., Hillier, S., Gadd, G.M., 1999. Lead mineral
transformation by fungi. Curr. Biol. 9, 691–694.
53
Schutzendubel, A., Schwanz, P., Teichmann, T., Gross, K., Langenfeld-Heyser, R.,
Godbold, D.L., Polle, A., 2001. Cadmium-induced changes in antioxidative
systems, hydrogen peroxide content, and differentiation in scots pine roots.
Plant Physiol. 127, 887–898.
Sharma, S.S., Dietz, K.J., 2006. The significance of amino acids and amino acid-
derived molecules in plant responses and adaptation to heavy metal stress.
J. Exp. Bot. 57, 711–726.
Singh, H.P., Batish, D.R., Kohli, R.K., Arora, K., 2007. Arsenic-induced root growth
inhibition in mung bean (Phaseolus aureus Roxb.) is due to oxidative stress
resulting from enhanced lipid peroxidation. Plant Growth Regul. 53, 65–73.
Smirnoff, N., Cumbes, Q.J., 1989. Hydroxyl radical scavenging activity of compatible
solutes. Phytochemistry 28, 1057–1060.
Srikanth, R., Rao, A.M., Kumar, C.S., Khanum, A., 1993. Lead, cadmium, nickel and
zinc contamination of ground water around Hussain Sagar lake, Hyderabad,
India. Bull. Environ. Contam. Toxicol. 50, 138–143.
Tao, Y.M., Chen, Y.Z., Liang, Y.L., Xu, M.Y., Xu, X.M., 2007. Cadmium-induced
membrane lipid peroxidation and changes in antioxidant enzyme activities
and peroxidase isoforms in Jerusalem artichoke seedlings. J. Plant Physiol. Mol.
Biol. 33, 301–308.
Todorova, D., Nedeva, D., Abrashev, R., Tsekova, K., 2008. Cd (II) stress response
during the growth of Aspergillus niger B 77. J. Appl. Microbiol. 104, 178–184.
Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant
systems in acid rain-treated bean plants. Protective role of exogenous poly-
amines. Plant Sci. 151, 59–66.
Vitoria, A.P., Lea, P.J., Azevedo, R.A., 2001. Antioxidant enzymes responses to
cadmium in radish tissues. Phytochemistry 57, 701–710.
Willmore, W.G., Storey, K.B., 2007. Purification and properties of glutathione
reductase from liver of the anoxia-tolerant turtle, Trachemys scripta elegans.
Mol. Cell Biochem. 297, 139–149.
Yan, J., Meng, X., Wancket, L., Lintner, K., Wu, H., Nelin, L., Chen, B., Smith, C., Rogers,
L., Liu, Y., 2011. The pivotal role of glutathione reductase in host defence against
Gram-negative bacteria. J. Immunol. 186, 111–125.
Zenk, M.H., 1996. Heavy metal detoxification in higher plants—a review. Gene 179,
21–30.
Zhang, F.Q., Wang, Y.S., Lou, Z.P., Dong, J.D., 2007. Effect of heavy metal stress on
antioxidative enzymes and lipid peroxidation in leaves and roots of two
mangrove plant seedlings (Kandelia candel and Bruguiera gymnorrhiza). Chemo-
sphere 67, 44–50.
Zhou, W.B., Qiu, B.S., 2005. Effects of cadmium hyperaccumulation on physiological
characteristics of Sedum alfredii Hans (Crassulaceae). Plant Sci. 169, 737–745.
Zhu, Y.L., Pilon-Smits, A.H., Jouanin, L., Terry, N., 1999. Overexpression of glu-
tathione synthetase in Indian Mustard enhances cadmium accumulation and
tolerance. Plant Physiol. 119, 73–79.