Biochemical characterization of a lead-tolerant strain of Aspergillus foetidus: An implication of bioremediation of lead from liquid media

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International Biodeterioration & Biodegradation xxx (2012) 1e9

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International Biodeterioration & Biodegradation

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Biochemical characterization of a lead-tolerant strain of Aspergillus foetidus:An implication of bioremediation of lead from liquid media

Shatarupa Chakraborty, Abhishek Mukherjee, Tapan Kumar Das*

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

a r t i c l e i n f o

Article history:Received 25 January 2012Received in revised form9 May 2012Accepted 10 May 2012Available online xxx

Keywords:Lead tolerantAspergillus foetidusOxidative stressThiolsBioremediation

Abbreviation: CD, Czapek Dox; CAT, catalase; GPsyringaldazine peroxidase; GR, glutathione reductaseacid phosphatase; ALP, alkaline phosphatase; DTNB, 5acid).* Corresponding author. Tel.: þ91 3473 238373,

fax: þ91 33 25828282.E-mail address: [email protected] (T.K. Das).

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a b s t r a c t

The fungus Aspergillus foetidus isolated from wastewater treatment center, Kalyani was identifiedprimarily as a lead (Pbþ2) tolerant strain by supplementation experiments with high concentrations of Pband confirmed the fungal strain as, A. foetidus MTCC 8876 after identification from the Microbial TypeCulture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India.Growth studies in liquid Czapek-Dox (CD) media with different Pb treatments showed an initial growthincrease up to 200 mg L�1 Pb treatment followed by gradual decrease at higher Pb doses. Protein andthiol leakage during growth experiments in CD broth containing Pbþ2 may be due to disturbance inmembrane structure as was further evident from lipid peroxidation induced by Pb. The strain wasefficient in removing Pb from the liquid growth medium by mycelial biosorption. Scanning electronmicroscopy (SEM) and energy dispersive X-ray spectroscopic (EDS) analysis confirmed the presence oftightly bound Pb as insoluble crystals outside fungal mycelia. Intracellular proline level and activities ofthe antioxidative enzymes increased up to a certain level to detoxify malondialdehyde and H2O2

produced by Pb toxicity. These data indicate that the test strain has some inherent mechanisms totolerate unusually high Pb doses and high Pb uptake potential, pre-requisite for acting as a suitablecandidate for Pb bioremediation from contaminated aqua-environment.

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1. Introduction

Currently one of the most serious problems that the world isfacing with is heavy metal deposition and its toxicities. Heavymetals have many deleterious effects which not only affect humanhealth but also affect other fauna and flora. Increased deposition ofheavy metals into the environment is caused by various anthro-pogenic activities. Among heavy metals like lead, chromium,mercury, uranium, selenium, zinc, arsenic, cadmium, silver, goldand nickel, Pb(II) is the most common and dangerous heavy metalcontamination found in the environment because it has practicallyno biological importance and it is biomagnified very easily. Pb(II)may be concentrated into the environment through differenthuman activities such as mining and smelting of lead ores, burningof coal, effluent from storage battery and industries that produce

X, guaiacol peroxidase; SPX,; LPO, lipid peroxidaton; ACP,,50-Dithio-bis(2-nitrobenzoic

þ91 9475944385 (mobile);

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borty, S., et al., Biochemicafrom liquid media, Interna

lead wires or pipes, automobile exhaust, metal plating operations,fertilizers, pesticides, lead based paints and additives in pigmentsand gasoline (Eick et al., 1999).

Lead is introduced to different trophic levels through precipi-tation or by ion exchange into soils and water, as heavy metalpollutants can localize and lay dormant. Metals are mobilized andcarried into food web as a result of leaching from waste dumps,polluted soils and water that cause bio-magnification (Paknikaret al., 2003). Unlike organic pollutants, heavy metals do not decayand thus pose a different kind of challenge for remediation.

There are several reports on the hazardous effects of Pb on plantand animals including humans. Pb can be directly accumulated inbone hampering the compositional properties and bone mechanicsthroughmineralization (Bjorå et al., 2001; Pain et al., 2007). It causesbrain and nervous system impairment, and particularly mentalretardation in children (Weisskopf et al., 2004) interferes withreproductive system, sperm motility, spontaneous abortion, prema-ture birth and low births (Borja-Aburto et al., 1999; Bonde, 2002). Italso hampers the circulatory system causing O2 absorption, decreaseand increase in blood pressure (Kalra et al., 2000) leading to respi-ratory tract, kidney, lungand stomachcancer (KaneandKumar1999).

Conventional physico-chemicalmethods such as electrochemicaltreatment, ion exchange, precipitation, osmosis, evaporation and

l characterization of a lead-tolerant strain of Aspergillus foetidus:tional Biodeterioration & Biodegradation (2012), http://dx.doi.org/

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S. Chakraborty et al. / International Biodeterioration & Biodegradation xxx (2012) 1e92

sorption followed for removal of heavy metal from soil and aquaticenvironment are not cost effective and eco-friendly (Mulligan et al.,2001; Kadirvelu et al., 2002). Nowadays process of bioremediation isa very promising, eco-compatible and economically feasible optionfor removal of heavy metals from the effluents contaminated withheavy metals. Bioremediation is associated with the high metalbinding capacity of biological agents with high efficiency to removeheavy metals from contaminated sites. Bioremediation showedpromising results in removing metals present even at very lowconcentrations where physico-chemical removal methods fail tooperate (Gadd and White, 1993).

Several biological tools like plants, bacteria, fungi, algae havebeen considered for bioremediation of Pb (Watanabe, 1997). Fila-mentous fungi are of prime interest in this respect due to their highmetal tolerance and intracellular metal binding capacity. Aspergillusspecies has been found to be the most efficient filamentous fungi inbioleaching of several heavy metals (Santhiya and Ting, 2006).

Hyper-tolerant strains of bacteria, fungi and algae have beenfound to be capable to volatilize or biotransform Pb(II) in liquidmedium up to a significant extent (Silver and Phung, 1996).However, the biochemical mechanism of hyper-tolerance is still notwell-understood.

Accordingly Pb-tolerant strain of Aspergillus foetidus, MTCC 8876was used to evaluate its bioremediation potentiality and Pb toler-ance limit. The biochemical mechanisms behind the abnormallyhigh Pb tolerance of the test strain were also evaluated. Livebiomass of the strainwas also considered to study its potentiality ofPb biosorption during growth in Pb-contaminated liquid medium.Electron microscopic studies were done in order to study whetherthe strain can transform soluble Pb into insoluble crystals and bindthem onto its mycelia surface.

2. Materials and methods

2.1. Source of microorganism and composition of growth media

The fungus Aspergillus sp. isolated from wastewater treatmentcenter, Kalyani was identified as a lead (Pbþ2) tolerant strain bysupplementation experiments with high concentrations of Pb andfinally it was identified as A. foetidus MTCC8876 by The MicrobialType Culture Collection and Gene Bank (MTCC), Institute ofMicrobial Technology (IMTECH), Chandigarh, India.

The isolation and enumeration of microorganisms were carriedout in solid CD medium as described by Raper and Thom (1949),that contained (per liter): KH2PO4 (1 g), NaNO3 (2 g), MgSO4 (0.5 g),KCl (0.5 g), FeSO4 (0.01 g), ZnSO4 (0.01 g), glucose (40 g). Hereglucose-1-phosphate was used instead of KH2PO4 as phosphatesource because lead phosphate precipitation was formed in pres-ence of inorganic phosphate in the medium. The pH of the mediumwas adjusted to 8 before autoclaving by suitable addition of 0.2 MHCl and 0.2 M NaOH solution (if needed). The medium was solid-ified with 2% agar as solid CD medium (CDA). Streptomycin wasadded to the medium for arresting the bacterial growth. All of theplates were allowed to incubate at 30 �C in an incubator for 72 h forthe fungal growth. The best grown fungal colony with black conidiawas primarily identified as high Pb(II) ion tolerant strain and thesame was preserved in CDA slant or CD containing 1000 mg L�1

Pb(NO3)2 for further purification.

2.2. Preparation of pure culture and its maintenance

The conidia of the preserved strain was taken in sterile watercontaining one/two drops of Tween 80 and shaken vigorously. Theproperly diluted conidial suspension was taken for spreading ontoCDA medium supplemented with Pb(NO3)2 and allowed to grow in

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an incubator at 30 �C for 72 h to get the countable colony. The bestgrown colony having black conidia was preserved in CDA slant andselected for the present study. The strainwasmaintained at 30 �C inCDA medium. Slant cultures were routinely sub-cultured every 1month prior to experimental use on the same medium; sporesuspension prepared from 8 day old culture was used as inoculum.

2.3. Growth of the PbR strain in liquid CD medium

A. foetidus MTCC 8876 was grown by the shake flask methodunder aerobic condition. Liquid CD brothwas used for the growth ofthe fungus 100 mL of sterile CD medium was transferred intoa series of 500 mL conical flasks. The conical flasks were inoculatedwith Spore suspension of the test strain (1010 conidia in 1 L) andshaken at 175 rpm at 32 �C in an orbital shaker. For enzymatic andbiomolecular studies, five different concentrations of Pb(II), namely200, 400, 600, 800, 1000 mg L�1 were added separately to thegrowth media. Biomass was harvested after 96 h growth period,filtered, washed with sterile de-ionized water and pre-weighedwith Whatman filter paper no.1. After that the biomass was keptat �20 �C until used.

2.4. Assay of total thiol (eSH) content

Total thiol was assayed by the modified method of Ellman(1959). 0.2 g of fungal mycelia (grown at different metal concen-trations) for each sample was taken and ground in a precooledmortar and pestle with 0.6 g of alumina and extracted with 4 mL of50mM ice-cold phosphate buffer (pH 7.0) with and without 50 mMEDTA. The homogenatewas centrifuged at 2000 g for 20min at 4 �Cand the supernatant was collected. Three milliliters of the super-natant was mixed with 2 mL phosphate buffer (pH 7.0) and 5 mLdistilled water and mixed well. 20 mL of 0.01 M DTNB solution wasadded to 3 mL of the reaction mixture, shakenwell and absorbancewas recorded at 412 nm. Thiol content was calculated usingextinction coefficient 13,600 M�1 cm�1 for DTNB at 412 nm.

2.5. Proline assay

The assay of Proline was carried out by the method of Chinard(1952). 0.2 g fresh mycelia was ground with 0.4 g alumina in anice-cold mortar and pestle and extracted with 4 mL of 3% sulfosa-licylic acid. The homogenate was centrifuged at 2000 g for 20 min.Two milliliters of the supernatant was pipetted into a 10 mL testtube and 2 mL glacial acetic acid and 2 mL acid-ninhydrin reagentswere added to the supernatant. The tube was stoppered witha glass marble and heated to 100 �C for 45 min and subsequentlyplaced in an ice bath to cool the tubes to room temperature and4 mL toluene was added to each tube. The tubes were sealed withglass stoppers and shaken vigorously for 2 min and allowed tostand for 15 min for completion of phase separation. Upper toluenelayer was separated and kept in room temperature for 10 min andthe intensity of red color was read at 520 nm against toluene blank.The proline concentration was determined from a standard curveand calculated on a fresh weight basis (mM proline/gFW).

2.6. Lipid peroxidation assay and determination of intracellularH2O2 content

The content of malondialdehyde (MDA), a final product of lipidperoxidation, was determined using the method as described byDhindsa et al. (1981). A 0.5 mL aliquot of extract was added to 1 mLof 20% (v/v) trichloroacetic acid and 0.5% (v/v) thiobarbituric acid.The mixture was heated in a water bath at 95 �C for 30 min andcooled to room temperature. It was centrifuged at 10,000 g for


Table 1Dry weight of mycelia after 96 h growth in liquid Czapek Dox media with differentPb(II)concentration.

Pb (II) concentrations (mg/L) Dry weight of mycelia (gm)

0 0.44 � 0.15200 0.55 � 0.21400 0.32 � 0.31600 0.28 � 0.1800 0.18 � 0.091000 0.11 � 0.08

Data were found to be significant at P < 0.05.

S. Chakraborty et al. / International Biodeterioration & Biodegradation xxx (2012) 1e9 3

10 min; the supernatant was read for absorbance at 532 and600 nm. The absorbance value for nonspecific absorption at 600 nmwas subtracted from the value at 532 nm. The amount of MDA (redpigment) was calculated using the adjusted absorbance and theextinction coefficient 155 mM�1 cm�1 (Heath and Packer, 1968).

H2O2 content was measured according to modified method ofVeliKova et al. (2000). Mycelia were digested and extracted with0.1% TCA (w/v) at 4 �C. The homogenate was centrifuged at 12000 gfor 15 min at 4 �C. 750 mL of the supernatant was added to 750 mL ofphosphate buffer (pH �7.0) and 1.5 mL 1 M KI. The absorbance wasread at 390 nm.

2.7. Assay of catalase (CAT) activity

0.5 g fresh mycelia was freeze-dried with liquid nitrogen andground with 1 g alumina in an ice-cold mortar and pestle andextracted by using 8 mL of 50 mM ice-cold phosphate buffer(pH 7.0) containing 1% polyvinylpyrrolidone in an ice bath. Thehomogenate was centrifuged at 15,000 g for 20 min at 4 �C. Thesupernatant was used for the assay of catalase activity .The activityof catalase enzyme was measured using the method of Chance andMaehly (1955). The reaction mixture (3 ml) contained 50 mMphosphate buffer (pH 7.0), 15 mM H2O2, and 0.1 mL of enzymeextract. Reaction was initiated by adding the enzyme extract.Because of the linear decline of absorbance at 240 nm within thefirst 3 min, changes of the absorbance were read every minute. Oneunit CAT activity was defined as the change of 0.01 units per min.

2.8. Assay of glutathione reductase activity

Glutathione reductase enzyme activity was determined spec-trophotometrically at 25 �C, according to the method of Carlbergand Mannervik (1985). Enzyme extraction procedure was thesame as mentioned for CAT activity determination. The assaysystem contained 500 mM potassium phosphate buffer (pH 7.3),including 1 mM EDTA, 1 mM GSSG and 4 mM NADPH solution. Oneenzyme unit is defined as the oxidation of 1 mmol NADPH per minunder the assay condition.

2.9. Assay of guaiacol peroxidases (GPX) and syringaldazineperoxidases (SPX) activity

Peroxidase activity towards guaiacol was assayed by the modi-fied method of Maehly and Chance (1954). This method consists ofthe assay of tetraguaiacol e a colored product of guaiacol oxidationin the investigated sample. The assay mixture contained 50 mMpotassium phosphate (pH 7.0), 100 mL enzyme extract, 20 mMguaiacol and 6 mM H2O2. Absorbance was recorded at 470 nm. Theactivity of the enzyme was expressed as Unit per 1 mg of protein.

Activity of peroxidase towards syringaldazine was assayedaccording to the modified method of Imberty et al. (1985). Theassay mixture contained 50 mM potassium phosphate buffer (pH7.0), 60 mL enzyme extract, 41.6 mM syringaldazine and 4 mMH2O2. Absorbance was recorded at 530 nm.

2.10. Protein measurement

The method of Lowry et al. (1951) was used to measure proteincontent in the above experiments.

2.11. Electron microscopic studies on Pb(NO3)2 treated Pb-resistantstrain

Samples were prepared for SEM according to Gharieb et al.(1995). In preparation of sample for electron microscopic studies,


freshly grown Pb(II) resistant mycelium was washed repeatedlywith phosphate buffer (pH-7.0) and fixed in 5% glutaraldehydeusing the same buffer of pH 7.0 for 4 h. The sample was washedagain with the same buffer and dried in graded alcohol solutionranging from 30% to 100% twice with each for half an hour timeinterval. The dried sample was mounted on carbon tape and coatedwith platinum for 45 s. The sample was then analyzed by a fieldemission scanning electronmicroscope JEOL JEM 6700F operated at5.0 KV.

2.12. Sample preparation for lead analysis

Sample preparation for flame AAS to detect Pb was done bya modified method of Greenberg et al. (1995) according to 1 g ofmycelia or 1 mL of spent medium was placed into an Erlenmeyerflask and 3 mL of concentrated HNO3 and 1 mL of concentrated HClwere added. This mixture was heated for 3 h at 85 �C until thesolubilization of the sample was complete and then diluted to25-mL volumewith deionized water. A blank digest was carried outin the same way.

0.2 g mycelia were ground using mortar and pestle with 0.6 g ofalumina and extracted with 4 mL of 50 mM phosphate buffer (pH7.0). The homogenate was centrifuged at 1000 g for 10 min at roomtemperature to discard the cell debris. The supernatant wascollected and prepared, as mentioned above, for measurement ofintracellular lead content by AAS.

Finally the lead was analyzed using PerkinElmer AAnalyst 200atomic absorption spectrometer equipped withmercury/hydride15and a quartz tube atomizer. Argon gas (ultrahigh purity 99.995%)was used to sheath the atomizer and to purge internally. Arsenichallow cathode lamp (PerkinElmer, USA) was used at a wavelengthof 193.7 nm with a slit width of 0.7 nm.

2.13. Statistical analysis

Each experiment was repeated three times. Statistical analysiswas done by one-way ANOVA followed by post-Hoc multiplecomparisons by Duncan’s method using spss 14. The difference wasconsidered as significant when P < 0.05.

3. Results

3.1. Growth of fungus

The toxic effect of Pb(II) on the test strain, A. foetidus wasmeasured by analyzing the dry weight of the mycelia grown for96 h in liquid broth containing different concentrations of Pb(II).Growth of the fungus initially increased by almost 20% at200 mg L�1 Pb(II) treatment in respect to control (no Pb in growthmedium) followed by gradual decrease with further increase inPb(II) treatment (Table 1). The growth of the strain became severelystunted at 1000 mg L�1 Pb treatment resulting in 75% growth


0

10

20

30

40

50

60

0 200 400 600 800 1000

Pro

tein

Con

tent

(µg

/gF

W)

Lead Content(mg L-1)

Fig. 1. Intracellular protein contents of the strain after 96 h growth in liquid CD brothwith different Pb (II) concentrations. Data were found to be significant at P < 0.05.

Table 2Intracellular proline contents of the strain after 96 h growth in liquid CD broth withdifferent Pb (II) concentrations.

Pb (II) concentration (mg L�1) Intracellular proline content (mg/gFW)

0 104.37 � 8.59200 127.42 � 15.56400 145.48 � 23.45600 178.57 � 8.021800 182.62 � 6.881000 240.9 � 5.38



reduction compared to control. No growth was observed beyond2000 mg L�1 Pb treatment. In solid medium the strain couldtolerate up to 5000 mg L�1 lead concentration.

3.2. Changes in intracellular protein content

Intracellular protein contents increased initially with Pb treat-ment and reached themaximumvalues at 400mg L�1 Pb treatment(16.73% increase with respect to control). Protein content was thendecreased gradually with further increase in Pb(II) treatment(Fig. 1). For the 1000 mg L�1 treatment group, the decrease wasfound to be 54.15% with respect to control.

3.3. Protein leakage

Leakage of intracellular proteins was observed in the spentmedium during growth of the strain along with Pb. As shown inFig. 2 protein leakage increased gradually with the increasingconcentration of Pb(II) in growth medium. For the 400, 600 and800 mg L�1 Pb treatment groups, the leakage of protein was not sosevere but at 1000 mg L�1 Pb treatment, the protein leakage wasfound to increase by almost 55.98% with respect to control.

3.4. Intracellular proline content

Intracellular proline content of the fungus was found to be(Table 2) gradually increased with the increase in Pb concentration.The values were found to be 22.08%, 39.39%, 71.09%, 74.97% for200mg L�1, 400mg L�1, 600mg L�1, 800mg L�1 lead nitrate treatedstrain respectively. At 1000 mg L�1 the increase in intracellularproline content was about 130.81% with respect to control (CD).

0100200300400500600700

0 200 400 600 800 1000

pro

tein

L

eak

ag

e co

nten

t (µ

g /g

FW

)

Pb content in mg L-1

Fig. 2. Protein leakage in spent media by the strain during growth in CD media withdifferent Pb (II) concentrations. Light shaded and deep shaded bars represent thiolcontents without and with EDTA in the extraction buffer. Data were found to besignificant at P < 0.05.


3.5. Changes in intracellular thiol (eSH) content

Intracellular thiol contents increased gradually with increase inPb stress during growth of the fungus in liquid medium. The strainsynthesized maximum amount of thiols at 1000 mg L�1 Pb treat-ment (1.75 fold higher than control). The experimental values forthiol content were found to be higher when EDTA was used in theextraction buffer than when there is no EDTA. However sucha differencewas not observed for thiol contents of the control group.

3.6. Thiol leakage

There was a significant amount of thiol leakage in the spentmedium during growth of the strain in Pb stress condition whichshowed a gradual increase with increase in Pb(II) stress. For the1000 mg L�1 Pb treatment group, there was 3.06 fold increases inthiol leakage in comparison to control group. The values for thiolcontent during leakage were found higher when EDTA was used inthe extraction buffer in comparison to that where there was noEDTA. Here again, no such difference in leakage values was notobserved for the control group.

3.7. Changes in catalase activity and extent of lipid peroxidationand H2O2 content

Catalase activity initially increased with increase in Pb(II)concentration and maximum activity was observed at 200 mg L�1

Pb treatment with an increase by 1.48 fold in respect to CAT activityof control. A gradual decrease was noticed with further increase inPb treatment. The decrease was almost 0.474 fold and 0.398 foldrespectively at 800 mg L�1 and 1000 mg L�1 lead concentration incomparison to control. The gradual decrease of enzyme activity asshown in the Fig. 5 suggests that, ROS formation due to Pb toxicitymay inhibit the activity of catalase enzyme.

Malondialdehyde, the final product of lipid peroxidation,increased gradually with increasing Pb(II) stress. For the 200,400,600 and 800 mg L�1 Pb treatment groups the MDA content wasfound to be increased 24.48%, 92.75%, 127.84% and 186.95%

020406080100120

0 200 400 600 800 1000

Thi

ol c

onte

nt in

µM

/gF

W

lead concentration in mg L-1

Fig. 3. Change in intracellular thiol content of the strain in response to Pb (II) treat-ment. Light shaded and deep shaded bars represent thiol contents without and withEDTA in the extraction buffer. Data were found to be significant at P < 0.05.


0

5

10

15

20

25

30

35

40

45

0 200 400 600 800 1000

Thi

ol le

akag

e(µM

/gF

W)

lead concentration in mg L-1

Fig. 4. Thiol leakage in spent media by the strain during growth in CD media withdifferent Pb (II) concentrations. Light shaded and deep shaded bars represent thiolcontents without and with EDTA in the extraction buffer. Data were found to besignificant at P < 0.05.

05101520253035404550

0 200 400 600 800 1000

Cat

alas

e A

ctiv

ity

(Uni

ts m

g-1pr

otei

n m

inut

e-1)

Lead content (mg L-1 )

Fig. 5. Catalase activity in mycelia of the strain grown at different Pb (II) concentra-tions. Data were found to be significant at P < 0.05.

0

5

10

15

20

25

30

35

0 200 400 600 800 1000

GR

A

ctiv

ity

(u

nits

m

g-1

pro

te

in

m

in

ute

-1)

Lead Content(mg L-1)

Fig. 6. Glutathione reductase activity of the strain grown at different Pb (II) concen-trations. Data were found to be significant at P < 0.05.

0

10

20

30

40

50

60

0 200 400 600 800 1000G

PX

act

ivit

y(u

nits

mg-1

prot

ein

min

ute- 1

)Lead Content(mg L-1)

Fig. 7. Guaiacol peroxidase activity in mycelia of the strain grown at different Pb (II)concentrations. Data were found to be significant at P < 0.05.

120

te

-1)


respectively in comparison to control. Maximum extent of lipidperoxidationwas found at 1000 mg L�1 Pb treatment with increasein MDA content by almost 253.53% in comparison to control. Thegradual increase of MDA content (Table 3) indicated significant risein ROS as well as H2O2 generation which consequently causes lipidperoxidation of membrane lipid and membrane damage.

Intracellular H2O2 contents increased gradually with increasingPb(II) stress. At 200 mg L�1 Pb treatment, there was no significantchange in H2O2 content. However with further increase in Pbtreatment, gradual increase in H2O2 production was noticed. Theincrease in H2O2 content was almost 37.87%, 120.00%, 144.25%,183.40% at 400 mg L�1, 600 mg L�1, 800 mg L�1, 1000 mg L�1 Pb(II)concentrations respectively, in comparison to control. Figs. 3 and 4.

3.8. Changes in Glutathione Reductase assay

Glutathione Reductase (GR) activity gradually increased withthe increase in Pb(II) concentration and reached maximum at1000 mg L�1 Pb treatment. The increase of GR activity at

Table 3MDA contents and intracellular H2O2 contents of the strain grown after 96 h growthin liquid CD broth with different Pb (II) concentrations.

Pb (II) concentrations(mg L�1)

Intracellular H2O2

content (mg/gFW)MDA content(mg/gFW)

0 2.35 � 0.166 6.568 � 0.58200 2.35 � 0.26 8.176 � 0.487400 3.24 � 0.08 12.66 � 0.632600 5.17 � 0.51 14.965 � 0.621800 5.74 � 0.45 18.847 � 1.1421000 6.66 � 0.63 23.22 � 1.529



1000 mg L�1 Pb concentration was about1. 85 fold with respect tocontrol (Fig. 6).

3.9. Changes in Guaiacol peroxidase and Syringaldazine peroxidaseactivity

GPX and SPX activities increased gradually with increase inPb(II) stress and showed maximum activities at 1000 mg L�1 Pbtreatment. GPX and SPX activities were almost 16.4 and 3.92 foldhigher at 1000 mg L�1 Pb treatment than that of control. Figs. 7e9

3.10Electron microscopic studies

The SEMmicrographs of the test strain are shown in Fig. 9. Cellswere grown aerobically in liquid CD broth containing 600 mg L�1

lead and harvested after 96 h incubation at 30 �C. Crystals in large

0

20

40

60

80

100

0 200 400 600 800 1000

SP

X activ

ity

(U

nits

m

g-1

pro

te

in

m

in

u

Lead content (mg L-1)

Fig. 8. Syringaldazine peroxidase activity in mycelia of the strain grown at different Pb(II) concentrations. Data were found to be significant at P < 0.05.


Fig. 9. Scanning electron micrographs of A. foetidus mycelia grown at 200 mg/L Pb (II) in liquid Czapek Dox medium. (a) & (b) showing bound crystals on the mycelial surface;(c) EDS analysis of crystal confirming the presence of Pb.


number were found to be bound tightly on to the mycelia surface.Energy dispersive X-ray spectroscopy confirmed the presence of Pbalong with carbon (C) and oxygen (O) within those crystals.

3.11. Pb content in total biomass

The total Pb content in fungal biomass was measured as anindicator of Pb absorption as well as adsorption by the straingrown in presence of Pb in liquid growth media. Results showedthat Pb(II) contents in total biomass increased with increasing Pb


concentration and maximum Pb content was found (512.35 gm/kgmycelia) for the 1000 mg L�1 Pb treatment group.

3.12. Pb content in spent medium

The Pb content in spent medium was taken as a measure ofinability of the fungal biomass to absorb or adsorb Pb from theliquid growth medium. Results showed that the fungal biomasscould take up large amount of Pb from the growth medium duringgrowth at 200, 400 and 600mg L�1 Pb concentrations. However, for800 and 1000 mg L�1 treatment groups (Table 1), large amount of



Pb was found to be left in the spent medium. At 800 and1000 mg L�1 the amount of Pb observed in spent medium was387.87 mg L�1 and 713.25 mg L�1 respectively.

3.13. Intracellular Pb uptake

The test strain was found to be able to absorb Pb intracellularly.Although intracellular Pb uptake gradually decreased with increasein Pb stress, significant amount of Pb was present inside the fungalcells as evident from figure. Minimum Pb uptake was 384.97 mg/kgmycelia, observed at 1000 mg L�1 Pb treatment and the maximumwas 1011.50 mg/kg, found at 200 mg L�1 Pb treatment.

3.14. Pb removal

The test strain could remove significant amount of Pb from theliquid growth medium as evident from Table 4. At 200 mg L�1 Pbtreatment, the strain could remove almost 94.57% Pb from themedium. However Pb removal efficacy of the strain decreased withfurther increase in Pb treatment. At 1000 mg L�1 Pb treatment thestrain could remove almost 28.67% of Pb from the liquid growthmedium.

4. Discussion

The data of the present study showed (Table 1.) that the teststrain, A. foetidus MTCC 8876 is highly tolerant to Pb(II). Growth ofthe strain was slightly stimulated at 200 mg L�1 Pb(II) concentra-tion, the lowest dose of the present study. Similar results werefound during the growth of Aspergillus nidulans with cadmiumdoses (Guelfi et al., 2003; Mukherjee et al., 2010). This may be dueto the fact that the low dose of Pb(II) evoked ArndteSchultz effect,according to which toxic substances (Pb in the present study) innon-lethal doses may induce instability in the cell membranepermeability that may cause more free flow of nutrients within thecell andmetabolic activity increases (Ahonen-Jonnarth et al., 2004).Though the biomass content may be gradually decreased at higherconcentration of lead but it is observed that the test strain canwithstand such a higher concentration of lead. So the test fungalstrain may be used as a bilogical tool for lead bioremediation as itcould tolerate high concentration of Pb(II).

In this present study, intracellular protein content initiallyincreased in Pb(II) stress and this may strengthen the support of thePb(II) tolerant property of the stain. During Pb (II) stress, the strainsynthesizedmore protein up to certain level of Pb treatment, for thesurvival strategy, which again, may reflect the possibility of Arndt-Schultz effect on the strain. At high concentration of Pb(II), intra-cellular protein contents decreased sharply which may be a resultof intolerance of the test strain to such unusually high leadconcentration.

Leakage of intracellular protein gradually increased with theincreasing concentration of Pb(II). From this result it indicates that

Table 4AAS measurement of Pb biosorption by mycelia, intracellular Pb contents and % Pbremoval. The % removal was calculated by measuring the Pb contents of the spentmedia.

Pb treatmentgroups (mg L�1)

Total Pb absorbed(gm Kg�1)

Intracellular Pbcontent (mg Kg�1)

% of Pb removal

200 68.8 � 0.98 1011.5 � 90.64 94.57 � 1.35400 223.678 � 5.61 778.6 � 61.99 93.45 � 0.26600 390.87 � 6.78 578.55 � 26.38 87.79 � 0.328800 457.92 � 15.21 520.93 � 77.456 51.523 � 1.71000 512.35 � 17.35 384.97 � 79.65 28.67 � 0.954



the stability of cell membrane was hampered with increase in Pbdoses. This may be due to enhanced lipid peroxidation caused byROS generated evoked by Pb toxicity .The gradual increase of MDAcontent with the increase of the concentration of Pb(II) strength-ened the conjecture that the enhancement of lipid peroxidation atthe level of cell membrane of the strain as shown in Table 3. Leakageof thiol-rich protein molecules at the extracellular level normallychelate Pb (II) and such complexes are effluxed by the fungal cells asa part of defense mechanism against Pb toxicity which could beconsidered as an evidence of bioremediation of Pb(II) as well as theevidence of a lead tolerance of the strain of fungus.

To minimize the toxic effect of the heavy metals, plants havesome intrinsic detoxifying mechanisms through bioremediation ofheavymetal which is chelation. The level of intracellular thiol(eSH)groups was found to be elevated during metal stress because thiolsare considered as well-known for metal chelation and detoxifica-tion (Schmoger et al., 2000). Phytocelatins, metallothionein,reduced glutathione etc, some important members of thiol familyare capable of binding heavy metal ions via thiolate coordination infungi (Pal and Das, 2005; Bellion et al., 2007). In the present study,the intracellular thiol content initially increased in treatment of thefungal strain with Pb(II). When EDTAwas used in extraction buffer,thiol content was found to be higher than that of the value whenEDTA was not used. As EDTA can chelate Pb(II), thus making thethiols free for reactionwith DTNB and when EDTAwas absent, thiolgroups were engaged in chelating Pb(II). Hence less number ofthiols reacted with DTNB and a decrease in value was observed.However, in both experiments the control group showed nodifference in thiol content. These findings clearly indicate thatformation of thiole Pb(II) complex took placewithin the fungal celland this mechanism may play a vital role in bioremediation ofPb(II).

Significant leakage of intracellular thiols was observed in spentmedium when the test strain was grown in liquid CD medium inpresence of Pb (II). Amount of thiol leakage increased with increasein Pb content in the medium. Here again, when EDTA was used inthe extraction buffer, thiol content was found to be higher than thatin absence of EDTA. These results indicate the fact that thiole Pb(II)complexes were formed within the test strain and the complexeswere moved out of the cell to minimize Pb content within thefungal cells. Hence the test strain might possess an inherentmechanism to chelate the intracellular Pb and to move suchcomplexes outside the cell.

Proline acts as an osmolyte, radical scavenger, stabilizers ofmacromolecules (Matysik et al., 2002). Proline has been found toaccumulate in Chlorella sp. under cupric ion stress (Wu et al., 1995).In this present study there was an increase in proline content inresponse to Pb(II) toxicity. This may suggest that proline plays animportant role in Pb(II) toxicity, probably to scavenge ROS gener-ated due to heavy metal toxicity.

A common consequence of heavy metal stress is the formationROS which may elevate the antioxidant defense system of the cell.Poly-unsaturated fatty acids present in cell wall as well as in thecytosolic environment are very much reactive to heavy metalgenerated ROS and undergo peroxidation producing different typesof cytotoxins like malondialdehyde (MDA) e a secondary lipidperoxidation product of cell and a reliable indicator of free radicalformation (Demiral and Türkan, 2005). Lipid peroxidation was re-ported from different types of living organism and this acts asa biomarker to indicate the presence of heavy metals (Zhang et al.,2007; Biswas et al., 2008). From the present study it was found thatPb toxicity caused significant amount of intracellular H2O2 gener-ation in the test strain. Moreover, lipid peroxidation was alsoinduced drastically within the test strain due to Pb toxicity asevident from high intracellular MDA contents during Pb(II) stress.



The reason may be that H2O2 generated during Pb stress causeddamage to the lipid layer of the cell membrane by peroxidation ofpoly-unsaturated fatty acids. During growth of the strain in CDbroth, significant amount of protein leakage was observed whichmay be due to damage of the cell membrane by Pb as evident fromhigh MDA contents.

To counterbalance the hazardous effects of ROS generated due totoxic conditions, the antioxidative enzymes present in cells act intandem.

Catalase uses hydrogen peroxide as a donor of hydrogen anda substrate in the catalytic decomposition of hydrogen peroxide toform oxygen and water. In our study, increased catalase activity atlow Pb(II) concentration indicated the involvement of this enzymein minimizing the enhanced amount of intracellular H2O2 gener-ated due to Pb(II) treatment and in recovery or repair of the damageof the cytosolic environment of the fungal cell due to oxidativestress (Imlay and Linn, 1988). Under such condition the test fungalstrain remain viable up to the tolerable concentration of Pb(II) andthe strain could be considered as an efficient tool for bioremedia-tion of Pb(II). But high concentrations of Pb(II) caused irreversibleoxidative damage to the fungal cell that was proved by the sharpdecrease in catalase activity.

Glutathione reductase (GR) also can remove H2O2 (Foyer andHalliwell, 1976) and maintains a balance between reduced gluta-thione (GSH) and oxidized glutathione (GSSG), as the latter is toxicto cells and this role of GR has been demonstrated by analyzingfungal mutants lacking this enzyme (Guelfi et al., 2003). In thepresent study GR activity gradually increased with the increase inPb(II) doses probably to minimize the toxic effects of H2O2 andother ROS generated due to Pb toxicity. Gradual increase of intra-cellular thiols during Pb treatment may reflect the role of GR inmaintaining stable GSH pool during toxic conditions. Hence GRmay play dual role for the test strain to minimize Pb toxicity.

Different types of peroxidase enzymes are found to be presentin higher as well as in lower organisms (Lepedus et al., 2005). GPXis one of the important peroxidases that play a vital role in celldetoxification. Enhanced activities of peroxidases in response toheavy metal stress are observed in, Phaseolus aureus Roxb (Singhet al., 2007). Haluskova et al. (2009) reported an increase inguaiacol peroxidase activity in barley root tip exposed to heavymetal stress. GPX acts upon H2O2 and breaks it down into waterand hydrogen to detoxify the cell. The gradual increase of GPXactivity due to Pb toxicity in the present study may reflect a part ofantioxidative defense mechanism of the test strain. SPX activityalso showed a gradual increase in Pb (II) stress. This increase mayresult in the breakage of excess H2O2 formed during Pb(II) stress,as a part of cellular antioxidative defense mechanism of the teststrain.

Scanning electron microscopic studies and EDX analysis clearlysuggest that the test strain could convert soluble Pb into insoluble Pbcrystals and retained themonto themycelial surface very tightly. Thismakes the strain suitable for subsequent growth and removal of Pbfrom Pb e contaminated sites. The biomass removed may thus takeaway a large amount of biosorbed Pb as well as Pb crystals stronglybound to the mycelial surface, from the contaminated site. This canprove to be a good strategy for easy, effective and lowcostmethod forPb bioremediation. Moreover, formation of insoluble Pb crystals mayindicate the ability of the strain to chelate Pb by synthesizing organicacids as was earlier reported for Aspergillus niger (Sayer and Gadd,1997) and Beauveria caledonica (Fomina et al., 2005).

Atomic absorption spectroscopic studies suggested that thestrain could biosorb large amount of Pb by total biomass. The straincould also take in Pb intracellularly up to a significant extent. Thestrain could grow and remove large amount of Pb from the liquidgrowth medium even at unusually high Pb treatments.


5. Conclusion

We have isolated the A. foetidus strainwith long-term adaptationto environment contaminated with heavy metals. High concentra-tions of Pb were used in the present study and the test strain ofA. foetidus could not only grow but remove large amount of Pb fromthe medium. Till date no other strain of fungi could tolerate sucha heavy concentration of lead in the medium. The strain was able totolerate the toxic effects of Pb(II) by modifying its enzymaticmachinery and synthesizing several biomolecules which couldchelate Pb(II) tominimize the toxic situation. The adaptive changes inmicrobial communities, their metabolic activities and biomassproduction capability lead them to survive and functionunder stress.The test strain could maintain its cellular functions under Pb(II)stress, suggesting the fact that the strain possesses some inherentcellular mechanisms to counteract Pb toxicity as a result of itsprevious adaptation to heavymetal contaminated environment. Thisproperty makes the strain more suitable for bioremediation of Pb.

Acknowledgment

The authors are thankful to University of Grants Commission(UGC) and DST of India, for their financial support. Authors are alsothankful to Prof. K. R. Samaddar (Retd.), University of Kalyani andDr. S. Das, University of Cambridge, London, U.K for their help inconstruction of English of the Manuscript.

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Biochemical characterization of a lead-tolerant strain of Aspergillus foetidus: An implication of bioremediation of lead from liquid media