A

PC

a

ARRAA

KAACpST

1

[nAtcmbncoscoffNsls

(

0d

Colloids and Surfaces B: Biointerfaces 81 (2010) 321–328

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

journa l homepage: www.e lsev ier .com/ locate /co lsur fb

lkali, thermo and halo tolerant fungal isolate for the removal of textile dyes

rachi Kaushik, Anushree Malik ∗

enter for Rural Development & Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India

r t i c l e i n f o

rticle history:eceived 15 April 2010eceived in revised form 6 July 2010ccepted 8 July 2010vailable online 22 July 2010

eywords:spergillus lentuluszo dyes

a b s t r a c t

In the present study potential of a fungal isolate Aspergillus lentulus FJ172995, was investigated for theremoval of textile dyes. The removal percentages of dyes such as Acid Navy Blue, Orange-HF, Fast Red A,Acid Sulphone Blue and Acid Magenta were determined as 99.43, 98.82, 98.75, 97.67 and 69.98, respec-tively. None of the dyes inhibited the growth of A. lentulus. Detailed studies on growth kinetics, mechanismof dye removal and effect of different parameters on dye removal were conducted using Acid Navy Bluedye. It was observed that A. lentulus could completely remove Acid Navy Blue even at high initial dyeconcentrations, up to 900 mg/L. Highest uptake capacity of 212.92 mg/g was observed at an initial dyeconcentration of 900 mg/L. Dye removing efficiency was not altered with the variation of pH; and biomass

oncentrationHaltemperature

production as well as dye removal was favored at higher temperatures. Dye removal was also efficienteven at high salt concentration. Through growth kinetics studies it was observed that the initial expo-nential growth phase coincided with the phase of maximal dye removal. Microscopic studies suggestthat bioaccumulation along with biosorption is the principle mechanism involved in dye removal by A.lentulus. Thus, it is concluded that being alkali, thermo and halo tolerant, A. lentulus isolate has a great

or the

potential to be utilized fsaline.

. Introduction

Azo dyes used in textile industries for dyeing are mutagenic1] and if ingested by humans, these dyes are reduced by intesti-al bacteria to form potentially carcinogenic aromatic amines [2].ryl amines which are the products of anaerobic microbial reduc-

ion, form highly reactive electrophiles in mammals that formovalent adducts with DNA and pose health hazards [3]. Treat-ent strategies based on various physico-chemical methods have

een developed [4,5]. Owing to their low cost and eco-friendlyature, studies on biological methods have gained ample signifi-ance which includes the use of various microbes either in livingr dead form [6–8]. Amongst the microbes, fungi are considereduperior as they have better capacity to adapt to environmentalonstraints as they produce large variety of extracellular proteins,rganic acids and other metabolites [9]. Studies conducted on dif-erent strains of Aspergillus sp. have reported various mechanismsor dye removal like biodegradation [10] and biosorption [11,12].

evertheless, the growth of fungi, enzyme production and sub-

equent dye decolourization is effected by the culture conditionsike pH, initial dye concentration, dye class, temperature and ionictrength [13]. Since, dye containing effluents are hot and alkaline

∗ Corresponding author. Tel.: +91 11 26591158; fax: +91 11 26591121.E-mail addresses: anushree@rdat.iitd.ac.in, anushree malik@yahoo.com

A. Malik).

927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2010.07.034

treatment of dye bearing effluents which are usually alkaline, hot and

© 2010 Elsevier B.V. All rights reserved.

and have high salt concentration [14]; therefore, microbes capableof surviving in such extreme conditions should be explored to treatdye waste water. To this date, most of the studies have reportedthe optimum dye removal at acidic pH (4–6) and mesophilic tem-perature (25–35 ◦C) by the fungi [13,15].

In the present study a strain of Aspergillus lentulus FJ172995was employed for removal of different dyes. In the previous study,authors have identified the nutritional requirements of the fungalisolate and formulated a low cost media required for an efficient dyeremoval from textile effluent [16]. In the present study, attemptshave been made to elucidate the mechanism of dye removal andto identify various functional groups present on the fungal cellwall that are involved in the process. Further, keeping in mind thesuitability for industrial applications, dye removal efficiency of thefungal isolate for broad range of various test parameters (initial dyeconcentration, pH, incubation temperature and salt concentration)was established. The present work indicates that its properties ofbeing alkali, thermo and halo tolerant make it a suitable candi-date for decolourization of textile effluents, which are often hotand alkaline.

2. Materials and methods

2.1. Dyes and chemicals

The dyes used in the experiment Acid Magenta (C.I. Acid Vio-let 19), Acid Navy Blue (C.I. Acid Blue 120), Acid Sulphone Blue

322 P. Kaushik, A. Malik / Colloids and Surfaces B: Biointerfaces 81 (2010) 321–328

Acid

(btSaaaeitA4awia

2

optot

Myes

2

w

the initial biomass concentration and k is a constant. The width ofouter active shell ω is calculated using the equation:

Fig. 1. Structures of the experimental dyes; (a) Acid Magenta, (b)

C.I. Acid Blue 89) and Fast Red A (C.I. Acid Red 88) were providedy Department of Textile Technology, IIT Delhi. Acid Magenta is ariarylmethane dye whereas Acid Navy Blue, Fast Red A and Acidulphone Blue are azo dyes. Orange-HF (C.I. Pigment Orange 34),disazo dye, was procured from small scale dyeing unit located

t Sanganer, Rajasthan (India). The chemical structures of the dyesre presented in Fig. 1. All these dyes are anionic in nature and findxtensive use in dyeing of woolen and silk fibers. Absorption max-ma for each dye was obtained by scanning the dye solution overhe visible range (Acid Magenta: 527 nm, Acid Navy Blue: 561 nm,cid Sulphone Blue: 572 nm, Fast Red A: 506 nm and Orange-HF:84 nm). Standard curves were plotted by measuring absorbancet absorption maxima for each dye. The stock solutions of 10 g/Lere prepared for each dye in distilled water. All the other chem-

cals used were of analytical grade and were obtained from Mercknd Qualigens.

.2. Microorganism and growing conditions

The experiments on dye removal were performed with a strainf A. lentulus FJ172995 previously isolated from the textile effluentrocured from Baddi (Himachal Pradesh, India) where large scaleextile units are located. The fungal isolate was maintained on slantsf Potato Dextrose Agar. Freshly revived cultures were used for allhe experiments.

The growth media having the composition: NH4NO3 0.5 g/L;gSO4·7H2O 0.1 g/L; K2HPO4 0.5 g/L; NaCl 1 g/L; glucose 10 g/L;

east extract 2.5 g/l and pH adjusted to 6.5, was used for all thexperiments unless otherwise stated. The flasks were agitated in ahaker (Scigenics Biotech, Orbitek) at 180 rpm and 30 ◦C.

.3. Removal of various azo dyes by A. lentulus

The efficiency of A. lentulus FJ172995 for removal of various dyesas determined using Acid Magenta, Acid Navy Blue, Acid Sul-

Navy Blue, (c) Acid Sulphone Blue, (d) Fast Red A, (e) Orange-HF.

phone Blue, Fast Red A and Orange-HF. Flasks having 100 mL mediawere prepared and autoclaved at 121 ◦C and 15 psi for 20 min. Ini-tial dye concentration for all the dyes in each flask was maintainedat 100 mg/L. The flasks were inoculated with 5% spore suspension(having spore concentration of 6.25 × 106/mL) aseptically and agi-tated in a shaker. Samples were withdrawn at 10 h interval for50 h. Absorbance of the supernatant of the withdrawn samples wasmeasured at absorption maxima of each dye with the help of a spec-trophotometer (Visiscan 167, Systronics) after centrifuging them at10,000 rpm for 15 min.

2.4. Kinetics of Acid Navy Blue removal by A. lentulus

Biomass production and dye removal by A. lentulus FJ172995was observed by examining its growth kinetics in presence of AcidNavy Blue at initial concentration of 100 mg/L. Flasks containingautoclaved media (100 mL each) were inoculated with 5% sporesuspension and agitated in a shaker. Flasks were removed at reg-ular intervals and the samples were analyzed for dye removal andamount of biomass produced. Control dye amended growth mediawithout inoculation of fungal spores was also run. Cube root kineticmodel [17] was used to describe the growth of the isolate using theequation:

M1/3 = M1/30 + kt (1)

where, M represents the biomass concentration at time ‘t’, M0 is

M = M0 +(

43

��n)1/3

ω�t (2)

where, � is the biomass density, n is the number of pellets and � isthe specific growth rate of the fungi.

rfaces

2A

ihttppSfltwarwp

2

tVaoe

D

wbc

itps(

dowstorf

2

p(wl

TE

Wa

P. Kaushik, A. Malik / Colloids and Su

.5. Effect of various test parameters on dye removal efficiency of

. lentulus

Effect of initial concentration of Acid Navy Blue on dye remov-ng efficiency of A. lentulus was established by inoculating the flasksaving sterilized growth media (pH 6.5) and different dye concen-ration (100–900 mg/L) with 5% spore suspension. For investigatinghe effect of pH on dye removal, flasks having growth media sup-lemented with dye (200 mg/L) were maintained at a fixed initialH ranging from pH 4 to 10 and inoculated with spore suspension.imilarly, effect of salt concentration was tested by maintaining theasks containing growth media at 0%, 0.5%, 1%, 2%, 5% and 10% (ini-ial dye concentration, 200 mg/L; pH 6.5). The flasks in all the casesere kept in a shaker at 30 ◦C and 180 rpm and the samples were

nalyzed as stated earlier. Effect of incubation temperature on dyeemoval by A. lentulus was established by monitoring dye removalith time at 30, 35, 40, 45 ◦C (initial dye concentration, 200 mg/L,H 6.5).

.6. Analytical techniques

The concentration of test dye was determined by measuringhe absorbance of samples through spectrophotometer (Systronicsisiscan 167) at 561 nm. A calibration plot between concentrationnd absorbance of the respective dye was used for determinationf dye concentration. Dye removal (%) was calculated using thequation:

ye removal (%) = (A0 − At) × 100A0

(3)

here A0 is the initial absorbance, At is the absorbance at incu-ation time, t. Test samples were analyzed for absorbance afterentrifuging (Sigma 4K5) the samples at 10,000 rpm for 10 min.

The dry cell weight of the biomass formed at the end of thencubation period was measured by filtering out the contents ofhe flasks through pre-dried and pre-weighed Whatman No. 1 filteraper and drying it overnight at 60 ◦C to a constant weight. Micro-copic photographs were taken through bright field microscopyLeica DM2500) at a magnification of 10×.

Fourier transform infrared (FTIR) spectroscopy analysis wasone for Acid Navy Blue dye, control biomass (grown in absencef dye) and dye laden biomass. For this, media free fungal biomassas first air dried and then mixed with KBr to form pellets. The FTIR

pectra were recorded using Spectrum One (Perkin Elmer) spec-rometer equipped with DTGS detector. IR absorbance data werebtained for the wave number range of 650–4000 cm−1. Data pointesolution of the spectra was 2 cm−1 and 4 scans were accumulatedor each spectrum.

.7. Statistical analysis

All the studies were conducted in triplicates and the results areresented as means of the replicates along with standard deviationrepresented as error bars). One-way and two-way ANOVA analysisas applied, as required, to statistically analyze the results with the

imit of significance set at P < 0.05 (SPSS Package, Version 16).

able 1fficiency of Aspergillus lentulus in removing various textile dyes (initial dye concentratio

Acid Magenta Acid Navy Blue

Dye removal (%) (20 h) 49.49 99.14fh

Dye removal (%) (50 h) 69.93 99.43foh

ithin a row, means followed with superscript letter have insignificant difference wherecolumn, h = insignificant difference between hours (P < 0.05).

B: Biointerfaces 81 (2010) 321–328 323

3. Results and discussion

3.1. Removal of various azo dyes by A. lentulus

Table 1 shows the dye removal (%) for 5 dyes under studyby A. lentulus FJ172995 after 20 and 50 h of incubation. Thedye removal efficiency of A. lentulus for different dyes after20 h of incubation was: Acid Navy Blue (99.14 ± 0.45%) > Fast RedA (98.33 ± 0.8%) > Acid Sulphone Blue (91.77 ± 1.22%) > Orange-HF(91.39 ± 1.38%) > Acid Magenta (49.49 ± 1.19%). When the biomassobtained in each case was measured, it was observed that therewas no significant difference between the values (P < 0.05). Thisimplies that none of the tested dyes inhibited the growth of A.lentulus. However, significant differences (P < 0.05) were observedin the rate of dye removal depending upon the class of the dye.The four azo dyes Acid Navy Blue, Acid Sulphone Blue, Fast Red Aand Orange-HF were efficiently removed (more than 90%) within20 h of incubation whereas 49% removal was observed in the flaskhaving Acid Magenta (triarylmethane) thereby suggesting that theuptake (of the dyes) by the fungal strain varies with the structure ofthe dye. Acid Magenta has a compact structure with three phenylrings bound to central C-atom. Extensive delocalization over threephenyl rings provides more stability to the structure of the dye.This might result in lower affinity of the dye for the fungal biomassand thus the reduced removal as compared to azo dyes. Since max-imum removal was observed for Acid Navy Blue, it was selected forcarrying out further experiments.

The dye removal efficiency of A. lentulus FJ172995 is consid-erably higher as compared to those reported in earlier studiesusing other microbial agents. Sumathi and Manju [11] reported95.99% colour removal from growth medium containing certainazo dyes in 48 h at 50 mg/L dye concentration using Aspergillusfoetidus. M. verrucaria and Ganoderma sp., are capable of decolour-izing dye containing wastewater with an efficiency of 99% after48 h of incubation through physical adsorption process [18]. Pasti-Grigsby et al. [19] reported that Phanerochaete chrysosporium andStreptomyces chromofuscus A11 needed 3.4 and 2.3 days, respec-tively, to demonstrate significant decolourization with a range ofazo dyes. A. lentulus brings about 98–100% removal of selected azodyes at 100 mg/L dye concentration within 20 h of incubation in dyecontaining growth medium.

3.2. Kinetics of Acid Navy Blue removal by A. lentulus

Fig. 2 shows the growth kinetics and dye removal using A. lentu-lus. Pelleted cultures are traditionally assumed to follow cube rootkinetics. Fig. 3 shows the plot of cube root of mycelial mass (M1/3)against time (t) till the process of dye removal was completed andequilibrium was achieved (21 h). This yields a straight line, the slopeof which gives the value of growth constant k = 0.08/h. A pellet isa spherical mass of non-growing mycelia surrounded by an outershell of active hyphae. Thus, a pellet increases in radius at a con-

stant rate through exponential growth of mycelia within the outershell of active hyphae. The width of the outer shell, ω, is determinedby the diffusion properties of material through the mycelium anddepends on the pellet structure (Papagianni 2004). For A. lentuluswidth of the outer active shell is 4.1 × 10−4 cm (from Eq. (2); where

n: 100 mg/L; pH: 6.5, temperature: 30 ◦C; agitation: 180 rpm).

Acid Sulphone Blue Fast Red A Orange-HF

91.77o 98.33nh 91.39s

97.67nfo 98.75nsoh 98.82nsf

n = Acid Navy Blue, s = Acid Sulphone Blue, f = Fast Red A, o = Orange-HF and within

324 P. Kaushik, A. Malik / Colloids and Surfaces

Fig. 2. Growth kinetics and removal of Acid Navy Blue by Aspergillus lentulus withtime (initial dye concentration: 100 mg/L; pH: 6.5, temperature: 30 ◦C; agitation:180 rpm, error bars represent the standard deviation at n = 3).

Fsa

�r(et

(dcwbbtppb

Ft1

ig. 3. Plot of cube root of mycelial biomass (M1/3) with time for Aspergillus lentulus;lope, k = 0.08/h (initial dye concentration: 100 mg/L; pH: 6.5, temperature: 30 ◦C;gitation: 180 rpm).

= 1.1356 g/cm3 and n = 4.8 × 105/L). Fig. 4 shows the plot of natu-al log of mycelial mass produced by A. lentulus [ln (M)] versus timet). The specific growth rate (�) of the fungus was 0.19/h during thexponential growth phase with a doubling time of 2.35 h (doublingime, td = ln 2/�).

As shown in Fig. 2, no dye removal was observed in the controluninoculated flasks) indicating that the dye removal was indeedue to organisms’ growth and not due to interaction with the mediaomponents. It is evident that complete dye removal occurredithin 18 h of incubation with the maximum removal taking place

etween 12 and 15 h. Positive correlation (R2 = 0.974) was observedetween dye removal (%) and biomass production during exponen-

ial growth phase. The results imply that actively growing biomasslays an important role in dye removal. This observation is com-arable to Sumathi and Manju [11] who observed that the youngiomass is important in dye removal.

ig. 4. Plot of natural log of biomass (ln M) produced with time during exponen-ial growth phase of Aspergillus lentulus; slope, � = 0.19 (initial dye concentration:00 mg/L; pH: 6.5, temperature: 30 ◦C; agitation: 180 rpm).

B: Biointerfaces 81 (2010) 321–328

As depicted in Fig. 2, substantial growth and biomass productionoccurred beyond 18 h, when the process of dye removal was alreadycomplete. Nevertheless, this additional biomass did not contributetowards dye uptake. Hence, it seems that the process time must beadjusted according to the initial concentration of the dye in order tooptimize the dye uptake and minimize the sludge production. Thisis also supported by the microscopic pictures (Fig. 5) which showsthe pellet morphology and load of the dye on pellets after 10 and48 h of incubation. The dye laden 10 h pellets appear to be smaller insize, under developed and completely saturated with dye (Fig. 5a).Whereas, 48 h old pellets are bigger in size, well formed and havemuch lower dye load that too concentrated on the central region.The peripheral regions of the mature fungal pellet which containsthe outer active shell appear white (Fig. 5b). These results also pro-vide an indication that young biomass posses high affinity for thedye which also gets bioaccumulated within the mycelium. This isevident from an unstained fungal hypha grown on solid agar platescontaining dye which appears blue due to bioaccumulation of theAcid Navy Blue dye (Fig. 5c). The mechanism of dye removal wasfurther studied by conducting desorption studies. If the dye wasbeing removed through biosorption without bioaccumulation asthe principle mechanism, then almost complete desorption of thedye from the fungal biomass should be achieved in the presenceof eluant. Desorption studies conducted with 0.1 M NaOH showsthat 50.75% dye could be desorbed in 30 min. Additional 22.67%dye could be desorbed when the dye biosorbed fungal pellets weredisintegrated with 0.5 M EDTA after carrying out surface desorptionwith NaOH. EDTA is a chelating agent and is used to disintegratethe fungal cell wall by making it more porous. Thus, disintegra-tion of cell wall resulted in the release of internalized dye. Hence,it seems that the dye removal is being effected simultaneously bybiosorption and bioaccumulation phenomenon. This is in contrastto the results obtained by Sumathi and Manju [11] for desorptionof Drimerene dyes from A. foetidus where more than 85% dye couldbe desorbed by 0.1 M NaOH and adsorption to cellular entities ofthe fungus was the principal mechanism. Similar reports indicatingbiosorption along with bioaccumulation of the dye by the micro-bial cells have earlier been reported [20,21]. Similar mechanismshave been proposed for the removal of metals from the solution[22].

The FTIR spectra for Acid Navy Blue dye, fungal biomass grownin the absence of dye and dye laden fungal biomass were obtainedin order to determine the groups that aid in the binding of dyemolecules to the fungal biomass. Spectra of the dye (Fig. 6a) showsdominant peaks at 1576.66 and 1516.66 cm−1 which representsthe primary and secondary amines and amides (N–H bending),respectively. The peak at 1343.33 cm−1 represents the sulphonylgroup (S O) which is representative for Acid Navy Blue dye whichis an anionic dye containing SO3

− group. Peaks at 1103.29 and1036.66 cm−1 represent C–N stretching vibrations. The spectra forcontrol biomass (Fig. 6b) show dominant peaks at 3279.33 cm−1

(representing bonded –COOH and –OH groups; primary andsecondary amine and amides, N–H stretching); 2926.66 cm−1

(C–H stretching); 1634.75 cm−1 (primary and secondary aminesand amides, N–H bending); 1546.66 cm−1 (amino group stretch-ing vibrations) 1405.41 cm−1 (C–H bending, CH3); 1236.66 cm−1

(C–O stretching of COOH; amines, C–N stretching); 1076.20 and1020 cm−1 (phosphate bonds, PO4

3−, P–OH; amines, C–N stretch-ing; C–O stretching of COOH) and 933 and 870 cm−1 (C–H bending).However, as compared to the control biomass, in the spectraof dye laden biomass (Fig. 6c), peaks corresponding to bonded

–COOH and –OH groups; primary and secondary amine and amides,N–H stretching (3280 cm−1); primary and secondary amines andamides, N–H bending (1640 cm−1) seems to have been reduced andpeaks representing C–H bending, CH3 (1405.41 cm−1); phosphatebonds, PO4

3−, P–OH; amines, C–N stretching; C–O stretching of

P. Kaushik, A. Malik / Colloids and Surfaces B: Biointerfaces 81 (2010) 321–328 325

F lue; (aa e (100

Chgpaapbs

ig. 5. Microscopic view of Aspergillus lentulus grown in the presence of Acid Navy bt 100 mg/L initial dye concentration, (c) fungal hypha grown in the presence of dy

OOH (1076.20 and 1020 cm−1); C–H bending (933 and 870 cm−1)as been masked or shifted. This suggests the probable role of theseroups of the fungal biomass in binding the dye molecules. Also, theeaks present in dye spectra in the region 1516.66–1576.66 cm−1

−1

nd at 1343.33 cm appear in dye laden biomass (prominent peakt 1536.66 cm−1 and in the region near 1300 cm−1 as several smalleaks), respectively. These peaks are all together absent in controliomass. This indicates that dye molecules are biosorbed on to thepecific groups of the fungal cell wall.

Fig. 6. Fourier transform infrared (FTIR) spectra of (a) Acid Navy Blue dye, (b) control

) 10 h grown mycelia at 100 mg/L initial dye concentration, (b) 48 h grown myceliamg/L initial dye concentration).

3.3. Effect of various test parameters

Effect of initial dye concentration on dye removing efficiencyof A. lentulus is depicted in Fig. 7. It was observed that process of

colour removal was slightly delayed with increase in initial dyeconcentration. At an initial dye concentration of 100 and 200 mg/L,complete dye removal was observed at 18 h interval. This timehowever increased to 21 h for initial concentration of 300 mg/L and24 h for initial concentrations of 400 and 500 mg/L. Parshetti et al.

biomass grown in the absence of dye, (c) biomass grown in the presence of dye.

326 P. Kaushik, A. Malik / Colloids and Surfaces B: Biointerfaces 81 (2010) 321–328

Ffa

[tt2l(scecuco

cflgbpatHn(aip9pmipptpwcidtcofa[F[m

Fig. 8. (a) Effect of initial pH on dye removing efficiency of Aspergillus lentulus (ini-tial dye concentration: 200 mg/L; temperature: 30 ◦C; agitation: 180 rpm, error barsrepresent the standard deviation at n = 3). (b) Change in pH profile during removal

ig. 7. Dye removal (%) and uptake capacity of Aspergillus lentulus obtained at dif-erent initial dye concentrations of Acid Navy Blue (pH: 6.5; temperature: 30 ◦C;gitation: 180 rpm, error bars represent the standard deviation at n = 3).

10] reported that time taken for complete adsorption of dye tohe mycelia of Aspergillus ochraceus increased from 24 to 120 h ashe initial concentration of Reactive Blue-25 was increased from00 to 500 mg/L. Hence, the extent of delay observed in case of A.

entulus (6 h) is quite lower as compared to that for A. ochraceus96 h). Moreover, greater than 98% dye removal was observed attill higher initial concentrations (600–900 mg/L) within 24 h indi-ating applicability of this strain for treating highly contaminatedffluents. Uptake capacity of the fungal biomass increased signifi-antly (P < 0.05) with increasing dye concentration (Fig. 7). Highestptake capacity of 212.92 mg/gm was observed at an initial dyeoncentration of 900 mg/L. This uptake capacity is slightly higherr at par with earlier reported strains [20,23].

Effect of pH on the dye removing efficiency of A. lentulus wasonducted at different pH values ranging from pH 4 to 10. Theasks were observed daily and it was found that simultaneousrowth occurred at all the tested pH resulting in considerableiomass production (5.3 ± 0.5 g/L) at 48 h. The pellets produced atH 4 were bigger (3–4 mm) as compared to pinhead sized pelletst rest of the tested pH. Enhanced aggregation of mycelia leadingo larger pellets is reported to be one of the toxicity mechanisms.ence, pH 4 should have contributed to stress generation but it didot adversely affect dye removal. Results suggest that eventuallyat 48 h) the dye removal was complete in all the cases (Fig. 8a)lthough the time taken for complete dye removal varied depend-ng upon the initial pH. While it took 46 h to obtain 99% removal atH 4 to 6, only 22 h were required to achieve the same at pH 7 to. At pH 10 also, 46 h of incubation was required to complete therocess of dye removal. Since, only the initial pH values were setanually, it was not possible to control the pH during the exper-

ment. As a result of this, a drop in pH was observed during therocess due to the production of fungal metabolites. This drop inH varied from case to case (Fig. 8b) and could have been a con-ributing factor in the delay caused in dye removal. It is seen that atH 4 and 5, the pH of the medium became more acidic (below pH 3)ith growth in fungal biomass which may have hindered the pro-

ess of dye removal. However, this reduction in pH was much lowern pH ranges from 7 to 9, and the prevailing pH range favoured theye removal process by the fungal isolate. However at pH 10, againhe pH of the medium became too extreme for the dye removal pro-ess, and thus the delay was observed. Nevertheless, the amountf fungal biomass required for the process to be completed wasormed in all the cases, almost complete dye removal was observed

t all pH ranges. Similar results were reported by Yesilada et al.24] where they reported efficient removal of Astrazon Red FBL byunalia trogii at pH ranges between pH 6 and 11. Meehan et al.25] reported that dye decolourization efficiency of Kluyveromycesarxianus remained unaltered between pH 3.0 to pH 5.5 with dye

of Acid Navy Blue by Aspergillus lentulus at different initial pH (initial dye concentra-tion: 200 mg/L; pH: 6.5; temperature: 30 ◦C; agitation: 180 rpm, error bars representthe standard deviation at n = 3).

decolourization ranging from 94 to 98% for different initial concen-trations of dye Remazol Black B (25, 50 and 100 mg/L). Howeverat 200 mg/L dye concentration dye decolourization at extreme pHvalues (pH 3.0 and 5.5) was reduced to 78% and 84% respectively.In contrast to this Asgher et al. [26] reported pH 4.5 as the opti-mal pH for removal of Solar Golden Yellow R dye by Schizophyllumcommune. In 6 days, 73% dye was decolourized at pH 4.5. However,decolourization reduced to less than 20% at pH 3 and 8% at pH 6.Aksu [20] also reported that Saccharomyces cerevisiae had a muchnarrow pH tolerance with optimum at pH 3.0 for the removal ofdye Remazol Blue. In view of the above reports, ability of A. lentulusto perform uniformly well in wide pH range (4–10) is remarkable.The pH range between pH 7 and 9 was considered optimum as thedifference between the dye removal values was insignificant whenlevel of significance was set at P < 0.05.

Studies conducted to evaluate the effect of temperature on dyeremoval predicted that higher temperature ranges promote higherdye removal. With increase in temperature from 30 to 45 ◦C, timetaken for complete dye removal decreased (data not shown). After12 h of incubation, 63.22% and 78.94% dye removal was obtainedat an incubation temperature of 30 and 35 ◦C, respectively (Fig. 9).But at 40 and 45 ◦C, dye removal increased to 93.63% and 93.98%,respectively, at 12 h of incubation which was significantly differentat P < 0.05. This can be attributed to the increased rate of fungalgrowth at higher temperature ranges. However at 20 ◦C (data notshown), fungal growth was delayed by 4 days. Similarly, Meehanet al. [25] reported increase in decolourization of Remazol Black B

by K. marxianus from 25 to 45 ◦C. Dye decolourization (%) increasedfrom 62% at 25 ◦C to 98% at 45 ◦C at an initial dye concentration of50 mg/L beyond which it started to decrease (10% decolourizationat 50 ◦C). In contrast to this, Arora et al. [27] reported 30 ◦C as the

P. Kaushik, A. Malik / Colloids and Surfaces

Fda

otbp

pocdhpiae[cdgal[a1p

psfg

Fl1

ig. 9. Dye removal (%) obtained by Aspergillus lentulus at 12 h of incubation forifferent incubation temperatures (initial dye concentration: 200 mg/L; pH: 6.5;gitation: 180 rpm, error bars represent the standard deviation at n = 3).

ptimum temperature for growth of Candida tropicalis and also forhe removal of Disperse dye 1. Lower dye removal was reportedeyond 35 ◦C. Thermal inactivation of the enzyme and low biomassroduction could have resulted in the decreased colour removal.

Effect of salt concentration on dye removal by A. lentulus isresented in Fig. 10. It is evident that complete dye removal wasbtained within 24 h for salt concentration up to 2%, optimum con-entration being 1%. In the case of 5% salt concentration, completeye removal was obtained in 64 h. The delay in dye removal atigher salt concentrations was caused due to the reduced biomassroduction. This can be caused due to the salt toxicity exhib-

ted through changes in osmotic balance, inhibition of metabolicctivities or hindrance of dye bioaccumulation by the salt due tolectrostatic interactions with the cell wall/membrane components11]. However, no growth occurred in the presence of 10% salt con-entration and whatever dye removal was obtained in this case wasue to the precipitation of the dye. Considering this, present fun-al isolate is equally halo tolerant when compared to the anaerobiccclimatized microbial consortia of Pseudomonas aeruginosa, Bacil-us circulans and two other unidentified strains NAD1 and NAD628]. Present result is also convincing over that obtained by Sumathind Manju [11] where they reported inhibition of approximately0% in removal of Drimarene Red (50 mg/L) by A. foetidus in theresence of 1% NaCl during the initial 20–40 h of incubation period.

The ability of A. lentulus to grow in alkaline pH and high tem-

erature may be explained by the behavior of two closely relatedpecies A. fumigatus (sibling species) and Neosartorya fischeri [29]. A.umigatus and N. fischeri are alkali tolerant fungi [30] that are able torow in alkaline pH. Thermotolerance of A. fumigatus and N. fischeri

ig. 10. Effect of salt concentration on dye removing efficiency of Aspergillus lentu-us (initial dye concentration: 200 mg/L; pH: 6.5; temperature: 30 ◦C; agitation:80 rpm, error bars represent the standard deviation at n = 3).

[

[[[[[[

[[[

[[[

[

[

B: Biointerfaces 81 (2010) 321–328 327

has been investigated extensively [31] and is attributed to THTAgene. A. lentulus also harbors salt responsive gene (srg) that maybe responsible to provide tolerance towards high salinity condi-tions. These properties render A. lentulus as a suitable candidate forremoving colour from dye wastewater. Studies conducted on realdye effluent suggest the suitability of the isolate for removing dyefrom effluents [16]. If suitable supplementation in the form of car-bon (glucose) and nitrogen (urea and ammonium chloride) sourcesis provided, as high as 66% removal can be obtained in unsterileconditions without diluting the effluent.

4. Conclusions

The ability of A. lentulus FJ172995 (isolated from textile efflu-ents) to remove selected dyes from synthetic solutions wasevaluated. It was observed that the strain could efficiently(97.67–99.43%) remove various azo dyes while another dye belong-ing to triphenyl methane class could be partially (69.98%) removed.Microscopic and desorption studies suggest that bioaccumulationalong with biosorption is the principle mechanism involved in dyeremoval by A. lentulus. Since, the dye containing effluents have alka-line pH and are hot; therefore, it is important that the test organismis able to remove dye at high pH ranges and is thermo-tolerant. A.lentulus FJ172995 isolate is capable of removing high concentra-tions of Acid Navy Blue (up to 900 mg/L) and produces efficientdye removal at wide pH ranges (pH 4–10) and high temperatures(up to 45 ◦C) as well. In addition to this, this isolate is capable oftolerating high salt concentration (up to 5%) which is a commonchemical auxillary found in dye waste water. Results show thatoptimum parameters required for efficient dye removal includealkaline pH (pH 8), high temperature (45 ◦C) and 1% salt concentra-tion. This strengthens the prospects of the strain to be developed forindustrial scale process for treatment of dye containing effluents.

Acknowledgements

Financial assistance from Department of Science & Technology,Government of India and CSIR Senior Research Fellowship to oneof the authors (PK), are gratefully acknowledged.

References

[1] N. Mathur, P. Bhatnagar, P. Bakre, Appl. Ecol. Environ. Res. 4 (2005) 111.[2] H. Xu, T.M. Heinze, S. Chen, C.E. Cerniglia, H. Chen, Appl. Environ. Microbiol. 73

(2007) 7759.[3] W.G. Tarpley, J.A. Miller, E.C. Miller, Cancer Res. 40 (1980) 2493.[4] H. Lata, V.K. Garg, R.K. Gupta, Dyes Pigments 74 (2007) 653.[5] S.D. Hosseini, F.S. Asghari, H. Yoshida, Water Res. 44 (2010) 1900.[6] I.M. Banat, P. Nigam, D. Singh, R. Marchant, Bioresour. Technol. 58 (1996) 217.[7] S. Deng, G. Yu, Y.P. Ting, Colloid Surf. B 44 (2005) 179.[8] C. Dogar, A. Gurses, M. Acikyildiz, E. Ozkan, Colloid Surf. B 76 (2010) 279.[9] L. Coulibaly, G. Gourene, S.N. Agathos, Afr. J. Biotechnol. 2 (2003) 620.10] G.K. Parshetti, S.D. Kalme, S.S. Gomare, S.P. Govindwar, Bioresour. Technol. 98

(2007) 3638.11] S. Sumathi, B.S. Manju, Enzyme Microb. Technol. 27 (2000) 347.12] Y. Fu, T. Viraraghavan, Adv. Environ. Res. 7 (2002) 239.13] P. Kaushik, A. Malik, Environ. Int. 35 (2009) 127.14] E.K. Dafnopatidou, N.K. Lazaridis, Ind. Eng. Chem. Res. 47 (2008) 5594.15] Y. Fu, T. Viraraghavan, Bioresour. Technol. 79 (2001) 251.16] P. Kaushik, A. Malik, World J. Microbiol. Biotechnol. (2010, In press),

doi:10.1007/s11274-010r-r0376-9.17] M. Papagianni, Biotechnol. Adv. 22 (2004) 189.18] D.G. Mou, K.K. Lim, H.P. Shen, Biotechnol. Adv. 9 (1991) 613.19] M.B. Pasti-Grigsby, A. Paszczynski, S. Goszczynski, D.L. Crawford, R.L. Crawford,

Appl. Environ. Microbiol. 58 (1992) 3605.20] Z. Aksu, Process Biochem. 38 (2003) 1437.

21] Z. Aksu, G. Donmez, Process Biochem. 40 (2005) 2443.22] L. Ruta, C. Paraschivescu, M. Matache, S. Avramescu, I.C. Farcasanu, Appl. Micro-

biol. Biotechnol. 85 (2010) 763.23] Z. Aksu, N.K. Kilic, S. Ertugrul, G. Donmez, Enzyme Microb. Technol. 40 (2007)

1167.24] O. Yesilada, S. Cing, D. Asma, Bioresour. Technol. 81 (2002) 155.

3 rfaces

[

[

[[

[29] S.A. Balajee, J.L. Gribskov, E. Hanley, D. Nickle, K.A. Marr, Eukaryotic Cell 4 (2005)

28 P. Kaushik, A. Malik / Colloids and Su

25] C. Meehan, I.M. Banat, G. McMullan, P. Nigam, F. Smyth, R. Marchant, Environ.

Int. 26 (2000) 75.

26] M. Asgher, S. Kausar, H.N. Bhatti, S.A.H. Shah, M. Ali, Int. Biodeter. Biodegr. 61(2008) 189.

27] S. Arora, H.S. Saini, K. Singh, Color. Technol. 121 (2005) 298.28] N. Dafale, N.N. Rao, S.U. Meshram, S.R. Wate, Bioresour. Technol. 99 (2008)

2552.

[

[

B: Biointerfaces 81 (2010) 321–328

625–632.30] T. Anthony, K.C. Raj, A. Rajendran, P. Gunasekaran, Enzyme Microb. Technol. 32

(2003) 647–654.31] Y.C. Chang, H.F. Tsai, M. Karos, K.J. Kwon-Chung, Fungal Genet. Biol. 41 (2004)

888–896.