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International Biodeterioration & Biodegradation 61 (2008) 189–193

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Optimization of medium for decolorization of Solar golden yellow R

direct textile dye by Schizophyllum commune IBL-06
Muhammad Asghera,, Shahnaz Kausara, Haq Nawaz Bhattia,
Syed Agha Hassan Shahb, Muhammad Alib
a
Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan
b
Department of Chemistry, University of Sargodha, Sargodha, Pakistan
Received 13 February 2007; received in revised form 18 July 2007; accepted 18 July 2007
Available online 21 September 2007

Abstract

The ability of two new white rot fungi Schizophyllum commune IBL-06 and Ganoderma lucidum IBL-05 to decolorize direct dye Solar
golden yellow R was investigated using Kirk’s basal salts medium. In initial time course study, the maximum decolorization (73%) of
Solar golden yellow R was caused by S. commune IBL-06 after 6 days of incubation at pH 4.5 and 35 1C. Different parameters like
incubation time, pH, temperature, and additional carbon and nitrogen sources were optimized to achieve maximum decolorization of
Solar golden yellow R by S. commune IBL-06 in minimum possible time period. Supplementation of the medium with additional carbon
sources enhanced dye decolorization to a variable extent. Addition of glucose (1%) gave the best results and caused a dramatic increase
in decolorization; complete decolorization (100%) of the dye was achieved after only 2 days of incubation under optimum conditions.
All the additional nitrogen sources showed an inhibitory effect on enzyme induction and dye decolorization. Manganese peroxidase
(MnP) was found to be the major enzyme (764 U/ml) secreted by S. commune IBL-06 followed by laccase and only a minor activity of
lignin peroxidase (LiP). The results suggest an excellent potential of S. commune IBL-06 for dye decolorization that can be enhanced by
careful optimization of process parameters.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Decolorization potential; Schizophyllum commune IBL-06; Direct textile dye; Medium optimization

1. Introduction The textile dyeing industries are facing an increasing

pressure from the environmentally concerned organiza-
Waste water from textile dying units contains dyes at tions to replace the conventional treatment technologies
concentrations ranging from 10 to 20 mg/l and causes with environment friendly ones. In this context, the
formation of toxic aromatic amines under anaerobic biological methods are receiving more attention since they
conditions in receiving media (Murugesan and Kalaichel- are considered as sustainable and eco-friendly (Hong et al.,
van, 2003). Color can be removed from waste water by 2000). Increasing number of experiments have been
physical and physiochemical methods, namely, adsorption, conducted on decolorization of dyestuffs by fungi,
coagulation, flocculation, oxidation, filtration and electro- especially white rot fungi. In recent studies, it has been
chemical methods (Lins and Peng, 1996). However, the demonstrated that several white rot fungi are capable of
complete breakdown of an organic molecule to inorganic oxidizing the various types of synthetic dyes (Glenn and
component is desirable outcome to avoid the persistence of Gold, 1983; Asgher et al., 2006). White rot fungi are
potentially hazardous compounds in the environment efficient in biodegradation of recalcitrant compounds like
(Tikoo et al., 1997). xenobiotics, lignin and dyestuffs by their extracellular
lignolytic enzymes (Brodkrob and Legge, 1992). This non-
Corresponding author. Tel.:+92 41 9200161/3309; specific extracellular enzyme system comprising mainly
fax: +92 41 9200764. lignin peroxidase (LiP), manganese peroxidase (MnP) and
E-mail address: [email protected] (M. Asgher). laccase can attack a wide variety of complex aromatic

0964-8305/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2007.07.009
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190 M. Asgher et al. / International Biodeterioration & Biodegradation 61 (2008) 189–193

structures including dyestuffs (Barelay et al., 1990; Jensen autoclave (Sanyo MLS-30200U, Japan) for 15 min. On cooling to
room temperature, 2 ml of inoculum was aseptically added to each flask
et al., 2003).
in laminar air flow (Dalton PAU. 1300BN, Japan). In the initial time
Direct dyes are mainly applied for dying cellulosic fibers course study, these flasks were incubated for 10 days at 120 rpm in a
and cellulosic component in fiber blends and viscous rayon. temperature controlled (35 1C) shaking incubator (Sanyo-Gallenkamp,
The direct textile dye Solar golden yellow R selected in this Plc., London, UK). The samples (2 ml) were removed after every 24 h and
study is extensively used in textile dyeing plants in centrifuged (TGL-16 HS centrifuge, China) at 10,000 rpm for 30 min.
Faisalabad, Pakistan. However, there is hardly any report Supernatants were collected and analyzed immediately for residual
dyestuff concentration.
on its decolorization by white rot fungi. The aim of this
study was to investigate the potential of two least studied
2.5. Process optimization/factors affecting dye biodegradation
white rot fungi Schyzophyllum commune IBL-06 and
Ganoderma lucidum IBL-05 for decolorization of direct The decolorization process was optimized by studying the effect of
dye Solar golden yellow R. This study may form the basis different factors on % decolorization of Solar golden yellow R by
for using these white rot fungi for development of effective S. commune IBL-06. The classical method for medium optimization was
decolorization process for textile industry effluents contain- followed, varying one parameter at a time and maintaining the pre-
optimized at constant level. Decolorization was carried out at varying pH
ing Solar golden yellow R and other structurally related
(3, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0). The pH was adjusted at different levels
dyes. using M HCl/M NaOH. In the next trial, the flaks adjusted at optimum
pH were incubated at varying temperatures (25, 30, 35, 40 and 45 1C) for 6
2. Materials and methods days. In subsequent experiments, the effect of different additional carbon
sources (1% glucose, molasses, sucrose, fructose, maltose and starch),
All the experiments were run in triplicate flasks. The data values in varying glucose (best carbon source) regimes (0.5%, 1.0%, 1.5% and
tables have been presented as mean7S.E. (standard error) and S.E. values 2.0%) and different nitrogen sources (1% ammonium nitrate, ammonium
have been shown as y-error bars in figures. sulfate, peptone, yeast extract and urea), was also studied to select the best
carbon and nitrogen sources for maximum decolorization of Solar golden
yellow R by S. commune IBL-06 within minimum possible time period.
2.1. Chemicals and dyestuff
2.6. Analytical
All the chemicals used in this study were of analytical grade and were
mainly from Fluka and Merck. The direct dyestuff Solar golden yellow R
was a gift from Clariant Pakistan Ltd., Faisalabad, Pakistan, for research 2.6.1. Dyestuff analysis
purpose. The residual concentration of the dyestuff was determined by
measuring its absorbance using UV/vis spectrophotometer (Hitachi
U2001, Tokyo, Japan). Wavelength resulting in maximum absorbance
2.2. Fungal culture and inoculum (lmax) was used for dyestuff analysis. Solar golden yellow R had lmax value
of 420 nm. The absorbance values of supernatants were compared to those
Pure cultures of two new strains of white rot fungi S. commune IBL-06 for standard dye solution (0.01% w/v) to calculate the percentage
and G. lucidum IBL-05 were obtained from the culture collection of decolorization. Standard dye solution was prepared by dissolving 0.01 g
Industrial Biotechnology Laboratory, Department of Chemistry, Uni- dye in 80 ml of Kirk’s medium and the volume was made to 100 ml mark.
versity of Agriculture, Faisalabad, Pakistan. The cultures were grown on For each experiment, separate standards of varying pH, with different
potato dextrose agar (PDA) medium at pH 4.5 and 35 1C for 3 days and sugars and additional nitrogen sources were prepared.
preserved at 4 1C in refrigerator. Spore suspensions for inoculating the
decolorization flasks were obtained by adding 1% (w/v) sterile glucose 2.6.2. Enzyme assays
solution to fungal culture. The slant was gently shaken to transfer spores Supernatants from the final optimum decolorization medium were
to the liquid medium. This spore suspension was passed through sterile analyzed for LiP, MnP and laccase activities to study the mechanism of
glass wool column to remove hyphal fragments. The spore concentration dye decolorization. LiP activity was determined by the method of Tien and
of the eluted suspension was determined by hemocytometer and adjusted Kirk (1988). The reaction mixture (2 ml) containing 4 mM veratryl alcohol
to give 1  108 spores/ml using 1% sterile glucose solution. in 10 mM sodium tartarate buffer (pH 3) was incubated with 100 ml of the
culture fluid at 30 1C. The reaction was initiated with addition of suitable
2.3. Culture media amount of 0.2 mM H2O2. The blanks contained buffer in place of varatryl
alcohol. One unit of LiP activity was defined as the amount of enzyme
Kirk’s basal salts medium (Tien and Kirk, 1988) with the following catalyzing the formation of 1 mmol of veratraldehyde per minute under the
composition (g/l)was used: ammonium tartarate, 0.22; KH2PO4, assay conditions.
0.2; MgSO4  7H2O, 0.005; CaCl2, 0.01; thiamine, 1 mg/l; 10 ml/l of 10% MnP activity was measured by monitoring the oxidation of 1 mM
(w/v) Tween-80 solution; 100 mM veratryl alcohol and 10 ml/l trace MnSO4 in 50 mM sodium melonate buffer (pH 4.5) in the presence of
elements solution was added. Trace elements solution had the following 0.1 mM H2O2 (Wariishi et al., 1992). One unit of MnP activity was defined
composition (g/l): CuSO4, 0.08; H2MnO4, 0.05; MnSO4  4H2O, 0.07; as the amount of enzyme required to produce an absorbance increase of
ZnSO4  7H2O, 0.043; and Fe2(SO4)3, 0.05. one per minute per milliliter of the reaction mixture. The blanks contained
all reagents except MnSO4.
Laccase activity was determined by monitoring the oxidation of 2,
2.4. Decolorization procedure 20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 420 nm
following the method of Shin and Lee (2000). An aliquot of the enzyme
Decolorization flasks (250 ml) were prepared in triplicate each set solution (100 ml) was incubated in 1 ml of 100 mM sodium succinate buffer
containing 100 ml of 0.01% (w/v) Solar golden yellow R dye solution in (pH 4.5) containing 2 mM ABTS at 30 1C. The blanks received the buffer
Kirk’s nutrient medium before sterilization. The dye (0.01 g) was dissolved in place of ABTS. One unit of enzyme activity was defined as the amount
in 80 ml of Kirk’s nutrient medium and final volume was made to 100 ml of enzyme required to produce an absorbance increase of one unit per
mark. The pH was adjusted to 4.5 before sterilization (121 1C) in an minute per milliliter of the reaction mixture.
 ARTICLE IN PRESS
M. Asgher et al. / International Biodeterioration & Biodegradation 61 (2008) 189–193 191

3. Results and discussion 100

80

Decolourization (%)
3.1. Initial time course studies
60
Results of initial time course study on decolorization of
Solar golden yellow R by S. commune showed that 40
maximum decolorization (73%) was noted on the 6th day
of incubation at pH 4.5 and 35 1C. The rate of color 20
removal kept on increasing within first 6 days and no
further decolorization occurred from 7 to 10 days (Fig. 1). 0
Decolorization of Solar golden yellow R by G. lucidum 3 3.5 4 4.5 5 5.5 6
followed almost the same pattern but maximum decolor- pH
ization (70%) was noted on the 7th day of incubation Fig. 2. Effect of pH on decolorization of Solar golden yellow R by
under the same conditions. The fungus showing better S. commune IBL-06.
decolorization of the dye was selected for optimization of
decolorization conditions. As maximum decolorization of
the dye was noted after 6 days, this optimum time period
was used for the subsequent experiments. Our results 90
favorably compare to our initial report (Asgher et al. 80

Decolourization (%)
(2006) showing 100% decolorization of Drimarene orange 70
K-GL by Phanerochaete chrysosporium in 8 days. Pasti- 60
Gribsby et al. (1992) found that P. chrysosporium needed 50
3–4 days to demonstrate a significant decolorization of a 40
range of azo dyes. 30
20
3.2. Effect of initial pH on dye decolorization 10
0
20 25 30 35 40 45 50
Results for the effect of pH on % decolorization of Solar
Temperature (°C)
golden yellow R showed that maximum decolorization
efficiency (73%) was observed in the medium adjusted at Fig. 3. Effect of varying temperatures on decolorization of Solar golden
pH 4.5 after 6 days (Fig. 2). The fungus decolorized the dye yellow R by S. commune IBL-06.
with lower efficiencies at pH 5–6 (59–8%). It means that
S. commune can work in acidic media. Maximum (77%) suggesting that the chemical nature of the dyes may affect
decolorization of Everzol turquise blue G by Coriolus optimum growth pH of the fungus.
versicolor at pH 4.5 has also previously been reported
(Kapdan et al., 2000). Most of the white rot fungi show 3.3. Effect of incubation temperature
maximum growth and dye decolorization in acidic pH
range (Kapdan et al., 2000; Asgher et al., 2006). However, The decolorization flasks (pH 4.5) were incubated at
Funalia troggii was found to efficiently decolorize Astrazon varying temperatures for 6 days. Results on the effect of
Red FBL in broad pH range of 6–11 (Yesilada et al. (2003) temperature on % decolorzation of Solar golden yellow R,
showed that maximum decolorization (78%) was obtained
at 35 1C (Fig. 3). Results are comparable to those of
80
Yesilada et al. (2003) who reported optimum color removal
of Astrazon Red FBL by Funalia troggii pellets at 30 1C.
70
Toh et al (2003) has also reported that decolorization of
Decolourization (%)

60 Remazol dyes by Trametes versicolor CNPR 8107 was

50 more favorable at 30 1C than at 37 1C.
40
30 3.4. Effect of additional carbon sources
S. commune IBL-06
20 G. Lucidum IBL-05
Decolorization experiments were carried out using
10
glucose, molasses, sucrose, fructose and starch as addi-
0 tional carbon sources and the flasks (pH 4.5) were
0 1 2 3 4 5 6 7 8 9 10
incubated at 35 1C for 6 days. A dramatic increase in
Incubation time (Days)
decoulurization of Solar golden yellow R was noted with
Fig. 1. Effect of incubation time on decolorization of Solar golden yellow glucoase (1%) addition and the medium was completely
R by S. commune IBL-06 and G. lucidum IBL-05. decolorized (100%) after only 48 h of incubation under
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192 M. Asgher et al. / International Biodeterioration & Biodegradation 61 (2008) 189–193

Table 1
Effect of different carbon sources on decolorization of Solar golden yellow R by S. commune IBL-06 under optimum conditionsa

Carbon source (1%) Decolorization (% mean7S.E.)

Days

1 2 3 4 5 6

Glucose 78.2071.28 100 100 100 100 100

Fructose 79.4071.44 75.3171.32 73.3371.42 68.9571.41 56.9571.28 43.9571.28
Maltose 83.5171.40 89.5771.42 88.2771.33 89.1871.94 87.1871.36 89.3371.88
Sucrose 80.1971.36 92.1171.38 93.2871.44 92.9470.45 92.307156 89.5571.74
Starch 65.3171.32 72.8171.56 84.5972.11 86.8572.01 86.8871.42 85.6471.48
Molasses 72.3671.29 78.7871.38 81.9370.68 80.5371.49 81.0971.53 80.1171.51
a
pH 4.5; temperature, 35 1C.

120 presence of 0.5 g/100 ml glucose as additional carbon

source and higher glucose level (0.2%) could cause only
100 65% dye removal.
Decolourization (%)

80
3.6. Effect of additional nitrogen sources
60
Effect of different additional nitrogen sources on %
40 decolorization was investigated under optimum conditions
of pH (4.5) and temperature (35 1C). After 2 days of
20 incubation, 87% decolorization was observed in the
medium supplemented with peptone (Table 2) that is also
0
1 2 3 4 5 6 lower as compared to the medium receiving no additional
Incubation time (Days) nitrogen source (100% decolorization). It means that all
0.5% Glucose 1.0% glucose 1.5% glucose 2.0% glucose additional nitrogen sources inhibited fungal growth and
dye decolorization. As the Kirk’s basal nutrient medium
Fig. 4. Effect of varying concentrations of glucose on decolorization of already contained a nitrogen source (ammonium tartarate),
Solar golden yellow R by S. commune IBL-06.
a further addition of nitrogen may have caused inhibition
of enzyme induction and dye decolorization.
optimum process conditions as compared to 78% decolor-
ization after 6 days when no additional carbon source was 3.7. Profile of lignolytic enzymes involved in decolorization
used. The decolorization efficiency showed a steady decline
on the 3rd day of incubation and so on (Table 1). All The supernatants from the finally optimized/decolorized
the carbon sources enhanced decolorization of Solar medium were subjected to lignolytic enzyme assays. The
golden yellow R. However, starch could cause only 86% finally optimized medium containing Kirk’s basal nutrient
decolorization after 5 days, followed by maltose (87% after medium supplemented with 1% glucose was adjusted at pH
3 days), sucrose (86% after 3 days), molasses (81% in 3 4.5 and was incubated for 2 days at 35 1C. Results showed
days) and fructose (79% after 1 day). that S. commune IBL-06 synthesized all the three lignolytic
enzymes. Maximum activity was noted for MnP (764 U/ml),
3.5. Effect of different glucose regimes followed by laccase (179 U/ml) and LiP (96 U/ml). Very
low LiP activity suggested that MnP and laccase are the
To find out the most suitable concentration of glucose major enzymes involved in oxidation of Solar golden
for optimum decolorization, different glucose regimes were yellow R by S. commune IBL-06. Different white rot fungi
used under optimum conditions. The results shown in have variable patterns and profiles of enzymes involved in
Fig. 4 demonstrate that complete dye decolorization was the decolorization of different dyes reflecting their different
obtained when 1% glucose (same as in previous experi- decolorization abilities (Yesilada et al., 2003; Chander
ment) was used. White rot fungi show better growth in lag et al., 2004). Pleurotus pulmonarius producing only laccase
phase on readily available energy sources and produce was able to decolorize dyes of different spectra (Zilly et al.
enzymes and secondary metabolites during log phase for 2002). MnP has also previously been found to play
biodegradation of dyestuffs in Kapdan and Kapdan the major role in decolorization of different dyes by
(2002). Results are comparable to those of Kapdan and T. versicolor CNPR 8107 (Toh, et al., 2003), Lentinula
Kargi (2002) who found that Everzol turquoise blue G was edodes (Boer et al., 2004) and Trametes trogii (Levin et al.,
optimally decolorized (77%) by C. versicolor in the 2005).
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Table 2
Effect of different nitrogen sources on decolorization of Solar golden yellow R by S. commune IBL-06 under optimum conditionsa

Nitrogen source (1%) Decolorization (% mean7S.E.)

Days

1 2 3 4 5 6

Ammonium nitrate 21.2971.29 68.9971.36 68.9971.18 69.9671.29 76.1671.81 65.2371.39

Ammonium sulfate 15.7171.18 18.4371.44 20.4871.36 32.7171.44 34.5671.46 31.1971.38
Yeast extract 25.5371.28 34.0971.29 41.4171.38 33.4171.49 33.1871.36 22.9171.34
Urea 31.2171.26 34.3971.20 49.1771.68 57.9671.49 73.5671.55 50.1171.48
peptone 26.9271.38 51.6671.41 68.9971.48 70.3271.55 77.5671.58 60.1171.59
a
pH 4.5; temperature, 35 1C; glucose, 1.0%.

4. Conclusion Jensen, K.A., Bao, W., Kaswai, S., Srebotnik, B., Hammel, K.B., 2003.
Manganese dependent cleavage of non phenolic lignin structures by
White rot fungus S. commune IBL-06 is a very efficient Phanerochaete chrysosporium. Journal of Biological Chemistry 267,
23688–23695.
decolorizer of Solar golden yellow R if the medium is Kapdan, I.K., Kapdan, F., 2002. Comparison of white rot fungi cultures
properly optimized and supplemented with additional for decolorization of textile dyestuffs. Bioprocess Engineering 22,
carbon/energy sources. The fungus seems to be highly 347–351.
promising for development of effective bioremediation Kapdan, I.K., Kargi, F., 2002. Biological decolorization of textile dyestuff
process for real industrial effluents that contain Solar containing waste water by Coriolus versicolor in a rotating biological
contractor. Enzyme and Microbial Technology 30, 195–199.
golden yellow R and other structurally related dyes. Kapdan, I.K., Kargi, F., McMullan, G., Marchant, R., 2000. Effect of
environmental conditions on biological decolorization of textile
Acknowledgments dyestuff by C. versicolor. Environmental Microbiology and Biotech-
nology 26, 381–387.
Levin, L., Forchiassi, F., Viale, A., 2005. Ligninolytic enzyme production
This manuscript is a part of a project funded by Higher
and dye decolorization by Trametes trogii: application of the
Education Commission (HEC), Islamabad, Pakistan. The Plackett–Burman experimental design to evaluate nutritional require-
financial support by HEC is highly acknowledged. We are ments. Process Biochemistry 40, 1381–1387.
highly thankful to the Department of Mycology, Uni- Lins, S.H., Peng, F.C., 1996. Continuous textile wastewater by combined
versity of the Punjab, Lahore, Pakistan, for technical help coagulation, electrochemical oxidation and activated sludge. Water
in isolation and characterization of fungi. Resources 30, 87–591.
Murugesan, K., Kalaichelvan, P.T., 2003. Synthetic dye decolorization by
White rot fungi. Indian Journal of Experimental Biology 41,
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