Biosorption of Textile Dyes and Effluents by Pleurotusflorida and Trametes Hirsutawith Evaluation of Their Laccase Activity

Document Type: Brief Report


1 Department of Industrial Biotechnology, Dr. M.G.R. Educational and Research Institute, Dr. M.G.R. University, Chennai-600 095, India

2 Center for Advanced Studies in Botany, University of Madras, Chennai-600 025, India


The rate and efficiency of decolorization of dyes like Blue CA, Black B133, and Corazol Violet SR were tested to evaluate white rot fungal strains. Trametes hirsuta and Pleurotus florida showed the greatest extent of decolorization on nutrient salt media. Maximum decolorization of 200 mg/l of Blue 133 was obtained by 4 days old incubated Pleurotus florida followed by Trametes hirsuta after 6 days. An attempt was made to improve the decolorization activity of both organisms with different concentrations of glucose 1 and 2% (w/v). The decolorization activity may be due to the laccase enzyme of white rot fungi. The production of this enzyme was estimated using solid state fermentation with rice bran as a substrate. It was found that P. florida exhibited 0.175 U/ml of laccase activity followed 0.126U/ml by T. hirsute, respectively. Decolourization was found to be more effective with P. florida in the presence of 2% (w/v) glucose. Crude extract containing the laccase enzyme was isolated and confirmed by SDS PAGE


Dyes are extensively used for several industrial applications and approximately 5% of them end up in effluents. Unfortunately, conventional wastewater treatments are ineffectual at removing dyes and involve
high costs, formation of hazardous by-products and
intensive energy requirements (Stolz, 2001).
Approximately, one lakh commercial dyes are manufactured which include several varieties of dyes such
as acidic, basic, reactive, azo, diazo and anthraquinone
based meta complex dyes. Over 10,000 dyes with an
annual production of over 7 × 105 metric tonnes are
commercially available (Campos et al., 2001).
Fungal laccase as part of the ligninolytic enzyme
system is produced by almost all wood and litter transforming Basidiomycetes. This group of N-glycosylated extracellular blue oxidases with molecular masses
of 60-390 kDa (Call and Mücke, 1997) contain four
copper atoms in the active site (as Cu2+ in the resting
enzyme) which are distributed among different binding sites (McGuirl and Dooley, 1999).
Laccases have been reported to oxidize many recalcitrant substances, such as chlorophenols (Fahr et al.,
1999), and polycyclic aromatic hydrocarbons ligninrelated structures (Bourbonnais et al., 1996),
organophosphorous compounds (Amitai et al., 1998),
nonphenolic lignin model compounds (Majcherczyk et
al., 1999), phenols, and aromatic dyes (Abadulla et al.,
2000). Laccases are able to oxidize polyphenols,
methoxy substituted polyphenols, diamines, and considerable range of other compounds. Besides reduction
of environmental pollution, enzymatic decolorization
of dyeing effluents has recently been shown to enable
reuse of the treated water in the dyeing process
(Abadulla et al., 2000).
The present study is to investigate the ability of
Trametes hirsuta and Pleurotus florida to carry out
biosorption of the reactive dyes at three different concentrations. The effect of glucose in improving the
production of laccase enzyme system was also studied

Pure cultures of T. hirsuta (MTCC-136) and P. florida
were obtained from the Microbial Type Culture
Collection (MTCC), Chandigarh and University of
Madras, India, respectively. T. hirsuta was maintained
in Yeast Extract Agar Media (YEA) and P. florida was
cultured in Potato Dextrose Agar media (PDA) and
stored at 4°C. Laccases, which are extracellular secre
tion of white rot fungus, were able to oxidize different
substrates such as guiacol, syringoldazine, and non
phenolic compounds. Oxidase enzyme system of
T. hirsuta and P. florida was checked based on Trejo
Hernandez et al. (2001).
Three different concentrations of the dyes (Blue
CA, Black B133, and Corazol Violet SR) at 25, 50, and
75 mg/l were prepared with 0.5% (w/v) glucose as a
carbon source. The effluent was collected from the
Thirupur dye house, Tamil nadu, India, which mostly
uses the reactive dyes. Glucose (1 and 2% (w/v) was
added to both the raw effluent (pH 11) and the pH
adjusted effluent (pH 6) which were inoculated with
T. hirsuta and P. florida and control was maintained.
They were kept at room temperature and observed for
decolorization. Kirk’s nutrient salt medium was pre
pared and inoculated with 5 discs of P. florida for the
determination of laccase activity. On the third day
5 µl of guiacol was added to 50 ml of medium and
0.05 g/100 µl of gallic acid was prepared and added to
50 ml of the medium. The same media was used for the
decolorization of these three dyes at the concentration
of 200 mg/l. The rate of dye decolorization was esti
mated by the following formula:
Rate of decolorization (%) =
Solid state fermentation was carried out in 500 ml
Erlenmeyer flasks with rice bran as substrate for laccase production. Extracellular enzyme was extracted
from solid cultures, which was obtained by means of
soaking rice bran substrate with mycelia in 10 mM
sodium acetate buffer (pH 6.0) for an hour, followed
by filtration using a 0.45 µ nylon membrane filter. The
filtrate was dialyzed and subsequently concentrated in
a lyophilizer. Extracellular laccase activity was
assayed spectrophotometrically as described by
Wolfenden and Wilson (1982) with 2, 2´- azino bis 3-
ethyl-benzothiazoline-6-sulphonate ABTS as substrate. One unit of enzyme activity was defined as 1
µmol of ABTS oxidized per minute at 25ºC (ε436=
29300 M-1CM-1)
The protein concentration was estimated according to the method of Bradford (1976).
SDS-PAGE [12% (w/v)] was performed according to
the protocol of Laemmli (1970) and samples were
treated with 1% SDS, β-mercaptorthanol and boiled
for 5 min. Proteins were visualized by staining with
silver nitrate. Laccase is mainly responsible for the
decolorization of aromatic compounds and can oxidize
substrates such as ABTS and guiacol. The dark reddish
brown zones appeared on both the culture plates in the
laccase assay.
Decolorization of Blue CA, Black B133, and
Corazol violet SR in 0.5% (w/v) glucose medium was
observed at 580, 590, and 530 nm, respectively using
a Beckman DU-40 spectrophotometer, up to 10 days at
an interval of two days. The visual decolorization was
observed within 24 h at a concentration of 25 mg/l by
both white rot fungi. Maximum decolorization was
found to be 93.54% and 92.17% on the 10th day of
incubation by P. florida and T. hirsuta, respectively
(Figure 1). The decolorization rate for 50 mg/l of dye

was found to be 61.27% by P. florida and 56.64% by
T. hirsuta on the 10th day of incubation. At a concentration of 75 mg/l this decolorization was found to be
52.42% and 39.45% by P. florida and T. hirsuta,
Decolorization of Black B133 was lower than the
other two dyes. At the given concentration of 25 mg/l,
maximum decolorization was 64.67% by P. florida and
57.21% by T. hirsuta (Figure 2). However, at a concentration of 50 mg/ml the maximum decolorization was
recorded as 33.94% and 29.97% by P. florida and T.
hirsuta, respectively. Very low decolorization of 75
mg/l of Black B133 by both the organisms on the 10th
day was found to be 28.57% by P. florida and 24.04%
by T. hirsuta.
An effective decolorization of 25 mg/l corazol violet SR was observed as indicated by 83.70% in the
presence of P. florida and 62.02% the presence of T.
hirsuta. The decolorization activity was found to be
69.12% by P. florida and 62.13% by T. hirsuta when
50 mg/l of corazol violet SR was used. At the 75 mg/l
concentration, the maximum decolorization rate was
58.04% and 43.48% by P. florida and T. hirsuta,
respectively. However, the extent of color removal is
not consistent with all the dyes and it depends upon
laccase activation by the dyes. This may be due to the
structural and chemical composition of dyes. Similar
observations regarding dye degradation by the white
rot fungus P. chrysosporium has been observed by
Cripps et al. (1990).
The rate of effluent decolorization was observed at
560 nm, on a daily basis up to five days of the incubation period. The pH adjusted (pH 6) effluent was
decolorized by P. florida and T. hirsuta by up to
45.62% and 42.07%, respectively. In the presence of
1% (w/v) glucose, the decolorization efficiency was
increased up to 56.86% and 52.57% by P. florida and
T. hirsuta, respectively. By the addition of 2% (w/v) of
glucose, the highest decolorization efficiency was
found as 67.86% and 64.1% by P. florida and T. hirsuta, respectively (Figure 3).
The mycelial growth of P. florida started from the
first day of inoculation in nutrient salt medium. On
third day, inducer guiacol and gallic acid were added
and the resulting mixture color change after 24 h indicates that laccase present in the medium. The results of
time course studies regarding decolorization of Blue
CA are shown in Figure 4. The P. florida culture produced approximately 100% decolorization after 4 days
of incubation and complete color removal was
observed after 6 days of incubation by T. hirsuta. In
comparison to the other two dyes, on the 6th day of
incubation, poor decolorization of Black 133 was
observed with 90% by P. florida and 70% in the presence of T. hirsuta (Figure 5). In case of Corazol violet
SR, P. florida showed 95% decolorization and T. hirsuta exhibited only 90% decolorization during 6 days
of incubation (Figure 6).
The maximum amount of protein was observed
(from P. florida and T. hirsuta amended Blue CA culture filtrate) to be 110 µg/l and 85 µg/l, respectively.
reference to 62 kDa of standard protein marker suggests the molecular weight of the partially purified laccase. In conclusion, P. florida was found to be more
effective than T. hirsuta in decolorizing the different
reactive dyes like CA, Black B133, Corazol Violet SR,
and the dye house effluent. Addition of glucose as a
carbon source increased the dye decolorization efficiency. Furthermore, both the test organisms produced
the laccase enzyme in the media which was confirmed
by suitable assays. Therefore, this biological system
may be applied to treatment of dye house effluents in
order to avoid environmental pollution.
The authors thank Prof. Anand, D.Sc., Director, for his moral
support, Mr. Arulmani Manavalan, Center for Advanced
Studies in Botany, University of Madras for his technical support and very grateful to Dr. M.G.R. Educational and Research
Institute , Chennai, India for providing the adequate laboratory facilities in the successful completion of this research work.


Abadulla E, Tzanov T, Costa S, Robra KH, Cavaco-Paulo A,
Gübitz GM (2000). Decolorization and detoxification of
textile dyes with a laccase from Trametes hirsuta. Appl
Environ Microbiol. 66:3357-3362.
Amitai G, Adani R, Sod-Moriah G, Rabinovitz I, Vincze A, Leader
H (1998). Oxidative biodegradation of phosphorothiolates by
fungal laccase. FEBS Lett. 438:195-200.
Bradford M (1976). A rapid and sensitive method for the quantification of protein using the principle of protein-dye binding.
Anal Biochem.72:248-254.
Bourbonnais R, Paice MG, Freiermuth B, Bodie E, Bornemann S
(1996). Reactivities of various mediators and laccases with
kraft pulp and lignin model compounds. Appl Environ
Microbiol. 63: 4627-32.
Campos R, Kandelbauer A, Robra KH, Artur Cavaco Paulo,and
Gubitz GM (2001). Indigo degradation with purified laccases
from Trametes hirsuta and sclerotim rolfsii. J Biotechnol.
Call HP, Mücke I (1997). Minireview: History, overview and appli

cations of mediated ligninolytic systems, especially laccasemediator-systems (Lignozym-Process). J Biotechnol. 53:163-
Cripps C, Bumpus JA, Aust SD (1990). Biodegradation of azo and
heterocyclic dyes by Phanerochate chrysosporium. Appl
Environ Microbiol. 56: 1114-1118.
Fahr K, Wetzstein HG, Grey R, Schlosser D (1999). Degradation of
2,4-dichlorophenol and pentachlorophenol by two brown rot
fungi. FEMS Microbiol Lett. 175:127-32.
Kirk TK, Farrel RL (1987) Decolorization of azo dyes with immobilized Pseudomonas luteola. Proc Biochem. 36: 757-763.
Laemmli UK (1970). Cleavage of structural proteins during the
assembly of head of bacteriophage T4. Nature 227:680-685.
Majcherczyk A, Johannes C, Hüttermann A (1999). Oxidation of
aromatic alcohols by laccase from Trametes versicolor mediated by the 2,2V-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) cation radical and dication. Appl Microbiol
Biotechnol. 51:267-76.
McGuirl MA, Dooley DM (1999). Copper-containing oxidases.
Curr Opin Chem Biol. 3:138-44.
Trejo Hernandez MR, Lopez Munguia A, Quintero Ramirez R
(2001).The evaluation of white rot fungi in the decolorization
of textile dyes. Enz Microbial Technol. 24: 130-137.
Stolz A (2001). Basic and applied aspects in the microbial degradation of azo dyes. Appl Microbiol Biotechnol. 56: 69-80.
Wolfenden BS, Wilson RL (1982). Color chemistry-synthesis,
properties and application of organic dyes and pigments.
VCH Publications. N.Y. pp. 92-102.