Process Biochemistry 40 (2005) 33373345

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Decolorization studies of synthetic dyes using

Phanerochaete chrysosporium and their kinetics
K.V. Radha, I. Regupathi, A. Arunagiri, T. Murugesan *
Department of Chemical Engineering, AC College of Technology, Anna University, Chennai 600025, India
Received 19 July 2004; received in revised form 2 February 2005; accepted 26 March 2005

Abstract
Treatment of effluents from dye-based industries poses a major problem and biotreatment with white rot fungi seems to be a viable option.
In this study, Phanerochaete chrysosporium, a commonly used white rot fungus, was used to biodegrade several synthetic dyes of varying
structures, namely azo, anthraquinone, thiazine and vat dyes. The decolorization potential of P. chrysosporium for seven dyes namely, Methyl
violet, Congo red, Acid orange, Acid red 114, Vat magenta, Methylene blue and Acid green was studied. The effect of various operational
parameters, namely dyes concentration (20400 mg/l), pH (27), temperature (2045 8C) and inoculum size (0.254 ml) on the maximum
percentage decolorization were investigated. Studies were carried out using free cells and fungal cell entrapped calcium alginate beads of
different sizes. The kinetics parameters Kdye and Vdye max for the decolorization process for all the seven dyes were estimated through
LineweaverBurk plots.
# 2005 Elsevier Ltd. All rights reserved.
Keywords: Phanerochaete chrysosporium; Decolorization; Dyes; Immobilization; Calcium alginate beads; Kinetics

1. Introduction
Synthetic dyes are used extensively for textile dyeing, paper
printing, leather dyeing, color photography and as additives in
petroleum products. With the increasing usage of the wide
variety of dyes in these industries, pollution from the effluents
has become increasingly alarming. The two major sources of
release of the dyes into the environment are the textile and
dyestuff manufacturing industries. Normally colors are
noticeable at a dye concentration of more than 1 mg/l and
an average concentration of 300 mg/l have been reported in
effluents from textile manufacturing processes [1,2]. Many
authors have reported physico-chemical treatments for the
removal of color from industrial wastewaters [35]. Even
though these procedures prove to be efficient, the operational
costs are relatively high and leads to other disadvantages like
sludge formation, biomass accumulation, etc. [6].
Microbial decolorization processes offer a complete
cleanup of pollutants in a natural way as it reduces the color
* Corresponding author. Tel.: +91 44 2220 3507; fax: +91 44 2235 2642.
E-mail address: tmgesan_57@yahoo.com (T. Murugesan).
1359-5113/$ see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2005.03.033

components to carbon dioxide, ammonia and water by

initiating cleavage of the bonds in the dyes rather than
creating possible toxic fragments of dyes [7]. Strains such as
Phanerochaete chrysosporium, Trametes versicolor, Pseudomonas luteola have been reported to be suitable for
decolorizing common dyes as these strains produce
peroxidases that oxidize the various structure of the dyes
and are found to be more suitable for decolorization [810].
The white rot fungi, more specifically, strains of P.
chrysosporium have been studied in the field of decolorization
of industrial effluents. This is due to the versatile ability of the
fungus to degrade, partially or completely various dyes such
as heterocyclic, azo, anthraquinone, vat and polymeric dyes
[1114]. P. chrysosporium displayed color reduction abilities
for all such dyes including the dyes used in newsprint, writing
and printing paper industries [15]. Extracellular peroxidases
such as lignin peroxidases, manganese peroxidases, hydrogen
peroxide and oxalates produced by the fungus catalyzes the
oxidative degradation of the pollutants [16]. These extra
cellular peroxidases, are non-specific towards the substrate so
that it can attack some recalcitrant chemicals of diverse
structures, including organic-pollutants. The lignolytic

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K.V. Radha et al. / Process Biochemistry 40 (2005) 33373345

enzymes of P. chrysosporium (due to their oxidative

mechanism) are considered responsible for the aerobic
decolorization of the dyes that not only decolorizes but also
detoxifies the effluents completely [17].
Recently, the application of immobilized cells has been
receiving increasing attention in the field of wastewater
decolorization. Many researchers have studied the effect of
immobilized whole cells and enzymes on decolorization
characteristics, since immobilization provides distinct
stability over free cells [12,18]. In this present work,
experiments on decolorization of the dyes were carried out
in batch mode using P. chrysosporium (MTCC 787) free
cells, to study the decolorization of seven structurally
different dyes and hence to find the optimum conditions viz.,
initial concentrations of the dyes, initial pH, glucose and
nitrogen concentrations, inoculum concentrations and
temperatures for the design of continuous reactors for
decolorization. Attempt have also been made for whole cells
immobilization using calcium alginate entrapment, due to
the gentle gelation procedure compared to that of chemical
polymerization procedures [19]. Similar to that of free cells,
studies were also carried out using immobilized beads of
varying bead sizes. The data obtained have been used to
study the kinetic effects on decolorization of individual dyes
since the kinetics studies are rarely reported with respect to
dyes decolorized by P. chrysosporium.

2. Materials and methods

2.1. Microorganism
The white rot fungus P. chrysosporium MTCC 787 was
obtained from the Culture Collection of Institute of
Microbial Technology, Chandigarh, India and the stock
cultures were maintained by periodic subculture on malt
agar medium at 4 8C.
2.2. Inoculum
The fungus P. chrysosporium was inoculated on malt agar
and incubated at 35 8C until extensive spore growth occurred.
The basal medium [20] used to study the fungal biomass and
decolorization test consists of: D-glucose, 5.0 g/l; KH2PO4,
2.0 g/l; NH4Cl, 0.050 g/l; MgSO4
7H2O, 0.5 g/l;
CaCl2
2H2O, 0.1 g/l; thiamine HCl, 100 mg; trace element
solution, 10 ml and the final pH of the medium was
maintained at pH 4.5. Trace element solution consisting of
MnSO4, 0.5 g/l; FeSO4
7H2O, 0.1 g/l; ZnSO4
7H2O, 0.1 g/l
was prepared separately and 10 ml was added to the medium.
2.3. Dyes and decolorization studies
Seven commercial dyes belonging to the groups of azo
(Acid orange, Acid red 114), triphenylmethane (Methyl
violet), anthraquinone (Acid green), thiazine (Methylene

blue), Vat (Vat magenta) and diazo group (Congo red) were
used. Individual dyes were added to Erlenmeyer flasks
(250 ml) containing 100 ml of the medium, which was
inoculated with approximately 3.2  105 cells. The experiments were carried out in an orbit shaker at 60 rpm for 7 days
at 35 8C. The dye concentrations were measured using
samples collected at regular intervals using a spectrophotometer [21] [UV/VIS shimadzu spectrophotometer
(model U2000)]. Control experiments for each test were
carried out using uninoculated medium with dye addition.
2.4. Enzyme assays
Enzyme activities were determined spectrophotometrically at 35 8C lignin peroxidase (LiP) activity was
determined by the oxidation of veratryl alcohol at 310 nm
as described by Tien and Kirk [22]. Manganese peroxidase
activity was assayed at 468 nm using dimethoxyphenol as
the substrate as suggested by Field et al. [23]. Laccase
activity was analyzed spectrophotometrically according to
Niku-Paavola et al. [24], with 2,20 -azino-di(3-ethyl-benzothiazolin-sulphonate) (ABTS) as substrate. One unit was
defined as the amount of enzyme that oxidized l mmol of
substrate per minute and the activities are reported as U/l.
2.5. Biomass determination
To determine biomass growth, a fixed volume of culture
broth was centrifuged at 1000 rpm for 45 min. The pellet
was removed, washed, filtered through a predried, preweighed filter paper. The filter paper was dried to a constant
weight and the dry weight of the biomass was determined as
gram per litre [25].
2.6. Biosorption studies
In order to study the role of mycelium in dye
decolorization, the mycelium and the supernatant were
separated when the fungus showed maximum activity. To the
mycelium the dye was added and tested for biosorption.
Enzyme activities were monitored during biosorption.
2.7. Immobilization
P. chrysosporium were grown into the stationary phase in
malt agar slants. Spore suspension of 2 ml (approximately
3.2  105 cells) was added to 100 ml of 2% sodium alginate.
The mixture was gently stirred at room temperature to
produce a uniform suspension and then dropped into 100 ml
of 20% calcium chloride solution. Five different nozzles
were used to form beads of uniform sizes (2, 3, 4, 5 and
6 mm). The beads so obtained were stored in calcium
chloride solution at 4 8C for 2 h to complete gel formation
[26]. The insoluble and stable immobilized P. chrysosporium alginate beads thus obtained were further used for the
decolorization studies.

K.V. Radha et al. / Process Biochemistry 40 (2005) 33373345

3. Results and discussion

3.1. Effect on initial concentrations of the dyes
P. chrysosporium was used to study the percentage
decolorization alongwith the maximum time requirements
for decolorization process. For initial experiments, keeping
the parameters such as initial pH and temperature as
constant, initial concentrations of the individual dyes were
varied from 0.02 to 0.4 g/l.
The maximum time taken for decolorization varies with
the nature of individual dyes and the longer time taken for
decolorization is a result of the production of extra cellular
peroxidases, which are available only after 23 days of the
growth of the fungus [27]. The enzyme production slowly
increased after 8 h of growth of the fungus and reached a
maximum of 234 U/l for LiP and 172 U/l for MnP by 120 h.
Thereafter, a drop in activity was observed (Fig. 1). Laccase
was tested for its activity from the day 1 but there was no
noticeable quantity of laccase production till the day 5, after
which 14 U/l was observed on day 7, further there was no
increase. From these results, it was observed that LiP and
MnP were the key enzymes responsible for the decolorization process. A similar trend was also observed by Sami and
Radhouane [28], using different medium for the production
of enzymes using the fungus P. chrysosporium. They have
also concluded that decolorization starts on the second day
and reaches a maximum during the fourth day but higher
activity of the enzyme was reported on the fifth day after
which it declined.
Acid orange and Vat magenta took less than 2 days for
decolorization which might be due to the fact that dyes acts
as suitable substrate for the peroxidases and oxidases
produced by the fungus [23,29]. Even though the same
amount of the inoculum was used for all the tested dyes, the
differences found in the decolorization characteristics for
the individual dyes are attributed to the dissimilarity in
specificities and structures of different dyes. Similar strains
of white rot fungi also decolorize the dyes in the same
manner like that of P. chrysosporium [30]. About 98% of
decolorization is achieved for Methyl violet and Acid

Fig. 1. Enzymes production during the growth of the fungus Phanerochaete

chrysosporium.

3339

orange, whereas Vat magenta, Methylene blue, Congo red

and Acid red 114 showed 8892% of decolorization.
Decolorization was far less for Acid green, which showed
only 75%. Fig. 2a and b show the maximal percentage
decolorization of the individual dyes and it is evident that P.
chrysosporium shows the potential to transform the dyes to
colorless substances.
Compared to all the dyes used in the present study
(Fig. 2a), Methyl violet had a high percentage of
decolorization due to the sequential demethylation with
the removal of penta, tetra and trimethyl groups [31]. The
tentative metabolic pathways of methyl violet decolorization
by different species are explained by Sarnaik and Kanekar
[32]. As concluded by Chizuko et al. [33], the presence of
hydroxyl group in the para position of the aromatic ring
leads to a faster cleavage of the bond by the organisms. This
could be the reason for the fast decolorization as the Acid
orange has a hydroxyl group in the para position. The
percentage decolorization of methyl violet upto an initial
concentrations of 0.2 g/l are at maximum and nearly uniform
(Fig. 2a), whereas for concentrations greater than 0.2 g/l, a
sudden drop in percentage decolorization was observed.
This may contribute to the fact that the fungus showed high
sensitivity and low tolerance to the dye [34]. For the case of
all other dyes a gradual decrease in the percentage
decolorization with respect to the initial concentration is
observed.

Fig. 2. Effect of initial concentration of the dyes on the percentage

decolorization (initial pH:4.5; T:35 8C; initial concentration (g/l): (a)
(^) 0.05; (&) 0.1; (~) 0.2; () 0.3; ( ) 0.4) and (b) (^) 0.02; (&)
0.04; (~) 0.05; () 0.06; ( ) 0.08; (*) 0.1).

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K.V. Radha et al. / Process Biochemistry 40 (2005) 33373345

In the present study, it is observed that P. Chrysosporium

showed lesser activity towards decolorization of Acid green
than other dyes tested (Fig. 2b). The organism was not able
to decolorize Acid green at a concentration greater than
0.08 g/l even though toleration in the medium was little
higher. The dye is expected to become toxic to the
microorganism at higher concentrations (>0.08 g/l), as a
result, an incomplete decolorization is observed [35]. Hence,
the present studies were made upto a concentration of
0.08 g/l only for Acid green, whereas for other dyes P.
chrysosporium was able to grow at higher concentrations
and an appreciable decolorization was observed.
Congo red and Acid red 114 showed a maximum
decolorization of upto 90% for an initial dye concentration
of 0.02 g/l. Even though there was an identical percentage
decolorization for an initial concentration of 0.02 g/l in
these two dyes, at higher concentrations, P. chrysosporium
proved to be more effective for the case of Congo red than
Acid red 114. A similar observation were also made for
Acid green and Methylene blue. As it is observed in all
these cases, beyond an optimum initial concentration of
dyes the rate of decolorization decreases and further
increase in concentration does not have any effect on
decolorization. This could be attributed to the fact that the
color removal depends on the destruction of the
chromophore. The peroxidases of the fungus needs to
attack one molecule of the dyes several times, a lower
concentration of the dye facilitate the destruction of the
molecules and the higher the concentration of the dyes the
slower the rate of color removal [36]. No general trend was
seen regarding the behaviour of dyes of different nature
(azo, anthraquinone, triphenylmethane, etc.) but the
structure of individual dye seems to an have influence
on the decolorization. The relevant factors that influence
the decolorization process are: (i) the structure of the dyes,
(ii) loss of the vital requirements by the organism and (iii)
might require an additional amount of veratryl alcohol
apart from the production by the microorganism itself
[37].
3.2. Biomass growth
To evaluate the effect of toxicity of the dyes on the
growth of P. Chrysosporium batch studies were conducted
with varying dye concentrations (0.020.1 g/l). A sample
graph is given for Congo red as the biomass growth was
found to have a similar pattern for all the dyes (Fig. 3). The
maximum biomass concentration decreased from 1.35 g/l
in the control to 1.25 and 0.75 g/l for the initial dye
concentration of 0.02 and 0.1 g/l, respectively. At an initial
dye concentration of 0.3 g/l there was complete inhibition
of growth. Further experiments were carried out under the
same conditions to produce biomass that is suitable for the
production of enzymes. The agitation was kept at 60 rpm
with the addition of Tween 80 in a nitrogen-limited
medium [38].

Fig. 3. Growth of biomass during decolorization process.

3.3. Biosorption studies

The role of mycelium in dye decolorization was
investigated by separating the mycelium and the supernatant
at the time at which the fungus showed maximum activity.
To this 0.05 g/l of Methyl violet was added and the time
course of the enzyme activity and dye decolorization was
studied and the results are shown in Fig. 4. The
decolorization was limited to 39% by physical adsorption
of the mycelium, as the enzyme activity was very low after
separation from the supernatent.
The experimental results clearly indicate that the mycelia
individually cannot produce significant decolorization [39].
As the fungal peroxidases appears to be extracellular
enzymes the maximum activity decreased to 58 U/l from the
initial value of 234 U/l for LiP and 36 U/l from 172 U/l for
MnP. Therefore, both the mycelium (biosorption) and the
extra cellular fungal enzyme (biodegradation) are necessary
for the dye decolorization.
3.4. Effect of pH and temperature
The growth of the P. chrysosporium and the corresponding decolorization process were essentially controlled by the
pH of the medium. The percentage decolorization of the
dyes, using free cells of P. chrysosporium at various initial

Fig. 4. Biosorption of the mycelium on decolorization.

K.V. Radha et al. / Process Biochemistry 40 (2005) 33373345

Fig. 5. Effect of initial pH of the dyes on the percentage decolorization

(initial concentration: Methyl violet, Acid orange, Vat magenta: 0. 05 g/l;
Acid red 114, Acid Green, Congo red, Methylene blue: 0. 02 g/l; T: 35 8C;
initial pH: (^) Methyl violet; (&) Acid orange; (~) Vat magenta; () Acid
green; ( ) Acid red 114; (*) Congo red; (j) Methylene blue).

pH conditions ranging from 2.0 to 7.0 were studied (Fig. 5).

Maximum decolorization for all the dyes studied in this
work was observed at a pH range of 4.05.0 and the
percentage decolorization decreased at both extremes of pH
(<4.0 and >5.0). Although the dyes were decolorized at a
pH range of 4.05.0, Methylene blue, Acid green, Congo red
and Vat magenta were mostly decolorized at around a pH
5.0. On the other hand, the maximum decolorization for
Acid orange, Methyl violet and Acid red 114 occurs at pH
4.04.5 and 4.05.0, respectively, above and below which
the decolorization process decreased remarkably. These

Fig. 6. Variation in pH during decolorization process (T: 35 8C; initial

concentration (0.05 g/l): (^) Methylene blue; (&) Methyl Violet; (~) Vat
magenta; () Congo red; ( ) LiP; (*) MnP) (T: 35 8C; initial concentration
(0.02 g/l): (^) Acid orange; (&) Acid green; (~) Acid red 114; ( ) LiP; (j)
MnP).

3341

Fig. 7. Effect of initial pH of the dye methyl violet on the percentage

decolorization using immobilized beads (initial concentration: 0.05 g/l; T:
35 8C; diameter of the bead: 2 mm; initial pH: (^) 2; (&) 3; (~) 4; ()
4.5;( ) 5; (*) 6; (j) 7).

observations indicate that the optimum pH for the fungus P.

chrysosporium depends on the nature of the substrate used as
observed and reported by Alberto et al. [40].
The variation in pH during the course of the decolorization using free cells are shown in Fig. 6. It is observed that,
for the case of Methyl violet, Vat magenta, Congo red and
Methylene blue even though there was a slight variation of
pH during the process of decolorization, the final pH was
maintained at 4.05.0. But for the case of Acid orange, Acid
green and Acid red 114, the final pH shoots upto 4.55.0
even though the initial pH was 2.5 (Acid green and Acid
orange) and 3 (Acid red 114). The decrease in initial pH did
not have any effect on decolorization [41]. As observed by
Dirk et al. and Ana et al. [42,43] the fungus produces organic
acids such as malonate, oxalate during the initial growth
period, which later decomposed by the enzyme (manganese
peroxidase). The fungus was biologically active during this
period.
The experiments were also carried out to study the
effect of initial pH on the percentage decolorization using
calcium alginate beads, since the medium pH is expected
to affect the ionization state of the functional groups on the
fungal cell walls and the entrapped fungus (carboxylic,
phosphate and amino groups). For example, Fig. 7 shows
the trend observed for Methyl violet, which is similar to

Fig. 8. Effect of glucose on dye decolorization (initial concentration of

Methyl violet: 0.05 g/l; T: 35 8C; concentration of glucose (g/l): (^) 1; (&)
2; (~) 2.5; () 5; ( ) 10; (*) 15).

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K.V. Radha et al. / Process Biochemistry 40 (2005) 33373345

Fig. 9. Effect of nitrogen on dye decolorization (initial concentration of

Methyl violet: 0.05 g/l; T: 35 8C; concentration of nitrogen (g/l): (^) 0; (&)
0.5; (~) 0.1; () 0.15; ( ) 0.2).

that of free cells. This may be possibly due to the same

functional group that gets reacted in both alginate and the
cell wall component of the mycelia [26].
In order to study the variation in temperature on
decolorization studies were carried out at temperatures
ranging from 20 to 45 8C. At higher (>35 8C) or lower
(<35 8C) temperatures the decolorization activity of the
fungus gets reduced, which indicate that either the fungus is
not able to produce the peroxidases for decolorization or
they get denatured. Hence, all further experiments were
carried out at 35  1 8C.
3.5. Effect of glucose and nitrogen
Earlier reports on decolorization studies [44] indicated
the necessity of glucose to enhance the decolorization
process. Hence, an attempt is made to study the effect of
glucose in basal medium. The initial rates of color reduction
significantly improved, as the concentration of glucose was
increased from 1 to 5 g/l. The results of the studies show that
the presence of glucose enhances the rate of decolorization
and a concentration of 5.0 g/l was found to be sufficient to
achieve the maximum decolorization percentage (Fig. 8). As
the concentration of glucose was increased from the
optimum level, the decolorization rate decreased which
may be due to the metabolic pathways, where there is more
prevalent growth of the fungus rather than the utilization of
the glucose by the fungus for the decolorization of the dyes
[45].

Fig. 10. Effect of inoculum size on dye decolorization (initial concentration

of Methyl violet: 0.05 g/l; T: 35 8C; inoculum size (ml): (^) 0.25; (&) 0.5;
(~) 1; () 2; ( ) 3; (*) 4).

Similarly, an optimum concentration of nitrogen for the

enhancement of decolorization of dyes was studied using
ammonium chloride as nitrogen source (00.2 g/l). The
decolorization percentage was observed to be 45% without
any nitrogen source in the medium and when the medium
was supplemented with 0.05 g/l of ammonium chloride the
percentage decolorization increased to 96%. From Fig. 9,
it is observed that the addition of nitrogen beyond the
optimum level of 0.05 g/l, leads to a rapid decrease in
percentage decolorization. The decrease in percentage
decolorization might be due to the reduction reaction
involving the nitrogen in the medium and the nitrogen in
the dyes [46].
3.6. Effect on size of inoculum
Experimental results on the estimation of optimum
concentration of the inoculum necessary to achieve
maximum decolorization, showed that the maximum
decolorization occurred at an inoculum size of 2 ml
(approximately 3.2  105 cell/ml) and the same size of
inoculum was used for all further studies. Since a similar
trend was observed for all the seven cases considered in
this work, a sample graph of Methyl violet is shown alone
in Fig. 10. Similarly, Shahvali et al. [47] have reported that
an inoculum size of within 10% was sufficient for the
decolorization of the textile wastewater, above which
there was no appreciable change in percentage decolorization.

Table 1
Effect on diameter of the immobilized beads
Beads diameter (mm)

Maximum percentage decolorization

Initial concentration: 0.05 g/l

2
3
4
5
6

Initial concentration: 0.02 g/l

120 h
Methyl viol

48 h
Acid orange

48 h
Vat magenta

120 h
Congo red

120 h
Acid green

96 h
Methylene

120 h
Acid red

95.17
92.15
87.32
81.49
78.47

90.14
88.33
86.52
84.91
83.90

95.90
93.95
92.82
88.72
85.64

85.35
84.85
84.34
83.83
82.53

66.83
63.32
61.81
61.31
60.80

86.87
82.32
81.82
79.80
78.28

83.33
81.31
74.75
73.74
72.73

K.V. Radha et al. / Process Biochemistry 40 (2005) 33373345

3.7. Studies on immobilized beads

Decolorization experiments with an estimated optimum
concentration (0.05 g/l for Methyl violet, Acid Orange and
Vat magenta and 0.02 g/l for Acid green, Acid red 114,
Congo Red and Methylene blue) were carried out with
immobilized calcium alginate beads of different sizes (2
6 mm). The maximum percentage decolorizations for
different sizes of beads are reported in Table 1. The
percentage decolorization decreased with increasing bead
diameter for all the dyes tested. The reason could be
attributed to the increase in the surface area in the smaller
beads compared to beads with larger diameters [19,48].
Basic experiments with alginate beads (without immobilization) showed an initial reduction of 20% of the color,
which is due to the absorption by the alginate beads and the
rest is being decolorized by P. chrysosporium and the time
taken for decolorization was nearly the same compared to
that of free cells.
3.8. Kinetic studies
The dye decolorization process is mainly an extra cellular
enzymatic process, hence studies have been made to find the
kinetics of the decolorization. For the purpose of establishing the kinetic parameters for the decolorization process
LineweaverBurk plots were used.


1
Km 1
1

V
Vmax s Vmax
The estimated values of Kdye and Vdye max are tabulated in
Table 2 for free cells and immobilized cells. For the case of
free cells the results shows the Kdye values in the range of
0.10.6 g/l for all the dyes tested except for Methyl violet
and Vat magenta which showed a Kdye value of 0.9304 and
0.9097 g/l, respectively. As Kdye is a measure of the enzyme
substrate complex, a high Kdye indicates a weak binding
[49]. For the case of Methyl violet the reason could be due to
the instant dissociation of the dye with the free cells as
explained by Bumpus and Brock [31]. The value of Kdye and
Vdye max for the decolorization of Azo dye (reactive red 22)
by P. luteola was found to be 0.156 g/l and 0.012 g/l/h) [50],
respectively, which is in good agreement with the present
range of results obtained using P. chrysosporium.
Similar experiments were carried out for immobilized
beads of diameter ranging from 2 to 6 mm to study the effect
on surface area. The entrapped P. chrysosporium in alginate
beads showed a low Kdye value for the dyes Methyl violet,
Congo red and Acid green and almost constant for Vat
magenta, Methylene blue and Acid red 114. For the case of
Acid orange the Kdye values are found to be quite high. The
variation in Kdye value might be due to the surface effects of
the immobilized cells and thereby affinity of the hydrophobic substrates either increases or decreases [51]. Similar
results of change in Kdye values from that of free cells to that

3343

Table 2
Estimated kinetic parameters using Phanerochaete chrysosporium free cells
and immobilized beads
Dyes

Kdye (g/l)

Vdye

Methyl violet
Free cells
2 (mm)
3 (mm)
4 (mm)
5 (mm)
6 (mm)

0.9327
0.8743
0.6550
0.6026
0.5771
0.5496

0.0131
0.0112
0.0083
0.0076
0.0075
0.0070

Acid orange
Free cells
2 (mm)
3 (mm)
4 (mm)
5 (mm)
6 (mm)

0.6884
0.8926
0.8335
0.8073
0.8162
0.8014

0.0078
0.0076
0.0072
0.0071
0.0068
0.0068

Vat magenta
Free cells
2 (mm)
3 (mm)
4 (mm)
5 (mm)
6 (mm)

0.9097
0.8898
0.8898
0.9806
0.9036
0.7777

0.0246
0.0224
0.0224
0.0248
0.0217
0.0219

Congo red
Free cells
2 (mm)
3 (mm)
4 (mm)
5 (mm)
6 (mm)

0.3029
0.2525
0.2460
0.2279
0.2090
0.2026

0.0042
0.0035
0.0034
0.0032
0.0031
0.0029

Acid green
Free cells
2 (mm)
3 (mm)
4 (mm)
5 (mm)
6 (mm)

0.4011
0.2529
0.2418
0.2385
0.2359
0.2197

0.0035
0.0029
0.0027
0.0027
0.0027
0.0025

Methylene blue
Free cells
2 (mm)
3 (mm)
4 (mm)
5 (mm)
6 (mm)

0.1328
0.1115
0.1078
0.1065
0.1009
0.1001

0.0021
0.0020
0.0020
0.0020
0.0018
0.0018

Acid red 114

Free cells
2 (mm)
3 (mm)
4 (mm)
5 (mm)
6 (mm)

0.1101
0.1362
0.1441
0.1383
0.1385
0.1433

0.0022
0.0021
0.0021
0.0019
0.0018
0.0018

max

(g/l h)

of immobilized are reported by Kuo-Cheng et al. [52].

Nurdan et al. [53] have observed higher degrading activity in
P. chrysosporium on immobilization. The reason for lower
Kdye compared to that of free cells could be attributed to the
fact that the calcium alginate used for entrapment might act
as a barrier for immediate dissociation of the dyes. As
observed from the results (Table 2) Vdye max value is lesser

3344

K.V. Radha et al. / Process Biochemistry 40 (2005) 33373345

for immobilized cells compared to that of free cells for all

the dyes tested and decreases with increase in the diameter
of the beads. This might be due to the fact that rate of
decolorization will be much faster in freely suspended cells
and at higher initial concentrations, the substrate inhibited
the decolorizing activity.

4. Conclusion
P. chrysosporium MTCC 787 is found to be suitable for
the decolorization of the different classes of dyes. An
optimum glucose and nitrogen concentration of 5 and
0.05 g/l, respectively, in the basal medium are found to be
useful for the enhancement of maximum decolorization.
Maximum percentage decolorization for all dyes studied in
this present work was found to be more than 75% at the
following optimum condition: temperature 35 8C, pH 45
and an inoculum size of 2 ml (approximately 1.6  105 cell/
ml) was obtained. Similar experiments were carried out for
entrapped cells in alginate beads in batch mode using shake
flasks. The MichaelisMenten kinetic parameters were
estimated for decolorization process using free cells and
immobilized cells. The kinetic parameters Kdye and Vdye
max using different diameter of immobilized beads can well
be used for the design and scale up of continuous reactors.

Acknowledgement
The authors wish to express their appreciation to Anna
University for the award of Research fellowship to K.V.
Radha for support of this investigation.

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