International Biodeterioration & Biodegradation 54 (2004) 215 223

www.elsevier.com/locate/ibiod

Biodegradation of synthetic dyes by Irpex lacteus

under various growth conditions
# ek Novotn'y , Kate#rina Svobodov'a, Aparna Kasinath, Pavla Erbanov'a
Cen#
Lab. of Experimental Mycology, Institute of Microbiology, Academy of Sciences of the Czech Republic,
V!"de#nsk!a 1083, 142 20 Prague 4, Czech Republic

Abstract
Degradability of azo-, anthraquinone- and Cu-phthalocyanine dye structures was studied using 39 strains of ligninolytic fungi, of
which 60% could attack azo dyes, compared with 8090% capable of attacking the other dyes. Irpex lacteus was shown to decolorize
a number of various azo-, anthraquinone-, thiazine-, triphenylmethane- and phthalocyanine dyes at a concentration of 200 mg l1 in
stationary liquid culture. Decolorization levels after two weeks were 60100%. Selective inhibition of manganese-dependent peroxidase
(MnP) and laccase by sodium azide and n-propyl gallate indicated the involvement of MnP in decolorization of anthraquinone- and azo
dyes. Immobilized on pinewood cubes, I. lacteus decolorized 100% of Remazol Brilliant Blue R (150 mg l1 ) within six days. It also
e:ciently decolorized textile industry e;uents containing color mixtures Drimarene Blue, Drimarene Red, Remazol Green and Acid
Black, achieving 100%; 80%; 45% and 35% decolorization, respectively, within 35 days.
? 2004 Elsevier Ltd. All rights reserved.
Keywords: Ligninolytic fungi; Synthetic dyes; Dye decolorization; Immobilized cultures; Textile industry e;uents; Manganese-dependent peroxidase;
Laccase

1. Introduction
Several tens of thousands of synthetic dyes are commercially available and large amounts of these compounds
are spilled into the environment in the form of e;uents
released by the dyestu@ and textile industries (Meyer,
1981). The elimination of such dye-containing e;uents is
mostly based on physical and chemical procedures, e.g.
adsorption, concentration, chemical transformation and incineration. These methods are rather costly and sometimes
produce hazardous byproducts, and therefore other alternatives, such as biodegradation, attract attention. However,
dye compounds are not readily biodegradable and are typically not removed from water by conventional wastewater
treatment systems (Shaul et al., 1991).
Ligninolytic fungi have been shown to possess a
remarkable potential for degrading various types of
dyes (McMullan et al. 2001; Jarosz-Wilkolazka et al.
2002; Toh et al. 2003). In nature these fungi colonize
Corresponding author. Tel.: +420-29644-2357; fax: +420-296442384.
E-mail address: novotny@biomed.cas.cz (C. Novotn'y).

0964-8305/$ - see front matter ? 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2004.06.003

wood and other lignocellulosic substrates and knowledge of

their capacity to remove recalcitrant dyes from contaminated
water and soil is rather poor. Di@erent extracellular peroxidases and laccase are able to oxidize a wide range of dye
compounds, such as azo, heterocyclic, triphenylmethane,
phthalocyanine and anthraquinone dyes (Paszczynski and
Crawford, 1991; Ollikka et al., 1993; Vyas and Molitoris,
1995; HeinJing et al., 1998). Development of e:cient dye
degradation biotechnology requires application of a suitable
selected strain and its use under favorable conditions to realize the degradation potential.
Irpex lacteus, a powerful lignin degrader (Capelari and
Zadrazil, 1997), is a white rot fungus typically inhabiting
dead hardwood trees (Ryvarden and Gilbertson, 1993). Phylogenetic analysis based on mitochondrial, small subunit and
ribosomal DNA sequences shows the fungus to be related to
the species Oxyporus latermaginatus and Hexagonia hydnoides, and, more distantly, to Ceriporia purpurea, Ceriporia viridans, Bjerkandera adusta and P. chrysosporium
(Ko and Jung, 1999). The pattern of ligninolytic enzymes of
I. lacteus, i.e. lignin peroxidase (LiP), manganesedependent peroxidase (MnP) and laccase (Novotn'y et al.,
2000), is typical of white rot species (Hatakka, 1994).

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C. Novotn!y et al. / International Biodeterioration & Biodegradation 54 (2004) 215 223

I. lacteus strain 238 was selected because of its capacity for

decolorization of the anthraquinone-based dyes Poly-R-478
and Remazol Brilliant Blue R (RBBR) and robust growth
# sek et al., 1998; Novotn'y
in deNned media and in soil (Sa#
et al., 2001).
The purpose of our study was to compare the potential
of I. lacteus to decolorize chemically di@erent dye structures with that of other ligninolytic fungi and deNne its
degradative power in liquid and immobilized cultures. Using selective inhibition of ligninolytic enzymes present and
measuring sorption capacity of mycelium in stationary culture, the mechanism of decolorization of model azo- and
anthraquinone dyes was characterized. Decolorization of industrial, dye-containing e;uents by I. lacteus was also documented.
2. Materials and methods
2.1. Microorganisms
The strains Agaricus bisporus 308, Armillaria mellea
330, Aurantioporus croceus 422/93, B. adusta 606/93,
Ceriporia metamorphosa 193/93, Coriolopsis polyzona
740, Daedalea quercina 528, Daedaleopsis confragosa
491/93, Dichomitus squalens 750, Flammulina velutipes
366, Fomes fomentarius 534, Fomitopsis pinicola 537,
Ganoderma lucidum 530/93, Hapalopilus nidulans 166/93,
Hymenochaete tabacina 227/93, Hypholoma fasciculare
813, Inonotus nidus-pici 189/93, Inonotus obliquus 615/93,
I. lacteus 238, I. lacteus 4, I. lacteus 902/II, Lentinula
edodes 648, Lentinus tigrinus 826, Merulius tremellosus
565, Mycena inclinata 422, Phanerochaete chrysosporium
(ME-446) 854, Phellinus pseudopunctatus 538/93, Phellinus punctatus 421/93, Piptoporus betulinus 585, Pleurotus
cornucopiae 464, Pleurotus cystidiosus 466, Pleurotus citrinopileatus 691, Pleurotus eryngii 471, Pleurotus ostreatus 106, Pleurotus pulmonarius 481, Pleurotus sajor-caju
666, Polyporus squamosus 676, and Pycnoporus cinnabarinus 787 were obtained from the Culture Collection of
Basidiomycetes, Academy of Sciences of the Czech Republic, Prague. Phanerochaete magnoliae was kindly provided
by Dr. M. Costa-Ferreira (Institute Nacional de Engenharia
e Tecnologia Industrial, Lisbon, Portugal). All were maintained on malt extract-glucose (MEG) agar slants at 4 C.
2.2. Chemicals
All chemicals used were of analytical grade, with
3-dimethylaminobenzoic acid (DMAB), 3-methyl-2benzothiazolinone-hydrazone hydrochloride (MBTH), veratryl alcohol, 2; 2 -azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS), n-propyl gallate (n-PG) and NaN3
being purchased from Sigma (USA). The following synthetic dyes were used: monoazo dyesMethyl Orange
(Lachema, Czech Republic), Methyl Red (Aldrich, USA),

Reactive Orange 16 (Aldrich, USA, Fig. 1); diazo dyes

Reactive Black 5 (Aldrich, USA), Congo Red (Merck,
Germany), Naphthol Blue Black (Aldrich, USA, Fig. 1),
Chicago Sky Blue 6B (Aldrich, USA); anthraquinone
dyesRemazol Brilliant Blue R (Sigma, USA, RBBR,
Fig. 1), Disperse Blue 3 (Aldrich, USA); heterocyclic
dyesFluorescein (Spolana, Czech Republic), Methylene Blue (Merck, Germany, Fig. 1); triphenylmethane
dyeBromophenol Blue (Lachema, Czech Republic, Fig.
1); metal-complex dyeCopper (II)-phthalocyanine-3; 4 ,
4 ; 4 -tetrasulfonic acid, tetrasodium salt (Aldrich, USA,
Fig. 1).
The speciNcation of samples of industrial e;uents removed from textile color baths was the following: Drimarene Blue sample containing Drimarene Violet and
LevaNx Royal Blue dyes (dye concentration 806 g ml1 );
Drimarene Red sample containing Drimarene Blue, Drimarene Brilliant Orange and LevaNx Brilliant Red dyes
(2300 g ml1 ); Acid Black sample containing Gelonyl
Black LD140Z dye (2300 g ml1 ); and Remazol Green
sample containing Yoracron Green and Remazol Brilliant Gel 6 dyes (1020 g ml1 ). Drimarene Blue and
Drimarene Red formulations also contained the additives
Skiantan and Zetesan N8 Neu ensuring equable coloration
of fabrics and Unitexol that prevented fracturing of Nbers
during the coloration process. The industrial, color-bath
e;uents were sterilized by Nltration (nitrocellulose Nlter,
pore size 0:2 m, Whatman, UK). The Nnal dilutions in the
culture medium of the individual color-bath liquids in the
biodegradation experiments were 10-fold.
2.3. Medium and growth
The following media were used: low-nitrogen, mineral
medium (pH 4.5, NMM) containing 2:4 mM diammonium
tartrate as the nitrogen source (Tien and Kirk, 1998); malt
extract-glucose medium MEG (pH 4.5) containing l1 malt
extract 5 g and glucose 1 g, and glucose-peptone-yeast extract medium GPY (pH 4.5) containing l1 glucose 1 g,
peptone 0:5 g and yeast extract 0:1 g all being sterilized at
120 C for 20 min. Filter-sterilized stock solutions of dye
in distilled water were added to ensure the appropriate dye
concentration. If necessary, the media were solidiNed with
agar (20 g l1 ).
A shallow stationary culture (500-ml Erlenmeyer Jask,
50 ml NMM medium), inoculated with two myceliumcovered 0:9 cm MEG agar discs, was grown at 28 C for 7
10 d, then aseptically homogenized in a Waring blender
(3 times, low speed, 20 s), and used as inoculum (10%,
v/v) for 20 ml dye-containing NMM in 250-ml Erlenmeyer
Jasks. Two types of control were included: heat-killed (biotic), 1-week old fungal cultures and (abiotic) uninoculated
controls containing the dyes. Heat inactivation was as described above. The fungal cultures were incubated statically
at 28 C for two weeks; aeration was by di@usion through

C. Novotn!y et al. / International Biodeterioration & Biodegradation 54 (2004) 215 223

217

Fig. 1. Chemical structure of major groups of dyes studied.

a cotton-wool stopper. At intervals, whole cultures were

sacriNced for analyses. The culture Juid was removed, pH
measured and adjusted to the initial value if necessary, and
color intensity determined spectrophotometrically (Perkin
Elmer Lambda 11 UV/VIS spectrophotometer, 1-cm optical
path) at the corresponding maximum wavelength. Fungal
biomass was harvested by Nltration, washed with distilled
water and dried (80 C) for gravimetric estimation of the
dry biomass.
2.4. Solid-phase bioreactor
I. lacteus was grown on 1-cm3 pinewood cubes as solid
support. Before use, the pinewood cubes were subjected to

three 30-min washes with hot distilled water and dried. To

set up a reactor 5-g dry cubes were autoclaved in 200 ml
2% malt extract broth medium (Oxoid, UK) in 500-ml
round-bottomed Jasks, inoculated with two 0.9-cm agar
plugs covered with fungal mycelium and incubated on a
rotary shaker at 110 rpm and 28 C for four days. After
the fungus colonized the solid support, the medium was
drained and the immobilized fungus aseptically transferred
to a reactor vessel.
The cylindrical glass reactors, working volume 27 ml, had
a bottom port for forced aeration by a room-aquarium aeration apparatus. The medium was circulated using a Jow
inducer (MHRE 22, Watson Marlow Ltd., UK) at a rate
of 1 ml min1 . On loading the reactor with the immobilized fungus, the mineral medium was circulated at 28 C for

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C. Novotn!y et al. / International Biodeterioration & Biodegradation 54 (2004) 215 223

Nve days to ensure even colonization of the support. Then

circulation of 100 ml mineral medium containing the dye
was started and the dye was allowed to adsorb to the supporting material for 24 h at 28 C. The unadsorbed dye was
then decanted and a fresh batch of dye-containing medium
was circulated in order to measure decolorization. Samples
were removed for spectrophotometric determination of decolorization with time. Before each decolorization cycle the
medium was Nlter-sterilized. No signiNcant sorption of dyes
to the Nlters was observed.
2.5. Enzyme assays
Crude culture Nltrates were used for the estimation of extracellular MnP, LiP and laccase activities.
The following methods used were, respectively; the 3dimethylaminobenzoic acid/3-methyl-2-benzothiazolinone
hydrazone hydrochloride assay (Vyas et al., 1994), veratryl
alcohol assay (Vyas et al., 1994) and 2; 2 -azino-bis-(3ethylbenzo-thiazoline-6-sulfonic acid) assay (Niku-Paavola
et al., 1998). Enzyme activities were measured at room
temperature, an enzyme unit being deNned as producing
1 mol min1 product under the assay conditions.
2.6. Enzyme inhibition
To examine the e@ects of selective inhibition of MnP and
laccase by sodium azide (Verdin et al., 2004) and n-propyl
gallate (n-PG) (Ku#cerov'a et al., 1999), respectively, on decolorization of RBBR or Reactive Orange 16, the assay system contained crude culture Nltrate, RBBR (20 g ml1 ),
H2 O2 (100 nmol ml1 ) and the inhibitor at an appropriate
concentration. The amount of dye decolorized after 4 h at
room temperature was used as a measure of the decolorization rate. Both biotic and abiotic controls were included in
the experiment.
2.7. Sorption of azo dye to fungal mycelium
Fungal biomass was separated by centrifugation (1100g;
4 C, 5 min; Medifriger-BL, Spain) and washed with 0:1 M
sodium lactate bu@er (pH 4.5). An amount of wet mycelium
equivalent to 5 mg dry biomass harvested from a 10-day
old culture was exposed to 5 ml Reactive Orange 16 dissolved in NMM at an indicated concentration for 120 min
at room temperature. The unabsorbed dye was removed by
centrifugation as mentioned above. Where mycelium was inactivated, sorption was carried out in the presence of 5 mM
NaCN.
2.8. Evaluation of results
In all experiments, the cultures and enzyme estimations
were run in triplicate, except for decolorization of color-bath
e;uents in the bioreactor when only one sample was removed at a time and evaluated.

3. Results and discussion

3.1. Decolorization by fungi growing on agar media
Using the set of 39 strains of ligninolytic fungi
pre-screened for e:cient decolorization of an anthraquinone
dye RBBR and a polymeric anthraquinone dye Poly R-478
# sek et al., 1998), we attempted to discover which dye
(Sa#
structures were more resistant to fungal decolorization
(Table 1). When representatives of azo-, anthraquinoneand phthalocyanine dyes were included in NMM and MEG
agar media and duplicates of each strain tested there were
minimal di@erences between these duplicates. The proportion of decolorizing strains, i.e. those able to decolorize the
dye-containing agar medium (to some extent at least), was
greater for the azo Congo Red and the anthraquinone Disperse Blue 3 dyes (Table 1) than for the azo Reactive Black
5 and the metal complex dye Cu-phthalocyanine. The number of strains capable of attacking the azo dyes under ligninolytic conditions, ensured by growth on the low-nitrogen
NMM, was higher than on MEG medium, but not in the
case of the anthraquinone and phthalocyanine dyes.
As another parameter of degradability, the mean time
necessary for complete decolorization of the dye-containing
plate was noted for those strains capable of complete decolorization (Table 1). Generally, the growth media did
not greatly inJuence the mean time necessary for complete
decolorization, the greatest di@erence, of almost two days,
being found in the case of Reactive Black 5. The mean time
necessary for complete decolorization of the anthraquinone
dye was mostly shorter by 37 days relative to the other
dyes, suggesting an easier decolorization by the strains capable of attacking all dyes tested (Table 1). This Nnding is in
keeping with a good degradability of another anthraquinone
# sek et al., 1998). Exdye, RBBR, by ligninolytic fungi (Sa#
cept for the anthraquinone dye, Disperse Blue 3, the mean
decolorization times detected for I. lacteus were shorter than
those observed with the other strains. In contrast, although
P. chrysosporium, a model ligninolytic fungus showed
greater decolorization rates on NMM medium (Table 1) it
decolorized many dyes only partially.
3.2. Decolorization by I. lacteus 238 growing on agar
media
A broader range of dyes than in Table 1, including
monoazo (Methyl Orange, Methyl Red, Reactive Orange
16), diazo (Reactive Blue 5, Congo Red, Naphthol Blue
Black), anthraquinone (RBBR, Disperse Blue 3), heterocyclic (Fluorescein, Methylene Blue) and triphenylmethane
dyes (Bromophenol Blue), was shown to be decolorized by
I. lacteus in a separate study using three di@erent growth
media, NMM, MEG and GPY (data not shown). In all
three media, the fungus was able to decolorize completely
all these dyes except Methylene Blue, which was not

C. Novotn!y et al. / International Biodeterioration & Biodegradation 54 (2004) 215 223

219

Table 1
Screening of dye-degrading strains of ligninolytic fungi on nitrogen-limited mineral and malt extract-glucose agar medium

Dye (200 mg l1 )

Proportion of decolorizing strainsa

Mean decolorization time (days)

NMMb

MEGc

NMMb

MEGc

Reactive Black 5

66.7

56.4

Congo Red

89.7

61.5

Disperse Blue 3

89.7

90.0

Cu-phthalocyanine

71.8

76.9

15.9
(17; 10)d
21.1
(24; p)d
14.4
(10; 17)d
17.5
(20; 13)d

18.7
(17; p)d
20.0
(20; p)d
13.9
(13; 20)d
18.4
(17; p)d

aA

total of 39 strains was used in the screening study.

mineral agar medium.
c MEGmalt extract-glucose agar medium.
d Numerals in parentheses indicate the number of days required for complete decolorization of plates inoculated with I. lacteus or
P. chrysosporium, respectively (p, only a partial decolorization within a period of 4 weeks was attained).
b NMMnitrogen-limited

decolorized on GPY despite good growth of the fungus

on this medium. Complete decolorization occurred within
14 weeks depending on the dye used. Mostly, di@erences
between decolorization times observed on individual media did not exceed 25%, with the exception of Methyl
Red, where the decolorization rate in NMM was 34 times
slower than in GPY and MEG, and Fluorescein, which was
decolorized 1.52 times slower in GPY in comparison with
the other two media.
3.3. Decolorization capacity of I. lacteus 238 in a liquid
medium
A study of the decolorization capacity of I. lacteus
growing in the colorless, NMM liquid medium containing
dyes (200 mg l1 ) in shallow stationary culture (Table 2)
revealed that all were 90% decolorized within two weeks,
except for Congo Red and Methylene Blue, which were,
respectively, decolorized by 65% and 80%. Such a broad
decolorization potential agrees with observations of
other authors working with various white rot fungi
(Pointing, 2001). In biological sorption controls (heat-killed
mycelium) included in all experiments, sorption was 20
30% of the initial dye concentration. The exceptions were
Congo Red, Chicago Sky Blue and Disperse Blue 3 where
sorption exceeded 60% (data not shown). Methylene Blue,
RBBR and Cu-phthalocyanine were most toxic to growth,
inhibiting growth yield by about 50% (Table 2).
When the extracellular MnP, LiP and laccase activity was
measured in cultures to document the e@ect of dyes on enzyme synthesis and, if possible, to correlate dye removal
with development of enzyme activities known to decolorize
synthetic dyes (Ollikka et al., 1993; HeinJing et al., 1998;
Pointing and Vrijmoed, 2000), some of the dyes tested stimulated production of MnP up to 5 and LiP by up to 5 and
10 times. On the other hand, RRBR and Bromophenol Blue
signiNcantly inhibited the synthesis of MnP (Table 3). The

levels of ligninolytic enzymes do not seem to be a limiting

factor in decolorization (Swamy and Ramsay, 1999; Moreira
et al., 2000). The overlap in time of enzyme presence with
decolorization of individual dyes was considered to be an essential condition of possible functional correlation. Accordingly overlap of the periods during which signiNcant enzyme
activity was present in the culture liquid and at least 90% of
the initial dye concentration was removed from the culture
was taken as a criterion of positive correlation (Table 2).
On this basis, in most cases the presence of MnP could be
correlated with the disappearance of dyes from the medium.
In the case of laccase, however, such a correlation was observed only with RBBR and Cu-phthalocyanine (Table 2).
LiP was absent in most cultures and, therefore, its role
in decolorization of synthetic dyes by I. lacteus seems to
be generally less important (Table 3). A similar conclusion
can be drawn even for decolorization of RBBR, since in
contrast to the data in Table 3 e:cient decolorization of
the dye has also been repeatedly observed without detection
of measurable levels of LiP in cultures (data not shown).
In the case of Chicago Sky Blue, the development of LiP
activity in stationary cultures of I. lacteus was found to
occur much later than the time by which decolorization was
accomplished.
3.4. Sorption and ligninolytic enzyme activities in
removal of azo and anthraquinone dyes
Both sorption by biomass and attack of dye by ligninolytic
enzymes were envisaged to participate in the process of
dye removal by I. lacteus, as Wang and Yu (1998) demonstrated that adsorbed dyes could also be degraded by living
mycelium and its extracellular enzymes. For the demonstration of the sorption capacity of stationary-grown I. lacteus
mycelium, the azo dye Reactive Orange 16 was chosen. The
kinetics of sorption of the dye, used at 50 mg l1 showed
that a maximum sorption of 4 mg dye g1 dry biomass was

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C. Novotn!y et al. / International Biodeterioration & Biodegradation 54 (2004) 215 223

Table 2
Decolorization of dyes by I. lacteus 238 and inhibition of fungal growth in stationary cultures growing in dye-containing, low-nitrogen mineral medium
at 28 C

Dye (type)

Decolorization
after 2 weeks
(%)a

Growth
yield (g l1 )

Time correlation of
decolorization with the
enzyme synthesisb;c

None
Reactive Orange 16
(monoazo)
Congo Red
(disazo)
Reactive Black 5
(disazo)
Naphthol Blue Black
(disazo)
Chicago Sky Blue
(disazo)
Remazol Brilliant Blue R
(anthraquinone)
Disperse Blue 3
(anthraquinone)
Methylene Blue
(thiazine)
Cu-phthalocyanine
(metal-complex)
Bromophenol Blue
(triphenylmethane)

95:8 0:0

4:3 0:2
3:7 0:0

MnP + =Lac

55:8 1:6

2:6 0:1

MnP + =Lac

98:4 0:5

4:7 0:3

MnP + =Lac

99:1 0:0

2:9 0:2

MnP + =Lac

98:6 0:1

3:0 0:0

MnP =Lac

95:1 0:1

2:1 0:1

MnP + =Lac+

94:0 0:6

3:0 0:0

MnP + =Lac

80:3 0:3

1:8 0:1

MnP =Lac

96:7 0:0

2:1 0:0

MnP + =Lac+

96:0 1:4

3:3 0:1

MnP + =Lac

a Decolorization related to an initial dye concentration of 200 mg l1 added to low-nitrogen, mineral medium (NMM); mean values standard
deviation are shown.
b Manganese-dependent peroxidase, MnP; laccase, Lac.
c Positive time correlation (+, signiNcant enzyme activity was present during the time period in the course of which at least 90% of the dye
added was removed); weak correlation (; inconclusive time overlap of the enzyme presence and decolorization); no correlation (; no time
overlap of the enzyme presence and decolorization).

accomplished after 30 min (data not shown). The sorption

curve measured for a concentration range 0750 mg dye l1
indicated that the mycelial capacity of sorption at the dye
concentration of 200 mg l1 used in the degradation experiments was about 15 mg dye g1 dry biomass (Fig. 2). The
adsorption capacity of a stationary culture thus represented
some 30% of the total dye added to the culture.
A further aim in the study was to obtain experimental
evidence for involvement of ligninolytic enzyme activities
in dye decolorization by I. lacteus cultures. Selective inhibition with sodium azide and n-propyl gallate was used
as a tool for proving the involvement of ligninolytic enzyme activities in decolorization of Reactive Orange 16
(azo dye) and RBBR (anthraquinone dye) by stationary
cultures of I. lacteus (Table 4). There was strong inhibition of MnP when sodium azide was used at 1 and
10 M, however, inhibition of laccase was negligible. Increasing inhibition of decolorization of both dyes with
increasing azide concentration suggested the involvement
of MnP, and also demonstrated di@erent sensitivity of the
two decolorization processes to partial inhibition of MnP
activity (Table 4). The results obtained with n-propyl gallate were less conclusive owing to the inhibition of both
MnP and laccase. Relatively minor inhibition of RBBR

decolorization at the lower concentration of this inhibitor, when laccase activity was decreased by 80%,
together with a considerable increase in inhibition of decolorization of the dye at the higher inhibitor concentration (but not a complete abolition of decolorization),
when the inhibition of MnP increased 2.6-fold and laccase activity was completely inhibited, conNrmed a major role for MnP in the decolorization of RBBR. The
results with n-propyl gallate might also suggest a role
for laccase in the decolorization of Reactive Orange 16.
This inhibition study supports the observation of Moreira
et al. (2000) that other factors limiting dye decolorization
may exist besides MnP, laccase and other extracellular enzyme activities, for instance the production of H2 O2 (see
Kotterman et al., 1996).
3.5. Decolorization in a packed-bed reactor
The stationary culture of I. lacteus demonstrated a capacity to decolorize several subsequent additions of RBBR
(Fig. 3). A similar result was also obtained with Reactive
Orange 16 (data not shown). Accordingly, a packed-bed
reactor in which the fungus was immobilized on a solid
support was constructed and used for decolorization. The

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221

Table 3
Ligninolytic enzymes secreted in stationary cultures of I. lacteus 238 in the presence of various dyes

Dye (type)

None
Reactive Orange 16
(monoazo)
Congo Red
(disazo)
Reactive Black 5
(disazo)
Naphthol Blue Black
(disazo)
Chicago Sky Blue
(disazo)
Remazol Brilliant Blue R
(anthraquinone)
Disperse Blue 3
(anthraquinone)
Methylene Blue
(thiazine)
Cu-phthalocyanine
(metal-complex)
Bromophenol Blue
(triphenylmethane)

Maximal enzyme activity (U l1 )a

Mn-dependent peroxidase

Laccase

Lignin peroxidase

76:2 4:4
(214)b
95:6 2:9
(214)b
79:8 2:6
(314)b
87:7 0:9
(314)b
338:0 5:9
(315)b
380:0 4:3
(715)b
43:7 0:0
(212)b
220:0 10:2
(315)b
218:0 29:7
(714)b
204:0 11:6
(314)b
49:2 1:9
(314)b

2:0: 0:0
(214)b
0

1:1 0:2
(215)b
0

0:3 0:0
(5)b
2:8 0:2
(15)b
1:8 0:2
(214)b
4:5 0:6
(515)b
0

0
12:5 1:3
(1115)b
15:9 4:3
(212)b
0
0

4:6 0:1
(314)b
0

0
0

nitrogen, mineral medium (NMM) containing optionally 200 mg dye l1 was used for growth; mean values of the enzyme activities
standard deviation values are shown.
b Numerals in parentheses indicate period in days during which the enzyme was detectable in the culture Juid.
a Low

Table 4
Selective inhibition of ligninolytic enzymes in crude culture Nltrate of a
stationary culture of I. lacteus 238 and its e@ect on dye decolorization

Inhibitora

None
NaN3 (1 M)
NaN3 (10 M)
n-PG (10 M)
n-PG (100 M)

Relative inhibition (%)

Enzyme activityb

Decolorizationc

MnP

Laccase

RBBR

RO16

0
55
79
26
68

0
0
3
83
100

0
0
50
10
60

0
31
62
40
98

a n-PG,

Fig. 2. Sorption of Reactive Orange 16 to stationary-phase fungal

mycelium at room temperature. Mean values standard deviation are
plotted against the dye concentration.

n-propyl gallate.
manganese-dependent peroxidase.
c RBBR, Remazol Brilliant Blue R (anthraquinone dye); RO16,
Reactive Orange 16 (monoazo dye).

immobilized fungus was able to remove all RBBR used as

a model dye at 150 mg l1 within six days (Fig. 4). MnP
and laccase activities were present during decolorization and
their levels were fairly stable at 318 and 12 U l1 , respectively (data not shown). The immobilized fungus also
e:ciently decolorized textile color-bath e;uents containing
the color mixtures Drimarene Blue, Drimarene Red, Remazol Green and Acid Black by 100%; 80%; 45% and 35%,
respectively, within 35 days (Fig. 4). Our experiments

thus demonstrated that, similarly to another white rot fungus

P. cinnabarinus (Schliephake et al., 1993), I. lacteus had
the potential for e:cient decolorization of various synthetic
dyes, when grown in solid-support, immobilized cultures.
After decolorization, the immobilized cultures could be regenerated in fresh medium without dye. Using RBBR, up to
eight subsequent cycles, each decolorizing 15 mg dye per
100 ml, could be achieved (data not shown).

b MnP,

222

C. Novotn!y et al. / International Biodeterioration & Biodegradation 54 (2004) 215 223

Czech Republic, Czech-Slovenian projects No. 2001/031

and 011-2004-05 of Ministry of Youth, Education and Sport
of the Czech Republic, and Institutional Research Concept
No. AV0Z5020903.
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Fig. 3. Repeated addition of RBBR (150 mg l1 ) into a stationary culture

of I. lacteus. Mean values standard deviation are plotted against time.

Fig. 4. Decolorization of RBBR and color-bath e;uents in a wood-cube

packed-bed bioreactor with immobilized I. lacteus.

4. Conclusions
I. lacteus showed a remarkable potential to decolorize a
broad spectrum of chemically di@erent, synthetic dyes. Compared with other ligninolytic fungal species, decolorization
was e@ective and rapid. Dye adsorption on mycelium and
the involvement of MnP activity were found to be important
features of the decolorization process. Immobilized cultures
of I. lacteus proved to be e:cient in removing of synthetic
dyes, including textile industry e;uents, and capable of periodical recovery in a fresh medium. These are promising
features for possible long-run applications.
Acknowledgements
The excellent technical assistance of L. Klanicov'a is
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Prague) and J. Hor'ak (Institute of Physiology, AS CR,
Prague) for kindly providing us with n-propyl gallate and
sodium azide. The work was supported by the following
grants: Project No. 526/00/1303 of Grant Agency of the

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