World J Microbiol Biotechnol (2008) 24:337–344

DOI 10.1007/s11274-007-9475-7

ORIGINAL PAPER

Cytotoxicity and genotoxicity evolution during decolorization

of dyes by White Rot Fungi
Sophie Vanhulle Æ Marie Trovaslet Æ Estelle Enaud Æ Mathias Lucas Æ
Marc Sonveaux Æ Cony Decock Æ Rob Onderwater Æ Yves-Jacques Schneider Æ
Anne-Marie Corbisier

Received: 27 March 2007 / Accepted: 8 June 2007 / Published online: 8 August 2007

Springer Science+Business Media B.V. 2007

Abstract White Rot Fungi (WRF) are able to decolorize
dyes through the use of relatively non-specific extracellular
oxidative enzymes. Nevertheless, decolorization does not
imply that the resulting metabolites are less toxic than the
parent molecules. The aim of the present study was to
evaluate the detoxification potential of six strains (Pyc-
noporus sanguineus, Perenniporia tephropora,
Perenniporia ochroleuca, Trametes versicolor, Coriolopsis
polyzona and Clitocybula dusenii) during decolorization of
dyes. Cytotoxicity assays were carried out on human Caco-
2 cells, which are considered as a validated model for the
human intestinal epithelium, and the results were compared
with those obtained on classical bacterial cells. Genotoxic
character was monitored through VITOTOX1 assays. The
biotransformation of an anthraquinonic dye (CI Acid Blue
62, ABu62) was studied. All tested strains were able to
decolorize extensively ABu62 (between 83 and 95%
decolorization), however, different cytotoxicity reduction
levels were reached (from 44 to 99%). Best results were
achieved with P. sanguineus strain and the major role of
laccases in cytotoxicity reduction was underlined. Based on
this result, efficiency of P. sanguineus strain was further
studied. Four azo and two anthraquinonic dyes were treated
by this strain. After WRF treatment, two dyes were found
to be more toxic in one or both toxicity assays. Genotoxic
character appeared during biotransformation of one dye,
however, it was removed by the addition of hepatic rat
extract to mimic liver transformation. These results stress
the importance of monitoring several parameters, such as
colour, toxicity and mutagenicity, to ensure the efficiency
of the bioremediation process.
Keywords Fermentation
Filamentous fungi

Laccase
Decolorization
Dyes
Cytotoxicity

Genotoxicity

Abbreviations
S. Vanhulle (&)
M. Trovaslet
E. Enaud
M. Lucas
ABTS 20 ,2-azino-bis-(3-ethylbenzo thiazoline-6-
M. Sonveaux
A.-M. Corbisier
Microbiology Unit, Université catholique de Louvain,
sulfonic acid)
Croix du Sud 3/6, 1348 Louvain-la-Neuve, Belgium ABu62 CI Acid Blue 62, CI 62045
e-mail: vanhulle@mbla.ucl.ac.be ABu113 CI Acid Blue 113, CI 26360
ABu281 CI Acid Blue 281
C. Decock
Faculté des Sciences Agronomiques, Mycothèque de
AOr116 CI Acid Orange 116
L’Université catholique de Louvain, 1348 Louvain-la-Neuve, AR266 CI Acid Red 266, CI 17101
Belgium AR299 CI Acid Red 299
BCCMTM/ Belgian Coordinated Collections of
R. Onderwater
Wetlands Engineering, rue Laid Burniat 6,
MUCL Microorganisms/Mycothèque de
1348 Louvain-la-Neuve, Belgium l’Université Catholique de Louvain
DMSO Dimethyl sulfoxide
Y.-J. Schneider MTT assay 3-(4,5-dimethylthiazol-2-yl)-2,5-
Cell Biochemistry Laboratory, Institut des Sciences de la vie,
Université catholique de Louvain, Croix du Sud 5/3, 1348
diphenyltetrazolium bromide assay
Louvain-la-Neuve, Belgium WRF White Rot Fungi

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Introduction
Since the discovery of the first synthetic dye mauvein by a
young English chemist William Perkin in 1856, the tradi-
tional dyes from animal (Hexaplex trunculus, Kerria lacca,
Kermes vermilio ...) or plant (Isatis indigota, Rubia cor-
difolia, Rhamnus lycioides...) origins have been
progressively replaced by synthetic dyes (Cardon 2003).
Acid dyes mainly comprise azo, anthraquinones and tri-
phenylmethane structures and may be used for the dyeing
of wool, nylon, leather, polyamide, silk and paper. During
the dyeing process, 5–20% of acid dyes are lost in the
effluent, and in numerous cases are flushed directly into
waterways (Trovaslet et al. 2007). This represents a world
effluent release ranging between 5,000 and 20,000 tons per
year, which constitutes a serious pollution as well as a non-
negligible risk of toxicity against living organisms. Indeed,
a survey of oral acute toxicity of 4,461 dyes, evaluated by
the lethal dose for 50% of treated rats, has revealed that azo
and cationic dyes are the most toxic (Clarke and Anliker
1980). There is ample evidence of the mutagenicity of
some dyes, especially azo dyes and amino-substituted dyes
such as 4-phenylazoanilin (Pasti-Grigsby et al. 1994; Ra-
jaguru et al. 1999). Epidemiological analyses indicated that
the regular and long term use of hair dyes can be associated
with the development of bladder cancer (Gago-Dominguez
et al. 2002). Toxicity may be due to the biosynthesis of
transformation products during biotransformation of the
dyes (Pasti-Grigsby et al. 1994; Soares Graça et al. 2002a,
b).
Increasingly restrictive regulations in Europe make
wastewater treatment obligatory (European Water Frame-
work Directive 2000/60/EC). Among wastewater
treatments currently under study, recent researches have
shown that decolorization processes using White Rot Fungi
(WRF) and their lignin-modifying enzymes are promising
(Eichlerova et al. 2005; Wesenberg et al. 2003; Zhang et
al. 1999). Nevertheless, decolorization does not imply that
the resulting molecules are less toxic than the parent ones.
On the contrary, it has been shown that anaerobic incu-
bation with rat intestinal microorganisms leads to reduction
and cleavage of the azo-bonds of dyes derived from ben-
zidine and the formation of potentially carcinogenic
aromatic amines (Cerniglia et al. 1982a, b).
Six WRF strains (Pycnoporus sanguineus, Perenniporia
tephropora, Perenniporia ochroleuca, Trametes versicolor,
Coriolopsis polyzona and Clitocybula dusenii), previously
shown to be efficient for dye decolorization (Vanhulle et
al. 2002; 2007b), were assayed for their capacity to reduce
the cytotoxicity using the model acid dye ABu62. As the
ingestion of contaminated water would lead to a direct
contact with the human intestine, toxicity assays were
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World J Microbiol Biotechnol (2008) 24:337–344
carried out on human Caco-2 cells, which are considered as
a validated in vitro model for the human intestinal epi-
thelium. It represents a useful and well-accepted tool for
studying toxicity of pollutants (Velarde et al. 1999). The
efficiency of the best detoxifying strain (P. sanguineus)
was further studied with six acid dyes. Indeed, biotrans-
formation of dyes with different structures will lead to the
formation of various metabolites with their own toxico-
logical characteristics. The bioremediation of four azo (CI
Acid Blue 113, CI Acid Orange 116, CI Acid Red 266, CI
Acid Red 299) and two anthraquinonic dyes (CI Acid Blue
62 and CI Acid Blue 281) was further studied and evolu-
tions of colour, toxicity and genotoxicity were monitored
upon WRF treatment. Cytotoxicity was evaluated on bac-
terial cells as well as on Caco-2 thus giving indications on
acute and subacute toxicity. The genotoxicity was followed
through VITOTOX1 test (Taylor et al. 2004), which is
based on SOS response (DNA repair system)-dependent
induction in genetically modified Salmonella typhimurium.
Material and methods
Strains
Coriolopsis polyzona MUCL 38443, Perenniporia tep-
hropora MUCL 41562, Perenniporia ochroleuca MUCL
41114, Pycnoporus sanguineus MUCL 41582 and Tra-
metes versicolor MUCL 38412 were obtained from the
BCCMTM/MUCL collection. Clitocybula dusenii DSM
11238 was obtained from the DSMZ collection. Stock
cultures were maintained at 4 C on malt agar 2% (w/v),
under a layer of mineral oil.
Medium and culture conditions
The strains were precultured on malt agar 2% (w/v) in Petri
dishes (diameter: 10 cm), incubated for 7 days at 25 C.
Five fragments of the mycelia were sampled at the margin
of the colonies with a hollow-punch of 3 mm diameter and
used to inoculate 50 ml of a 2% malt extract broth sup-
plemented with 1.75 mM of dye into a 250-ml shaken
flask. Dyes were courteously supplied by Yorkshire Europe
(Yorkshire Europe, Tertre, BE). The cultures were incu-
bated at 25 C for 14 days, on a rotary shaker at 125 rev/
min, with a daily sampling.
Decolorization measurement
Absorbance spectra of the filtered broth taken from the
shaken flask cultures were recorded between 200 and
World J Microbiol Biotechnol (2008) 24:337–344 339

800 nm using a Spectronic Genesys 2 PC (Analis, Gent, [3-ethylbenzthiazoline-6-sulfonate] (ABTS) in 100 mM

BE). The biotransformation of dyes was analysed spec- tartrate pH 4.5 at 414 nm (emax = 34 450 M1 cm1). One
trophotometrically at the wavelength of maximum unit of activity (U) was defined as the amount of enzyme
absorbance. For decolorization of ABu62 by purified lac- that oxidizes 1 lmol of ABTS min1.
cases, 1 ml solution of 1.75 mM ABu62 in 100 mM
tartrate buffer (pH 4.5) was treated by 30 U l1 of purified
Toxicity measurement on Caco-2 cells
laccases from P. sanguineus MUCL 41582 at 25 C (static
conditions).
Caco-2 cells (ATCC, Rockville, MD) were routinely cul-
tured in plastic tissue culture flasks (175 cm2, Greiner
Labortechnik, Frickenhausen, DE) as described by Halleux
Laccase production and purification
and Schneider (1991) using a serum free nutritive medium
(Schneider 1989) composed of a 5:5:1 (v/v/v) mixture of
The medium contained (per l) 10 g glucose, 2 g KH2PO4,
Iscove’s modified Dulbecco’s, Ham’s F12 and NCTC 135
0.5 g (MgSO4
7H2O), 0.1 g CaCl2, 0.5 g ammonium
media (GIBCO BRL, Paisley, UK). In this study, the Caco-
tartrate, 0.25 g yeast extract and 2.2 g sodium succinate
2 cell-line culture and the exposure were both under serum
and its pH was adjusted to 4.5 prior to sterilization. Liquid
(protein) free conditions. Therefore, no differences in
medium (150 ml) was sterilized at 121 C for 20 min. The
bioavailability of the parent dye or its metabolites were
medium was supplemented with xylidine (330 lM) and
incurred because of serum protein binding. Cells were
Cu2+ (125 lM as CuSO4), which were added to stimulate
cultured at 37 C for 3-4 days between passages. Cyto-
laccase production. The inoculum was taken from a 7-day
toxicity screening was performed in 96-well microplates
malt extract agar plate cultures grown at 25 C. Half of the
(Greiner). Cells were seeded at a density of 5 · 104 cells
fungal mycelia grown on the Petri dishes was homogenized
cm2 and incubated for 24 h at 37 C. The samples of
aseptically in 50 ml of sterile deionized water using an
ABu62 (at initial concentration 1.75 mM) and its bio-
IKA Ultraturrax T25 (Janke and Kuntel, Staufen, DE).
transformation products were added at increasing dilutions
Each medium-containing flask was inoculated with 5%
(from 0 to 20%, v/v, corresponding to 0–350 lM ABu62)
(v/v) of this solution. The fermentations were carried out at
and incubated for 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-
28 C, in the dark, under static conditions (Vanhulle et al.
diphenyltetrazolium bromide (MTT) assays were per-
2002; Wesenberg et al. 2002, 2003).
formed as described previously (Berger et al. 2003;
Purified laccases was prepared as described below by
Halleux et al. 1991). The intensity of the purple colour
Dr. D. Wesenberg and F. Buchon from the Unit of Bio-
(formazan) was measured to determine cell viability after
logical Engineering of the Catholic University of Louvain
solubilisation of the crystals in DMSO on a microplate
(Louvain-la-Neuve, BE). Liquid culture medium was
reader at a wavelength of 540 nm. The percentage of
replaced periodically (each 2 or 3 days) by fresh medium.
inhibition was determined by comparing cell viability in
The collected media were frozen at 20 C to precipitate
the absence or presence of the sample tested. The per-
polysaccharides. After thawing, they were filtered on glass
centage of detoxification was calculated as described in
fibres. Filtrates were concentrated by ultrafiltration (Min-
Equation (1):
isette Omega open channel filter cassette, 10 kDa
molecular weight cut-off, Pall Filtron, Dreieich, DE). The %inhinitial  %inhfinal
resulting concentrate was fractionated by FPLC (Gilson, d% ¼ ð1Þ
%inhinitial
Roissy, FR) on QA-CIM disk (Quaternary amine-Con-
vective interaction Media disk monolithic column, BIA where d% is the percentage of detoxification, % inhinitial
Separations, Ljubljana, SL), under a linear saline gradient and % inhfinal are respectively the percentage of inhibition
(0.1 M NaCl, buffered at pH 5.5 using 10 mM sodium determined before and after WRF treatment.
succinate) with a 3 ml min1 flow. A second fractionation Results presented are an average of at least six
was performed under the same conditions on DEAE-CIM replicates.
(Diethylaminoethyl-CIM, BIA Separations, Ljubljana, SL).
The fractions obtained were concentrated and preserved at
20 C. Purity of fractions was determined by SDS- Toxicity measurement on bacterial cells (Lumis test)
PAGE. Characteristics of these enzymes were described by
Vanhulle et al. (2002). The determination of acute toxicity was carried out on
Laccase activity was determined using a Spectronic freeze-dried luminescent bacteria (Vibrio fischeri NRRL-
Genesys 2PC spectrophotometer (Analis, Namur, BE), by B-11177) from Dr. Lange (LCK 484, Dr. Lange, Düssel-
measuring the oxidation of 25 mM 2,20 -azino-bis- dorf, DE). The measurements were determined using the

123
340
LUMISmini instrument (Dr. Lange). For the coloured
samples, colour correction cuvettes (LPZ 340, Dr. Lange)
were used. The procedure for testing the toxicity of dyes
and intermediates was done following the instructions of
the manufacturer. The inhibition of the natural light pro-
ducing strain was determined against a non-toxic control
and the range of linearity was comprised between 10% and
90% inhibition.
Genoxicity measurement
Genotoxicity assays were performed as described in
Verschaeve et al. (1999) with the VITOTOX1 test
(Thermo Electron, Courtaboeuf, FR) and according to the
manufacturer’s instructions. Dyes, at an initial concentra-
tion of 1.75 mM were tested before and after fungal or
enzymatic treatment, at dilutions from 0 to 10%. The
VITOTOX1 assay is based on a recombinant Salmonella
typhimurium strain (TA 104 recN2) containing the lux
operon of Vibrio fisheri under the transcriptional control of
recN gene, which is a part of the SOS system. In the case of
damage to the DNA of the strain TA 104 recN2-4 caused
by the presence of a genotoxic compound, the SOS
response system is activated as well as the luciferase gene.
Luciferase activity is measured by the light emission and is
dependent on the genotoxicity of the compound tested. The
signal/noise ratio (S/N) represents the light production of
the exposed cells divided by the light production of non-
exposed cells. Some compounds (e.g. aldehydes) can
interfere with light production or enhance the metabolism
of the bacteria, therefore creating false positive results. The
response measured was therefore compared to the one
obtained in the presence of a constitutive light producing
bacterial strain (TA 104 pr1) with a lux operon under the
control of the constitutive pr1 promoter and in which light
production is not influenced by genotoxic compounds. A
dose-dependent response of the light produced by TA 104
recN2-4 divided by the light from TA 104 pr1 (called
later in this paper ‘‘rec/pr1’’) higher than 1.5 indicates
Table 1 Comparison of the percentage of decolorization and detoxification of ABu62 by different White Rot Fungal strains
Strain % decolorization(1)
Coriolopsis polyzona 83 ± 4
Perenniporia ochroleuca 93 ± 4
Clitocybula dusenii (nd)
Trametes versicolor 95 ± 5
Perenniporia tephropora 90 ± 4
Pycnoporus sanguineus 92 ± 4
Colour was measured by absorbance at the maximum wavelength of ABu62 (595 nm). Toxicity was measured on human intestinal cells (Caco-2
line) during the biotransformation of ABu62 by 6 strains. Percentage of decolorization at 595 nm and detoxification were determined after (1)
7 days and/or (2) 14 days of culture. (nd) = not determined
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World J Microbiol Biotechnol (2008) 24:337–344
the mutagenicity of the sample. As a positive control,
4-nitroquinoline-N-oxide was used in the absence of
metabolizing S9 hepatic rat fraction, while benzo[a] pyrene
was used in the presence of S9. Analysis in the presence of
S9 hepatic rat extract were carried out as described by
Verschaeve et al. (1999) to mimic liver transformation.
Results and discussion
Comparison of decolorization and cytotoxicity
reduction (Caco-2 cells) potentials of different White
Rot Fungal strains
The ABu62 decolorization ability of six WRF strains
(Coriolopsis polyzona, Perenniporia tephropora, Perenni-
poria ochroleuca, Pycnoporus sanguineus, Trametes
versicolor and Clitocybula dusenii) was investigated and
the percentage of decolorization obtained at 595 nm after
one week of culture was compared (Table 1). Different
WRF have already been investigated for their potential to
decolorize dyes (Robinson et al. 2001; Wesenberg et al.
2003). However, most of the strains in this study were
investigated for the first time. These strains were selected
by our group through a screening of 350 WRF for their
efficiency in decolorizing seven industrial dyes (Vanhulle
et al. 2002). It should be noted that through the selection,
the ability of the strains to make the first step of decolor-
ization was quite fast (5 days) as compared with other
studies (Conneely et al. 1999a, b; Kirby et al. 2000) where
it is reported that between seven to twenty days are usually
required to achieve 90% decolorization.
During the studied biotransformations, the cytotoxicity
evolution on Caco-2 cells was monitored as well as the
colour reduction. It was shown that T. versicolor, P. och-
roleuca, P. sanguineus and P. tephropora led to very good
decolorization levels, whereas the detoxification ability
differed significantly among the strains (between 44 and
99% after two weeks of culture). The weakest decolor-
ization was obtained with C. polyzona (83%), but this
% detoxification(1) % detoxification(2)
30 ± 2 74 ± 4
32 ± 2 45 ± 3
34 ± 2 44 ± 3
37 ± 3 75 ± 4
45 ± 3 86 ± 5
64 ± 4 99 ± 5
World J Microbiol Biotechnol (2008) 24:337–344
strain showed a good cytotoxicity reduction (74%) after
two weeks. On the contrary, P. ochroleuca was a good
decolorizer (93% after one week), but low detoxifier (45%
after two weeks). C. dusenii led also to a low cytotoxicity
reduction, although it was previously demonstrated to be an
efficient decolorizer (Wesenberg et al. 2003). T. versicolor
was found to be one of the best decolorizers, and also a
good detoxifier. Highest levels of decolorization and
detoxification were obtained with P. tephropora and
P. sanguineus (respectively 86% and 99% of detoxification
after two weeks of culture). These results clearly demon-
strate that decolorization is not directly correlated to the
detoxification potential and it is in agreement with those
reported previously (Abadulla et al. 2000). It could be
suggested that cytotoxicity should be studied as an
important parameter for wastewater treatment efficiency.
ABu62 biotransformation by P. sanguineus
Further studies were carried out using P. sanguineus as it
was the best decolorizer and detoxifier among the strains
tested. As previously demonstrated (Vanhulle et al. 2007a;
b), the blue colour of the ABu62-containing medium turned
red before complete decolorization. This phenomenon is
represented in Fig. 1 by the decrease of absorbance at
595 nm between day-2 and day-5 with a concomitant
increase of absorbance at 500 nm. In the same time, a three
times reduction in the cytotoxicity on Caco-2 cells was
observed. Thereafter, neither colour nor cytotoxicity evo-
lution was observed for three days. After day 8, absorbance
Fig. 1 Biotransformation of ABu62 by P. sanguineus: evolution of
absorbance and cytotoxicity for Caco-2 cells (MTT assays).
% Absorbance: Percentage of maximum absorbance at 595 nm (–•–)
and at 500 nm (––); Percentage of inhibition of Caco-2 mitochon-
drial succinate dehydrogenase activity (–j–)
341
at 500 nm began to decrease concomitantly with a cyto-
toxicity reduction. These results may reflect the fact that
the fungus alters its enzymatic system to adapt to new
substrates.
Laccases were observed in the medium during ABu62
biotransformation (Vanhulle 2007b) and their implication
was shown in several dye decolorizations (Chagas and
Durrant 2001). The cytotoxicity of ABu62 (1.75 mM)-
containing media was hence investigated after 1 h or 24 h-
laccase treatment and compared with the 7-day fungal
treatment. Cytotoxicities of the initial and treated solutions
were evaluated on Caco-2 cells as a function of the initial
dye concentration expressed in % (Fig. 2). The ABu62
concentration inhibiting 50% of the Caco-2 cells mito-
chondrial succinate dehydrogenase activity (IC50) was
about 0.6% of the initial dye concentration, which means
that IC50 was 9.5 lM, under the conditions of this exper-
iment, which is quite similar to the IC50 of phenol in the
same conditions (10 lM). After a 7-day P. sanguineus
treatment, the culture medium dilution responsible for 50%
inhibition of Caco-2 cells viability was 4%. This represents
a significant reduction in cytotoxicity. After 24 h of in vitro
biotransformation by a purified laccase of P. sanguineus,
cytotoxicity reduction reached the level obtained with an
in vivo 7 days biotransformation. This information is quite
important for an industrial wastewater treatment develop-
ment. Actually, laccase treatment could lead to
considerable reduced contact time required to ensure the
same reduction in cytotoxicity as the one obtained with a
whole cell culture.
Fig. 2 Inhibition of Caco-2 cells viability (MTT assays) in the
presence of increasing concentrations of dye. Initial concentration in
ABu62 was 1.75 mM
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Colour and cytotoxicity (on Caco-2 cells and on Vibrio while it is reported as being 13.1 lM in the case of LLC-
fisheri cells) during biotransformation of four azo and PK1 kidney cells and 34.4 lM in an OK kidney cell line
two anthraquinonic dyes (Bondy and Amstrong 1998). Our results on dyes showed
that the use of prokaryotic bacterial model can lead to
As P. sanguineus was identified as an efficient detoxifying opposite interpretations when compared with the results
strain, its bioremediation potential was further evaluated in obtained with the eukaryotic human model, as demon-
the presence of different acid dyes. In this study, two an- strated in the case of ABu113. In addition, the conditions of
thraquinonic dyes (ABu62 and ABu281), a mono-azo the experiment can lead to evaluation of acute, subacute,
(AR266) and three di-azo dyes (AOr116, AR299 and chronic toxicity or genotoxicity. In this study, the LUMIS
ABu113, with similar 3-(4-arylazo-naphtalen-1-ylazo)- test led to the determination of acute toxicity, while the
benzenesulfonic acid moiety) were tested during a period Caco-2 test gave indications on subacute toxicity. All these
of two weeks (Fig. 3, Table 2). studies are important and complementary, each of them
WRF treatment decreased AR266 cytotoxicity against representing only one aspect of a very complex process.
human Caco-2 cells, while an increase of the toxicity was
observed against Vibrio fisheri bacterial cells. On the
contrary, no change in cytotoxicity of ABu113 was Genotoxicity during biotransformation of four azo
observed against Caco-2 cells, while its toxicity against and two anthraquinonic dyes
bacteria was reduced. In the case of AOr116, a very low
decolorization was observed, however the dye was effi- Since it was shown that some bacteria bioactivate dyes into
ciently detoxified according to both cytotoxicity models. carcinogenic compounds (Cerniglia et al. 1982 a, b), we
AR299 was decolorized, while an increase of the cyto- decided to investigate the genotoxicity through VITO-
toxicity was observed on both bacterial and human cells. TOX1 test (Taylor et al. 2004) during dyes
The two anthraquinonic dyes (ABu62 and ABu281) were biotransformation (Table 2). This short term assay (4 h),
efficiently decolorized and detoxified on both cytotoxicity based on SOS response-dependent induction of light pro-
models. duction by genetically modified Salmonella typhimurium
Evaluating the toxicity of wastewater is complicated, bacteria, is less time-consuming, less expensive and easier
since the results greatly depend on the type of organism to handle than the Ames test. Therefore, it was well
(e.g. daphnia, fish, algae, bacteria) or cells (e.g. LLC-PK1 adapted to the objectives of this study. The rec/pr1 values
kidney cells, Caco-2 cells, hepatocytes) selected for the of AR299 were between 0.95 ± 0.05 and 1.25 ± 0.06 (data
study. Different models can lead to significant differences not shown) under the range of concentration studied (0–
in the values obtained. As an example, the IC50 of ochra- 175 lM), indicating no genotoxic effect (Table 2). After
toxin A is 0.4 lM in Caco-2 cells (Berger et al. 2003), its biotransformation, the rec/pr1 value at the highest
concentration reached 1.6 ± 0.4, indicating that mutage-
AR299
nicity appeared during the biotransformation. Upon
ABu62
O NH2 exposure to a xenobiotic, mammalian cells, mainly he-
SO3- patocytes, enterocytes and kidney cells, metabolize most of
N C2H4OH
N
N N them into more suitable water-soluble products to facilitate
N C2H4OH
O HN their excretion by the kidney into the urine or by the liver
SO3-
ABu281
into the bile. In this process, their genotoxic character may
O NH2 be altered by metabolic activity. For this reason, the test
SO3-
AOr116 was coupled with an analysis of the sample after incuba-
O
tion with an exogenous metabolic extract (S9, hepatic rat
O HN
N N extract). In the presence of S9, the rec/pr1 values was
N N
O
O S O SO -
0.9 ± 0.05 after biotransformation of AR299, which indi-
3
O cates that genotoxic effect was no longer observed in
conditions that mimic the liver microsome biotransforma-
AR266 ABu113 tion. None of the other dyes studied presented a mutagenic
NH2
character before or after biotransformation. In earlier car-
N N
N CF3
N N cinogenicity studies, the carcinogenic character appeared
N N H
SO3- OH
SO3-
when azo type dyes were cleaved by bacterial azo-reduc-
SO3-
tases, leading to the formation of aromatic amines (Velarde
Cl
et al. 1999). On the contrary, WRF bioconversion of dyes
Fig. 3 Chemical structures of the dyes studied were shown to be mainly performed by oxidative enzymes

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Table 2 Comparison of the
Dyes % Decolorization
decolorization, detoxification
(Caco-2 cells and bacterial
cells) and genotoxicity of
several dyes after 14 days of AR266 85 ± 4
incubation with P. sanguineus ABu113 91 ± 4
AOr116 7±1
AR299 78 ± 3
ABu62 99 ± 5
ABu281 71 ± 3
such as laccases, LiP, MnP and to produce metabolites with
reduced toxicity compared with the parent dye (Heinfling
et al. 1997). However, testing WRF-treated dyes and
wastewater for potential genotoxic activity seems essential
to ensure the safety of newly developed wastewater treat-
ment technologies.
Conclusion
This study was carried out in order to evaluate the potential
of white rot fungal strains to decolorize and detoxify acid
dyes. The results showed that (i) decolorization by culture
was achieved after two weeks of incubation with a con-
comitant cytotoxicity reduction between 45% and 99%,
depending on the strain used; (ii) mutagenic character
appeared during the biotransformation of one among the
six dyes studied; (iii) there was no link between the
decolorization and the detoxification levels; (iv) one day
in vitro enzymatic treatment with purified laccases of P.
sanguineus resulted in a cytotoxicity reduction comparable
to that obtained in 7 days by a culture.
When developing a coloured wastewater bioremediation
process, decolorization is the easiest parameter to monitor,
and is also the most striking feature in the public opinion.
However, our results clearly demonstrated that more
extensive studies are required to ensure the efficiency of
the process. Indeed, three different methods were applied
to provide information on the toxicity, and their comple-
mentary information were demonstrated. Many other
toxicological issues such as the apparition of endocrine
disruptors may also be relevant. It is suggested that
wastewater treatment efficiency should be evaluated
through complementary toxicological parameters.
Acknowledgements This work was supported by the Directorate
for Technology, Research and Energy of the Walloon Regional
Government of Belgium (BIOVAL 981/3870 and CHAMBOIS) as
well as the European Commission, Sixth Framework Program (SO-
PHIED contract NMP2-CT2004-505899). The authors acknowledge
for their efficient collaboration: V. Mertens (Wetlands Engineering,
Belgium), S. Agathos and D. Wesenberg (Unit of Biological Engi-
neering of the catholic University of Louvain, Belgium), S. Taghavi,
343
% Detoxification Genotoxicity
Caco-2 bacteria  S9 +S9
90 ± 4 25 ± 3  
1±1 17 ± 2  
100 ± 5 15 ± 2  
40 ± 2 37 ± 3 + 
99 ± 5 11 ± 1  
92 ± 4 12 ± 1  
L. Regniers, L. Verschaeve and D. van der Lelie (Vlaamse Instelling
voor Technologische Onderzoek, Belgium). P. Villers, A. Bastiaens,
F. Gouardères and the Musketeers are greatly acknowledged for their
kind and continuous help.
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