Journal of Environmental Protection, 2010, 1, 85-94
doi:10.4236/jep.2010.12011 Published Online June 2010 (http://www.SciRP.org/journal/jep)
Sustainable Discoloration of Textile Chromo-Baths
by Spent Mushroom Substrate from the Industrial
Cultivation of Pleurotus ostreatus
Simona Di Gregorio1, Francesco Balestri1, Maria Basile2, Valentina Matteini2, Francesco Gini1,
Stefania Giansanti2, Maria Grazia Tozzi1, Riccardo Basosi2, Roberto Lorenzi1
1Department of Biology, University of Pisa, Pisa, Italy; 2Department of Chemistry, University of Siena, Siena, Italy.
Received January 24th, 2010; revised April 11th, 2010; accepted April 12th, 2010.
ABSTRACT
Synthetic dyes are recalcitrant to degradation and toxic to different organisms. Physical-chemical treatments of textile
wastewaters are not sustainable in terms of costs. Biological treatments can be more convenient and the lig-
nin-degrading extracellular enzymatic battery of basidiomycetes are capable to discolor synthetic dyes. Many basidio-
mycetes are edible mushrooms whose industrial production generates significant amount of spent mushroom substrate
(SMS) with residual high levels of lignin-degrading extracellular enzymatic activities. We have demonstrated that the
low cost organic substrate, the SMS deriving from the cultivation of the basidiomycetes Pleurotus ostreatus, is able to
discolor anthraquinonic, diazo and monoazo-dyes when incubated in dying chromo-reactive and chromo-acid baths
containing surfactants and anti-foams, where the concentrations of the different dyes are exceeding the one recovered
in the corresponding wastewaters. Laccase was the lignin-degrading extracellular enzyme involved in the discolouring
process. The exploitation of the low cost SMS in the treatment of textile wastewaters is proposed. Accordingly, a toxi-
cological assessment, based on a cyto-toxicity test on a human amnion epithelial cell line (WISH) and the estimation of
the germination index (GI%) of Lactuca sativa, Cucumis sativus and Sorghum bicolor, has been performed, showing
the loss of toxicity of the chromo-baths after being discoloured by the SMS.
Keywords: Anthraquinonic Dye, Diazo Dye, Laccase, Monoazo Dye, White Rot Fungi, Spent Mushroom Substrate
1. Introduction
Synthetic dyes find application in different industrial
divisions including textile industry. An annual con-
sumption of about 0.7 million tons of synthetic dyes
has been reported [1]. These compounds result to be
recalcitrant to degradation and toxic for higher animals
[2] and surface aquifers [3]. Textile industry alone ac-
counts for two-thirds of the total dyestuff market. Ac-
cordingly, discoloration of textile wastewaters is one of
the major environmental concerns since last decades.
Physical-chemical treatments such as coagulation/adsor-
ption, electrolysis or ozonation are sometime unsuc-
cessful or very expensive and frequently producing
large amounts of toxic wastes [4]. At the same time, the
strong electron-withdrawing groups characterizing the
chemical structure of dyes protect them by the attack of
bacterial oxygenases [5], affecting the treatment of
textile wastewaters by conventional activated sludge
plants. On the other hand, anaerobic digestions of
various dyes produce toxic amines [6], which make the
processes not recommended, unless combined with
subsequent aerobic treatments to oxidize the toxic in-
termediates [7].
In this scenario, the already reported capacities of
fungi [8,9] with respect to aerobic oxidation of synthetic
azo-dyes [10,11] anthraquinones [12], and phthalo-
cyanins [13], result to be of extreme interest. Ligni-
nolytic basidiomycetes causing white rot on wood, were
shown to be the most promising fungi because of their
capacity to produce a complex array of lignin-degrading
extra-cellular enzymes, with very low substrate specific-
ity towards xenobiotics [9]. The ligninolytic battery of
extracellular enzymes of the basidiomycetes P. ostreatus
has been described as capable to transform a very broad
spectrum of waste substrates [14-16] including textile
dyes [17]. However, it is worth to mention that P.
ostreatus and many other basidiomycetes are edible

Sustainable Discoloration of Textile Chromo-Baths by Spent Mushroom Substrate from the Industrial Cultivation of Pleurotus ostreatus
86
mushrooms and their industrial cultivation produces a
significant amount of spent mushroom substrate (SMS),
reported as harboring high levels of residual oxidative
enzymatic activity [18]. Actually, an average of 5 kg of
SMS, are produced for 1 kg of mushrooms on the market,
and the P. ostreatus production has increased over 500%
in the last ten years, ranking the second or the third posi-
tion in the context of edible mushroom industrial produc-
tion in the world [19]. Thus, SMS would be a low cost
source of ligninolytic enzyme [20-22]. However, the
costs of purification of the latter can still negatively in-
fluence the exploitation of the spent matrix in pollutants
biodegradation. As far as we know, with the exception of
the description of potential of the spent mushroom com-
post of Pleurotus pulmonarius in synthetic pentachlo-
phenol contaminated waters [23], no report on the poten-
tial of the SMS from the cultivation of edible mushrooms
as an oxidising matrix for the treatment of industrial ef-
fluents has been reported.
The aim of this work was the recover and direct ex-
ploitation of the low cost organic substrate, the SMS
from the industrial production of P. ostreatus, to dis-
colour complex chromo-baths used in textile industry.
The feasibility of the process has been verified incu-
bating the SMS directly in reactive and acid chromo-
baths, containing anthraquinonic, mono and diazo dyes,
as single dyes or as a mix. In addition to dyes, the
chromo-baths contained surfactants and anti-foams that
are used as auxiliaries in the colouring process and
normally released in textile wastewaters. Although
usually not specifically investigated in terms of disap-
pearance and generally used as biodegradable ingredi-
ents, surfactants and anti-foams might interfere with
the discolouring process or contribute to the toxicity of
the textile effluents. The progressive discolouration of
the different complex chromo-baths by the SMS were
recorded in parallel with the evaluation of the loss of
the here recorded toxicity. This latter has been moni-
tored by a cyto-test on a human amnion epithelial cell
line (WISH) and the estimation of the germination in-
dex (GI%) of Lactuca sativa, Cucumis sativus and
Sorghum bicolor in presence of untreated and discol-
oured chromo-baths. The ligninolytic enzymes poten-
tially involved in the discolouring and detoxifying
process have been monitored.
2. Materials and Methods
2.1 Chemicals, SMS, Seeds and Epithelial Cell Line
Analytical grade chemicals were purchased from Sigma
Aldrich (Milan, Italy). The chromo-reactive and chromo-
acid baths were provided by manufacturers, chemical
components are reported in Table 1. The real structure of
the different dyes and auxiliaries were not disclosed by
the manufactures, that categorized the different chromo-
baths in relation to the general classification of an-
traquinonic, mono and diazo-group. Surfactants (Setavin
group) and anti-foams (Kolassol group) were respec-
tively classified as alkylamine ethoxylated and silicon
based chemicals. The SMS of Pleurotus ostreatus was
obtained by a local mushroom farm. The human amnion
epithelial cell line (WISH) was kindly provided by Prof.
Francesco Sgarrella, University of Sassari (Italy). Plant
seeds were obtained from the USDA-ARS North Central
Regional Plant Introduction Station, Iowa State Univer-
sity, Ames, IA, USA.
2.2 Discoloration of the Chromo-Baths by the SMS
The capacity of the SMS to discolor the chromo-baths
was tested at 21 2◦C, in static and dynamic conditions
(orbital shaking at 250 rpm) in sterile 2 L glass flasks
incubated with plugs of SMS (0.3 cm thick), visually
homogenously colonized by the fungal mycelium. The
Table 1. Chemical composition of the chromo-baths
Bath name Dye name g/l Auxiliaries
Name g/l % vol/vol λmax
Kollasol
LO-BD
4
Black Reactive
mix (BRM)
Yellow 39
5-10% wt/vol
(monoazo)
Red 136
47-52% wt/vol
(monoazo)
Black DM 5594 43%
wt/vol (disazo)
110
Setavin RE
80%
Acetic acid
20
4
590
Kollasol
LO-BD
5
Setavin RE 20
Blue Reactive
Bath
(BRaB)
Blue 3G
(anthraquinonic)
11
80%
Acetic acid 1.5
609
Setavin MSN 7.5
Blue Acid
bath
(BAaB)
Blue BLI
(anthraquinonic)
11
NH4SO4 30
637
and
595
Setavin MSN 7.5
Acid
red
(RAmaB)
Red FGN
(monoazo)
11
NH4SO4 30
505

Sustainable Discoloration of Textile Chromo-Baths by Spent Mushroom Substrate from the Industrial Cultivation of Pleurotus ostreatus 87
SMS was collected for a total amount of 130 g of fresh
weight and submerged in the glass flask containing 500
mL of the coloring bath diluted in 150 mM NaCl solution
in water. The Black chromo-Reactive Mix (BRM) was
10 and 5-fold diluted, the Blue chromo-Reactive an-
thraquinonic Bath (BRaB), the Blue chromo-Acid an-
thraquinonic Bath (BAaB) and the Red chromo-Acid
monoazo Bath (RAmaB) were 5-fold diluted. Two flasks
(one set) were prepared for each dilution. One set of
flasks was not inoculated with SMS, the other was in-
oculated with SMS previously autoclaved at 121◦C, 1 atm
for 20 minutes, respectively control sets for abiotic dis-
coloration and for adsorption of the dye onto the SMS. A
further set of flasks was prepared in sterile 150 mM NaCl
solution in water to evaluate the contribution of the re-
lease of pigmentation from SMS to the increase in the
spectral absorbance of flask supernatants in the visible
range. At each time-point, three volumes of supernatant
of each flask were collected and analyzed for UV-Vis
absorption between 400-800 nm (Beckman Instruments,
Fullerton, CA, USA). All the samples at time zero were
diluted to give an absorbance < 1 and the same dilution
has been used for samples corresponding to the succes-
sive time points. The 150 mM NaCl solution served as
blank. Percentage of discoloration with time of incuba-
tion was quantified as the decrease of areas under the
absorbance spectra of flask supernatants at the different
time-points of analysis and calculated against a baseline
defined by the absorbance of supernatants from flasks
without chromo-bath addition, at the corresponding time
of analysis. The areas under the absorbance spectra were
calculated by using PeakFit, Systat Software Inc., San
Jose, California, USA. Decrease of the area under the
spectra of supernatants deriving from flasks inoculated
with autoclaved SMS with respect to the areas under
spectra of supernatants from flask not inoculated with
SMS was interpreted as percentage of adsorption of the
dye onto SMS.
2.3 Enzymatic Activity
Ligninolytic oxidative capacity related to laccase (EC
1.10.3.2), manganese peroxidase (EC1.11.1.13), lignin
peroxidase (EC1.11.1.14) and versatile peroxidase
(EC1.11.1.16) activity were quantified, by a spectropho-
tometer based method, in sample volumes of each flask
supernatant collected in triplicate. Enzymatic activities
were calculated as specific activity. Total proteins were
determined according to [24], using bovine serum albu-
min as standard. All enzyme assays were performed at 37
± 0.5◦C. Linearity with time and protein concentration
was observed for all enzyme activities assayed. Laccase
activity was determined as described by [25]. Manganese
peroxidase and versatile peroxidase activity were deter-
mined as described by [26]. Lignin peroxidase activity
was measured as described by [27].
Enzymatic activities were detected before chromo-bath
amendments, at the moment of the amendment of
chromo-baths and at the end of the discoloring process.
Laccase stability at different pH of incubation was
monitored on 20 µl of control flask supernatant (no
chromo-baths amended) incubated in different buffers
and quantified using standard conditions at the time zero,
after 3 and 24 hours of incubation. Laccase stability at
different temperature was monitored on 50 µL of control
flask supernatant incubated for 15’ at different tempera-
ture. The effect of ionic strength on laccase activity was
monitored in presence of increasing amounts of NaCl in
the laccase mixture assay.
2.4 Isolation of P. ostreatus Mycelium
The mycelium of P. ostreatus has been aseptically col-
lected from the fungal fruiting body, streaked on a sterile
potato dextrose agar plate and incubated for 1 week at
28◦C in the dark. The mycelium was maintained, through
periodic transfer at 4◦C on potato dextrose agar plates in
the presence of 0.5% yeast extract. Laccase activity has
been monitored in liquid cultures prepared incubating 3
plugs (1 cm diameter) of the aseptically collected myce-
lium, grown on the maintaining agar plates, in shaking
flask (125 rpm/min) containing 250 ml potato dextrose
(24 g/L) broth with 0.5% yeast extract at 28◦C in the dark.
At successive times, 1 mL of supernatant was collected
in triplicate and analyzed for laccase activity.
2.5 Native Protein Gel Electrophoresis in
Gradient of Polyacrylamide
A 5-30% polyacrylamide gradient gel was performed at
an alkaline pH under non-denaturing conditions. The
separating gel contained a gradient of acrylamide from 5
to 30%, while the stacking gel contained 4% of acryla-
mide. The electrode reservoir solution was 25 mM
Tris-190 mM glycine (pH 8.4). A total of 60 ng of pro-
teins deriving from the supernatants of 1) P. ostreatus
mycelium grown in potato dextrose broth; 2) SMS in-
cubated in control flasks; 3) SMS incubated in 5 fold
diluted blue chromo-reactive bath (BRaB), were loaded
on the gel that has been stained for laccase activity us-
ing 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS) as substrate. Sample supernatants were col-
lected at the time of maximum laccase activity for 1).
2.6 Seed Germination Test
The germination indexes (GI%) of Lactuca sativa, Cu-
cumis sativus, Sorghum bicolor seeds in presence of ali-
quots of undiluted different flask supernatants before and

Sustainable Discoloration of Textile Chromo-Baths by Spent Mushroom Substrate from the Industrial Cultivation of Pleurotus ostreatus
88
after the treatment with the P. ostreatus SMS were com-
pared. Seed germination tests were performed in sterile
Petri dish plates containing Whatman N.1 ashless filter
imbibed with 10 ml of 1) control solution, 150 mM NaCl
in water; flask supernatants 2) before and 3) after the
SMS incubation. The plates were kept for 120 hrs in the
dark at 25 ± 1◦C. The germination indexes were calcu-
lated from the number of germinated seeds and the cor-
responding root length values, according to the formula:
GI% = (Gs × Ls) / (Gc × Lc) × 100, where Gs was the
mean number of germinated seeds and Ls the mean root
length of germinated seeds in the sample; Gc was the
mean number of germinated seeds and Lc the mean root
length of the germinated seed in the control. The analy-
ses were done in triplicate in plates containing 15 seeds
for each set.
2.7 Cytotoxic Test
The established WISH cell line derived from human am-
nion epithelium has been routinely grown in RPMI me-
dium with 10% FBS and 2% antibiotics at 37 ± 0.5◦C in a
humidified 5% CO2/95% air atmosphere. The experi-
ments were performed on 35 mm plates containing ap-
proximately 1.000.000 of cells in RPMI complete me-
dium maintained for 24 hours in the absence (control) or
presence of volumes of the different flask supernatants
corresponding to the dilutions of the chromo-baths used
for the SMS discoloration. After incubation, plates were
evaluated for the number of adhering cells to plates and
for the trypan blue method [28] to evaluate the acute
toxicity of the chromo-baths before and after discolora-
tion with SMS.
2.8 Statistical Treatment of Data
The analysis of variance of the data (ANOVA), has been
performed by using GraphPad InStat version 3.00 for
Windows 95, GraphPad Software, San Diego California
USA, to evaluate the effects of different parameters of
incubation on the discolouring process. The means of the
significantly different main effects were compared by the
Duncan’s test at the 5% level using the Statistic program
(Statsoft Inc., 1997).
3. Results
3.1 Discoloration of the Chromo-Baths
In order to evaluate the potential of P. ostreatus SMS to
discolor the black chromo-reactive mix (BRM), two dif-
ferent dilutions of the latter were incubated in presence
of the SMS. The incubation of the 10-fold diluted BRM
was operated both in static and dynamic conditions. Dis-
coloration of 10-fold diluted BRM, matched with a visi-
ble effect to the naked eye. The 97% of discoloration
occurred in 24 hrs (Figure 1(a)). The process has been
observed only in presence of not autoclaved SMS and in
dynamic incubation. In static condition a maximum of
8% of discoloration has been recorded.
Due to the high percentage of discoloration observed in
dynamic incubation with the 10-fold dilution, a 5-fold di-
lution of BRM has been inoculated with the SMS only in
dynamic incubation, because of the evidence that the oxy-
genation was mandatory for discoloration. After 24 hrs,
the 65% of discoloration has been observed; the 80% was
recorded after 110 hrs of incubation (Figure 1(b)).
Figure 1. Panel (a) discoloration by the SMS of the 10-fold
BRM, in static (grey bar) and dynamic incubation (black
bar); Panel (b) discoloration of the 5-fold diluted BRM
(black bar) and BRaB (dashed bar) in dynamic incubation;
Panel (c) discoloration of 5-fold diluted BAaB, (grey bar)
and RAmaB (dashed bar) in dynamic incubation

Sustainable Discoloration of Textile Chromo-Baths by Spent Mushroom Substrate from the Industrial Cultivation of Pleurotus ostreatus
89
3.2 Enzymatic Activities and Native Gel ouring process (Table 2). An increase of laccase specific
activity either in presence or absence of chromo-baths
has been recorded during the discolouring process (Table
2). No enzymatic activities have been detected in the
supernatants of autoclaved SMS.
Electrophoresis
Enzymatic activities of lignin-degrading enzymes, measu-
red before the addition of the chromo-baths to the dis-
coloring sets of flasks, revealed that the lignin degrading
battery of enzymes was limited to laccase. In fact no lig-
nin peroxidase activity has been detected and the re-
corded laccase activity was predominant on the manga-
nese and versatile peroxidase (0.294 ± 0.65 U/mg for
laccase; 0.039 ± 0.03 U/mg and 0.034 ± 0.017 U/mg for
manganese and versatile peroxidase respectively). The
same enzymatic activity profile was recorded after the
chromo-baths amendment and at the end of the discol-
The laccase recovered from the supernatant of SMS
incubated in the 5-fold diluted BRaB has been collected
to be compared by native gel electrophoresis to 1) the
laccase recovered from the set of flasks of control; 2) the
supernatant of actively growing mycelium of P. osteauts
in rich medium. Results obtained showed a very similar
profile for the laccases recovered from the three super-
natants (Figure 2(d)).
Table 2. Laccase activity recovered in the supernatants of the set of flasks of control (C) and the different discolouring flasks
Laccase
activity U/mg C flasks BRM 10-fold diluted
BRM
5-fold diluted
BRaB
5-fold diluted
BAaB
5-fold diluted
RAmaB
5-fold diluted
T0 0.267 ± 0.042 0.276 ± 0.008 0.363 ± 0.040 0.267 ± 0.004 0.248 ± 0.007 0.334 ± 0.002
TE 0.396*** ± 0.020 0.328* ± 0.028 0.581*** ± 0.057 0.346** ± 0.007 0.393*** ± 0.005 0.387* ± 0.010
T0, time of chromo-baths amendment; TE, end of the discoloring process. Statistical significance of difference recorded for laccase activity at the end
vs. the beginning of the discoloring processes: ***p < 0.0001; **p < 0.001; *p < 0.05. BRM, black reactive mix; BRaB, blu reactive bath; BAaB,
blue acid bath, RAmaB red acid bath.
Figure 2. Residual laccase activity assayed in standard condition after exposure at different pH as function of time (panel a),
after exposure for 30 min at different temperature (panel b) or assayed at different ionic strength (panel c). The laccase activ-
ity was measured on supernatant of SMS of set of flasks of control. Panel a: citrate buffer, pH 3 (◊), acetate buffer, pH 4 (■),
glycine buffer, pH 10 (Δ), tris buffer, pH 8 (●), acetate buffer, pH 5 (▲), sodium phosphate buffer, pH 6 (□), sodium phos-
phate buffer, pH 7 (♦). Statistical significance *** p < 0.0001; ** p < 0.001; * p < 0.05. Panel d shows the zymogram of laccase
isoenzymes derived from the supernatant of: lane 1, mycelium of P. ostreatus actively growing in potato dextrose broth; lane
, SMS in control flasks, 150 mM NaCl in water, lane 3, SMS incubated in 5-fold diluted BRaB 2

Sustainable Discoloration of Textile Chromo-Baths by Spent Mushroom Substrate from the Industrial Cultivation of Pleurotus Osteatus 90
The stability of the laccase recovered from the set of
flasks of control has been monitored with respect to pH
and temperature, furthermore the effect exerted by ionic
strength has been evaluated (Figures 2(a)-(c)). The en-
zyme, whose optimum pH was between 4 and 5 (results
not shown), showed a significant stability in a range of
pHs between 5 and 10 and after 30 min of incubation up
to 60◦C. The laccase activity resulted to be 50% inhibited
by 200 mM NaCl.
3.3 Germination and Cytotoxicity Test
As a result of the germination tests, all the chromo-baths
were extremely toxic because of the significant inhibition
of seed germination that was nearly absent in the case of
incubation in chromo-reactive baths for all the three plant
species analyzed (Figures 3(a)-(d)). Acid baths were
also extremely toxic (GI% < 50%), even though a certain
percentage of germination has been observed (20%) with
the exception of Lactuca sativa that was strongly inhibited
in germination by the Red chromo-acid bath (Figures
3(a)-(d)). The discoloration of all the chromo-baths de-
termined the loss of toxicity of the discolored baths with
a recover of the GI% to values (nearly 100%) communi-
cated for the selected plant seeds by the USDA-ARS
North Central Regional Plant Introduction Station (Fig-
ures 3(a)-(d)).
A human amnion epithelial cell line (WISH) has been
used to determine the effect exerted by the chromo-baths
on the cell viability. The WISH cells subjected to incuba-
tion with chromo-baths showed a significant decrease of
the number of adhering (healthy) cells (Figure 3(e)) and
a significant increase of the percentage of dead cells
(stained by trypan blue) (Figure 3(f)) compared to con-
trol cells incubated in PBS 5-fold diluted in RPMI me-
dium (control medium). The discoloration of the chromo-
baths by SMS incubation removed their toxic effect: the
number of adhering cells and the percentage of dead cells,
were not significantly different as compared to the con-
trol (Figures 3(e)-(f)).
4. Discussion
Both azo and anthraquinonic dyes are associated to a
high ecotoxicity and high recalcitrance to discoloration
[12,29]. Our results demonstrated that, in aerated (oxida-
tive) conditions, a low cost organic substrate, the SMS
from P. ostreatus cultivation, discolored industrial
chromo-baths containing either monoazo, diazo and an-
thraquinonic dyes, either as single dyes or as a mix of
them. Being interested in SMS exploitation for discol-
ouring textile wastewaters, the capacity of the organic
substrate has been tested on industrial chromo-baths,
instead of aqueous solution of single synthetic dyes. In
fact, the formers have a higher similarity to the real in-
dustrial wastewaters, since containing auxiliaries of the
dying process (mainly surfactants, anti-foams and salts),
that actually are also released in real wastewaters and
potentially can interfere with the discolouring process.
The dilution of the chromo-baths, here tested for discol-
oration, contained the different chemicals at concentra-
tions definitely higher than the one recovered in the real
wastewaters. In fact, they contained dyes and auxiliaries
in concentration up to the 20% of the initial chromo-bath
mass used in the dying process, while it is estimated that
the amount of textile dyes released in wastewaters during
the industrial process accounts for the 10% of the total
used [30]. Thus, the SMS was capable to discolour dif-
ferent chromo-baths at similar or higher concentrations
than the one recovered in real textile wastewaters. The
efficiency of the SMS in the described process was dif-
ferent in relation to the different chromo-baths. Reactive
baths resulted more recalcitrant than acid one, even
though a comparison is either questionable because of
the difference in chemical components of the different
chromo-baths, and difficult since the real structures of
the different dyes and chromo-baths auxiliaries, indicat-
ing their putative recalcitrance to biodegradation, were
not provided by the manufacturers. However, in 24 hrs
the discoloration of every chromo-baths tested accounted
for the 70-90% of the total. Abiotic discoloration and
adsorption were excluded by the persistence of colours in
sets of flasks not incubated with the SMS and in sets of
flasks where every biological activity has been elimi-
nated by autoclaving. Thus, the occurrence of a biotic
process has been explored monitoring the ligninolytic
activity of fungal extracellular enzymes, eventually asso-
ciated to the grow of P. ostreatus on the SMS. Laccase
resulted to be either the only enzymatic activity measur-
able from the incubation of the SMS in a saline solution
and in presence of the different chromo-baths and the
only enzymatic activity recovered during the time inter-
val corresponding to the discoloration process. Actually
during discoloration, laccase activity increased either in
presence or absence of chromo-baths. Results obtained
indicated the ligninolytic laccase as the enzymatic activ-
ity involved in the discolouring process. The hypothesis
was confirmed by the nearly absence of discoloration in
static condition, because in accordance with the known
mechanism of catalytic reaction of the enzyme that uses
molecular oxygen as electron donor to catalyze reactions
consequently occurring in oxidative (aerated) condi-
tions. The laccase recovered in the supernatant of dis-
colouring baths showed peculiar traits. Laccases from
basidiomycetes are generally considered acidic enzymes
not active at basic pH, characteristic actually not con-
venient for the exploitation of the enzyme in the treat-
ment of textile wastewaters, which are essentially asso-
ciated to neutral up to basic pH. However, the laccase
produced by P. ostreatus growing on the SMS, although
displaying an acidic optimum of pH, showed activity and
igh stability at neutral to strong basic pH. A second h

Sustainable Discoloration of Textile Chromo-Baths by Spent Mushroom Substrate from the Industrial Cultivation of Pleurotus Osteatus 91
Figure 3. Effect of the 5-fold diluted BRM (panel a), 5-fold diluted BRaB (panel b), BAaB (panel c) and RAmaB (panel d) on
the germination Index (GI%) and on the cell viability (panel e and f) before and after (+SMS) the discolouring process. Lc:
Lactuca sativa; Cs: Cucumis sativus; Sb: Sorghum bicolour. Statistical significance of germination index of seeds treated vs.
seeds with no SMS treatment: ***p < 0.0001. Panel e, indicates the number of adhering cells, panel f, the percentage of dead
cells evaluated by the trypan blue staining technique. Statistical significance of cell viability vs. cells with no chromo-bath
ddition (C: control): ***p < 0.0001; **p < 0.001; *p < 0.05; ns, not significant a

Sustainable Discoloration of Textile Chromo-Baths by Spent Mushroom Substrate from the Industrial Cultivation of Pleurotus ostreatus
92
interesting trait of the here investigated enzyme was re-
lated to the capacity to catalyse discoloration at high
ionic strength. Also this characteristic results to be of
interest because of the nature of textile wastewaters that
are often associated to high ionic strengths. In fact, the
laccase from SMS discoloured acid chromo-baths in in-
cubation media containing two different salts, NaCl 150
mM and NH4SO4 227 mM. The enzyme resulted to be
50% inhibited by NaCl 200 mM, but the discoloration of
acid baths occurred in only 24 hrs. The enzyme showed
also a significant stability with respect to the temperature
of incubation, and all the characteristics previously de-
scribed make the laccase from P. ostreatus SMS ex-
tremely attractive for industrial application, suggesting
the exploitation of the SMS not only as a low cost sub-
strate with oxidative capacity, but also as a source of
robust laccase. In this context, the induction of laccase
production could be advantageous for enzyme-purifying
purpose, even though, in the experimental condition here
explored, an effect of induction of the chromo-baths on
the ligninolytic battery of enzymes eventually associated
to the SMS was absent. Actually dyes induction of oxi-
dative enzyme, comprising laccase, has been observed in
fungi [31]. However, the process can not be defined a
generic effect of dyes on fungal metabolisms [32]. On
the other hand, results obtained comparing the isoforms
of laccases from different supernatants by native elec-
trophoresis showed that P. ostreatus was able to produce
the enzyme (with similar profile) in very different growth
conditions, spanning from the one related to the presence
of an easily available carbon source (rich broth where the
fungus grows in axenic culture), to growth condition with
a more complex carbon source, eventually not sustaining
the production of fruiting body and in presence of
chromo-baths (onto SMS). Laccases from basidiomy-
cetes have been described as capable to quite efficiently
decolorize azo-dyes and, in relation to anthraquinonic
dyes, the enzymes are reported as more efficient than
other oxidases [33]. However, as far as we know, this is
the first report on putative laccase capacity to discolour
azo and anthraquinonic dyes in complex mixtures of
chemicals as chromo-baths, containing high concentra-
tions of different surfactants, anti-foams and salt. In rela-
tion to the experimental condition adopted, the extremely
versatile laccase oxidative capacity here observed, can be
interpreted as related to the co-presence of the enzyme
and eventually of chemical redox mediators deriving
from the ligninolytic activity of the basidiomycetes. Me-
diators act as electron transfer between the enzyme and
very different substrates characterised by high redox state
[34,35]. They can also accelerate oxidative processes in
terms of kinetics of reactions involved [36,37]. Actually,
intermediates of the process of lignin degradation, de-
fined as natural mediators, are reported as the best one to
use for dye discoloration by laccase [38]. With reference
to the here described discolouring incubations, it is rea-
sonable to assume that the ligninolytic activity of P.
ostreatus, growing on a lignin containing substrate as the
SMS, released these or analogous intermediates in the dis-
colouring supernatants. Thus, the occurrence of natural
mediators might be the reason of the peculiar capacity of
the P. ostreatus SMS to discolour a plethora of different
chromo-baths, with different chemical compositions, by
the quite efficient kinetics of reactions here observed.
Moreover, as a matter of fact, the Annex I of the Dan-
gerous Substances Directive in the European Union (EU)
describe some dye as toxic or mutagenic and others as
skin sensitizers for consumers [Council Directive 67/
548/EEC on the approximation of laws, regulations, and
administrative provisions relating to the classification,
packaging, and labeling of dangerous substances. Offi-
cial Journal of the European Communities, June 27, 1967;
Vol. 196, p 1.]. At the same time, some discolouring
process are associated to the production of toxic interme-
diates of dyes oxidation process [4,6]. Thus, any ap-
proach for the treatment of textile wastewaters must be
focused either on discolouring and detoxifying processes.
Accordingly, the opportunity to exploit the SMS from
P. ostreatus in the treatment of textile wastewaters has
been verified by the toxicological assessment of the
process, based on a cyto-toxicity test on a human amnion
epithelial cell line (WISH) and a phyto-toxicity test on
germinating seeds of Lactuca sativa, Cucumis sativus
and Sorghum bicolor. The results obtained showed a
strong toxicity of the chromo-baths related either to the
cyto-toxicity exerted on cultured human amnion epithe-
lial cells (WISH) and to the phyto-toxicity exerted on
seed of different plant species. The discoloration of
chromo-baths by the SMS was associated to the loss of
toxicity either on WISH cells and plant seeds.
In conclusion, the SMS from P. ostreatus resulted to
be capable to either discolour and detoxify a plethora of
different chromo-baths containing complex mixtures of
chemicals with recognised toxic effect for the environ-
ment. The versatility of the low cost organic substrate is
related at least to the laccase activity deriving from P.
ostreatus, even though other elements favouring oxida-
tion cannot be excluded. All in all, the development of
processes of treatment of textile wastewaters based on
the exploitation of the substrate, either as a source of
robust enzymes or as a versatile low cost organic sub-
strate with oxidative capacity, results to be sustainable in
terms of costs and eventually profitable for the design of
an integrated management of the disposal of the SMS as
an organic waste.
5. Acknowledgements
This work was supported by grants from Assessorato
all’Istruzione, Formazione e Lavoro, Regione Toscana
(Project MECHOS, POR Ob3 2000/2006 Toscana, Pro-
getti integrati di ricerca Mis. D4) and from MIUR

Sustainable Discoloration of Textile Chromo-Baths by Spent Mushroom Substrate from the Industrial Cultivation of Pleurotus ostreatus 93
(Progetti di Rilevante Interesse Nazionale, PRIN 2007 to
RB).
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Abbreviations:
SMS, Spent Mushroom Substrate;
BRM, Black chromo-Reactive Mix;
BRaB, Blue chromo-Reactive anthraquinonic Bath;
BAaB, Blue chromo-Acid anthraquinonic Bath;
RAmaB, Red chromo-Acid monoazo Bath;
ABTS, 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid.