ARTICLE IN PRESS

International Biodeterioration & Biodegradation 57 (2006) 16

www.elsevier.com/locate/ibiod

Biodegradation of a model azo disperse dye by the white rot fungus

Pleurotus ostreatus
Xueheng Zhaoa,, Ian R. Hardinb, Huey-Min Hwanga
a

Department of Biology, Jackson State University, Jackson, MS 39217, USA

Department of Textiles, Merchandising, and Interiors, University of Georgia Athens, GA 30602, USA

Received 2 June 2005; accepted 17 October 2005

Available online 5 December 2005

Abstract
Disperse Orange 3, 4-(4-nitrophenylazo)aniline, was chosen as a model to study biodegradation of azo dyes by the white-rot fungus
Pleurotus ostreatus (strain Florida) which was grown in submerged culture under controlled conditions. Degradation was investigated
using a commercial preparation of Disperse Orange 3 that contained 20% dye plus dispersing agents, and an high-performance liquid
chromatography puried preparation of the dye. The metabolites generated by Pleurotus ostreatus were identied as 4-nitroaniline, 4nitrobenzene, 4-nitrophenol, and 4-nitroanisole. Veratryl alcohol, a redox mediator for lignin peroxidase of white-rot fungi, and its
oxidant veratraldehyde were also detected in cultures grown in the presence of Disperse Orange 3. 4-Nitroanisole was the major
metabolite when 4-nitrophenol was incubated with Pleurotus ostreatus. Kinetic proles of these degradation products were determined
and a partial degradation pathway is proposed.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Azo dye; Biodegradation; Products; White rot fungi

1. Introduction
Between 10% and 15% of the total dye consumed in
dyeing processes of textile industries pollutes massive
quantities of the wastewater generated (Jarosz-Wilkolazka
et al., 2002). Dyes in textile wastewater present aesthetic
problems and can pose threats to public health (Chung,
1983; Achwal, 1997), although most liquid and solid
efuents from textile industries are treated before being
discharged into the environment. In wastewater the textile
dyes can be physically or chemically removed by occulation, adsorption, ltration and oxidation. Most of the
physical methods, however, simply accumulate and concentrate dyes and create solid waste, and so the problem of
disposal still exists. Chemical oxidation with either
peroxide or ozone can destroy dyestuffs but this approach
is costly (Robinson et al., 2001a). The possibility of using
white-rot fungi to decolorize wastewater containing dyes
has received much attention because their ligninolytic
Corresponding author. Tel.: +1 601 979 1226; fax: +1 601 979 2778.

E-mail address: xueheng.zhao@jsums.edu (X. Zhao).

0964-8305/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2005.10.008

enzymes have the ability to degrade many recalcitrant

pollutants, including synthetic dyes. Biocatalytic processes
based on white-rot fungi provide alternative methods to
decolorize textile efuents (Swamy and Ramsay, 1999;
Selvam et al., 2003).
The basidiomycete Phanerochaete chrysosporium is one
of the ligninolytic fungi that was extensively used to
degrade dyes (Cripps et al., 1990; Goszczynski et al., 1994;
Spadaro et al., 1992; Wesenberg et al., 2003). This fungus
produces several extracellular ligninolytic enzymes that
have been associated with the degradation of dyes (JaroszWilkolazka et al., 2002; Wesenberg et al., 2003). Other
species of ligninolytic fungi, such as Bjerkandera adusta,
Pleurotus ostreatus and Irpex lacteus also showed potential
for decolorization of various dyes including azo dyes
(Novotny et al., 2001; Robinson et al., 2001b; Zhao, 2004),
however, the biochemical pathways involved in azo dye
degradation and decolorization by white rot fungi are still
unclear (Wesenberg et al., 2003).
Disperse azo dyes, account for about 70% of the total
commercial disperse dyes used in the textile industry
(Zollinger, 1991). Therefore understanding the pathway

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X. Zhao et al. / International Biodeterioration & Biodegradation 57 (2006) 16

NO2

NH2

Fig. 1. Structure of Disperse Orange 3 (C.I. 11005).

by which these dyes are metabolized by white-rot fungi, is

of importance for the application of fungi in the treatment
of textile efuents. In this study, Disperse Orange 3, 4-(4nitrophenylazo)aniline, a monoazo dye, was chosen as a
model compound to investigate the pathways involved in
the metabolism of an azo dye by Pleurotus ostreatus. It has
a simple structure (Fig. 1) that is typical of many disperse
azo dyes.

A set of identical cultures grown under the same conditions was used to
investigate the degradation of the non-puried commercial preparation of
Disperse Orange 3 (20% colorant content) at a concentration of
200 mg l
1 in the cultures. Samples (3 ml) were taken from the culture daily
for 9 days to monitor substrate depletion and metabolite production. The
same amount of liquid medium containing 200 mg l
1 of commercial
disperse dye was added after each sampling to keep a constant volume in
the culture ask. Four replicate asks with the same dye concentration
were used for the study and results are reported as the mean. An equal
volume of methanol was mixed with the culture samples to ensure
complete solubilization prior to measurement (Zhao and Hardin, 2002).
The concentration of Disperse Orange 3 in the growth medium was
monitored spectrophotometrically at its maximum wavelength of absorption (lmax 415 nm). The initial concentration of Disperse Orange 3
corresponded to 100% of the dye. During the fungal degradation of
Disperse Orange 3 metabolites were monitored by HPLC analysis. No
signicant variation (o5%) in concentration was induced by photodegradation, and no degradation products were detected in control
samples.

2.3. GCMS analyses

2. Materials and methods
2.1. Chemicals
Disperse Orange 3 (Color Index No. 11005, 20% colorant content) and
Disperse Orange 3 (90% colorant content) used in this work were
purchased from Aldrich Chemical Co. (Milwaukee, Wis.). The commercial
form of this dye contains dispersing agents and surfactants. Compounds
used as standards: 4-nitroaniline, 4-nitrophenol, 4-nitroanisole, nitrobenzene, veratryl alcohol, veratraldehyde, 1,4-phenylenediamine, 1,4-dinitrobenzene, 4-nitrocatechol, 2-amino-5-nitrophenol, aniline, 2-nitroaniline,
and 3-nitroaniline were analytical grade reagents and were obtained from
Aldrich. Acetonitrile (Aldrich) and methanol (EMD Chemicals, Gibbstown, NJ) used in high-performance liquid chromatography (HPLC)
analysis and sample preparation were of HPLC grade. Phosphoric acid
(85%), potassium dihydrogen phosphate and sodium hydroxide pellets
were analytical grade (J.T.Baker, Phillipsburg NJ). All other chemicals
used throughout this study were reagent-grade chemicals. Puried water
was obtained from an ion exchange and membrane ltration system from
US Filter (Warrendale, PA).

2.2. Culture conditions and biodegradation assays

Pleurotus ostreatus (strain Florida) was provided by K.-E. Eriksson
(University of Georgia, USA) and was maintained on malt agar medium
and subcultured every month as described previously (Zhao and Hardin,
2002). A 10-mm agar plug taken from a fungal colony growing on a malt
agar plate was used to inoculate 250-ml Erlenmeyer ask containing
125 ml Kirks medium (Kirk et al., 1978) at pH 5.0. The cultures were
incubated for 3 days at 30 1C, and shaken at 200 rpm.
Commercial disperse dyes always contain dispersing agents and
surfactants, so, to eliminate interference from such impurities, technical
grade Disperse Orange 3 (90% content) was further puried by
recrystallization with acetonitrile. The puried product was free of
impurities when examined by HPLC. For the degradation assays, the
puried colorant was nely ground and added to 3-day fungal cultures to
a nal concentration of 80 mg l
1 (w/v). The shaking rate of the cultures
was reduced to 150 rpm. Controls without dyes and/or inoculum were run
under identical conditions. Duplicate cultures were sampled daily for 5
days. At the time of sampling, the entire contents of each culture (120 ml)
was gravity ltered (Fisher P8 lter paper) and the ltrate from each ask
extracted three times with methylene chloride (10 ml). The combined
organic layers, approximately 30 ml, were concentrated to about 1 ml
using a rotary vacuum evaporator (Buchi Analytical, New Castle, DE)
after drying with sodium sulphate. The methylene chloride extract was
analyzed by GCMS.

GCMS was performed using a QP5000 mass spectrometer (Shimadzu)

tted with a GC17A gas chromatograph (Shimadzu). The ionization
voltage was 70 eV. Gas chromatography was conducted in the temperature-programming mode with a Restek column (0.25 mm  30 m; XTI-5).
The initial column temperature was 40 1C for 4 min, then increased linearly
at 10 1C min
1, to 270 1C, and held for 4 min. The temperature of the
injection port was 275 1C and the GC/MS interface was maintained at
300 1C. The helium carrier gas ow rate was 1.0 ml min
1. Degradation
products were identied by comparison of retention time and fragmentation pattern with known reference compounds, as well as with mass
spectra in the NIST spectral library stored in the computer software
(version 1.10 beta, Shimadzu) of the GCMS.

2.4. High-performance liquid chromatography (HPLC)

A Hewlett-Packard 1100 series HPLC system (Hewlett-Packard
GmbH, Germany), consisting of a model G1311A quaternary pump, a
model G1322A degasser and a model G1315A diode array detector, was
used for liquid chromatography. HP ChemStation software (version 3.1)
was used for data processing and reporting. A stainless steel ODS column
5 mm packing (Phenomenex, Ultracarbs, 150 mm  4.6 mm I.D.) and a
RP-C18 guard column was used in the analysis. The volume of each
injection was 100 ml delivered by a model HP1313A automatic injector.
The separation temperature was xed at 25 1C. The mobile phase was a
gradient 0.025 M phosphate buffer (pH 3.0) (A); acetonitrile (B): from 0
to 20 min, B increased linearly from 5% to 25%, then increased to 40% in
the next 10 min, B was kept at 40% for 10 min and then decreased to 5%
in 5 min. The ow rate was 1 ml min
1 and the sample was ltered through
a 0.45 mm membrane lter prior to HPLC analysis.
The identity of products was conrmed by comparison of both
retention time and spectrum with those of standard compounds and was
consistent with the GCMS analysis. For quantication, standards were
prepared by dilution of stock solutions that were prepared in methanol or
water (1 mg ml
1) and were stored at 4 1C, to give concentrations of 0.05,
0.10, 0.25, 0.50, and 1.25 mg ml
1. Calibration curves were constructed by
regressing peak area against concentrations of the standard solutions.
Concentration of products in the samples was calculated using linear
regression equations from the calibration curves. Peak areas based on the
lmax of each compound were used for determination. All calibration
curves were linear (r2 40:999) over the concentration range investigated.

3. Results and discussion

Total ion chromatograms (TIC, Fig. 2) obtained from
GCMS analysis of the degradation products of puried

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X. Zhao et al. / International Biodeterioration & Biodegradation 57 (2006) 16

Concentration of products (m)

5.00

100

4.00

80

3.00

60

2.00

40

1.00

20

% remain of dye

and accounted for about 1015% of total decolorization.

One of the products, 4-nitroaniline (II), reached a
maximum on day 2 and then decreased, indicating that 4nitroaniline was not the nal product in the degradation.
From day 2 onwards, nitrobenzene concentration increased
markedly and linearly for 9 days. Another product, 4nitrophenol, was detected after 4 days along with its
methylated product, 4-nitroanisole. Taking into consideration the absorption of the substrate by the mycelium and
the loss of products during sample transfer, it was
estimated that these four metabolites contributed to
approx. 1520% of the Disperse Orange 3 that was
removed after incubation for 9 days. Other possible
products were sought by extraction of fungal mycelium
and using acid or basic extraction conditions. However, no
other products could be found by these methods.
Oligomeric coupling of azo dye by peroxidases was
reported by Spadaro and Renganathan (1994). A similar
reaction might produce some dimers or oligomers of
Disperse Orange 3 in the fungal transformation, which
could not be identied in this work.

Disperse Orange 3 (I) colorant revealed the presence of

several peaks other than the dye. Mass spectral analysis of
the metabolites produced from the culture of Pleurotus
ostreatus incubated with Disperse Orange 3 for 3 days
allowed the identication of 4-nitroaniline (II), nitrobenzene (III), 4-nitrophenol (IV) and 4-nitroanisole (V) which
are probable metabolites of the dye. Both retention times
and mass spectra of these products matched those of
authentic standards. None of these compounds was
detected in the control samples. 4-Nitroaniline (II) was
detected in a previous investigation when Pleurotus
ostreatus was grown under similar conditions (Zhao and
Hardin, 2002). Veratryl alcohol (VI) and its oxidation
product, veratraldehyde (VII), were also detected on the
GCMS chromatogram, (Fig. 2). The other peaks in the
TIC shown in Fig. 2 were also present in the control
cultures. In order to verify the presence of cell-bound
metabolites, the fungal mycelium was extracted with
methylene chloride. When the supernatant was extracted
with methlyene chloride under acid (pH 2) and basic (pH
13) conditions, no metabolites other than those obtained
when the extraction was carried out under neutral
conditions, were detected.
Owing to the unsatisfactory recovery rate of degradation
products from Disperse Orange 3 by liquidliquid extraction (with methylene chloride), GCMS was not used to
quantify these compounds. Instead, HPLC analysis was
employed to quantify the metabolites because of its
accuracy and simplicity.
HPLC analysis veried that commercial Disperse Orange 3 and puried dye produced identical products during
degradation (data not shown). Therefore, commercial
Disperse Orange 3 (20% colorant content) was used in
the remaining studies.
The kinetic proles of the dye and its four degradation
products during a period of 9 days are shown in Fig. 3.
More than half the added dye was seen to be removed from
the medium during this period by monitoring the
maximum wavelength at 415 nm. Adsorption of dye by
the fungal mycelium contributed in part to this removal

0.00
0

10

Time (days)
4 - Nitroaniline

4 - Nitrobenzene

4 - Nitroanisole

DO3

4 - Nitrophenol

Fig. 3. Partial kinetic proles of some degradation products of Disperse

Orange 3 by HPLC.

4.00E + 06
VI

TIC

3.00E + 06
C

2.00E + 06
C

1.00E + 06

VII

III

II
IV

0.00E + 06
3.00 5.00 7.00 9.00 11.00 13.00 15.00 17.00 19.00 21.00 23.00 25.00 27.00 29.00 31.00
Time (min)
Fig. 2. Total ion chromatogram of the metabolites generated from puried Disperse Orange 3. Peak I, Disperse Orange 3; Peak II, 4-nitroaniline; Peak
III, nitrobenzene; Peak IV, 4-nitrophenol; Peak V, 4-nitroanisole; Peak VI, veratryl alcohol; Peak VII, veratraldehyde; Peaks C, peaks also detected in
control samples. Sample was taken after degradation for 3 days by Pleurotus ostreatus, supernatant extracted by methylene chloride.

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X. Zhao et al. / International Biodeterioration & Biodegradation 57 (2006) 16

1.00E + 07

TIC

IV

8.00E + 06
6.00E + 06
4.00E + 06

0 Day

2.00E + 06
0.00E + 00
3
1.00E + 07

11

13

15

17

19

21

23

25

27

29

31

TIC

8.00E + 06
6.00E + 06

IV

4.00E + 06
2.00E + 06
0.00E + 00
3

5 Days
5

11

13

15

17

19

21

23

25

27

29

31

Time (min)
Fig. 4. Identication of 4-nitroanisole as the major transformation product of 4-nitrophenol by GCMS.

To examine a possible pathway responsible for 4nitroanisole production, a separate experiment was conducted, in which 4-nitrophenol was added to the cultures
and its degradation was examined by GCMS. The only
metabolite identied was 4-nitroanisole (Fig. 4). This
shows that methylation occurs in the Pleurotus ostreatus
culture. Methylation of phenolic compounds by Phanerochaete chrysosporium has also been reported by other
researchers (Valli and Gold, 1991; Valli et al., 1992).
Compared with Phanerochaete chrysosporium, Pleurotus
ostreatus produces laccases instead of LiP as the major
enzyme responsible for the biodegradation of lignin and
textile dyes (Robinson et al., 2001b; Rodriguez et al., 1999).
However, in Kirks medium, Pleurotus ostreatus produced
all three types of essential extracellular enzyme, LiP, MnP,
and laccases (Robinson et al., 2001b). The activity of
laccases was also detected in this study (data not shown
here). Veratryl alcohol (VI), a natural product synthesized
by fungi, is the mediator in the action of LiP. In a previous
investigation (Paszczynski and Crawford, 1991), LiP
compound I oxidized azo dyes and was converted to
compound II, which was then reduced by veratryl alcohol
to complete the catalytic cycle of the enzyme. The pathway
to re-oxidize veratryl alcohol involves two successive oneelectron oxidations to form veratraldehyde (Tien, 1987;
Zapanta and Tien, 1997). Furthermore, Paszczynski and
Crawford (1991) found that lignin peroxidase was only
capable of catalyzing the oxidization of recalcitrant azo
dyes effectively when veratryl alcohol was present.
Although the data are not shown, in this study, it was
found that veratryl alcohol (VI) was detectable in the
culture before nitrobenzene (III), 4-nitrophenol (IV),
and 4-nitroanisole (V). These observations suggest that
veratryl alcohol was involved in the degradation of this
azo dye although more biochemical investigations will

be required to demonstrate its role in the conversion of

azo dye.
A partial degradation pathway is proposed in Fig. 5,
showing the results expected from both reduction and
oxidation of Disperse Orange 3 (I) by Pleurotus ostreatus.
It is presumed that Pleurotus ostreatus peroxidases and
laccases were also involved in the degradation of Disperse
Orange 3 (I) and the methylation of 4-nitrophenol (IV)
observed in this study. Spadaro and Renganathan (1994)
proposed a degradation pathway for another disperse azo
dye, Disperse Yellow 3, by lignin peroxidase suggesting an
oxidation mechanism involving carbonium ion and phenyl
radicals. These results agree with their pathway for
nitrobenzene (III) production. Hydroxylation of aromatic
ring concomitant with cleavage of azo bond (NQN) is
presumed to be responsible for 4-nitrophenol (IV) production.
The 4-nitroaniline (II) detected on the rst day of
degradation does not seem to be generated by an
extracellular lignolytic enzyme. A reduction procedure
rather than an oxidation might be responsible for its
generation through a symmetric breakage of the azo bond.
Rodriguez et al. (1999) suggested that enzymes other than
those of the ligninolytic system were engaged in the dye
decolorization by Pleurotus ostreatus. The formation of 4nitroaniline may also be related to the reduction ability of
this fungus. However, this needs conrmation since no 1,4phenylenediamine, another likely reduction product, was
found in our investigation. This pathway may result from
the presence, in the azo dye, of an amino functional group
which is a weaker electron donor than a hydroxyl group,
and is thus more difcult to oxidize.
Data showed that white-rot fungi degrade the azo dye
Disperse Orange 3 via an oxidative mechanism, which is
different from that in anaerobic bacteria, and aromatic

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X. Zhao et al. / International Biodeterioration & Biodegradation 57 (2006) 16

O2N

NH2

I
Veratryl alcohol
Symmetric azo bond cleavage
Veratraldehyde

LiP
O2N

NH2
II

OH

O2 N

O2N
III

IV
LiP/Laccases

OCH3

O2N
V

Fig. 5. Proposed degradation pathway of Disperse Orange 3 by Pleurotus ostreatus.

amines are not exclusive products. Exactly the same

degradation products were released from Disperse Red 1,
which is structurally similar to Disperse Orange 3, by
cultures of Pleurotus ostreatus (Lu and Hardin, 2005). This
indicates that the degradation pathway of Disperse Orange
3 can be used to assess degradation and predict the
degradation pathway of other azo dyes with similar
structure. Further study of the biodegradation of these
products is continuing in order to understand the complete
degradation pathway of azo dyes by white-rot fungi.
Acknowledgment
Authors appreciate the discussion of results and help
from Professor George L. Baughman at the University of
Georgia.
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