Journal of Biotechnology 101 (2003) 49 /56

www.elsevier.com/locate/jbiotec

The toxicity of textile reactive azo dyes after hydrolysis and

decolourisation
Anna Gottlieb a, Chris Shaw b, Alan Smith a, Andrew Wheatley b,
Stephen Forsythe a,*
a

Department of Life Sciences, The Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK
Department of Civil Engineering, Loughborough University of Technology, Loughborough LE11 3TU, UK

Received 11 December 2001; received in revised form 10 September 2002; accepted 24 September 2002

Abstract
The toxicity of C.I. Reactive Black 5 and three Procion dyes, as found in textile effluents, was determined using the
bioluminescent bacterium Vibrio fischeri . Hydrolysed Reactive Black had a slightly greater toxicity than the parent
form (EC50 11.49/3.68 and 27.59/4.01 mg l
1, respectively). A baffled bioreactor with anaerobic and aerobic
compartments was used to decolourise hydrolysed Reactive Black 5 in a synthetic effluent. Decolourisation of
hydrolysed Reactive Black resulted in an increased toxicity (EC50 0.29/0.03 mg l
1). Toxicity was not detectable when
decolourised Reactive Black 5 was metabolised under aerobic conditions. No genotoxicity was detected after the
decolourisation of either the parent or the hydrolysed reactive dyes, either in vitro or in the bioreactor. The toxicity and
genotoxicity of decolourised C.I. Acid Orange 7 was due to the production of 1-amino-2-naphthol (EC50 0.19/0.03 mg
l
1).
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Textile; Azo; Dyes; Decolourisation; Toxicity; Genotoxicity

1. Introduction
Reactive azo dyes occur in textile dyehouse
wastewater in concentrations ranging from 5 to
1500 mg l
1 due to their poor fixation to fabrics
(Pierce, 1994). Moreover, reactive azo dyes are not
degraded by conventional aerobic sewage treatment plants as they are resistant to biological

* Corresponding author. Tel.: /44-115-848-3529; fax: /44115-848-6636.

E-mail address: stephen.forsythe@ntu.ac.uk (S. Forsythe).

oxidative degradation (Easton, 1995). Decolourisation can be achieved using either anaerobic
digestion, microbial generation of oxygen radicals,
i.e. laccase activity of white-rot fungi (Schliephake
et al., 2000) or expensive physico-chemical treatments (ONeill et al., 2000). Although reactive
textile dyes can be decolourised under anaerobic
conditions due to reduction of the azo bond, the
resultant aromatic amines resist further degradation and may be toxic or genotoxic (Sweeney et al.,
1994). Toxicity could be eliminated through bacterial fission of the aromatic ring structure, a
process that requires oxygen. Therefore, secondary

0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
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A. Gottlieb et al. / Journal of Biotechnology 101 (2003) 49 /56

aerobic treatment can be used to detoxify dyes

(Seshadri et al., 1994).
Previous workers have studied the parent form
of textile reactive dyes. However, this may have
different chemical and biological properties to the
hydrolysed form which is the actual form found in
dyehouse effluents. This paper reports the toxicity
(Hao et al., 1996) of various azo dyes (parent and
hydrolysed forms) following anaerobic decolourisation using pure bacterial cultures, and a mixed
microbial consortium in a laboratory scale baffled
reactor.

2. Materials and methods

2.1. Textile and non-textile azo dyes
Industrial grade textile dyes C.I. Reactive Black
5, Procion Yellow H-EXL, Procion Navy H-EXL
and Procion Crimson H-EXL were provided by
Welbeck Ltd UK. Hydrolysed forms of the textile
dyes were prepared by refluxing for 3 h after
adjustment to pH 11 with 1 M sodium hydroxide
and were neutralised using 1 M hydrochloric acid
before testing. C.I. Acid Orange 7, C.I. Food
Yellow 3 (Sunset Yellow FCF) and naphthol
compounds were purchased from Sigma Ltd.

were harvested by centrifugation (10 000/g for 20

min) in the exponential phase and concentrated in
potassium phosphate buffer (100 mM, pH 7) to a
final cell density of 5 mg cell wet weight per ml.
Bacterial decolourisation was achieved by incubating the dyes (0.1 ml, 100 mg l
1) with bacteria (0.3
ml) and glucose (0.1 ml, 1% w/v) in potassium
phosphate buffer (4.5 ml, 100 mM, pH 7) at
37 8C. The incubation period was 10 min for
non-reactive dyes and 60 min for Reactive Black 5.
The absorption spectra of decolourised samples
were measured over the range 200 /700 nm (scan
speed 4 nm s
1, 1 cm path length) using a Unicam
SP1800 Ultraviolet Spectrophotometer against a
reference blank of sample matrix. All samples (1
ml) were centrifuged in a microfuge at 11 600/g
for 5 min before analysis. The requirement for
reducing equivalents for decolourisation activity
was demonstrated by the replacement of glucose
from the assay with additional potassium phosphate buffer. Where necessary bacterial cultures
and reactions were incubated in a Compact
anaerobic cabinet (Don Whitley Scientific Ltd.,
UK). Chemical decolourisation was achieved by
the addition of 6 or 8 mg sodium dithionite to 10
ml hydrolysed and parent Reactive Black (500 mg
l
1 in phosphate buffer), respectively.

2.2. Dye decolourisation assays

2.3. Toxicity assessment

Two bacterial strains were used for the decolourisation assays Enterococcus faecalis as previously used by Sweeney et al. (1994) and
Clostridium butyricum which was isolated from
the effluent stream of a textile dyehouse. Both
organisms had initially been screened for azo dye
decolourisation (as opposed to adsorption) by
plating onto tryptone soya agar plates and Schaedler anaerobe agar plates, respectively, containing
1% (w/v) Reactive Black 5 followed by anaerobic
incubation at 37 8C for 2 days. Decolourisation of
the agar without colony colouration indicated
azoreductase activity which was confirmed using
the following quantitative assay.
E. faecalis and C. butyricum were grown
anaerobically in tryptone soya broth and Schaedler anaerobe broth at 37 8C, respectively. They

Toxicity to the bioluminescent organism Vibrio

fischeri was assayed using the Merck ToxAlert
100 (Merck Eurolab, UK) according to BS EN
ISO 11348:1998. All samples were serially diluted
in 2% w/v NaCl, and each assay was performed in
duplicate at pH 7.0, 15 8C. Sodium chloride (2%
w/v) was used as the negative control. The sample
concentration that inhibited 50% of the light
output after a 5 min exposure period (EC50) was
automatically determined using the WINTOX software supplied with ToxAlert 100. Where appropriate the EC50 value refers to the concentration
(mg l
1) of the sample under test. However, for
bioreactor samples where the toxic component was
unidentified, the units are expressed as percent
dilution of original sample. Colour correction was
according to the manufacturers instructions.

A. Gottlieb et al. / Journal of Biotechnology 101 (2003) 49 /56

2.4. Genotoxicity assessment

Genotoxicity was assayed according to Forsythe
(1990). The percentage survival of the repair
deficient mutant Escherichia coli strain CM871
(uvrA recA lexA ) to the wild type WP2 was
measured using the Rapid Automated Bacterial
Impedance Technique (Don Whitley Scientific
Ltd). All decolourised and bioreactor samples
were clarified by centrifugation (10 000/g for 20
min) and each assay was performed in triplicate.
Saline (0.85% w/v NaCl) was used as the negative
control. This technique expresses genotoxicity
values as a ratio (Coefficient of Survival, CS) of
the DNA repair deficient E. coli strain viability
compared with the repair proficient wild type
strain. A compound giving a CS value of B/0.3
was deemed a positive genotoxin, 0.3 /0.75 was
weakly positive, and /0.75 was negative (Sweeney et al., 1994).
2.5. Laboratory scale baffled reactors
Two laboratory scale bioreactors (total volume
10 l) were constructed so that specified terminal
compartments could be aerated (0.4 l air per min)
as necessary (Shaw et al., 2002). Each compartment was divided into a downflow section (10 mm
wide) by a hanging baffle, and an upflow section
(35 mm) wide by a standing baffle. The tips of the
hanging baffles were angled at 458 to direct the
flow to the centre of the upflow section. Additional compartments (1 l) were added when
necessary. The bioreactors were run at 379/1 8C
as continuous flow systems. The bioreactors were
fed a synthetic feed which represented a typical
textile dyehouse effluent (mg l
1); 2670 starch, 400
polyvinyl alcohol, 270 carboxymethyl cellulose,
500 hydrolysed Reactive Black 5, 26 700 NaCl,
1000 Na2CO3 and 110 NaOH, pH 9.0. They were
seeded using anaerobic granules from a full scale
upflow anaerobic sludge bioreactor treating potato wastewater, which had not previously been
exposed to azo dyes. The two bioreactors were run
in parallel; one being fed the synthetic feed but
without the hydrolysed textile dye as a control.
The bioreactors were run with a hydraulic retention time of 6 h per cell in three phases (i) day 1/

51

76, eight anaerobic cells (ii) day 77 /154, seven

anaerobic cells and three aerobic cells (iii) day
155/204, seven anaerobic cells and three aerobic
cells plus the addition of activated sludge (volatile
solids concentration 3.05 g l
1) from a works
treating combined sewage and textile wastes.
Colour removal was measured at the lmax (597
nm) for hydrolysed Reactive Black 5 after filtration through a 0.45 mm polyvinylidene fluoride
membrane filter.

3. Results and discussion

3.1. Relationship of azo dye toxicity and structure
The structures of the azo dyes C.I. Food Yellow
and C.I. Acid Orange 7 showing their constituent
aromatic amines are illustrated in Fig. 1. Both
compounds generate sulphanilic acid following
azo bond reduction (decolourisation), but different
amino-naphthols. No decolourisation was recorded in incubation periods up to 2 h if glucose
was omitted from the assay. Hence glucose was
required in the decolourisation assays as a source
of reducing equivalents (Sweeney et al., 1994). The
toxicity of Acid Orange 7 and Food Yellow were
similar before reduction (Table 1). However, after
azo bond reduction by E. faecalis (10 min incubation) the toxicity of Food Yellow slightly decreased (EC50 22.19/2.47 and 32.59/1.53 mg l
1)
but the toxicity of Acid Orange 7 increased nearly
100-fold (EC50 15.79/2.68 and 0.29/0.0 mg l
1,
respectively). Standard 1-amino-2-naphthol was
very toxic (EC50 0.19/0.03 mg l
1) compared
with its sulphonated analogue (1-amino-2naphthol-6-sulphonate, EC50 3919/21.92 mg
l
1). The toxicity of sulphanilic acid was equivalent to that of the unreduced dyes. Hence the
increased toxicity of Acid Orange 7 after reduction
was probably due to the liberation of 1-amino-2naphthol. The lack of a sulphonate group probably increases its lipid solubility and hence uptake
by the V. fischeri tester strain. The toxicity of
naphthol compounds varied according to the type
and position of their substitution groups (Table 1).
For example naphthalene sulphonic acid was less
toxic than when the sulphonic group occurs in the

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A. Gottlieb et al. / Journal of Biotechnology 101 (2003) 49 /56

Fig. 1. The structure of azo dyes and related compounds used in this study. The underlined region of Reactive Black 5 indicates the
portion of the vinyl sulphone group altered in the hydrolysed dye form, with OSO3
replaced by OH
.

A. Gottlieb et al. / Journal of Biotechnology 101 (2003) 49 /56

Table 1
Toxicity and genotoxicity of non-textile azo dyes and related
naphthol compounds
Compound

Azo dyes
Acid Orange 7
Acid Orange 7 (E. faecalis )d
Food Yellow
Food Yellow (E. faecalis )

EC50
(mg l
1)a

15.7
0.2
22.1
32.5

(2.68)c
(0.0)
(2.47)
(1.53)

CS valueb

1.1 (0.04)
0.04 (0.05)
0.9 (0.07)
0.4 (0.18)

53

2-naphthol-6-sulphonate with 1-amino-2-naphthol

confirmed that sulphonation reduces genotoxicity,
which is most likely to be due to a decrease in
membrane permeability (Rosenkranz and Klopman, 1989).
3.2. Toxicity and genotoxicity of Reactive Black
and Procion textile dyes

EC50 refers to the concentration of compound (mg l
1)

required to inhibit the light output of V. fischeri by 50% after a
5 min exposure period.
b
CS value is the ratio of DNA-repair proficient to DNArepair deficient E. coli cells surviving exposure to the test
compound. Values less than 0.3 indicate genotoxicity, values
0.3 /0.7 indicate weak genotoxicity. All values are quoted for
100 mg l
1 of compound.
c
Number in parenthesis indicates S.D. values.
d
Decolourisation achieved using anaerobic incubation with
E. faecalis .

The structures of the reactive textile azo dye

Reactive Black 5, diamino H-acid and the vinyl
sulphone groups are given in Fig. 1. The structures
of the related Procion dyes are proprietary.
Bacterial decolourisation of the dyes, as opposed
to adsorption, was demonstrated by the loss of
colour in the visible region and the increase in UV
adsorption (see Fig. 2). Hydrolysis of Reactive
Black 5, as would occur during the dying process,
resulted in a slightly more toxic compound (EC50
11.49/3.68 compared with 27.59/4.01 mg l
1,
respectively, Table 2). The toxicity of Reactive
Black 5 (parent and hydrolysed forms) increased
after decolourisation with the bacteria E. faecalis
and C. butyricum under anaerobic conditions and
chemically with sodium dithionite. For example E.
faecalis reduced parent Reactive Black 5 toxicity
(EC50 mg l
1) was 0.79/0.09 compared with
unreduced dye at 27.59/4.01 (Table 2). Glucose

1? position than the 2? position (29.19/2.46 and

8.39/0.23 mg l
1, respectively).
The genotoxicity of the naphthol compounds
was compared at 100 mg l
1 using the E. coli
differential lethality test (Table 1). Acid Orange 7
and Food Yellow were non-genotoxic (CS value
1.1 and 0.9, respectively). Azo fission of Acid
Orange 7 (by anaerobic incubation with E. faecalis ) resulted in the generation of genotoxic compound(s). The genotoxicity of reduced Acid
Orange 7, as with V . fischeri toxicity, was probably due to the production of 1-amino-2-naphthol
since this compound was very genotoxic (CS value
B/0.01). The decolourisation of Food Yellow also
decreased the CS value to 0.4 which is deemed as
weakly genotoxic. Sulphanilic acid and the other
naphthol compounds tested did not show any
genotoxicity. Comparing the results for 1-amino-

Fig. 2. Absorbance spectra of Reactive Black 5 after anaerobic

incubation with E. faecalis .

Predicted azo fission products and other naphthol compounds

Sulphanilic acid
21.5 (6.72)
0.8 (0.06)
1-Amino-2-naphthol
0.1 (0.03) B/0.01
1-Amino-2-naphthol-6-sulpho391.0 (21.92)
1.0 (0.03)
nate
1-Naphthalene sulphonic acid
29.1 (2.46)
1.2 (0.8)
2-Naphthalene sulphonic acid
8.3 (0.23)
1.3 (0.3)
4-Amino-1-naphthalene sulpho28.3 (3.82)
0.8 (0.25)
nic acid
H-acid
48.6 (9.03)
1.2 (0.15)
a

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A. Gottlieb et al. / Journal of Biotechnology 101 (2003) 49 /56

Table 2
Toxicity and genotoxicity of textile dyes
Compound

EC50 (mg l
1)a

Parent

Azo dyes
Reactive Black 5
Reactive Black 5 (E. faecalis )d
Reactive Black 5 (C. butyricum )
Reactive Black 5 (sodium dithionite)
Procion Navy
Procion Navy (E. faecalis )
Procion Navy (C. butyricum )
Procion Yellow
Procion Yellow (E. faecalis )
Procion Yellow (C. butyricum )
Procion Crimson
Procion Crimson (E. faecalis )
Procion Crimson (C. butyricum )

27.5
0.7
2.2
4.9
18.9
0.5
9.1
71.0
0.8
9.1
34.7
19.9
8.1

(4.01)c
(0.09)
(0.71)
(1.45)
(5.65)
(0.17)
(0.53)
(6.00)
(0.17)
(1.04)
(0.27)
(0.10)
(0.66)

CS valueb
Hydrolysede

Parent

Hydrolysed

11.4
0.2
0.3
0.2
27.9
0.5
8.6
66.4
0.5
5.6
37.7
18.2
9.1

1.5
1.4
0.6
0.6
/
/
/
/
/
/
/
/
/

0.7
1.3
0.8
0.6
/
/
/
/
/
/
/
/
/

(3.68)
(0.03)
(0.01)
(0.09)
(3.28)
(0.07)
(1.19)
(0.93)
(0.28)
(0.89)
(1.72)
(2.3)
(1.04)

(0.33)
(0.16)
(0.26)
(0.37)

(0.05)
(0.27)
(0.34)
(0.23)

No toxicity detected at highest concentration (given) tested.

a
EC50 refers to the concentration of compound (mg l
1) required to inhibit the light output of V. fischeri by 50% after a 5 min
exposure period.
b
CS value is the ratio of DNA-repair proficient to DNA-repair deficient E. coli cells surviving exposure to the test compound.
Values less than 0.3 indicate genotoxicity, values 0.3 /0.7 indicate weak genotoxicity. All values are quoted for 100 mg l
1 of
compound.
c
Number in parenthesis indicates S.D. values.
d
Decolourisation achieved using either anaerobic incubation with E. faecalis , C. butyricum or chemical reduction using sodium
dithionite.
e
Parent dye hydrolysed by fluxing for 3 h at pH 11, followed by neutralisation.

was required by both organisms as a source of

reducing equivalents (i.e. NADH and FADH) for
the reduction of the azo bonds (/N /N/). The
omission of glucose from the assay resulted in the
absence of decolourisation even after prolonged
incubation periods (18 h). The products from the
reduced hydrolysed form were more toxic than
those from the parent form. For example the
toxicity of sodium dithionite reduced Reactive
Black 5 was EC50 4.99/1.45 and 0.29/0.09 mg
l
1 for the parent and hydrolysed forms, respectively (Table 2).
Parent Reactive Black 5 was not genotoxic
whereas the hydrolysed form was weakly genotoxic (Table 2). Some genotoxicity was apparent
after decolourisation of parent Reactive Black 5 by
C. butyricum and sodium dithionite, but not by
reduction with E. faecalis . The structurally related
compound H-acid (commercial product) was not
genotoxic (Table 1). It is plausible that any

genotoxic potential of the reduced dye was due

to the vinyl sulphone side chain, however, it is also
possible that different methods of dye reduction
produce different end products, and further work
is required to resolve this.
Decolourisation of the proprietary textile dyes
(Procion Navy, Procion Yellow and Procion
Crimson) increased toxicity though the extent
differed according to bacterial strain used (Table
2). Reduction with C. butyricum conferred similar
toxicity (EC50 9.19/0.53, 9.19/1.04 and 8.19/0.66
mg l
1, respectively). Reduction with E. faecalis ,
however, conferred similar toxicity with Procion
Navy and Procion Yellow (EC50 0.59/0.17 and
0.89/0.17 mg l
1) but Procion Crimson was less
toxic (EC50 19.99/0.10 mg l
1). This suggests that
different methods of dye reduction produced
different end products from the same dye. The
toxicity of hydrolysed Procion Navy was less than
the parent form, whereas the toxicity of the two

A. Gottlieb et al. / Journal of Biotechnology 101 (2003) 49 /56

forms of Procion Crimson were similar and

Procion Yellow showed increased toxicity in the
hydrolysed form, as per Reactive Black 5. Therefore, there is no common trend in the affect of
hydrolysis on reactive azo dyes. Although the
structures of the Procion dyes have not been
published, it is plausible that the toxicity of the
end products will depend on substituted naphthol
compounds similar to those previously studied
(Table 1).

3.3. Toxicity and genotoxicity of hydrolysed

Reactive Black 5 in baffled bioreactors
Laboratory scale baffled bioreactors were designed to decolourise a synthetic textile dyehouse
wastewater containing hydrolysed Reactive Black
5, and were run for 205 days. During this period
the bioreactors were operated under anaerobic and
anaerobic /aerobic phases. The bioreactors
achieved an average of 84.1 /87.6% colour removal
throughout the period of operation. Decolourisation, as opposed to adsorption to biomass, was
evident by changes in the absorption spectra
(increase in UV absorbance) already reported in
Shaw et al. (2002). The toxicity of the effluent
from the bioreactors is shown in Fig. 3. When dye
was omitted from the synthetic textile wastewater
it was non-toxic at the highest concentration (50%
dilution) that was possible to test, whilst the

55

addition of hydrolysed Reactive Black 5 (500 mg

l
1) resulted in a detectable toxicity (EC50 5.09/
1.22%). Effluent collected from the control bioreactor (no added dye) was toxic (EC50 7.09/4.5%)
during anaerobic operation (Part A, Fig. 3) but
this toxicity was removed when an aeration phase
was included (Parts B and C, Fig. 3). This effect
was most likely due to the aerobic removal of
hydrogen sulphide which had formed by microbial
metabolism under anaerobic conditions. Synthetic
wastewater containing hydrolysed Reactive Black
5 was toxic when decolourised during anaerobic
operation (EC50 0.59/0.17%, Part A, Fig. 3). This
toxicity decreased slightly during anaerobic /aerobic operation, and reduced further after the
addition of an acclimated activated sludge to the
aerobic stage (Parts B and C, Fig. 3). Hence the
decolourisation of hydrolysed Reactive Black 5 in
the baffled bioreactors resulted in the production
of toxic end products, such as aromatic amines
and vinyl sulphone, which could be further degraded under aerobic conditions. This finding
agrees with the work of ONeill et al. (2000) who
demonstrated that anaerobic decolourisation of a
synthetic wastewater containing a hydrolysed
reactive dye in an upflow anaerobic sludge blanket
bioreactor produced a toxic effluent, and that
subsequent passage of the effluent through an
aerobic bioreactor eliminated the toxicity. Alternative means of removing toxicity via oxidative

Fig. 3. Toxicity of effluents from the laboratory scale baffled reactor for three consecutive periods; day 1 /76 anaerobic operation (A),
day 77 /154 anaerobic /aerobic operation without additional biomass (B), day 155 /205, anaerobic /aerobic operation with additional
biomass (C). -"-, Control, synthetic wastewater without hydrolysed Reactive Black 5; -j-, synthetic wastewater with hydrolysed
Reactive Black 5 (500 mg l
1).

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A. Gottlieb et al. / Journal of Biotechnology 101 (2003) 49 /56

processes may be achievable using white-rot fungi

(Verma and Madamwar, 2002) but this has yet to
be achieved on a large scale. There was no
significant genotoxicity in either the synthetic
wastewater or the bioreactor effluents under any
of the conditions tested, which suggests that either
the mechanisms utilised to decolourise the dye did
not result in the formation of genotoxic endproducts or that any end-products were further
degraded within the bioreactor.

Acknowledgements
The authors wish to express their gratitude to
the EPSRC (GR/L54738/01) for funding of this
project, Vanessa Kennerley and Cynthia Carliell
for their assistance and Dr Michael RaynerBrandes (Merck Speciality Chemicals Ltd) for his
constructive criticism of the V. fischeri work.

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