Journal of Biotechnology 89 (2001) 141 145

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Decolorization of textile indigo dye by ligninolytic fungi

Doralice S.L. Balan a,*, Regina T.R. Monteiro b
a

Faculdade de Tecnologia de Americana, FATEC/CEETEPS/UNESP, A6. N.S. Fatima 567, 13478 -540 Americana, SP, Brazil
b
CENA/USP, Caixa Postal 96, 13400 -970 Piracicaba, SP, Brazil
Received 26 June 2000; received in revised form 6 April 2001; accepted 17 April 2001

Abstract
The indigo dye is extensively used by textile industries and is considered a recalcitrant substance, which causes
environmental concern. Chemical products used on textile processing, which affect the environment through effluents,
can be voluminous, colored and varied. Vat textile dyes, like indigo, are often used and dye mainly cellulosic fibers
of cotton. Decolorization of this dye in liquid medium was tested with ligninolytic basidiomycete fungi from Brazil.
Decolorization started in a few hours and after 4 days the removal of dye by Phellinus gil6us culture was in 100%,
by Pleurotus sajor-caju 94%, by Pycnoporus sanguineus 91% and by Phanerochaete chrysosporium 75%. No color
decrease was observed in a sterile control. Thin layer chromatography of fungi culture extracts revealed only one
unknown metabolite of Rf=0.60, as a result of dye degradation. 2001 Published by Elsevier Science B.V.
Keywords: Ligninolytic fungi; Indigo; Textile industries; Decolorization

1. Introduction
Chemical products used on textile process,
which affect the environment through effluents
can be voluminous, colored and varied. Vat textile
dyes, like indigo, are very popular and largely
employed on cellulosic fibers like cotton. Dyes are
colored substances used on several substrates in
food, cosmetics, paper, plastic, and textile industries among others. They are retained on the
substrates by physical adsorption, by making
compounds with metals and salts by mechanical
retaining and solution or by making covalent
* Corresponding author. Fax: + 55-19-4681049.
E-mail addresses: balanitl@uol.com.br (D.S.L. Balan), monteiro@cena.usp.br (R.T.R. Monteiro).

bonds. Synthetic dyes are extensively used. About

10 000 different dyes have an assured future.
World consumption of these dyes for cellulosic
fibers represents 60 000 ton year 1, being 5% of
this amount for indigo (Peter, 1991; Spadaro et
al., 1994).
In the Americana, SP area, there are about 700
textile industries, and few of them are concerned
about treatment of their effluents and most of
them use dyeing or printing processes. Textile
waste drainage uses the canals of the city or the
rivers of Piracicaba, Atibaia and Jaguari which
comprise the river basin that serves the whole
area. Effluents are either treated by a biological
process at the industries or treated together with
the domestic wastewater in the municipal water
treatment center (Sanin, 1997).

0168-1656/01/$ - see front matter 2001 Published by Elsevier Science B.V.

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D.S.L. Balan, R.T.R. Monteiro / Journal of Biotechnology 89 (2001) 141145

In this biological treatment, the effluent of

secondary sludge is partially decolorized, however dyes are not degraded and stay adsorbed
on the biomass and in the sludge from the treatment center (Conchom et al., 1997; Morais et
al., 1999). The sludge turns out to be the colored pollutant agent generated in wet base
about 1 10 ton day 1 in a medium size industry (consumption of 50 m3 water per h).
Decolorization of industrial textile wastewater
can be obtained by ozonization (50 60% of
color reduction); floculation filtration (up to
80% of color removal) and by alkalinization
with calcium hydrossulfite. These methods can
be pre-treatments or can be employed after biological depuration (Ginocchio et al., 1985).
The treatment of wastewater containing dyes
and its decolorization involves serious problems.
A wide range of several pH intervals, salt concentrations and chemical structures very often
add to the complications. Among low cost viable alternatives available for effluent treatment
and decolorization, the biological systems seem
to be the best ones. Nowadays, biological systems are recognized by their capacity to reduce
biochemical oxygen demand and chemical oxygen demand by conventional aerobic biodegradation. But there is a problem with its inability
to remove color (ONeill et al., 2000).
Although the decolorization is a challenge for
textile industry as well for wastewater treatment
systems the literature suggests that there is a
great potential for developing microbiological
decolorization systems with total color removal,
in some cases within few hours (Balan, 1999;
Balan and Monteiro, 2000).
Colored compounds are just a small fraction
of wastewater organic content and their color
make them aesthetically unacceptable.
The employment of microorganisms to decontaminate polluted places by bioremediation has
increased in the last years.
Fungi from the basidiomycete group, known
as wood white-rot fungi, have been indicated as
capable of degrading several pollutants of diverse structures by a complex ligninolytic enzymatic system. Aromatic hydrocarbons, pesticides

organochlorates, explosives, dyes and others are

effectively degraded to carbon dioxide (Barr and
Aust, 1997; Young and Yu, 1997).
Several dyes have been studied and the ability
of white-rot fungi to decolorize them indicates
that these organisms can be used for wastewater
treatment in dye-employing industries (Shin et
al., 1997). A well-known enzymatic system for
lignin degradation includes peroxidases, Mn dependent peroxidases (Mnps), and oxidases occurs in Phanerochaete chrysosporium, Pleurotus
ostreatus, Trametes hirsuta and other basidiomycete.
Vat dyes, where the main groups are indigoids
and antraquinone, are applied to cellulosic fibers
like leuco-soluble salts, after reduction in an alcaline bath; following fiber exhaustion they are
reoxidized to the ceto-insoluble form.
The utilization of ligninolytic basidiomycete in
biotechnological processes for the environmental
area has recently been emphasized aiming at the
recovery of soils contaminated by xenobiotics
and another application of great emphasis is related to pharmacological properties.
Our investigation has the purpose to verify
the decolorization potential of some basidiomycete fungi native from Brazil upon the
textile indigo dye.

2. Materials and methods

2.1. Dye
The synthetic liquid indigo-blue dye (CI Vat
Blue I), 96% pure, chemical formula C12H8O2N2
and molecular structure (Fig. 1).

Fig. 1. Chemical structure of CI Vat Blue I.

D.S.L. Balan, R.T.R. Monteiro / Journal of Biotechnology 89 (2001) 141145

2.2. Microorganisms
Fungi used were Phellinus gil6us (CCB 254), Ph.
chrysosporium (CCB 539), Pycnoporus sanguineus
(Morr) (CCB 458) and Pleurotus sajor-caju (CCB
020). The fungal cultures were received as gifts
from culture collection of botanical garden of Sa o
Paulo. They were on malt extract agar and stored
at 4 C.

2.3. Culti6ating conditions

Fungi were grown for 7 days in plates with malt
extract. Discs of 0.7 mm containing mycelium and
spores (0.6 mg dry weight) were removed from
borders of colonies, then added to Erlenmayer
flasks (250 ml) with 60 ml of minimum medium
MM (Pontecorvo et al., 1997) with the dye as the
unique source of Carbon, in 0.02% v/v
concentration.
The dye was diluted in sterile water and added
to the flasks, right after they were inoculated with
three discs of agar with isolate fungi.
The flasks were incubated in the dark, in static
culture, at a temperature of 2530 C for 4 days.

2.4. Decolorization
After the addition of dye, at intervals of 24 h, 1
ml of extracellular media was removed from the
flasks and diluted ten times in distilled water.
Absorbance readings in spectrophotometer were
taken at wavelengths 580 and 680 nm which
represents the maximum length of indigo absorption spectrum.

2.5. Thin layer chromatography

The mycelium was separated from the medium
by centrifugation at 6000 g for 20 min, after
incubation in MM for 4 days.
The dye was extracted with chloroform and
concentrated to 0.5 ml in evaporator (model Heidolpheh WV 2000) and then transferred to vials
of 2 ml and kept in a freezer (18 C) for
conservation and later analysis.
Extracts obtained were analyzed qualitatively in
regards to dye biodegradability, by thin layer

143

chromatography on plates of silica gel 60 F254

(Merck).
The automatic applicator CarmagLinomat IV
was used for application in plates. With the assistance of a micro syringe 100 ml of centrifuged
mycelium extract and of supernatants of cultures
grown in the presence or absence of dye and 25l
ml of indigo dye were placed in the points of
application. The solvent systems used were benzene/ethanol/chloroform (94:4:1) according to
Gogna et al. (1992).
After development of plates up to a neighboring of 15 cm, they were dried in the air and stains
observed under short and long wave ultra violet
light.
In order to evaluate the relation between the
solvent front and the distance that test substances
ran (Rf), the Rf values were determined by
Touchstone and Dobbins (1978).
3. Results and discussion
In the first 24 h, a visible decolorization of the
medium occurred with a decrease in the absorption values at 580 and 680 nm. No decolorization
was presented by the sterile control (Table 1).
The decolorization was expected due to high
capacity of dye absorption by mycelium of fungi
as well as the reduction of dye intensity in solution because of changes caused by them.
One quantity of indigo was removed by absorption to mycelium, which dyed them of blue. The
decolorization of mycelium occurred and new
mycelial mass did not present color indicating the
absorption of dye and the degradation of chromophore group. Considering that the dye was
used as unique source of carbon in medium of
culture it served as a nutritional sub-extract for
fungi; the increase of biomass was observed and
409 2 mg of dry weight was found, after 14 days
of growth for all fungi.
According to Table 1, the biggest color removal
occurred in the first 24 h, and the measurements
were followed for 4 days.
The percentage of dye removal from liquid
medium shows that the best decolorization activity was presented after 4 days by Phellinus gil6us
with a total color reduction (100%).

D.S.L. Balan, R.T.R. Monteiro / Journal of Biotechnology 89 (2001) 141145

144

Table 1
Decolorization on liquid medium for the wavelengths 580 and 680 nm, during the incubation period of 4 days
Organism treatment

1 day (%)

2 days (%)

3 days (%)

4 days (%)

Phellinus gil6us
580 nm
680 nm

64
70

88
88

92
91

100
100

Ph. chrysosporium
580 nm
680 nm

35
75

47
51

60
68

70
75

Py. sanguineus
580 nm
680 nm

75
73

81
83

86
87

90
91

Pl. sajor-caju
580 nm
680 nm
Controla

70
74
0

87
87
0

90
91
0

94
94
0

Culture medium without inoculum.

The basidiomycete Ph. chrysosporium, with numerous references and well known ligninolytic
system, presented a reduction of 70 75%; Py.
sanguineus 91% and Pl. sajor-caju 94%.
The decrease of the values by absorbance allowed the perception of significant spectrum
changes. These data show that decolorization was
caused by degradation of dye. These observations
agree with those from classical studies of decolorization like Glenn and Gold (1983). They concluded that decolorization of several polymeric
dyes by Ph. chrysosporium in liquid medium is a
process of secondary metabolism and the degradation system of lignin or part of it is responsible
for decolorization. While the adsorption of
mycelium to fungus and changes caused by it
reduce the quantity of dye in solution, adsorption
measurements were taken in two wavelengths.
The proportional decrease in adsorption values
indicates adsorption. When there are many
changes in adsorption rates in two wavelengths
the selected dye is being degraded. The polymeric
nature of dyes assures that initial decolorization
phases, at least, are extra cellular.
Laccase, lignin peroxides, manganese peroxidase and H2O2 dependent peroxidases are functional extracellular enzymes by fungi in
biodegradation of lignin and dyes (Arora and
Gill, 2000). Some white rot fungi produce all these

enzymes while others produce only one or two of

them.
Laccase is an important ligninolytic enzyme
and has great biotechnological potential. Laccases
are a diverse group of multi-copper proteins that
catalyze the oxidation of a variety of aromatic
compounds, including dyes.
Qualitative determination of laccase and peroxides production by the strain Phellinus gil6us
(CCB 254), Py. sanguineus (CCB 458) and Pl.
sajor-caju (CCB 020) was reported by Okino
(1996). The laccase activity in these strains, began
on the first day of cultivation, however Ph.
chrysosporium began only after three days of cultivation. These results are in accordance with decolorization data presented here (Table 1).
These results showed that good color removal
occurred on the first day, however the measurements taken on the fourth day presented maximum decolorization by Phellinus gil6us, Py.
sanguineus and Pl. sajor-caju, and these are extremely practical, because this is the time for
textile effluent retention in conventional stations
of biological treatment.
As indigo was used as a unique source of
carbon available to microorganisms in this assay,
our results can indicate the possibility that indigo
undergoes a biodegradation process, being a nutritional sub-extract for fungi. The ligninolytic

D.S.L. Balan, R.T.R. Monteiro / Journal of Biotechnology 89 (2001) 141145

cultures of several white-rot fungi have been

documented by their capacity to degrade and
decolorize several dyes. However, it is expected
that fungi differ in this ability based on their
qualitative and quantitative differences for enzymatic production.
Degradation was also demonstrated by thin
layer chromatography of supernatants and
mycelium extract of decolorization assay. The
dye did not have any mobility, staying in the
place of application, without migrating on the
plate, presenting Rf= 0. Only one unknown
metabolite, Rf= 0.60 was detected in the extracts where indigo was present, being detected
in all mycelium, and also in all supernatants.
This metabolite was not detected in mycelium
grown without dye presence and did not have
color in the visible spectrum; it is maybe a subproduct of indigo degradation that does not
have chromophore units but present the structure benzene rings, maybe detectable in long ultra violet light.
Very few reports are available on the
biodegradation products or intermediates of indigo dyes. The degradation of indigo by laccases
produces isatin (indole-2,3 dione) which was further degraded to anthranilic acid (2-aminobenzoic acid) by HPL analysis.
According to conditions above described, we
can state that Phellinus gil6us, Ph. chrysosporium, Py. sanguineus and Pl. sajor-caju will contribute to the decolorization and degradation of
effluents containing indigo dye and their action
will reduce the pollutant discharge and toxicity
related to the presence of dyes. Different potentials for degradation among several basidiomycetes tested can suggest a possible
cooperation among them in treatment systems as
a manner to accelerate or increase the level of
color removal in industrial effluents.

References

Acknowledgements
FUNDUNESP proj. 540/94; FAPESP proj.
98/09769/3; FATEC Americana.

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