Microbial decolourization of textile waste water N.F. Alia,*, R.S.R. El-Mohamedyb aDyeing and Printing Department, National Research Center, Dokki,… [601968]

ORIGINAL ARTICLE
Microbial decolourization of textile waste water
N.F. Alia,*, R.S.R. El-Mohamedyb
aDyeing and Printing Department, National Research Center, Dokki, Giza, Egypt
bPlant Pathology Department, National Research Center, Dokki, Giza, Egypt
Received 24 November 2010; accepted 30 November 2010
Available online 15 December 2010
KEYWORDS
Fungi;
Decolourization;Waste water;
Reactive dyes;
Acid dyesAbstract Six fungal isolates belong to Aspergillus niger, Penicillium spp., and Pleurotus osstreatus
were used for decolourization activities of some acid and reactive dyes, after they screened for opti-
mum efficiency and the condition for temperature and pH were optimized The results obtained indi-cated that Pleurotus osstreatus andAspergillus niger 1 and 2 are more efficient than Penicillium spp.,
with the two kinds of dyes used. The results also revealed that, the maximum degradation activitiesof these isolates for acid dyes was at pH 5 after 9 days incubation period and at pH 5-6, for reactivedyes. Simulated and actual waste water samples were used in the experiments. Fungi decolouriza-tion of synthetic dyes according to their life state group 1: (living cells) to biodegradation and bio-
sorb dyes. The major mechanism is biodegradation because they can produce the lignin modifying
enzymes, lactose, manganese peroxides (Lip) to mineralize synthetic lignin of dyes. Group 2: deadcells (Fungal biomass) to adsorb dyes.
ă 2011 King Saud University. Production and hosting by Elsevier B.V.
1. Introduction
The textile industry is one of the industries that generate a high
volume of waste water and creates potential for water pollu-
tion. Among the many chemicals in textile waste water, dyesare considered as important pollutants ( Huantian and Ian,
2001).
The removal of dye from textile effluents is one of the most
significant environmental problems ( Kim et al., 2004; Park
et al., 2006). Dyes are used in large quantities in many indus-tries including textile, leather, paper, printing, plastic, food,etc. to colour their products ( Garg et al., 2004 ).
The extensive use of dyes often passes pollution problems.
The presence of very low concentrations of dyes in large waterbodies is highly visible and indisputable and also reduces light
penetration and photosynthesis. In addition some dyes are
either toxic or mutagenic and carcinogenic ( Gong et al.,
2005; Nigam et al., 2000; Birhanli and Oznen, 2005; Degon
et al., 2005).
Dye wastewater from textile and dyestuff industries is diffi-
cult to treat. This is because dyes usually have a synthetic ori-
gin and complex aromatic structures which make them more*Corresponding author. Tel.: +20 23370931; fax: +20 23370951.
E-mail address: [anonimizat] (N.F. Ali).
1319-6103 ă2011 King Saud University. Production and hosting by
Elsevier B.V.
Peer review under responsibility of King Saud University.
doi:10.1016/j.jscs.2010.11.005
Production and hosting by ElsevierJournal of Saudi Chemical Society (2012) 16, 117–123
King Saud University
Journal of Saudi Chemical Society
www.ksu.edu.sa
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Open access under CC BY-NC-ND license.
Open access under CC BY-NC-ND license.

stable and more difficult to biodegrade ( Stolz, 2000 ). Azo dyes
are the most widely used as they account for over 60% of the
total number of dye structures known to be manufactured ( Al-
len, 1971).
Recently, a number of studies have focused on microbial
biodegradation of dye waste water. In this respect, Fu and
Viraghavan (2002) reported that Aspergillus niger was capable
of removing dyes from an aqueous solution and biosorption of
dyes was influenced by the functional groups in the fungal bio-
mass and chemical structure of the dyes. Donmez (2002) stud-
ied bioaccumulation of the reactive textile dyes Ramazol Blue,
Reactive Black and Reactive Red by the yeast species Candida
tropicolis growing in molasses medium and found that the in-
crease in dye concentration inhibited growth of yeast andcaused a long lag period ( Chivukula and Renganathan, 1995 ).
Interest in the pollution potential of textile dyes depends on
their possibility of toxicity or carcinogenity, cleavage of azo
dyes into the corresponding amines many of which are carcin-
ogenic ( Chivukula et al., 1995; Chung and Stevens, 1995;
Thurston, 1994). Azo reductases have been shown to be very
specific enzymes thus cleaving only the azo bonds of azo dyes.
In contrast the phenoxidases lignin peroxidase, manganese
peroxidase and laccase act more unspecifically on the aromaticring and have the potential to degrade a wide range of aro-
matic structures ( Atlas, 1993 ).
A great number of white rot fungi have been reported to
produce the lignin-degrading enzymes LiP, MnP, and laccase,
or at least one of these enzymes ( Fu and Tiraraghavan, 2001 ).
In this study, the acid, reactive and exhausted dyebaths
were treated by microbial isolates from Egyptian soil. The dec-olourization efficiency for these dyes was investigated.2. Experimental
2.1. Materials
2.1.1. Dyes
The dyes used in this study were: C.I. Reactive Blue 19, C.I.Reactive Blue 81, Acid Red 27, and C.I. Acid Red 151, thecommercial names, the maximum wave length ( k
max) and the
chemical structures of these dyes are illustrated in Table 1.
The dyes were added to cultures as aliquots of concentrated
stock solutions. Decolourization was measured spectrophoto-
metrically at the wavelength of peak absorbance of each dye
using UV–Vis recording spectrophotometer.
2.2. Preparation of dye solution
The dye stock solution was prepared by dissolving accurately
weighed dyes in distilled water to the concentration of
500 mg/l. Different concentrations were prepared from the
stock solution (0.02–0.1 g/l).
2.3. Fungal isolates
Six fungal isolates belong to Aspergillus niger, Penicillium spp.,
andPleurotus arteubus were used in this study. Aspergillus ni-
gerisolate no. 1 and 2, Penicillium spp. isolate no. 1 and 2 were
kindly obtained from plant pathology department, National
Research Centre, Egypt. These isolates. Meanwhile isolate of
Plouritus arteubus were obtained from Faculty of Agriculture,
Ain Shames University, Egypt. The above fungal isolates were
chosen for their high dye decolourization potential towards
Table 1 The commercial names, the maximum wave length ( kmax) and the chemical structures of the dyes under investigation.
The commercial names kmax The chemical structure
C.I. Acid Red 27 520
SO3NaSO3Na
N NHO
NaO 3 S
C.I. Acid Red 151 519 NO H
NN N NaSO3
C.I. Reactive Blue 19 583
OONH2
NHSO3Na
SO2CH2CH2OSO3Na
C.I. Reactive Blue 81 590N
NN N H OH
SO3Na SO3NaCl
Cl N N NH
SO3Na118 N.F. Ali, R.S.R. El-Mohamedy

many industrial dyes after a screening on solid PDA medium
made with 60 isolates including Aspergillus and Penicillium
genera and five isolates of P. arteubus. All fungal isolates were
grown on savoured dextrose Agar (SDA): 18 g Agar, 10 g pep-
tone, 40 g glucose and one litre distilled water. This medium
were autoclaved at atmospheric pressure 1.5 for 30 min
(Chung and Stevens, 1995 ).
Table 3 Decolourization potential of reactive dyes fungal
isolates.
Isolates Incubation period
3 days 6 days 9 days
% Decolourization
Aspergillus niger (1) 39 50 80
Aspergillus niger (2) 35 52 78
Aspergillus niger (3) 38 49 65
Penicillium spp. (1) 40 58 63.6
Penicillium spp. (2) 45 54 70
Pleurotus ostreatus 50 57 90Table 2 Decolourization potential of acid dyes fungal
isolates.
Isolates Incubation period
3 days 6 days 9 days
% Decolourization
Aspergillus niger (1) 40.4, 54.5 70
Aspergillus niger (2) 40, 54 65
Aspergillus niger (3) 38.5 50 58
Penicillium spp. (1) 40.9 51 60.5
Penicillium spp. (2) 46 56 65
Pleurotus ostreatus 52.5 59 85
01020304050607080
conc.0.02 0.04 0.06 0.08 0.1decolourization %dye 1
dye 2dye 3
dye 4
Figure 1 Effect of concentration (g/l) of dyes on the % of
decolourization (for 9 days incubation) of the acid and reactive
dyes solution using Aspergillus niger (1). Dye 1: Acid Red 27; dye
2: Acid Red 151; dye 3: Reactive Blue 19; dye 4: Reactive Blue 81.0.02 0.04 0.06 0.08 0.1
conc.0102030405060708090decolourization% dye 1
dye 2dye 3
dye 4
Figure 2 Effect of concentration (g/l) of dyes on the % of
decolourization (for 9 days incubation) of the acid and reactive
dyes solution using Aspergillus niger (2). Dye 1: Acid Red 27; dye
2: Acid Red 151; dye 3: Reactive Blue 19; dye 4: Reactive Blue 81.
010203040506070
0.02 0.04 0.06 0.08 0.1
conc.decolourization %
dye 1
dye 2dye 3
dye 4
Figure 3 Effect of concentration (g/l) of dyes on the % of
decolourization (for 9 days incubation) of the acid and reactive
dyes solution using Aspergillus niger (3). Dye 1: Acid Red 27; dye
2: Acid Red 151; dye 3: Reactive Blue 19; dye 4: Reactive Blue 81.
010203040506070
0.02 0.04 0.06 0.08 0.1
conc.decolourization %
dye 1
dye 2dye 3
dye 4
Figure 4 Effect of concentration (g/l) of dyes on the % of
decolourization (for 9 days incubation) of the acid and reactive
dyes solution using Penicillium spp. (1). Dye 1: Acid Red 27; dye 2:
Acid Red 151; dye 3: Reactive Blue 19; dye 4: Reactive Blue 81.Microbial decolourization of textile waste water 119

2.4. Dye decolourization
The experiments were carried out in 250 ml flasks containing
100 ml of SDA medium, at different concentrations of tested
dyes (0.02–1 g/l). The pH was adjusted (3–7), and then the
flasks were autoclaved at atmospheric pressure 1.5 for30 min. The autoclaved flasks were incubated with 5 mm discs
of 7 day old fungi of cultures of the tested isolates. They incu-
bated for 3, 6 and 9 days; after the end of this period 20 ml ofthe dye solution was centrifuged at 5000 rpm for 15 min. Thenthe maximum absorption ( k
max) was measured using spectro-
photometer. Non-incubated flasks of each dye concentrationwere set as control (check treatment).
2.5. Decolourization assay
The decolourization assay was expressed in the terms of deco-
lourization % using spectrophotometer. The decolourization010203040506070
0.02 0.04 0.06 0.08 0.1
conc.decolourization %dye 1
dye 2dye 3
dye 4
Figure 5 Effect of concentration (g/l) of dyes on the % of
decolourization (for 9 days incubation) of the acid and reactive
dyes solution using Penicillium spp. (2). Dye 1: Acid Red 27; dye 2:
Acid Red 151; dye 3: Reactive Blue 19; dye 4: Reactive Blue 81.
0102030405060708090100
0.02 0.04 0.06 0.08 0.1
conc.decolourization %
dye 1
dye 2dye 3
dye 4
Figure 6 Effect of concentration (g/l) of dyes on the % of
decolourization (for 9 days incubation) of the acid and reactive
dyes solution using Pleurotus ostreatus . Dye 1: Acid Red 27; dye 2:
Acid Red 151; dye 3: Reactive Blue 19; dye 4: Reactive Blue 81.
01020304050607080
34567Decolourization %
pH1Dye
2Dye 3Dye
4Dye
Figure 7 Effect of pH of dyes on the % of decolourization (for
9 days incubation) of the acid and reactive dyes solution using
Aspergillus niger (1). Dye 1: Acid Red 27; dye 2: Acid Red 151; dye
3: Reactive Blue 19; dye 4: Reactive Blue 81.01020304050607080
34567
pHDecolourization %Dye 1
Dye 2Dye 3
Dye 4
Figure 8 Effect of pH of dyes on the % of decolourization (for
9 days incubation) of the acid and reactive dyes solution using
Aspergillus niger (2). Dye 1: Acid Red 27; dye 2: Acid Red 151; dye
3: Reactive Blue 19; dye 4: Reactive Blue 81.
01020304050607080
34567
pHDecolourization %
Dye 1
Dye 2Dye 3
Dye 4
Figure 9 Effect of pH of dyes on the % of decolourization (for
9 days incubation) of the acid and reactive dyes solution using
Aspergillus niger (3). Dye 1: Acid Red 27; dye 2: Acid Red 151; dye
3: Reactive Blue 19; dye 4: Reactive Blue 81.120 N.F. Ali, R.S.R. El-Mohamedy

% in dye concentration was calculated for each treatment (dye
concentration and fungal isolate) as follows:
%Decolourization ¼100/C2ðC0/C0CtȚ=C0
where C0is the initial concentration of the dye (control) and Ct
is the concentration at time t.
3. Results and discussion
Fungi decolourization of synthetic dyes according to their life
state group 1: living cells to biodegradation and biosorb dyes.The major mechanism is biodegradation because they can pro-duce the lignin modifying enzymes, lactose, manganese perox-
ides (Lip) to mineralize synthetic lignin of dyes ( Stolz, 2001;
Wesenberg et al., 2003). Group 2: dead cells (fungal biomass)
to adsorb dyes. The mechanism in bio sorption which involves
physico-chemical interactions such as adsorption, deposition
and ion-exchange ( Husseiny, 2008 ).
The colour removal of the reactive and direct dyes can be
achieved by treating with A. niger andPenicillium spp. A. niger
was the most efficient fungus to cause biodegradation of thecolour of Direct Red 81 dye, while Penicillium spp. A. niger
was the most efficient fungal to cause biodegradation of the
colour Reactive Red 120 dye ( Husseiny, 2008; Zheng et al.,
1999).3.1. Effect of initial time of incubation on decolourization % of
acid and reactive dyes
The results obtained indicated that decolourization efficiency
of acid dyes (Acid Red 27, Acid Red 151 and Reactive Blue
19, Reactive Blue 81) for 3, 6 and 9 days intervals using twoinocula sizes of 400 and 800 mg dry weight of microbe is
shown in Tables 2 and 3 with 0.9 g inoculum size, most of fun-
gal isolates were capable of removing approximately the high-
est % of dye colour after 9 days incubation.
The nature of the substituent on the aromatic ring has been
shown to influence enzymatic oxidation. Electron donatingsubstituents as methyl and methoxy and amino groups en-hance enzymatic degradation of azo phenols while electron
withdrawing substituents as chloro, nitro and hydroxyl inhib-
ited oxidation ( Zheng et al., 1999 ). Hydroxy and amino groups
enhance decolourization. The presence of Lip in addition to
Laccase in the P. ostreatus had positive effect on the degree
of decolourization for all tested dyes.
3.2. Effect of initial dye concentration on decolourization % of
acid and reactive dyes
The effect of initial dye concentration of dye in solution on re-
moval of dyes was studied. We used different concentration of
dyes (0.02, 0.04, 0.08 and 0.1 g/l). The removal of dyes wasclearly dependent on the initial dye concentration of the solu-
tion ( Figs. 1–6 ).
Figs. 13 and 14 show the photographs of decolourization of
acid and reactive dyes before and after 3, 6 and 9 days incuba-tion consequently.
The isolate of P. ostreatus produced the highest biomass
accumulation after 9 days and it is the most efficient one in
the removal of the reactive and acid dyes at low
concentrations.
3.3. Effect of the pH on decolourization % of acid and reactive
dyes
Figs. 7–12 show the effect of pH of the dye solution on the dec-
olourization % of microorganisms within the range of (3–7).
The results showed that the decolourization reached maximumat pH 5 for acid dyes while it reached maximum at pH 6 for
reactive dyes for all isolates used. Many authors stated that
pH is very important for fungal growth.020406080
345 6 7
pHDecolourization %
Dye 1
Dye 2Dye 3
Dye 4
Figure 10 Effect of pH of dyes on the % of decolourization (for
9 days incubation) of the acid and reactive dyes solution using
Penicillium spp. (1). Dye 1: Acid Red 27; dye 2: Acid Red 151; dye
3: Reactive Blue 19; dye 4: Reactive Blue 81.
Decolourization %
020406080
34567
pHDye 1
Dye 2Dye 3
Dye 4
Figure 11 Effect of pH of dyes on the % of decolourization (for
9 days incubation) of the acid and reactive dyes solution using
Penicillium spp. (2). Dye 1: Acid Red 27; dye 2: Acid Red 151; dye
3: Reactive Blue 19; dye 4: Reactive Blue 81.020406080100
34567
pHDecolourization %Dye 1
Dye 2Dye 3
Dye 4
Figure 12 Effect of pH of dyes on the % of decolourization (for
9 days incubation) of the acid and reactive dyes solution using
Pleurotus ostreatus . Dye 1: Acid Red 27; dye 2: Acid Red 151; dye
3: Reactive Blue 19; dye 4: Reactive Blue 81.Microbial decolourization of textile waste water 121

Fungi can grow at low pH from 4 to 5. The optimum pH
for decolourization by unidentified white rot fungus was
pH 4–5 ( Zheng et al., 1999 ).
4. Conclusion
Fungi decolourization of acid and reactive dyes can be
achieved by treating with ( A. niger ,Penicillium spp., and P.
ostreatus ). It can be stated that most of fungal isolates were
capable of removing the highest percent of dye colour after9 days incubation. The isolate of Plouritus ostreatus produced
the highest biomass accumulation after 9 days and it is the
most efficient one in the removal of the reactive and acid dyes
at low concentrations. The results showed that the decolouri-
zation reached maximum at pH 4 for acid dyes while it reacheda maximum at pH 5 for reactive dyes for all isolates used.
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