Middle-East Journal of Scientific Research 17 (12): 1695-1700, 2013

ISSN 1990-9233
© IDOSI Publications, 2013
DOI: 10.5829/idosi.mejsr.2013.17.12.81146

Biological Degradation of Reactive Dyes by

Using Bacteria Isolated from Dye Effluent Contaminated Soil

N. Sriram, D. Reetha and P. Saranraj

Department of Microbiology, Annamalai University,

Chidambaram – 608 002, Tamil Nadu, India

Abstract: The textile industry is one of the industries that generate a high volume of waste water. Strong colour
of the textile waste water is the most serious problem of the textile waste effluent. The disposal of these wastes
into receiving water causes damage to the environment. Dyes may significantly affect photosynthetic activity
in aquatic habitat because of reduced light penetration and may also be toxic to some aquatic life due to the
presence of aromatics, metals, chlorides and other toxic compounds. The present study was carried out to
degrade the textile Reactive azo dyes by using bacteria isolated from dye contaminated soil. Three different
bacterial isolate such as, Bacillus sp., Escherichia coli and Pseudomonas fluorescens were isolated from textile
dye effluent contaminated soil sample and used for the degradation study. It was noticed that there was a
decrease in the OD in all the three species of all the five dyes as the incubation period increased. Pseudomonas
fluorescens was more effective followed by Bacillus and Escherichia coli. It was found that all the isolated
bacteria were efficient decolourizers of Reactive textile azo dyes.

Key words: Degradation Decolourization Reactive Azo Dyes Effluent Contaminated Soil Bacteria

INTRODUCTION Dyeing of textile request water and generates a

substantial quality of effluents containing mineral salts
Dyes are recalcitrant by design and not readily and dyes at high concentration. An estimated 700000 tons
amendable to common treatment methods, imposing a of dyes are produced annually worldwide of which
challenge for closed water systems. Extensive research in 60-70% are azo dyes [3]. Chronic effects of dyestuffs,
the field of biological azo dye decolourization has shown especially of azo dyes, have been studied for several
promising results, but much of this work has been done decades. Azo dyes in purified form are mutagenic or
with single model compounds [1]. However, industrial carcinogenic, except for some azo dyes, leads to formation
textile wastewater presents the additional complexity of of aromatic amines and several aromatic amines are known
dealing with unknown quantities and varieties of many mutagens and carcinogens to human beings. In mammals,
kinds of dyes [2], as well as low BOD/COD ratios, which metabolic reduction of azo dye is mainly due to bacterial
may affect the efficiency of the biological decolourization. activity in the anaerobic parts of the lower gastrointestinal
India's dye industry produces every type of dyes and tract. Various organs, especially the liver and the kidney
pigments. Production of dye stuff and pigments in India also reduce azo dyes. After azo dye reduction in the
is close to 80,000 tones. India is the second largest intestinal tract, the released aromatic amines are absorbed
exporter of dye stuffs and intermediates after China. by the intestine and are excreted in the urine. The acute
The textile industry accounts for the largest consumption toxic hazard of aromatic amines is carcinogenesis,
of dyestuffs, at nearly 80%. Industrialization is vital to a especially bladder cancer. International Agency for
nation’s economy because it serves as a vehicle for Research on Cancer (IARC) summarized the literature on
development. However, there are associated problems suspected azo dyes, mainly amino-substituted azo dyes,
resulting from the introduction of industrial waste fat soluble dyes and benzidine azo dyes, also a few
products into the environment. sulphonated azo dyes [4].

Corresponding Author: N. Sriram, Department of Microbiology, Annamalai University,

Chidambaram – 608 002, Tamil Nadu, India. Mob: 91+87606 78545.
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Dyes are stable against breakdown by many Dyes: Reactive azo dyes used in this research are,
microorganisms and most dyes do not biodegrade under Reactive Orange – M2R ( m = 493 nm), Reactive Blue –
the aerobic biological treatments in a municipal sewage M58 ( m = 572 nm), Reactive Yellow – M4G ( m = 413 nm)
plant. Many dyes, including the azo dyes, degrade under and Reactive Black - B ( m = 574 nm).
anaerobic conditions and the aromatic amines thus formed
have been found to degrade further aerobically. Out of Isolation and Identification of Dye Bacteria from Dye
several methods that are used in the treatment of textile Effluent Contaminated Soil: Pour plate technique was
effluents to achieve decolourization, including used for the isolation of dye decolourizing bacteria from
physiochemical methods like filtration, specific dye effluent contaminated soil. Well grown bacterial
coagulation, use of activated carbon and chemical colonies were picked and further purified by streaking.
flocculation some of the methods are effective but quite The isolated strains were maintained on Nutrient agar
expensive. Biotreatment offers a cheaper and slants and stored in refrigerator at 4°C. Identification of
environmentally friendlier alternative for colour removal in the bacterial isolates was carried out by the routine
textile effluent [5]. Bioremediation is a pollution control bacteriological methods i.e., By the colony morphology,
technology that uses biological systems to catalyze the preliminary tests like Gram staining, capsule staining,
degradation or transformation of various toxic chemicals endospore staining, motility, catalase and oxidase, plating
to less harmful forms. This natural process, on selective medias and performing biochemical tests.
bioremediation, includes bioengineering the capabilities
of intrinsic microorganisms, to clean up the environment Screening of Bacterial Isolates for Textile Direct Azo
is an effective alternative to conventional remediation Dye Degradation
methods [6]. Inoculum Preparation: The suspension of 2 days old
Investigations to bacterial dye biotransformation cultures of bacteria was used to investigate their abilities
have so far mainly been focused to the azo dyes. The to decolourize dyes. They were prepared in saline solution
electron withdrawing nature of the azo linkages obstructs (0.85% Sodium chloride). A loopful of bacterial cultures
the susceptibity of azo dye molecules to oxidative were inoculated into 50 ml of saline and incubated at 37°C
reaction. Therefore, azo dyes generally resist aerobic for 3 hours.
bacterial biodegradation. Only bacteria with specialized
azo dye reducing enzymes (Azoreductase) were found Dye Decolourization Experiment: Fifty milliliter of
to degrade azo dyes under fully aerobic conditions. Nutrient agar sterile medium was amended separately with
This anaerobic reduction implies decolourization of the each of the textile dyes (200 mg/l) and subsequently
dyes are converted to usually colourless but potentially inoculated with 2% bacterial suspension. The suspension
harmful aromatic amines. Aromatic amines are generally contained 2.5 x 106 cfu/mL spores. The flasks were kept in
not further degraded under anaerobic conditions. mechanical shaker and incubated at 30±1°C for 8 days.
Anaerobic treatment must therefore be considered merely Samples were drawn at 2 days intervals for observation.
as the first stage of the complete degradation of azo dyes. The samples were centrifuged at 10000 rpm for 10 minutes
The second stage involves conversion of the produced and decolourization was assessed by measuring
aromatic amines. For several aromatic amines, this can be absorbance of the supernatant with the help of
achieved by biodegradation under aerobic conditions. spectrophotometer at wavelength maxima ( m) of
The present study was carried out to degrade the textile respective dye. Two control flasks (Dye + medium without
Reactive azo dyes by using bacteria isolated from dye inoculums and medium with inoculums without dye) were
contaminated soil. maintained.

MATERIALS AND METHODS Dye Decolourization Assay: Dye decolourization assay

was measured in the terms of percentage decolourization
Sample Collection and Preservation: The dye house using UV-Spectrophotometer. The percentage
effluent soil was collected from the surroundings of dye decolourization was calculated from the following
industry, Tirupur region, Tamil Nadu, India. The sample equation,
was collected in a plastic container. Then the sample was
brought to the laboratory as early as possible and was Intitial OD - Final OD
% Decolourization = x 100
subjected for various microbiological studies. Initial OD

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RESULTS AND DISCUSSION Reactive Yellow – M4G and Reactive Black-B were
studied against five bacterial isolates which have been
Azo dyes are the largest group of dyes. More than isolated from the dye effluent sample by Pour plate
3000 different varieties of azo dyes are extensively used in method and percentage decolourization was shown in the
the textile, paper, food, cosmetics and pharmaceutical figures accompanying the results. Three different bacteria
industries [7]. Azo dyes are characterized by the presence were isolated from the textile dye effluent. Based on
of one or more azo groups – N = N -, which are preliminary tests, plating on selective media and
responsible for their colouration and when such a bond is biochemical tests, they were identified as Bacillus sp.,
broken the compound loses its colour. They are the Escherichia coli and Pseudomonas fluorescens.
largest and most versatile class of dye, but have structural Olukanni et al. [10] isolated eighteen textile effluent
properties that are not easily degradable under natural adapted bacterial isolates belonging to the genera,
conditions and are not typically removed from water by Bacillus, Acinetobacter, Staphylococcus, Legionella and
conventional waste water system. Azo dyes are designed Pseudomonas were investigated for the potential of textile
to resist chemical and microbial attacks and to be stable in effluent adapted bacteria in decolourizing it. Bacillus and
light and washing. Legionella were found to have use in effluent treatment.
A number of azo dyes including reactive dyes are Ajibola et al. [11] checked the ability of Staphylococcus
used in textile dying operations. This leads to effluent aureus, Bacterioides fragilis, Bacillus subtilis, Bacillus
streams containing intense colour due to the presence of cereus, Clostridium perifringens, Escherichia coli and
azo dyes. The removal of azo dyes from effluents is Peptostreptococcus sp. to reduce and stabilize textile
important due to their mutagenicity and carcinogenicity effluents containing predominantly Indigo Blue.
together with their intense colouration. Both Saranraj et al. [12] isolated five different bacterial
physicochemical and biological methods for the removal isolates from the textile dye effluent sample and identified
of dyes have been investigated widely. The as Bacillus subtilis, Pseudomonas aeruginosa, Proteus
physicochemical dye removal techniques such as mirabilis, Klebsiella pneumoniae and Escherichia coli.
flocculation–coagulation, adsorption, electrochemical Manivannan et al. [13] also isolated three different
oxidation, photocatalytic oxidation, electro- Fenton bacteria were isolated from the textile dye effluent. Based
oxidation appear to face several technical and economic on preliminary tests, plating on selective media and
limitations [8]. On the other hand, biological methods biochemical tests, they were identified as Bacillus sp.,
such as activated sludge process and anaerobic treatment Escherichia coli and Pseudomonas fluorescens.
have been applied to control pollution of aquatic The decolourization efficiency of Bacillus sp.,
environment. Lower cost of treatment and amenability Pseudomonas fluorescens and Escherichia coli was
to scale up easily are the merits of biological methods. studied by measuring the optical density after 0, 4, 8, 12,
The present study was focused on decolourization of 16 days of incubation and the results were showed in
textile azo dyes and biodegradation of textile dye effluent Figure – 1, 2, 3 and 4. It was noticed that there was a
by using bacteria isolated from textile dye effluent decrease in the optical density (OD) in all the three
contaminated soil. species in all the five colours as the incubation
In Tamil Nadu many of the districts are known for period increased. Pseudomonas fluorescens was more
textile industries. Tirupur is one among them. These effective followed by Bacillus sp. and Escherichia coli.
industries discharge the coloured effluents with dyes and The percentage of decolourization of colours by the
toxic compounds into the open environment. Textile and bacteria was also calculated. It was found that all the
dyeing industry are among those which contribute much isolated bacteria were efficient decolourizers of Reactive
to water and soil pollution. They consume substantial Orange – M2R. The decolourization of dye amounted to
volumes of water and chemicals. Further, about 10,000 59, 77 and 79 respectively within 16 days. Reactive Yellow
different dyes and pigments are being used. Among these – M4G was recalcitrant to decolourization, the O.D. value
azo-dyes are widely used. Apart from chemicals nearly from an initial value of 0.6912 was reduced only to 0.303
10-15% of the dye is lost as effluent during the dyeing and from 0.746 to 0.218, 1.236, to 1.33 by Pseudomonas
process [9]. fluorescens, Bacillus sp. and Escherichia coli
Biodegradation of commercially available textile dyes respectively. Percentage decolourization was 43%, 15%,
namely Reactive Orange – M2R, Reactive Blue - MR, 90% respectively.

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100
90
80

Decolourization (%)
70
60
50
40
30
20
10
0
Bacillus sp. E. coli Pseudomonas
fluorescens

Fig. 1: Decolourization percentage of Reactive Orange – M2R by bacterial isolates

100
90
80
Decolourization (%)

70
60

50
40

30
20
10
0
Bacillus sp. E. coli Pseudomonas
fluorescens
Fig. 2: Decolourization of Reactive Blue - MR by bacteria
100
90
80
Decolourization (%)

70
60

50
40

30
20
10

0
Bacillus sp. E. coli Pseudomonas
fluorescens

Fig. 3: Decolourization of azo dye Reactive Yellow – M4G by bacteria

100
90
80
Decolourization (%)

70
60
50
40
30
20
10
0
Bacillus sp. E. coli Pseudomonas
fluorescens

Fig. 4: Decolourization of azo dye Reactive Black - B by bacteria

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The decolourization of textile reactive azo dyes by further environmental damage. Hence, economical and
Clostridium biofermentans isolated from a contaminated eco-friendly techniques using bacteria can be applied for
site was studied under aerobic conditions. Clostridium fine tuning of waste water treatment. Biotreatment offers
biofermentans decolourized the dyes Reactive red 3B-A, easy, cheaper and effective alternative for colour removal
Reactive black 5 and Reactive yellow 3B-A, by over 90% of textile dyes. Thus, by this present study it is concluded
after 36 hours post-inoculation spectrophotometric that the bacterial isolates like Bacillus sp., Pseudomonas
analyses of the reactive dyes showed no distinct peak fluorescens and Escherichia coli can used as a good
indicating aromatic amines. The results suggested that microbial source for waste water treatment.
Clostridium biofermentans was a suitable bacterium for
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