Journal of Applied & Environmental Microbiology, 2013, Vol. 1, No.

1, 1-5
Available online at http://pubs.sciepub.com/jaem/1/1/1
Science and Education Publishing
DOI:10.12691/jaem-1-1-1
Microbial Decolorization of the Azo Dye Methyl Red by
Enterobacter spp. ETL-1979
Maulin P Shah
*
, Kavita A Patel, Sunu S Nair, A M Darji
Industrial Waste Water Research Laboratory, Applied & Environmental Microbiology Lab, Enviro Technology Limited (CETP),
GIDC, Ankleshwar, Gujarat, India
*Corresponding author: [email protected]
Received September 26, 2013; Revised November 27, 2013; Accepted December 08, 2013
Abstract Bacterial isolates, obtained from dye-contaminated sludge, decolorized the toxic azo dye methyl red
(MR). Enterobacter spp. ETL-1979 was selected because of its better abilities to completely decolorize MR under
aerobic conditions. Effects of physicochemical parameters (temperature, stirring, concentration of glucose and pH of
the synthetic medium) on the MR decolorization by the selected bacterium were studied. Under optimal conditions,
Enterobacter spp. ETL-1979 completely decolorized 100 mg/l of MR within 6 h of incubation in synthetic medium.
The high MR decolorization ability and low nutrient and environmental requirements of Enterobacter spp. ETL-
1979 enable this bacterium to be used in the biological treatment of industrial effluent containing azo dyes.
Keywords: azo dye, methyl red, bacterial decolorization, Enterobacter
Cite This Article: Maulin P Shah, Kavita A Patel, Sunu S Nair, and A M Darji, Microbial Decolorization of
the Azo Dye Methyl Red by Enterobacter spp. ETL-1979. Journal of Applied & Environmental Microbiology 1,
no. 1 (2013): 1-5. doi: 10.12691/jaem-1-1-1.
1. Introduction
The structural diversity of dyes derives from the use of
different chromophoric groups (azo, anthraquinone,
triarylmethane and phtalocyanine groups) and different
application technologies (reactive, direct, disperse and vat
dyeing) [3]. Azo dyes are the most common types of
synthetic dyes and constitute the largest class of dyes used
commercially [3]. Synthetic azo dyes are used extensively
as dyes for textiles, food and cosmetics. More than
800,000 tons of these dyes are produced annually
worldwide [3]. Most of the azo dyes, which are released
into the environment, originate from the textile industry
and the dyestuff manufacturing industry [2]. Azo dyes are
a group of compounds characterized by the presence of
one or more azo bonds (-N=N) in association with one or
more aromatic systems [1]. This makes them relatively
resistant to biological and chemical degradations.
However, several studies have shown azo dyes to be toxic
and/or carcinogenic [5,6,7,8] Most biological degradations
of azo dyes are carried out by anaerobic bacteria
[2,13,14,21]. Generally, azo dyes are resistant to attack by
bacteria under aerobic conditions. Only some studies
reported that bacteria under aerobic conditions could
degrade azo dyes [3,4,9,16,18,20]. In both cases (aerobic
and anaerobic conditions) the initial step in the
biodegradation is the cleavage of the azo bond.
Azoreductase catalyzes reductive cleavage of the azo bond
to produce aromatic amines. Under aerobic conditions, the
initial step of azo bond cleavage is typically followed by
hydroxylation and ring opening of the aromatic
intermediates [7,15]. In previous studies, Acetobacter
liquifaciens and Klebsiella pneumoniae were shown to
decolorize the toxic azo dye methyl red (MR) under
aerobic conditions and yield two colorless compounds
(Figure 1), 2-aminobenzoic acid (ABA) and N,N-
dimethyl-p-phenylenediamine (DMPD) [16,18]. In the
present study, we report the aerobic decolorization of MR
by Enterobacter spp. ETL-1979 growing on a synthetic
medium. This bacterial strain, isolated from dye
contaminated sludge, decolorizes MR faster than A.
liquefaciens and K. pneumoniae. The effects of various
physicochemical parameters on the decolorization of MR
by Enterobacter spp. ETL-1979 were studied and
compared.

Figure 1. Reductive cleavage of the azo dye methyl red (MR) (Wong
and Yuen 1996)
2. Materials and Methods
2.1. Isolation of Methyl Red Decolorizing
Bacteria

2 Journal of Applied & Environmental Microbiology
Dye-contaminated sludge was collected from a
wastewater outlet of an industrial area in Ankleshwar
GIDC, Gujarat, India. Approximately, 1 g of sludge was
suspended in 10 ml of sterile sodium chloride solution
0.85% (w/v) and mixed thoroughly. The mixture was
serially diluted with sterile sodium chloride solution
0.85%. Aliquots of 0.1 ml of 10
1
, 10
2
and 10
3
dilutions
were spread onto minimum medium (MM) plates
containing: K
2
HPO
4
, 1.36 g/l; MgSO
4
, 0.1 g/l; SO
4
(NH4)2,
0.6 g/l; CaCl
2
, 0.02 g/l; NaCl, 0.5 g/l; MnSO
4
, 1.1 mg/l;
ZnSO
4
, 0.2 mg/l; CuSO
4
, 0.2 mg/l; FeSO
4
0.14 mg/l; MR,
100 mg/l; agar, 15 g/l. pH was adjusted to 7 with 1 M HCl.
All plates were incubated at 37 C for 3 days. Colonies
surrounded by decolorized zones were picked and
streaked onto MM plates containing 100 mg/l of MR. The
plates were again incubated at the same conditions to
confirm their abilities to decolorize MR.
2.2. Decolorization of Methyl Red by Selected
Bacterium in Liquid Medium
One bacterial isolate with higher MR decolorization
abilities was selected. A preculture of this bacterium was
prepared by growing a single colony in 10 ml of MM
containing 1% of glucose at 37 C in a rotary shaker at
100 rpm for 18 h. An aliquot of 1 ml of precultures was
inoculated into 100 ml of MM with 1% of glucose and
100 mg/l of MR. This culture was incubated under the
same conditions for 24 h. After incubation, an aliquot of
10 ml of this culture was sampled and cells were pelleted
by centrifugation at 12,000 g at 4 C for 15 min in a
Sigma 3K15 refrigerated centrifuge. The absorbance at
430 nm (the absorption maximum of the MR) of the
culture supernatant was determined using a Shimazu 1800
UV/Visible spectrophotometer.
2.3. Presumable Identification of the Selected
Bacterium
The selected bacterial isolate was subject to the Gram
stain and then tentatively identified by biochemical
analysis according to the standardized micromethod API
20 E (bioMrieux, Inc.). All reagents and accessories were
purchased from the bioMrieux Inc. and used according to
the instructions of the manufacturer.
2.4. HPLC Analysis of Methyl Red and Its by-
Products
The selected bacterium was grown in 100 ml of MM
with 100 mg/l of MR and 1% of glucose at 37 C. After 6
h of incubation, the culture was centrifuged to remove
bacterial cells and the supernatant was concentrated to
about 20 ml in a rotary evaporator at 60 C. A
concentrated sample was then extracted three times with
equal volume of dichloromethane (DCM). The DCM
extracts were pooled and evaporated to 5 ml at 40 C in a
rotary evaporator and then transferred to a test tube. The
remaining DCM was removed by evaporation at room
temperature (20 0.5 C) and placed in a hood overnight.
The extracted residue was dissolved in 10 ml acetonitrile
and filtered through a 0.2 m nylon filter. Twenty l was
analyzed by a J asco HPLC system equipped with a model
875 variable wavelength detector (detection wavelength
was 254 nm) and a reverse phase C18 column (25 cm 4
mm) packed with 5 m particle size. The mobile phase
was sodium phosphate buffer (25 mM, pH 3.0) and
acetonitrile (40/60, v/v) with a flow rate of 0.2 ml/min. A
control test without the bacterium and containing 100 mg/l
of MR was prepared and analyzed under the same
conditions. The standards of methyl red (MR) (BDH
Chemicals, England), N,Ndimethyl-p-phenylenediamine
(DMPD) (Fluka, Switzerland) and 2-aminobenzoic acid
(ABA) (Fluka, Switzerland) were injected for comparison
and confirmation.
2.5. Effects of Physicochemical Parameters on
Methyl Red Decolorization and Bacterial
Growth of Selected Bacterium
Pre cultures of the selected bacterium were prepared by
growing a single colony in 10 ml of MM containing 1% of
glucose for 18 h at 37 C with shaking. Aliquots of 2 ml
were inoculated in culture media containing 200 ml of
MM with 1% of glucose and 100 mg/l of MR. The
inoculated cultures were then incubated at different
experimental conditions to determine the effects of
physicochemical parameters on the MR decolorization and
the bacterial growth. Effect of temperature: The inoculated
cultures were incubated at various temperatures (24, 37
and 44 C) in rotary shakers running at 100 rpm. Effect of
stirring: The inoculated cultures were incubated at 37 C
without shaking (static condition) and in a rotary shaker
running at 100 rpm (shaking condition).
Effect of glucose concentration: The culture medium
used to determine the effect of the concentration of
glucose was MM containing 100 mg/l of MR and various
concentrations of glucose (0, 1, 2 and 4%). The inoculated
cultures were incubated at 37 C under shaking condition
(100 rpm).
Effect of pH of culture medium: Procedures to
determine the effect of pH were similar to those cited for
the glucose concentration effect study, except the culture
medium used was MM with 1% of glucose and 100 mg/l
of MR. The pH of the culture media was adjusted to 3, 5,
7 or 9 with 1 M HCl. At several time intervals, 2 ml
aliquots of each culture were sampled and their cell
densities were measured spectrophotometrically at 600 nm
in order to determine the bacterial growth. Cells were then
pelleted by centrifugation and the supernatant was 10-fold
diluted and the concentration of MR was determined
spectrophotometrically at 430 nm. All experiments were
carried out in triplicate.
3. Results and Discussion
3.1. Isolation of Methyl Red Decolorizing
Bacteria
Preliminary selection of MR decolorizing bacteria was
based on the decolorization of MR on MM plates because
most azo dye decolorizing microorganisms cleave the azo
bond of respective azo dye and produce colorless by-
products. Nine bacterial isolates, which decolorized MR
on MM plates, were isolated from dye-contaminated
sludge. Among these bacteria, one bacterial isolate with
higher MR decolorization ability (i.e. larger decolorized
halo surrounding the colony) in MM plates was selected

Journal of Applied & Environmental Microbiology 3
for further study. We have used in the present work MM
as a culture medium with low levels of carbon and
nitrogen sources because previous studies have indicated
that bacteria had better azo dye decolorizing abilities
under such nutritional conditions (Ogawa et al., 1986;
Ogawa and Yatome, 1990).
3.2. Presumable Identification of the Selected
Bacterium
The selected bacterial isolate was subject to the Gram
stain. It was Gram negative and it was then tentatively
identified by the standardized micromethod API 20 E. The
results indicated the selected bacterium to be
Enterobacters with about 88% of identification
probability. This bacterial isolate belongs to the family of
Enterobacteriaceae. Most members of this family are
characterized by their minimum requirements for growth
[10].
3.3. Identification of Methyl Red and Its by-
Products by HPLC Analysis
The HPLC analysis of the control test without the
bacterium exhibits one peak corresponding to MR at a
retention time of 8.05 min (Figure 2A). The same
retention time was obtained when MR standard was
injected. Whereas, the HPLC analysis of the culture
supernatant of Enterobacter, after 6 h of incubation in the
presence of MR, indicated the disappearance of the MR
peak (8.06 min) and the appearance of two major peaks
X1 and X2 with the retention times of 2.12 and 4.21 min,
respectively (Figure 2B). Tentative assignment of the by-
products (X1 and X2) was made on the basis of the
retention times of standards, supported by previous
knowledge of MR degradation processes [16,17,18].
Therefore, products X1 and X2 were identified as
N,Ndimethyl-p-phenylenediamine (DMPD) and 2-
aminobenzoic acid (ABA), respectively. Similar
compounds were reported as metabolites of MR
degradation by other bacteria [16,17,18]. Consequently,
these results suggest that Enterobacter completely
decolorized MR and produced at least two metabolic by-
products N,Ndimethyl-p-phenylene-diamine (DMPD)
and 2-aminobenzoic acid (ABA) (Figure 1).

Figure 2. HPCL analysis of MR (methyl red) (A) and its product of decolorization (X1 & X2) by Enterobacter spp. ETL-1979 after 6 hrs of incubation
(B) X1 & X2were identified,in comparision to standards, as, N,Ndimethyl-p-phenylenediamine (DMPD)
3.4. Effects of Physicochemical Parameters on
Methyl Red Decolorization and Bacterial
Growth of Selected Bacterium
Enterobacter spp. ETL-1979 had high MR
decolorization at 37 C. However, at 24 C and at 44 C
the MR decolorization was slower. Thus, the bacterium
decolorize 92% of MR within 6 h of incubation at 37 C
whereas only 20% of MR was decolorized at this time
period by the bacterium incubated at 24 C and at 44 C
(Figure 3A). This can be explained by the variations of the
bacterial growth rates of E. agglomerans according to the
temperature of incubation (Figure 3B). Indeed, the
bacterial growth is much faster and more significant at
37 C than at 24 C. Whereas, at 44 C the bacterial
growth was very poor. A larger population of cells could
certainly decolorize more molecules of MR. (Figure 3C)
shows that the stirring of the culture medium do not have
an influence on the decolorization of MR by Enterobacter
Indeed, the evolution of the rates of decolorization of MR
by Enterobacter under shaking and static conditions is
almost similar and it reaches 92% after 6 h of incubation
(Figure 3C). However, the stirring of the culture medium
slightly influences the kinetics of the bacterial growth of E.
agglomerans (Figure 3D). Enterobacter spp. ETL-1979
decolorizes approximately 90% of MR after 6 h of
incubation in the presence of 1, 2 and 4% of glucose
(Figure 4A). Whereas, in absence of glucose, the rate of
decolorization of MR by Enterobacter is only 30% and
this after 10 h of incubation. This can be explained by the
very weak growth of Enterobacter in absence of glucose
(Figure 4B). Under these conditions, the bacterium uses
MR as a sole source of carbon and energy for its growth.
Whereas in the presence of glucose the bacterium utilized
most of the energy generated from the glucose degradation
for the reductive cleavage of MR. It should be noted also
that in the presence of 4% of glucose the bacterial growth
is slightly slower than in the presence of 1% or 2% of
glucose (Figure 4B). Probably, the presence of glucose in
excess in the culture medium slows down the bacterial
growth of Enterobacter. Enterobacter decolorizes 90% of

4 Journal of Applied & Environmental Microbiology
MR at pH 5, 7 and 9 but with variable rates (Figure 4C).
Indeed, at pH 5 the rate of decolorization of MR reaches
90% after only 4.5 h of incubation, whereas at pH values
of 7 and 9, 90% of decolorization was reached after 6 h
and 8 h, respectively. On the other hand, at pH 3 no
decolorization of the MR was observed. This could be
explained by the inhibition of the bacterial growth at this
acid pH (Figure 4D). The bacterial growth of
Enterobacter is more significant at pH 9 than at pH 5
(Figure 4D) while the decolorization of MR is faster at pH
5 than at pH 9. This can be explained by the fact that at
acid pH the MR is more easily reducible than at alkaline
pH.

Figure 3. Effect of temperature on the MR decolorization (A) and on the bacterial growth (B) of E.spp. ETL-1979 incubated at 24 C(--), 37 C(-)
and 44 C(--). Effect of stirring on the MR decolorization (C) and on the bacterial growth (D) of E. spp. ETL-1979 incubated under shaking condition
100 U/ml (--) and under static condition(--)

Figure 4. Effect of glucose concentration on the MR decolorization (A) and on the bacterial growth (B) of E. spp. ETL-1979 incubated in MM
containing 0% (--) 1% (-) 2% (--) et 4%(--) of glucose. Effect of pH on the MR decolorization (C) and on the bacterial growth (D) of E. spp.
ETL-1979 incubated in MM at pH 3 (--), 5 (--),7(--) and 9 (--)

Journal of Applied & Environmental Microbiology 5
4. Conclusion
Enterobacter spp. ETL-1979 has a higher aerobic MR
decolorization ability compared to other azo dye
decolorizing bacteria incubated under similar conditions
[11,16,18,19]. This bacterial strain, isolated from dye-
contaminated sludge collected from an industrial area in
Casablanca city, completely decolorized 100 mg/l of MR
in the synthetic medium. The MR decolorization by
Enterobacter spp. ETL-1979 was widely much faster
pneumoniae [16,18]. Indeed as shown by the HPLC
analysis, Enterobacter completely decolorizes the MR
after only 6 h of incubation to yield the corresponding
aromatic amines. This bacterial decolorization of MR by
the isolated bacterium required fewer nutrients and was
less sensitive to the environmental changes. These
advantages allow the use of this bacterium in the treatment
of azo dyes in industrial effluents with ever-changing
physicochemical properties. We could then suggest the
immobilization of this bacterium on various carriers to be
used in a continuous process for the decolorization of azo
dyes in industrial effluents.
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