Chemosphere 68 (2007) 394–400

www.elsevier.com/locate/chemosphere

Technical Note

Decolourization of azo dye methyl red by Saccharomyces cerevisiae

MTCC 463
J.P. Jadhav, G.K. Parshetti, S.D. Kalme, S.P. Govindwar *

Department of Biochemistry, Shivaji University, Kolhapur 416 004, Maharashtra, India

Received 13 May 2006; received in revised form 11 December 2006; accepted 15 December 2006
Available online 9 February 2007

Abstract

Saccharomyces cerevisiae MTCC 463 decolourizes toxic azo dye, methyl red by degradation process. Methyl red (100 mg l1) is
degraded completely within 16 min in plain distilled water under static anoxic condition, at the room temperature. Effect of physico-
chemical parameters (pH of medium, composition of medium, concentration of cells, concentration of dye, temperature and agitation)
on methyl red decolourization focused the optimal condition required for decolourization. Biodegradation (fate of metabolism) of
methyl red in plain distilled water was found to be pH dependent. Cells of Saccharomyces cerevisiae could degrade methyl red efficiently
up to 10 cycles in plain distilled water. Analysis of samples extracted with ethyl acetate from decolourized culture flasks in plain distilled
water (pH 6.5) and at pH 9 using UV–VIS, TLC, HPLC and FTIR confirm biodegradation of methyl red into several different meta-
bolites. A study of the enzymes responsible for the biodegradation of methyl red in the control and cells obtained after decolourization in
plain distilled water (pH 6.5) and at pH 9 showed different levels of the activities of laccase, lignin peroxidase, NADH-DCIP reductase,
azoreductase, tyrosinase and aminopyrine N-demethylase. A significant increase in the activities of lignin peroxidase and NADH-DCIP
reductase was observed in the cells obtained after decolourization in plain distilled water (pH 6.5), however cells obtained at pH 9 shows
increased activities of azoreductase, tyrosinase, lignin peroxidase and NADH-DCIP reductase. High efficiency to decolourize methyl red
in plain distilled water and low requirement of environmental conditions enables this yeast to be used in biological treatment of industrial
effluent containing azo dye, methyl red.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Saccharomyces cerevisiae; Methyl red; Azo dyes; Decolourization; Biodegradation; Yeast; Azoreductase

1. Introduction
Large numbers of chemically different dyes are used for
various industrial applications and significant proportion
appears in the form of wastewater and is spilled into the
environment (Meyer, 1981). Among these, azo dyes are
prominent class of colourants used in tattooing, cosmetics,
printing, consumer’s products as well as in textile dying
because of their chemical stability and versatility. Their
durability however causes pollution once the dyes are
released in to the environment. In addition, some azo dyes
are toxic and mutagenic (Holme, 1984). Several physical
*
Corresponding author. Tel.: +91 231 2609152; fax: +91 231 2691533.
E-mail address: spg_biochem@unishivaji.ac.in (S.P. Govindwar).
and chemical methods have been suggested for the treat-
ment of dye contaminated wastewater but not widely used
because of the high cost and secondary pollution that can
be generated by excessive use of chemicals. In contrast,
microbial degradation of these dyes does not have similar
problem so it is necessary to establish biological wastewa-
ter treatment of azo dye considering enzymes involved in
it. Degradation of azo dye is feasible only if the azo linkage
is first cleaved.
Saccharomyces cerevisiae cells also showed bioaccumula-
tion of diazo reactive textile dye (Remazol Blue, Remazol
Black B and Remazol Red RB) during the growth in molas-
ses (Aksu, 2003). In comparative study on biosorption
capacities of different kinds of dried yeasts for Remazol
Blue, Candida lipolytica showed highest dye uptake capacity

0045-6535/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2006.12.087
 J.P. Jadhav et al. / Chemosphere 68 (2007) 394–400
up to 250 mg g1 cells (Donmez, 2002; Aksu and Donmez,
2003). The highest adsorption capacity found for aniline
blue was 200 mg g1 dried magnetic adsorbent on magnet-
ically modified S. cerevisiae cells (Šafařı́ková et al., 2005),
while thermo tolerant yeast, Kluyveromyces marxianus
IMB3 have capability to decolourize Remazol Black B
(Meehan et al., 2000). S. cerevisiae cells have a capacity to
decolourize malachite green within 7 h in plain distilled
water by biodegradation (Jadhav and Govindwar, 2006)
and magnetized cells have higher efficiency to absorb mala-
chite green.
This study encouraged us to find out the possibility of
S. cerevisiae cells for biosorption/biodegradation of azo
dye-methyl red. As yeast cells of genus Saccharomyces
are non-pathogenic, easily available and an economical
biomass may be used for removal of toxic azo dye methyl
red from textile industry effluent.
2. Materials and methods
2.1. Organism and culture conditions
S. cerevisiae MTCC 463 strain used in the present study
was obtained from Institute of Microbial Technology
Chandigarh, India, and was routinely maintained on 5%
(w/v) glucose, 1% peptone, 1% yeast extract and 3% agar.
The cells used in dye degradation studies were always
grown in medium contain 5% (w/v) glucose, 1% peptone,
1% yeast extract, for 23 h after addition of inoculum 10%
(v/v) at room temperature (30 ± 2 °C). The cells were col-
lected by centrifugation at 5000 rpm for 7 min under cold
conditions (+4 °C). Cells were rinsed once with distilled
water to remove the medium.
2.2. Dyes and chemicals
Methyl red dye was obtained from Himedia laboratories
India, ABTS (2,2 0 -Azino-bis (3-ethyl benzothiazoline-6-sul-
fonic acid) and lysing enzyme (Rhizoctonia solani), NADH
purchased from Sigma Chemical Company (USA),
whereas n-propanol, catechol, tartaric acid and other fine
chemicals were obtained from SRL Chemicals, India.
2.3. Decolourization experiments
All decolourization experiments were carried out at sta-
tic anoxic condition, at room temperature (30 ± 2 °C),
methyl red concentration (100 mg l1) and 2 g cells unless
otherwise stated. The time required for complete decolou-
rization (measured at 420 nm, OD was 0.0) of methyl red
was noted.
Effect of different methyl red concentrations on biode-
gradation by S. cerevisiae cells was studied taking three dif-
ferent methyl red concentrations 100, 200 and 300 mg l1
in a set of three flasks. To study the effect of cell mass on
degradation, 0.1, 0.5, 1 and 2 g cell weights were used in
a set of four flasks. Five different medium compositions
395
were used to study the effect of medium on degradation
as: (100 ml): (a) plain distilled water; (b) distilled water with
5% glucose; (c) distilled water with 1% glucose, 0.1% pep-
tone and 0.1% yeast extract; (d) distilled water with 0.1%
peptone; (e) distilled water with 0.1% yeast extract. Degra-
dation was studied under shaking condition at 150 rpm.
In the study of effect of temperature and pH on methyl red
degradation, the flasks were kept at different temperatures 0,
30, 40 and 50 °C for 30 min and pH 3, 5, 7 and 9. pH of
the medium was adjusted with diluted HCl and NaOH.
2.4. Preparation of cell free extract
(1 ! 3)-b-glucanase with protease activity was used to
lyse the cell wall since it is made up of a-glucan, b-glucan,
mannan and chitin. This treatment inactivates lignin perox-
idase, azoreductase hence another extraction method was
used for the preparation of enzyme. Standard methods
were used for preparation of enzyme extract in case of
NADH-DCIP reductase and aminopyrine N-demethylase
activity (Jadhav and Govindwar, 2006)
2.4.1. Enzymatic treatment
Cells collected from nutrient medium contained 5% glu-
cose, 1% peptone and 1% yeast extract (control cells). These
cells, after decolourization, in plain distilled water (pH 6.5)
and at pH 9 were used for the determination of enzyme
activity. Lysing enzyme from Rhizoctonia solani was pre-
pared by dispersing 10 mg lyophilized powder
in 10 ml 0.1 M phosphate buffer (pH 7.4), which was centri-
fuged and the supernatant was used. Five hundred milli-
gram of cells were suspended in 2 ml lysing enzyme and
the mixture was incubated at 37 °C for 30 min. The
reaction mixture was chilled thoroughly and sonicated
(Sonics-vibracell ultrasonic processor), keeping sonifier
output at 60 A and giving five strokes each of 10 s, at
1 min intervals, at 4 °C. The extract was centrifuged for
3000 rpm for 10 min and used as enzyme source. The super-
natant obtained by this method was used for testing laccase
and tyrosinase activity in both control and in cells obtained
after decolourization in both the condition (Jadhav and
Govindwar, 2006).
2.4.2. Sand homogenization
S. cerevisiae cells (1 g) were homogenized with sand in
1 ml 50 mM phosphate buffer (pH 7.4) using a mortar
and pestle, centrifuged and the supernatant was used for
testing lignin peroxidase and azoreductase activity in both
the control and cells obtained after decolourization.
2.4.3. Sonication
S. cerevisiae cells (500 mg) were suspended in 5 ml
50 mM phosphate buffer, chilled properly and sonicated
keeping sonifier output at 60 A, giving three strokes each
of 30 s, at 2 min intervals, at 4 °C. The cell suspension
was used for studying NADH-DCIP (Dichlorophenol indo-
phenol) reductase and aminopyrine N-demethylase activity.
396 J.P. Jadhav et al. / Chemosphere 68 (2007) 394–400
2.5. Enzyme assays
Laccase activity was determined by taking 10% 2,2 0 -
Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)
in 0.1 M acetate buffer (pH 4.9) at room temperature. Oxi-
dized ABTS was measured at 420 nm. The volume of reac-
tion mixture maintained was 2 ml (Hatvani and Mecs,
2001). Tyrosinase activity was determined in a reaction
mixture containing 0.01% catechol in 0.1 M phosphate buf-
fer (pH 7.4). The formed catechol quinone was measured at
410 nm at room temperature by keeping volume of the
reaction mixture at 2 ml (Zhang and Flurkey, 1997). Lignin
peroxidase activity was determined by monitoring the for-
mation of propanaldehyde at 300 nm in a reaction mixture
containing 100 mM n-propanol, 250 mM tartaric acid,
10 mM H2O2 in 2.5 ml reaction mixture (Shanmugam
et al., 1999). In all assay procedures blank contained all
components except enzyme. One unit of enzyme activity
was defined as a change in absorbance units min1 ml1
of enzyme.
NADH-DCIP reductase activity was determined by
using a procedure reported earlier (Salokhe and Govind-
war, 1999). The assay mixture contained 50 lM DCIP,
28.57 mM NADH in 50 mM potassium phosphate buffer
(pH 7.4) and 0.1 ml of enzyme solution (sonicated cells
suspension) in a total volume of 5.0 ml. The DCIP reduc-
tion was calculated using the extinction coefficient of
19 mM1 cm1.
The azoreductase activity was determined spectrophoto-
metrically at 25 °C using Hitachi UV–VIS spectrophotom-
eter by monitoring NADH disappearance at 340 nm based
on the procedure described by Zimmerman et al. (1982).
Enzyme preparation was added to 50 mM potassium phos-
phate buffer (pH 7.4) containing 0. 350 mM NADH and
110 lM methyl red in a total volume of 1.0 ml. One unit
of enzyme activity was defined as 1 mg of protein catalyzes
the oxidation of 1 lmol of NADH min1.
The assay of aminopyrine N-demethylase activity
included incubations in 50 mM N-2-Hydroxymethyl piper-
azine-N-2 ethane sulfonic acid buffer (pH 7.8), containing
NADPH-generating system (NADPH 2.6 mM, glucose
6-phosphate 12.5 mM, Glucose 6-phosphate dehydroge-
nase 4 units) and 0.5 ml cell suspension (50 mg cells) ml1
of incubation medium. After the addition of 0.4 ml of ami-
nopyrine (80 mM) to the incubation medium, the reaction
mixture was incubated at 37 °C for 10 min and adding 1 ml
of ice cold 20% trichloroacetic acid solution terminated the
reaction. The amount of formaldehyde liberated was deter-
mined colourimetrically using Nash reagent (Salokhe and
Govindwar, 1999).
2.6. Analytical procedure
2.6.1. HPLC, UV–VIS spectral analysis, FTIR and TLC
The metabolites produced during the biodegradation of
methyl red at 0 h (control) after 15 min in plain distilled
water (pH 6.5) and after 1 h at pH 9 were extracted with
equal volumes of ethyl acetate. The extracts were dried over
anhydrous Na2SO4 and evaporated to dryness in rotary
evaporator. The crystals obtained were dissolved in small vol-
ume of HPLC-grade methanol and the same sample was used
for HPLC, UV–VIS spectral analysis, Fourier Transform
Infrared Spectroscopy (FTIR) and Thin Layer Chromatog-
raphy (TLC) analysis. HPLC analysis was carried out on a
Waters model equipped with a dual UV–VIS detector and
a C18 column; the mobile phase used was acetonitrile:
water in a proportion of 50:50 with flow rate 0.5 ml min1
for 10 min. UV–VIS spectral analysis was carried out using
Hitachi UV–VIS spectrophotometer (UV 2800) and
changes in its absorption spectrum (400–800) were
recorded. FTIR analysis was carried out using Perkin
Elmer 783 Spectrophotometer and changes in percentage
transmission at different wavelengths were observed. TLC
analysis was carried out on silica gel using mobile phase
of methanol and distilled water in a proportion of 9:1.
The samples of 0 h (control), after 15 min in plain distilled
water (pH 6.5) and after 1 h at pH 9 were analyzed and the
spots were developed using an iodine chamber.
3. Results and discussion
Yeast cells represent an inexpensive and promising
material for removal of azo dye methyl red from textile
dye effluents. When decolourization capacity of S. cerevi-
siae MTCC 463 cells in plain water without adding any
organic or inorganic compounds tested for methyl red
(100 mg l1) at room temperature (30 °C ± 2) under static
anoxic condition, cells decolourize methyl red completely
within 16 min by biodegradation, however, it took 60 min
at shaking condition. Autoclaved cells did not show deco-
lourization activity (data not shown). Microscopic obser-
vation of cells obtained during decolourization shows no
adsorption of methyl red on cell membrane as observed
in case of malachite green (Jadhav and Govindwar, 2006)
indicates decolourization might be due to extracellular
azoreductase. The activities tested in the supernatant after
decolourization, showed only presence of azoreductase
(0.0047 units min1 mg protein1).
In the next part of our studies we have tried to find out
effect of various conditions on the decolourization of methyl
red (Table 1). Methyl red was decolourized completely
within 16 min when the concentration of the dye was
100 mg l1, while higher dye concentration (200 and
300 mg l1) requires more time for complete decolouriza-
tion. The time required for the complete decolourization
of methyl red was significantly reduced as cell mass increases
(100 mg–2 g). Effect of various medium compositions on the
decolourization of methyl red (100 mg l1) when tested
showed complete decolourization at different time intervals.
The best results were obtained, when plain distilled water
was used as the medium, it takes only 16 min. The results
indicate influence of medium composition and faster bio-
degradation of methyl red when compared to complete de-
colourization of methyl red 100 mg l1 within 6 h by
 J.P. Jadhav et al. / Chemosphere 68 (2007) 394–400 397

Table 1
Time required for decolourization of methyl red by Saccharomyces cerevisiae at different methyl red concentrations, cell concentrations, in different media,
different pH and different temperature in static condition
Different methyl red concentration (mg l1) 100 200 300
Time required (min) 17 ± 2 21 ± 3 25 ± 2
Different cell wet weight (g) 0.1 0.5 1 2
Time required (min) 1440 ± 10 100 ± 5 45 ± 3 17 ± 2
Different medium A B C D
Time required (min) 18 ± 2 25 ± 3 30 ± 2 45 ± 2
Different temperature (°C) 0 30 40 50
Time required (min) 45 ± 3 16 ± 2 09 ± 1 06 ± 1
Different pHa 3.0 5.0 7.0 9.0
Time required (min) 17 ± 2 17 ± 2 45 ± 3 60 ± 3
All experiments were performed using 100 ml distilled water in 250 ml conical flask at room temperature (30 °C ± 2), pH 6.5 and static condition using 2 g
cells and 100 mg l1 methyl red concentration unless otherwise stated.
(A) Distilled water with 5% glucose, (B) Distilled water with 1% glucose, 0.1% peptone and 0.1% yeast extract, (C) Distilled water with 0.1% peptone and
(D) Distilled water with 0.1% yeast extract.
a
pH is adjusted using 0.1 N HCl or NaOH.

Enterobacter agglomerans under aerobic condition in syn-
thetic medium (Moutaouakki et al., 2003a). Decolouriza-
tion ability of cells when tested by repeated use of the
same cells, it could degrade methyl red efficiently through
ten cycles but the time required was 30 min for the last cycle.
Decolourization of methyl red (100 mg l1) at different tem-
perature at 0, 30, 40 and 50 °C showed change in time
required as 45, 16, 9 and 6 min respectively. More time
required at 0 °C may be because of less activity of the
enzymes. These results indicate temperature effect on meta-
bolic rate and enzymes potential to express in a wide range
of temperatures (0–50 °C) so this strain can be used at low
temperatures. pH of the medium also affects the decolouri-
zation time (methyl red, 100 mg l1) and fate of metabolism
by S. cerevisiae cells (Table 1).
3.1. Enzymatic analysis
The major mechanism of biodegradation in living cells
is realized because of the lignin modifying enzymes, lac-
case, manganese peroxidase, lignin peroxidase, tyrosinase
and to some extent by N-demethylase to mineralize syn-
thetic dyes (Raghukumar et al., 1996). The relative contri-
butions of laccase, manganese peroxidase, lignin
peroxidase to decolourization of dyes may be different
for each fungus viz. lignin peroxidase in Phanerochaete
chrysosporium (Pasti-Grigsby et al., 1992; Ollikka et al.,
1993).
Previous studies on biological decolourization of the azo
dyes mainly focused on the azoreductases present in micro-
organisms since they catalyses reductive cleavage of azo
groups (–N=N–) primarily responsible for the biodegrada-
tion. Reductive cleavage of methyl red and related dye was
due to the azoreductase (Moutaouakki et al., 2003b), which
is a monomer with molecular weight 28 000, and requires
NADH. Degradation of methyl red by mixed culture iso-
lated from a domestic waste water treatment plant was
found to remove the colour of azo dye methyl red within
18 h, it degrades 700 mg l1 of methyl red in presence of
glucose while in absence it could degrade only 100 mg l1
(Vijaya and Sandhya, 2003). Decolourization of methyl
red by mixed culture of Bacillus sp. and Pseudomonas

Table 2
Activities of laccase, lignin peroxidase, NADH-DCIP reductase, tyrosinase, azo reductase and aminopyrine N-demethylase in control Saccharomyces
cerevisiae cells and cells obtained after decolourization in plain distilled water at pH 6.5 and pH 9.0
Enzymes Control cells Cells obtained after decolourization at Cells obtained after decolourization at
pH 6.5 in plain distilled water pH 9.0 in plain distilled water
Laccasea 0.196 ± 0.009 0.095 ± 0.010*** 0.006 ± 0.003***
Lignin peroxidasea 0.072 ± 0.013 0.25 ± 0.026*** 0.16 ± 0.006*
NADH-DCIP reductaseb 5.67 ± 0.080 7.47 ± 0.150*** 6.70 ± 0.050**
Tyrosinasea NA NA 0.025 ± 0.003***
Azo reductasec 0.0066 ± 0.001 0.0105 ± 0.002*** 0.0138 ± 0.001***
Aminopyrine N-demethylased 5.09 ± 0.140 2.23 ± 0.150*** NA
NA – No activity.
Values are mean of three experiments ± SEM, Significantly different from control cells at *P < 0.05, **P < 0.01 and ***P < 0.001 by two tail P – values
comparison test.
a
Units min1 ml1.
b
lg of DCIP reduced min1 mg cells1.
c
Units min1 mg protein1.
d
nmol of formaldehyde liberated min1 mg protein1.
398 J.P. Jadhav et al. / Chemosphere 68 (2007) 394–400

stutzeri in LB medium was also reported (Itoh et al., 2002).

Methyl red was degraded (56%) by Irpex lacteus and Pleu-
rotus ostreatus within 14 d in liquid medium (Novotny
et al., 2001). The microorganisms reported earlier having
capacity to degrade azo dyes are Acetobactor liquefaciens
(So et al., 1990) and Klebsiella pneumoniae RS-13 (Wong
and Yuen, 1996). Decolourization and detoxification of
methyl red by aerobic bacteria from wastewater treatment
plant have capacity to decolourize complete dye at pH 6
within 6 h at methyl red concentration 5 mg l1 (Adedayo
et al., 2004). While E. agglomerans completely decolourizes
100 mg l1 methyl red with in 6 h under aerobic condition
in synthetic medium (Moutaouakki et al., 2003a) and this is
the fastest degradation reported earlier. As most of the
information on biodegradation of synthetic dyes by ligno-
lytic fungi has been obtained with P. chrysosporium (Cripps
et al., 1990; Paszczynski and Crawford, 1995).
Data showed in Table 2 represents the enzymatic activ-
ities present in the control cells, the cells obtained after dec-
olourization in plain distilled water (pH 6.5) and at pH 9.
Azoreductase, NADH-DCIP reductase, laccase, lignin per-
oxidase and aminopyrine N-demethylase activity was Fig. 2. TLC analysis of metabolites of methyl red extracted with ethyl
found to be present in the control cells. An induction in acetate at 0 h (control), products formed after 15 min at pH 6.5, and
the activity of NADH-DCIP reductase and lignin peroxi- products formed after 1 h at pH 9.0 after addition of S. cerevisiae cells.
dase was observed in the cells obtained after decolouriza-
tion in plain distilled water (pH 6.5) however, cells at pH In the present study we have analyzed the products of
9 showed increased activities of azoreductase, NADH- biotransformation of methyl red by HPLC, UV–VIS spec-
DCIP reductase, tyrosinase and lignin peroxidase. A tral analysis, FTIR and TLC. The absorption spectrum
significant increase in the activities of azoreductase and obtained for control and sample obtained after decolouri-
tyrosinase in the cells at pH 9 might be responsible for zation at pH 6.5 and 9 showed no change in wavelength
change in the biodegradation fate of methyl red when com- shift. The absorption shift of methyl red was observed in
pared to that of cells from plain distilled water (pH 6.5).
The results indicate prominent role of azoreductase in the 0.12
0.10
a
decolourization process at this set of conditions. This sup-
0.08
ports earlier observations (Wong and Yuen, 1996; Chen AU 0.06
et al., 2005). 0.04
0.02
0.00
1
0.07 b
0.9 0.06
0.05
0.8 0.04
AU 0.03
0.7 0.02
Optical density

0.6 0.01
0.00
0.5
0.45 c
0.40
0.4 0.35
0.30
0.3 0.25
AU 0.20
0.2 0.15
0.10
0.1 0.05
0.00
0
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0
400 440 480 520 560 600 640 680 720 760 800 Min
Wavelength in nm
Fig. 3. HPLC analysis of products extracted with ethyl acetate formed by
Fig. 1. UV–VIS spectra of extracted sample at 0 h at pH 6.5 (n) and pH degradation of methyl red: (a) at 0 h (control), (b) products formed after
9.0 (h) (control), products formed after 15 min at pH 6.5 (m) and 15 min at pH 6.5 and (c) products formed after 1 h at pH 9.0 after addition
products formed after 1 h at pH 9.0 (j). of S. cerevisiae cells.
 J.P. Jadhav et al. / Chemosphere 68 (2007) 394–400 399

peak from 490 nm to 430 nm due to change in pH (Fig. 1).
Decrease in the absorption indicates that decolourization
of this dye occurred by degradation in addition to the
visual observation of decolourization. The products col-
lected at 0 h (control), after 16 min in plain distilled water
(pH 6.5) and 1 h at pH 9 after addition of cells were
extracted with ethyl acetate, crystallized, dissolved in meth-
anol and used for further analysis. TLC analysis showed
the appearance of three spots in sample of 1 h at pH 9,
immediately after decolourization compared to control
(0.93), RF (Retardation factor) values at 0.90, 0.81, 0.70
and only one spot with RF value at 0.90 in the sample of

75

70

Control methyl red 579.81

2922.52 543.03
65 726.03
635.75
688.20
60 1547.47 941.14 765.81
1717.14 1528.65 1230.28 889.23
%T 1482.82 827.69
1465.45
55

50 1311.45
1276.86
1112.91
45 1364.11
1600.76 1145.91
a
41

80

79

78 Metabolites at pH 6.5

77

1512.23
%T 76
2847.82 1197.90 1020.86
1717.92 1421.91 1168.99
75

2924.50
74

73 3422.39
b
72

84

83

82

%T 81

Metabolites at pH 9.0
80 9

c
79
3434.82 1371.32 1230.41 1020.86
2924.04 1615.08

78
4000 3000 2000 1500 1000 450
-1
cm
Fig. 4. FTIR spectra of products extracted with ethyl acetate formed by degradation of methyl red: (a) at 0 h (control), (b) products formed after 15 min at
pH 6.5 and (c) products formed after 1 h at pH 9.0, after addition of S. cerevisiae cells.
400 J.P. Jadhav et al. / Chemosphere 68 (2007) 394–400
15 min at pH 6.5 (Fig. 2). HPLC analysis showed a pro-
minent peak at retention time 1.75 in distilled water sample
(pH 6.5) and one peak at retention time 2.60 along with
three shoulder peaks at 2.94, 3.76 and 3.95 in the sample
of 1 h at pH 9 which are different than control methyl red
retention time (2.1) (Fig. 3a–c). Results of FTIR analysis
of both the samples obtained after decolourization showed
absence of peak at 1600 cm1 indicates breakdown of azo
bond, might be due to action of azoreductase. Absence of
peaks at 688 cm1, 726 cm1, 765 cm1 and 827 cm1 indi-
cates loss of aromaticity or benzene ring. FTIR spectrum of
sample at pH 6.5 shows peaks at 1168 cm1 and 1197 cm1
indicating production of primary and secondary amines.
However peaks at 1421 cm1, 1512 cm1 shows further
conversion of aliphatic primary and secondary amines into
nitroalkanes (Fig. 4b). FTIR analysis of the sample
obtained after decolourization at pH 9 showed peaks
at 1615 cm1 and 1371 cm1 indicating synthesis of ali-
phatic secondary amines and dimethyl groups, respectively
(Fig. 4c). It seems that azoreductase catalyses the reductive
cleavage of the azobond of methyl red and produces 2-
aminobenzenzoic acid and N,N 0 -dimethyl-p-phenylenedi-
amine using NADH as electron donor (Moutaouakki
et al., 2003b). These products are further degraded into ali-
phatic amines that might be facilitated by oxidative
enzymes such as lignin peroxidase and laccase.
Thus it may be concluded that S. cerevisiae MTCC 463
effectively decolourizes methyl red due to biodegradation
and it shows different fate of metabolism at different pH.
Enzymatic studies indicate the involvement of azoreductase
as prominent enzymes for the biodegradation at this set of
conditions. This study provides the initial step of a biodeg-
radation process, with further detoxification by different
micro flora present in the environment.
Acknowledgements
The study is supported by Department of Science and
Technology, New Delhi, India. (SERC Fast track Young
Scientist Scheme to Dr. (Mrs.) J. P. Jadhav).
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