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RESEARCH ARTICLE
Microbial Degradation and Detoxification of Synthetic DyeMixture by Pseudomonas sp. SUK 1
Amit S. Chougule • Shekhar B. Jadhav •
Jyoti P. Jadhav
Received: 16 August 2013 / Revised: 4 December 2013 / Accepted: 17 January 2014
� The National Academy of Sciences, India 2014
Abstract Textile industry is the major source of colored
effluents constituting a number of complex dyes affecting
terrestrial and aquatic ecosystems which necessitates their
removal from environment. Bioremediation offers an
inexpensive and eco-friendly approach hence, Pseudomo-
nas sp. SUK 1, which is already known for its potential in
degradation of individual dyes is used to degrade synthetic
dye mixture of eight structurally different dyes. The bac-
terium was capable to remove 94.3 % American Dye
Manufacturing Institute value of dyes mixture, within 24 h
under static condition at pH 7 and 30 �C temperature. The
results from the batch experiments revealed the ability of
the tested bacterium to remove the synthetic dye mixture
after repeated exposure. Induction in the activities of var-
ious biotransformation enzymes like laccase, veratryl
alcohol oxidase, nicotinamide adenine dinucleotide-2,
6-dichlorophenol-indophenol reductase and tyrosinase was
observed, indicating the significant role of these enzymes
in biodegradation. After treatment significant decrease in
chemical oxygen demand and biological oxygen demand
was observed. The biotransformation of synthetic dye
mixture was confirmed by UV–Vis spectroscopy, high
performance liquid chromatography and Fourier transform
infrared spectroscopy analyses of samples before and after
decolorization. The toxicity analysis of degraded metabo-
lites formed after biotransformation was carried out on
Triticum aestivum, Phaseolus mungo and Sorghum vulgare
crop plants with respect to germination ability, shoot and
root length analysis. However oxidative stress study was
carried out on Allium cepa L. proved that degraded
metabolites were less toxic. Thus biodegradation of com-
plex synthetic dye mixture to non-toxic metabolites using
Pseudomonas sp. SUK 1 would be a better option for
biological treatment of textile effluents.
Keywords Biodegradation � Enzyme assay �Oxidative stress � Phytotoxicity � Synthetic dye mixture
Introduction
The revolutionary research in 18th century gave birth to
synthetic dyes which proved to be a breakthrough in dyeing
and textile processing. However, increased use of dyes in
textile industries has resulted into severe problems of water
pollution due to effluents, contaminated with dyestuffs.
Throughout the world annual consumption of dyes is
around 7 9 104 tones and out of which 50 % of dyes are
lost in processing and manufacturing units [1]. Dyes are
designed to be chemically stable thus they introduce
potential danger of bioaccumulation that eventually affects
human beings by transport through the food chain. Due to
discharge of colored effluents into natural water bodies, the
sunlight penetration is decreased which reduces both pho-
tosynthetic activity and dissolved oxygen concentration [2]
and disturbs ecology of water. Structural changes in red
and white blood cells of fishes were recorded due to stress
caused by such eco-toxic effluents [3]. Toxic effects of
A. S. Chougule
Department of Biotechnology and Bioinformatics, Padmashree
Dr. D.Y. Patil University, Navi Mumbai 400614, India
S. B. Jadhav � J. P. Jadhav
Department of Biochemistry, Shivaji University,
Kolhapur 416004, Maharashtra, India
J. P. Jadhav (&)
Department of Biotechnology, Shivaji University,
Kolhapur 416004, Maharashtra, India
e-mail: [email protected]
123
Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.
DOI 10.1007/s40011-014-0313-z
textile dyes are not only limited to aquatic flora and fauna
but there are some reports which are indicative of transport
of dyes through food chain. Contamination of hot chilli,
baked foods and other spices with azo dyes [4] which are
known as carcinogenic [5] and genotoxic [6] in nature
leading to exposure in the human gastrointestinal tract.
Increased amount of such effluents and their hazards gen-
erated a need to concentrate upon the proper waste man-
agement tasks.
Several physico-chemical decolorization methods have
been developed during past two decades which are still
being used as routine practice in textile industries [7, 8].
These methods have major disadvantages as they are
highly expensive, coupled with formation of sludge and
emission of toxic substances which result into secondary
pollution [9, 10]. Therefore, cost-effective and eco-friendly
methods were required to treat the effluents before their
discharge into water bodies. Biological treatment fulfills
these requirements along with several other attractive
benefits [11, 12]. Thus microbial decolorization has
received much attention due to the ability of microbes to
survive under extreme conditions and its cost-effectiveness
[13]. Recently, several reports showed that microorganisms
have ability, not only to decolorize dyes but also to
detoxify them [14–16]. Thus currently an extensive
research work is carried out to find out optimal microbial
biomass which is versatile and capable of treatment of
large volume of effluents [17]. Many studies are focused on
decolorization of single textile dye. However, the effluent
released from textile industry contains mixture of different
dyes thus there is a need to study the capability of single
microorganism to degrade variety of dyes.
Present study is focused on decolorization and biodegra-
dation of a synthetic dye mixture (SDM) by using the bacterial
strain Pseudomonas sp. SUK 1. Furthermore, not only the
effects of various physico-chemical parameters (temperature,
pH and SDM concentrations) on degradation capability were
studied but also the role of enzymes in decolorization of SDM
was determined. Various analytical techniques such as UV–
Vis spectroscopy, high performance liquid chromatography
(HPLC) and Fourier transform infrared spectroscopy (FTIR)
were used to confirm the degradation of dyes present in SDM.
Additionally chemical oxygen demand (COD), biological
oxygen demand (BOD) and toxic nature of SDM before and
after dye decolorization was determined.
Material and Methods
Chemicals and Textile Dyestuffs
All chemicals required were of analytical grade and
obtained from Sigma Aldrich. Solvents were purchased
from Hi-media Laboratories Pvt. Ltd., Mumbai, India and
Sisco Research Laboratory (SRLs), India. The textile dyes
viz. Remazol Orange 3R, Scarlet RR, Brown 3REL,
Golden yellow HER, Remazol Red, Cotton Blue, Ama-
ranth and Orange 2RX were obtained from textile indus-
tries in Ichalkaranji, India.
Media Preparation and Maintenance of Microorganism
The strain Pseudomonas sp. SUK 1 procured from
Department of Biochemistry, Shivaji University, Kolhapur
was chosen for the study because of its high efficiency in
dye decolorization. The pure culture was maintained on
nutrient agar slants at 4 �C. The decolorizing medium
consists of peptone 5 g, NaCl 5 g, yeast extract 1.5 g and
vegetable extract 1.5 g per liter of distilled water and has
pH 7.4 ± 0.2. Prior to addition of SDM the medium was
sterilized by autoclaving at 121 �C and 15 lb pressure for
20 min.
SDM Decolorization
The degradation ability of microorganism towards various
dyes was evaluated and dyes showing better decolorization
(Remazol Orange 3R, Scarlet RR, Brown 3REL, Golden
yellow HER, Remazol Red, Cotton Blue, Amaranth,
Orange 2RX) were selected for the preparation of SDM.
The stock dye mixture of 2,400 mg l-1 concentration was
prepared by adding 15 mg of each of these dyes in 50 ml
distilled water. The concentration of SDM in the nutrient
media was 80 mg l-1 during the study. Decolorization of
SDM was monitored using American Dye Manufacturing
Institute (ADMI) 3WL tristimulus method reported earlier
by Waghmode et al. [18]. Abiotic controls (with died cells
and without cells) were always included.
COD and BOD of SDM
The BOD and COD [19] was measured before and after
SDM degradation by using Hanna BOD meter and auto-
mated COD analyzer (Spectralab CT 15, India)
respectively.
Effect of Various Physico-chemical Parameters
The 24 h grown culture broth of Pseudomonas sp. SUK 1
in the nutrient medium at 30 �C, under static condition was
used to study effect of physico-chemical parameters. The
SDM was added into culture broth and kept at static and
shaking (120 rpm) conditions. The decolorization activity
was recorded in both conditions. Effect of variable pH (4,
5, 6, 7, 8, 9 and 10) on the decolorization performance was
studied. The pH of media was adjusted with the help of
A. S. Chougule et al.
123
0.1 M NaOH and 0.1 N HCl by using pH meter (Thermo
scientific, Eutech instruments model-pHTestr20) prior to
addition of SDM. Similarly the effect of various tempera-
tures (10, 20, 30, 40 and 50 �C) on the decolorization
performance was studied. Culture flasks were kept at
respective temperatures for 30 min before SDM addition to
attain the temperature and after SDM addition, incubation
was continued at respective temperatures. Effect of initial
SDM concentrations (26.6–133 mg l-1) on the decolor-
ization performance was studied.
Effect of Fed Batch Culture
A fixed concentration of SDM (80 mg l-1) was added into
the 24 h grown culture of Pseudomonas sp. SUK 1. After
decolorization, again fixed concentration of SDM was
added continuously into the decolorized broth (without
further addition of supplement) until the microorganism
loses its decolorization ability.
Enzyme Assays
The 24 h grown culture of Pseudomonas sp. SUK 1 was
centrifuged at 6,786 g for 25 min at 4 �C. Supernatant was
used as the source of extracellular enzymes. The biomass
of microorganism was separately resuspended in 50 mM
potassium phosphate buffer (pH 7.4) and homogenized,
which results into cell rupture. It was sonicated (sonics-
vibracell ultrasonic processor, 7 strokes of 30 S each for
30 min interval based on 40 amplitude output) at 4 �C.
This sample was further centrifuged (4 �C, at 6786 g for
25 min) and used as a source of intracellular enzymes.
Similar procedure was used to quantify the enzyme activ-
ities after SDM decolorization. The extracellular (cell free
broth) and intracellular (cell extract) activities of dye
degrading enzymes such as laccase, veratryl alcohol oxi-
dase (VAO) [20] and tyrosinase [21] were assayed at room
temperature (25 ± 2 �C). Laccase activity was monitored
with o-tolidine (50 mM) in a 2.1 ml reaction mixture
containing 1.8 ml buffer (acetate buffer, 0.1 M and pH
4.8), 0.2 ml o-tolidine and 0.2 ml enzyme. For VAO assay,
the 2 ml reaction mixture contained 4 mM veratryl alcohol
in 0.05 M citrate phosphate buffer, pH 3 and 0.2 ml
enzyme to start the reaction. For tyrosinase assay the 3 ml
reaction mixture contained 50 mM of catechol and 2.1 mM
of ascorbic acid in 50 mM potassium phosphate buffer (pH
6.5) equilibrated at 25 �C. The DA265 nm was monitored
until constant and then 0.1 ml of the supernatant from the
reaction mixture was added. The formation of o-benzo-
quinone and dehydro-ascorbic acid and decrease in optical
density was measured at 265 nm. One unit of tyrosinase
activity was equal to a DA265 nm of 0.001 per min at pH
6.5 at 25 �C in a 3 ml reaction mixture containing L-
catechol and L-ascorbic acid. The nicotinamide adenine
dinucleotide-2, 6-dichlorophenol-indophenol (NADH–
DCIP) reductase activity was assayed by modifying earlier
reported method [22]. DCIP reduction was monitored at
590 nm and calculated using an extinction coefficient of
0.019 lM-1 cm-1. The reaction mixture (5 ml) prepared
contained 25 lM substrate (DCIP) in 50 mM potassium
phosphate buffer (pH 7.4) and 0.1 ml enzyme. From this,
2 ml reaction mixture was assayed at 590 nm by addition
of 250 lM NADH. All the enzyme assays were run in
triplicates.
SDM Biodegradation Analysis
SDM decolorization was monitored after 24 h by taking
UV–Vis spectrophotometer (Shimadzu UV-1800 Spectro-
photometer, Tokyo, Japan) wavelength scan (400–800 nm).
The metabolites formed after decolorization of SDM were
extracted with two different solvent systems which were
ethyl acetate: toluene and dichloromethane: acetonitrile at
1:1 proportion, dried, dissolved in HPLC grade methanol
and used for HPLC and FTIR analysis [18].
Toxicological Studies
The phytotoxicity study was carried out using Phaseolus
mungo, Sorghum vulgare and Triticum aestivum seeds at
room temperature by watering 5 ml of dye solution
(1,200 ppm) and its formed metabolites (1,200 ppm)
obtained after degradation. Control set was carried out
using distilled water (daily 5 ml watering) at the same
time. Germination (%) and length of shoot and root was
recorded after 7 days. Germination % was calculated by
following formula as:
Germination ð%Þ ¼ No: of seeds germinated
No: of seeds sowed� 100
The oxidative stress, with respect to antioxidant enzymes
and lipid peroxidation, was studied in the bulbs of A. cepa.
The bulbs were cleaned and exposed to water for the
development of the roots. These roots developed bulbs which
were then grouped into three sets as (a) control (distilled
water treatment), (b) treated with SDM and (c) treated with
metabolites obtained after SDM degradation [23]. The bulbs
in each case were exposed to the respective treatment for
72 h. Antioxidant enzymes namely catalase (CAT, E.C.
1.11.1.6), superoxide dismutase (SOD, E.C. 1.15.1.1) and
guaiacol peroxidase (GPX, E.C. 1.11.1.7) were analyzed by
spectrophotometric measurements, using the procedure
reported earlier [24]. Lipid peroxidation was measured by
the method reported earlier [24], with slight modification.
Briefly, samples prepared from different target species as
described above were homogenized in 4 ml reaction mixture
Synthetic Dye Mixture Decolorization by Pseudomonas sp. SUK 1
123
containing 20 % (w/v) trichloroacetic acid (TCA) and 0.5 %
(w/v) thiobarbituric acid (TBA). The homogenate was
incubated at 95 �C for 30 min. The reaction was stopped
by placing the homogenate in ice. Afterwards homogenate
was centrifuged at 8,378 g for 15 min, followed by
measurement of absorbance of resulting supernatant at 532
and 600 nm. The nonspecific absorbance at 600 nm was
subtracted from the absorbance at 532 nm. The
concentration of malonyldialdehyde (MDA) was
calculated using an extinction coefficient (e = 155 mM-1
cm-1) and expressed in nmol g-1 FW.
Statistical Analysis
Data was analyzed by one-way analysis of variance
(ANOVA) and Turkey-Kramer Multiple Comparison Test.
Level of significance was studied at P value 0.5.
Results and Discussion
Decolorization of SDM
After screening various textile dyes, the SDM of eight
structurally different dyes as Remazol Orange 3R, Scarlet
RR, Brown 3REL, Golden Yellow HER, Remazol Red,
Cotton Blue, Amaranth, Orange 2RX was prepared and
taken for further study. The decolorization of dye mixture
was determined in terms of the ADMI value [25]. The
ADMI value provides an accurate measurement of water
color, independent of hue thus used in effluent and dye
mixture decolorization study [26]. There was no significant
decolorization of SDM in abiotic control (without cells). It
was also notable that there was no adsorption of dyes on
heat killed cells which indicates that only bacterial action
was responsible for the decolorization of SDM.
Effect of physico-chemical parameters (static; shaking
condition, pH, temperature and initial SDM
concentration) on decolorization
Bacterium showed 84 and 8.90 % ADMI removal at static
and shaking (120 rpm) conditions respectively after 24 h of
incubation which suggests that under the static condition
Pseudomonas sp. SUK 1 shows enhanced decolorization of
SDM than in shaking condition. Similar results were
observed in early reported studies with bacterial strains such
as P. desmolyticum and S. marcescens which show better
decolorization of dye under static condition than the shak-
ing condition [27, 28]. In the presence of shaking condition
only oxidative enzymatic system is present, however under
the static anoxic condition synchronized action of oxidative
as well as reductive enzymatic systems work for efficient
decolorization of dyes [29]. The maximum decolorization
was observed at the pH 7.0 (Fig. 1), however slight acidic
and basic shift of pH affected the percent ADMI removal
capacity of the bacterium steeply. However, maximum
94.3 % ADMI removal was recorded at temperature 30 �C
(Fig. 2). Maximum potential of Pseudomonas sp. to
decolorize Malachite green, Fast green, Brilliant green,
Congo red and Methylene blue [30] and Red BLI [31] was
noticed at 30 �C. Higher ADMI removal is achieved using
Pseudomonas sp. SUK 1 than previous reports on SDM
[18]. Optimum pH, temperature for decolorization is 7 and
30 �C respectively which are easy to maintain at static
condition for large scale treatment.
Different concentrations of the mixture were treated by
Pseudomonas sp. SUK 1 to prove its degradation potential.
With increasing initial SDM concentrations as 26.6, 53.2,
80 and 106 mg l-1 time required was observed to be
increased respectively (Fig. 3). Increasing concentrations
of SDM repressed the % ADMI removal and also
decreased the decolorization rate. The % ADMI removal
0
20
40
60
80
100
0 2 4 6 8 10
% A
DM
I R
emov
al
pH
Fig. 1 Effect of pH on decolorization of synthetic dye mixture
0
20
40
60
80
100
0 20 40 60
% A
DM
I R
emov
al
Temperature (°C)
Fig. 2 Effect of temperature on decolorization of synthetic dye
mixture
A. S. Chougule et al.
123
was strongly inhibited at 133 mg l-1 dye in the medium.
The increased dye concentrations might be inhibitory to the
bacterial enzyme system as it was observed in previous
study [20]. Reduction of color removal capacity of bacteria
due to increasing dye concentrations has been reported
earlier [32, 33].
Effect of Fed Batch Process
Consecutive 4 cycles of dye decolorization were studied by
the repeated additions of SDM (80 mg l-1) in the medium.
It showed the effective dye decolorization during these
cycles. The time required for the decolorization was
increased steadily. The decolorization occurred for first
cycle within 24 h and after that the subsequent cycles
required 30, 34 and 44 h respectively (Fig. 4). Similar
observations have been recorded previously for decolor-
ization of reactive dyes [34, 35]. It might be due to
decreased number of viable cells and exhaustion of nutri-
ents, in the medium. Thus, Pseudomonas sp. SUK 1 shows
the ability to decolorize repeated addition of SDM. It can
decolorize different types of dyes at the same time and at
several times, which is noteworthy for its commercial
applications.
BOD and COD Reduction
Generally textile wastewater has higher BOD and COD
values because of complex and recalcitrant nature of dyes
present in it. Efficient treatment of textile wastewater is
essential to decrease the BOD and COD values [36]. Ini-
tially recorded BOD of SDM containing microbial broth
was 700 mg l-1, which was reduced considerably to
500 mg l-1. Observed COD reduction of 25 % showed
partial mineralization of SDM. Considerable amount of
reduction in BOD and COD was recorded after 24 h of
microbial treatment.
Enzymatic Analysis
Enzymatic studies (Table 1) indicated the differences
between the enzyme activities present in the control and in
sample obtained after decolorization. Intracellular activi-
ties of laccase, NADH–DCIP reductase, VAO and tyrosi-
nase were present in the control cells. A significant
increase in the activities of NADH–DCIP reductase,
tyrosinase and laccase were observed in the cells obtained
after decolorization. In extracellular samples activities of
laccase, NADH–DCIP reductase and VAO were found to
be induced. It can be presumed that the major mechanism
of decolorization in the cells is mostly because of the
biotransformation enzymes viz. laccase, VAO, NADH–
DCIP reductase and tyrosinase. The relative contributions
of different biotransformation enzymes in decolorization of
dyes may be different for different microorganisms [37].
The induction of NADH–DCIP reductase, laccase, VAO
and tyrosinase enzymes showed their predominant role in
the decolorization process. This supports the earlier
observations of Kalme et al. [38]. The enzyme activity
without adding SDM after reaction was reduced which may
be due to limited availability of nutrients, cells entering in
death phase or enzyme turnover.
SDM Biodegradation Analyses
UV–Vis Spectral Analysis
UV–Vis analysis (400–800 nm) of supernatants obtained
after treatment of SDM with Pseudomonas sp. SUK 1 for
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25
% A
DM
I R
emov
al
Time (hr)
Fig. 3 ADMI removal (%) of synthetic dye mixture at different initial
concentrations ( 26.6 mg l-1, 53.2 mg l-1, 80 mg l-1,
106 mg l-1, 133 mg l-1)
0
20
40
60
80
100
0
10
20
30
40
50
1 2 3 4
% A
DM
I Rem
oval
Tim
e (h
r)
Cycles
Fig. 4 Synthetic dye mixture decolorization in fed batch process (
h, % ADMI removal)
Synthetic Dye Mixture Decolorization by Pseudomonas sp. SUK 1
123
24 h showed the decolorization and decrease in dye con-
centration (Fig. 5). Initially due to the presence of dyes in
control SDM UV–Vis spectrum showed higher absorbance
at kmax 510 nm but after the microbial treatment absor-
bance was significantly decreased.
High Performance Liquid Chromatography (HPLC)
Analysis
The HPLC analysis of the control SDM showed retention
time of 1.923, 2.117, 3.005, 2.883, 3.279 3.436 and
4.406 min (Fig. 6a), whereas metabolite sample obtained
after the biodegradation of dyes present in SDM (after
24 h) showed complete different profile. The retention time
of metabolites was found to be 2.466, 2.649, 3.251, 3.805
and 5.515 min which was different from the control sample
(Fig. 6b). Thus significant variations in the retention time
before and after microbial treatment was observed. This
confirmed the biodegradation of different dyes present in
the mixture into different metabolites.
FTIR Analysis
FTIR analysis was carried out to detect the presence of
various functional groups in SDM which are transformed
or removed after treatment by Pseudomonas sp. SUK 1
(Fig. 7). FTIR spectrum of the SDM showed the peaks at
3,232.80, 2964.69, 2887.53, 2349.38, 1666.55, 673.18,
1575.97 and 1408.08 cm-1 which suggest the presence
of free or bonded O–H stretching or N–H trans stretching,
–CH3 stretching, C–H stretching, NH3? stretching, C–S
stretching, C–S stretching, C–S stretching and S=O
stretching or C–OH deformation respectively. The FTIR
spectrum of metabolites obtained after degradation of the
SDM showed the peaks at 3261.74, 2949.26, 1442.80,
2351.30, 1680.05, 1535.39, 1267.27 and 690.54 cm-1
which suggest the presence of O–H stretching, –CH3
stretching, –CH3 stretching, NH?, C=C stretching, C=N
stretching, conjugated C–O–C stretching and C–S stretch-
ing. Metabolites obtained after decolorization of SDM by
Pseudomonas sp. SUK 1 showed differential FTIR spec-
trum than control SDM suggests the biodegradation of
SDM. Similar analysis of biodegradation of dyes using
FTIR has been reported earlier [36].
Toxicity Study
Phytotoxicity Study of SDM and its Degradation Product
Despite the fact that untreated dyeing effluents may cause
the serious environmental and health hazards, they are
being disposed off in water bodies and this water is used
for the agriculture purpose. Thus, assessment of phyto-
toxicity of the SDM before and after degradation becomes
necessary. Phytotoxicity studies on the germination of
Table 1 Enzyme activities in control (0 h), decolorized state (after 24 h) and without SDM (24 h)
Enzyme Intracellular Extracellular
Before
decolorization (0 h)
After decolorization
(24 h)
Without SDM
(24 h)
Before
decolorization (0 h)
After decolorization
(24 h)
Without SDM
(24 h)
Laccasea 0.14 ± 0.02 5.09 ± 0.03** 0.05 ± 0.02 0.14 ± 0.08 0.24 ± 0.03* 0.04 ± 0.02
VAOa 1.16 ± 0.06 1.17 ± 0.03 0.29 ± 0.02 0.82 ± 0.08 1.21 ± 0.11* 0.31 ± 0.04
Tyrosinasea 245 ± 31.8 553 ± 20.5** 92.3 ± 12.2 503 ± 21.3 532 ± 21.3 108 ± 17.1
NADH–DCIP reductaseb 322 ± 2.56 452 ± 3.61*** 114 ± 1.63 210 ± 3.92 292 ± 6.09** 48.2 ± 2.03
a Units mg-1 protein min-1
b lg of DCIP reduced mg-1 protein min-1
Values are mean of three experiments (±) SD. Significantly different from control (before decolorization) at * P \ 0.05, ** P \ 0.01, ***
P \ 0.001 by one-way analysis of variance (ANOVA) with Tukey–Kramer comparison test
0
0.2
0.4
0.6
0.8
1
1.2
400 500 600 700 800
Abs
orba
nce
Wavelength (nm)
Fig. 5 Visible range scan of synthetic dye mixture decolorization
(horizontal line after decolorization, dashed hyphen before
decolorization)
A. S. Chougule et al.
123
plant seeds Triticum aestivum (Wheat), Sorghum vulgare
(Jowar) and Phaseolus mungo (Green gram), which are
important plants in Indian agriculture, were studied. The
relative sensitivities towards the SDM and its degradation
products in relation to these plant seeds are presented in
Table 2. Phytotoxicity study with Sorghum vulgare, Triti-
cum aestivum and Phaseolus mungo seeds treated with
metabolites formed after SDM degradation showed 60, 70
and 80 % germination rate respectively which is (ca. 40 %)
higher than germination rate of respective seeds treated
with SDM. Significant growth in the plumule and radical
was recorded in all plant seeds treated with metabolites as
compared to the SDM treated seeds. This study indicates
that the toxicity of SDM was reduced after its treatment
with Pseudomonas sp. SUK 1.
Oxidative Stress Studies and Lipid Peroxidation Assay
Analysis of antioxidant enzyme activities (SOD, CAT and
GPX) and lipid peroxidation from the root cells of A. cepa
exposed to SDM and its degradation products was carried
out. Achary et al. [24] have studied the antioxidant
enzymes during the toxicity of Al in A. cepa root cells.
According to them up/down regulation of these enzymes
can be taken as indication of oxidative stress. In this study
SDM treated samples showed increase in CAT activity as
compared to CAT activity in water treated roots (control).
SDM might be leading to generate H2O2 radicals in high
amount and it can be efficiently scavenged by CAT. On the
other hand activities of SOD and GPX enzymes decreased
in the SDM treated samples as compared to control. This
might be because, SDM could not generate the substrate for
them. Also the H2O2 in the presence of catalytic iron ions
might have inactivated SOD [39]. In all the three cases
enzyme activity values of metabolite treated samples were
close to the control values indicating their less toxic nature
(Table 3).
Oxidative stress subsequently accompanies with lipid
peroxidation. It was observed that the level of lipid per-
oxidation was increased in SDM treated root sample and
the biodegraded metabolite treated sample which showed
almost similar level of LiP with respect to control which
defines reduction in the toxicity. Lipid peroxidation chain
reaction is a strong indicative of generation of oxidative
stress. Thus it can be concluded that textile dyes induce
oxidative stress on plants.
Fig. 6 HPLC elution profile of
the synthetic dye mixture before
(a) and after its decolorization
(b)
Synthetic Dye Mixture Decolorization by Pseudomonas sp. SUK 1
123
Fig. 7 FTIR spectra of
synthetic dye mixture (a) and
metabolites extracted after 24 h
(b)
Table 2 Phytotoxicity study of
synthetic dye mixture and its
degradation product
a Water treated sampleb 1,200 ppm
Values are mean of three
experiments, SEM (±),
significantly different from the
control (seeds germinated in
water) at * P \ 0.05, **
P \ 0.01 and *** P \ 0.001 by
one-way analysis of variance
(ANOVA) with Tukey–Kramer
comparison test
Plants Parameter
Germination Plumule (cm) Radical (cm)
Triticum aestivum
Controla 80 12.2 ± 1.62 6.01 ± 0.31
SDMb 30** 3.66 ± 0.32*** 3.53 ± 0.24*
Metaboliteb 70 9.03 ± 1.64 5.43 ± 0.73
Sorghum vulgare
Controla 80 5.93 ± 0.32 5.33 ± 0.61
SDMb 30** 3 ± 0.89** 1.66 ± 0.21**
Metaboliteb 60 4.33 ± 0.43 4.28 ± 0.54
Phaseolus mungo
Controla 100 11.5 ± 0.81 3.86 ± 0.21
SDMb 30*** 7.76 ± 0.76** 1.86 ± 0.11*
Metaboliteb 80 9.91 ± 1.15 2.95 ± 0.43
A. S. Chougule et al.
123
Conclusion
In conclusion, Pseudomonas sp. SUK 1 possesses high
decolorization efficiency, reusability and stability for
SDM. The maximum 94.3 % ADMI removal obtained after
24 h at pH 7 and temperature 30 �C under static condition
which is easy to obtain for large scale treatment of textile
waste. The observed COD reduction was 25 % and
reduction in BOD was recorded more than 28 % within
24 h. The decolorization and degradation of SDM by
Pseudomonas sp. SUK 1 might be because of activity of
laccase, VAO, NADH–DCIP reductase and tyrosinase. The
analytical study confirms biodegradation into different
products. The phytotoxicity and oxidative stress study
suggests that these products are less toxic in nature and
toxicity of SDM is drastically reduced. Thus the bacterium
is suitable for industrial application and further study can
be focused on designing a bioreactor and immobilization of
these cells which will scale up the process and reduce the
disruption of cells.
Acknowledgments First author would like to thank Department of
Biotechnology and Bioinformatics, Padmashree Dr. D. Y. Patil Uni-
versity, Navi Mumbai and Department of Biotechnology, Shivaji
University, Kolhapur for providing research facilities.
Conflict of interest Authors declare that they have no conflict of
interest.
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