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Department of Biotechnology, Sathyabama University , Jeppiaar Nagar, Old Mamallapuram Road, Sholinganallur, Chennai 600 119, Tamil Nadu, India
Department of Chemical Engineering, Sathyabama University, Jeppiaar Nagar, Old Mamallapuram Road, Sholinganallur, Chennai 600 119, Tamil Nadu, India
Abstract
A bacterial consortium was constructed using five different bacterial strains isolated from the effluent with the
ability to degrade Acid Blue 113, a diazo dye. These organisms were identified as Citrobacter freundii (2 strains),
Moraxella osloensis, Pseudomonas aeruginosa using 16S rRNA analysis and Pseudomonas aeruginosa CLRI
BL22. The consortium was found to degrade 90% of the dye by 22 h in 80% diluted textile effluent supplemented with
glucose and ammonium nitrate. Optimization studies using Response Surface Methodology have confirmed that the
degradation process was predominantly influenced by agitation and pH where as glucose was found to have negative
effect. TLC analyses indicated the presence of metanilic acid and peri acid in 24 h sample which disappeared by 48
h. The GC-MS analysis has confirmed the presence of methyl salicylic acid, catechol and -ketoadipic acid with the
RT values of 7.71, 10.88 and 15.04 respectively confirming the complete degradation of Acid Blue 113.
Abbreviations
rRNA: Ribosomal Ribonucleic Acid; RT: Retention Time; rDNA:
Ribosomal Deoxyribonucleic Acid; EMBL: European Molecular
Biology Laboratory; DDBJ: DNA Databank of Japan; BLASTN: Basic
Local Alignment Search Tool; FTIR: Fourier Transform Infrared; TLC:
Thin Layer Chromatography; GC-MS: Gas Chromatography-Mass
Spectroscopy; RSM: Response Surface Methodology; CCD: Central
Composite Design; Rf : Relative Front; CN: Consortia Number; TCA:
Tricarboxylic Acid Cycle
Introduction
An estimated 120 crore people in the world today do not have
access to safe drinking water. Water pollution due to industrial
effluents and sewage mixing is a major cause of this malady. Colored
effluent discharge from tanneries, textile and dye stuff industries
to neighboring water bodies and waste water treatment systems is a
major environmental hazard in this regard causing significant health
concerns to environmental regulatory agencies. While colored organic
compounds per se generally impart only a minor fraction of the organic
load to waste water, their colour renders it aesthetically unacceptable.
The colour of the industrial effluents is mainly due to the presence of
azo dyes which are aromatics substituted with azo groups (-N=N-).
Therefore, colour from the industrial effluents has become a subject
of great scientific interest as evidenced from the flurry of research
papers published in recent years in this niche segment [1-3]. Despite
the existence of a variety of chemical and physical treatment processes,
bioremediation of the effluents is seen as an attractive solution due to its
reputation as low-cost, environment friendly and publicly acceptable
treatment technology.
Microorganisms are frequently the sole means, biological
or nonbiological, in detoxifying these synthetic chemicals by
converting them into inorganic products. Microorganisms play a
significant role in soils, water and sediments because of their ability
to mineralize anthropogenic compounds [4,5]. Natural communities
of microorganisms harbour an amazing physiological versatility and
catabolic potential for the breakdown of an enormous number of
organic molecules [6].
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Citation: Nachiyar CV, Sunkar S, Kumar GN, Karunya A, Ananth PB (2012) Biodegradation of Acid Blue 113 Containing Textile Effluent by Constructed
Aerobic Bacterial Consortia: Optimization and Mechanism. J Bioremed Biodeg 3:162. doi:10.4172/2155-6199.1000162
Page 2 of 9
Acid Blue 113, we cannot solely depend on them for the treatment of
dye containing effluent which warrants the construction of consortia.
Therefore, an attempt has been undertaken to construct an aerobic
bacterial consortium capable of degrading Acid Blue 113 in textile
effluent. Further, optimization of culture conditions for the maximum
degradation of this dye and identification of intermediate products
have been carried out.
Effluent collection
Effluent samples were collected from Suntex processing mills,
Gummidipoondi; Professional fabrics, Tirupur and Kafer Textile mills,
Tirupur.
Microorganisms
The bacteria present in the effluent were isolated by serial dilution
method and their dye degrading ability was checked by pour plate
method on nutrient agar containing Navitan Fast blue S5R. All the
bacterial cultures were maintained on nutrient agar slants and were
used for biodegradation studies after preculturing in nutrient broth
for 12 h. The bacterial cells were cultivated in nutrient broth for 24 h
and the cells after centrifugation were resuspended in 20% glycerol and
stored at 20C as stock cultures. The purity of the glycerol stocks were
checked on nutrient agar plates before sub culturing on nutrient agar
slants for inoculum preparation.
Consortia development
The consortia was developed by aseptically transferring the
2% inoculum containing approximately 2 x 108 cells per ml of 12 h
grown culture of each individual strains in different combinations in
250 ml Erlenmeyer flasks containing 50 ml of medium and 50 mg/l
dye solution at optimized conditions [26]. Individual strains were
inoculated with 2% (v/v) aliquots of 12 h grown culture, respectively, to
maintain the same cell count in the pure culture and in the consortium.
The bacterial strains in the consortia are identified by the standard 16S
rRNA analysis by isolating and sequencing the 16S rDNA sequence
followed by comparison with the public databases (Genbank, EMBL
and DDBJ) and BLASTN sequence match routines.
Experimental design
Evaluation and optimization of culture medium was carried out
using Response Surface Methodology (RSM). RSM consists of a group
of empirical techniques devoted to the evaluation of relations existing
between a cluster of controlled experimental factors and the measured
responses, according to one or selected criteria. A prior knowledge
and understanding of the process and the process variables under
investigation are necessary for achieving a more realistic model [29].
This design of experiment was done using Minitab 16. Based on the
results obtained in preliminary experiments, glucose, agitation and pH
were found to be major variables in the dye degradation [26]. Hence,
these variables were selected to find the optimized condition for higher
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Identification of metabolites
50 ml of effluent containing 100 mg l-1 of dye was inoculated with
2% inoculum and kept in the orbital shaker at 30C. 5 ml samples were
drawn at regular intervals and centrifuged at 12,000 rpm for 20 min.
The culture supernatant was lyophilized and kept in a desiccator. TLC
analyses for the breakdown products were done on fluorescent silica
plates (Polygram Sil G/UV, 40 X 80 mm, Germany). For identification
of metanilic acid, the solvent system used was Chloroform:
Dichloromethane:Ethanol (5:5:5) [31] and for the identification of
peri acid, the solvent system used was Chloroform: Ethanol (9:1) [32].
The compounds were identified by comparing their Rf values with that
of standards. Fourier Transform Infrared analysis of the lyophilized
sample was carried out in Perkin Elmer RX1 FTIR spectrophotometer.
The culture medium after the incubation period was centrifuged
and the supernatant was extracted thrice, with equal volume of ethyl
acetate, dried over anhydrous Na2SO4, and then the solvent was
evaporated in a rotary evaporator. GCMS analysis of the ethyl acetate
extract was performed by using GC-MS-QP 2010 [SHIMADZU] MS
spectrometer. The column used was VF-5 ms, 30 m x 0.250 mm dia with
the film thickness of 0.25 m and the column oven was programmed
between 70 and 300C at the rate of 10C per minute with the injection
temperature of 240C. Mass spectra were recorded under scan mode in
the range of 401000 m/z. Compounds were identified using WILEY8.
LIB.
Citation: Nachiyar CV, Sunkar S, Kumar GN, Karunya A, Ananth PB (2012) Biodegradation of Acid Blue 113 Containing Textile Effluent by Constructed
Aerobic Bacterial Consortia: Optimization and Mechanism. J Bioremed Biodeg 3:162. doi:10.4172/2155-6199.1000162
Page 3 of 9
CN1
2,3,4,6,7,10
95.11
97
CN2
1,2,3,4,6,7
80.87
84.05
CN3
1,2,3,4,6,9
80.83
87.31
CN4
1,2,4,6,7,9
71.29
81.29
CN5
1,2,3,4,6,9,11
78.70
78.69
CN6
1,2,3,4,5
80.70
82.74
CN7
1,2,3,4,6
80.85
83.09
CN8
1,3,4,6,9
80.58
80.57
CN9
1,2,4,6,7
82.99
85.74
CN10
1,2,3,4
80.79
83.17
CN11
2,3,4,6
84.75
89.25
CN12
1,2,4,5
77.33
83.57
CN13
2,3,4
76.60
83.35
CN14
1,2,4
81.38
86
CN15
1,3,6
75.38
81
CN16
2,6,7,11
74.51
82.35
CN17
3,6,7,11
68.63
78.43
CN18
3,6,9,11
70.37
80.39
CN19
2,6,9,11
66.67
83.3
CN20
1,11,5,3,2,6,7,4
68.75
81.25
CN21
1,11, 2,3,4,6,7
72.73
72
CN22
1,11,6,7
69.64
83.92
CN23
2,3,4,6,7
73.47
79.59
CN24
10,11, 9
76.47
76.47
CN25
10,11,9,1,6
67.92
81.13
CN26
4,6,1,10,11
67.92
79.24
CN27
2,6,11,3,10
70.00
84
CN28
11,10,9,7
74.51
84.31
CN29
6,11,3,7
71.43
80.85
CN30
6,4,7,11,10
77.27
84.48
Decolorization (%)
Consortium
100
90
80
70
60
50
40
30
20
10
0
10
) 24 h, (
) 48 h
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
11
24 h
48 h
Citation: Nachiyar CV, Sunkar S, Kumar GN, Karunya A, Ananth PB (2012) Biodegradation of Acid Blue 113 Containing Textile Effluent by Constructed
Aerobic Bacterial Consortia: Optimization and Mechanism. J Bioremed Biodeg 3:162. doi:10.4172/2155-6199.1000162
Page 4 of 9
100
80
80
150
60
40
20
0
20
30
Time(h)
40
50
100
150
60
100
40
50
20
0
0
10
20
30
40
0
0
50
100
150
60
100
40
50
20
0
10
20
30
Time(h)
40
50
40
50
200
150
60
100
40
50
20
0
10
20
30
Time(h)
40
50
Consortia
100
180
160
Decolourization(%)
200
80
30
20
80
Time(h)
100
10
Time(h)
200
80
50
20
10
100
40
20
60
Decolourization(%)
40
Decolourization(%)
200
120
60
140
80
Decolourization(%)
100
160
Decolourization(%)
180
100
2). All the individual organisms and also consortia started to degrade
the dye in their late lag phase and 90% degradation was achieved after
26 h (Citrobacter freundii), 30 h (Moraxella oslensis), 28 h (Citrobacter
freundii), 42 h (Pseudomonas aeruginosa) but the consortia could
degrade 90% of the dye by 22 h reaching a maximum of 96% by 24 h.
Biomass production was found to be proportional to glucose utilization.
The reducing equivalents from glucose metabolism might have been
involved in the dye degradation [43]. With reference to growth, the
consortia reached its decline phase by 44 h when compared to the
individual organisms which remained in their late stationary phase
by that time. Khehra et al. [44] have reported a similar result for the
decolorization of a group of azo dyes by consortia HM-4 containing
Bacillus cereus, Pseudomonas putida, Pseudomonas fluorescens and
Stenotrophomonas acidaminiphila. They have attributed the higher
decolorization efficiency of consortium to the concerted activities of
the constituent strains. The individual cultures might have transformed
the dye to intermediates which acted on the redox mediators for
80
140
120
60
100
80
40
60
20
40
20
0
0
10
20
30
Time(h)
40
50
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
Citation: Nachiyar CV, Sunkar S, Kumar GN, Karunya A, Ananth PB (2012) Biodegradation of Acid Blue 113 Containing Textile Effluent by Constructed
Aerobic Bacterial Consortia: Optimization and Mechanism. J Bioremed Biodeg 3:162. doi:10.4172/2155-6199.1000162
Page 5 of 9
Variables
Trials
pH
Glucose (g/l)
Agitation (rpm)
Observed
Predicated
Residual
5.0
0.1
150.0
0.0820
0.0802
-0.0018
8.0
0.6
75.0
0.0665
0.0665
0.0000
11.0
0.1
0.0
0.0744
0.0731
-0.0012
13.0
0.6
75.0
0.0727
0.0716
-0.0012
8.0
-0.2
75.0
0.0836
0.0836
0.0000
8.0
0.6
75.0
0.0665
0.0665
0.0000
8.0
0.6
-51.1
0.0564
0.0555
-0.0009
8.0
0.6
75.0
0.0665
0.0665
0.0000
11.0
1.0
150.0
0.0661
0.0659
-0.0001
10
11.0
1.0
0.0
0.0524
0.0528
0.0004
11
5.0
0.1
0.0
0.0669
0.0671
0.0002
12
8.0
0.6
75.0
0.0685
0.0665
-0.0020
13
5.0
1.0
0.0
0.0446
0.0468
0.0022
14
8.0
0.6
201.1
0.0776
0.0775
-0.0001
15
8.0
0.6
75.0
0.0665
0.0665
0.0000
16
8.0
1.3
75.0
0.0494
0.0494
0.0001
17
3.0
0.6
75.0
0.0603
0.0615
0.0012
18
8.0
0.6
75.0
0.0655
0.0665
0.0010
19
5.0
1.0
150.0
0.0616
0.0599
-0.0017
20
11.0
0.1
150.0
0.0824
0.0862
0.0038
Table 2: Variables in Central composite design with experimental and predicted values of dye decolourisation.
Predictor
Co-efficient
SE Co-efficient
T-value
P-value
Constant
0.064349
0.001301
49.48
0.0010
pH
0.001003
0.0001352
7.42
0.0025
Glu
-0.02256
0.0009011
-25.03
0.0005
Agi
8.73E-05
5.41E-06
16.15
0.0010
Citation: Nachiyar CV, Sunkar S, Kumar GN, Karunya A, Ananth PB (2012) Biodegradation of Acid Blue 113 Containing Textile Effluent by Constructed
Aerobic Bacterial Consortia: Optimization and Mechanism. J Bioremed Biodeg 3:162. doi:10.4172/2155-6199.1000162
Page 6 of 9
12
response
10
pH 8
0.08
0.07
0.06
0.05
3
0.0
pH
0.5
1.0
12
1.5
Glu
0.00
0.25
0.50
Glu
0.75
1.00
1.25
1.25
response
<
0.05 0.06 0.07 >
0.75
0.08
0.07
0.50
0.06
0.05
0.25
0.0
0.5
1.0
0.00
0
1.5
200
100
Agi
-50
50
Agi
100
150
200
response
<
0.05 0.06 0.07 >
12
10
response
0.05
0.06
0.07
0.08
0.08
Glu
response
1.00
0.05
0.06
0.07
0.08
0.08
0.08
0.05
0.06
0.07
0.08
0.08
pH 8
0.07
0.06
0.05
12
9
100
Agi
200
pH
4
-50
50
Agi
100
150
200
Figure 3: Response surface plot showing the effect of variables on dye degradation a) Effect of glucose concentration and pH b) Effect of glucose concentration and
agitation c) Effect of agitation and pH.
TCA cycle. These reports clearly indicated that this consortium has also
taken up a degradation pathway similar to Pseudomonas aeruginosa in
degrading Acid Blue 113 [11].
Conclusion
Water pollution control is presently one of the major areas of
scientific activity. Effluent discharge from textile and dyestuff industries
to neighboring water bodies and wastewater treatment systems
is currently causing significant health concerns to environmental
regulatory agencies. Color removal, in particular, has recently become
of major scientific interest, as indicated by the multitude of related
research reports. As microbes, the powerful weapon of bioremediation
process, offer advantages of completely mineralizing the pollutants,
investigations are warranted in the present research work focused on
the possibilities of using constructed aerobic microbial consortium for
decolorization and complete mineralization of azo dyes in dye laden
Citation: Nachiyar CV, Sunkar S, Kumar GN, Karunya A, Ananth PB (2012) Biodegradation of Acid Blue 113 Containing Textile Effluent by Constructed
Aerobic Bacterial Consortia: Optimization and Mechanism. J Bioremed Biodeg 3:162. doi:10.4172/2155-6199.1000162
Page 7 of 9
10.0
20.0
80
23.0
120
90
22059
21075
17100
17178
16581
16931
15340
16079
15773
14872
15041
14144
13130
13370
12227
7319
5.0
100
19259
10000
12112
7.7 min
70
60
152
92
50
40
30
20
65
10
53
40
30
100
40
90
80
76
50
60
70
137
105
80
90
100
110
120
130
140
154
150
49
70
m/z
10.9 min
41
73
60
50
56
40
84
30
110
20
123
10
127
100
30
40
90
80
50
60
70
80
90
100
110
120
152
154
130
140
79
43
150
m/z
15.04 min
60
70
60
41
129
50
40
30
185
85
20
101
10
30
50
70
90
115
110
143
130
150
157
228
171
199
170
190
219
210
230
m/z
Figure 4: GC-MS spectra of ethyl acetate extract of the culture filtrate containing the degradation products of Acid Blue 113 (a) Gas chromatogram (b) mass spectra
for the peak with RT value of 7.71 min (c) mass spectra for the peak with RT value of 10.9 min (d) mass spectra for the peak with RT value of 15.04 min.
textile effluent. This consortium was effective in degrading the azo dye
Acid Blue 113 to -ketoadipic acid which can be utilized by the bacteria
for their normal metabolism.
Acknowledgement
The authors would like to thank the Department of Biotechnology, India for
the financial support and also the Management of Sathyabama University for the
encouragement and opportunity to carry out this work.
J Bioremed Biodeg
ISSN: 2155-6199 JBRBD, an open access journal
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Citation: Nachiyar CV, Sunkar S, Kumar GN, Karunya A, Ananth PB (2012) Biodegradation of Acid Blue 113 Containing Textile Effluent by Constructed
Aerobic Bacterial Consortia: Optimization and Mechanism. J Bioremed Biodeg 3:162. doi:10.4172/2155-6199.1000162
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Citation: Nachiyar CV, Sunkar S, Kumar GN, Karunya A, Ananth PB (2012) Biodegradation of Acid Blue 113 Containing Textile Effluent by Constructed
Aerobic Bacterial Consortia: Optimization and Mechanism. J Bioremed Biodeg 3:162. doi:10.4172/2155-6199.1000162
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Special features:
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ISSN: 2155-6199 JBRBD, an open access journal