International Journal of Advancements in Research & Technology, Volume 2, Issue 7, July-2013
ISSN 2278-7763
182
Decolourization of Dye Compounds by Selected Bacterial Strains isolated from Dyestuff
Industrial Area
M.H.Fulekar*, Shrutika L.Wadgaonkar and Anamika Singh
Department of Life Sciences, University of Mumbai, Santacruz (E), Mumbai-400 098, India
*
School of Environment and Sustainable development, Central University of Gujarat, Gandhinagar-482030, India
*
Email: mhfulekar@yahoo.com
Abstract:
In the present study, the effluent and soil sample collected from dyestuff industrial area have
been examined for microbial characteristics. Six bacterial strains, namely, Aeromonas
hydrophila, Pseudomonas putida, P. plecoglossicida, Lysinibacillus fusiformis, P. monteilii
and Comamonas testosterone have been isolated and identified from dyestuff industrial area
for the decolourization of selected dye compounds-Methyl Orange, Acid Orange, Malachite
Green, Methylene Blue and Rhodamine B. Each compound was applied separately at 20 mg ll
in minimal salt medium to evaluate decolourization efficiency of the identified bacterial
strains. The result shows that the selected bacterial strains have potential to decolourize dye
compounds after 7 days of incubation period. The highest decolourization (91%) was found
for the dye malachite green by Pseudomonas putida after 5 days of incubation. Comamonas
testosterone was found to decolourize 85% of methyl orange after 7 days of incubation. P.
putida was also found to decolourize 85% and 69% of acid orange II and methylene blue,
respectively after 7 days of incubation. 56% of Rhodamine B was decolourized by P.
monteilii after 3 days of incubation. The investigation proved that the microorganisms found
in industrial area have ability to decolourize dye compounds. The potential of these bacteria
can be exploited for the removal of residual dyes from the wastewater streams for
environmental cleanup and restoration of ecosystem.
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Keywords: effluent, bacteria, decolourization, dye compounds, potantial
1. Introduction
The wastewater generated from the textile, dye and dyestuff industries is a complex
mixture of various organics, like chlorinated compounds, pigments, dyes and inorganic
compounds. Dyes usually have a synthetic origin and complex aromatic molecular structures
which make them more stable and more difficult to biodegrade [1]. The textile industry
utilizes about 10000 different dyes and pigments. The worldwide annual production of dyes
is over 7x105 tons [2,3]. The dyestuff usage has been increased day by day because of
tremendous increase of industrialization and man’s urge for color [4]. It is reported that
approximately 15% of the dyestuffs are lost in the industrial effluents during the
manufactoruing and processing operations [5]. The effluents of these industries are highly
colored and the disposal of these wastes into receiving waters causes damage to the
environment. The presence of dye compounds in the effluent, even at low concentrations (15 ppm) is highly visible and toxic to the biotic life. Dyes are resistant towards conventional
methods of wastewater treatment. The conventional methods are less efficient, costly, of
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183
limited applicability and produce secondary pollutants, which are very difficult to dispose off.
In this regard, researchers are now focussing on biological approaches for removal of dyes
from wastewater. In the biological methods, the microbes such as bacteria, fungi and algae
are being used for the wastewater treatment, which could be a viable option as low-cost and
eco-friendly technology. There are various microorganisms found in the contaminated
environment, have potential to decolourize and even completely mineralize many dyes from
the wastewater efficiently under certain environmental conditions. Decolorization study of
dyes by microorganisms isolated from industrial effluent area have been reported by various
researchers[6, 7, 8, 9, 10, 11, 12]. Several bacterial strains that can aerobically decolourize
dyes have been isolated during the past few years. Many of these strains require organic
carbon sources, as they cannot utilize dye as grwoth substrate [13].
The present research study portrays on collection of effluent and soil samples from
the dyestuff industrail area followed by their microbial characterizaion. The microorganisms
which are surving in the contaminated area have been isolated and identified from the soil
samples. The microorganisms have been further tested for their ability to decolourize the
dyes, namely Methyl Orange, Acid Orange, Malachite Green, Methylene Blue and
Rhodamine B. The decolourization of selected dyes by different bacterial strains,
viz.Aeromonas hydrophila, Pseudomonas putida, P. plecoglossicida, Lysinibacillus
fusiformis, P. monteilii and Comamonas testosteroni was carried out in incubator-shaker. The
aliquot of 5 ml was withdrawn from each dye solution at the intervals of 6 hr, 1d, 2d, 3d, 4d,
5d, 6d and 7d to check the decolourization pattern by selected bacterial strains. The samples
were centrifuged and subjected to UV-Visible spectrophotometer for the optical density of
the selected dyes. The decrease in concentration from the solution is attributed to the
decolourization of dyes by the selected bacterial strains.
The ojectives of the present reasearch are: i) microbial characterization of soil
samples; ii) isolation and identification of bacterial strains from contaminated soil samples &
iii) to check the potential of bacterial strains for decolourization of various dyes.
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2. Materials and Methods
2.1. Chemicals
Dyes commonly used in textile and leather dyeing industries viz., Methyl Orange,
Acid Orange II, Malachite Green, Methylene Blue and Rhodamine B (table 1) were procured
from sd-fine chemicals ltd/ Merck, Mumbai. The Nutient agar and nutrient broth for culture
and isolation of bacteria were obtained from Himedia.Gram staining kit (K001-1KT, HiMedia) was used for the bacterial characterization study. KB003 Hi25TMEnterobacteriaceae
Identification Kit was procured from Himedia, Indiafor the biochemical characterization of
selected bacteria. Other Analytical gradechemicals were obtained from sd-fine chem ltd/
Merck.
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Table 1: Characteristics of selected dyes
Dyes
Structural formula
Methyl
orange
Acid
Orange II
Malachite
green/
Methylene
blue
Rhodamine
B
Molecular
Formula
Molar Mass
C 14 H 14 N 3 NaO 3
S
327.33
g/mol
C 16 H 11 N 2 NaO 4
S (sodium salt)
350.32
g/mol
C 23 H 25 ClN 2
(chloride)
364.911
g/mol
(chloride)
C 16 H 18 N 3 SCl
319.85
g/mol
Group
Anionic
dye
Absorption
Maxima
(λ max)
470nm
orange
Anionic
dye
481nm
Cationic
dye
617nm
red light
orange
intense
green color
Cationic
dye
665nm
blue
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Cationic
dye
C 28 H 31 ClN 2 O 3
Appearance
554nm
479.02
red to violet
powder
2.2. Sample Collection
In the present research, dye pigment manufacturing industry located at Ratnagiri,
Mahad, Maharashtra has been selected for collection of soil samples. The soil sample was
collected from various sites of the dyestuff industry for the isolation of potential
microorganisms for decolourization of dye compounds. All the samples were collected in
sterile sterile glass-screw cap tubes and preserved at 4oC in refrigerator.
2.3. Microbial characterization of soil samples for Isolation of bacterial strains
Soil samples were serially diluted to 10,000 folds and plated on nutrient agar. 1ml
bacterial culture was inoculated in nutrient broth and further, in 250ml Erlenmeyer flasks
containing 100ml minimal media with a dye concentration of 10mg L-1. Minimal media
comprised of Na 2 HPO 4 , NH 4 Cl, glucose blended with 0.6ml of trace elements solution.
Trace elements solution contains CaCl2 .2H 2 O, MgSO 4 , MnSO 4 .7H 2 O, and FeSO 4 .7H 2 O.
Flasks were kept in incubator shaker at37°C, 150rpm. pH was adjusted to 7.0 before
inoculation. Optical density at 600nm was measured daily to analyze bacterial growth. Grown
cultures were serially diluted and spread on a sterile nutrient agar plate containing dye. Plates
were incubated at 37 °C for 48 h after which isolated colonies were selected for further
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isolation. Individual isolated colonies were re-streaked on nutrient agar plates for
identification.
2.4. Identification of the microorganisms
2.4.1. Gram Staining
The isolated bacterial culture was transferred on a non greasy glass slide to form the
smear and heat fixed. Further the culture was gram stained as per the manufacturer’s
instructions using gram staining kit (K001-1KT, Hi-Media) and observed under the light
microscope [14].
2.4.2. Biochemical characterization
The isolated microorganisms after preliminary isolation and identification were
further identified by conventional microbiological and biochemical techniques as described in
Bergey’s Manual of Determinative Bacteriology [15]. The different biochemical tests like
oxidase production, catalase production, lactose utilization, glucose utilization, saccharose
utilization, adonitol utilization, xylose utilization, citrate utilization, indole production, nitrate
production, urease activity, lysine decarboxylation, β-galactosidase activity etc. were
performed using KB003 Hi25TMEnterobacteriaceae Identification Kit procured from
Himedia, India [16].
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2.5. Decolourization of dye compounds
The organisms were grown in 100 ml Minimal Salt medium (MSM) containing
selected dye compounds in conical flask containing at 25°C and continuous shaking at 100
rpm. The composition of MSM is yeast extract (1g/L), (NH 4 ) 2 SO 4 (2.5g/L), KH 2 PO 4
(13.3g/L), Na 2 HPO 4 (21.6 g/L) and glucose (1.25g/L). The experiment was carried out using
20 mg L-1 of Methyl orange, Acid orange, Malachite green, Methylene blue and Rhodamine
B dyes separately in each MSM separately. The organisms were inoculated in the sterile
liquid minimal media in conical flasks and incubated at room temperature using shake-flask
method. Samples were withdrawn at the intervals of 0hr, 6hrs, 1 d, 2d, 3d, 4d, 5d, 6d and 7d
during the incubation and checked for decolourization of dye compounds using
spectrophotometric analysis. A small aliquot of the media was extracted in sterile conditions.
The aliquot was subjected to centrifugation at 10000rpm for 5 min. The supernatant was
collected and the absorbance was noted using the sterile un-inoculated media without dye as a
blank. The absorbance for different dyes was noted at different wavelengths.
The decolourization of the media indicates the degradation of the dye by the bacteria. The
efficiency of degradation of the dye can be calculated using the following formula:
% Degradation = A0 – A 1 x 100
A0
Where, A 0 = Initial absorbance of the media
A 1 = Absorbance of the media at the interval of t time.
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3. Results and discussion
In the present research, dye pigment manufacturing industry located at Ratnagiri,
Mahad, Maharashtra has been selected for collection of effluent and soil samples. The soil
sample was collected from various sites of the dyestuff industry for the isolation of potential
microorganisms for decolourization of dye compounds.
3.1. Microbial characterization of soil samples
3.1.1. Isolation and Identification of the microorganisms
The soil samples were analysed for microbial characteristics. The microbes were
cultured in Nutrient broth and bacterial colonies were isolated on Nutrient agar medium. Six
organisms out of 15 were found to have potential for decolourization of dye compounds.
Grams staining, colony and biochemical characteristics of the organisms were carried out and
the results are presented in table 2-4, respectively. Out of six, five isolates were gram
negative bacteria and one was gram- positive bacteria (Table 2). The colony characters on
nutrient agar and microscopic features are presented in table 3. The colony morphology was
found differ in size, colour and opacity. All of them were circular in shape, smooth textured
and having straight margin.
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Table 2: Gram staining nature of selected bacteria
S.No.
Organism 1
Organism 2
Organism 3
Organism 4
Organism 5
Organism 6
Gram nature
Gram negative Rods
Gram negative Rods
Gram negative Rods
Gram positive Rods
Gram negative Rods
Gram negative Rods
Table 3: Colony Characteristics of the selected bacteria
Characters
Organism Organism Organism 3
1
2
Size
2mm
2mm
1mm
Shape
Circular
Circular
Circular
Colour
Cream
Off white
White
Texture
Smooth
Smooth
Smooth
Margin
Straight
Straight
Straight
Opacity
Opalescent Opalescent Transparent
Consistency
Soft
Soft
Soft
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Organism Organism 5 Organism 6
4
1mm
2mm
3mm
Circular
Circular
Circular
Cream
Yellow
White
Smooth
Smooth
Smooth
Straight
Straight
Straight
Opaque Translucent Translucent
Soft
Hard
Soft
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Table 4: Biochemical Characteristics of the selected bacteria
Sr.
No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Biochemical Tests
ONPG
Lysine Utilization
Ornithine Utilization
Urease
Phenylalanine Deamination
Nitrate Reduction
H 2 S Production
Citrate Utilization
VogesProskauer’s
Methyl Red
Indole
Malonate Utilization
Esculin Hydrolysis
Arabinose
Xylose
Adonitol
Rhamnose
Cellubiose
Melibiose
Saccharose
Raffinose
Trehalose
Glucose
Lactose
Oxidase
Organism
1
+
+
+
+
V
+
-
Organism
2
+
+
+
+
+
Organism
3
+
+
+
+
+
Organism
4
V
V
+
V
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Organism
5
+
+
V
+
+
Organism
6
+
+
+
+
+
V
-
3.2. Decolourization of dyes by the selected bacteria
Six bacterial strians- Aeromonas hydrophila, Pseudomonas putida, P. plecoglossicida,
Lysinibacillus fusiformis, P. monteilii and Comamonas testosterone have been isolated from
the soil samples collected from dyestuff industrial area. In the present investigation, six
bacteria were tested for their ability to decolourize 20 mg L-1 of five dyes, namely-Methyl
Orange, Acid Orange, Malachite Green, Methylene Blue and Rhodamine B. The MSM was
spiked with 20 mg L-1 concentration of each dye separately in a conical flask, followed by
inoculation of selected bactrial strains and placed on a shaker-nucubator. Decolourization of
each dye by bacterial strains was ensured by checking the concentration of dye in MSM at the
intervals of 0hr, 6hr, 1d, 2d, 3d, 4d, 5d, 6d and 7d. an aliquote of 5ml was withdrawn from
the MSM at regural intervals and analysed by UV-VIS spectrophotometer at specific
wavelength (Table 1) for each dye. The dye concentraion was decreasing with increasing
period of time.
After seven days of incubation period, significant dye decolourization by selected
bacteria was seen (table 5). The overall decolorization of 56% to 91% was achieved up to an
initial dyeconcentration of 20 mg L-1 in 3-7 days of incubation. The percentage degradation of
various dyes- Methyl orange, Acid orange II, Malachite green, Methylene blue and
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Rhodamine B by selected bacteria could be seen in fig. 1-5. The results indicate that all the
selected dyes were decolourized by selected bacteria after 3-7 days of incubation.
Table 5: Percentage degradation of various dyes by six selected bacteria after 7 days of
incubation
Name of Bacterial isolates
Aeromonas hydrophila
Pseudomonas putida
P. plecoglossicida
Lysinibacillusfusiformis
P.monteilii
Comamonas testosterone
Methyl
orange
83.38
70.27
8.86
79.78
80.98
85.13
Acid
orange II
66.01
85.47
16.41
66.73
55.34
44.13
Malachite
green
86.95
91.01#
49.61
76.74
18.1
14.3
Methylene
blue
40.19
69.29
33.73
25.07
46.83
39.66
Rhodamine
B*
49.46
54.51
54.51
45.94
56.27
55.93
*% degradation after 3 days of incubation; #% degradation after 5 days of incubation
The results indicate that Aeromonas hydrophila was able to degrade 83.38%,
66.01%, 86.95%, 40.19% and 49.46 % of Methyl orange, Acid orange II, Malachite green,
Methylene blue and Rhodamine B, respectively after 3-7 days of incubation. The capability
of Aeromonas hydrophila for degradation of textile dyes had exploited by Chen et al [17].
Chimezie and Thomas have reported bioremediation and detoxification of synthetic
wastewater containing triarylmethane dyes by Aeromonas hydrophila isolated from industrial
effluent [18]. Pseudomonas putida has able to decolourize 70.27%, 85.47%, 91.01%, 69.29%
& 54.51% of Methyl orange, Acid orange II, Malachite green, Methylene blue and
Rhodamine B, respectively after 3-7 days of incubation. Whereas P. plecoglossicida has
decolourized 8.86, 16.41, 49.61, 33.73 and 54.51% of Methyl orange, Acid orange II,
Malachite green, Methylene blue and Rhodamine B, respectively after 3-7 days of incubation.
The third species of Pseudomonas, i.e. P. monteilii showed 80.98, 55.34, 18.1, 46.83 and
56.27% decolourization of Methyl orange, Acid orange II, Malachite green, Methylene blue
and Rhodamine B, respectively after 3-7 days of incubation.The dye decolorizing potential of
Pseudomonas putida MTCC 102 for Acid Orange 10 was reported by Tripathi and Srivastava
[19]. They concluded that due to its high degrading potential, Pseudomonas putida MTCC
102 could be used successfully in treatment of textile waste waters as they contain high
concentration of azo dyes [19].
In case of Lysinibacillus fusiformis 79.78, 66.73, 76.74, 25.07 and 45.94% of Methyl
orange, Acid orange II, Malachite green, Methylene blue and Rhodamine B, respectively
decolourization was observed after 3-7 days of incubation. Our results show that Comamonas
testosterone has decolourized 85.13, 44.13, 14.3, 39.6 and 55.93% of Methyl orange, Acid
orange II, Malachite green, Methylene blue and Rhodamine B, respectively after 3-7 days of
incubation. The potential of Comamonas sp. VS-MH2 was exploited for its ability to degrade
a synthetic dye mixture (SDM) (comprising of four azo reactive dyes) under static conditions.
The isolate showed high metabolic activity towards SDM and degraded it completely (100
mg L-1) within 30 h at pH 7 and 35 °C [20].
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It was observed that after 3d of incubation no significant decolourization was found in
case of Rhodamine B. The growth of all selected bacteria was retarded; hence the
decolourization study was continued till 3d of incubation. The findings of the present
research work showed that the highest decolourization (91%) was found for the dye
malachite green by Pseudomonas putida after 5 days of incubation (fig.3). Comamonas
testosterone was found to decolourize 85% of methyl orange after 7 days of incubation
(fig.1). P. putida was also found to decolourize 85% and 69% of acid orange II and
methylene blue, respectively after 7 days of incubation (fig.2 & 4). 56% of Rhodamine B was
decolourized by P. monteilii after 3 days of incubation (fig.5).
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Fig 1: Decolourization of Methyl orange by selected bacteria at 350C after 7 days of
incubation
Fig 2: Decolourization of Acid orange II by selected bacteria at 350C after 7 days of
incubation
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Fig 3: Decolourization of Malachite green by selected bacteria at 350C after 7 days of
incubation
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Fig 4: Decolourization of Methylene blue by selected bacteria at 350C after 7 days of
incubation
Fig 5: Decolourization of Rhodamine B by selected bacteria at 350C after 3 days of
incubation
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4. Conclusion
The present study demonstrates that the bacterial strains present in the dyestuff
industrial area have potential to decolourize various types of dyes used in such industries.
The potential of these bacteria can be exploited for the removal of residual dyes from the
wastewater streams for environmental cleanup and restoration of ecosystem.
5. Acknowledgements
Authors are grateful to Department of Biotechnology (DBT), Ministry of Science and
Environment, Government of India for sponsoring the research project and rendering
financial assistance.
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