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R E S E A R C H C O M M U N II C A T II O N
ABSTRACT
____________________________
VSRD-TNTJ, Vol. 2 (5), 2011, 238-248
Biodegradation of Textile Dyes
By Pseudomonas Species and E.Coli
1 Tom Sinoy E.S.*, 2 Dhade Anuradha Mohan and 3 Humera Shaikh
In the present study, an attempt was made to examine the potential of aerobic mixed culture for decolourization
of Brilliant green Malachite green, carbol fuchsin and crystal violet dyes. The effect of pH, temperature,
inoculums and initial concentration of dye was studied with an aim to determine the optimal conditions required
for maximum decolourization and degradation. The culture exhibited maximum decolourization ability at pH
between7-8 and at 30°C.10% (v/v) inoculums were found to be the optimum for decolourization. A maximum
of 98% decolourization was observed at 25 ppm initial concentration of dye after 18 hours of incubation period.
At higher dye concentration of 300 ppm, the removal of color was found to be 75% in 48 hours of incubation
period. The results show that the enriched mixed culture from activated sludge has good potential in removal of
Brilliant green and crystal violet dyes from wastewater under aerobic conditions.
Keywords : Decolourization, Brilliant Green, Malachite Green, Carbol Fuchsin and Crystal Violet, Aerobic
Conditions.
1. INTRODUCTION
Synthetic dyes are coloring agents mainly used in textile industries which generate a huge amount of wastewater
in the process of dyeing. It is estimated that these industries discharge around 280,000 tonnes of dyes worldwide
every year into the environment. A very small amount of dye in water (10-50 mg L-1) affects the aesthetic
value, transparency of water and gas solubility of water bodies. The effluents from these industries are complex;
contain a wide variety of dyes and other products such as dispersants, acids, bases, salts, detergents, humectants,
oxidants, etc. Discharge of these colored effluents into rivers and lakes results into reduced dissolved oxygen
concentration, thus creating anoxic conditions that are lethal to resident organisms.
Many reports indicate that textile dyes and effluents have toxic effect on the germination rates and biomass
concentration of several plant species which play many important ecological functions such as providing the
13 Assistant Professor, 2 Research Scholar, 123 Department of Biotechnology, Yeshwant College of IT (Bi-Iinformatics & Bio-
Technology), Nagpur, Maharashtra, INDIA.
*Correspondence : biolichen@gmail.com

Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (5), 2011
habitat for wildlife; protecting soil from erosion and providing bulk of organic matter that is significant to soil
fertility.
The toxicity of effluent is because of the presence of dye or its degraded products which are mutagenic or
carcinogenic. Therefore, the treatment of industrial effluents contaminated with dye becomes necessary prior to
their final discharge to the environment. Various kinds of physico-chemical methods are in use for the treatment
of wastewater contaminated with dye. These methods are not environment friendly and cost-effective and hence
become commercially unattractive.
Many microorganisms belonging to the different taxonomic groups of bacteria, fungi, actinomycetes and algae
have been reported for their ability to decolorize azo dyes. However, maintaining the purity of single cultures in
the large scale application and their inability to degrade all different dyes present in the actual effluent are the
more potential for large scale application at field level. The syntrophic interactions present in the mixed
communities lead to complete mineralization of azo dyes. In the present study, an attempt has been made for the
degradation and decolourization of an azo dye using aerobic mixed culture in flasks. Effect of various process
parameters like pH, temperature, inoculums concentration and initial concentration of dye was studied.
2. MATERIALS AND METHODS
2.1. Location of Soil Sample And Source of Inoculums
Soil sample collected from a small dying industry from where the colored effluent passes was used as the
parent source of inoculums in the present study.
2.2. Enrichment
For enrichment of the culture, the heterogenous population was first grown aerobically in a medium containing
1% (w/v) glucose (COD= 1100 - 1250mg/L) as the carbon and energy source. During acclimatization period,
the amount of glucose was regularly checked and maintained at 1%.
The culture was gradually exposed to increasing concentrations of Brilliant green, crystal violet, Carbol Fuchsin
and malachite green dyes in order to acclimatize the microbial culture. Successive transfers of culture into
sterile nutrient broth containing higher concentrations of Brilliant green and crystal violet dyes upto 300 ppm
was done at 37°C in rotary condition. This acclimatized microbial culture was for all experiments.
2.3. Isolation and Screening of Dye Decolorizing Microorganism
The collected soil samples were used for screening of dye decolorizing cultures by enrichment technique using
nutrient broth amended with dye as a sole source of carbon and energy. Dye containing media (100ml) in 250ml
Erlenmeyer flasks were inoculated with 10ml soil suspension (10%w/v) and incubated in orbital shaker under
static condition (150rpm). After 48 hours of incubation, 1.0ml of the culture broth was appropriately diluted and
plated on nutrient agar containing 100mg/l Brilliant green and crystal violet dye. After incubation
morphologically distinct bacterial isolates showing clear zone around their colonies due to the decolourization
of dye.
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2.4. Identification
Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (5), 2011
A pure colony of the unknown isolates were named as B1 and B2, these isolates were identified presumptively
on the basis of the following features gram staining, motility and biochemical reaction.
2.5. Dye Decolourisation Studies
Frequently used dyes were collected from dye house industry and one color was selected for better
decolurization study using isolated bacterial organism.
2.6. Inoculation of Bacterial Biomass
The identified organisms B1 and B2 were grown in nutrient broth. About 5% (V/V) of 18 hour old culture was
added for decolurization studies.
2.7. Decolourisation Experiment
The MSM medium constitutions amended with 100ppm of Brilliant green and crystal violet was inoculated with
bacterial culture (5% v/v) and incubated at 37°C under static condition. Samples were withdrawn at a particular
interval time (not exceed 7days) and analyzed for Decolorization.
2.8. Analysis of Decolourisation Rate
The decolourisation rate was monitored spectrometrically. The absorbance was monitored at 510nm for Brilliant
green, 470nm for malachite green, 570nm for crystal violet and 520 for carbol fuchsin dyes. After incubation
5ml culture was taken from flask and centrifuged using bench top centrifuge (5000rpm for 20 minutes) and the
mean value was taken. Medium without dye and inoculums was used as blank. Medium with dye but without
inoculums was used as control. The Decolorization efficiency was expressed as per the following equation or
formula.
Decolorization % = I – F × 100 I
Where, I: initial absorbance: F: final absorbance
2.9. Thin Layer Chromatography
Confirmation of dye degradation /Decolorization of dyes which was done as follows:
Different solvent system used for TLC where water: ethanol: methanol (4:3:3) and ethanol: methanol (5:5) and
water: methanol (4:3).But proper separation was obtained in ethanol: methanol (5:5) solvent system for
detection of degradation of the dyes. The TLC plates were prepared by making slurry of silica gel G in carbon
tetra chloride and were dried. The culture broth was centrifuged and the supernatant (5ml) was applied to TLC
plates by using small capillary tube and solvent system. The plates were developed by using iodine as locating
reagent in iodine chamber.
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3. RESULT AND DISCUSSION
3.1. Isolation and Screening of Dye Decolorizing Microorganism
Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (5), 2011
About five different morphologically distinct bacteria isolated from the soil. Among them two bacterial isolate
showed higher Decolorization and it was used for further study. The two isolates were identified as
Pseudomonas species and E. coli based on the gram staining, motility and biochemical reaction and the results
were tabulated on Table1.
4. Colony Characteristics
Brilliant Green Crystal Violet
Size 2mm
Shape Circular
Color Off white
Margin Regular
Elevation Elevated
Opacity Opaque
Consistency Sticky
Gram’s Character Gram – v e
Morphology Short rod
Motility Motile
E. Coli
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Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (5), 2011
Size 2mm
Shape Circular
Color white
Margin Entire
Elevation Raised
Opacity Translucent
Surface Smooth
Gram’s Character Gram – v e
Morphology Cocci
Motility Motile
5. Biochemical Characteristics of Isolates
Pseudomonas Species
Character Reaction
Methyl red Positive
Urease Negative
H2S Negative
KCN Negative
Indole Positive
Citrate Negative
Gas production Positive
5.1. Decolourisation of various dyes by mixed culture of Pseudomonas and E. coli:-
Four dyes were selected and decolourization pattern was measured by mixed culture of Pseudomonas and E.
coli in nutrient broth. This mixed culture showed significant ability to decolorize all the dyes viz. Brilliant green
(96%), crystal violet (94%), Carbol Fuchsin (93.3%) and malachite green (98%) in 24 hours.
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Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (5), 2011
BEFORE AFTER AFTER BEFORE
Decolourization of Brilliant Green Decolourization of crystal violet
AFTER BEFORE AFTER BEFORE
Decolourization of Carbol fuchsin Decolourization of Malachite Green
5.2. Effect of various parameters on decolourization of dyes
5.2.1. Effect of pH
Effect of pH (5-9) on decolourization with time of Brilliant green, crystal violet, Carbol Fuchsin and malachite
green dyes at 100 ppm initial concentration of dye with 10 % inoculum is shown in figure 2.4. The figure clearly
shows that the percentage removal of dye increased with increase in time irrespective of pH.
pH % of decolourisation
BG CV CF MG
5 50.65 55.7 47.52 49.32
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5.2.2. Effect of Temperature
Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (5), 2011
6 96.41 97.4 94.1 83.19
7 98.26 98.3 83.62
8 89.26 71.1 68.5 72.13
Profile of pH
Figure 2.3 shows decolourisation of dyes with time at different temperatures (20, 30, 35 and 45) at 100 ppm
initial dye concentration and 10 % inoculum. It is clear from the figure that percentage removal of dye increased
with an increase in temperature from 20 °C to 30°C. The percentage removal of dye decreased with further
increase in temperature up to 45°C.
Temperature
in 0 C
% of decolourisation
BG CV CF MG
20 70 67.99 75.2 72.3
30 98 94.26 86.3 96.1
35 98.55 94.00 90.1 95.6
45 42.23 45 82.5 51.1
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5.2.3. Effect of Inoculums Concentration
Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (5), 2011
Figure 2.5 shows the effect of inoculum concentration (5-20 %) with time on decolourization of dye at 100 ppm
initial concentration. It is clear from the figure that percentage removal of dye increased with an increase in time
for all concentrations of inoculum.
5.2.4. Effect of Initial Concentration of Dye
Inoculums conc. in % % of decolourisation
BG CV CF MG
5 75 71 68 72
10 86 90 77 92
15 90 94.00 89 95
20 95 96 94 97
Figure 2.2 shows the effect of initial concentration of dye ranging from 25–300 ppm of dye at pH 7, 10 %
inoculum concentration and at 30°C. It is clear from the figure that percentage removal of dye increased with an
increase in time irrespective of initial dye concentration. Further, percentage removal of dye decreased with an
increase in dye concentration.
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Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (5), 2011
Concentration of dye ppm % of decolourisation
BG CV CF MG
50 98 94 96 97
100 94 92 89 91
150 88 89 75 84
200 75 72 69 73
5.2.5. Confirmation of Dye Degradation/Decolourization
The result of biodegradation of Brilliant Green, crystal violet, malachite green and Carbol Fuchsin was
confirmed by Thin Layer Chromatography (TLC) as spots with different RF values were obtained as
compared to spot of standard dye sample i.e. control.
Result Analysis By Thin Layer Chromatography
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6. CONCLUSION
Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (5), 2011
The present study reveals that enriched aerobic mixed culture can be used successfully for decolourizing.
Brilliant green, crystal violet, Carbol Fuchsin and malachite green. The culture exhibited maximum
decolourization ability at pH between 7-8 and 30° C .Moreover, 10% (v/v) inoculum and 1 % glucose
concentrations were found to be optimum for decolourization. At 25 ppm initial dye concentration a maximum
of 98% removal of color was observed after 18 hours. At higher dye concentration of 300 ppm, the removal in
color was found to be 75 after 48 hours of incubation period. On the basis of the results of the present study
suitable strategy can be developed for the treatment of waste water contaminated with dye.
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