J. Env. Bio-Sci., 2015: Vol.

29 (2):331-336
(331) ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

COMPARATIVE STUDY ON DECOLORIZATION OF CRYSTAL VIOLET DYE BY

ASPERGILLUS FUMIGATUS (MTCC 3002) AND ASPERGILLUS NIGER (MTCC 478)
Shishir Vind Sharma1, Gopal Krishan Sahu1 and Anil Kumar*
1
Department of life science, MATS University, Pandri, Raipur (C.G.)
*Department of biotechnology/Zoology, Govt. V. Y. T. P .G. Auto. College, Durg, Chhattisgarh, India- 491001
[Corresponding author E-mail*: aimum_aishley@yahoo.co.in]

Received: 28-05-2015 Accepted: 27-11-2015

Crystal violet (CV), which has been extensively used as a biological stain and a commercial textile dye, is a recalcitrant molecule.
Aspergillus has the ability to decolorize the crystal violet dye and it is useful in bioremediation. The aim of this research work was
to comparatively optimize the potential of Aspergillus fumigatus MTCC 3002 and Aspergillus niger MTCC 478. The best result of
decolorization was obtained in Aspergillus fumigatus MTCC 3002 which was found to decolorize 79% of crystal violet dye from 20
to 100µM at pH-3 with additional carbon and nitrogen source and Aspergillus niger MTCC 478 was found to decolorize 55% to
crystal violet dye from 20 to 100 µM at pH-2 with additional carbon and nitrogen source within 7 days. Aspergillus fumigatus was
found more efficient than Aspergillus niger.

The Crystal Violet (C.V.) is a triphenylmethane dye and these
types of dye are extensively used in textile industries for dying
nylon, silk, wool, cotton, medicine and biological stain9-13.
Paper and leather industries are also major consumers of
triphenylmethane dyes. This group of dyes is also used for
dying plastics, varnish, oil, and wax etc14. Due to inefficiencies
of the industrial dyeing process, 10-15% of the dyes are lost
in the effluents of textile units4. It is observed that 280,000
tons of textile dyes are discharged in such industrial effluents
every year worldwide6. They are highly visible and affecting the
transparency and gas solubility of water bodies. They disturb
reflection and absorbance of the sunlight entering water, thereby
interfering the aquatic life, growth and affecting
photosynthesis11.
Crystal violet has been classified as a recalcitrant compound,
and it is poorly metabolized by microbes and, consequently,
it has capacity to persist in environments for long period and
introduce in food chain8. It was also reported that Crystal violet
accumulates in soil and river as sediments that could be traced
back to the dumping of improperly treated chemical wastes.
Some research works showed that Crystal violet are potent
clastogens and responsible for promoting tumor growth in some
species of fish5. Decolorization of this type of dyes by physico-
chemical method such as chemical oxidation and reduction,
physical flocculation and precipitation, adsorption, photolysis,
electrochemical treatment, reverse osmosis advanced
oxidation, and biodegradation has been investigated1. But the
physico-chemical treatments are cost-effective and produce
large amount of sludge and degradative product which are also
hazardous for environment3. Therefore, biological wastewater
treatment systems are the point of investigation now-a-days,
because it is effectively removing Crystal Violet from the
wastewater, resulting in its dispersal into the environment. The
report on bacterial biodegradation is scanty. Earlier workers
demonstrated a relatively successful biodegradation of C.V.
using various species of fungi and were considered to be
impractical for use in possible commercial biodegradation
systems.
The aim of the present work was to investigate the efficiency
of decolorization of crystal violet dye by Aspergillus species
and optimize the best parameter for bioremediation of crystal
violet.
MATERIAL AND METHODS
Dye and chemicals: Crystal violet dye was purchased from
Merck and all chemical were used of analytical grade. The 1
mM stock solution was prepared from solid form of crystal
violet diluted to 1 mM.
Culture of fungal species: The two species of Aspergillus
i.e. Aspergillus fumigatus MTCC 3002 and Aspergillus niger
MTCC 478 were obtained from MTCC, Chandigarh. Fungal
strains were sub cultured and maintained in Czapek yeast
extract medium, the composition of the medium was K2HPO4

NAAS Rating (2016)-4.20

 COMPARATIVE STUDY ON DECOLORIZATION
-1.0gm, Yeast extract-5.0gm, Sucrose-30.0gm, Agar-15.0 gm,
NaNO3 -30.0gm, KCL -5.0gm, MgSO4.7H2O -5.0gm, FeSO4-
7H2O -0.1gm, Distilled water- 1000.0ml. After inoculation of
fungal species, the czapek yeast agar slants were incubated
for 3-7 days and after spores' formation slant of fungal species
were stored in refrigerator at 4°C.
Decolorization Studies: The experiment was performed with
optimization of pH, dye concentration and study of additional
carbon nitrogen source.
Batch decolorization studies were conducted in 100 ml of 7
Erlenmeyer flasks containing 50 ml of basal medium (Potato
dextrose broth) was prepared and the dye (Crystal Violet) was
diluted from stock solution to make 10µM concentration of
dye. The effect of pH was studied at different values of pH- 2,
3, 4, 5, 6, 7 and 8 was maintained by 1.0 N NaOH and 1.0 N
HCL. Aspergillus fumigatus (MTCC 3002) and Aspergillus niger
(MTCC 478) were inoculated and incubated at 25 °C for 7
days (Fig-2).
For measuring the optimum concentration of dye and toxic
level, 20 Erlenmeyer flasks (100 ml) with 50 ml basal PDB
medium in each flask was prepared with various increasing
concentrations (20,40,60,80,100,120,140,160,180 and 200 µM)
of Crystal Violet dye. Both Aspergillus fumigatus and
Aspergillus niger species were inoculated at specific pH-3 and
2 that was optimized by preliminary experiment. These batch
cultures were incubated for 7days. This secondary experiment
was helpful to measure the toxic concentration of dye that
was affecting the growth and decolorization capability of fungal
species.
The addition of glucose and sodium nitrate in 10:1 as a carbon
and nitrogen source was used to improve the potential of both
Aspergillus species to decolorize Crystal Violet as a next
experiment, that was conducted on 20 flasks (100 ml)
containing 50 ml basal PDB medium, prepared with various
concentrations (20,40,60,80,100,120,140,160,180 and 200 µM)
of Crystal Violet dye. Both Aspergillus fumigatus and
Aspergillus niger species were inoculated at specific pH-3 and
2 respectively that was optimized by preliminary experiment
and culture was incubated for 7 days.
After 7 days broth of fungal cultures were separated by
Whatmann filter paper- 1 and filtered broth was measured by
(332)
spectrophotometer of Elico-india 159, at a wave length of 592
nm. The percentage of decolorization was calculated by using
the formula12.
Percentage of Decolorization % = I ab - F ab× 100/I ab
Whereas, I ab - Initial absorbance
F ab - Final absorbance
RESULTS AND DISCUSSION
Effect of pH:- In the present study the two species of
Aspergillus fumigatus and Aspergillus niger were investigated.
Our result showed that, better decolorization was obtained by
Aspergillus fumigatus (MTCC 3002) and was found to
decolorize 79.89% at pH-3, and 74.43%, 61.97%, 48.01%
and 44.26% was optimized at pH-4, 5, 6 and 7 respectively
(Fig-1). At pH-3 Aspergillus fumigatus MTCC 3002 showed
the best decolorization result, but when pH was increased the
percentage of decolorization was decreased. In case of
Aspergillus niger MTCC 478 good decolorization result 69.33% at
pH-2 was found and 65.37%, 63.71%, 60.00% & 47.74% results
were optimized at the pH-3, 4, 5, and 6 respectively(Fig-3). Low
percentage of decolorization (41.71% and 20.38%) was
measured by pH-7 and 8 respectively. Comparatively
Aspergillus fumigatus showed the best result (79.89%) than
Aspergillus niger (65.37%) at pH-3. Whereas, the better result
of Aspergillus niger (69.33%) was obtained than Aspergillus
fumigatus (67.93%) at pH-2. Consequently the pH-3 was good
for Aspergillus fumigatus to decolorize crystal violet. But in
case of Aspergillus niger good decolorization at pH-2 was
reported (Fig-2 and Table-2).
Effect of dye concentration:-Further experiment was
conducted in batch culture to optimize the toxic concentration
of dye that affect decolorization and inhibited the growth of
fungal species and point out the decolorization potential of
fungal species between 20-200 µM dye concentrations. The
result of this experiment showed that, Aspergillus fumigatus
(MTCC 3002) was able to decolorize 77.04%, 70.49%, 65.14%,
60.89% and 55.22% of crystal violet dye at a concentration of
20, 40, 60, 80, and 100 µM respectively (Fig-3). The good
result was obtained at 20 µM concentration and on increasing
the concentration of dye there was a decline in decolorization
property, therefore, the decolorization result of 120,140 and
160 µM was found lowered as 48.46%, 37.04% and 32.88%
(333) SHARMA, SAHU AND KUMAR

Table-1.The effect of pH on crystal violet (C.V.) degradation by Aspergillus fumigatus and Aspergillus niger.

Table-2.The effect of concentration on decolorization of crystal violet by Aspergillus fumigatus and

Aspergillus niger

Table-3. Decolorization of crystal violet by Aspergillus fumigatus and Aspergillus niger additional
carbon and nitrogen source

Figure-1 . Showing effect of pH on decolorization of crystal violet by Aspergillus fumigatus (MTCC 3002)

Figure-2 . The effect of pH on crystal violet (C.V.) degradation by Aspergillus fumigatus and Aspergillus niger.
 COMPARATIVE STUDY ON DECOLORIZATION (334)

Figure-3 . Showing effect of pH on decolorization of crystal violet by Aspergillus niger (MTCC 478)

Figure-4 . Showing decolorization of crystal violet based on concentration variance by Aspergillus fumigatus
(MTCC 3002) (4.1) and Aspergillus niger (MTCC 478) (4.2).

Figure-5. Decolorization of crystal violet by Aspergillus fumigatus and Aspergillus niger based on concentration
of dye.
(335) SHARMA, SAHU AND KUMAR

Figure-6. Decolorization of crystal violet by Aspergillus fumigatus and Aspergillus niger based on additional
carbon and nitrogen source.
respectively. Very low decolorization (28.39% and 22.78%) and 200 µM, it was properly inhibitory for growth (Fig-6 and
was measured at 180 and 200 µM which was the toxic level of Table-3).
fungal growth and inhibited of decolorization. Whereas,
In the present study, it was observed that both Aspergillus
Aspergillus niger (MTCC 478) showed the decolorization result
fumigatus and Aspergillus niger were competent to decolorize
as 68.34%, 64.87%, 60.32%, 58.52% and 46.87% at
crystal violet, but Aspergillus fumigatus showed more
concentration of 20, 40, 60, 80 and 100 µM respectively (Fig-4).
promising result at pH-3 than Aspergillus niger. Aspergillus
The good result was obtained at 20 µM and when the
niger has decolorized crystal violet (20-100 µM) more than
concentration is in increasing order, the percentage of
55% at pH-2. Whereas, Aspergillus fumigatus achieved more
decolorization continuously decrease, therefore the
than 79% decolorization at same concentration but at pH-3.
decolorization result of 120,140 and 160 µM was found low
(43.88%, 35.40% and 30.81%) and 180 and 200 µM of dye Previously some authors have tried to decolorize crystal violet
was found inhibitor for growth and decolorization. Comparatively, by using some other species of Aspergillus not by niger and
Aspergillus fumigatus showed the best result of decolorization fumigatus. Some workers have achieved 57% decolorization
of Crystal Violet than Aspergillus niger (Fig-5 and Table-2). of crystal violet by using Aspergillus ochraceus11. Some earlier
researchers have also reported decolorization of crystal violet
Effect of additional carbon and nitrogen source:- Glucose
up to 80% by using Pseudomonas putida within one week4.
and sodium nitrate (10:01) were used as an additional carbon
Earlier workers have also reported decolorization of crystal
nitrogen source in basal medium to improve the potential of
violet up to 98% by using Fusarium solani2. Some other workers
decolorization. In this decolorization study, result showed that,
have achieved similar result up to 78% by using white rot fungi7.
Aspergillus fumigatus was able to decolorize Crystal Violet
Earlier researchers had applied Phanerochaete chrysosporium to
up to 84.44%, 83.41%, 82.14%, 81.54% and 79.77% at pH-3
decolorize crystal violet and concluded conversion of crystal violet
with the dye concentration of 20,40,60,80,and 100µM
into 3 metabolites (N,N,N',N',N"-pentamethylpararosaniline,
respectively. A good co-effect also showed that in Aspergillus
N,N,N',N"-tetramethylpararosaniline, and N,N',N"-trimethylpararo-
niger (MTCC 478), it was 70.42%,64.15%,62.84%,59.76% and
sanizline)3.
55.92% at pH-2 with the dye concentration of 20,40,60,80,and
100 µM respectively. But in the case of 120, 140, and 160 µM Besides fungus, some authors have also applied several
concentration of dye the decolorization was very low and 180 bacteria like Agrobacterium radiobacter (MTCC 8161) to
 COMPARATIVE STUDY ON DECOLORIZATION (336)

decolorize crystal violet and they found promising result within 2. Abedin, R.M.A. (2008). Amrican Eurasian journal of botany.,17.
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crystal violet is an important task now-a-days before the 4. Boer, C.G., Obici, L., Souza, C.G. and Peralta, R.M. (2004).
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Biotechnol Lett., 391:396.
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Present study describes that Aspergillus fumigatus (MTCC 1147:1149.
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In this study we observed that Aspergillus fimigatus was more Journal of Biotechnol., 407:410.
13. Willian, A.U., Pathak, S. and Hsu, T.C. (1978). Mutat. Res.,
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efficient and eco-friendly. The regenerated biomass can be
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