Indo American Journal of Pharmaceutical Research, 2014
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ISSN NO: 2231-6876
INDO AMERICAN
JOURNAL OF
PHARMACEUTICAL
RESEARCH
TO STUDY THE BIOREMEDIATION OF MONOCROTOPHOS AND TO ANALYZE THE
KINETICS EFFECT OF TWEEN 80 ON FUNGAL GROWTH
Bhawana Pandey1, Priyanka Singh Baghel2 and Shikha Shrivastava3
Department of Biotechnology and Microbiology, Bhilai Mahila Mahavidyalaya,
Hospital Sector, Bhilai (C.G.), India.
ARTICLE INFO
Article history
Received 18/01/2014
Available online
24/02/2014
Keywords
Biodegradation And
Bioremediation,
Microorganisms,
In Situ or Ex Situ,
Surfactants,
Immunodeficiency.
ABSTRACT
Biodegradation and bioremediation both are same processes which are based on the
metabolism of pesticide and insecticides by microorganisms. Microorganisms which are used
for the process of bioremediation are known as bioremediators.
In this experiment we used an organophosphorus compound i.e. Monocrotophos (MCP)
pesticide. Aspergillus fumigatus is one of the Aspergillus species to cause disease in human
being with an immunodeficiency. Addition of Tween 80 stimulates the utilization of hexamer.
When Aspergillus niger combined with Tween 80 this mixture effectively enhanced the
solubility and degradation of MCP. Bioremediation of Monocrotophos at different
concentration by using Aspergillus fumigatus and the kinetics effect of Tween 80 on fungal
growth were studied and found that the A. fumigatus degrade the MCP till its 1%
concentration.
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Please cite this article in press as Bhawana Pandey et al.To study the bioremediation of monocrotophos and to analyze the kinetics
effect of tween 80 on fungal growth. Indo American Journal Of Pharm Research.2014:4(02).
925
Corresponding author
Bhawana Pandey
Bhilai Mahila Mahavidyalaya,
Hospital Sector, Bhilai (C.G.), India
[email protected].
09424118308
Vol 4, Issue 02, 2014.
Bhawana Pandey et. al.
ISSN NO: 2231-6876
INTRODUCTION
The pollution is a problem for human being. Microorganisms play a major role in the breakdown of these pollutants
(Alexander, 1981). Bioremediation process is very effective method for biodegradation in contaminated soils (McLaughlin, 2001;
Swannell et. al., 1996). Physiochemical and biological method can be used for the remediation but because of certain disadvantage
like very expensive technology, very long time taken researchers were also focusing towards the biological methods (Atlas, 1995;
Hoff, 1993; Swannell et al., 1996).
Those microorganisms which are used to perform the function of bioremediation are known as bioremediators. Technologies can be
generally classified as in situ or ex situ. Some examples of bioremediation technologies are phytoremediation, bioventing, bioleaching,
land farming, bioreactor, composting, bioaugmentation and biostimulation etc. (Fragoeiro,2005).
Biodegradation and bioremediation are matching process to an extent that both of these are based on the conversion or
metabolism of pesticide by microorganisms. The biodegradation is the natural process where as bioremediation is a technology.
Temperature, pH and amount of pesticides act as limiting factor for pesticide degrading microorganism (Fogarty and Tuovinen, 1990).
An ideal pesticide should have the ability to destroy any pest quickly and also should be able to degrade non-toxic substances as
quickly as possible (Vaccari et. al., 2006).
In the present study Monocrotophos (MCP) pesticide was used which is an organophosphorus compound. Organophosphorus
compound covers about 70% of the pesticide used worldwide. Natural degradation of MCP takes place over a period of 12-16 days
and the process could be expatiated through bioremediation (Manohar Das et. al. 2007)
Aspergillus fumigatus is one of the most common Aspergillus species to cause disease in individuals with an immunodeficiency, it
plays an important role in carbon and nitrogen recycling. Colonies of this fungus produce from conidiophores thousands of minute
conidia (2–3 μm) that readily become airborne.
The synthetic surfactant Tween 80 is a non-toxic and inert for soil microorganisms and soil matrix and enhanced dissolution
rate for single compounds (Angela et. al. 2000). The synthetic surfactant are useful for bioremediation of hydrophobic pollutants at
low concentrations (Aronstein et. al. 1993). The addition of Tween 80 with Aspergillus fumigatus effectively enhanced the solubility
and degradation of MCP and they also reported that Tween 80 is biodegradable (Wong et. al. 2004). The oxygen uptake rate of
bacterial cells was not affected in the presence of surfactants (Volkering et. al. 1995)
The objectives of the present study were: Maintenance of culture by regular sub culturing.
 To study the effect of MCP at different concentration on fungal growth.
 To study the kinetic effect of Tween 80 on fungal growth and study the bioremediation of MCP with the help of
fungi.
Materials and Methods :
Media preparation :Sterilized by autoclaving at 121°C for 15 minute and add 1% MCP in media.
Table 1: Complete media
SN
Chemical
Amount
(in gm)
Na2NO3
2
2
KCl
0.5
3
MgSO4
0.5
4
K2HPO4
1
5
FeSO4
0.01
6
Sucrose
30
7
MCP1%
10 ml
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Bhawana Pandey et. al.
ISSN NO: 2231-6876
Table 2: Incomplete media
SN
Chemical
Amount
(in gm)
1
Na2NO3
2
2
KCl
0.5
3
MgSO4
0.5
4
K2HPO4
1
5
FeSO4
0.01
6
MCP (1%)
10 ml
Table 3: Czapek Dox Agar Media
SN
Chemical
Amount
(in gm)
1
Na2NO3
2
2
KCl
0.5
3
MgSO4
0.5
4
K2HPO4
1
5
FeSO4
0.01
6
Sucrose
30
7
Agar
15
For kinetics study – 1% Tween 80 was added in complete and incomplete media.
Methodology
1. Maintainance of culture:- Preparation of Czapek Dox Agar Media (pH – 7).
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3. Measurement of fungal growth by Mycelia Dry Weight Method: Czapek Dox broth medium was prepared and sterilized at 121°C for 15 minutes.
 5-10 agar blocks of 5 mm diameter was cut from actively growing margin of A. fumigatus and transferred 5 blocks into each 3
flask with the help of inoculating loop.
 The inoculated flasks were incubated at 25°C for 10 - 15 days.
 The mycelial mat was filtered through pre-weighed whattman filter paper and dried at 80°C for 24 hours. Weighed dried mycelia
mat and recorded dry weight.
 The same procedure was done for each concentrations of MCP and for kinetics study by Tween 80.
927
2. Measurement of fungal growth by Colony Diameter Method: Grown the test fungus ( Aspergillus fumigatus) on Czapek Dox Agar Medium in advance.
 Cut agar blocks of 5 mm diameter from actively growing margin of Aspergillus fumigatus and transfer single block in the centre of
Czapek Dox Agar plates.
 Incubated the inoculated plates at 25°C for about 5 days by putting upside down.
 The same procedure was done for each concentrations of MCP and for kinetics study by Tween 80.
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Bhawana Pandey et. al.
ISSN NO: 2231-6876
Table 4: Table for dry weight method : For
MCP at different concentration
Medi
a(ml)
NO.
Control
Flask 1
Flask 2
Flask 3
Fungal Concentration of
culture MCP (ml)
1% 2% 3%
-nt
1
+nt
2
+nt
-ml
3
+nt
-ml
ml
100
99
98
97
Table 5:For kinetics test by using Tween 80
Medi
a
(ml)
100
99
98
97
NO.
Contro
l
Flask
1
Flask 2
Flask
3
Fungal
culture
Tween
80(ml)
-nt
+nt
+nt
+nt
1
1
1
1
Concentration
of MCP (ml)
1
2% 3%
%
1
-ml 2
ml
3 ml
Table 6: Observation: 1. By Fungal Zone Detection
Day interval
7 days
14 days
21 days
(16/03/2013)
(22/03/2013)
(28/03/2013)
H (in cm)
V (in cm)
H (in cm)
V (in cm)
H (in cm)
V (in cm)
M1
M1 + M2
-
-
-
-
-
-
M1 + M2 + T + F
2.0
2.2
3.0
3.0
3.4
3.4
M1 + M2 + F
M1(C) + F
M1(C) + T + F
1.5
2.4
4.5
1.6
2.2
4.0
2.5
3.6
4.7
2.4
3.7
4.2
3.0
4.1
5.0
2.5
3.8
5.0
Plates
In the presence of Tween 80 the fungal growth is increased. H = Horizontal,T
M1 = Media, F = Fungus, M2 = Monocrotophos
= Tween 80, V = Vertical, C = Complete,
ZONE DIAMETER METHOD
14 days
21 days
Fig.1
Fig.2
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Fig.3
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928
7 days (control)
Vol 4, Issue 02, 2014.
Bhawana Pandey et. al.
ISSN NO: 2231-6876
Table 7: By Dry Weight Determination
Day interval
After
filteration
( dry weight in gm )
Paper weight (in gm)
Plates
M1 + M2
1.070
1.071
M1(C) + M2
M1(C) + F
M1(C) + T +F
M1 + M2 + F
M1 + M2 + T+ F
1.080
1.100
1.000
1.240
1.360
1.080
1.760
2.070
1.260
1.520
Fungus weight
(in gm)
0.001
0.000
0.660
1.070
0.020
0.160
In the presence of Tween 80 the fungal growth is increased. H = Horizontal, T = Tween 80, V = Vertical, C = Complete,
M1 = Media, F = Fungus, M2 = Monocrotophos
FOR DRY WEIGHT METHOD
A) For MCP
Fig.4
B) For Tween 80
Fig.5
Fig.6
Fig.7
WEEK
2
ND
3
RD
MCP Degradation Rate
Different Concentration
WEEK
WEEK
at
Determining Kinetics Effect
Through Tween 80
MCP Degradation Rate at
Different Concentration
Determining Kinetics
Through Tween 80
Effect
0.3
0.2
0.01
0.01
0.6
0.4
0.01
0.01
0.5
0.4
0.01
0.01
0.8
0.6
0.01
0.01
0.7
0.6
0.01
0.01
0.9
0.7
0.01
0.01
RESULT AND DISCUSSION
In the present investigation it was found that A. fumigatus has capability to degrade the organophosphorus pesticide
Monocrotophos. The present work was carried out at a different concentration (1%, 2% and 3%) of MCP and it was found that A.
fumigatus can degrade and tolerate only 1% MCP and show no growth at higher concentration that is 2% and 3% MCP.
Present investigation was also carried out at kinetic level using Tween 80 as a growth enhancer and it was found that the growth rate
increases in the presence of Tween 80.
Mycellial dry weight and colony diameter growth methods were used in the present investigation to measure the fungal
growth.
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1
ST
MCP Degradation Rate at
Different Concentration
Determining Kinetics Effect
Through Tween 80
929
Table 8: Determining MCP Degradation Rate at Different Concentration and Determining Kinetics effect through Tween
80
CONTROL
EXPERIMENTAL (MCP
(complete media)
as carbon source in gms)
DAY
1%
2%
3%
CONDITIONS
( in gms)
INTERVAL
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Bhawana Pandey et. al.
ISSN NO: 2231-6876
R. Jayashree et. al. discussed on their research that the degradation of alpha and beta endosulfan by Pseudomonas aeruginosa
with Tween 80 was higher than without Tween 80. The addition of synthetic surfactant Tween 80 enhanced the solubility and
degradation of endosulfan. But in the present investigation the bioremediation of organophosphorus bioremediation insecticides such
as Monocrotophos at different concentration by using Aspergillus fumigatus with Tween 80 was studied and the same result was
found. Ansaruddin et. al. had reported that exposure to chlorpyrifos and its metabolites have been related to a variety of nerve
disorders in humans. They had found a diverse range of microorganisms that are responsible for chlorpyrifos degradation.
CONCLUSION
In present investigation the bioremediation of organophosphorus insecticides such as Monocrotophos at different
concentration by using Aspergillus fumigatus and the kinetics effect of Tween 80 on fungal growth were studied and found that the A.
fumigatus can also degrade the MCP till its 1% concentration and the growth of microorganism increased in the presence of Tween 80.
With the help of present investigations researchers may identify and isolate other fungal species which are responsible for
biodegradation of different types of pesticides.
Authors’ Statements
Competing Interests
The authors declare no conflict of interest.
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