Research Journal of Environmental and Earth Sciences 3(5): 614-619, 2011
ISSN: 2041-0492
© Maxwell Scientific Organization, 2011
Received: June 27, 2011
Accepted: August 08, 2011
Published: August 10, 2011
Detoxification of Chlorpyriphos by Micrococcus luteus NCIM 2103, Bacillus
subtilis NCIM 2010 and Pseudomonas aeruginosa NCIM 2036
Madhuri V. Bhuimbar, Ashwini N. Kulkarni and Jai S. Ghosh
Department of Microbiology, Shivaji University, Vidyanagar, Kolhapur 416004, India
Abstract: This investigation aims to explore mechanisms of microbiological detoxification of residual
organophosphorus pesticide - chlorpyriphos using certain soil heterotrophs like Pseudomonas aeruginosa
NCIM 2036, Bacillus subtilis NCIM 2010, Bacillus cereus NCIM 2156, Micrococcus luteus NCIM 2103 and
Galactomyces geotrichum MTCC 1360 by comparing with dimethoate and monocrotophos. Organophosphorus
class of pesticides has evolved after the gross misuse of organochlorine pesticides like DDT having a half life
of 5 years in agricultural soils. Therefore, the pharmacodynamics of the residues often led to effects like
mutagenesis, carcinogenesis and teratogenesis in higher animals including humans. It was observed that
common heterotrophs like Bacillus subtilis NCIM 2010, Bacillus cereus NCIM 2156, Micrococcus luteus
NCIM 2103 brought about degradation of Monocrotophos, Chlorpyriphos and Dimethoate along with
Galactomyces geotrichum MTCC 1360. However, though Pseudomonas aeruginosa NCIM 2036 was found
to degrade Monocrotophos, dimethoate but could not detoxify chlorpyriphos.
Key words: Bacillus, chlorpyriphos, micrococcus, pseudomonas
action is mostly on contact though sometime it is effective
after ingestion. These chemicals inhibit cholinesterase, an
enzyme that is essential for synaptic nerve ends of both
humans and insects, thus polarizing the nerves (Skripsky
and Loosli, 1994). Chlorpyriphos may affect the
development of central nervous system (Slothkin et al.,
2006) and also known to affect the endocrine secretion
(Rawlings et al., 1998). Monocrotophos is also commonly
used to protect plants from certain pests. Its toxic effect in
humans involves the central nervous system (Horrigan
et al., 2002). Monocrotophos is highly toxic to birds while
it is moderately toxic to fish (Meister, 1992). Most of
these organophosphorous compounds are slightly soluble
in water and have high oil-water partition coefficient and
low vapour pressures. These are mostly degraded by
photolysis, hydrolysis, yielding water-soluble products
(Gallo, 1991). The compounds used for agricultural
purposes are available mainly as emulsifiable
concentrates. The 3 common organophosphorus pesticides
include monocrotophos, dimethoate, Chlorpyriphos
(Fig. 1).
This investigation attempts to study the microbial
detoxification of chlorpyriphos by degradation and
bioaccumulation under different experimental conditions.
INTRODUCTION
The word pests include a wide range of life forms
including plants and animals. Insects form the largest
group of pests. Likewise insecticides form a group of
pesticides used to control insect pests. Since 2000BC,
humans have utilized pesticides to protect their crops.
These agents are not only used to improve the agronomy
of any crop, but are widely used to check public health by
controlling insects carrying disease causing germs either
as passive vectors or zoonotic vectors. The term pesticide
is also used for substances intended for use as a plant
growth regulator, defoliant, desiccant or agent for
thinning fruit or preventing the premature fall of fruit also
used as substances applied to crops either before or after
harvest to protect the commodity from deterioration
during storage and transport (FAO, 2002). The first
known pesticide was elemental sulfur followed by arsenic,
mercury, lead, pyrethrin, rotenone, DDT, synthetic
pesticides. There are various types of pesticides used
depending on pest.
Organophosphate pesticides are potent toxicants used
in agriculture. These are synthetic in origin and are
normally esters, amides, or thiol derivatives of
phosphoric, phosphonic, phosphorothioic or
phosphonothioic acids (FAO, 2002). The indiscriminate
use results in accumulation of residual pesticides and
causes hazardous effects on soil fertility and ecology. One
such pesticide in common use is dimethoate. This is
moderately toxic by ingestion, inhalation and dermal
absorption (Carman, 1982). Another such chemical is
chlorpyriphos; which is a broad spectrum insecticide. Its
MATERIALS AND METHODS
The study was conducted between August 2010 and
February 2011.
Microorganisms used: Cultures of Bacillus subtilis
NCIM 2010, Bacillus cereus NCIM 2156, Micrococcus
Corresponding Author: Dr. Jai S. Ghosh, Department of Microbiology, Shivaji University, Vidyanagar, Kolhapur 416004, India
614
Res. J. Environ. Earth Sci., 3(5): 614-619, 2011
Fig. 1: Chemical structures of, (a) dimethoate, (b) Chlorpyriphos, (c) monocrotophos
luteus NCIM 2103, Galactomyces geotrichum MTCC
1360 and Pseudomonas aeruginosa NCIM 2036 were
obtained from NCIM Pune, India. Cultures of Bacillus
subtilis and Bacillus cereus were grown and maintained
on peptone agar, cultures of Micrococcus luteus and
Pseudomonas aeruginosa were grown and maintained on
nutrient agar while culture of Galactomyces geotricum
was grown and maintained on yeast extract mannitol agar
(Atlas, 2004).
Galactomyces geotrichum and Pseudomonas aeruginosa.
The growth was measured in terms of optical density at
530 nm after 12 h interval till constant consecutive
readings.
Analysis of pesticides or their degradation product:
Samples from medium, with and without with pesticides,
showing growth of the respective microorganisms were
collected from late log phase of growth. These were
subjected for centrifugation at 8000×g for 15 min. to
separate the cells. Clear supernatant of the cell free media
were collected. The cell pellet was resuspended, in sterile
saline and centrifuged likewise to remove traces of media
and pesticides. The washed cells were homogenized by
ultra Sonication. The homogenized suspension was
centrifuged at 10,000×g for 20 min at 4ºC to remove all
cell debris. Now the pesticides were seperated from both
the cell free medium and the homogenized cell suspension
by the method described above.
Extraction of pure pesticides: Commercially available
preparations are not pure compounds, as these contain
other ingredients like emulsifiers, stabilizers etc. Hence it
is essential to carry out extraction of pure pesticide from
commercially available formulations. The extraction of
Monocrotophos, Chlorpyriphos, and Dimethoate is carried
out from Phoskill (36% E.C), Lethal (21.50% E.C), and
Rogor (30% E.C.), respectively.
Few drops of these formulations were taken in 5ml of
acetone and shaken well. To this then 5 mL of chloroform
and 2 mL of distilled water was added and shaken
vigorously. The tubes were allowed to stand till the
chloroform layer seperated clearly which was carefully
removed and passed through anhydrous sodium sulfate to
remove traces of water. This was then evaporated at 3035oC to get fine crystals or amorphous powders of the
respective pesticide. The residues obtained were washed
with chloroform- water mixture a few times to remove all
impurities. The purity was checked by GCMS (results not
shown).
Gas chromatography mass spectroscopy: Samples of
cell free supernatants were mixed with diethyl ether. The
organic phase was separated, dried and redissolved in
methanol, and used for mass spectroscopy studies. The
samples were also checked spectrophotometrically, for
absorption maxima.
RESULTS
The degradation of these pesticides occurs depending
on the nature of active compounds present in it. The entire
degradation protocol of monocrotophos, chlorpyriphos
and dimethoate might be started from dephosphorylation
followed by demethylation. The degradation was clearly
observed after spectrophotometric analysis of samples for
absorption maxima. It is still not clear whether the
degadation mechanism is either secondary or tertiery
metabolism. Some organisms showed intracellular
accumulation of pesticides too.
Bacillus subtilis, Pseudomonas aeruginosa, Bacillus
cereus, Micrococcus luteus and Galactomyces geotrichum
were incubated with chlorpyriphos for 3 days. It can be
Growth pattern of the microorganisms in presence
and absence of pesticides: Normal growth pattern of the
organisms were studied by inoculating fresh culture of
organisms in sterile liquid nutrient medium (Atlas, 2004),
without pesticides. The growth was measured in terms of
optical density at 530 nm at 30 min interval till constant
consecutive readings.
The effect of the pesticides on growth was checked
by using 200 ppm concentration of monocrotophos,
chlorpyriphos and dimethoate separately in sterile liquid
nutrient medium which were inoculated with Bacillus
subtilis, Bacillus cereus, Micrococcus luteus,
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Res. J. Environ. Earth Sci., 3(5): 614-619, 2011
120
100
80
Residue in medium(%)
60
40
20
80
40
60
20
s
Ga
l
a
ge o c t o
tric m y
h um c e s
Mi
lut cro co
e us c c
u
Ba
cer cillus
e us
B
100
60
ntr
o
us
Ga
geo lacto
tric my
h u ces
m
Co
Mi
lut croco
eus cc
Ba
cer cillus
eu
s
Ps
aer edom
ug on
ino as
sa
Ba
sub cilus
tili
s
ntr
ol
Co
Ga
geo lacto
tric m y
h u ces
m
0
us
0
Mi
lut croco
eus cc
20
l
20
Ba
cer cillu
eu s s
40
Ps
aer ed om
ug o n
ino as
sa
60
80
Ba
sub cilu s
t ili
s
Accumulation(%)
40
120
120
C
100
C
Degradation(%)
100
80
60
40
80
40
60
20
Ba
cer cillus
eu
s
Mi
lut croc
eu occ
s
us
Ga
geo lacto
tric my
hu ces
m
Ga
ge lacto
otr m
ich yce
um s
Ba
ce cillu
reu s
s
Mi
c
lut roc
eu oc
cu
s
s
Ba
sub cilus
tili
s
Ps
ed
aer om
ug on
ino as
sa
Co
ntr
ol
0
Pse
aer dom
ug on
ino as
sa
20
Ba
sub cilus
ti li
s
0
-20
Co
ntr
ol
Degradation(%)
Ps
aer ed om
ug on
i no a s
sa
120
80
-20
Ba
s u b c i l us
tili
s
nt r
ol
-20
B
100
Accumulation(%)
0
Ga
ge lacto
otr m
ich yce
um s
120
Ba
cer cillu
eu s
s
Mi
c
lut roco
eus cc
us
Ps
e
aer dom
ug on
ino as
sa
Ba
sub cilus
tili
s
Co
ntr
ol
0
Co
Residue in medium(%)
100
-20
A
120
A
Fig. 3: (a) Residual dimethoate detected in cell free medium,
(b) dimethoate accumulated inside cells, (c) Extent of
degradation of dimethoate by individual organisms
Fig. 2: (a)Residual chlorpyriphos detected in cell free medium,
(b)Chlorpyriphos accumulated inside cells, (c) Extent of
degradation of Chlorpyriphos by individual organisms.
organisms with exception of Pseudomonas aeruginosa.
On examining Fig. 2c, it can be noted that Bacillus
subtilis brought about 87.5% degradation of the pesticide.
Pseudomonas aeruginosa did not do any degradation. The
other microorganisms viz. Bacillus cereus, Micrococcus
luteus and Galactomyces geotrichum brought about 75,
58.75 and 72.50% degradation respectively.
Bacillus subtilis NCIM 2010, Pseudomonas
aeruginosa NCIM 2036, Bacillus cereus NCIM 2156,
Microccocus luteus NCIM 2103 and Galactomyces
geotrichum MTCC 1360 were incubated with dimethoate
seen from Fig. 2a that the amount of residual
chlorpyriphos was reduced in the medium, with exception
of that in which Pseudomonas aeruginosa was growing.
Here the entire amount of the pesticide remained intact as
residue in the medium. Whereas, in the medium in which
Bacillus subtilis was growing, there was no
residualpesticide lef inthe medium Fig. 2b showed varied
amount accumulated chlorpyriphos inside the cells of all
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Res. J. Environ. Earth Sci., 3(5): 614-619, 2011
120
A
Residue in medium(%)
100
80
60
40
20
Ba
cer cillus
eus
Mi
c
l ut roc
eus occ
us
Ga
l
a
geo cto
tric m y
hum ces
Ps
aer edom
ugi on
n o as
sa
Ba
sub cilus
til i
s
Co
ntr
ol
0
B
120
Fig. 5: General pathway
Accumulation(%)
100
with the exception of Galactomyces geotrichum. Bacillus
cereus and Micrococcus luteus were completely removed
the pesticide from the medium. Fig. 4b shows that varied
amount of pesticide accumulated inside cells of all
organisms. On observing Fig. 4c, it can be noted that
Micrococcus luteus brought about 62.50% degradation.
Again Galactomyces geotrichum did not bring about any
degradation,though Bacillus cereus and Pseudomonas
aeruginosa shows 37.50 and 13.75%, respectively.
80
60
40
20
s
Ga
l
a
c
geo to
tric m y
h u c es
m
Mi
lu t croco
eu s cc
u
120
Ba
cer cill u
eu s s
Ba
su b cilus
t il i
s
Ps
aer ed om
ug o n
ino as
sa
Co
ntr
ol
0
DISCUSSION
100
C
Degradation(%)
80
Many of the organophosphorus compounds are ester
or thiol derivatives of phosphoric, phosphonic or
phosphoramidic acid. Their general formula contains
mainly the alkyl group, which can be directly attached to
a phosphorus atom or via oxygen (phosphates) or a
sulphur atom (phosphothioates) while the X group can be
diverse and may belong to a wide range of aliphatic,
aromatic or heterocyclic groups, also known as “leaving
group” because on hydrolysis of the ester bond it is
released from phosphorus as shown in Fig. 5 (Sogorb,
Vilanova and Carrera, 2004).
Some microorganisms are capable of degrading these
compounds. The enzymes involved in the degradation
mainly are esterases, hydrolases and phosphotases
(Bhadbhade et al., 2002). Microbial metabolic routes of
biodegradation of monocrotophos (Bhadbhade et al.,
2004) and dimethoate (Deb-Mandal et al., 2008) had been
proposed previously based on various experimental
results.
40
60
20
Ga
ge lacto
o tr m
ich yce
um s
Mi
lut croc
eu occ
s
us
Ba
cer cillu
eus s
Pse
aer dom
ug on
ino as
sa
Ba
sub cilus
tili
s
-20
Co
ntr
ol
0
Fig. 4: (a) Residual monocrotophos detected in cell free
medium, (b) monocrotophos accumulated inside cells,
(c) Extent of degradation of monocrotophos by
individual organisms.
for 3 days. It is very clear from Fig. 3a that residual
dimethoate is reduced in medium in which the organisms
were growing. Bacillus cereus and Micrococcus luteus
shows 100% accumulation of dimethoate inside the cells
as compared to Bacillus subtilis, Pseudomonas
aeruginosa and Galactomyces geotrichum (Fig. 3b). It can
be seen from Fig. 3c that Bacillus subtilis brought about
37.50% dimethoate degradation and Pseudomonas
aeruginosa, Galactomyces geotrichum brought about 1.25
and 3.75% degradation of the pesticide, respectively.
It can be seen from Fig. 4a residual amount of
monocrotophos was reduced in medium, by all organisms
CONCLUSION
Biodegradation of chlorpyriphos has so far not been
attempted. Its degradation by Micrococcus luteus NCIM
2103, Bacillus subtilis NCIM 2010 and Bacillus cereus
NCIM 2156 is being proposed in this study. Microbial
hydrolysis of chlorpyriphos, releases 3, 5, 6-trichloro
2 pyridenol (TCP), as a majo r intermediate. Further
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Res. J. Environ. Earth Sci., 3(5): 614-619, 2011
Fig. 6: Proposed mechanism of degradation of chlorpyriphos
degradation of TCP was observed with Micrococcus spp.
and Bacillus spp. to dichlorodihydroxy pyridine. This was
identified by the method of Feng et al. (2002) which had
studied the mechanism using Pseudomonas spp. ATCC
700113. Dehalogenation reaction takes place with this
compound to form 3, 6 dihydroxy pyridine 2, 5 dione.
Jingquan et al. (2010 isolated TCP degrading strain
Ralstonia spp.producing same metabolite 3, 6dihydroxypyridine-2, 5-Dione. This dione accumulates in
the cells. Proposed pathway for this is as given in Fig. 6:
The dione compounds are very difficult to biodegrade as
there are two ketonic groups. Hence the degradation stops
at this step.During growth of Pseudomonas aeruginosa
NCIM 2036 in presence of chlorpyriphos there was no
siderophore activity (results not shown).
The siderophore of this organism is the pigment
pyoverdin which maintain the balance of ferric ions by
chelating them which helps and enhances the production
of rhamnolipids (Ochsner et al.,1995) which would have
been essential for accumulation of chlorpyriphos
intracellularly. This is very unlike the degradation of
monocrotophos and dimethoate wherein the first step is
the formation of methyl amine, which is further converted
to ammonia. Such a compound is important
agronomically. The entire detoxification of these two
compounds ultimately lead to the formation of either
acetic acid or valeric acid, which are completely
metabolized by microorganisms. The mechanism of
degradation of monocrtophos and dimethoate has been
well illustrated by Bhadbhade et al. (2004).
ACKNOWLEDGMENT
The authors are very grateful to the Department of
Microbiology, Shivaji University, Kolhapur, for extending
the laboratory facilities to complete the investigation.
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