Abstract: levels of these pesticides are reported

Abstract:
Agriculture is the major
occupation of people in India, around 60-70% of its population is engaged in
agriculture. Annually millions
and tons of pesticides are used in field which in turn accumulates in
our ecosystem and food chain as well. The toxicity levels of these pesticides
are reported in amphibians, aves, fishes and even human beings. Malathion
(S-(1, 2 dicarethoxyethyl)-O, O-dimethyldithiophosphate) which cause inhibition
of acetylcholine esterase, there by leading to increase of the
acetylcholine concentration at the synaptic junction resulting in muscle
disfunction, paralysis and ultimately death. Malathion could be degraded either chemically or
biologically. Chemical degradation leads to production of malaxon which is 50
times more toxic then the parent compound. Our present study aim at
biodegradation of malathion using yeast. Around twenty organisms were isolated from different
sources like idli batter, paneer, cheese etc. Plate assay results
revealed that only one isolate i.e. I1 isolated from idli batter was able to
grow at very high concentrations of malathion 6,400 mg/L. Thin layer
chromatography result suggested that, there is some degradation of malathion by
organisms I1 when compared with standard. Further by morphological analysis using
phase contrast microscopy and monochrome staining and molecular
characterization using 18s gene sequencing and by phylogenetic analysis this
organisms was identified
as Candida tropicalis.It is
first study which reveals that Candida
tropicaliscould degrade malathion at such high concentration.

 

Keywords:
Biodegradation, Organophosphate, Malathion, Candida
tropicalis.

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INTRODUCTION

The excessive
use of natural resources
and large scale synthesis of xenobiotic compounds have generated a number of
environmental problems such as contamination of air, water and terrestrial
ecosystems, harmful effects on different biota, and disruption of
biogeochemical cycling 5.Organophosphorus compounds (OP), are  group of highly toxic agricultural chemicals
3. Some of
these pesticides have also been reported to be
toxic, mutagenic,
carcinogenic and tumorogenic 13.

Malathion, S-(1,2-dicarbethoxyethyl)-O,O-dimethyldithiophosphate,
is an organophosphate insecticide that has been used for some time as a DDT substitute for the control of
field crop pests, household insects, flies and animal parasites. It has been known as the first
organophosphorous insecticide with high selective toxicity 2.
Malathion is powerful inhibitors of acetyl cholinesterase, an important enzyme
involved in neurotransmission, in the form of acetylcholine substitutes 16.
Environmental hazards and health risks caused by pesticides could therefore
potentially affect human health
and environment 16.

Therefore, remediation of contaminated sites
is currently underway in order to develop safe, convenient and economically
feasible methods for pesticide detoxification 19. Bioremediation is an
environmental cleanup process that currently being investigated for use on a
wide variety of chemical 16. The
use of microbes in the bioremediation and
detoxification of many toxic xenobiotics, especially toxic pesticide is an
efficient tool for the
remediation of contaminated sites in the environment 16. They have the
capacity to utilize
virtually all naturally
and synthetically occurring compounds as their sole carbon and energy
source 13. Bacteria, fungi and some plants have potency to degrade malathion
12. Malathion
degradation products include dimethyl phosphate,
dimethyldithiophosphate, dimethylthiophosphate, isomalathion, malaoxon and due to cutinase,
carboxylesterase, phosphatase
enzymatic activity, malathion is degraded into malathion mono and
dicarboxylic acid 1.Mycodegradation is a deterioration phenomenon causing
degradation by fungi in a wide variety of materials and compounds 6.Studies
have been reported, the occurrence of several yeast such as Saccharomyces species, Candida species in various fermented
dairy or dairy related product like curd, cheese, idli, dosa, jalibi, fruit,
juices and brewery products
17.This paper focuses on biodegradation of Malathion by yeast isolated from
idli batter.

 

 

Materials & Methods

 

• Sample collection

Different samples (Curd, Paneer, Cheese, Buttermilk,
Soya sauce, Honey, Raw cow and buffalo milk, Idli batter) were collected in
sterile tube and stored in refrigerator until use 18.

 

• Isolation procedure

Sample was inoculated in 1% concentration
in 50ml of MRS broth and incubated under microaerophilic condition at 37°C for
24 hours. Ten fold dilutions were made using sterile saline and the last three
dilutions i.e. 10-8, 10-9, 10-10 were spread
on MRS agar and incubated under microaerophilic condition at 37°C for 24 hours.
Isolated colonies were streaked on MRS agar plate and they were further used
for screening of pesticide degraders 18.

 

·        
First enrichment

Isolated colony was inoculated in
10ml of MRS broth and incubated under microaerophilic condition at 37°C for 24
hours.

 

·        
Second enrichment

Enriched broth obtained from first
enrichment step was dispensed in sterile centrifuge tube and centrifuged at
5000 rpm for 10 minutes. Pellet was washed with sterile saline and 0.1 ml of
resuspended cells was inoculated in Davis Minimal broth containing 0.1% glucose
and 1.5 mg/L Malathion. Malathion was added after autoclaving and incubated
under microaerophilic condition at 37°C for 48 hours.

 

·        
Screening of Malathion degraders

Screening was done by streaking on
Davis Minimal agar plate containing 1.5 mg/L malathion. Further growth was
assessed on different concentration of Malathion ranging from 1.5mg/L to
12800mg/L 10.

 

·        
Morphological observation

The morphology of isolate was
observed using monochrome staining and phase contrast microscopy.

 

·        
Biochemical Test

Biochemical tests such as sugar
fermentation, sugar utilization, Urease test were performed using the following
protocol

 

·        
Checking growth of I-1 isolate in
soil sample

To check whether our isolate I-1 is
able to grow in presence of soil microflora flora following protocol was
carried out 15: Soil sample was collected in a petri plate, air dried and
grounded into powder form. Both autoclaved and 
unautoclaved soil was spiked with malathion under aseptic condition. One
set of autoclaved and unautoclaved soil was inoculated with isolate and other
set was used as control. Incubate the plate at 37ºC for 24 hours.  20g of test and control soil sample was
dispense in sterile saline and tenfold dilutions were subsequently made using
sterile saline.0.1 ml were spread on Minimal medium containing pesticide and
Incubated at 37ºC for 72 hours. Plates were checked for growth of I-1 isolate.

 

 

·        
Thin layer chromatography

 

0.1 ml of Enriched
broth was inoculate in Davis Minimal broth containing 6400 mg/L malathion and
incubated for 48 hrs under microaerophilic condition. Then 5mL of this broth
was taken in centrifuge tube and centrifuged at 5000 rpm for 10 minutes.
Supernatant was transfer in new centrifuge tube and equal volume of ethyl
acetate was added. Tube was inverted several times and was then transfer in
separating funnel. Two layer was obtained aqueous layer was discarded and
organic layer was filter through Whatman filter paper No. 1 containing sodium
sulphate.Filtrate was collected in centrifuge tube.TLC was run, solvent system
used were Hexane : Ethyl acetate(20:80) 8.

Visualization
with Iodine-Azide Procedure

After developed and air
dried, plate were sprayed with spraying solution( 6% sodium azide ,0.25% starch
at pH 6.5), they were exposed to iodine vapors for 15 seconds. The plate was
left for 15 minutes to let the iodine-azide reaction proceed.Malathion showed
white spot in yellow background 11.

 

·        
Isolation of genomic DNA

The isolate was streaked on Sabouraud
Dextrose agar (SDA) and incubated at 37°C for 24 hours. The resulting isolate
was used for extraction of DNA by Edward method 9: 50 mg of cells was taken
in 1.5mL microfuge tube to which 200µL of Edward buffer was added. Cells were
manually crushed with plastic pestle for 5 minutes. 200µL of Edward buffer was
further added and cells were crushed for additional 5 minutes. Volume was made
up to 1000µL by of Edward buffer. Vortexing was carried out for 15 seconds and
incubated at 100°C for 10 minutes and then centrifuged at 2000 rpm for 10
minutes. 500µL of supernatant was transferred in new microfuge tube and
centrifuged at 2000 rpm for 10 minutes. 400µL of resulting supernatant was
transferred in new microfuge tube to which 400µL of ice cold isopropanol was
added. Tube was inverted gently for 5 times and incubated at room temperature
for 10 minutes. Centrifugation was carried out at 14000 rpm for 10 minutes.
Discard the supernatant and air dry pellet at 37°C till fully dried. Wash dried
pellet with 70% ethanol and centrifuge at 14000 rpm for 10 minutes. Suspend the
dried pellet in 100µL of TE buffer. Concentration of DNA was checked by using
Nano drop spectrophotometer.

 

 

 

·        
PCR amplification & purification
of PCR product

For PCR all components such as 16.5µL
of Millipore water, 2.5µL of 10X buffer, 1µL of dNTPs, 1.5µL of MgCl2, 1µL of
specific primers (ITS1, ITS4, LROR, and LR5), and 0.5µL of Taq polymerase were
added. PCR conditions were as follows: First cycle at 95°C for 3 minutes &
then 30 cycle as follows: 60 seconds of denaturation at 94°C, 30 seconds of
annealing at 55°C & 45 seconds of primer extension at 72°C. Following the
last cycle, an additional 10 minutes of incubation at 72°C was carried out to
ensure the complete polymerization of any remaining PCR product 7. PCR
product so formed was run on agarose gel & loading of gel was done in
triplicates(like ITS I, ITS O, ITS N& LSU I, LSU O, LSU N) Visualization of
bands were done using syngene system. PCR product was then purified using
FLAVOGEN kit 40µL of PCR product was taken in microfuge tube to which 200µL of FADF
buffer was added and vortexed. FADF column was placed in collection tube &
mixture was transferred into the column, centrifuged at 11000×g for 30 seconds,
flow through was discarded. 750µL of wash buffer was added to FADF column,
centrifuged at 11000×g for 30 seconds & flow through was discarded.
Centrifugation was carried out at 18000×g for additional 3 minutes to dry the
column matrix. FADF column was placed in new microfuge tube to which 40µL of
elution buffer was added. Stand the column for 1 minute and centrifuge at
18000× g for 1 minute to elute DNA.

 

·        
Sequencing and phylogenetic tree
construction

 

Purified
PCR product was sequenced using ITS and LSU primers. For sequencing following
components were added: 0.2µL of specific primer (ITS1, ITS4, LROR, LR5), 1.8µL
of Millipore water, 1µL PCR product & 2µL of Big dye terminator. Data
alignments were made and sequences were compared to sequences in public
database with BLAST search tool on the National Center for Biotechnology
Information (NCBI) website (http://www.ncbi.nlm.nih.gov/)
to identify the isolate 14. For phylogenetic tree construction, the 18S rRNA
gene sequence of the isolate was compared to references of 18S rRNAgene
sequence of other yeast isolates retrieved from NCBI database. Phylogenetic
analysis was performed using Mega 5.05 software by neighbor-joining method
14.

 

Results                         

Isolation:
About
20 different organisms were isolated from various sources

Table
1: Total number of organism isolated from various sources

 

Screening
of Malathion Degraders: Among the 20 isolates, only 1
isolate.i.e I1 was able to grow at 1.5 mg/L of malathion. Further result
revealed that isolate I1 was able to grow at malathion concentration ranging
from 1.5 ppm to 6400 ppm. However growth was inhibited at a concentration of
about 12800 ppm

 

Table 2: Growth of
twenty isolates in 1.5 mg/L malathion concentration. (+) indicate growth
and (-) indicate
no   growth

 

 

 

Fig:
1 Isolate I-1 growing at different malathion concentration ranging from 1.5
mg/L to 6400 mg/L.
 

 

 

 

·        
Morphological
observation: By monochrome staining, only one out of
20 isolates was found to be yeast and average size of the cell was found to be
8µm by phase contrast microscopy.

Fig: 2
Morphological analysis using monochrome staining (a) and Phase contrast
microscopy at 100X (b).

 

 

·        
Biochemical Test:

 

a.      
Sugar fermentation

 

 

‘A’:  Acid production

‘-‘  :  No
acid production

Table 3:
Result of sugar fermentation for I-1 isolate

 

 

 

b.Sugar utilization

 

 

‘+’:
Growth

 

Table 4:
Result of sugar utilization for I-1 isolate

‘-‘: No
growth

 

 

 

 

c.Urease test

 

 

‘-‘:
Negative test  ‘+’: Positive test

Table
5: Result of urease test for I-1 isolate

 

 

 

 

·        
Checking growth of isolate I-1 in
soil sample

‘+’:
Growth

Table
6: Growth of I-1 isolate in soil sample

‘_’: No growth

 

 

·        
Thin
layer Chromatography: Result of TLC revealed that, there
is decrease in number of spots in test as compared to the standard.

 

 

 

Fig: 3 Thin layer
chromatography. a Standard (Malathion) b Supernatant c Broth (Test)

 

 

 

 

 

·        
PCR
amplification: The PCR products were run on 1.5% Agarose gel and documented
using syngene system. Bright bands were observed.

 

Fig
4: Agarose gel electrophoresis of PCR products
 

 

 

 

·        
Sequencing
and Phylogenetic analysis

All
sequences were edited manually and trimmed to remove ambiguous region and the
18S rRNA sequence were BLAST search against GenBank database in the NCBI 14.
The sequence showed 99% similarity to Candida
tropicalis. The sequences were aligned and phylogenetic tree was
constructed using Mega 5.05 software 14. The phylogenetic analysis revealed
that our isolate was closely associated with Candida tropicalis.

 

Fig
5: Phylogenetic tree for strain IB-1 showing close association with Candida tropicalis. Bootstrap values
were indicated as percentage at all branches.
 

 

 

 

Discussion

Extensive
use of organophosphate compounds in agriculture makes it one of the most
important and widespread pollutant in our environment2. Organophosphorus
compound poisoning is a world health problem with about 3 million poisoning and
2, 00,000 deaths annually 4. Organophosphorus insecticide like malathion are
considered to be hazardous and have been known to potentially cause adverse
effect on human health by inhibition of acetyl cholinesterase activity in the
body19. Biological removal of chemo pollutant becomes the method of choice,
since microorganism can use variety of xenobiotic compounds including pesticide
for their growth and mineralize and detoxify them 13. Bioremediation, which
involves the use of microorganism to detoxify and degrade pollutants, has
received attention as an effective biotechnological approach to clean up
polluted environment 4. The biological method of detoxification is preferable
than physical and chemical methods due to its advantage at low operational
cost, low investment and also environmental friendly 2. It has been reported
that several bacterial genera such as Acinetobacter
2, Pseudomonas 4, Bacillus & Enterobacter 19, Serratia,
Flavobacterium, Sphingomonas and Agrobacterium
2 and fungi may participate in efficient degradation of organophosphate.
There are some reports on Malathion degradation by microorganism 12, 1,
6, 19, 4, 2. In this study 20 organisms were screened for their ability
to degrade malathion out of which only one isolate i.e. I-1 was able to grow on
minimal medium supplemented with 6400 mg/L malathion as the sole source of
carbon. Result of TLC revealed that there is decrease in number of spots in
test when compared to standard which may indicate that our isolate is able to degrade
malathion. Further based on morphological observation, molecular
characterization by 18S gene sequencing and phylogenetic tree analysis our
isolate showed 99% similarity with Candida
tropicalis. This is the first study were C.tropicalis can be used to degrade malathion up to 6400 mg/L.
Looking at its high Malathion degradation ability, C.tropicalis can be used as a bioremediation tool at environmental
polluted sites.