Abstract: isolated from idli batter was able to

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Abstract: isolated from idli batter was able to

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 tropicalis could 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

 

Sr.No

Source

No. of isolates

1.      
 

Idli batter (I)

2

2.      
 

Cheese 
(CH)

2

3.      
 

Buttermilk (BM)

2

4.      
 

Colostrum(K)

5

5.      
 

Honey (H)

1

6.      
 

Yakult (YAK)

2

7.      
 

Yogurt (Y)

2

8.      
 

Raw milk (BU)

 

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

 

 

Concentration of malathion

Isolates

 
1.5 mg/L

I1

I2

H1

YAK1

YAK2

Y1

Y2

CH1

CH2

K1

+

 

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

 

Isolate

Dextrose

Maltose

sucrose

Lactose

Galactose

xylose

Sorbital

Fructose

I-1

A

A

A

A

A

A

 

 

‘A’:  Acid production     ‘-‘:  No acid production

 

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

 

 

 

 

 

 

 

 

b. Sugar utilization

 

 

Isolate

Dextr
ose
 

Malto
se

Sucro
se

Lacto
se

Galacto
se

Xylo
se

Sorbi
tal

Fructo
se

I-1

A

A

A

A

A

A

 

‘+’: Growth

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

‘-‘: No
growth

 

 

 

 

c.       
Urease test

 

Isolate

Obsevation

Result

I-1

No color change

 

 

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

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

 

 

 

 

Checking
growth of isolate I-1 in soil sample:

Isolate

Autoclaved
Soil

Autoclaved
Soil( C )

Unautoclaved soil

Unautoclaved soil ( C )

I-1

+

+

 

‘+’: Growth    ‘_’: No growth    ‘C ‘: Control

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

 

 

 

 

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.

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