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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.

References

1.       A.Ratna Kumari et al “Malathion degradation by Bacillus spp. isolated from soil” IOSR Journal of Pharmacy, Volume 2 Issue 4, PP.37-42, July-August 2012.

2.       Ahmed F. Azmy et al “Biodegradation of Malathion by Acinetobacter baumannii Strain AFA Isolated from Domestic Sewage in Egypt” International Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering Vol: 9, No: 1, 55-65, 2015.

3.       Ali Mohammad Latifi et al “Isolation and characterization of five chlorpyrifos degrading bacteria” African Journal of Biotechnology Vol. 11(13), pp. 3140-3146, 14 February, 2012.

4.       Amal E. Saafan et al “Isolation and characterization of two malathion degrading Pseudomonas sp. in Egypt”. African Journal of Biotechnology, Vol. 15(31), pp. 1661-1672, 3 August, 2016.

5.       Brajesh K. Singh et al “Microbial degradation of organophosphorus compounds” FEMS Microbiol Rev 30, 428–471, April 2006.

6.        Chalamala Ramadevi et al “Mycodegradation of Malathion by A Soil Fungal Isolate, Aspergillus Niger” International Journal of Basic and Applied Chemical Sciences, Vol. 2 (1), pp.108-115, January-March, 2012.

7.       J.M. Garc?´a Mart?´nez et al, “Identification of pathogenic yeast species by polymerase chain reaction amplification of the RPS0 gene intron Fragment”. Journal of Applied Microbiology ISSN 1364-5072, Oct, 2009.

8.       Joseph C. O’ Kelley et al ” Degradation of pesticide by algae” U.S. Environmental Protection Agency, Office of Research and Development, Environmental Research Laboratory,1- 42 March,1976.

9.       K.Edward et al, “A simple and rapid method for the preparation of plant genomic DNA for PCR analysis”. Nucleic Acid Research, Vol.19, No. 6, Jan, 1991.

10.    M.M.Gurjar et al “Pyrethroid-Cypermethrin Degradation using Microorganisms Isolated from Rhizospheric Soil” Int.J.Curr.Microbiol.App.Sci, Special Issue-2: 97-102, May 2015.

11.    Monika Skowron et al, “Application of image analysis technique for the determination of organophosphorus pesticides by Thin Layer Chromatography”, .Journal of planar chromatography, DOI: 10.1556/1006, 2016.

12.    Prathamesh S.Kamalapure et al “Biodegradation of Organophosphate Insecticide Malathion by Microorganisms Isolated from Soils of Sholapur Region, India” Gjra – Global Journal For Research Analysis, Volume-4, Issue-8, 72-73, August-2015.

13.    S. Kavi Karunya et al “Efficiency of Bacterial Isolates in the Degradation of Malathion and Parathion” International Journal of Pharmaceutical & Biological Archives, 3(3):659-665, 17 Jun 2012.

14.    S.F.Maleeka Begum et al, “Isolation, Characterization and Identification of Dimethoate degradation bacteria from soil series of Tamil Nadu”. International Journal of Advanced Scientific and Technical Research. Issue 6, Vol. 3, ISSN 2249-9954, Jun 2016.

15.    Shamsa Akbar et al ” Soil bacteria showing a potential of chlorpyrifos degradation and plant growth enhancement” Brazilian journal of microbiology 47 , 563–570, 20 April 2016.

16.    Soni Yadav et al “Isolation and Characterization of Organophosphate Pesticides Degrading Bacteria from Contaminated Agricultural Soil” OnLine Journal of Biological Sciences, 15 (1): 113.125, 2 June, 2015.

17.    Syal P et al ” Probiotic Potential Of Yeast Isolated From Traditional Indian Fermented Foods” International Journal of Microbiology Research,ISSN:0975-5276 & E-ISSN: 0975-9174, Volume 5,Issue 2,pp-390-398,March 21,2013.

18.    V.Bali et al ” Isolation ,Screening and Evaluation of Antimicrobial Activity of Potential Bacteriocin Producing Lactic acid Bacteria Isolate”,Microbiology Journal,1-7, 2011.

19.    Zenith K. Mohamed et al “Isolation and molecular characterisation of malathion-degrading bacterial strains from waste water in Egypt” Journal of Advanced Research (1), 145–149, 6 March 2010.

 

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