Abstract: levels of these pesticides are reported

Abstract:Agriculture is the majoroccupation of people in India, around 60-70% of its population is engaged inagriculture. Annually millionsand tons of pesticides are used in field which in turn accumulates inour ecosystem and food chain as well. The toxicity levels of these pesticidesare reported in amphibians, aves, fishes and even human beings. Malathion(S-(1, 2 dicarethoxyethyl)-O, O-dimethyldithiophosphate) which cause inhibitionof acetylcholine esterase, there by leading to increase of theacetylcholine concentration at the synaptic junction resulting in muscledisfunction, paralysis and ultimately death.

Malathion could be degraded either chemically orbiologically. Chemical degradation leads to production of malaxon which is 50times more toxic then the parent compound. Our present study aim atbiodegradation of malathion using yeast. Around twenty organisms were isolated from differentsources like idli batter, paneer, cheese etc.

Plate assay resultsrevealed that only one isolate i.e. I1 isolated from idli batter was able togrow at very high concentrations of malathion 6,400 mg/L. Thin layerchromatography result suggested that, there is some degradation of malathion byorganisms I1 when compared with standard. Further by morphological analysis usingphase contrast microscopy and monochrome staining and molecularcharacterization using 18s gene sequencing and by phylogenetic analysis thisorganisms was identifiedas Candida tropicalis.It isfirst study which reveals that Candidatropicaliscould degrade malathion at such high concentration. Keywords:Biodegradation, Organophosphate, Malathion, Candidatropicalis.

   INTRODUCTION The excessiveuse of natural resourcesand large scale synthesis of xenobiotic compounds have generated a number ofenvironmental problems such as contamination of air, water and terrestrialecosystems, harmful effects on different biota, and disruption ofbiogeochemical cycling 5.Organophosphorus compounds (OP), are  group of highly toxic agricultural chemicals3. Some ofthese pesticides have also been reported to betoxic, 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 offield crop pests, household insects, flies and animal parasites. It has been known as the firstorganophosphorous insecticide with high selective toxicity 2.Malathion is powerful inhibitors of acetyl cholinesterase, an important enzymeinvolved in neurotransmission, in the form of acetylcholine substitutes 16.Environmental hazards and health risks caused by pesticides could thereforepotentially affect human healthand environment 16.

Therefore, remediation of contaminated sitesis currently underway in order to develop safe, convenient and economicallyfeasible methods for pesticide detoxification 19. Bioremediation is anenvironmental cleanup process that currently being investigated for use on awide variety of chemical 16. Theuse of microbes in the bioremediation anddetoxification of many toxic xenobiotics, especially toxic pesticide is anefficient tool for theremediation of contaminated sites in the environment 16. They have thecapacity to utilizevirtually all naturallyand synthetically occurring compounds as their sole carbon and energysource 13. Bacteria, fungi and some plants have potency to degrade malathion12. Malathiondegradation products include dimethyl phosphate,dimethyldithiophosphate, dimethylthiophosphate, isomalathion, malaoxon and due to cutinase,carboxylesterase, phosphataseenzymatic activity, malathion is degraded into malathion mono anddicarboxylic acid 1.Mycodegradation is a deterioration phenomenon causingdegradation by fungi in a wide variety of materials and compounds 6.

Studieshave been reported, the occurrence of several yeast such as Saccharomyces species, Candida species in various fermenteddairy or dairy related product like curd, cheese, idli, dosa, jalibi, fruit,juices and brewery products17.This paper focuses on biodegradation of Malathion by yeast isolated fromidli batter.  Materials & Methods • Sample collectionDifferent samples (Curd, Paneer, Cheese, Buttermilk,Soya sauce, Honey, Raw cow and buffalo milk, Idli batter) were collected insterile tube and stored in refrigerator until use 18.

 • Isolation procedureSample was inoculated in 1% concentrationin 50ml of MRS broth and incubated under microaerophilic condition at 37°C for24 hours. Ten fold dilutions were made using sterile saline and the last threedilutions i.e. 10-8, 10-9, 10-10 were spreadon 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 usedfor screening of pesticide degraders 18.

 ·        First enrichmentIsolated colony was inoculated in10ml of MRS broth and incubated under microaerophilic condition at 37°C for 24hours. ·        Second enrichmentEnriched broth obtained from firstenrichment step was dispensed in sterile centrifuge tube and centrifuged at5000 rpm for 10 minutes. Pellet was washed with sterile saline and 0.1 ml ofresuspended cells was inoculated in Davis Minimal broth containing 0.1% glucoseand 1.

5 mg/L Malathion. Malathion was added after autoclaving and incubatedunder microaerophilic condition at 37°C for 48 hours. ·        Screening of Malathion degradersScreening was done by streaking onDavis Minimal agar plate containing 1.5 mg/L malathion. Further growth wasassessed on different concentration of Malathion ranging from 1.5mg/L to12800mg/L 10. ·        Morphological observationThe morphology of isolate wasobserved using monochrome staining and phase contrast microscopy. ·        Biochemical TestBiochemical tests such as sugarfermentation, sugar utilization, Urease test were performed using the followingprotocol  ·        Checking growth of I-1 isolate insoil sampleTo check whether our isolate I-1 isable to grow in presence of soil microflora flora following protocol wascarried out 15: Soil sample was collected in a petri plate, air dried andgrounded into powder form.

Both autoclaved and unautoclaved soil was spiked with malathion under aseptic condition. Oneset of autoclaved and unautoclaved soil was inoculated with isolate and otherset was used as control. Incubate the plate at 37ºC for 24 hours.

  20g of test and control soil sample wasdispense in sterile saline and tenfold dilutions were subsequently made usingsterile saline.0.1 ml were spread on Minimal medium containing pesticide andIncubated at 37ºC for 72 hours. Plates were checked for growth of I-1 isolate.  ·        Thin layer chromatography 0.1 ml of Enrichedbroth was inoculate in Davis Minimal broth containing 6400 mg/L malathion andincubated for 48 hrs under microaerophilic condition. Then 5mL of this brothwas taken in centrifuge tube and centrifuged at 5000 rpm for 10 minutes.Supernatant was transfer in new centrifuge tube and equal volume of ethylacetate was added.

Tube was inverted several times and was then transfer inseparating funnel. Two layer was obtained aqueous layer was discarded andorganic layer was filter through Whatman filter paper No. 1 containing sodiumsulphate.Filtrate was collected in centrifuge tube.TLC was run, solvent systemused were Hexane : Ethyl acetate(20:80) 8.Visualizationwith Iodine-Azide ProcedureAfter developed and airdried, plate were sprayed with spraying solution( 6% sodium azide ,0.25% starchat pH 6.

5), they were exposed to iodine vapors for 15 seconds. The plate wasleft for 15 minutes to let the iodine-azide reaction proceed.Malathion showedwhite spot in yellow background 11.

 ·        Isolation of genomic DNAThe isolate was streaked on SabouraudDextrose agar (SDA) and incubated at 37°C for 24 hours. The resulting isolatewas used for extraction of DNA by Edward method 9: 50 mg of cells was takenin 1.5mL microfuge tube to which 200µL of Edward buffer was added.

Cells weremanually crushed with plastic pestle for 5 minutes. 200µL of Edward buffer wasfurther added and cells were crushed for additional 5 minutes. Volume was madeup to 1000µL by of Edward buffer. Vortexing was carried out for 15 seconds andincubated at 100°C for 10 minutes and then centrifuged at 2000 rpm for 10minutes. 500µL of supernatant was transferred in new microfuge tube andcentrifuged at 2000 rpm for 10 minutes. 400µL of resulting supernatant wastransferred in new microfuge tube to which 400µL of ice cold isopropanol wasadded. Tube was inverted gently for 5 times and incubated at room temperaturefor 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 driedpellet with 70% ethanol and centrifuge at 14000 rpm for 10 minutes. Suspend thedried pellet in 100µL of TE buffer.

Concentration of DNA was checked by usingNano drop spectrophotometer.   ·        PCR amplification & purificationof PCR productFor PCR all components such as 16.5µLof Millipore water, 2.5µL of 10X buffer, 1µL of dNTPs, 1.5µL of MgCl2, 1µL ofspecific primers (ITS1, ITS4, LROR, and LR5), and 0.5µL of Taq polymerase wereadded. 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 ofannealing at 55°C & 45 seconds of primer extension at 72°C.

Following thelast cycle, an additional 10 minutes of incubation at 72°C was carried out toensure the complete polymerization of any remaining PCR product 7. PCRproduct so formed was run on agarose gel & loading of gel was done intriplicates(like ITS I, ITS O, ITS N& LSU I, LSU O, LSU N) Visualization ofbands were done using syngene system. PCR product was then purified usingFLAVOGEN kit 40µL of PCR product was taken in microfuge tube to which 200µL of FADFbuffer 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 thecolumn matrix. FADF column was placed in new microfuge tube to which 40µL ofelution buffer was added. Stand the column for 1 minute and centrifuge at18000× g for 1 minute to elute DNA.

 ·        Sequencing and phylogenetic treeconstruction PurifiedPCR product was sequenced using ITS and LSU primers. For sequencing followingcomponents were added: 0.2µL of specific primer (ITS1, ITS4, LROR, LR5), 1.8µLof Millipore water, 1µL PCR product & 2µL of Big dye terminator. Dataalignments were made and sequences were compared to sequences in publicdatabase with BLAST search tool on the National Center for BiotechnologyInformation (NCBI) website (http://www.ncbi.

nlm.nih.gov/)to identify the isolate 14.

For phylogenetic tree construction, the 18S rRNAgene sequence of the isolate was compared to references of 18S rRNAgenesequence of other yeast isolates retrieved from NCBI database. Phylogeneticanalysis was performed using Mega 5.05 software by neighbor-joining method14. Results                          Isolation:About20 different organisms were isolated from various sources Table 1: Total number of organism isolated from various sources  Screeningof Malathion Degraders: Among the 20 isolates, only 1isolate.

i.e I1 was able to grow at 1.5 mg/L of malathion.

Further resultrevealed that isolate I1 was able to grow at malathion concentration rangingfrom 1.5 ppm to 6400 ppm. However growth was inhibited at a concentration ofabout 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.      ·        Morphologicalobservation: By monochrome staining, only one out of20 isolates was found to be yeast and average size of the cell was found to be8µm 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’-‘  :  Noacid 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 ‘-‘: Nogrowth    c.Urease test   ‘-‘:Negative test  ‘+’: Positive test Table 5: Result of urease test for I-1 isolate     ·        Checking growth of isolate I-1 insoil sample ‘+’:Growth Table 6: Growth of I-1 isolate in soil sample ‘_’: No growth  ·        Thinlayer Chromatography: Result of TLC revealed that, thereis 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)      ·        PCRamplification: The PCR products were run on 1.

5% Agarose gel and documentedusing syngene system. Bright bands were observed.  Fig 4: Agarose gel electrophoresis of PCR products      ·        Sequencingand Phylogenetic analysisAllsequences were edited manually and trimmed to remove ambiguous region and the18S rRNA sequence were BLAST search against GenBank database in the NCBI 14.The sequence showed 99% similarity to Candidatropicalis. The sequences were aligned and phylogenetic tree wasconstructed using Mega 5.

05 software 14. The phylogenetic analysis revealedthat 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.

     DiscussionExtensiveuse of organophosphate compounds in agriculture makes it one of the mostimportant and widespread pollutant in our environment2. Organophosphoruscompound poisoning is a world health problem with about 3 million poisoning and2, 00,000 deaths annually 4. Organophosphorus insecticide like malathion areconsidered to be hazardous and have been known to potentially cause adverseeffect on human health by inhibition of acetyl cholinesterase activity in thebody19. Biological removal of chemo pollutant becomes the method of choice,since microorganism can use variety of xenobiotic compounds including pesticidefor their growth and mineralize and detoxify them 13. Bioremediation, whichinvolves the use of microorganism to detoxify and degrade pollutants, hasreceived attention as an effective biotechnological approach to clean uppolluted environment 4. The biological method of detoxification is preferablethan physical and chemical methods due to its advantage at low operationalcost, low investment and also environmental friendly 2.

It has been reportedthat several bacterial genera such as Acinetobacter2, Pseudomonas 4, Bacillus & Enterobacter 19, Serratia,Flavobacterium, Sphingomonas and Agrobacterium2 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 abilityto degrade malathion out of which only one isolate i.e.

I-1 was able to grow onminimal medium supplemented with 6400 mg/L malathion as the sole source ofcarbon. Result of TLC revealed that there is decrease in number of spots intest when compared to standard which may indicate that our isolate is able to degrademalathion. Further based on morphological observation, molecularcharacterization by 18S gene sequencing and phylogenetic tree analysis ourisolate showed 99% similarity with Candidatropicalis. 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 environmentalpolluted sites.