Chemical pesticides are mostly used in different types of harvests all over the universe. This peculiar survey trades with the mycoremediation of the pesticide Monocrotophos ( MCP ) which is mostly used for the protection of hard currency harvests. For the survey of its bio-degradation, the fungal strain which can degrade MCP was isolated from the dirt contaminated with the pesticide by agencies of enrichment civilization. The molecular word picture was done by 18S rRNA sequencing. 300mgL-1 monocrotophos along with its major metabolites dimethyl phosphate ( DMP ) and N-methylacetoacetamide were wholly degraded within 24 hours of incubation in the mineral medium. In dirt enriched with monocrotophos and foods ( C, phosphoric, N ) , the fungous strain was able to degrade monocrotophos and its metabolite in 24 and 48 hours severally. Again, the dirt was spiked with monocrotophos ( 300mgL-1 ) which was devoid of foods and the fungus was able to degrade monocrotophos and its metabolite in 24 and 48 hours severally. High public presentation liquid chromatography every bit good as Fourier transform infrared analysis confirmed the debasement procedure of monocrotophos by the fungus. These consequences showed that the peculiar fungus had a great potency to degrade the monocrotophos contaminated dirt even in the absence of foods.
Keywords: Biodegradation, monocrotophos, fungous strain
A big figure of pesticides are used in agricultural harvests throughout the universe ( Shukla et al.2006 ) and bulk of them have some toxic effects to both animate beings and worlds. Furthermore, inordinate usage of pesticides adversely affects the dirt birthrate and can alter the dirt chemical science which might go wholly unfit for turning harvests. The organophosphorous pesticides interfere with the activity of an enzyme acetylcholinesterase which plays an of import function in the normal transmittal of nervus urge. Most of the organophosphorous pesticides have a chemical construction incorporating three phosphoester linkages. The toxicity of the pesticides is greatly reduced when the hydrolysis of one of the phosphoester bonds takes topographic point, therefore extinguishing their acetylcholinesterase-inactivating belongingss ( Horne et al. 2002 ) .
Monocrotophos [ DimethylA ( E ) -1-methyl-2- ( methylcarbamoyl ) vinyl phosphate ] , a common pesticide is mostly used in agricultural harvests. It is a chlorinated organophosphorous insect powder, acaricide and termicide against cutworms, saddle sore midge, leaf booklet, leaf hopper, etc. The half life of monocrotophos has been reported which varies from 10-120 yearss. This half-life mostly depends upon some environmental factors like temperature, pH, wet content, pesticide preparation and organic C content. The major debasement merchandise of monocrotophos is more H2O soluble than monocrotophos itself and therefore it causes a high scope of taint in dirts and in aquatic environment.
Different engineerings have been implemented including physicochemical and biological interventions to work out the job of dirt and H2O pollution. It is worthy to advert that it causes toxicity to worlds and animate beings and greatly affects the Central Nervous System. Biological intervention utilizing microorganisms has been evaluated to be the best technique of cut downing dirt pollution as it is environment friendly and cheap. Many studies have been shown the efficiency and potency of micro-organisms to degrade pesticides ( Singh et al. 1999 ) . Initially, the debasement of pesticide was observed in alkaline dirts and its phenomenon was related to its hydrolysis in high pH. However, complete hydrolysis of monocrotophos was observed with high pH in dirt under unfertile status which indicated the engagement of dirt micro-organisms ( Racke et al. 1996 ) .
Pseudomonas aeruginosa, Clavibacter michiganense Arthrobacter atrocyaneus, Agrobacterium radiobacter, Bacillus megaterium and Pseudomonas mendocina ( Ramanathan and Lalithakumari, 1999, Bhadbhade et al. , 2002b, Horne et al. , 2002, Singh and Singh, 2003 ) have been reported to degrade MCP in solutions and dirts. But, really limited information is available about the fungus which can degrade pesticides.
In the present survey, a fresh fungus was isolated which has the capableness of degrading non merely monocrotophos, but besides its major metabolite. The biodegradation of monocrotophos and its major metabolite in mineral medium and dirt were observed and investigated. This survey really aims the possible elucidation of the stray fungal strain for the bioremediation of the monocrotophos- contaminated environment.
2 Materials and Methods
The pesticide monocrotophos, whose mycodegradation is studied was purchased from Sigma Aldrich ( St. Louis, MO, USA ) as was of analytical class ( 36 % ) . All other reagent used here were of high pureness and analytical class.
2.2 Soil sample
Soil sample was collected from the top bed Internet Explorer. 0-20cm of the Paddy field which had been exposed to monocrotophos pesticide in Vellore territory, Tamil Nadu, India. The dirt sample was greatly dried at the room temperature in the research lab upto the point of dirt wet was suited for screening.
2.3 Enrichment Procedure and Isolation of Fungal Strain
Isolating of fungous strain were carried out in Czapek Dox Broth harmonizing to the method of Anwar et Al. ( 2009 ) . Monocrotophos degrading fungous strain were obtained by enrichment civilization in the Czapek Dox broth incorporating barm infusion, 3g/L, peptone 10g/L, dextrose 2g/L and monocrotophos 100mg/L. Approximately 5g of dirt sample contaminated with monocrotophos was used to inoculate 50 milliliter of Czapek Dox broth incorporating Monocrotophos ( 100mg/L ) and cultured in 250 milliliter of Erlenmeyer flasks on the rotary shaker ( at 100 revolutions per minute ) which was incubated at room temperature. Following this incubation in agitating status for about 8 yearss, fungous settlement was isolated by streaking the enriched sample on the Czapek Dox medium. After the incubation of streaky Czapek Dox plates for about 2-3 yearss at 25degree celcius, stray fungous civilization was maintained on agar incline on the sample medium incorporating Chlorpyrifos.
2.4 Gradient Plate Assay and Minimum Inhibitory Concentration for Fungal Isolates
The enrichment experiments which resulted in the isolates were so furthered screened for monocrotophos tolarence capacity of the undermentioned gradient home base method. The monocrotophos concentration gradient was prepared by adding base bed of 20 milliliter of Czapek Dox agar without pesticide to the petriplate tilted at 30 degree angle. The agar was allowed to solidify at room temperature into the cuneus shaped bed. The other half of the petriplate was now subjected to the add-on of 20 milliliter of same media agar incorporating monocrotophos ( 100mg/L ) to give monocrotophos gradient across the home base surface. Following the gradient home base readying the spore suspension of fungous isolates was prepared in 0.1 % Triton X-100 and streaked along the unfertile cotton swab. Petri home bases were incubated at 30+/- 2 degree celcius for 8 yearss. After that the length of the fungous growing along the gradient was recorded. ( Bhalero and Puranik 2007 )
Minimum Inhibitory Concentration ( MIC ) and tolarence to monocrotophos were checked for the fungous isolates utilizing broth check. A series of 250ml of Erlen-meyer incorporating 100ml of M1 medium composed of NaNo3 2g, KCl 0.5g, MgSO4. & A ; H2O 0.5g, glucose 10g, FeCl3 10g, BaCl2 0.2g and CaCl2 0.5 g per liter at ph 6.8 were taken. The flask were in bend amended with increasing concentration of chlorpyrifos. The flask were so inoculated with fungous spore suspension of about 1ml in each. The fungous spore suspension were prepared in 0.01 % Triton X-100. The flask were incubated at 30A± 2 degree Celsius on a rotary shaker at 120 revolutions per minute. Mycelial growing was maintained in the flask after 10 yearss of incubation. Whatman filter no.1 was used for the filtration of the mycelial mass in order to divide. Then the mycelial mass was wased with deionised H2O. The dry weight of the fungous biomass was so determined by drying at a changeless weight for 80 degree celcius in pre weighted aluminium foil cups. The MIC was noted as the concentration of monocrotophos ensuing in the complete suppression of mycelial growing in flask ( Bhalerao and Puranik 2009 ) .
2.5 Growth Dynamicss
For the finding of growing pattern 1ml of spore suspension ( 10^8 spores per milliliter ) of the identified fungal strain was inoculated in a series of flask incorporating Czapek Dox stock ( 100 milliliter in 250 milliliter of erlen meyer flask ) with and without monocrotophos ( 100mg/L ) the flask was subjected at changeless shaking in a rotary shaker at 120 revolutions per minute at 30A±2 degree celcius. After that remotion of 1 flask from each series at intervels of 12-,24-,48-,72-,96- and 120- H intervels. The mycelial mass from each series was so separated by filtration utilizing Whatman filter paper no.1 and washed with deionised H2O. Biomass finding was done by drying the fungous biomass for a changeless rate at 80 degree celcius in pre weight aluminum foil cups.
2.6 Biodegradation of Monocrotophos in Mineral Medium
Degradation of the pesticide monocrotophos was performed in 250 milliliter of erlen meyer flask incorporating 100 milliliter of M1 medium amended with 100 mg/L monocrotophos as the exclusive C beginning and incubated with a 1 milliliter of spore suspension of fungous strain ( 10^8 spores/ml ) . Flask were so subjected to incubation at 30A±2 degree celcius on a rotary shaker 120rpm. Samples were taken at 12,24,48,72 and 96 H from cultured flasks and the remotion of monocrotophos was analysed by High Performance Liquid Chromatography ( HPLC ) .
2.7 Biodegradation of Monocrotophos in dirt
For the finding of the ability of fungous strain to degrade monocrotophos in the same dirt sample in which the Fungi was isolated, the same dirt sample was taken from the top bed ( 0-15cm ) was air dried at room temperature before continuing for the analysis, the dirt was sterilised by three full autoclaving during 30 min at 120 degree celcius. After that two interventions were carried out: ( 1 ) pesticide add-on, stray fungal spores and foods ( C, N and phosphoric ) and ( 2 ) pesticide add-on and stray fungal spores without add-on. 100 gram of dirt was placed in erlen-meyar flask and 30 mlof a solution incorporating 10^8 spores per milliliter of fungous spores and 100 mg/L of monocrotophos, N, phosphoric and glucose were added under unfertile conditions. The sums of C, N and phosphoric which was added were calculated utilizing the relationship C/N/P 100:10:1. The beginnings of C, N and phosphoric were glucose, ( NH4 ) 2SO4 and K2HPO4 severally ( Martin et.al,2007 ) . As a dirt trial control, samples sterilised in an sterilizer were used to verify that the debasement was mainlu due to the action of microorganism instead than abiotic factor ( Pino and Penuela 2011 ) .
2.8 Extraction of Samples ( Pesticide Residues ) from Mineral Medium and Soil
On the twenty-four hours of extraction 5-10 milliliter of aquous samples were recovered from the culted flask and was subjected to centrifugation at 7200Xg for 10 mins to obtained cell free medium. Extraction of monocrotophos from supernatant utilizing equal volume of dichlomethane ( DCM ) twice. Organic bed of DCM were aspirated pool and evaporated at room temperature under N. The residues were dissolved in HPLC class acetonitrile ( 1ml ) and so filtered through filter membrane ( 0.22AµmFH ) to take any atoms and find the concentration of HPLC. ( Anwar et al 2009 ) .
Collection of dirt samples was done from each intervention trails with and without amendments of foods for pestiside analysis.10 g of dirt sample were weight into 250ml erlen meyer flask and 20ml of HPLCgrade acetonitrile was in turn addedand was subjected to agitating on rotary shaker at 120 revolutions per minute for 30 mins. After that the samples were allowed to stand until the dirt had settled and clear supernatant was used for the finding of pesticide concentration by HPLC.
2.9 Recovary analysis
Recovery experiment was conducted in the M1 medium and dirt for the survey on extraction efficiency of the method established. Assorted known concentrations of monocrotophos in 50 milliliter of M1 medium ( 100,200 & A ; 300 mg/l ) and 50 gram of dirt ( 100,200 & A ; 300 mg/kg ) were spiked.
2.10 Scanning of Electron Microscopy
Preparation of fungous mycelial samples for scanning negatron microscopy ( SEM ) before and after monocrotophos debasement was done. The freezing dried fungous mycelium treated with 100 mg/L monocrotophos and those non treated after 120 hour of incubation were mounted on specimen stubs with dual dided adhesive tape and coated with gold in a spatter coater ( Hitachi, Model E-1010 Ion Sputter ) to avoid bear downing and examined under SEM ( Hitachi, Model S-3400N )
2.11 Analytic Methods
Varian HPLC was used to analyze the extracted sample which was equipped with binary pump, programmable variable wavelength UV sensor, and ODS2 C18 reversed phase column. A gradient nomadic stage of methanol/water ( 85:15, v/v ) was prepared for carry oning the analysis of the pesticide residue. 20 I?l of the sample was taken and the nomadic stage was programmed at a flow rate of 1ml min-1. At 230nm wavelength, the remotion of monocrotophos and the accretion of its major metabolite were detected by HPLC. The keeping clip for monocrotophos and its major metabolite was 13.2 and 7.6 severally.
Infrared ( IR ) spectra of the parent compound ( Monocrotophos ) and sample after fungous debasement were recorded at room temperature in the frequence scope of 4,000-400 cma?’1 with a Fourier transform infrared ( FTIR ) spectrophotometer ( 8400 Shimadzu, Japan, with Hyper IR-1.7 package for Windows ) with a helium-neon optical maser lamp as a beginning of IR radiation. Pressed pellets were prepared by crunching the extracted samples with K bromide in a howitzer with 1:100 ratio and instantly analyzed in the part of 4,000-400 cma?’1 at a declaration of 4 cma?’1.