Chemical pesticides are largely used in different types of crops all over the world. This particular study deals with the mycoremediation of the pesticide Monocrotophos (MCP) which is largely used for the protection of cash crops. For the study of its bio-degradation, the fungal strain which can degrade MCP was isolated from the soil contaminated with the pesticide by means of enrichment culture. The molecular characterization was done by 18S rRNA sequencing. 300mgL-1 monocrotophos along with its major metabolites dimethyl phosphate (DMP) and N-methylacetoacetamide were completely degraded within 24 hours of incubation in the mineral medium. In soil enriched with monocrotophos and nutrients (carbon, phosphorous, nitrogen), the fungal strain was able to degrade monocrotophos and its metabolite in 24 and 48 hours respectively. Again, the soil was spiked with monocrotophos (300mgL-1) which was devoid of nutrients and the fungus was able to degrade monocrotophos and its metabolite in 24 and 48 hours respectively. High performance liquid chromatography as well as Fourier transform infrared analysis confirmed the degradation process of monocrotophos by the fungus. These results showed that the particular fungus had a great potential to degrade the monocrotophos contaminated soil even in the absence of nutrients.
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Keywords: Biodegradation, monocrotophos, fungal strain
A large number of pesticides are used in agricultural crops throughout the world (Shukla et al.2006) and majority of them have some toxic effects to both animals and humans. Moreover, excessive use of pesticides adversely affects the soil fertility and can change the soil chemistry which might become totally unfit for growing crops. The organophosphorous pesticides interfere with the activity of an enzyme acetylcholinesterase which plays an important role in the normal transmission of nerve impulse. Most of the organophosphorous pesticides have a chemical structure containing three phosphoester linkages. The toxicity of the pesticides is greatly reduced when the hydrolysis of one of the phosphoester bonds takes place, thus eliminating their acetylcholinesterase-inactivating properties (Horne et al. 2002).
Monocrotophos [DimethylÂ (E)-1-methyl-2-(methylcarbamoyl)vinyl phosphate], a common pesticide is largely used in agricultural crops. It is a chlorinated organophosphorous insecticide, acaricide and termicide against cutworms, gall midge, leaf folder, leaf hopper, etc. The half-life of monocrotophos has been reported which varies from 10-120 days. This half-life largely depends upon some environmental factors like temperature, pH, moisture content, pesticide formulation and organic carbon content. The major degradation product of monocrotophos is more water soluble than monocrotophos itself and thus it causes a high range of contamination in soils and in aquatic environment.
Different technologies have been implemented including physicochemical and biological treatments to solve the problem of soil and water pollution. It is worthy to mention that it causes toxicity to humans and animals and greatly affects the Central Nervous System. Biological treatment using microorganisms has been evaluated to be the best technique of reducing soil pollution as it is environment friendly and inexpensive. Many reports have been shown the efficiency and potentiality of microorganisms to degrade pesticides (Singh et al. 1999). Initially, the degradation of pesticide was observed in alkaline soils and its phenomenon was related to its hydrolysis in high pH. However, complete hydrolysis of monocrotophos was observed with high pH in soil under sterile condition which indicated the involvement of soil microorganisms (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 soils. But, very limited information is available about the fungus which can degrade pesticides.
In the present study, a novel fungus was isolated which has the capability of degrading not only monocrotophos, but also its major metabolite. The biodegradation of monocrotophos and its major metabolite in mineral medium and soil were observed and investigated. This study actually aims the possible elucidation of the isolated 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 grade (36%). All other reagent used here were of high purity and analytical grade.
2.2 Soil sample
Soil sample was collected from the top layer ie. 0-20cm of the paddy field which had been exposed to monocrotophos pesticide in Vellore district, Tamil Nadu, India. The soil sample was greatly dried at the room temperature in the laboratory upto the point of soil moisture was suitable for sieving.
2.3 Enrichment Procedure and Isolation of Fungal Strain
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Isolating of fungal strain were carried out in Czapek Dox Broth according to the method of Anwar et al. (2009). Monocrotophos degrading fungal strain were obtained by enrichment culture in the Czapek Dox broth containing yeast extract, 3g/L, peptone 10g/L, dextrose 2g/L and monocrotophos 100mg/L. Approximately 5g of soil sample contaminated with monocrotophos was used to inoculate 50 ml of Czapek Dox broth containing Monocrotophos (100mg/L) and cultured in 250 ml of Erlenmeyer flasks on the rotary shaker ( at 100 rpm) which was incubated at room temperature. Following this incubation in shaking condition for about 8 days, fungal colony was isolated by streaking the enriched sample on the Czapek Dox medium. After the incubation of streaked Czapek Dox plates for about 2-3 days at 25degree celcius, isolated fungal culture was maintained on agar slope on the sample medium containing Chlorpyrifos.
2.4 Gradient Plate Assay and Minimum Inhibitory Concentration for Fungal Isolates
The enrichment experiments which resulted in the isolates were then furthered screened for monocrotophos tolarence capacity of the following gradient plate method. The monocrotophos concentration gradient was prepared by adding base layer of 20 ml 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 wedge shaped layer. The other half of the petriplate was now subjected to the addition of 20 ml of same media agar containing monocrotophos (100mg/L) to give monocrotophos gradient across the plate surface. Following the gradient plate preparation the spore suspension of fungal isolates was prepared in 0.1% triton X-100 and streaked along the sterile cotton swab. Petri plates were incubated at 30+/- 2 degree celcius for 8 days. After that the length of the fungal growth along the gradient was recorded. (Bhalero and Puranik 2007)
Minimum Inhibitory Concentration (MIC) and tolarence to monocrotophos were checked for the fungal isolates using broth assay. A series of 250ml of Erlen-meyer containing 100ml of M1 medium composed of NaNo3 2g, KCl 0.5g, MgSO4.&H2O 0.5g, glucose 10g, FeCl3 10g, BaCl2 0.2g and CaCl2 0.5 g per litre at ph 6.8 were taken. The flask were in turn amended with increasing concentration of chlorpyrifos. The flask were then inoculated with fungal spore suspension of approximately 1ml in each. The fungal spore suspension were prepared in 0.01% Triton X-100. The flask were incubated at 30Â± 2 degree Celsius on a rotary shaker at 120 rpm. Mycelial growth was maintained in the flask after 10 days of incubation. Whatman filter no.1 was used for the filtration of the mycelial mass in order to separate. Then the mycelial mass was wased with deionised water. The dry weight of the fungal biomass was then determined by drying at a constant weight for 80 degree celcius in pre weighted aluminium foil cups. The MIC was noted as the concentration of monocrotophos resulting in the complete inhibition of mycelial growth in flask (Bhalerao and Puranik 2009).
2.5 Growth Kinetics
For the determination of growth pattern 1ml of spore suspension (10^8 spores per ml) of the identified fungal strain was inoculated in a series of flask containing Czapek Dox broth (100 ml in 250 ml of erlen meyer flask) with and without monocrotophos (100mg/L) the flask was subjected at constant shaking in a rotary shaker at 120 rpm at 30Â±2 degree celcius. After that removal 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 then separated by filtration using Whatman filter paper no.1 and washed with deionised water. Biomass determination was done by drying the fungal biomass for a constant rate at 80 degree celcius in pre weight aluminium foil cups.
2.6 Biodegradation of Monocrotophos in Mineral Medium
Degradation of the pesticide monocrotophos was performed in 250 ml of erlen meyer flask containing 100 ml of M1 medium amended with 100 mg/L monocrotophos as the sole carbon source and incubated with a 1 ml of spore suspension of fungal strain ( 10^8 spores/ml). Flask were then subjected to incubation at 30Â±2 degree celcius on a rotary shaker 120rpm. Samples were taken at 12,24,48,72 and 96 h from cultured flasks and the removal of monocrotophos was analysed by High Performance Liquid Chromatography (HPLC).
2.7 Biodegradation of Monocrotophos in soil
For the determination of the ability of fungal strain to degrade monocrotophos in the same soil sample in which the fungi was isolated, the same soil sample was taken from the top layer (0-15cm) was air dried at room temperature before proceeding for the analysis, the soil was sterilised by three full autoclaving during 30 min at 120 degree celcius. After that two treatments were carried out: (1) pesticide addition, isolated fungal spores and nutrients( carbon, nitrogen and phosphorous) and (2) pesticide addition and isolated fungal spores without addition. 100 gms of soil was placed in erlen-meyar flask and 30 mlof a solution containing 10^8 spores per ml of fungal spores and 100 mg/L of monocrotophos, nitrogen, phosphorous and glucose were added under sterile conditions. The amounts of carbon, nitrogen and phosphorous which was added were calculated using the relationship C/N/P 100:10:1. The sources of carbon, nitrogen and phosphorous were glucose, (NH4)2SO4 and K2HPO4 respectively( Martin et.al,2007). As a soil test control, samples sterilised in an autoclave were used to verify that the degradation was mainlu due to the action of micro-organism rather than abiotic factor( Pino and Penuela 2011).
2.8 Extraction of Samples ( Pesticide Residues) from Mineral Medium and Soil
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On the day of extraction 5-10 ml 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 using equal volume of dichlomethane(DCM) twice. Organic layer of DCM were aspirated pool and evaporated at room temperature under nitrogen. The residues were dissolved in HPLC grade acetonitrile(1ml) and then filtered through filter membrane(0.22ÂµmFH)to remove any particles and determine the concentration of HPLC.( Anwar et al 2009).
Collection of soil samples was done from each treatment trails with and without amendments of nutrients for pestiside analysis.10 g of soil sample were weight into 250ml erlen meyer flask and 20ml of HPLCgrade acetonitrile was successively addedand was subjected to shaking on rotary shaker at 120 rpm for 30 mins. After that the samples were allowed to stand until the soil had settled and clear supernatant was used for the determination of pesticide concentration by HPLC.
2.9 Recovary analysis
Recovery experiment was conducted in the M1 medium and soil for the study on extraction efficiency of the method established. Various known concentrations of monocrotophos in 50 ml of M1 medium(100,200 & 300 mg/l) and 50 gm of soil(100,200 & 300 mg/kg) were spiked.
2.10 Scanning of Electron Microscopy
Preparation of fungal mycelial samples for scanning electron microscopy(SEM) before and after monocrotophos degradation was done. The freeze dried fungal mycelium treated with 100 mg/L monocrotophos and those not treated after 120 hr of incubation were mounted on specimen stubs with double dided adhesive tape and coated with gold in a sputter coater ( Hitachi,Model E-1010 Ion Sputter) to avoid charging and examined under SEM ( Hitachi, Model S-3400N)
2.11 Analytical Methods
Varian HPLC was used to analyse the extracted sample which was equipped with binary pump, programmable variable wavelength UV detector, and ODS2 C18 reversed phase column. A gradient mobile phase of methanol/water (85:15, v/v) was prepared for conducting the analysis of the pesticide residue. 20 Î¼l of the sample was taken and the mobile phase was programmed at a flow rate of 1ml min-1. At 230nm wavelength, the removal of monocrotophos and the accumulation of its major metabolite were detected by HPLC. The retention time for monocrotophos and its major metabolite was 13.2 and 7.6 respectively.
Infrared (IR) spectra of the parent compound (Monocrotophos) and sample after fungal degradation were recorded at room temperature in the frequency range of 4,000-400 cmâˆ’1 with a Fourier transform infrared (FTIR) spectrophotometer (8400 Shimadzu, Japan, with Hyper IR-1.7 software for Windows) with a helium-neon laser lamp as a source of IR radiation. Pressed pellets were prepared by grinding the extracted samples with potassium bromide in a mortar with 1:100 ratio and immediately analyzed in the region of 4,000-400 cmâˆ’1 at a resolution of 4 cmâˆ’1.