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Keywords:

  • organophosphorus;
  • biodegradation;
  • HPLC;
  • FTIR;
  • Fusarium oxysporum JASA1

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

The present work was focused in isolating fungus which possessed the special ability to degrade malathion. The pesticide of choice was malathion as it is being used to control a variety of pests in the agricultural fields in India. The fungal strains with the potential to degrade malathion was isolated by enrichment technique from malathion contaminated soil. The molecular characterization of 18S rRNA sequence homology confirmed its identity as Fusarium oxysporum. The isolate was able to degrade 400 mg L−1 malathion completely in mineral medium. In soil spiked with malathion and with addition of nutrients (carbon, nitrogen, phosphate), the isolate was capable of degrading malathion at 8th day of incubation. Where as in the second trial, in the absence of nutrients, JASA1 was able to degrade 400 mg L−1 malathion on the 9th day of incubation. The course of the degradation process was studied using High performance liquid chromatography (HPLC) and Fourier transform infrared (FTIR) analyses which confirmed the degradation potential of the fungus. © 2014 American Institute of Chemical Engineers Environ Prog, 34: 112–116, 2015


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

Malathion [S-(1,2-dicarbethoxyethyl)-O,O-dimethyldithiophosphate], is otherwise called carbophos, maldison, and mercaptothion. It is one of the first organophosphorus insecticide with selective toxicity. Organophosphate pesticides are largely being employed in many countries for public health and agricultural purposes [1]. The most toxic metabolite of malathion is the oxidation product malaoxon which is formed in the presence of air by oxidation and is responsible for the insecticidal activity of the parent compound. Malaoxon can be found either as an impurity in malathion or generated during the oxidation of malathion in air or soil and it breaks down more rapidly than malathion in alkaline and moist soil. Malathion is absorbed by practically all routes including the gastrointestinal tract, skin, mucous membranes, and lungs [2]. Toxic effect of malathion has been shown to affect the central nervous system of invertebrates, immune system of higher vertebrates, reproductive functions of vertebrate, adrenal glands, and tissues of fish [3-8].

The magnitude of microbial degradation as compared to chemical degradation is found to increase with increasing soil organic matter and is directly dependent on pH of soil. Chatterjee et al. [6] studied the biodegradation of malathion with Rhizopus oryzae biomass and concluded that 85% of malathion was degraded from its aqueous solution as against 47–68% by other fungal biomasses. In another investigation [9], Bacillus thuringiensis MOS-5(Bt) was isolated from agricultural waste water and it was able to degrade malathion cometabolically. The major degraded products were Mal-monocarboxylic acid (MMA) and Mal-dicarboxylic acid (MDA). Xie et al. [10] employed Acinetobacter johnsonii MA19 to degrade malathion by using it as an sole carbon source. Biodegradation of malathion with Brevibacillus sp. strain KB2 and Bacillus cereus strain PU was conducted by Singh et al. [11] both the strains were cultured in the presence of malathion under aerobic and energy-limiting conditions. Both strains grew well in the medium with malathion concentration up to 0.15%. Reverse-phase high-Performance liquid chromatography (HPLC)-UV analysis indicated that strain KB2 was able to degrade 72.20% of malaoxon (an analogue of malathion) and 36.22% of malathion, whereas strain PU degraded 87.40% of malaoxon and 49.31% of malathion, after 7 days of incubation. Singh et al. [12] demonstrated degradation of malathion with Lysinibacillus sp. isolated from soil, which was able to tolerate 0.15% of malathion under aerobic conditions utilizing it as a sole carbon source. The degradation rate recorded was 20% malathion and 47% malaoxon out of the initial concentration of malathion. Two metabolites, mal-monocarboxylic acid and mal-dicarboxylic acid, were detected within 7 days at 30°C [12]. The present study reports on the isolation, molecular characterization and biodegradation of malathion by Fusarium oxysporum.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

Chemicals

Analytical grade malathion (97.2%) was purchased from Sigma Aldrich (St. Louis, MO). Technical grade malathion of 25% emulsifiable concentrate was used for this study which was obtained from Insecticides (India) Limited, India. Yeast extract, peptone, dextrose, sucrose, dipotassium sulfate, ferrous sulfate, agar agar, Triton X-100, MgSO4, NaNO3, KCl, MgSO4.7H2O, FeCl3, BaCl2, CaCl2, (NH4)2SO4, and K2HPO4 were purchased from Hi-media, Mumbai, India. HPLC grade acetonitrile was obtained from and Merck, India.

Soil Sample

Sugarcane field with previous exposure to malathion over a period of 3 years was selected and soil was collected from the top layer of depth 0–23 cm. In the laboratory, the soil was air dried at room temperature and sieved at a particle size of <2 mm.

Isolation of Fugal Strain and Enrichment Technique

Fungal strain was isolated from sugarcane field soil sample by screening it in Czapek Dox broth [13]. Isolated fungal strain which was capable of degrading malathion was obtained by enrichment culture in the Czapek Dox broth containing yeast extract 3 g L−1; peptone 10 g L−1; dextrose 2 g L−1; along with malathion 100 mg L−1; and 20 g of soil collected from agricultural field with previous exposure to malathion. In this enrichment technique 100 mL of the media was spiked with 100 mg L−1 malathion and kept at 100 rpm on a rotary shaker at room temperature for 5 days. The enriched samples were transferred to sterile medium containing malathion 100 mg L−1 as the only carbon source and 5 mL of inoculum was subcultured and incubated for 2–4 weeks. Moisture content was maintained throughout the experiment by regular addition of sterile distilled water. After 2 weeks, the fungal colonies were isolated by streaking the enriched samples on Czapek Dox medium of sucrose 30 g; magnesium sulfate 0.5 g; sodium nitrate 2 g; dipotassium sulfate 0.35 g; ferrous sulfate 0.001 g; malathion 100 mg; and agar agar 30 g L−1 at pH 6.8. Isolated fungal culture was maintained on agar slopes of the same medium containing malathion.

Gradient Plate Assay

The isolate obtained from the enrichment experiments were screened for malathion tolerance by following the gradient plate method. Malathion concentration gradient was prepared by adding a base layer of 20 mL of modified Czapek Dox agar without malathion to a petriplate tilted at an angle of 30°. The agar was allowed to solidify at room temperature into a wedge-shaped layer. Onto the set base, another 20 mL of agar containing malathion 400 mg L−1 was poured to give malathion a gradient across the surface of the plate. Spore suspension of fungal isolates was prepared in 0.1% Triton X100 and streaked along the malathion gradient using a sterile cotton swab. Petri plates were incubated at 30 ± 2°C for 8 days. After incubation, the length of fungal growth along the gradient was measured [14].

Minimum Inhibitory Concentration

The isolated fungus was subjected to broth assay for evaluating minimum inhibitory concentration (MIC) and tolerance to malathion. A series of 250-mL Erlenmeyer flasks containing 100 mL of the M1 medium composed of NaNO3 2 g; KCl 0.5 g; MgSO4.7H2O 0.5 g; glucose 10 g; FeCl3 10 mg; BaCl2 0.2 g; and CaCl2 0.5 g; per liter at pH 6.8 was spiked with increasing concentration of malathion. The flasks were inoculated with 1 mL of fungal spore suspension prepared in 0.01% Triton X-100 and incubated at 30 ± 2°C on a rotary shaker 120 rpm. After 10 days of incubation, the flasks were observed for mycelial growth. Mycelial mass from each flask was separated by filtration using Whatman filter paper no.1 and washed with deionized water. The dry weight of fungal biomass was determined by drying at a constant weight for 80°C in preweighed aluminum foil cups. The MIC was noted as the concentration of malathion resulting in inhibition of mycelia growth in flasks [14].

Identification of Fungal Strain

The isolated fungal strain was identified by 18S rRNA sequence analysis. The fungal genomic DNA was isolated by using AMpurE fungal gDNA Mini kit. In this kit detergent and other noncorrosive chemicals were used to break open the cellulosic cell wall and plasma membrane to extract DNA from fungal cells. The 18S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal primers CGW CGR AAN CCT TGT NAC GAS TTT TAC TN and AWG CTA CST GGT TGA TCC TSC CAG N. PCR reaction mix of 50 µL final volume contained: 50 ng sample gDNA, 100 ng forward primer, 100 ng reverse primer, 2 µL dNTP's mixture (10 mm), 5 µL 10X Taq polymerase buffer, 3 U Taq polymerase enzyme, and PCR grade water to make up the volume. Amplified PCR product was sequenced by using ABI3730xl genetic analyzer (Amnion Biosciences, Bangalore, India). The result was submitted to the GenBank National Centre for Biotechnology Information (NCBI) database and the accession number was obtained.

Growth Rate

In order to determine the growth pattern, 1 mL of spore suspension was inoculated in a series of flask containing Czapek Dox broth (100 mL in Erlenmeyer flask) with and without 400 mg L−1 of malathion. The flasks were incubated at 30 ± 2°C on a rotary shaker at 120 rpm. After incubation the mycelial mass was removed at regular time intervals and separated by filtration using Whatman filter paper no.1 and washed with deionized water. Biomass determination was done by drying the fungal biomass for a constant weight at 80°C in preweighed aluminum foil cups.

Biodegradation of Malathion in Liquid Medium and Soil

Studies on degradation of malathion in liquid medium, was performed by adding 100 mL of M1 medium supplemented with 400 mg L−1 of malathion as the sole carbon source and incubated with 1 mL fungal spore suspension of JASA1 strain. Flasks were incubated at 30 ±2°C on a rotary shaker at 120 rpm and samples were taken at regular time intervals. In order to determine the ability of JASA1 to degrade malathion, further studies were conducted in the same soil sample. Two trails were carried out: (1) Addition of pesticide 400 mg L−1, fungal spore, and nutrients (carbon, nitrogen, and phosphorous), and (2) Addition of pesticide (400 mg L−1) and fungal spore without nutrients. The amount of carbon, nitrogen and phosphorus was calculated using the relationship C/N/P 100:10:1. The sources of carbon, nitrogen and phosphorous were glucose, (NH4)2SO4 and K2HPO4, respectively. The utilization of malathion was determined by HPLC.

Analytical Methods

The extracts were analyzed on a Varian HPLC equipped with a binary pump, programmable variable wavelength UV detector and ODS2 C18 reverse phase column. The liquid samples from malathion degradation flasks were extracted with equal volume of acetonitrile. Ten grams of soil samples were collected from each treatment trails with and without nutrients. The soil samples were extracted with 20 mL of acetonitrile in order to determine the pesticide concentration by HPLC. The isocratic mobile phase composed of acetonitrile:water (50:50, V: V), which was pumped through the column at a flow rate of 0.1 mL/min. Malathion and its metabolite were detected at 215 nm. Infrared spectra of the malathion parent compound and sample after fungal degradation was recorded at room temperature in frequency range of 4000–400cm−1 with FTIR. Spectrophotometer (8400 Shimadzu, Japan with Hyper IR-1.7 Software for windows) with helium-neon laser lamp as a source of IR radiations. Pressed pellet were prepared by grinding the extract samples with potassium bromide in motor with ratio of 1:100 immediately analyses in the region of 4000–400 cm−1 at a resolution of 4 cm−1.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

In the present study, fungal strain JASA1 was isolated from sugarcane field soil sample using enrichment technique and three morphologically different strains were isolated on Czapek Dox agar plate containing malathion. Malathion gradient plate assay was applied to screen the isolates for highest tolerance to malathion. The growth performance was recorded as the length of fungal growth in centimeters across the malathion gradient. Among all the three strains JASA1 showed a growth of above 8 cm across the gradient plates, which was further confirmed using broth assay. The MIC of malathion was performed for JASA1 strain which could tolerate upto 500 mg L−1 and had a confluent growth at 400 mg L−1 of malathion. In a similar study, Aspergillus oryzae ARIFCC 1054 and Aspergillus terreus JAS1 showed growth upto 900 and 400 mg L−1 of organophosphorus pesticides, respectively [14, 15].

The molecular characterization of 18S rRNA sequence and BLAST results exhibited close relationship and 99% similarity to that F. oxysporum. Multiple sequence alignments and phylogenetic tree (Figure 1) revealed the strain JASA1 cluster with Fusarium sp. Therefore, JASA1 isolate was designated as F. oxysporum JASA1 and the sequence result was submitted to GenBank NCBI database and accession number KF175514 was obtained.

image

Figure 1. Phylogenetic relationship of Fusarium oxysporum JASA1 based on 18S rRNA gene nucleotide sequences.

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Growth kinetics of F. oxysporum JASA1 in the presence and absence of malathion as a function of time is presented in Figure 2. The metabolism of malathion by F. oxysporum JASA1 was assessed by the increase in mycelial growth. Initially, the growth was found to be suppressed in presence of malathion, but after acclimatization to malathion, the culture was capable of growing rapidly exhibiting high growth rate. In later stage, the amount of biomass produced in the medium containing pesticide was much higher than the growth in the absence of malathion (control). This could be because of the availability of additional carbon and sulfur upon degradation of malathion in the medium. Growth kinetic study was done to learn the patterns of growth of the efficient JASA1 strain. Czapek Dox broth was spiked with malathion as sole carbon source and the JASA1 strain grew luxuriantly in it. While comparing the patterns with test and control, there was increased biomass in the test conditions.

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Figure 2. Growth performance of Fusarium oxysporum in the presence and absence of malathion (400 mg L−1). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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HPLC was used to monitor the degradation of malathion and the results are presented in Figure 3. Recovery experiment was conducted in the M1 medium and soil for the study on extraction efficiency of the methods established. Different known concentrations of malathion were spiked in 50 mL of the M1 medium (100, 200, 300, and 400 mg L−1) and 50 g of soil (100, 200, 300, and 400 mg kg−1). Average recoveries of malathion from the M1 medium at levels of 100, 200, 300, and 400 mg L−1 were measured to be 96.2 ± 4.3, 97.6 ± 4.8, 98.4 ± 3.2, and 97.6 ± 1.2 %, respectively. The corresponding recoveries from the soil at levels of 100, 200, 300, and 400 mg kg−1 were 94.7 ± 3.6, 96.7 ± 3.2, 95.3 ± 2.9, and 94.2 ± 0.6 %, respectively. These data indicate that HPLC for malathion determination has a high accuracy, and the extraction procedures are efficient in extracting the malathion residues from the M1 medium and soil. The HPLC analysis revealed that JASA1 strain was capable of growing in M1 medium containing malathion as the sole source of carbon and energy, and confirmed the degradation of malathion. F. oxysporum degraded malathion in the aqueous medium to an undetectable level in 5 days (Figure 3b), which was compared with the HPLC peaks for the malathion standard (Figure 3a). The degradation dynamics of malathion in the soil are presented in Figure 4. The strain JASA1 was inoculated in soil with 400 mg kg−1 of malathion and nutrients (carbon, nitrogen, and phosphorous) were amended, after 8 days of incubation it showed 100% degradation of malathion (Figure 4a). There was no appreciable difference in the soil inoculated with JASA1 strain in the absence of nutrients and the 100% degradation was recorded after 9 days (Figure 4b). In previous study [11], it was reported that, malathion was degraded after 7 days of incubation by strain KB2 which degraded 72.20% of malaoxon and 36.22% of malathion, whereas strain PU degraded 87.40% of malaxon and 41.30% of malathion. Singh et al. [16] demonstrated degradation of malathion by B. thuringiensis/cereus bacteria by performing two systems, soil slurry system and soil box system. After 4 days of incubation, it was found that 65.87% of malaoxon and 30.93% of malathion was degraded and in case of soil box system 74.75% of malaoxon and 26.12% of malathion was degraded. These results indicated that degradative pathway of malathion might be facilitated by the activity of esterase enzyme [17]. In few studies the gene encoding carboxylesterase was cloned and the recombinant protein was expressed [18, 19]. The carboxylesterase family comprises a group of esterases hydrolyzing carboxylic ester bonds, which is present in malathion, with relatively broad substrate specificity. They show a high degree of sequence similarity and are believed to be involved in the detoxification of many xenobiotics [20].

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Figure 3. (a) The HPLC chromatogram of malathion at standard condition. (b) HPLC chromatogram of biodegradation of malathion in aqueous medium by Fusarium oxysporum JASA1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Figure 4. (a) Biodegradation of malathion in soil with nutrients and (b) soil without nutrients. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Comparison of FTIR spectrum of control with extracted metabolites after complete degradation of malathion by F. oxysporum strain JASA1 which clearly indicated the degradation of malathion (Figure 5a and 5b). The infrared spectrum of malathion degraded sample showed a band at 3446 cm−1 which corresponds to N[BOND]H stretch. The peak at 1641 cm−1 represents C[DOUBLE BOND]C stretch. The acid dimer band of malathion degraded sample in the aqueous medium by JASA1 was found to be at 991cm−1 and the peak at wave number 1085 cm−1 indicated PH bend. The acid dimer band support the malathion degradation to malathion mono-acid or malathion diacid may occur through the action of carboxylesterase [21]. The C[BOND]H deformation band was seen in the malathion degraded sample at 678 cm−1. However, in earlier study on the biodegradation of chlorpyrifos [15], the bands at 659, 696, and 699 cm−1 are the characteristics of C[BOND]H deformation which strongly supports our observations. The overall observation confirms the degradation of malathion in the sample.

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Figure 5. (a) Control and (b) FTIR spectrum of biodegradation of malathion in aqueous medium.

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CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED

Malathion degrading fungus was isolated and characterized as F. oxysporum strain JASA1 which could efficiently degrade malathion in both liquid medium and soil. The effectiveness of degradation was confirmed by HPLC and FTIR studies. Moreover, this study confirms that the F. oxysporum strain JASA1 could be used efficiently for the remediation of malathion contaminated environment.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. CONCLUSION
  7. LITERATURE CITED
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    Singh, B., Kaur, J., & Singh, K. (2012). Transformation of malathion by Lysinibacillus sp. Isolated from soil, Biotechnology Letters, 34, 863867.
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    Anwar, S., Liaquat, F., Khan, Q.M., Khalid, Z.M., & Iqbal, S. (2009). Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol by Bacillus pumilus strain C2A1, Journal of Hazardous Materials, 168, 400405.
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    Singh, B., Kaur, R., & Singh, K. (2009). Degradation of Organophosphate pesticide, malathion, by Bacillus sp. isolated from soil, Proceedings of the 11th International Conference on Environmental Science and Technology, Chania, Crete, Greece, 3–5 September.
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    Kim, Y.H., Ahn, J.Y., Moon, S.H., & Lee, J. (2005). Biodegradation and detoxification of organophosphate insecticide, malathion by Fusarium oxysporum f. sp. pisi cutinase, Chemosphere, 60, 13491355.
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    Kim, H.E., Lee, I.S., Kim, J.H., Hahn, K.W., Park, V.J., Han, H.S., & Park, K.R. (2003). Gene cloning, sequencing, and expression of an esterase from Acinetobacter lwoffii I6C-l, Current Microbiology, 46, 291295.
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    Kakugawa, S., Fushinobu, F., Wakagi, T., & Shoun, H. (2007). Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritime, Applied Microbiology and Biotechnology, 74, 585591.
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    Jakoby, W.E., & Ziegler, D.M. (1990). The enzymes of detoxification, The Journal of Biological Chemistry, 265, 2071520718.
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