Mineralization of Malathion by Fusarium

oxysporum Strain JASA1 Isolated from Sugarcane

FieldsLogeswari Peter, Anudurga Gajendiran, Deepa Mani, Sugitha Nagaraj, and Jayanthi AbrahamMicrobial Biotechnology Laboratory, School of Biosciences and Technology, VIT University, Vellore 632014, Tamil Nadu, India;jayanthi.abraham@gmail.com (for correspondence)

Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ep.11970

The present work was focused in isolating fungus whichpossessed the special ability to degrade malathion. The pesti-cide of choice was malathion as it is being used to control avariety of pests in the agricultural fields in India. The fungalstrains with the potential to degrade malathion was isolatedby enrichment technique from malathion contaminated soil.The molecular characterization of 18S rRNA sequencehomology confirmed its identity as Fusarium oxysporum. Theisolate was able to degrade 400 mg L21 malathion completelyin mineral medium. In soil spiked with malathion and withaddition of nutrients (carbon, nitrogen, phosphate), the iso-late was capable of degrading malathion at 8th day of incu-bation. Where as in the second trial, in the absence ofnutrients, JASA1 was able to degrade 400 mg L21 malathionon the 9th day of incubation. The course of the degradationprocess was studied using High performance liquid chroma-tography (HPLC) and Fourier transform infrared (FTIR)analyses which confirmed the degradation potential of thefungus. VC 2014 American Institute of Chemical EngineersEnviron Prog, 00: 000–000, 2014

Keywords: organophosphorus, biodegradation, HPLC,FTIR, Fusarium oxysporum JASA1

INTRODUCTION

Malathion [S-(1,2-dicarbethoxyethyl)-O,O-dimethyldithio-phosphate], is otherwise called carbophos, maldison, andmercaptothion. It is one of the first organophosphorus insecti-cide with selective toxicity. Organophosphate pesticides arelargely being employed in many countries for public healthand agricultural purposes [1]. The most toxic metabolite ofmalathion is the oxidation product malaoxon which is formedin the presence of air by oxidation and is responsible for theinsecticidal activity of the parent compound. Malaoxon can befound either as an impurity in malathion or generated duringthe oxidation of malathion in air or soil and it breaks downmore rapidly than malathion in alkaline and moist soil. Mala-thion is absorbed by practically all routes including the gastro-intestinal tract, skin, mucous membranes, and lungs [2]. Toxiceffect of malathion has been shown to affect the central nerv-ous system of invertebrates, immune system of higher verte-brates, reproductive functions of vertebrate, adrenal glands,and tissues of fish [3–8].

The magnitude of microbial degradation as compared tochemical degradation is found to increase with increasingsoil organic matter and is directly dependent on pH of soil.Chatterjee et al. [6] studied the biodegradation of malathionwith Rhizopus oryzae biomass and concluded that 85% ofmalathion was degraded from its aqueous solution asagainst 47–68% by other fungal biomasses. In another inves-tigation [9], Bacillus thuringiensis MOS-5(Bt) was isolatedfrom agricultural waste water and it was able to degrademalathion cometabolically. The major degraded productswere Mal-monocarboxylic acid (MMA) and Mal-dicarboxylicacid (MDA). Xie et al. [10] employed Acinetobacter johnso-nii MA19 to degrade malathion by using it as an sole car-bon source. Biodegradation of malathion with Brevibacillussp. strain KB2 and Bacillus cereus strain PU was conductedby Singh et al. [11] both the strains were cultured in thepresence of malathion under aerobic and energy-limitingconditions. Both strains grew well in the medium with mal-athion concentration up to 0.15%. Reverse-phase high-Per-formance liquid chromatography (HPLC)-UV analysisindicated that strain KB2 was able to degrade 72.20% ofmalaoxon (an analogue of malathion) and 36.22% of mala-thion, whereas strain PU degraded 87.40% of malaoxon and49.31% of malathion, after 7 days of incubation. Singh et al.[12] demonstrated degradation of malathion with Lysiniba-cillus sp. isolated from soil, which was able to tolerate0.15% of malathion under aerobic conditions utilizing it as asole carbon source. The degradation rate recorded was 20%malathion and 47% malaoxon out of the initial concentra-tion of malathion. Two metabolites, mal-monocarboxylicacid and mal-dicarboxylic acid, were detected within 7 daysat 30�C [12]. The present study reports on the isolation,molecular characterization and biodegradation of malathionby Fusarium oxysporum.

MATERIALS AND METHODS

ChemicalsAnalytical grade malathion (97.2%) was purchased from

Sigma Aldrich (St. Louis, MO). Technical grade malathion of25% emulsifiable concentrate was used for this study whichwas obtained from Insecticides (India) Limited, India. Yeastextract, peptone, dextrose, sucrose, dipotassium sulfate, fer-rous sulfate, agar agar, Triton X-100, MgSO4, NaNO3, KCl,VC 2014 American Institute of Chemical Engineers

Environmental Progress & Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep Month 2014 1

MgSO4.7H2O, FeCl3, BaCl2, CaCl2, (NH4)2SO4, and K2HPO4

were purchased from Hi-media, Mumbai, India. HPLC gradeacetonitrile was obtained from and Merck, India.

Soil SampleSugarcane field with previous exposure to malathion over

a period of 3 years was selected and soil was collected fromthe top layer of depth 0–23 cm. In the laboratory, the soilwas air dried at room temperature and sieved at a particlesize of <2 mm.

Isolation of Fugal Strain and Enrichment TechniqueFungal strain was isolated from sugarcane field soil sam-

ple by screening it in Czapek Dox broth [13]. Isolated fungalstrain which was capable of degrading malathion wasobtained by enrichment culture in the Czapek Dox brothcontaining yeast extract 3 g L21; peptone 10 g L21; dextrose2 g L21; along with malathion 100 mg L21; and 20 g of soilcollected from agricultural field with previous exposure tomalathion. In this enrichment technique 100 mL of the mediawas spiked with 100 mg L21 malathion and kept at 100 rpmon a rotary shaker at room temperature for 5 days. Theenriched samples were transferred to sterile medium contain-ing malathion 100 mg L21 as the only carbon source and 5mL of inoculum was subcultured and incubated for 2–4weeks. Moisture content was maintained throughout theexperiment by regular addition of sterile distilled water. After2 weeks, the fungal colonies were isolated by streaking theenriched samples on Czapek Dox medium of sucrose 30 g;magnesium sulfate 0.5 g; sodium nitrate 2 g; dipotassium sul-fate 0.35 g; ferrous sulfate 0.001 g; malathion 100 mg; andagar agar 30 g L21 at pH 6.8. Isolated fungal culture wasmaintained on agar slopes of the same medium containingmalathion.

Gradient Plate AssayThe isolate obtained from the enrichment experiments

were screened for malathion tolerance by following the gra-dient plate method. Malathion concentration gradient wasprepared by adding a base layer of 20 mL of modified Cza-pek Dox agar without malathion to a petriplate tilted at anangle of 30�. The agar was allowed to solidify at room tem-perature into a wedge-shaped layer. Onto the set base,another 20 mL of agar containing malathion 400 mg L21 waspoured to give malathion a gradient across the surface of theplate. Spore suspension of fungal isolates was prepared in0.1% Triton X100 and streaked along the malathion gradientusing a sterile cotton swab. Petri plates were incubated at30 6 2�C for 8 days. After incubation, the length of fungalgrowth along the gradient was measured [14].

Minimum Inhibitory ConcentrationThe isolated fungus was subjected to broth assay for eval-

uating minimum inhibitory concentration (MIC) and toler-ance to malathion. A series of 250-mL Erlenmeyer flaskscontaining 100 mL of the M1 medium composed of NaNO3 2g; 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 spikedwith increasing concentration of malathion. The flasks wereinoculated with 1 mL of fungal spore suspension prepared in0.01% Triton X-100 and incubated at 30 6 2�C on a rotaryshaker 120 rpm. After 10 days of incubation, the flasks wereobserved for mycelial growth. Mycelial mass from each flaskwas separated by filtration using Whatman filter paper no.1and washed with deionized water. The dry weight of fungalbiomass was determined by drying at a constant weight for80�C in preweighed aluminum foil cups. The MIC was notedas the concentration of malathion resulting in inhibition ofmycelia growth in flasks [14].

Identification of Fungal StrainThe isolated fungal strain was identified by 18S rRNA

sequence analysis. The fungal genomic DNA was isolated byusing AMpurE fungal gDNA Mini kit. In this kit detergentand other noncorrosive chemicals were used to break openthe cellulosic cell wall and plasma membrane to extract DNAfrom fungal cells. The 18S rRNA gene was amplified by poly-merase chain reaction (PCR) using the universal primersCGW CGR AAN CCT TGT NAC GAS TTT TAC TN and AWGCTA CST GGT TGA TCC TSC CAG N. PCR reaction mix of 50mL final volume contained: 50 ng sample gDNA, 100 ng for-ward primer, 100 ng reverse primer, 2 mL dNTP’s mixture (10mm), 5 mL 10X Taq polymerase buffer, 3 U Taq polymeraseenzyme, and PCR grade water to make up the volume.Amplified PCR product was sequenced by using ABI3730xlgenetic analyzer (Amnion Biosciences, Bangalore, India).The result was submitted to the GenBank National Centre forBiotechnology Information (NCBI) database and the acces-sion number was obtained.

Growth RateIn order to determine the growth pattern, 1 mL of spore

suspension was inoculated in a series of flask containingCzapek Dox broth (100 mL in Erlenmeyer flask) with andwithout 400 mg L21 of malathion. The flasks were incubatedat 30 6 2�C on a rotary shaker at 120 rpm. After incubationthe mycelial mass was removed at regular time intervals andseparated by filtration using Whatman filter paper no.1 andwashed with deionized water. Biomass determination wasdone by drying the fungal biomass for a constant weight at80�C in preweighed aluminum foil cups.

Biodegradation of Malathion in Liquid Medium andSoil

Studies on degradation of malathion in liquid medium,was performed by adding 100 mL of M1 medium supple-mented with 400 mg L21 of malathion as the sole carbonsource and incubated with 1 mL fungal spore suspension ofJASA1 strain. Flasks were incubated at 30 62�C on a rotaryshaker at 120 rpm and samples were taken at regular timeintervals. In order to determine the ability of JASA1 todegrade malathion, further studies were conducted in thesame soil sample. Two trails were carried out: (1) Additionof pesticide 400 mg L21, fungal spore, and nutrients (carbon,nitrogen, and phosphorous), and (2) Addition of pesticide(400 mg L21) and fungal spore without nutrients. Theamount of carbon, nitrogen and phosphorus was calculatedusing the relationship C/N/P 100:10:1. The sources of car-bon, nitrogen and phosphorous were glucose, (NH4)2SO4

and K2HPO4, respectively. The utilization of malathion wasdetermined by HPLC.

Analytical MethodsThe extracts were analyzed on a Varian HPLC equipped

with a binary pump, programmable variable wavelength UVdetector and ODS2 C18 reverse phase column. The liquid sam-ples from malathion degradation flasks were extracted withequal volume of acetonitrile. Ten grams of soil samples werecollected from each treatment trails with and without nutrients.The soil samples were extracted with 20 mL of acetonitrile inorder to determine the pesticide concentration by HPLC. Theisocratic mobile phase composed of acetonitrile:water (50:50,V: V), which was pumped through the column at a flow rate of0.1 mL/min. Malathion and its metabolite were detected at 215nm. Infrared spectra of the malathion parent compound andsample after fungal degradation was recorded at room temper-ature in frequency range of 4000–400cm21 with FTIR. Spectro-photometer (8400 Shimadzu, Japan with Hyper IR-1.7Software for windows) with helium-neon laser lamp as a

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source of IR radiations. Pressed pellet were prepared by grind-ing the extract samples with potassium bromide in motor withratio of 1:100 immediately analyses in the region of 4000–400cm21 at a resolution of 4 cm21.

RESULTS AND DISCUSSION

In the present study, fungal strain JASA1 was isolatedfrom sugarcane field soil sample using enrichment techniqueand three morphologically different strains were isolated onCzapek Dox agar plate containing malathion. Malathion gra-dient plate assay was applied to screen the isolates for high-est tolerance to malathion. The growth performance wasrecorded as the length of fungal growth in centimeters acrossthe malathion gradient. Among all the three strains JASA1showed a growth of above 8 cm across the gradient plates,which was further confirmed using broth assay. The MIC ofmalathion was performed for JASA1 strain which could toler-

ate upto 500 mg L21 and had a confluent growth at 400 mgL21 of malathion. In a similar study, Aspergillus oryzaeARIFCC 1054 and Aspergillus terreus JAS1 showed growthupto 900 and 400 mg L21 of organophosphorus pesticides,respectively [14,15].

The molecular characterization of 18S rRNA sequence andBLAST results exhibited close relationship and 99% similarityto that F. oxysporum. Multiple sequence alignments and phy-logenetic tree (Figure 1) revealed the strain JASA1 clusterwith Fusarium sp. Therefore, JASA1 isolate was designatedas F. oxysporum JASA1 and the sequence result was submit-ted to GenBank NCBI database and accession numberKF175514 was obtained.

Growth kinetics of F. oxysporum JASA1 in the presenceand absence of malathion as a function of time is presentedin Figure 2. The metabolism of malathion by F. oxysporumJASA1 was assessed by the increase in mycelial growth. Ini-tially, the growth was found to be suppressed in presence ofmalathion, but after acclimatization to malathion, the culturewas capable of growing rapidly exhibiting high growth rate.In later stage, the amount of biomass produced in themedium containing pesticide was much higher than thegrowth in the absence of malathion (control). This could bebecause of the availability of additional carbon and sulfurupon degradation of malathion in the medium. Growthkinetic study was done to learn the patterns of growth of theefficient JASA1 strain. Czapek Dox broth was spiked withmalathion as sole carbon source and the JASA1 strain grewluxuriantly in it. While comparing the patterns with test andcontrol, there was increased biomass in the test conditions.

HPLC was used to monitor the degradation of malathionand the results are presented in Figure 3. Recovery experi-ment was conducted in the M1 medium and soil for thestudy on extraction efficiency of the methods established.Different known concentrations of malathion were spiked in50 mL of the M1 medium (100, 200, 300, and 400 mg L21)and 50 g of soil (100, 200, 300, and 400 mg kg21). Averagerecoveries of malathion from the M1 medium at levels of100, 200, 300, and 400 mg L21 were measured to be

Figure 1. Phylogenetic relationship of Fusarium oxysporumJASA1 based on 18S rRNA gene nucleotide sequences.

Figure 2. Growth performance of Fusarium oxysporum inthe presence and absence of malathion (400 mg L21). [Colorfigure can be viewed in the online issue, which is availableat wileyonlinelibrary.com.]

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

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96.2 6 4.3, 97.6 6 4.8, 98.4 6 3.2, and 97.6 6 1.2 %, respec-tively. The corresponding recoveries from the soil at levels of100, 200, 300, and 400 mg kg21 were 94.7 6 3.6, 96.7 6 3.2,95.3 6 2.9, and 94.2 6 0.6 %, respectively. These data indicatethat HPLC for malathion determination has a high accuracy,and the extraction procedures are efficient in extracting themalathion residues from the M1 medium and soil. The HPLCanalysis revealed that JASA1 strain was capable of growingin M1 medium containing malathion as the sole source ofcarbon and energy, and confirmed the degradation of mala-thion. F. oxysporum degraded malathion in the aqueousmedium to an undetectable level in 5 days (Figure 3b),which was compared with the HPLC peaks for the malathionstandard (Figure 3a). The degradation dynamics of malathionin the soil are presented in Figure 4. The strain JASA1 wasinoculated in soil with 400 mg kg21 of malathion andnutrients (carbon, nitrogen, and phosphorous) wereamended, after 8 days of incubation it showed 100% degra-dation of malathion (Figure 4a). There was no appreciabledifference in the soil inoculated with JASA1 strain in theabsence of nutrients and the 100% degradation was recordedafter 9 days (Figure 4b). In previous study [11], it wasreported that, malathion was degraded after 7 days of incu-bation by strain KB2 which degraded 72.20% of malaoxonand 36.22% of malathion, whereas strain PU degraded87.40% 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 systemand soil box system. After 4 days of incubation, it was foundthat 65.87% of malaoxon and 30.93% of malathion wasdegraded and in case of soil box system 74.75% of malaoxonand 26.12% of malathion was degraded. These results indi-cated that degradative pathway of malathion might be facili-tated by the activity of esterase enzyme [17]. In few studiesthe gene encoding carboxylesterase was cloned and therecombinant protein was expressed [18,19]. The carboxyles-terase family comprises a group of esterases hydrolyzing car-boxylic ester bonds, which is present in malathion, withrelatively broad substrate specificity. They show a high

degree of sequence similarity and are believed to beinvolved in the detoxification of many xenobiotics [20].

Comparison of FTIR spectrum of control with extractedmetabolites after complete degradation of malathion by F. oxy-sporum strain JASA1 which clearly indicated the degradationof malathion (Figure 5a and 5b). The infrared spectrum of mal-athion degraded sample showed a band at 3446 cm21 whichcorresponds to NAH stretch. The peak at 1641 cm21 repre-sents C@C stretch. The acid dimer band of malathion degradedsample in the aqueous medium by JASA1 was found to be at991cm21 and the peak at wave number 1085 cm21 indicatedPH bend. The acid dimer band support the malathion degrada-tion to malathion mono-acid or malathion diacid may occurthrough the action of carboxylesterase [21]. The CAH deforma-tion band was seen in the malathion degraded sample at 678cm21. However, in earlier study on the biodegradation ofchlorpyrifos [15], the bands at 659, 696, and 699 cm21 are thecharacteristics of CAH deformation which strongly supportsour observations. The overall observation confirms the degra-dation of malathion in the sample.

CONCLUSION

Malathion degrading fungus was isolated and character-ized as F. oxysporum strain JASA1 which could efficientlydegrade malathion in both liquid medium and soil. The effec-tiveness of degradation was confirmed by HPLC and FTIRstudies. Moreover, this study confirms that the F. oxysporumstrain JASA1 could be used efficiently for the remediation ofmalathion contaminated environment.

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

Figure 4. (a) Biodegradation of malathion in soil withnutrients and (b) soil without nutrients. [Color figure can beviewed in the online issue, which is available at wileyonline-library.com.]

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