Pleurotus ostreatus as a Biodegradator for Organophosphorus Insecticide Malathion

Organophosphorus compound poisoning is a worldwide health problem with around three million poisonings and 200000 deaths annually [1,2]. Continuous and excessive use of organophosphorus compounds has led to the contamination of several ecosystems in different parts of the world [3-5]. Malathion [S-(1,2dicarbethoxyethyl)-O,O-dimethyldi-thiophosphate], also known as maldison, carbophos and mercaptothion is a nonsystemic, widespectrum organophosphorus insecticide used to control the household and agricultural pests. According to Singh et al. [6] malathion was recognized as the first organophosphorous insecticide with highly selective toxicity. Malathion is an organophosphate insecticide and acaricide that has been suited for the control of sucking and chewing insects on field crops, fruits, vegetable, livestock, and also extensively used to prevent mosquitoes, flies, household insect, animal parasites, and head body lice as substitute for DTT compound [7,8]. Malathion in soil undergo a variety of transformations that provide a complex pattern of metabolites and its fate in soil is controlled by chemical, biological and physical dynamics of this matrix. Products resulted from malathion degradation include dimethyl phosphate, dimethyldithiophosphate, dimethylthiophosphate, isomalathion, malaoxon and malathion mono and dicarboxylic acid due to enzymatic activity of cutinase, carboxylesterase, phosphatase [9]. Malathion itself is of low toxicity, it mainly concentrates in peel and may not readily removed by washing in water alone [10], but easily enter the body through absorption through the skin, ingestion and inhalation results in its metabolism to malaoxon which is substantially more toxic [11]. Long-term exposure to oral ingestion of malaoxon in rats, showed 61 times more toxic than malathion.


Introduction
Organophosphorus compound poisoning is a worldwide health problem with around three million poisonings and 200000 deaths annually [1,2]. Continuous and excessive use of organophosphorus compounds has led to the contamination of several ecosystems in different parts of the world [3][4][5]. Malathion [S-(1,2dicarbethoxyethyl)-O,O-dimethyldi-thiophosphate], also known as maldison, carbophos and mercaptothion is a nonsystemic, widespectrum organophosphorus insecticide used to control the household and agricultural pests. According to Singh et al. [6] malathion was recognized as the first organophosphorous insecticide with highly selective toxicity. Malathion is an organophosphate insecticide and acaricide that has been suited for the control of sucking and chewing insects on field crops, fruits, vegetable, livestock, and also extensively used to prevent mosquitoes, flies, household insect, animal parasites, and head body lice as substitute for DTT compound [7,8]. Malathion in soil undergo a variety of transformations that provide a complex pattern of metabolites and its fate in soil is controlled by chemical, biological and physical dynamics of this matrix. Products resulted from malathion degradation include dimethyl phosphate, dimethyldithiophosphate, dimethylthiophosphate, isomalathion, malaoxon and malathion mono and dicarboxylic acid due to enzymatic activity of cutinase, carboxylesterase, phosphatase [9]. Malathion itself is of low toxicity, it mainly concentrates in peel and may not readily removed by washing in water alone [10], but easily enter the body through absorption through the skin, ingestion and inhalation results in its metabolism to malaoxon which is substantially more toxic [11]. Long-term exposure to oral ingestion of malaoxon in rats, showed 61 times more toxic than malathion.
Today, microorganisms are used in bioremediation of environmental pollutants. Using microorganisms is natural process, very effective when compared to the other methods and applicable for insitu bioremediation. Biodegradation of organophosphate compounds releases phosphate as one of the end product [12]. Fungi [13] are capable of degrading malathion. Ahmed et al. [14] found that the degradation rate of malathion by bacterial was slower than the fungal isolates. White rot fungi as a group include Phanerochaete chrysosporium, Trametes versicolor, Pleurotus ostreatus, Bjerkandera adusta, and Pleurotus tuberregium. On the basis of their ability to secrete extracellular lignin-degrading enzymes, these fungi have been applied in bioremediation studies [15]. White-rot fungi can degrade a wide spectrum of recalcitrant organopollutants, often carcinogenic, mutagenic or toxic, that arise from industrial operations, petroleum released into environment and plant protection [16]. Pleurotus ostreatus is a commercially important edible mushroom [17]. Most of the white-rot fungi secrete various extra cellular enzymes that degrade a large range of natural and anthropogenic compounds that have structural and chemical similarities to the lignin substructure. Among them, peroxidase or ligninolytic enzymes, predominantly lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. Presence of nonspecific enzyme system, enable fungi to degrade natural complex aromatic polymers of lignin as well as complex aromatic polymers

Abstract
Excessive and continuous use of organophosphorus insecticides as well as malathion has led to the contamination of ecosystems in several rejoins of the world. For its degradation in the ecosystems usage of the microbes has been proved to be the effective method for controlling ecosystems pollution. Biodegradation of pesticides by using microbes have more advantages over the conventional methods. In the present investigation therefore attempts have been made to make use of white rot fungus Pleurotus ostreatus for malathion degradation. Malathion rest quantity at 5, 15 and 25 days was 40.5, 22.6 and 11.8 (µg/100 mL) with degradation % 19.0, 54.8 and 76.4 respectively at initial concentration (50 µg/100 mL). Lignin peroxidase, manganese peroxidase, and laccase production was stimulated with the presence of malathion. At 25 µg/100 mL malathion, productivity of lignin peroxidase, manganese peroxidase, and laccase was 0.51, 0.57 and 4.30 U/ml respectively compared with the control (growth medium without malathion). At low concentrations of malathion (25 and 50 µg) P. ostreatus productivity of these enzymes was more, but at high concentration of malathion the productivity was less than control. Addition of lignin compound induced the productivity of ligninolytic enzymes and therefore the biodegradation of malathion was increased. GC/MS analysis revealed the presence of many products as a result of malathion biodegradation including diethyl mercaptosuccinate, methy l,2-(Dimethoxyphosphoryl)-2-(1,2,3thiadiazol-4-yl) acetate, 3,4-dihydrothienyl (3,4,b)-5-carboxythiophene, Butanedioic acid, 2,2'-Thiodisuccinic acid; 1,1-Dimethyltetradecyl hydrosulfide and Disulfide, di-tert-dodecyl. that share structure with lignin, such as pesticides, PAHs, PCBs and dyes [18]. Several studies stated that potential applications of whiterot fungi and their enzymes are gaining increasing importance in the detoxification of industrial waste waters, biodegradation, biopulping, degradation and detoxification of recalcitrant substances [19][20][21][22][23]. The present work was aimed at studying the ability of a white-rot fungus Pleurotus ostreatus, as a biodegratator of insecticide malathion. Optimal conditions for the biodegradation were also explored.

Biodegradator used
Pleurotus ostreatus (obtained from agricultural ministry, Egypt) was cultivated on Malt agar plates for 10 days at 30°C. Mycelium agar plugs 9 mm in diameter (cut along the edge of an actively growing colony) were used as inocula.

Fungal degradation of organophosphrus insecticides at different incubation period
Mineral salt liquid medium supplemented with 50 µg/100 mL concentration of analytical grade standard of Malathion ( Figure 1) was used for biodegradation test at 5,15 and 25 days.

Effect of different concentrations of malathion on fungal growth
Different concentrations of malathion 25, 50 and 100 µg/100 mL were added to growth medium. Fungal mycelia (disc 0.5 cm) were transferred to growth medium and incubated at 28 ± 2°C for 10 days.

Lignin as inducer to malathion degradation at different concentrations and ligninolytic enzymes detection
Mineral salt liquid medium supplemented with various concentrations of malathion (25, 50 and 100 µg/100 mL) was autoclaved. Then, 0.2 g % of lignin was added to growth medium, in order to test the ability of the fungus to produce ligninolytic enzymes which enhance the biodegradation of malathion. Flasks containing medium were inoculated with fungus and then incubated at 30°C for 15 days. After incubation period the malathion rest quantity was extracted for quantitative analysis and the metabolized medium was filtrate through filter paper, and used for assay of three enzymes lignin peroxidase (LIP), laccase (phenol oxidase), manganese peroxidase (MnPase). LIP activity was assayed using veratryl alcohol as a substrate. LIP catalyzes the oxidation of veratryl alcohol by H 2 O 2 to veratryl aldehyde. The aldehyde was absorbed strongly at 310 nm after incubation for 1 h at 37 °C. The reaction medium contained: 1 mL of the enzyme solution (culture filtrate), 0.2 mL of 2 mmol/L veratryl alcohol, 0.2 mL of 0.4 mmol/L H 2 O 2 , 0.2 mL of 0.25 mmol/L tartaric acid. Laccase activity is determined by the oxidation of 2, 2¢-azino-bis (3-ethylthiazoline-6-sulfonate), i.e., ABTS at 37°C at A420 [24]. One unit of enzyme activity is defined as the amount of enzyme oxidizing 1 mMol of ABTS per minute. Reaction mixture of 1 ml contained 2 mM of ABTS in a Mcllvaine buffer (pH 5.0). To the assay mixture, 100 µl of centrifuged extracellular supernatants were added. The enzymatic activity was estimated in IU by monitoring the adsorbance change at 420 nm (ABTS), Є=36 Mm -1 cm -1 by spectrophotometer (UVikon XS/60/99-90289; Volt 110/240 V) at 30°C. MnPase activity was carried out in 1 cm quartz cuvette. The reaction mixture of 1 ml contained 2 mM of ABTS and 1mM Mn 2+ in a Mcllvaine buffer (pH 5.0). To the assay mixture, 100 µl of centrifuged extra cellular fluids (supernatants) were added. The peroxidase activity was then initiated by the addition of 0.4 mM H 2 O 2 [25,26]. The enzymatic activity was estimated in IU by monitoring the adsorbance change at 420 nm (ABTS), Є=36 mM -1 cm -1 by spectrophotometer at 30°C.

Gas Chromatography/Mass Spectrometry (GC/MS) analysis of malathion degradation
The concentrated extract of metabolized medium was extracted with using methanol, then filtrated and concentrated into 1 ml and was placed in GC auto-sampler vials until they were analyzed. A Varian Star 3400 Cx Ion Trap GC/MS Shimadzu GCMS-QP 5050 A. software class 5000. Searched library: Wiley 229 LIB. Column: DBI, 30 m, 053 mm ID; 1.5 um film. Carrier gas: Helium (flow rate 1 ml/min.). Ionization mode: El (70 ev). Temperature program: 70 (static for 2 min) then gradually increasing (at a rate of 2 /min) up to 220 (static for 5 min). Detector temperature 250 injector temperature 250 The chromatographs were compared and individual peaks were identified by comparing mass spectra to the library references. At the Regional Center for Mycology and Biotechnology AL-Azhar University).

Results and Discussion
Investigation of microbial degradation is useful for developing insecticide degradation strategies using microorganisms. In the current study, Pleurotus ostreatus growth decreased with increasing malathion concentration (Table 1 and Figure 2). At 25, 50 and 100 µg/100 mL malathion, the inhibition % was 4.62, 35.48 and 64.62% respectively. Recently, Abbas et al. [27] found that the efficacy of the pesticides on the inhibition of the fungal growth and spore germination showed that they were both reduced with the increasing concentration of the insecticide.
GC/MS analysis of malathion degradation (Figure 3) with using Pleurotus ostreatus at different incubation periods up to 25 days indicated that biodegradation increased with increasing incubation period. Malathion rest quantity at 5, 15 and 25 days was 40.5, 22.6 and 11.8 (µg/100 mL) with degradation % 19.0, 54.8 and 76.4 respectively at initial concentration (50 µg/100 mL) (Figure 4). Chalamala et al. [13] worked on mycodegradation of Malathion by using A. niger and got 86.72% of degradation. Recently, research activities in this area have shown that a diverse range of microorganisms are capable of degrading malathion [28]. White-rot fungi tolerate considerable amounts of toxic pollutants, have high surface growth rates [29] and produce extracellular enzymes and other mediators that can reach contaminants that are poorly bioavailable [30].
Pleurotus ostreatus secretes enzymes such as lignin peroxidase, manganese peroxidase, and laccase. These ligninolytic enzymes have been reported to be versatile and nonspecific in their degradation characteristics and can be used in degrading a broad range of aromatic contaminants [31]. In the current study, lignin peroxidase, manganese peroxidase, and laccase production was stimulated with the presence of malathion. At 25 µg/100 mL malathion, productivity of lignin peroxidase, manganese peroxidase, and laccase was 0.51, 0.57 and 4.30 U/ml respectively compared with the control (growth medium without malathion) ( Table 2). In previous studies [32,33], production of lignolytic enzymes is indeed known to be stimulated by toxic phenols released during lignin degradation or by toxins released by other organisms and by the presence of aromatic xenobiotic compounds. At low concentrations of malathion (25 and 50 µg) P. ostreatus productivity of these enzymes was more, but at high concentration of malathion the productivity was less than control. Addition of lignin compound induced the productivity of ligninolytic enzymes and therefore the biodegradation of malathion was increased ( Table 2). According to many studies [34][35][36] biodegradation and bioremediation of a large array of organic pollutants, including polycyclic aromatic hydrocarbons, insecticides, polychlorinated aromatic compounds, and synthetic dyes with using white-rot fungi have been successfully. Enzymatic systems of white-rot fungi are capable of oxidative depolymerization and subsequent mineralization of lignin-related compounds [37]. Fungi and bacteria produce intracellular or extracellular enzymes including hydrolytic enzymes, peroxidases, oxygenases [38,39]. According to Xie et al. [40], malathion was completely decomposed within 29 h at the concentration of 12.860 g/L. and decreased from 100 to 0 mg/L in 84 h with using Acinetobacter johnsonii.

Conclusions
White-rot fungus possesses complex and efficient lignolytic enzyme system. They have been successfully applied in treatment and