Isolation of Chlorpyrifos Degrading Bacteria from Garden Soil

Introduction

Pesticides are chemical agents which kill pests especially insects. Pesticides have numerous beneficial effects which include crop protection, preservation of food materials and to control vector-borne diseases. Chlorpyrifos is widely used for pest control in agriculture, gardening and soil treatment [1]. Among pesticides, Organophosphate pesticides are a highly toxic group of agricultural chemicals which widely used to increase plant protection and production. Abundant usage of pesticide resulted in an accumulation of pesticide residues in crops, soils, and biosphere creating an environmental stress [2].

Chlorpyrifos, (O, O-diethyl O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) is one of the most commonly used broad-spectrum Organophosphates, and its phosphorus is linked to a sulfur with a double bond (P =S). It is used globally to control a variety of chewing and sucking insect pests and mites on the economically important crops including rice, bananas, vegetables, and cotton etc. Applicability of Chlorpyrifos also causes serious damage to non-target organisms [3]. A consequence of the persistent usage of chlorpyrifos causes contamination in an aquatic and terrestrial environment [4]. In previous studies, chlorpyrifos may affect all the physiological systems due to its high mammalian toxicity [5, 6]. Compounds of this organophosphate pesticide are hydrolyzed spontaneously and cause major neurotoxicity in mammals [7].

The half-life of chlorpyrifos is 60–120 days in soil and its rate of degradation is 50 to 570 days based on the nature of the soil [8]. During degradation the chlorpyrifos converted into 3, 5, 6-trichloro-2-pyridinol (TCP), an intermediate compound accumulate in the natural environment, which is recalcitrant to microbial degradation [9]. TCP has an antimicrobial property that prevents the proliferation of chlorpyrifos degrading microbial population [10].

Detoxification of chlorpyrifos intermediates using microorganisms is comparatively a best alternative approach of pesticide degradation because other methods of removing chlorpyrifos residues are impractical or costly or are themselves hazardous to the environment [11].

Biosurfactants are amphipathic in nature. It has a characteristic feature of hydrophilic-lipophilic balance, which specifies the portion of hydrophilic and hydrophobic constituents in surface-active substances. Because of their amphiphilic structure, biosurfactants increase the bioavailability of hydrophobic contaminants by changing the properties of the bacterial cell surface [12].

In the present study, Pseudomonas aeruginosa was isolated from pesticide-treated garden soil invitro biodegradation of Chlorpyrifos was performed. Degradation capacity of isolated Pseudomonas aeruginosa was identified by HPLC analysis and its biosurfactant ability was also assessed by blue agar plate method and penetration assay.

Discussion

Results of the present study were based on the biodegradation potential. The organism isolated from the garden soil was identified as Pseudomonas aeruginosa based on the morphological and biochemical characterizations. The rate of biodegradation is increased by the Pseudomonas aeruginosa which has the capacity to produce biosurfactants.

For this study, chlorpyrifos resistant bacterium was isolated by enriching soil sample which is contaminated with the organophosphate chlorpyrifos. The isolated microorganism is allowed to grow on the Pseudomonas isolation agar medium. This medium is very useful for isolating Pseudomonas species. Pseudomonas Isolation Agar includes Irgasan which has a potent antimicrobial activity but not active against Pseudomonas species and it selectively inhibits Gram-positive and Gram-negative bacteria other than Pseudomonas species. This medium is discerning and prepared to enhance the formation of blue or blue-green pyocyanin pigment because this confirms the presence of Pseudomonas aeruginosa. The production of pyocyanin pigment was promoted by the presence of Magnesium Chloride and Potassium Sulfate in the medium [17].

The rate of degradation of methyl parathion which is an organophosphorus pesticide was found out by using physical techniques such as UV-visible spectroscopy, FTIR spectroscopy, and HPLC. The extent of biodegradation was evident from the reduction of the peak and it is also observed from the decrease of area of the peak [18].

In the present study, the peak area of the experimental sample reduced when compared to the control. Pesticide concentration is one of the major factors that determine the fate of biodegradation. Very high concentration leads to the failure of biodegradation as microbes are not resistant against that. On the other hand, very low pesticide concentration shows a strong affinity with soil particles thus become non-available to microbes [19]. The rate of degradation is based on the concentration of Chlorpyrifos. Hence, the area measurement is a more reliable measure of the concentration of chlorpyrifos. The media contained a 1ppm concentration of Chlorpyrifos had higher peak area when compared to the 5 and 9 ppm concentration of chlorpyrifos. In each concentration, the peak area decreased gradually from 5th day to 15th day. This indicated that the rate of chlorpyrifos degraded gradually by Pseudomonas aeruginosa. Chlorpyrifos was used as a carbon source, phosphorous and energy source by the microorganisms. There is no additional energy source added in this present study to assure the degrading capability of bacterial isolates [20].

Another reason of efficient degradation is the Pseudomonas aeruginosa has the capacity to produce biosurfactants. Pseudomonas species, Bacillus species, and Acinetobacter species have potential to produce biosurfactant for acceleration of biodegradation of pesticides. It increased the biodegradation efficiency of chlorpyrifos by improving the bioavailability of the xenobiotic compound by developing critical micellar concentration. The complete detoxification of the pesticide occurs with those microbes that also degrade its metabolites and avoid its accumulation in the environment. The biosurfactant production, mineralization ability, immobilization of cells, nutrient supplementation, etc. increased the activity of microbial strains in chlorpyrifos degradation [21].

Pseudomonas aeruginosa showed the highest production of biosurfactant in both cell-free culture and pellets [22]. Similarly, in the present study, Pseudomonas aeruginosa produce biosurfactant. The production of biosurfactant by Pseudomonas aeruginosa was determined by using two qualitative methods such as penetration assay and blue agar plate method. The penetration assay relies on the contacting of two insoluble phases which lead to a color change. In this assay, the color changed within 15 minutes from clear red to cloudy white. The described effect relies on the phenomenon that silica gel is entering the hydrophilic phase from the hydrophobic paste much more quickly because of the presence of biosurfactants producing capacity. In penetration assay, the presence of biosurfactant was confirmed by the experiment done in Nocardiopsis sp. with remarkable bio-surfactant and biodegradation capacity [23].

In the blue agar plate method dark blue halo zone was formed after 48 hours of incubation. This is because of the production of rhamnolipid by Pseudomonas aeruginosa. The dark blue halo zone in the methylene blue agar plate supplemented with CTAB confirmed the presence of anionic biosurfactant. The assay was developed based on the property that the concentration of anionic surfactants in aqueous solutions can be determined by the formation of insoluble ion pairs with various cationic substances. The insoluble ion pair precipitates are formed in the agar plate containing methylene blue exhibited dark blue color. The diameter of the dark blue region has been shown to be semi-quantitatively proportional to the concentration of the rhamnolipid biosurfactants [24].

The biosurfactant produced by microorganism was found to be specific to organophosphates and it also plays a major role in enhancing the degradation processes of organophosphates [25, 26]. In this study, the Pseudomonas aeruginosa degrades chlorpyrifos gradually. The rate of degradation is increased with the help of biosurfactant properties.

Conclusion

The Pseudomonas aeruginosa isolated from the garden soil effectively degraded the chlorpyrifos in all the concentration when the duration of exposure is increased. From the studies, it has been concluded that chlorpyrifos can be biodegradable and Pseudomonas aeruginosa utilized carbon, nitrogen and phosphorous from chlorpyrifos pesticide for its growth. This strain can be used for bioremediation of chlorpyrifos contaminated sites.

Acknowledgement

I am thankful to The American College for providing me with the facilities and allowing me to carry out my project work. I take this opportunity to express my sincere gratitude to my guide Prof. M. Rajesh, for inspiring, guidance, affectionate encouragement, care and support throughout my project. Though words are not enough, it is my duty and heartiest wish to greatly acknowledge and thank Dr. Ted Adams for providing me educational support.