Efficiency of Pseudomonas putida in bioremediation of chlorpyrifos toxicity

At present, extensive varieties of pesticides are being used but the demand for Organophosphorus pesticide is increasing globally to control insects. Chlorpyrifos is a broad-spectrum, highly toxic, and chlorinated organophosphate insecticide that is synthetic in origin and is normally ester or thiol derivatives of phosphoric. The mode of action involves inhibiting acetyl-cholinesterase leading to the accumulation of acetylcholine causing neurotoxicity. Bacteria capable of degrading the pesticide, chlorpyrifos, were isolated from soil contaminatied with pesticiedes. In this way, three distinct chlorpyrifosdegrading strains of Pseudomonas putidia were isolated, and were characterized using morphological and biochemical analysis. Strain PB1 exhibited the greatest chlorpyrifos degradation rate reach to 100% and was consequently selected for further investigation. Degradation of chlorpyrifos by strain PC1 was rapid at 20 and 37C. Strain PB1 was able to effectively degrade chlorpyrifos in sterilized medium using high inoculum levels. The maximum degradation rate of chlorpyrifos was calculated as 0.08µg/ml to 0.002µg/ml during 6-12 days. Bacteria such as strain PB1, that use chlorpyrifos as a carbon source, could be employed for the bioremediation of sites contaminated with pesticides.


1.Introduction
Chlorpyrifos (O,O-diethyl-O-3,5,6-trichlor-2-pyridyl phosphorothioate; CPF) is a broad spectrum organophosphate insecticide (OP) that is commercially used to control insects especially termite [1]. It was first developed by the Germans in the 1930s and first introduced into the marketplace in 1965, has been widely used globally as an insecticide to control crop pests in agriculture, reduce household pests such as termites, reduce insect damage to turf on lawns, and for mosquito control [2,3].
A variety of studies have shown that exposure to CPF elicits acute toxicity through inhibition of acetylcholinesterase (AChE) throughout the central and peripheral nervous systems, leading to acetylcholine accumulation and long-term stimulation of cholinergic receptors [4]. Chlorpyrifos works basically the same way both in insects and other animals including humans through impairment of nervous system. during development can cause persisting neurobehavioral dysfunction, even with low doses that do not elicit acute toxicity [5].
The fate of chlorpyrifos is affected not only by its own physicochemical properties , but also by characteristics of the soil, management practices and environmental conditions [6]. Its persistence increases with decreased temperature, decreased pH, and decreased light. The half-life of chlorpyrifos showed that it remained stable even after 12 months in the soil [5] and Chlorpyrifos is moderately soluble in water, the half-life of chlorpyrifos in water ranges from 35 to 78 days [2].
It has also been reported to have short to moderate persistence in the environment as a result of several non-biological methods including volatilization, photolysis, abiotic hydrolysis, and also by microbial degradation that might occur concurrently [5].
Chlorpyrifos has been of a great concern due to persistence, toxicity and accumulation in soils and ground waters [7] and It was observed may be causes altered the structure of the gills and liver of fishes [8].
The unsafe misuse of pesticides has caused widespread harm to human health and the environment, because it is one of the most common causes of poisoning around the world [9], as well as the appearance of its residues in crops and soil, and has proven many Reports, including the Environmental Protection Organization, that pesticides cause  [10]. When pesticides are dispersed in the environment, they become pollutants, with ecological effects that require remediation [11].
It has become important to treat these pollutants, and there are many different methods of treatment, including chemical and physical treatment, which have faced many criticisms because of the main problems they cause such as the production toxic and radioactive acids and bases, in addition to these methods are uneconomic and ineffective and not suitable for large areas [12].
Lately, research activities in this area have demonstrated that microorganisms are potential tool in decaying insecticides into less harmful and non-toxic metabolites through a process known as bioremediation [13]. Bioremediation has now emerged as an innovative technology that is critically important for the clean-up of polluted sites.
In summary, due to the pervasive nature throughout Pseudomonas putida, the potential non-pathogenic effect on fish, and efficiency in CPF metabolism, Pseudomonas species were considered a vital potential agent for testing in bioremediation. The aim of this study was to determine the extent to which Pseudomonas putida acts as an effective biomarker, reducing CPF toxicity by breaking down CPF into non-toxic metabolites, and which degrade and utilize chlorpyrifos as sole carbon source [14]. Bacillus sp. And Micrococcus sp. possess potential to degrade chlorpyrifos [2].

2-1 sample collection
The soil samples used for the isolation of pesticide degrading bacteria were collected from agricultural fields, residential buildings, and Garden yards from the Iraq-Al Diwaniyah where chlorpyrifos pesticide is used extensively. This study included a total of 50 samples were collected at a depth of 5cm to 10cm from different regions.
Commercial chlorpyrifos (48%) pesticide was procured from local pesticide shop . Stock solution was prepared and stored in refrigerator for further use. The samples were collected using sterile petri dish and plain tube and 5 grams of soil samples were taken and added Normal line to it, for the purpose of purification and isolation of bacteria.

2-2 The instruments that used in determining removal of chlorpyrifos and detection of bacteria
2-High Performance Liquid Chromatography (Hplc) (Knuaer , Germany ).

2-3 Preparation of stock pesticide
Commercial grade chlorpyrifos (48%) pesticide was procured from local pesticide shop. Stock solution was prepared and stored in refrigerator for further use. To estimate the amount of residual chlorpyrifos concentrations after removal by bacterial isolates.
Standard was prepared for the active ingredient CP by selecting more than one manufacturer of the pesticide containing the active ingredient chlorpyrifos at a concentration of 48%, then 5 mg was taken from each package and analyzed by high performance chromatography according to [15]. The cultivated bacterial isolates were incubated on the nutrient medium for 24 hours at a temperature of 37 ° C and the bacteria were diagnosed by a modern device called VITEK2 SYSTEM from the company (Biomerieux, France). VITEK2 SYSTEM was examined against three bacteria as shown in (table. 1).

2-5 Treating the medium with the pesticide
Nutrient broth is prepared for the purpose of treatment with chlorpyrifos and consists of peptone 5 g / liter, sodium chloride 5 g / liter, meat extract B 1.5 g / liter, yeast extract 1.5 g / liter. Addion 800 ml of distilled water and dissolve 10.2 grams from the medium, then heat the mixture by microwave and sterilize with an autoclave at 15lbs (121° C) for 15min and then cool to 45° C.

Result and discussion
The results showed, three distinct chlorpyrifosdegrading strains of Pseudomonas putidia were highly efficient in the biological removal of organic phosphorous pesticides (chlorpyrifos) at a concentration of 120 µg/l, thus the possibility of using it in the bioremediation of pollutants. PB1 strain removal 98% of CP with at 6 days, the residual chlorpyrifos concentration was 0.08μg/ml to 0.002 µg /mL, while PB2 and PB3 degraded CP within 6 days from 0.23μg/ml to 0.067 μg/ml and 0.54μg/ml to 0.429 μg/ml (table 2).
The PB1 could rapidly degrade chlorpyrifos compare with other species during the logarithmic phase of growth. The percentage degradation in first 6 day was higher and rapidly increased thereafter to 100% within 12 day compare with control ( Fig. 2,3,4).
These results are consistent with many studies and research that have confirmed the ability of microorganisms (bacteria and fungi) to consume a wide range of pesticides and in most cases their ability to consume one or more compounds as a source of carbon and energy [2,11]. The strain could utilize CP as the sole source of carbon for growth. In HPLC analysis of sample uninoculated (without bacteria) with 200 µl CP, two peaks appeared in HPLC separation (Fig.1), which were further identified as CP by comparing with their authentic standard. HPLC analysis showed that CP could be degraded by PB1 to TCP and diethylthiophosphoric acid.    Only a few organisms capable of degrading chlorpyrifos have been reported till date. The maximum rate of chlorpyrifos removal was observed from 6 to 12 days, which coincided with the period of maximum proliferation. This might indicate that the pseudomonas putida utilized CP as a source of energy during its growth phase. It was noted that on reaching lower concentrations of residual pesticide in the medium, the degradation rate decreased significantly as previously observed by.
The reported minimum incubation time required for complete removal of chlorpyrifos was 6 h by Cupriavidus sp. DT-1 in a liquid medium [16]. Most of the common CP degrading bacteria were reported to degrade the pesticide within 48 h in liquid media [17]. Chlorpyrifos has been shown to be degraded cometabolically in liquid media by bacteria [14]. P.putida is the most common Gram negative bacterium found in soil. Isolates of this bacterium have been found to have potential to degrade chlorpyrifos[18].  [19]; these strain of bacteria with its high degradation potential, fast growth, minimal nutrient requirements and tolerance to high concentrations of pesticide can play a vital role in decontamination of polluted soil. The rapid removal of CP from liquid medium within 6 day by the pB1 strain thus could be one of the fastest reported for effective CP degradation and first of its kind amongst the Pseudomonads. .
To demonstrate the capacity of the recombinant P.putida strain to degrade CP, the degradation experiments were performed by inoculating the recombinant strain into a nutrient broth medium supplemented with CP. High-performance liquid chromatography (HPLC) analysis indicated that CP were completely degraded within 6 and 12 days respectively. Moreover, the concentration of 3,5,6-trichloro-2-pyridinol (TCP) in the medium increased gradually with the decrease in CP concentration. CP was degraded quickly in the first 6 days, which accounted for 48% of the amount of the initially added pesticides respectively. Maybe a reduction in the degradation rate after 12 days may be due to the accumulation of TCP, which have antimicrobial activity and are toxic to the bacterial growth and metabolism[20, 21].

CONCLUSION
The present study shows that use of the pseudomonas putida culture could effectively degrade chlorpyrifos pesticide. Removal of xenobiotic compounds which are a major threat to environmental pollution are of major concern as they enter the food chain and are the major causes of various diseases. The study shows that the bacterial isolate is able to use chlorpyrifos as sole source of carbon. Therefore these bacteria culture can be used effectively for Bioremediation of contaminated sites. Future studies aim in studying the biotechnology of pesticide degradation and its significance in fieldconditions.