Isolation and Characterization of Chlorpyrifos Degrading Bacteria from Contaminated Agricultural Lands

Previous literature has confirmed that pesticides are posing serious threats to the lives of human beings and animals alike because of their use in agricultural crops. Pesticides have also increased pollution in aquatic and terrestrial environment. In this study, chlorpyriphos degrading bacterial strains were isolated from pesticide contaminated agricultural soils by the selective enrichment method. Bacterial isolates were biochemically characterized for the strain identification. The effect of different environmental factors on optimum growth was also checked. These factors included pH, temperature, and concentration of pesticide. Isolated strains were also ribotyped for identification. Metal resistance profiling was performed for different heavy metals, that is, ZnSO4.7H2O (Zinc sulphate), CdCl2.H2O (Cadmium chloride), CuSO4 (Copper sulphate), and K2CrO4 (Potassium chromate). It was found that strains NW3O and NW3T were sensitive to cadmium except NR2 and NR4, which were resistant at the concentration of 100 and 200 µg ml-1. NR4, NW4 and NW3G were resistant to chromium upto the concentration of 100 µgml-1, while NR2, NW3O and NW3T tolerated 200 µgml-1 of the metal. All the strains were resistant to zinc at different concentrations. NR2 showed maximum resistance to copper by showing growth on all concentrations of the metal. Thin layer chromatography was performed for the detection of different intermediates formed during the degradation of chlorpyrifos. Minimum inhibitory concentration of pesticide was estimated in M9 medium containing chlorpyriphos. The current study resulted in the isolation of efficient chlorpyrifos degrading strains with a wide range of pH and temperature tolerance that can utilize chlorpyrifos upto 700mg/L during lab scale degradation tests (growth on chlorpyrifos supplemented minimal agar and broth).

chlorpyriphos. The current study resulted in the isolation of efficient chlorpyrifos degrading strains with a wide range of pH and temperature tolerance that can utilize chlorpyrifos upto 700mg/L during lab scale degradation tests (growth on chlorpyrifos supplemented minimal agar and broth).

Introduction
Since before 2500 BC, pesticides are used by humans to protect the crops. Synthetic pesticides are produced in large quantity since 1940's. Pesticides target species, including non-target species, bottom sediments, water, food and air. However just 95% herbicides and 98% sprayed insecticides reach a destination (1). The usage of pesticides for protection of the crops, firstly, recorded about 4500 years ago. Paul Muler discovered DDT as a very effective insecticide in 1993. Pesticides in large amount are used in agricultural activities all over the world that are toxic for humans and also for animals. Aquatic and terrestrial ecosystems are contaminated due to inappropriate use of pesticides. Many pesticides are used instantly on different agricultural crops, that increased pollution (2).
In 1989, pesticides consumption was fivefold increase in one year, because public sector transferred the sale of pesticides to private sector. About 80% pesticides are used on cotton plants, and others are applied on fruits, vegetables and paddy tobacco (3). It has been assessed that throughout the world about 2.5 million tons pesticides are used every year and it is also increasing day by day (4). Pakistan shows the same trend. In Pakistan and other Asian countries, nitrates and pesticides causes the contamination in the shallow groundwater (5-7).
Conventional methods using for degradation of Chlorpyrifos results in accumulation of unmanageable residual and many toxic products. So, biodegradation for its removal from environment by using native microorganisms is quite attractive. The most common Chlorpyrifos biodegradation products such as 3, 5, 6trichloro-2-pyridinol (TCP) has antimicrobial activity (8), and by the US environmental protection agency (EPA) it has been persistent, mobile and toxic with a half-life about 65 to 360 days in soil (9). It causes large scale contamination of aquatic and soil environments, because, due to its greater water solubility it is more portable than the parent molecule. So, study of TCP biodegradation is also important (10). Sphingomonas sp. Dsp-2, Enterobacter strain B-14 and Stenotrophomonas sp. YC-1 have been described as Chlorpyrifos degrading bacteria (11)(12)(13). Chlorpyrifos degrading fungi such as Vertocollium, Phanerochaete chrysosporium, and Aspergillus terreus sp. have also been reported (14)(15)(16). Unfortunately, TCP accrued in the medium without further metabolism and degradation of bacteria was only partial. Only Pseudomonas sp. (TCP mineralizing bacterium) has been describe earlier (17), less data is available on the microbial metabolism of TCP (18). Aim of the presented study is to isolate Chlorpyriphos degrading bacterial strains Bioscientific Reviews Volume 1(2) from pesticide contaminated agricultural soils as well as to biochemically characterize the isolated bacterial strains. The effect of different environmental factors on optimum growth have also been checked these factors include pH, temperature, concentration of pesticide etc. Isolated strains were also ribotyped for identification.

2.Methodology
Agricultural soil samples with pesticide usage history were collected from different cities of Punjab as shown in table 1. By using sterile scalpel the sampling of soil attained from the depth of 5cm ( Figure  1) and moved into polythene bag to store at 4ºC until used.

Characterization and isolation of Chlorpyriphos degrading Bacteria
The bacterial strains were isolated from different agricultural land fields that had earlier history of Chlorpyriphos contamination (Table 1)   NR2, NR4, NW4, NW3T, NW3G and NW3o were selected which had the capability to use the pesticide as the sole source of energy and carbon. These strains were finally purified and maintained under stress condition.

MIC of the pesticide
The isolated strains were examined for degradation of pesticide at dissimilar concentrations such as; 100, 200, 300 and 500 µg/L. All the strains showed growth in the presence of 100 and 200 µg/L of the pesticide. Only NR2 showed the growth on 300 and 500 µg L -1 of the pesticide and NR4 also showed the growth on 300 µg/L 1 of the pesticide (Table 2 & Figure 2).

Colony morphology
Colony morphology of the bacteria strains were examined on N-agar. The strain NR2 showed off-white colonies and is thus characterized as Bacillus spp. Greenish, flat and opaque colonies were observed in the case of strains NR4 and NW3G, which shows that it belongs to Pseudomonas spp. While all other showed off-white colonies, as shown in the table 3 and figure 3. NR2 + + + +

Triple Sugar Iron test
To examine the fermentation pattern of the isolated pesticide degrading bacterial strain the triple sugar iron test was done. The slants color changed so, results of triple sugar iron tests are understood. The results of triple sugar iron test are took by the change in color of the slants. Strain NR2, NW4, NW3O, NW3G and NW3T did not ferment glucose except NR4 which gave yellow butt indicated that it had the ability to ferment glucose. All the strains showed red slants which indicated that none of them can ferment lactose and sucrose (Table 5 & Figure 5).    (Table 6 and figure 6).

Antibiotic Resistance Profiling
NR2 was sensitive to Ampicillin and Tetracycline. However, it was weak positive to Erythromycin, Gentamycin and Chloremphenicol at 50 µg ml -1 . NW4 was sensitive towards Erythromycin and 100 µg ml -1 concentration of Gentamycin, Chloremphenicol and Tetracycline. However, it showed strong positive and weak positive growth on 50 and 100 µg ml -1 of ampicillin. NW3T and NR4 were sensitive to all. NW3O was strongly positive against 50 µg ml -1 concentration of Ampicillin, while it was weak positive against 50 µg ml -1 of Erythromycin, Chloremphenicol and Tetracycline. NW3G was sensitive to all; however, it showed resistance towards results at 50 µg ml -1 of Chloremphenicol (Table 7).

Thin layer chromatography
Degradation experiment of Chlorpyrifos was preceded for 7 days. After 7 days extraction, running and development of TLC for the detection of different intermediates formed during degradation of Chlorpyrifos was done ( Figure 6).

Discussion
It has been predicted that between 1995 and 2020 the food grain demand is likely to be doubled; for vegetables more than 2.5 times and for fruits 5 times. Consequently, the increase in the consumption of pesticides is likely to be at least 2 to 3 times more in the years to come. Extensive use of pesticides is inevitable since they provide a sure cover to the farmer to protect his investment in seeds, fertilizers, irrigation and his own hard labor from the insects and pests. Environmental pollution due to pesticide residues, therefore, will continue and strategies like biodegradation and bioremediation will have to be followed (19 Breaking down of toxic pesticides into nontoxic compounds and in som case, breakdown into the original elements from which they derived is described as pesticide degradation. Three types of pesticide degradation are microbial, chemical, and photo degradation. Degradation in soil is commonly carried out by microorganisms, mainly bacteria and fungi. Although, a lot of work has been reported on biodegradation, particularly of parathion and methyl parathion, meager data is available on the application of microbial cultures in bioremediation of soil contaminated with organophosphorous. In this work, bacterial strains were isolated from different pesticides contaminated agricultural soils which have the ability to degrade pesticides. A well-known technique 'selective enrichment' was used to isolate the pesticide degrading bacteria (20). Six strains NR4, NR2, NW4, NW3O, NW3G and NW3T were isolated from the pesticide contaminated sites and were found to be highly efficient in degrading the pesticide used in this study, that is, chlorpyriphos. These strains had the ability to utilize chlorpyriphos as their sole source of carbon and energy. These isolated pesticide degrading bacteria were maintained on minimal agar containing pesticide, so that their ability of pesticide degradation was not lost. A bacterial strain Bacillus sp., capable of degrading chlorpyrifos at concentration as high as 1g/l, was also reported in several studies. chlorpyrifos was also reported for the  (29). Spots with Rf value approximately 0.66 indicate the presence of TCP, which is the most common degraded product of chlorpyriphos. As TCP is more polar than CP, so its Rf value is higher as compared to CP. On the whole, this study resulted in the isolation of efficient chlorpyrifos degrading strains with a wide range of pH and temperature tolerance that can utilize chlorpyrifos upto 700mg/L.

Conclusion
The curent study resulted in isolation of efficient chlorpyrifos degrading strains with a wide range of pH and temperature tolerance that can utilize chlorpyrifos upto 700mg/L during lab scale degradation tests (growth on chlorpyrifos supplemented minimal agar and broth).