Biodegradation of Malathion Using Pseudomonas stutzeri (MTCC 2643)

Pesticides are applied in agricultural fields for controlling pest population to achieve crop protection. But they cause damage to nontarget organisms and affect the quality of environment including water, air and soil. The present study has been designed to test the efficiency of Pseudomonas stutzerion the degradation of malathion. The bacterial strain was subjected to 50, 100, 150 and 200 ppm of malathion in minimal broth for 30 hours and changes in orthophosphate levels, pH and turbidity were monitored for every six hours. Efficiency of free and immobilized cells were compared for orthophosphate release. Influence of different sugars on degradation was also compared. Degradation of 150 ppm of malathion was confirmed with UV-Visible spectrophotometric analysis and HPLC analysis. The data were subjected to two way analysis of variance and the results are discussed.


Introduction
Pesticides are used to prevent the plants from pests. They are most commonly abundant environmental chemicals present in soil, water, food and so on, due to their extensive applications in agriculture. Pesticides applied in the soil affect the fertility and productivity of soil and sometimes cause pollution in aquatic ecosystems by entering into them from the agricultural fields making water unsuitable for human consumption.
They persist in the environment for a long period of time, which causes severe problems to the environment and also to human beings. Therefore, it is inevitable to discover appropriate strategies to overcome the problems caused by pesticides [1,2,3,4,5].
The use of bioremediation technology to degrade organic contaminants including hazardous pesticides has achieved a great deal of attention in recent years. Research has revealed that microbial degradation process to detoxify pesticide contaminants can be effectively used to overcome the pollution problems [6,7,8]. Bacterial strains with the capacity of degrading several pesticides have been isolated from soil. They include metamitron-degrading Rhodococcus sp. [9], chlorpyrifos-degrading Flavobacterium sp.

Pesticide Used
Malathion, an orthophosphate pesticide was selected for the present study based on its broad range of application in the agricultural fields and present market trends. to 600 nm in a spectrophotometer (Elico SL: 159) and analysed for specific absorption in the spectrum.

High Pressure Liquid Chromatographic (HPLC) Analysis
The samples from 150 ppm concentration of malathion before and after 30 hours of treatment period were subjected to HPLC analysis by UV detection.

Statistical Analysis
Two way analysis of variance (ANOVA) was performed on the factors like orthophosphate released, turbidity, and pH for the two variables namely treatment period and malathion concentration using MS-Excel

Results and Discussion
Malathion, one of the world's most wide-spread general-purpose organophosphorus insecticides with high selective toxicity is mostly used for the control of Similarly, the pH of the assay mixtures showed the change in pH from 7.2 to 6.0 -6.1 at the end of three hours, which confirmed the breakdown of malathion and the formation of acidic intermediates [26]. It is observed that, initially, at 0 hour, the inoculated cell population was low and then started increasing slowly until 18 hours and P. stutzeri entered into the phase of positive acceleration. P. stutzeri utilized malathion as a sole source of carbon and phosphorus for their growth.
Turbidity measurement (Figure 3)    Kannan and Vanitha [25] reported that the assay mixture containing mineral salts medium at the end of three hours showed two characteristic absorption regions i.e. one at 230-235nm and the other one at 240-245nm in spectrometric analysis. The degradation of malathion by P. stutzeri was clearly observed that there was an increase in absorbance till 12 hours and gradually declined in absorbance with increase in incubation period ( Figure 5). The specific absorption peak of malathion was observed at 200-300 nm in minimal medium that extended from UV to visible region but the spectrum obtained after 6 hrs of incubation showed absorption peaks with the total disappearance of peaks specific for malathion. HPLC is a most powerful method used for pesticide analysis for a long period of time [35]. In the present study, HPLC was performed to confirm the bacterial degradation of malathion at 30  Malathion by free and immobilized cells of P. stutzeri  Figure 6). The peak value in Figure 7 shows