Isolation And Degrading Characters Of Chlorpyrifos Degrading Bacteria

In this dissertation, taking chlorpyrifos as a model pollutant, a strain of bacterium XZ-3 capable of highly degrading chlorpyrifos was screened from the soil sample collected from a pesticide plant after taming and enrichment. Based on analysis of phenotype, physiological and biochemical characters and 16S rDNA, XZ-3 was identified as Arthrobacter sp.. Degrading-bacteria enrichment and isolation, characterization of its biodegradation and degradative enzyme were studied systematically. The effect of earthworms on chlorpyrifos degradation and chlorpyrifos affecting the earthworms'physiology index had been studying on. The results could be summarized as follows:1. The author introduced the use situation, pollution, mobility in environment, degradation and metabolism, ecological toxicological characteristics and pollutant management of chlorpyrifos, which has been applied extensively in the world and recently in China; summarized the research of earthworm affect the degradation of chlorpyrifos in the soil, accordingly author presents the issues that would be investigated.2. The chlorpyrifos degrading-bacteria were isolated by direct way and selective enrichment from soil exposed to pesticides and soil of pesticides production, and then researched on the bacteria in our laboratory conservation. Three high efficiently degrading strains XZ-3, XZ-4 and QZ-11 were isolated by selective enrichment, and their degrading rates under the condition of 30℃,pH 7.0 in culture midia containing 50mg·L-1 of chlorpyrifos in 48h were all above 70%. In contrast, the degrading rates of strains XZ-3 was the highest, based on analysis of phenotype, physiological and biochemical characters and 16S rDNA, XZ-3 was identified as Arthrobacter sp., we choose the strain for further study.3. The effects of carbon source, pH, temperature and chlorpyrifos concentration on degradation was determined. The results showed that the degradation rate of chlorpyrifos by XZ-3 was 86.8% in 24h. The biodegradation rates were the highest when the additional carbon source was 0.3%, pH value was 9.0, chlorpyrifos concentration was 100mg·L^-1, and cultivated temperature was 30℃. The growth of bacteria increased with carbon source concentration increased, higher with pH from 8.0 to 10.0, and the highest when temperature was 30℃, chlorpyrifos concentration was 800mg·L^-1. The strain could survive when chlorpyrifos concentration was 1000mg·L^-1 and the removed amount of chlorpyrifos increased with chlorpyrifos concentration.4. Degradation characteristics of chlorpyrifos insecticides was determined by the crude enzyme extracted from the isolated strain XZ-3 (Arthrobacter sp. ). The objective of this study was to initially ascertain the best separating condition and the degrading characteristic of chlorpyrifos. The content of this study included the form and orientation of the degrading enzyme for chlorpyrifos; rate of degradation for chlorpyrifos by its intracellular enzyme, extracellular enzyme and cell fragment was 50.5%, 24.2% and 34.9% respectively. The degrading enzyme was extracted after this strain was incubated for several generations in the condition of non-inducement, and its enzymic activity lossed less, the results showed that this enzyme was an intracellular and connatural enzyme. The solubility protein of the crude enzyme was determined with Albumin (bovine scrum) as standard protein and the solubility protein of the crude enzyme was 1.894mg·ml^-1. The pH optimum for crude enzyme was 9.0 for enzymatic degradation of chlorpyrifos and the optimum temperature for enzymatic activity was at 30℃, it still had comparatively high activity in the rage of temperature 20-40℃. The crude enzyme showed Km value for chlorpyrifos of 0.8633mmol·L-1, and the maximal enzymatic degradation rate was 0.2411μmol·(mg·min)-1. Additional experimental evidence suggested that the enzyme had the stability of endure for temperature and pH.5. Two treatments had been set up, one add some concentration chlorpyrifos but not add earthworms (Eisenia foetida), the other add some concentration chlorpyrifos and earthworms (Eisenia foetida). The research was on the effect of earthworms to chlorpyrifos degradation, at the same time chlorpyrifos affecting the earthworms'physiology index had been studying on. On one hand by measuring the earthworms'total weight before and after addding chlorpyrifos, we can conclude that in total the earthworms'total weight decreasing as the time increasing except on the 10d. On the other hand, the degradation of chlorpyrifos in soil was increased as a result of earthworms, so by adding some earthworms in soil can work on the degradation of chlorpyrifos.