Screening And Identification Of Three Efficient Degradation Bacteria Of Avermectin And Their Study Of Degradation Characteristics

A high performance liquid chromatography (HPLC) method was developed for detecting Avermectin (AVM) levels in the media or soil, three efficient degradation bacteria of AVM were screened, and the characteristics of degradation of three bacteria were studied in the thesis.Acetonitrile was used for extracting AVM from the media or soil, methanol:H2O (92:8, V/V) was the mobile phase which was used in the HPLC analysis. The ultraviolet detecter (250nm) was used, column temperature was30℃, and flow rate of mobile phase was1.0mL/min. The linear calibration curves were obtained over the concentrations ranging from1～80mg/L and coefficients were more than0.999. At the added doses of1,10,50mg/L of AVM, the average recovery of AVM in the media exceeded85%and the intra-batch and inter-batch precisions were less than5%; the average recovery of AVM in the soil was around90%, the intra-batch precisions were less than3%and inter-batch precisions were less than5%. The limit of detectability (LOD) and quantitation (LOQ) of AVM in the media were0.5mg/L and1mg/L, respectively.A screening model which was used for obtaining highly efficient degradation bacteria of AVM in the media was built. By using initial screening, bacteria were isolated, then depurated and domesticated, after re-screening, three bacteria were founded finally. By using Gram staining and16S rDNA identification of molecular biology, the bacteria were identified to be Bacillus subtilis, Bacillus prodigiosus and Bacillus cereus and were called G1、G6and G10in the thesis, respectively.The degradation of AVM and yield of three bacteria in media were affected by some factors, including pH, temperature, ventilatory capacity, concentration of substrate, bacterial load, added carbon and nitrogen and added metal ion. The results suggested that G1showed degradation ability doughtily of AVM under the conditions of pH6,35℃, ventilatory capacity of80mL, bacterial load of0.1%, concentration of substrate of100mg/L, content of sucrose and yeast extract of0.2%and Fe3+and Cu2+of0.1%; G6showed degradation ability doughtily of AVM under the conditions of pH6, 40℃, ventilatory capacity of40mL, bacterial load of0.05%, concentration of substrate of150mg/L, content of sucrose and beef extract of0.2%and Fe3+and Cu2+of0.1%; G10showed degradation ability doughtily of AVM under the conditions of pH6,40℃, ventilatory capacity of120mL, bacterial load of0.1%, concentration of substrate of150mg/L, content of starch and yeast extract of0.2%and Fe3+and Cu2+of0.1%.The simulation test of the three bacteria in the soil was done in the lab. The soil in this test was sterilized and non-sterilized, with continuous cultivation for60days under concentration of10mg/kg,25mg/kg and50mg/kg,6500Lux,12h light and12h dark at25℃. The results showed that the optimum degradation time of AVM was between40d and60d in the non-sterilized soil, and there was seldom degradation of AVM in the sterilized soil.