Characterization, Fermentation Of Atrazine Degrading Bacterium HB-5 And Elementary Purification Of Its Degrading Enzyme

In this dissertation, taking atrazine degradation bacterium HB-5 as a primary model, and its identification, optimized culture medium and fermentation conditions and enzyme purification were studied systematically. The results could be summarized as follows:1. The situation of application, pollution and mobility in environment, ecological toxicological characteristics and pollutant biological management of atrazine, which had been applied extensively all over the world and recently in China, was introduced. The knowledge of bacterium identification by 16S rRNA, flask-waves fermentation and enzyme purification were also expounded. Accordingly the issues that would be investigated was presented by the author.2. Classification of isolated HB-5 was definited by molecular classification. BLAST analysis of its 16S rDNA gene sequence suggested that the identity of with Arthrobacter sp. AD3. According to polyphasic taxonomical principle,strain HB-5 is denominated as. Arthrobacter sp.3. An atrazine bio-degradation bacterium (Arthrobacter sp.), named as HB-5,was isolated from waster water of pesticides in our laboratory. The optimum fermentation conditions and fermentation culture medium of HB-5 were selected by uniform experimental design and orthogonal experiment, and the amount of enzyme production from HB-5 were estimated by the degradation rate of atrazine. The experimental results were analyzed by the SAS system. It was found that the ideal composition of culture medium of HB-5 included saccharose 3.0 g?L-1, atrazine 0.38 g?L-1, K2HPO4 1.2 g?L-1, K2HPO4 0.5 gL-1, MgSO4?7H2O 1.2 g?L-1,NaCl 0.1g·L-1,microelement 3.8mL L-1. The optimum culture conditions of HB-5 were: the culture time was 48h, the inoculum size was 2%, the initial pH was 9.0 and the amount of culture medium was 80ml. The degradation rate of atrazine by the optimum fermentation liquid reached up to 91.64%, and increased 125% as compared with the original culture medium (40.67%).4. The degradation enzyme from HB-5 was purified. After ammonium sulfate precipitation with 30%～70% saturation, dialysis, an ion-exchange chromatography with DEAE-Sepharose FF, and gel filtration with Sephacryl S-300HR, a protease was purified partly.