Biologic Characteristics Of Genetically Engineered C.testosteroni And Their Preliminary Application In Bioremediation

Recently, more and more stable compounds such as steroids, PAHs, pesticide and phenol are poured into the environment, which are not only difficult to degrade but greatly destroy the ecosystem and human's health. Steriods such as estradiol and prophylactic is one kind of the 70 kinds of compounds with estrogen activity. Estradiol-like enviromental hormones are poured into environment with human's daily activity and destroy the stability of ecosystem. It was reported that enviromental hormones, ozonosphere destroy and greenhouse effect were the three great global environment problems. Natural estradiol and artificial compound estradiol mainly come from the excretion of livestock and human. Nowadays, as the development of livestock rising, more and more artificial compound estradiol is adopted, which largely aggravates the estrogen pollution. Meanwhile, prophylactic is widely used as the increasing number of people and fast-moving urbanization, which lead human being an important factor of estrogen pollution.The methods of degrading these materials usually include physical, chemical, and bio-remediation methods.But due to the low degrading rate and long degrading time of traditional methods, using gene engineering strains (GES) to degrade stable compounds have higher efficiency.It was reported that Commamonas testosteroni can degrade steroids. So in this study, two strains (Cp7, Ca3) that have higher degrading efficiency had been screened out due to their better temperature applicability, enzyme activity and degarading rate to cholesterol. Meanwhile, the abilities of Cp7 and Ca3 degrading steroids in chicken waste and pond mud were also been studied. The main results were as follows:1. In the six temperatures, the four GES's active cell counts were significantly more than Ct, showing that GES had stronger temperature applicability, especially Cp4, Ca3 and Cp7.GES can be alive for more than one month under lower- temperature environment. But in higher temperatures such as 40℃, they can only be alive for several days or less. So these GES can be applied in most areas of China, especially in the Northern areas, and the best degrading temperature was 30℃.2. Enzyme activity determination of 3a-HSD showed that GES were significantly higher than Ct, especially Cp7, which had the highest enzyme activity among the five strains, 50% higher than Ct. Meanwhile, after point mutation, Cp7 and Cp4 showed higher enzyme activities. And Cp7 was significantly higher than the stable strain Ca3.3. Using HPLC to determine cholesterol's concentration concequently determine GES's degarading rate of cholesterol. The chromatogram condition was C18 colum, Waters2996 as the detecter, 100% methanol as the mobile phase, and the colum temperature was 30℃, velocity of flow was 1ml/min.The results suggested that five strains were able to grow on cholesterol as the sole carbon source and energy source. In different concentration substrates, viability of Ca3, Cp4 and Cp7 in adversity were weaker than Ct,but their degrading ability were significantly stronger than Ct, of which Ca3 is 78.68%, 11.3 % higher than Ct,Cp7 and Cp4 take second place.4. In the experiment of chicken waste degrading by GES,the average degrading rate of Ca3 reached 27.1 %,4.3% higher than Ct; About the degrading time, when it reached 20d,the degrading rate got to the maximum 23.5%.5. Steriods existed in the mud from pond breeding big fish and sewage without management, whose contents were about 1.5mg/g, not varing greatly with the time;wild strain of Commamonas testosteroni were detected from the two muds.This result indicated that the wild type in nature can't degrade steroids effectively.After adding GES,the degrading rates of Cp7 and Ca3 were siginificantly higher than Ct,of which degrading rate of Cp7 reached 34.4%,1.97 times of Ct,and that of Ca3 reached 31.4%, 1.79 times of Ct.6. The method to extract steroids quickly and determine in a large number was primarily built.The detailed process was as follows:sampling→drying→grinding with mortar→sifting out if necessary→measure sample→adding 2mol/l vitriol 30ml, ultrasonic 2h→filtrating,collecting residue and drying→bundling the residue with filter parer,putting them into round bottom flask→adding chloroform 50ml→ultrasonic 1h→filtering and collecting filtrate when needed→taking 3ml, adding vitriol- acetic anhydride 2ml→50℃electric water bath rings 40min→taking out and determing OD₄₆₀ when it colded to room temperature.The results upwards provided academic basis for learning gene engineering strains of C. testosteroni,establishing basic knowledge for amplying GES into bio-remediation.