Research Of Degradation Characteristic And Plasmid Characteristic Of Chlorobenzene-degrading Bacterium CB-2

A highly effective chlorobenzene-degrading strain is selected from the waste water. It was primarily identified as enterobactor.The strain is studied on the resistant, optimal degradation conditions, elimination of p1asmid,degeneration characteristic of eliminated-plasmid strains.(一)Degeneration characteristic of chlorobenzene-degrading strain1,Separation and appraisal of the strainA strain that had a capability to degrade chlorobenzene was isolated from the waste water sample. The strain could use chlorobenzene as the sole carbon source.It was primarily identified as enterobactor based on morphological ,physio-biochemical characteristics and 16SrDNA analysis of sequence. Resistance of this strain to antibiotic is detected. The result shows this strain has resistance to streptomycin,but others.2,The optimal degeneration conditions of chlorobenzene-degrading strainThe optimal degeneration conditions are strate concentration 120mg/L, pH 7.0,temperature 30℃, rotating rate 120rpm,and inoculation amount 5%.(二)P1asmid characteristic of chlorobenzene-degrading bacterium CB-21,Extraction of the p1asmidThe plasmid extraction from wild strains needs an appropriate method.The study used three different methods to extract plasmid : alkaline lysis method,boiling method and SDS method.The experimental results show that:for large plasmids,the SDS method is better than the other two methods.SDS is a relatively mild plasmid extraction method suitable for the extraction of large plasmids.The plasmid approximately is 23 kb. Although the operation of boiling method is fast, it seriously damaged the large plasmid, without plasmid extracted. Alkaline lysis method damaged plasmid,so the effect is also not ideal.2,Elimination of the plasmidTo determine funetion of the plasmid,the experiment is done to cure the plasmid from the cell. In this paper,we used acridine orange, SDS, sodium benzoate to eliminate the plasmid. The results showed that SDS could eliminate plasmid without any strains growth on the selective medium containing chlorobenzene.The sodium benzoate and acridine orange method could not eliminat plasmid.3,Degeneration characteristic of eliminated-plasmid strainsTolerance and degradation rate of eliminated-plasmid strains to chlorobenzene were measured.The results showed that the eliminated-plasmid strains grew difficultly on chlorobenzene inorganic salts culture medium.Strains to the tolerance of chlorobenzene concentration and degradation rate of chlorobenzene decreased significantly. With the elimination of the plasmid,the performance of degradation chlorobenzene is also lost.4,Transformation of plasmidThe experiment transformed the plasmid into E.coli.The transformants can grow on inorganic salts medium of chlorobenzene as sole carbon source, so we determine the plasmid is degradating plasmid.The plasmid was related with the metabolism process of chlorobenzene degradation.