The Growth Characteristics And Degradation Gene Screening Of Ibuprofen Degradable Bacteria

In spite of the great effect on pain relief, ibuprofen also has a bad influence on environment. In order to alleviate the environmental problems, the author do research into microbial degradation with three ibuprofen degradation bacteria selected, expecting to find the theoretical and practical basis in microbial remediatin of ibuprofen. Three degradation bacteria which used ibuprofen as a sole source of carbon in the cultural medium had been isolated from Xi’an sewage treatment plant water. Through building gene library to select ibuprofen degradation gene, the author got a positive clone that can produce brown catechol metabolites after building three bacteria’s Fosmid libraries successfully. Based on the mutant library of this kind of clone strain, researcher got seven mutant strains that wouldn’t calculate brown metabolites any longer. Sequencing analysis of disfunction strains, respectively, we could preliminary determinate the ibuprofen degradation genes of degradation bacteria. The main conclusions are as follows:(1) Identified the specific of three ibuprofen degradation bacteria by microscopic morphology observation, physiological and biochemical characteristics experiment, 16 s rDNA identification and constructing the phylogenetic tree, respectively, this study ascertains that I-2 is Klebsiella sp,I-4 is Pseudomonas sp and I-14 is Acinetobacter beijerinckii。(2) Through the growth characteristics experiment on three ibuprofen degradation bacteria to explore the optimum growth conditions, result shows that the optimum temperature of three bacteria are 30℃, the optimal p H of I-2, I-14 are 7, the optimal pH of I-4 is 6. I-2 have the highest tolerance for ibuprofen substrate concentration, which is 1000 mg/L, I-4 can resistance 900 m g/L ibuprofen, I-14 is weakest, but also to 500 mg/L.(3) We successfully constructed three Fosmid libraries using ibuprofen degradation bacteria’ genomic DNA. Through screening libraries, a strain 2H10 which could accumulate catechol metabolites in I-2 library was selected. 2H10 strain could grow using ibuprofen as a sole carbon source, which accouted for that 2H10 strain may be contain ibuprofen degradation genes.(4) Building Tn5 insertion mutant library of 2H10 strain and screening the mutant library, we got 7 strains which lost ibuprofen degradation function. By sequencing and analysising of these strains, we screened five coding genes, and preliminary deternined the degradation genes of I-2.