Study On Cloning Method And Diversity Of Function Genes In Wastewater Treatment Bioreactor

Heterocyclic aromatic hydrocarbons are major pollutants in industrial wasterwater treatment. Many efficient pollutant treating techniques have been reported, but we know little about the microbial major functional species and degrading-related genes. In this study, molecular microbial ecology technology and genomic technology were used to analyze the microbial communities in heterocyclic aromatic hydrocarbons (such as quinoline) containing industrial wasterwater treatment. Functional species were studied and a promoter trap plasmid system was constructed for cloning degrading-related genes.Quinoline is the typical pollutants in industrial wasterwater treatment. Due to its strong water solubility, it has become widespread pollutant. We had set up a lab scale denitrifying Quinoline-degrading bioreactor. After 12 months of acclimation, it reached high quinoline removal efficience of about 80%. The study showed that in the process of bioreactor, there are high diverse bcrA and oxoO genes at the bioreactor. The number of bacteria with oxoO gene decreased while the bacteria with bcrA gene increase gradually during the running of the bioreactor. It indirectly proved that bcrA gene participates in the anaerobic degradion of quinoline. Because the bundance of these functional genes closely related with the degradation rate in the process of bioreactor, we can use these two genes as a biomarker to monitor the quinoline degradation in wastewater treatment system.In order to intensively investigate the functional genes related to heterocyclic aromatic hydrocarbons degrading, a promoter trap plasmid system was constructed. The screening method was initially explored for cloning gene fragments with different promoter activity from the wasterwater treatment system based on the substrate-induced gene expression screening. Two types of clones were screened out from the clone library containing 8121 clones. One type of clone was screened due to the IS2 insertion fragment which has destroied the suicide gene function of plasmid. Because the IS2 from E.coli K-12 strains, the result indicate the choose of E.coli strain will be a important factor should be considered for the similar research in future. Another type of clone had the insertion and showed promoter activity.But the structure of promoter in this insertion fragment still unknown due to the limit of software for promoters searching.