Study On Heterocyclic Aromatic Hydrocarbon Degrading Bacteria And Method Of Clonging Degrading Genes

Heterocyclic aromatic hydrocarbons are major pollutants in industrial wasterwater treatment. Many efficient pollutant treating techniques have been developed, but we know little about the microbial community composition,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 and indole) containing industrial wasterwater treatment. Functional species were studied and a promoter trap plasmid system were constructed for cloning degrading-related genes.Two lab scale bioreactors treating indole-containing wasterwater were acclimated using the same seeding sludge. They ran in denitrifying and anaerobic conditions, respectively. After 6 weeks acclimation, both of them reached the stable stage. The average indole removal efficiency was 80.4± 1.7% in denitrifying reactor and 52.0±4.5% in anaerobic reactor. The community structures of both reactors were investigated by clone library and DGGE methods after they reached steady-state.The results showed a distinct discrepancy among seeding sludge and two bioreactors. Beta-proteobacteria was the most dominant group of bacteria in denitrifying bioreactor. Among them, bacteria from genera Alicycliphilus, Acaligenes and Thauera might be the main degraders of indole, whereas the Clostridia and Actinobacteria became the dominant bacteria and probably also the main degrading members in anaerobic bioreactor. Although the diversity index of bacteria at OTU level kept in stable in two acclimation processes, the microbial composition both at class and genus level of those samples was distinctly different. The only difference between two reactor's acclimation, nitrate added or not, had lead to the formation of two different communities.The comparison of quinoline and indole acclimated denitrifying bioreactor showed that, despite the same seeding sludge and identical acclimating condition was used, molecular ecological analysis showed distinctly different communities of two reactors. All OTUs of 16S rDNA clone library of quinoline acclimated bioreactor were affiliated to Beta-proteobacteria, whereas the percentage of this subclass in library of indole acclimated bioreactor was only 56.3%. Results showed that microbial diversity in indole acclimated community was higher. 73% clones in quinoline acclimated community was Thauera related OTUs from the Rhodocyclaceae family. But OTUs from the families of Comamonadaceae, Alcaligenaceae and Rhodocyclaceae were dominant OTUs in indole acclimated community. Its most dominant OTU from Comamonadaceae was 28.7% in clone library. These results showed that the type of pollutants in the wastewater had a strong effect on the selection of population in microbial community. Our study was the first report comparing the microbial structure of two effective denitrifying communities which could efficiently degrade quinoline and indole.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 functional genes from the degraders and biofilms of wasterwater treating bioreactor on the basis of substrate-induced gene expression screening.