Studies On Microbial Ecology Toxicity Of Phenanthrene And Isolation, Characterization Of Phenanthrene Degrader As Well As Cloning And Expression Of Degrading Genes

Polycyclic aromatic hydrocarbons are widespread pollutants and can be released to atmosphere, soils, foods and water. Exposure to PAHs would be hazardous to human health and environments, and therefore, their elimination is considered a priority in many countries. Phenanthrene is one of the simplest aromatic hydrocarbons, that contains a "bay-region" and a "K-region", which are of importance in terms of reactivity and carcinogeniciry. So it has been taken as a model compound for studies on PAHs. Work on phenanthrene biodegradation not only is beneficial to eliminate the contamination of phenanthrene from environments, but also can be valuable in understanding the biodegradation mechanism and feasibility of other PAHs, providing control measurements for bioremediation of soil and other environment contaminated by PAHs.In this thesis, soil microbial physiological and ecological characteristics in submerged paddy soil contaminated by phenanthrene were studied using traditional incubation. This thesis also deals with the isolation and identification of two phenanthrene-degrading bacteria from soil contaminated by oil, the factors influencing growth of phenanthrene-degrading bacteria and degradation of phenanthrene, the routes of phenanthrene degradation and the cloning and expression of phenanthrene degrading gene of two strains. The results will certainly supply useful reference for building up alert index systems in paddy soil polluted by phenanthrene, for environmental quality evaluation and for the using of phenanthrene-degrading bacteria strain. Here are presented the main results of this study:1. Influence of phenanthrene on change processes of culturable microbial populations, and the enzyme activities in submerged paddy soil were studied using traditional incubation method. The results showed that various concentrations of phenanthrene could all stimulate the growth of aerobic bacteria and the activity of urease; but appeared toxic, especially higher concentrations togrowth of fungi and the activity of dehydrogease during whole treatment. All concentrations of phenanthrene showed a short term toxicity on actinomycetes, anaerobic nitrogen-fixing bacteria and methanogenic bacteria during 14days of treatment and the concentration of l00mg/kg dried soil could inhibit the growth of actinomycetes during whole treatment. The effect on diversity of eubacteria in submerged paddy soil with phenanthrene was also studied using the denaturing gradient gel electrophoresis (DGGE) technique. The result showed that phenanthrene had the impact on the microbial community structures, what's more the effect would be enhanced with the increase of concentrations of phenanthrene.2. Two strains which could use phenanthrene as sole carbon source were isolated from oil-contaminated soil. Both strains were considered to belong to Sphingomonas based on morphology, physio-biochemical identification, whole cell fatty acid analysis and the phylogenetic analysis of 16S rDNA and ITS sequences. Strain ZX4 was identified as Sphingomonas paucimobilis, while strain EVA17 was presumed as a new species of the genus, due to 93%~98% homology with other all species in the genus.3. These two strains had wide pH range for growth and degradation, and could tolerate high concentration of phenanthrene, indicating that the strain should be a potential one used for bioremediation of oil-contaminated environments. Furthermore, strain ZX4 could transform indole to blue indigo, suggesting it could also be an engineering bacterium used to produce indigo. The growth on salicylic acid and catechol showed that the strain degrades phenanthrene via salicylate pathway. The salicylate hydroxylase and catechol 2, 3-dioxygenase were detected in cell-free lysates, suggesting the pathway for phenanthrene catabolism furthermore. The measurement of phenanthrene degradation by strain ZX4 indicated that the rate of phenanthrene degradation was the highest at the initial pH 7 in medium, and the degradation rate of phenanthren was larger when (NH4)2SCX4 as nitrogen sour...