Isolation, Identification, Degradation Characteristics Of And Cloning, Expression Of Catechol 2, 3-Dioxygenase Gene Of Phenanthrene-Degrading Bacteria

Phenanthrene is one of three-aromatic PAHs and distributes in environments widely, which causes diseases of human respiration system and skin, and induces mutation, aberrance and carcinogen of fish and other animals. Phenanthrene has the special structure K-region and Bay-region which can arise carcinogens. Therefore, 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 phenanthren from environments, but also can be valuable to understand the biodegradation mechanism and feasibility of other PAHs, providing theorically and practically knowledge and control measurements for bioremediation of soil and other environment contaminated by PAHs.This thesis deals with the isolation and identification of three phenanthrene-degrading bacteria from soil sample contaminated by oil, the factors influencing growth of phenanthrene-degrading bacteria and degradation of phenanthrene, the routes of phenanthrene degradation, the cloning and expression of catechol 2,3-dioxynase gene of ZX4 strain. The results obtained are as follows:1. Three phenanthrene-degrading bacteria were isolated from soil samples contaminated by oil, which are capable of using phenanthrene as sole carbon and energy sources, and were identified to be belonged to Sphingomonas, strain ZX4 as S. paucimobilis, strain ZX6 as S. aromaticivorans and strain EVA 17 not being identified to species, respectively, based on the results of physio-biochemical tests and the phylogenetical analyses of 16S rDNA sequence. ZX4 strain was the best one on growth and phenanthren degradation among the three strains identified, with degradation rate of 98.74% within 14 d at concentration of l000mg phenanthrene ?L-1 in medium by strain ZX4.2. The optimal temperature was 35 for growth of ZX4 strain. There existed differencesbetween the effect of initial pH6 and that of initial pH7 in media on growth and phenanthrene degradation of ZX4 strain. Compared with initial pH7 of medium, the growth was better in medium at initial pH6, and phenanthrene degradation, however, stagnated almost after 2d of incubation, madding against the complete degradation of phenanthrene. As increasing phenanthrene content, substrate saturation appeared in medium at initial pH6, but not in medium at initial pH7 for growth of ZX4 strain. The addition of surfactant Tween-80 inhibited the growth of ZX4 strain in medium with initial pH6, but promoted significantly in medium with initial pH7. The effects of NH4NO3, (NH4)2SO4,NaNO3 (NH4)2HPO4 were weaker on growth of ZX4 strain, but the degradation rate of phenanthren was larger when utilized (NH4)2SO₄ as nitrogen source than utilized other nitrogen sources. Addition of yeast extraction, or peptone or glucose could promote the growth and phenanthren degradation of ZX4 strain at different degree. Compared to phenanthren alone, the mixture of naphthalene and phenanthren in medium not only promoted the growth, but also enhanced the phenanthren degradation rate of ZX4 strain. Comparing to the pure culture of single ZX4 strain, the mixture of ZX4 strain and EVA 17 strain had very little effective on total biomass, but could enhance the phenanthren degradation rate remarkably. The results showed that co-metabolism and multi-strain mixture is probably more effective and feasible for bioremediation of PAHs contaminated environments.3. ZX4 strain could utilize catechol and salicylate, but not o-phthalate as sole carbon and energy source. Research data have showed that phenanthren is degraded by bacteria through one of two different routes via a common inter-metabolite l-hydroxy-2-naphthoate. In one route, l-hydroxy-2-naphthoate is further degraded via salicylate, while in the other route, the ring of l-hydroxy-2-naphthoate is cleaved and further metabolized via o-phthalate. Catechol is an intermedia of salivylate route and o-phthalate is an intermedia of o-phthalate route. So the results of degradable substrates illuminated that ZX4 strain probably deg...