| Phenanthrene is one of polycyclic aromatic hydrocarbons(PAHs), which contains three benzene ring structure and widely exist in the soil, water and air contaminated with fossil raw materials such as coal, oil. The chemical properties of phenanthrene are stable and shows strongly hydrophobicity, which lead to a large amount of phenanthrene accumulate in the environment then enter to human body through food chain and finally cause cell canceration. Currently, microbiological deterioration has widely been paid attention to repair PAHs pollution in the environment. Especially the works that screening high efficient PAHs-degrading bacteria isolated have become a hotspot for research.Therefore, this subject deals with the isolation of a phenanthrene-degrading bacteria from coking wastewater sludge filter, identification on the basis of strains morphology observation, physiological and biochemical properties and 16 s rDNA sequence determination, the optimal growth conditions by response surface experiment, the properties of biodegradation of the bacteria and the routes of phenanthrene degradation by HPLC, GC-MS and FT-IR. The results obtained are as follows:(1) A phenanthrene-degrading bacteria was isolated from coking wastewater sludge filter, named P-1 which was capable of using phenanthrene as sole carbon and energy sources. Morphological observation showed that the strain is a gram positive bacteria; the colonies is pale yellow, rounded, tidy of margin, hemispherical, Smooth and moist; and bacteria is small, short rod single permutation, bacteria size is about 0.2 ~ 0.3 × 0.5 ~ 0.8 μm. 16 s rDNA identification results showed that the strain P-1 may be sphingobium sp., but can not be identified to species.(2) The study optimized the optimal growth conditions of the strain P-1 by single factor experiment, the Plackett- Burman experiment, the steepest climbing experiment and response surface experiment. The result showed when keep temperature as 36 ℃, initial pH value as 7.3, fluid volume as 33 ml, the strain grew best and growth forecast value was 0.602(OD600). Under the optimal growth conditions, the growth actual value was 0.613(OD600) which was very close to the predicted value.(3) The degradation rate of phenanthrene(1000 mg·L-1) by strain P-1 was 99.99% within 14 days under the optimal growth conditions in wastewater treatment. The addition of surfactant Tween-80 promoted significantly the growth of P-1 strain and the degradation rate of phenanthrene. The rate was 99.75% within 6 days under the above condition which was 2.3 times in contrast to not addition of Tween-80. Besides, Compared to phenanthrene alone, the mixture of anthracene and phenanthrene in medium not only promoted the growth, but also enhanced the phenanthrene degradation rate of P-1 strain about 12.65%, while the mixture of naphthalene and phenanthrene had a opposite effect which inhibited the rate about 41.7% and in the same as mixed above three compounds which about 22.82%.(4) The strain P-1 was capable of using naphthalene, salicylic acid and catechol as sole carbon and energy sources, which showed that the strain degraded phenanthrene via salicylate pathway. And salicylic acid and phthalic acid were been detected by GC-MS thereby indicated the strain metabolic phenanthrene by both salicylate pathway and phthalic acid pathway. What’s more, salicylic acid and phthalic acid were been detected and quantitated by HPLC, which not only further proved the above speculation, but also indicated salicylate pathway was a major approach. Besides, the colony of P-1 quickly become a deep yellow after sprayed catechol solution on the surface, which suggested the pathway that catechol 2,3-dioxygenase catalyzed catechol ortho fracture.(5) The strain P-1 will produce yellow pigment in the growth process. The results of GC-MS and FT-IR indicted that the xanthein xanthophyl in medium was β-carotenoids and the pigment would gradually accumulate with the increase of incubation according to the consequences of ultraviolet spectroscop. |
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