Isolation And Characterization Of A Phenanthrene-Degrading Bacterial Strain

The purpose of this dissertation is to investigate the characteristics of a newly isolated Burholderia sp. AFF with the ability to degrade phenanthrene. The physiobiochemical characterization and 16S rRNA molecular identification were carried out. The characteristics of strain AFF growing and degrading phenanthrene were also investigated and the possible metabolic pathway of phenanthrene degradation by strain AFF was presumed. Meanwhile, the characteristic of the crude enzyme from strain AFF was studied, and catechol 2, 3 -dioxygenase in the process of degradation was purified.Strain AFF is a high-effective strain phenanthrene-degrading strain with high efficincy. It was identified as Burholderia belonging to Proteobacteriaαsubclass based on morphological and 16S rRNA sequence analysis. The 16S rRNA sequence of strain AFF shared high similarity (98%) with that of type strain AJ544691. The 16S rRNA sequence of strain AFF was submitted for GenBank with the number EF506612.The characteristics of strain AFF growth and phenanthrene degradation were investigated. Strain AFF is resistant to streptomycin and tetracycline and sensitive to other antibiotics. The optimal conditions for both growth and degradation process is as follows: temperature 30℃, pH7.0, shaking speed 150r/min and inoculation 10%. Phenanthrene (100mg/L) was removed 98.4% within 84 hours by this strain. It proved that strain AFF was a high efficient degradation strain to phenanthrene. Phenanthrene concentration is determined using HPLC. Biomass is determined using Comass method.The degradation mechanism of strain AFF was determined. Literatures have reported that phenanthrene degradation by bacteria including two pathways, salicylic acid pathway and o-phthalic acid pathway. It suggests that strain AFF possesses the salicylic acid pathway by UV-VIS, enzyme determination, HPLC and HPLC-MS methods.Strain AFF was capable of degrading catechol via the meta-cleavage pathway. When catechol was used as substrate, the kinetic constants K_m and V_max were 10.93μM and 106.38μM/min, receptively. It indicates that the affinity of the crude enzyme to catechol is great and catechol is a better substrate. Crude enzyme from the strain was affected largely by pH and temperature. Crude enzyme were affected little by main group monovalue cations such as K⁺ and Na⁺ and main group divalue cations such as Ba²⁺, Ca²⁺, Mg²⁺ and Al³⁺; crude enzyme were affected to some extent by transition metal ions such as Mn²⁺, Ni²⁺, Cu²⁺, Co²⁺, Zn²⁺, Fe²⁺, Pb²⁺ and Fe³⁺. Finally, the effects of oxidant, reductant, KI, SDS and acrylamide to crude enzyme were determined. Catechol 2, 3-dioxygenase was purified to homogeneity from the cell extract of strain AFF by Anion-exchange Column and detected by SDS-PAGE. And the yield was about 27.6%.