Isolation And Identification Of P.chlororaphis SY-02, Lanser Irradiation And Petroleum Hydrocarbon Degrading Characteristics

The petroleum hydrocarbon contamination in the environment is a serious threat to human health and ecosystem safety. In this study, a strain SY-02 degrading petroleum hydrocarbon was isolated and identified from an oil contaminated soil sample, which might contribute to the bioremediation of oil pollution in the environment. This study included four parts:the factors influencing bacterial growth, degradation of the petroleum hydrocarbon, mutation breeding and cloning the key gene for degradation.Strain SY-02 could use petroleum hydrocarbon as sole carbon soure, which was identified as Pseudomonas chlororaphis (GenBank FJ638892) based on morphological, physiological, G+C content and 16S rRNA gene analysis. The concentration of petroleum hydrocarbon affected the growth of SY-02. The optimal concentration of petroleum hydrocarbon for growth of SY-02 was 50-800 mg/L. The optimal growth and degradation inculum, temperature, pH and the medium/flask volume ratio for SY-02 were 1%,30?,7-8 and 50/250 mL in liquid minimal medium with petroleum hydrocarbon, respectively. The plasmid elimination and pure hydrocarbon degradation test showed that the key enzyme genes lies in a degradative plasmid,pHL1(approximate 19 kb).The mutation of P.chlororaphis SY-02 obtained from He-Ne laser irradiating, with the optimal irradiation parameters:8 mW 12.5 min,10 mW 10 min and 12 mW 8.33 min. In the biodegradation tests of petroleum hydrocarbon, a positive mutant named PS 2 showed the efficient biodegradation potential and mutation stability. The comparison of full-length sequence of naphthalene dioxygenase gene in the strain SY-02 and its mutant strain PS 2 showed that four nucleotides were the mutant sites which induced the amino acid of naphthalene dioxygenase in the strain PS 2 were changed.On the basis of biodegradation experiment, various model parameters of Haldane equation were acquired by data fitting. The cell growth and substrate degradation kinetics model of the strain SY-02 and its mutant strain PS 2 were established, respectively. The comparison and test results showed that the regression model results were consistent with the experimental data.