Study On Identification And Characterization Of A Pseudomonas Aeruginosa Strain DN1 For Fluoranthene Biodegradation

Fluoranthene is a memberof representative high molecular weight polycyclic aromatic hydrocarbons (HMW PAHs) that contains a four ring condensed with naphthalene and benzene ring, which is widespread in environment and belongs to the majority of HMW PAHs in petroleum-contaminated soil. Its molecular stability, hydrophobicity and low bioavailability appear to be some of the main factors that contribute to their persistence in the environment and exert potentially carcinogenic effect on human. Demand for seeking an efficient way to eliminate it from environment is increasing at present. Compared to traditional physical or chemical methods, microbial activities represent one of the promising processes because of their high efficiency, low cost and environment-friendly. Research in regard to biodegradation of fluoranthene has continued to advance worldwide.In this study, a bacterial strain named DN1, which can use fluoranthene as the sole carbon and energy source, was isolated by enrichment culture from a petroleum-contaminated soil sample. This strain was identified as Pseudomonas aeruginosa through morphological observation, physiological and biochemical determination and 16S rRNA gene homological sequencing. The optimum growth temperature and the optimum pH of the strain was 34℃-37℃ and 5.5-7.5, respectively. Strain DN1 has capability of producing rhamnolipids, the best rhamnolipid yield could reach to 22.9 g/L when the strain was cultured under optimum conditions for 7 days in BPLM, which is able to form highly stable emulsion of fluoranthene in aqueous solution and promote its biodegradation. The fluoranthene degradative rate of strain DN1 was up to 90.2% when it was cultivated with 0.5g/L fluoranthene as the sole carbon and energy source for 14 days, and gas chromatographic detection showed fluoranthene was almost entirely eliminated. Genes of catechol 1,2-dioxygenase(C12O) and catechol 2,3-diocygenase(C23O) were verified by PCR, and the change of enzyme activitiy was detected when it was induced with 0.2g/L fluoranthene. The activity of both enzymes enhanced when induced after 24h and droped after 48h. The activity of C12O was significantly higher than that of C23O, which indicated that C12O played a major role in fluoranthene degradation. A library contains more than 12,000 transposon-mutants was established, and in which 11 fluoranthene-degradation negative mutants were screend. Sequences nearby insertion sites of transposon in mutants were obtained by random PCR, products were sequenced thereupon genes relate to fluoranthene biodegredation were identified. Genes including alginate synthesis protein, FeⅢ-citric transport protein Fec A, transport regulator protein, tryptophane synthesis protein and AraC(cytosine arabinoside)family transcriptional regulator were obtained while the function for these genes needs to be further investigated.In conclusion, DN1 is identified as a Pseudomonas aeruginosa strain which has high efficiency in degrading fluoranthene and can be cultured in broad conditions. Its high efficiency in degrading fluoranthene may be consistant with the excellent capability of producing rhamnolipids. Enzyme activity of both C12O and C23O enhanced and C12O played a major role in fluoranthene degradation. Some genes that relate to degradation of fluoranthene have been obtained by transposon-mutantion, yet efforts to exucide the molecular background for those genes have not been initiated, which may expand our understanding of the potential of Pseudomonas aeruginosa DN1 for bioremediation application.