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A Preliminary Study on a PAHs Degrading Marine Bacterium with Floating Character

Author: XuYan
Tutor: HuZhong
School: Shantou University
Course: Biochemistry and Molecular Biology
Keywords: Rhodococcus sp Polycyclic aromatic hydrocarbon (PAHs) Biodegradation floating
CLC: X172
Type: Master's thesis
Year: 2007
Downloads: 4
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Abstract


As the development of economy and petrochemical industry, industrial wastewater andindustrial waste gas directly discharged into the sea, which caused severe PAHs pollution. Thecharacters of PAHs, such as stability of structure, lipophilic property and carcinogenicityendangered human health, damaged the ecosystem and deteriorated the water quality. Thegovernments and many researchers paid more attention on PAHs pollution. Microbialbioremediation was considered to be the most efficient way to remove environmental pollutionsbecause of its economical, efficiency and mineralization. Based on the isolation of marine PAHsdegrading bacteria with higher efficiency, a preliminary study on its mechanism of PAHsdegradation was carried out, which could be helpful to develop a new method to environmentremediation.PAHs-degrading strains (named as P1, P2 and P14) isolated from Xiamen harbor had beenidentified as Corynebacterium sp., Ochrobactrum sp., and Rhodococcus sp. by ERIC and16SrDNA sequence analyses. The analysis of the degrading rate in 30d with 50mg/L Pyreneindicated that strain P14 had the best degrading ability in the strains tested. Combined with theSEM and BiOLOG Microbial Identification System analysis, we denominated strain P14 toRhodococcus sp. P14. Strain P14 can degrade 43％phenanthrene, 34％pyrene and 30％Benzo(a)pyrene in 30d.Strain P14 had been found to possess the floating character when culturing in oil. Thus it hadthe ability to collect oil into balls or woolly masses. Meanwhile, it tended to locate in theinterface between the water and oil to form air bags.Total protein of Rhodococcus sp. P14 was extracted and analyzed by SDS-PAGE duringPAHs biodegradation. Some special bands related with PAHs degradation were observed. Whenstrain P14 was cultivated in MSM containing pyrene and benzo[a]pyrene as the sole carbon andenergy sources, C120 (1,2-Catechol Dioxygenase) was detected with the specific enzymeactivity of 1.16179Units/mg and 2.633Units/mg, respectively. However, when culturing inphenanthrene or 2216E, there was no (2120 enzyme activity.The genomic library of Rhodococcus sp.P14 was constructed by using pSK plasmidtransformed to E. coli DH5α. A positive clone pBluescriptSK-P14-817 was screened out by Blue/White Cloning, PAH screening pressure, and the indigo synthetic experiment whichindicated the clone took gene encoding the dioxygenase for degrading PAH.A positive clone pBV220-Pyr1-38 was screened from a pyrene degrading bacterium genomiclibrary in previous study. The method of DOUGLAS G was used to extract the plasmid.Restriction enzyme digestion analysis of the recombinant plasmid showed there were twoenzyme digest sites in the insert segment. Three parts of Pyr1/pBV220-38 plasmid insertsequence were obtained by RAPD. One of them was related to the gene encoding PAHsdegrading enzyme. Then SON PCR was applied to get the flank sequence.

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