| Isolation and screening of PAHs-degradating bacteria is an important aspect at the biodegradation and bioremediation of PAHs. The high efficient PAHs-degradating bacteria is the premise of bioremediation of polluted soils. Two PAHs-degradating Mycobacterium isolated from the contaminated soil utilizing the plate method were studied on their PAHs-degrading effect in solid plate, broth and contaminated soil condations. The results indicated that two strains both have high capability of biodegradation. Two primer pairs of aromatic-ring dioxygenase genes were designed based on the sequences reported, gene sequences coding big subunit and small subnit of dioxygenase were amplificated. For further research about PAHs-degrading genes, genomic library of strain M11 was constructed, and positive clones possessing PAHs-degrading genes were achieved.Fifty-one bacteria that had the ability to degradate PAHs were isolated from 103 PAHs-contaminated soil samples. Out of 51 strains, two high efficient strains were selected, named M11 and M16. Those two strains can grow strongly on medium with acenaphthene, fluorine, phenanthrene, anthracene, fluorethene, pyrene as a sole carbon and energy sources. They were identified as Mycobacterium sp. according to the results of morphology, physiology, fatty acid analyse, the phylogenetical analyses of 16S rDNA and ITS sequences, but not the same species.Both Mycobacterium sp.M11 and Mycobacterium sp.M16 degradated PAHs high efficiently on agar plates and in liquid medium. In vivo, the degradation rate of phenanthrene (1.24×10-2mg/d )and fluoranthene (0.09×10-2mg/d) of M16 were higher than M11, but the degradation rate of pyrene (1.00×10-2mg/d)of M11 was higher than M16. In liquid medium, the degradation rate of Mycobacterium sp.M11 and Mycobacterium sp.M16 for phenanthrene reached 98% within 5d, pyrene was degradateed 80% and fluoranthene had only 50% residue at the sixteenth day. The degradation rate of M11 was 76.9%, 91.8% and 79.23% in liquid medium at the level of 50mg/L,100mg/L and 200mg/L pyrene. In liquid medium with 100mg/L pyrene, M11 could grow well and degradated pyrene in pH 5～9. The degradation ability of Ml 1 was inhibited by high concentration glucose.Addition of the nitrogen and phosphorus exhibited a stimulative effect for bacteria to degradate PAHs in contaminated soil .Whereas potassium had restraining effect .The pot experiment shown that Mycobacterium sp.M11 and Mycobacterium sp.M16 associated with crops had a better effect. Inoculation of Mycobacterium sp.M11 and Mycobacterium sp.M16 indicated a excellent results than without bacteria . In the unsterilized soil pyrene existed less than sterilized soil. The roots and shoots of crops have an accumulation of pyrene in soil, the crops in treatment with strain M11 and M16 absorbed less pyrene than no inoculation. When inoculation with bacteria,the concentratin of pyrene in the crops decreased, the crops grown in the sterilized soil have much more pyrene content than in the crops from unsterilized soil.Genomic library of strain M11 was constructed in the cosmid vector pLAFR3 using E. coliDH5αas the host strain. About 10,000 clones obtained from selective culture. Recombinants were screened from the plates containing phenanthrene and PCR amplificated gene sequences coding big subunit of dioxygenase. |
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