Microbioal Toxicity Of Phenanthrene And Isolation, Characterization Of Phenanthrene Degraders As Well As Cloning And Expression Of Degradation Genes

In this study, two phenanthrene-degrading bacterial and another bacterial with can improve the phenanthrene degradation speed when inoculated with phenanthrene degrading strains were isolated using traditional incubation method. Their identification was taken by studying its biochemical and genetic character. The factors influencing growth of phenanthrene-degrading bacteria and degradation of phenanthrene, the phenanthrene degradation pathway, the cloning and expression of phenanthrene degrading gene of two strains, the membrane toxicity mechanism of phenanthrene were also tested in this paper. The results was expected to supply useful reference for building up alert index systems in soil polluted by phenanthrene, for environmental quality evaluation and for bioremediation of PAHs pollution.Here are presented the main results of this study:1. Two phenanthrene-degrading bacteria strains ZP1 and ZP2 and another strain ZP5 which can improve ZP1's phenanthrene degradation speed were isolated from soil in oil refinery field in Shanghai. They were identified as belong to genus Sphingomonas, Pseudomonas and Tistrella, respectively based on Gram staining, morphology, oxydase reaction, biochemical tests, FAME analysis, G+C content and 16S rDNA gene sequence analysis. Strain ZP1 was identified as Sphingomonas paucimobilis, while strain ZP2 was identified as Pseudomonas stutzeri which is the first representative of Pseudomonas stutzeri sp., able to degrade phenanthrene very fast at high experimental concentration.2. Both ZP1 and ZP2 has wide temperature, pH range for growth and degradation, and could tolerate high concentration of phenanthrene. The optimal growth conditions of strain ZP1 and ZP2 were determined to be at pH 7.0, 30℃and pH 8.0, 37℃, respectively. Addition of yeast extraction, peptone or glucose could promote the growth and phenanthrene degradation ability of both ZP1 and ZP2 to diferent degree. The phenanthrene degradation speed was nearly the same when (NH₄)₂SO₄, NH₄Cl, NH₄NO₃ were tested as different nitrogen resorce. Strain ZP1 can remove more than 90% of phenanthrene at any concentrations ranged from 250 to 1000ppm in 8 days while ZP2 can nearly consume them all in 6 days. Both Brij 30 and Trition 100 can inhibit the phenanthrene degradation speed of ZP2 at high concentration, but has no obvious effect on ZP1. Tween 80 can promote the degradation of phenanthrene by both ZP1 and ZP2.3. Both ZP1 and ZP2 show high salicylate hydroxylase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activity especially catechol 2,3-dioxygenase. Two key products 1-Hydroxy-2-naphthoic acid and 2-Hydroxy-1-naphthoic acid were found in the phenanthrene degradation process, which could suggest the isolates degrade phenanthrene via phthalic acid pathway.4. The gene pha-ZP1 encoding a subunit of aromatic hydrocarbon dioxygenase was cloned from strain ZP1, it's accession number in Genbank is EU082776. The determination and sequence analysis of the gene indicated that the DNA fragment was 1287 bp in length, encoding 383 amino acids. Two conserved regions: the [2Fe-2S] Rieske center and the mononuclear iron binding domain were found at the expected positions by deducing the protein using Cn3D4.1 software and Swiss Model Workspace web instrument. The amino acid sequence of the protein showed the highest similarity with that of Sphingomonas yanoikuyae B1 and Sphingomonas sp. P2.5. The gene phn-ZP1 and phn-ZP2 encoding catechol 2,3-dioxygense was cloned from strain ZP1 and ZP2 respectively, their accession number in Genbank were EU082777 and EU082778. The determination and sequence analysis of the gene indicated that phn-ZP1 fragment was 797 bp in length, encoding 233 amino acids, phn-ZP2 fragment was 1047 bp in length, encoding 331 amino acids. The structure of these two enzymes deduced by using Cn3D4.1 software and Swiss Model Workspace web instrument suggested they were mostly composed by a helix and (3 strand.6. The gene phn-ZP2 of C230 of strain ZP2 was recombined and expressed in Rosetta successfully. The activity of recombined enzyme protein expressed by recombinant obtained was much more than that by the original ZP2 strain in primary detection.7. Lecithin liposome as the simulation membrane was used to investigate the interactions with PAHs such as phenanthrene. Results reveal that apolar compounds obeyed the Nerst partition law. The partition coefficient of phenanthrene was calculated and its' binding bonds in liposomes were clarified. Effects of electrolyte, temperature, acidity of solution were analyzed as well as effect of the molecular structure was discussed in detail. Besides, the inverse partition of apolar compounds from liposome to an analogue cytosol was first proposed. Transmembrane distribution difference of phenanthrene between Eschericha coli and phenanthrene-degrading strain Sphingomonas sp. ZP1 was investigated. The apolar compounds penetrated in membrane phospholipid bilayer by distribution effect and into cytosol by anti-distribution way. Results show that compare to E. coli, phenanthrene was easier to enter into cytoplasm of phenanthrene-degrading bacteria. The accumulation of phenanthrene in cell membrane cause the barrier effect, so it maybe the reason of toxicity of PAHs pollutants. The transmembrane barrier-structure effect (TBSE) was advanced and it will provide a very helpful experimental strategy for toxicity assessment of a lipophilic compound.