Study On The Degradative Characteristics And Pathway Of PAHs Degrading Bacteria

The presence of polycyclic aromatic hydrocarbons (PAHs) in the environment is a considerable public health hazard because of their intrinsic chemical stability, high recalcitrance to different types of degradation and high toxicity to living organisms. The principle processes for their successful removal are currently believed to be microbial transformation and degradation.This research aimed at finding bacteria capable of breaking down the PAHs, identifying the functional genes and enzymes involved in the biodegradation, and elucidating PAHs biodegradative pathway.The main results were as follows:1. A microbial consortium was obtained from the enrichment culture of sea water samples collected from Botan oil port in Xiamen, China, using the persistent high concentration of high molecular weight polycyclic aromatic hydrocarbons (HMW PAHs) enrichment strategy. Denaturing Gradient Gel Electrophoresis (DGGE) was adopted to investigate the bacterial composition and community dynamic changes based on 16S rRNA genes PCR amplification during enrichment batch culture. Using the spray-plate method, three bacteria namely BL01, BL02 and BL03, which corresponded to the dominant bands in the DGGE profiles, were isolated from the consortium. Sequences analysis showed BL01, BL02 and BL03 were phylogenetically closer to Ochrobactrum sp., Stenotrophomonas maltophilia and Pseudomonas fluorescens respectively. The degradation of Benzo(a)Pyrene (BaP), a model HWM-PAH compound by individual isolate, mixture of three isolates and the microbial consortium(BL) isolated from sea water were examined. Results show that the degradation capability of bacteria represented the order of consortium(BL), mixture of three isolates, individual isolate for high to low, the consortium BL showed the highest PAH degradation capability for degrading HMW-PAH compounds, 44.07% of benzo(a)pyrene was degraded by consortium BL after 14 days incubation. Our results pointed out that the high selective pressure strategy is feasible and effective to enrich the HMW PAH-degraders from the original sea water sample.2. Using modified sublimation method, an effective and more convenient method, two phenanthrene-degrading bacteria, identified as Sphingomonas sp. B2-7 and Mycobacterium sp. S8, were isolated from a mixed culture that had been enriched under the selective pressure of PAHs. They can utilize a wide range of PAHs made of 2-4 aromatic rings as the sole carbon source and degrade these PAH compounds. Both of these two strains can degrade phenanthrene to undetectable levels in 72 h. Further experiments were carried out to optimize the degradation of HMW-PAHs and results showed that non-ionic surfactant Tween-80 and glucose can significantly increase the biodegradation percentages.3. Strain B2-7 was used to study the enzymes involved in PAHs degradation using genomic and proteomic methods. The genes encoding two key enzymes were cloned and one of them was overexpressed in Escherichia coli BL21 (DE3). One is ring-hydroxylating dioxygenase a subunit (phnA) , the enzyme that catalyzes the initial step in PAHs degradation, and the other is catechol-2,3-dioxygenase (C23O), catalyzes the extradiol ring-cleavage of catechol. These results indicated the main catabolic pathway of strain B2-7 degrades PAHs.4. Phenanthrene, a tricyclic PAH, was selected as the model PAH compound to determine the degradation pathway. Two-dimensional (2D) gel electrophoresis of phenanthrene-induced proteins from cultures of strain B2-7 was used to detect proteins that increased after phenanthrene exposure. Comparison of proteins from phenanthrene-induced and uninduced cultures on 2D gels indicated that at least ten major proteins were expressed. Proteins newly induced by phenanthrene were then analyzed from the gels by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Most of them shared a high degree of similarity with the existing database and were responsible for phenanthrene or naphthalene degradation, such as hydroxylating dioxygenase, dihydrodiol dehydrogenase, oxovalerate aldolase and isocitrate dehydrogenase. A complete phenanthrene degradation pathway for Sphingomonas sp. B2-7 based on these results was proposed.