Screening Of A High-efficient Pyrene-degrading Bacterial Strain And Its Degradation Pathway

Polycyclic aromatic hydrocarbons(PAHs) are a class of ubiquitous, persistent, toxic, and carcinogenic pollutants that pose significant risks to environmental and human health. At present, it has been widely accepted that microbial degradation is a major way to remove PAHs from contaminated environment. Separation and screening high-efficient PAHs-degrading strains is the key work of this method. Proteomics becomes a hot spot during the process of PAHs biodegradation. Enzyme systems with new metabolic fuction can be produced by microorganism through inducement. To study the proteins/enzyme expression, which is involved in environmental pollutant metabolism, will be helpful to further understand the machanism of bioderadation.Pyrene was chosen as a target compound for studies on the biodegradation of PAHs. Pyrene-degrading strains were screened from oil-contaminated soil(PAHs) in the area nearby Sinopec Guangzhou Petrochemical Complex and activated sludge from coke wastewater treatment plant of SGIS Songshan Co., Ltd, respectively. Further study was held in a high-efficient pyrene-degrading bacterium that was selected from those screened strains. The effects of different incubation conditions on pyrene degradation by strain CP13 were investigated in this study. And the difference of total proteins expression between control sample and pyrene-induced samples were analyzed by 2D-gel, in combination with the intermediate products of pyrene degradation which were detected by GC-MS, the pyrene-degrading pathway of CP13 was primarily deduced. The maim conclusions of this study are as follow:(1) Strain CP13, which is isolated from activated sludge of coke wastewater treatment plant, with the best degrading effect among the screened degrading strains that can remove more than 90% of pyrene(50mg/L) after 7 d. Base on the partial 16 S r DNA gene fragment(about 1408 bp) and sequence alignment, strain CP13 was tentatively classified as Mycobacterium gilvum strain. The Gen Bank accession numbers of strain CP13 is KF378755. Strain CP13 was also preserved in China General Microbiological Culture Collection Center(CGMCC), CGMCC No. 7963.(2) The effect of incubation conditions on pyrene degradation by CP13 was investigated, such as initial pyrene concentration, temperature, initial p H and so on. The results showed that the pyrene degradation were over 88% when CP13 was incubated under the conditions of initial pyrene concentration ≤75 mg/L, temperature range from 30 to 35 ℃, p H range from 7~10. That guarantees the degradation effect of its application in the different environment.(3) According to the results of GC-MS analysis, four metabolites were detected in the process of pyrene degradation by strain CP13, i.e. phthalic acid, 1-naphthol, 4-phenanthrenol and 4-phenanthrenecarboxylic acid. Especially, 4-phenanthrenol was accumulated during pyrene biodegradation.(4) The differences of total proteins expression between control sample and pyrene-induced samples were analyzed by 2D-gel. The results show that proteins related to physilogical activities mantainance, such as glycosyl transferase group1 and GMC oxidoreductase family, were involved during the biodegradation process. Besides, an important enzyme, putative extradiol dioxygenase which is related to pyrene biodegradation, was detected in the p H=9 incubation condition.(5) Based on the results of GC-MS analysis and 2-DE analysis, a probable pathway of pyrene degradation was proposed: under oxygenase of strain CP13, pyrene was oxidated to 4,5-dihydroxypyrene, then cleaved and subsequently metabolized to 4-phenanthrenecarboxylic acid. Afterwards 4-phenanthrenecarboxylic acid was decarboxylated to 4-phenanthrenol or to 3,4-dihydroxyphenanthrene, which were later transformed to 1-naphthol, then cleaved to phthalic acid and finally accessed to TCA-cycle exhaustive degraded to CO2 and H2 O.