Isolation And Identification Of Salt-tolerant PAHs Degrading Bacteria And The Response Mechanism To Salt Stress

Polycyclic aromatic hydrocarbons(PAHs),an important component of petroleum hydrocarbons,are typical persistent organic pollutants with "three causes" effect.There is a high overlap between onshore oilfield area and salinization area,which results in the dual stress of PAHs pollution and salinization.Because high salt environment has a significant negative impact on the survival of microorganisms with poor tolerance,which affects the natural dissipation of PAHs pollution and the efficiency of artificial biorestition,it is of great significance to screen salt-tolerant microorganisms with high PAHs degradation ability.However,at present,there is a lack of salt-tolerant microbial resources at home and abroad,and the knowledge about mechanisms of their responses to the salt stress.In this thesis,the oil sludge from the Shengli Oilfield was used as the source of bacteria.The high-throughput screening technology was used to screen and isolate the salt-tolerant and efficient PAHs degrading strains,and 51 PAHs degrading strains were obtained.The growth curves,changes of degradation efficiency,cell membrane fatty acid and compatible solute(tetrahydropyrimidine)of these salt-tolerant degrading microorganisms were analyzed under different salt stresses.,at different salinities were also discussed.Therefore,the physiological response mechanism of salt-tolerant PAHs degrading microorganisms to salt stress was explored.Finally,by analyzing the protein differences and expression changes of metabolic functional genes,,the response mechanism to salt stresses of degrading microorganisms at the functional level was discussed.The main research contents and results are as follows:1.Fifty-one strains of PAHs-degrading bacteria were preliminarily isolated via high-throughput screening technology.The cyclic PAHs degradation efficiency of the degraded strains was further determined.According to the capacity of PAHs degradation,the high-efficient strains were screened.Then,the salt-tolerant and high-efficient bacteria were further screened by analyzing the PAHs degrading ability of the strains under different Na Cl concentrations of 0 ～ 3%.Through 16 S r DNA identification of the obtained salt-tolerant and phenanthrenivorans degradation bacteria,A-5 and A-11 were identified as Pseudarthrobacter phenanthrenivorans and Microbacterium paraoxydans,respectively.After the whole genome sequencing of the strain A-5,which has a higher degradation efficient to PAHs,multiple PAHs degrading genes and salt-tolerance related genes were obtained from the whole genome sequence.2.That the strain A-5 was revealed growing well in LB medium with salt concentrations under 7%,but its growth rate was inhibited comparing with those in lower salinities to some extent.In LB medium with different salinities,the content of saturated fatty acids in cell membrane of the strain A-5 decreased with the increase of salinities,while the content of unsaturated fatty acids increased accordingly.Meanwhile,the contents of tetrahydropyrimidine in cell membrane the of strain A-5also showed an increasing trend with the increase of salinities.3.The phenanthrene degradation rate of A-5 was 79.54% when Na Cl concentration was 3%,while decreased significantly to 2.98% and 1.56%,respectively,when Na Cl concentrations were 5% and 7%.In the inorganic salt medium with phenanthene as the only carbon source,the expression levels of PAH-RHDα(hydroxylated dioxygenase α subunit)in the strain A-5 decreased with the increase of salinities.In LB medium and LB added 7% Na Cl,the strain A-5 was studied for differential proteomics analysis.A total of 435 differentially expressed proteins were screened between groups,including 134 up-regulated and 301 down-regulated proteins.Ten proteins related to salt tolerance were identified among the up-regulated differentially expressed proteins.Through the structural domains,GO and KEGG,the function of these differentially expressed proteins and the involved metabolic pathways were further analyzed,which revealing the cellular adaptive response process of P.phenanthrenivorans A-5 facing salt stress from the functional level.