Screening Of PAHs-Degrading Microbes In Salt Soil And Basic Study Of Their Application On The Bioremediation Of Pollutants

Many oil fields in China are located in the salt-alkali soil, with the development of prtroleum industry, the pollution of nearby soil by polycyclic aromatic of oil fields is getting more serious; In addition, the frequent happening of oil spills recently at coastal area also leads to the PAHs contamination of salt-alkali soil. Thus, the PHAs pollution of salt-alkali soil is getting more and more attention. Microbial degradation is considered to be the major approach for the removal of PAHs in the environment. However, dual stresses of the salt-alkali environment and the aromatic compounds make it difficult for the microbes to survive and grow, which result in the difficulty of the microbial rehabilitation of PAHs pollution in salt-alkali soil. We isolated some PAHs degrading bacteria from the long-standing Oil-polluted coastal salt-alkali soil of Dagang field in Tianjin.The infuluence of different salt concentration, pH, PAHs concentration and other factors are studied systematically using liquid culture method, The salt soil of Dagang oil field was taken as the experimental soil to study systematically the degradation of PAHs after the immobilization of degrading strain and the addition of surface active agen to the polltuted soil. On this basis, by measuring the degrading enzyme activity of PAHs-degrading bacteria Pantoea sp.TJB5 and expression of related genes, we drew some conclusions on the degradation mechanism of the PAHs on enzymetic and molecular level. The results showed that this strain has a strong activity on PAHs degradation and is possible to be applied on salt-alkali soil rehabilitation. The main results obtained are as follows.1. Taking mixed samples of pehnanthrene and pyrene as sole carbon and energy source, we isolated 5 strains of bacteria and 3 strains of fungi from the salt-alkali soil. It was showed that all the 8 strains isolated could degrade PAHs, but the degradation capacities were different. The 5 strains of bacteria isolated were named as TJB1、TJB2、TJB3、TJB4 and TJB5, the 3 strains of fungi were named as TJF1、TJF2、TJF3. TJB1 was leaf spp(Phyllobacterium sp.), TJB2 and TJB3 were Pseudomonas (Pseudomonas sp.), TJB4 was the salt unit cell(Halomonas sp.), TJB5 was pan-genus(Pantoea sp.); TJF1 was Penicillum(Penicillium sp.), TJF2 was Hyperbolic sp(Sigmoidea sp.), TJF3 was anthrax spores for the plastic case (Colletotrichum sp.).2. Bacteria Pantoea sp.TJB5 and fungus Penicillium sp. TJF1 with high degradation ability and 8 strains mixture (TJM) were tested using the liquid culture, Taking PAHs-phenanthrene and pyrene as the research object, the effect of pH, salt content, the initial concentration of PAHs and suppling levels of glucose on the strains’s degradation rate on PAHs. The results showed that the degradation efficiency of the tested strains on PAHs decreased with increasing initial concentration of PAHs, but Pantoea sp.TJB5 had better effect on pehnanthrene, and TJM had better effect on pyrene; Pantoea sp.TJB5, Penicillium sp. TJF1 and TJM were more tolerant to salt-alkali. When the initial concentration of phenanthrene and pyrene was 50 mg/L, salt concentration was 2%, pH was 8.6, the degradation rate on phenanthrene of them reached 93.9%、41.3%、56.6% and 20.1%、27.9%、52% respectively; Addition of appropriate amount of glucose as co-metabolic substrate can promote the degradation of PAHs, for example, the degradation rate of Pantoea sp.TJB5, Penicillium sp.TJF1 and TJM on phenanthrene and pyrene increased by 1.2%、5.5% and 22.5%、29.3% and 35.5%、13.8%.3. The functional degrading enzyme of Pantoea sp.TJB5 on PAHs was showed to be intracellular enzyme through positioning measurement. The IR spectra analysis showed that the intracellular enzymes containing groups as OH、NH、C=O、C—O、and C=S, these groups were connected with some enzymes and Carbohydrates, and played some roles in the oxidation, decarboxylation and reduction, and thus make PAHs more soluble and easy degrading. Catechol-2,3-dioxygenase (C230) is a key enzyme in the ring cleavage of PAHs, we amplified C230 partial gene sequence of 543 bp from Pantoea sp.TJB5, indicating that the key enzyme-- C230 of PAHs degrading exists.4. Degrading effect of bacterium Pantoea sp.TJB5, Penicillium sp. TJF1 and flora TJM on phenantheren and pyrene was significant after immobilization or addition of the surface-active agent in the salt-alkali soil remediation. The results showed that the tested strains has a high removal rate of pollutants on phenanthren, pyrene in salt-alkali soil after addition of immobilized micro-organisms and surface-active agent to the contaminated soil, which proved that it’s practical in the repair of contaminanted alkali soil. Immobilization of the tested strains, and addition of surfactrant for 50 days could improve the degradation rate of pyrene by more than 20% compared with control of sole bacteria; The effect of immobilized bacteria mixture was best, the phenanthrene content in salt-alkali soil was close to the background value after 20 days, the removal rate of pyrene 50 days was 97.4%, higher than the control treatment of free bacteria’s 25.8%.5. The remediation effect of immobilized strain and rhamnolipid treated immobilized strain was remediation of PAHs in salt soil; Removal rate of 4 rings PAHs in soil was 94.9% 5 months after the addition of rhamnolipid, higher than in the treatment of free bacteria.