Study On Isolation And Characteristics Of Polycyclic Aromatic Sulfur Heterocycles Degrading Bacteria

As the major sulfur-containing chemical compounds of crude oils,polycyclic aromatic sulfur heterocycles（PASHs）seriously threat human health and the entire ecosystem with their potential toxicity,carcinogenicity,mutagenicity and trouble bioavailability.In this study,PASHs-degrading bacteria were isolated and identified.The metabolic pathways and degradation characteristics of other representative pollutants were also analyzed.In addtion,the chemical structures and physicochemical properties of biosurfactant produced by degrading bacterium were determined.And this bacterium was applied in bioremediation of contaminated soil.This research would provide a theoretical basis for reducting or eliminating the potential impact of PASHs on environment.Dibenzothiophene has been chosen as a model of PASHs in this study.5 highly efficient dibenzothiophene degrading strains were isolated from contaminated soil samples in Shengli oil field.These isolates were identified to genus level by physiology biochemistry experiments and 16 S rDNA sequence analysis.The phylogenetic tree was established to analyze the relationship of phylogeny.The cultivating conditions of Pseudomonas sp.LKY-5 were optimized by using response surface methodology.A second-order regression model was developed.The variance analysis,residual analysis and experimental verification illustrated that the regression model was reasonable and acceptable.The substrate concentration was most important factor in biodegradation by significant analysis.Strain LKY-5 could degrade almost all of dibenzothiophene（100mg/L）at temperature of 25℃³5℃,agitation rate of 140rpm¹80rpm and pH of 6.5⁸.5 in 7 days.The metabolic intermediates produced by Pseudomonas sp.LKY-5 were analyzed by GC-MS.Two completely mineralized pathways were proposed,which were named extended 4S pathway and extended Kodama pathway,respectively.These pathways were significant for bioremediation.Hexadecane,phenanthrene,pyrene and carbazole could be degraded by strain LKY-5 in single system.The biodegradability first was hexadecane,then phenanthrene,next carbazole,and last pyrene.The degradation of DBT was promoted with hexadecane or phenanthrene,and inhibited with carbazole or pyrene.The degradation of hexadecane,phenanthrene,pyrene and carbazole were inhibited by DBT in mixed system.The sulfur compounds in different types of diesel were degraded variously by Pseudomonas sp.LKY-5.Due to the analysis of some long chain alkanes and fatty acids,the biosurfactant produced by Pseudomonas sp.LKY-5 were analyzed and determined by TLC and HPLC-ESI-MS.The major constituent of biosurfactant was di-rhamnolipid with chemical structure of Rha-Rha-C8-C8:1.Besides,some free fatty acids were also contained in the biosurfactant,such as C₁₈,C_18:1,C₁₉,C_19:1,C_20:1,C₂₀.The critical micelle concentration,hydrophilic-lipophilic balance value and the emulsifying property of the biosurfactant were also confirmed.The biosurfactant was stable in extreme conditions,such as high temperature,high pH value and high salinity.The impact of pollutant levels,degrading bacteria,initial moisture content,N/P and saw dust were discussed by applying Pseudomonas sp.LKY-5 to enhanced bioremediation,and the most suitable bioremediation conditions were established.The theoretical basis and technical assistance would be provided for in-situ bioremediation of PASHs-contaminated soil.