Isolation And Characteristics Of Alkane Degrading Bacterium In Tiabjin Sea Region And Analysis Of Its Main Degradation Pathway

In recent years,the oil pollution in the marine environment is becoming increasingly serious.The management of the marine environment is extremely urgent.In the existing measures to control marine oil pollution,the bioremediation method has the advantages because of no seconding pollution and safety.The oil degrading microorganisms play an important role in bioremediation.In this paper,six strains of alkane degrading strains were isolated from Tianjin sea area.The six strains was named as Citrobacter sp.TUST-S1?Citrobacter sp.TUST-S6(Registration number of GeneBank is KM 186143?KM 186148)according to their physiological and biochemical characteristics as well as 16S rRNA phylogeneti-c analysis.In order to further understand the ability of the strains to degrade petroleum hydrocarbons,the degradation characteristics,emulsifying activity and hydrophobicity of the above six strains were explored.The degradation characteristics of those strains were determined by GC-MS.Extraction of residual oil in the culture medium with hexane before and after degradation.Samples were stored at 4 ? after removed of water and filtation.The standard production of C7-C30 and squalane were diluted into a final concentration of 100 mg/L.The C7-C30 and squalane after dilution was mixed with same volume,and then the same volume of squalane was added to a sample,and the GC-MS analysis was carried out.The results show that the degradation rate of the strains were very different,The lowest degradation rate of these strains was 39%for Citrobacter sp.TUST-S6 while the highest degradation rate was 87%for Citrobacter sp.TUST-S5.it was found that the strain Citrobacter sp.TUST-S5 could degrade alkane of C11-C28,and the degradation rate of alkane that carbon chain length was greater than C20 was more than 80%.After mixing of the bacterial suspension and hexadecane of same volume with vortex oscillator,the mixture was stratifited in room temperature.The emulsifying activity and hydrophobicity of these strains were calculated with related formula.Results show that the emulsification activity and hydrophobicity for Citrobacter sp.TUST-S5 was highest which respectively is 47.86%and 38.30%,and the emulsification and hydrophobic interaction of strains could be to promote the degradation process.In view of relatively high degradation rate of Citrobacter sp.TUST-S5,Citrobacter sp.TUST-S5 was used as the experimental material to study the relationship between the growth and degradation of the strain,the expression pattern of the key enzyme gene(alkB)and the main degradation pathway.After determining the growth and degradation curve of the strain,it is found that the concentration of bacteria is closely related to its degradation rate.The expression pattern of key enzyme gene(alkB)was determined by Q-PCR.The results show that the expression of the gene is the highest at 8h after inoculation.The possible degradation pathway was speculated by GC-MS.The results show that the main degradation pathway is mainly in the way of terminal oxidation and secondary end oxidation.