Isolation And Identification Of High Efficiency Diesel Degradation Strain In Oil Sludge

The continuous development of global industry had brought about the exploitation,processing and use of oil on a global scale,which had made the problem of oil leakage and pollution in the environment more and more serious.Bioremediation technology had the characteristics of low cost,simple operation,and no secondary pollution,it had been a research hotspot for oil pollution control in many countries.However,for different contaminated plots and environmental media,it was still necessary to use different characteristics microorganisms to degrade and continuously expand strain resources and genetic engineering resources for high efficiency degradation of microorganisms.For a diesel contaminated site in Nanjing,in order to screen different kinds and characteristics of high-efficiency diesel degrading strains and study their degrading mechanism in different environmental medium,7 high-efficiency diesel degrading bacterial strains were isolated from the sludge sediments in sewage pipes of Nanjing Yangzi Petrochemical Plant in this experiment.Morphological,physiological and biochemical indexes and molecular identification of 16S rDNA were performed on the selected highly effective diesel degradation bacteria.The identification results showed that J-1 belonged to the genus Micrococcus,J-2 belonged to the genus Acinetobacter,and J-3 belonged to the genus Serratia,J-4 belonged to the genus Psychrobacter,J-6 and J-7 belonged to Bacillus,and J-8 belonged to the genus Paenibacillus.After optimizing the growth conditions of these strains and degradation effects of these strains on diesel,The test results found that the degradation rates of strains J-1 and J-3 exceeded 50.0%of diesel,and the application of strain J-3 in diesel pollution remediation was less studied.Therefore,the strain J-3 was selected as the experimental strain to further study its degradation conditions and degradation mechanism.The degradation efficiency of diesel and the degradation mechanism of strain J-3 were studied by hydroponic and soil culture methods combined with gas chromatography-mass spectrometric analysis(GC-MS)and genome comparison techniques.The results showed that the strain could efficiently grow using diesel as its sole carbon source,produce surfactant,have stable emulsifying properties and the ability to reduce liquid surface tension.The strain J-3 grew well under the temperature of 20-40℃ and the initial pH of 5-9,while enduring concentration of 15 g/L Nacl and 5 g/L diesel.The degradation efficiency of diesel under the optimal growth conditions(pH 7,25℃,1 g/L Nacl and 0.5 g/L diesel)was 62.0%.GC-MS spectrum analysis confirmed that J-3 had the ability to degrade all components in diesel oil,the degradation rate of some medium and long straight-chain alkanes exceeded 99.0%,and the degradation rate of macromolecules such as alcohol,ether and other macromolecules exceeded 58.0%.Based on the whole-genome DNA sequence analysis of the strain J-3,two genes associated with alkane oxidation were found,namely alkB,an alkane hydroxylase gene,and almA,a Long-chain alkane monooxygenase gene.These two oxidase genes may play a role in the degradation of diesel oil by strain J-3.In addition,when the highly degraded strain J-3 and the currently well-researched strain J-7(Bacillus)were applied to the soil remediation test of diesel pollution,the degradation efficiency of diesel were studied by Research on single bacteria and the coexistence of single bacteria and indigenous microorganisms combined with ultraviolet spectrophotometry and gas chromatography-mass spectrometric analysis(GC-MS).It was found that under the conditions of this experiment,the indigenous microorganism played a major role in the degradation of diesel fuel in soil.The degradation rates of diesel in soil were 33.8%and 20.1%respectively under the conditions that the strains J-3 and J-7 were symbiotic with indigenous microorganisms;The degradation rates of single bacteria to diesel in the soil were 48.9%and 36.7%respectively under the conditions that the strains J-3 and J-7 were added to the pasteurized soil;GC-MS spectrum analysis confirmed that the strains J-3 and J-7 had the degradation ability of medium chain length components of diesel,which could effectively degrade diesel in soil.Under different control treatments,the degradation ability of the strain J-3 to diesel in soil was higher than that of J-7,indicating that the strain J-3 was a high-efficiency diesel degradation bacterial strain.The strain J-3 could be well used for growth in both water and soil,and could be applied to the actual bioremediation project of diesel pollution.