Isolation,Identificationand Degradation Characteristics Of Alkane-degrading Strain From Oil-contaminated Soil

With n-hexadecane as sole carbon source,in this study 14 strains that belonged to the 9 genus were isolated.Strain LAM1007 as the major object in this study because its highest degradation.Furthermore,Single factor experiment were carried out on different environment factors（temperature,pH,biomass,shaking speed）and different concentration of n-hexadecane（0.1%,0.3%,0.5%,1.0%,1.5%,2.0%,v/v）to revealtheir influence on the degradationg of n-hexadecane by strain LAM1007.Meanwhile,We further studied the degradation ability of strain LAM1007 to the high concentration of diesel（2.0%,v/v）and parraffin liquid（0.3%,v/v）to preliminary reveal the degradation characteristics adout the each alkane components in the samples.Based on the high degradation ability of strain LAM1007,the cloning and expression of function gene（alk B）were did,the optimization of expression conditions of recombinant alkB was also studied in order to lay the foundation for the subsequent application of genetic engineering bacteria and the study of the following enzymaticcharacteristics.In this study,strain LAM1007 was preliminary identified as a likely novel because its low 16 S rRNA sequence similarity and more research on phylogenetic classification were did.Strain LAM-WHM-D11 was also classified with the methodof polyphasic taxonomy.The main conclusionsare as follows: First,With n-hexadecane as sole carbon source,in this study 14 strains that belonged to the genus of Aquabacterium,Sphingomonas,Acinetobacter,Pseudomonas,Brucella,Spingopyxis,Kosakonia,Gordoniaand Arenimonas has been isolated from the oil-contaminated soil sample.Second,the results of degradation characteristics of n-hexadecane showed that the optimum degrading condition in the medium were 30°C,pH 7.0,1%（v/v）biomasss,180 r/min.The degradation of strain LAM1007 for 0.3% and 2.0%（v/v）concentration of n-hexadecane within 60 h at optimum degrading condition were 90% and 53%,respectively.And The 16 S rRNA gene sequence of strain LAM1007 showed the closest similarity of 98.8 % with Acinetobacter junii LMG 998 T and 97.0 % with Acinetobacter venetianus LMG 19082 T,and with analyses of colony morphology preliminary identified that strain LAM1007 belong to the genus of Acinetobacter and using the method of polyphasic taxonomy further confirmed that it would possibly be an novel.And strain LAM-WHM-D11 that with the ability of having a growth in the inorganic salt medium（ISM）thatsupplemented with 0.3 %（v/v）hexadecane and toleranceing different kinds of heavy metalswas confirmed as a novel with analyses of polyphasic taxonomy.Meanwhile,with diesel as sole carbon source,the data of GC-MS showed that the strain LAM1007 had a significant degradation effect on alkane components C₁₃-C₁₉,especially C₁₆,which showed the similar regularity when degrading the parraffin liquid.Finally,the alkB gene was successfully amplified and E.coli BL21/pET28a-alk B was constructed successfully.When the temperature was 37 °C,30 °C 25 °Cthe soluble protein AlkB was not expressed induced at IPTG 0.1-1.0 mM,but which were expressed at temperature 16°C,however,there was no significant difference in the content.