| Microbial remediation technology is one of the safest and most effective methods to deal with oil pollution in the environment.Therefore,it is the first prerequisite for the construction of the later engineering strains to obtain the microorganisms with efficient alkane degradation function and to study their metabolic process.In this study,Dietzia sp.DQ12-45-1b,which was isolated and selected from Daqing Oilfield and domesticated in the laboratory for many years,was selected as the target strain.By knockout fadD,which plays an active role in the degradation of alkanes,the metabolic process of the mutant strain and the original strain in the degradation of octadecane was studied.Firstly,the mutant strain was obtained by gene knockout method,and the cell activity and degradation rate of the strain in octadecane were studied.Secondly,the changes of surfactants and cellular characteristics that play a role in the efficient utilization of alkanes by microorganisms were studied in different medium.Finally,the mechanism of fadD in Dietzia sp.DQ12-45-1b reduction of ultra-long chain alkane was preliminary explored through the background of fadD and the analysis of the whole genome function of the mutant as well as the detection of metabolites of the mutant.The main results are as follows:1.The mutant strain A12 constructed by gene knockout did not affect its utilization of anhydrous sodium acetate,but affected the degradation process of hexadecane and octadecane.The mutant strain A12 produced a degradation rate lower than the original strain WT in the hexadecane medium.However,the degradation rate of A12 was higher than that of WT in the process of degradation of octadecane.The comparison results showed that fadD did not affect the?-oxidation process of alkanes in octodecane,and it was speculated that the removal of fadD might change the transport process of alkanes.2.With hexadecane and anhydrous sodium acetate respectively two kinds of carbon sources for comparison,comparing the index activity and cell characteristics of the mutant strain A12 and the original strain WT in the octadecane culture medium,it was found that the lack of fadD resulted in:(1)In the process of degradation of octadecane,the maximum diameter of the oil circle of mutant strain A12 reached24±1.3 mm at 27 days,which was 2.7 times higher than that of the original strain WT.(2)The lowest surface tension of WT decreased to 54.49±1.32 m N·m-1.The surface tension of mutant strain A12 decreased from 75 m N·m-1 to 42.85±1.97 m N·m-1from the initial stage to the stable stage,which was at least 21.37%lower than that of the original strain WT.(3)The maximum emulsifying activity of WT was 44.49±1.9%,and the maximum emulsifying activity of mutant A12 was 36.3%higher than that of WT.(4)The maximum cell surface hydrophobicity of mutant strain A12 increased by59.6%compared with that of the original strain WT.(5)Generally speaking,the activities of above indexes produced by the strain were the highest in octadecane culture medium,suggesting that the mutant strain might produce more surfactant in the octadecane medium.In addition,the activity index and cell characteristic index of anaplerotic strain A12R in octadecane tended to the original bacteria WT,indicating that fadD did regulate the metabolic process of Dietzia sp.DQ12-45-1b in octadecane.3.Combining the background of fatty acyl-CoA synthase biosynthesis analysis and the whole genome analysis of mutant strain A12,it is speculated that the mechanism of fadD affecting the degradation of ultra-long chain alkanes is mainly three points:(1)fadD affected the activation process of degradation of ultra-long chain octadecane.(2)The hydrophobicity of cell surface was changed by influencing the membrane transport process,and the contact area between mutant A12 and alkanes was increased.(3)fadD affected the type and content changes of metabolites in the process of alkane metabolism.Mutant A12 produced two glycolipids and two lipopeptides,which changed the degradation rate of ultra-long chain octadecane. |
PDF Full Text Request