Screening,Mutagenesis Breeding And Fermentation Condition Optimization Of A High Yield Microbial Oil-producing Strain

In recent years,microbial oils have been widely used in food,medicine and daily necessities industries.Currently,the type of strains used in the industrial production of microbial oils are relatively single,and only a few strains have truly achieved industrial production.Therefore,it is important to breed more high yield microbial oil-producing strains with good growth conditions.In this thesis,an oil-producing wild strain was isolated and screened,the strain was induced by atmospheric and room temperature plasma(ARTP)mutation,and an excellent strain with high yield microbial oil-producing was screened.The fermentation conditions for oil production were optimized by response surface tests.The main research contents are as follows:1.A total of 140 strains of fungi were isolated and purified from oil-producing plants and rhizosphere soil samples.Microscopic observation by sudan black staining showed that there were black oil droplets in the mycelia of 39 strains.The strains were successively cultured in shake flasks,and the residual sugar,biomass,oil content and oil yield of the fermentation broth were detected respectively.The results showed that the MD-3 strain had the highest oil content and yield,and the best utilization of glucose.According to morphological observation and molecular identification,this strain was identified as Mortierella elongata.The acid-heating extraction was used to extract intracellular oil,and the oil content and yield of MD-3 strain were determined to be 30.15%and 4.67 g/L,respectively.2.ARTP was used to further enhance the oil production of the strain.The optimum treatment conditions were as follows:ARTP power 110 W,exposure distance 2 mm,mutagenesis time 60 s.An efficient method for screening mutant strains was established,in which the working concentration of cerulenin was 1.8?mol/L.Finally,a stable mutant strain MD-3-7 was obtained.The oil content of the mutant strain was 41.69%and the oil yield was 6.62 g/L.Compared with the original strain,which was increased by 38.27%and41.76%,respectively.By observing the morphological changes of mycelia during the cultivation process,it was found that most of the mycelial balls of the mutant were villous,the mycelia became shorter and the density increased,which was conducive to the accumulation of oil.3.In order to maximize the oil production of mutant strain MD-3-7,response surface methodology was used to optimize the composition of fermentation medium for oil production of this mutant strain.The results showed that the optimal medium for oil production was as follows:glucose 110.57 g/L,Ca Cl₂ 0.32 g/L,KH₂PO₄ 1.88 g/L,yeast flour 8 g/L,KNO₃ 1 g/L,Mg SO₄·7H₂O 0.3 g/L,IAA 3 mg/L,Fe Cl₃·6H₂O 0.015 g/L,Zn SO₄·7H₂O 0.0075 g/L,and Cu SO₄·5H₂O 0.0005 g/L.The optimized medium was used for fermentation,and the oil yield was 11.83 g/L,which was 78.70%higher than that of the original strain.4.Two key genes in fatty acid synthesis pathway,acetyl Co A carboxylase(ACC)and fatty acid synthase(FAS)were detected.The relative expression levels of these two genes in the mutant were increased by 9.25 and 4.37 times,respectively.The increase of oil yield was positively correlated with the expression level of these two genes.According to the third method of GB 5009.168-2016,the content of linoleic acid,arachidonic acid and eicostrienoic acid were increased by 55.31%,42.62%and 142.56%,respectively.In this experiment,a high-oil-producing strain Mortierella elongata was isolated and identified from oil-producing plants and rhizosphere soil samples.After ARTP mutagenesis,a mutant strain MD-3-7 was obtained.Its oil content and yield are 41.69%and 6.62 g/L,respectively.After optimizing its medium composition through response surface,the fermentation oil yield reached 11.83 g/L.The ACC and FAS genes of the mutant strains were up-regulated 9.25 and 4.37 times,respectively,and the contents of linoleic acid,arachidonic acid,and eicosatrienoic acid in the mutant strains were increased by 55.31%,42.62%,and 142.56%,respectively.This study provides a technical reference for the breeding of oil producing microorganisms,and at the same time provide a valuable source for the development and utilization of oil producing microorganisms.