Mutagenesis Breeding And Fermentation Regulation Of Oleaginous Yeast For Lipid Production

Microbial lipid has high nutritional value,small toxic effects,and fatty acid composition similar to vegetable oils,so it can be used as a cheap material to produce biodiesel,which is of great significance to solve the problem of energy shortage.However,the current insufficient lipid synthesis capacity of oleaginous yeast has resulted in microbial fermentation cost,which becomes a big challenge to microbial lipid industrialization development.In this thesis,oleaginous yeast Trichosporon dermatis L7 preserved in my laboratory was mutated to improve its lipid production capacity.The fermentation process was optimized and regulated by selecting some growth additives and co-factors,and strengthened with effective fed-batch fermentation strategy on lipid production capacity.The main contents obtained are as follows:（1）T.dermatis L7 was subjected to ARTP-DES compound mutagenesis and screened by microplate reader.Then,a high--lipid--yielding strain G5 with good tolerance to high sugar concentration was obtained.When the sugar concentration of this strain was 120 g·L^-1,the cell mass and lipid titer of fermentation increased by 7%and 12%,respectively,compared with the starting strain L7,and the fermentation time was shortened by 3 days.（2）The experiment enhanced the fermentation level of mutant strain G5 by means of optimization of fermentation conditions,nutrient restriction and addition of exogenous substances to improve lipid accumulation.Results showed that the culture conditions in shake flasks were temperature 25℃,p H=6,inoculum amount 10%（v/v）,liquid volume 50 m L/250m L,rotation speed 200 rpm.The ratio of essential nutrients was C/N=300,C/P=188,C/S=5682.The optimal concentration of each additive was Mg Cl₂ 1.12 g·L^-1,trehalose 1.15 g·L^-1,sodium malate 0.67 g·L^-1,sodium citrate 0.50 g·L^-1,glycerol 6.0 g·L^-1,biotin 0.4 mg·L^-1.N-dodecane of 2%（v/v）was added as an oxygen carrier in the early fermentation stage to shorten the fermentation time by 20%.Coupled with the above process optimization and regulation,the cell mass,lipid titer and lipid content of the strain reached 33.4 g·L^-1,19.4g·L^-1 and 58%for 9 days of the fermentation,which increased by 22%,32%and 8%,respectively,compared with that before the optimization.（3）The fed-batch fermentation method was optimized,finding that the initial sugar concentration was 120 g·L^-1,together with addition of 0.5 g·L^-1 of soy peptone at 24 h of fermentation.The carbon source was fed by intermittent decreasing feeding method,in which the feed concentration was 30 g·L^-1,20 g·L^-1,10 g·L^-1,respectively,feeding at the 4^th,5^th and6^th day of the fermentation.After 12 days of the fermentation,the cell mass,lipid titer and lipid content achieved at 50.1 g·L^-1,28.4 g·L^-1 and 57%,respectively.In a 7-L fermenter,the microbial lipid fermentation with this mutant attained 64.9 g·L^-1 of the cell mass and 37.0g·L^-1 of the lipid titer for 10 days which increased by 50%as compared to the batch fermentation.