Screening Of High Lipid-yield Strain By Rhodosporidium Toruloides And Its Medium Optimization

Microbial Lipid has a wide range of use in people’s daily life and industrialproduction. On the one hand, it contains high value-added functional lipids, which arewidely used in food, medicine and chemistry industries, such as health food functionalfactors, some synthetic drug sprecursors, important components of cosmetics, etc;Onthe other hand, it is the main component of triglyceride and can be used as rawmaterial for biodiesel production. Compared to plant and animal lipids or fats, themicrobial lipid has a wide range of sources of raw materials, continuous massproduction, components oriented after the biological treatment, small occupied space;the disadvantages is that lipid production is still relatively low, the lipid extraction isdifficult.This study takes Rhodosporidium toruloides purchased from the China GeneralMicrobiological Culture Collection Center as the starting strain. After a series ofmutation breeding, we get the strain D_5-2with high-yield lipid and stable geneticproperties, and then the fermentation medium of D_5-2was optimized, and discuss thefermentation kinetics. Results obtained were given as follows:（1） Considering the operation difficulty, extraction time and cost, thermal-acidmethod, Soxhlet extraction, organic solvent, acid heat ultrasonic coupling of severallipid extraction methods were compared. We selected the acid method as experimentalof lipid extraction method.（2） Through UV mutation, microwave mutation, two ethyl sulfate mutagenesis, ahigh-yield lipid strains of D_5-2were finally got, and its biomass was12.63±0.13g/L,and lipid production was3.32±0.06g/L.（3） After a series of single factor optimized, the Rhodosporidium toruloides bestlipid medium was pH5.5、temperature28℃、the volume of liquid60mL, inoculationamount10%, shaking speed180r/min; after the response experiment optimized,thebest optimize fermentation medium components were:yeast powder0.049%, calcium chloride0.043%, potassium dihydrogen phosphate0.1%, magnesium sulfate0.071%,manganese sulfate0.02%, nitrate sodium0.06%, glucose6%, zinc chloride0.02%.Lipid production reached to5.20±0.07g/L, increased by89.71%than that beforeoptimization.（4） Effects of precursors on Rhodosporidium toruloides lipid-producing wereinvestigated. After the response surface experiments and single factor optimization, theoptimal concentration of each factor is: coenzyme A5.82mg/L, glycerine0.32g/Lsodium, citric acid is0.69g/L. In order to test the accuracy of the regression modelprediction, under the optimized conditions, oil production is6.17±0.09g/L, and themodel prediction is6.24g/L, increased by18.65%.（5） Under the optimized fermentation conditions, The fermention curve wasdrawed.From the curve can be seen, with the increase of fermentation time, the strainwas in lag period from1d to2d, and the biomass and lipid content increased, but thegrowth was relatively slow. From2d to4d, strains were into the rapid growth period,and lipid content increased. From4d to6d, biomass growth was slow, and the lipidcontent increased greatly, indicated that the strains were into the stable period with alarge number of lipids beginning to synthetize. After7d, the biomass and lipid contentdecreased, which demonstrated that strains entered into the phase of decline. A largenumber of strains began to deaths and cell autolysis, meanwhile, duing to lack ofcarbon source, living strains began to decompose the lipid as the carbon source andenergy metabolism. During the whole fermentation process, the consumption ofglucose was always inversely proportional to the biomass growth rate, indicated that itwas the main carbon source for microbial growth and fat synthesis. In the wholeprocess, pH of fermentation liquid was relatively stable （always between4.7and5.5）,which is the appropriate pH interval of Rhodosporidium toruloides growth and lipid-producing.