Study On The Use Of Crude Glycerol For Microbial Oil Fermentation By Oleaginous Yeasts

Microbial oils, named as single cell oils, which have similar fatty acid composition to vegetable oils, are promising lipid material for biodiesel production. However, the cost of traditional medium for microbial oil fermentation is too high, and using lignocellulose and industrial and agricultural by-products as raw material for microbial oil fermentation can efficiently lower its production cost. Crude glycerol is the major by-product in biodiesel production and the amount of which is increasing greatly with the expansion of biodiesel. Thus it has become a key problem to effectively recycle crude glycerol. Use of crude glycerol as medium for fermentation can not only reduce production cost of microbial oil but also realize the efficient recycle of crude glycerol. Hence, comparative study on microbial oil production with crude glycerol by different oleaginous yeasts was performed and the effect of surfactant on the cell growth and lipid accumulation of which was investigated. Furthermore, the pilot-scale culture in a 5 L fermentor by the above oleaginous yeast with the best fermentation performance was explored in this study. It will provide therotical and practical basis for using crude glycerol for microbial oil production by oleaginous microorganisms.The effects of media components and culture conditions on the microbial oils fermentation by Trichosporon fermentans, Trichosporon cutaneum and Lipomyces starkeyi with crude glycerol as carbon source were investigated. The optimal glycerol concentration for T. fermentans, T. cutaneum and L. starkeyi were 50 g/L, 70 g/L and 70 g/L, respectively. The suitable nitrogen source, C/N ratio, inoculum amount and pH for the three yeasts were yeast extract + peptone, 60, 10.0% and 6.0, respectively. The optimal culture temperature for T. fermentans, T. cutaneum and L. starkeyi were 28oC, 30oC and 30oC, respectively. Under the optimal conditions, the maximum biomass, lipid content, lipid yield and lipid coefficiency for T. fermentans, T. cutaneum and L. starkeyi were 16.04 g/L; 32.35%; 5.19 g/L and 0.16 g/g, 17.35 g/L; 32.15%; 5.58 g/L and 0.17 g/g, 21.06 g/L; 35.70%; 7.25 g/L and 0.17 g/g, respectively. The results showed that the above three yeasts could efficiently use crude glycerol for lipid production. Compared with T. fermentans and T. cutaneum, more biomass, lipid content and lipid can be achieved by L. starkeyi on crude glycerol. Moreover, the effect of methanol present in the crude glycerol on the lipid production by the three yeasts was investigated. It was found that the biomass and lipid content of T. fermentans, T. cutaneum and L. starkeyi haven’t reduced obviously indicating that the methanol present in the crude glycerol had no significant inhibiton on the cell growth and lipid accumulation of oleaginous yeasts. Hence, crude glycerol is a potential medium for microbial oil fermentation.To further improve the microbial oil production ability of yeasts, the effects of surfactants(non-ionic, cationic and anionic surfactants) on the microbial oil production by the three yeasts were studied. It was found that in the non-ionic surfactants, Tween 80 had no significant influence on the cell growth and lipid accumulation of the three yeasts. The biomass and lipid content of T. fermentans and L. starkeyi decreased with the increase of PEG-200 concentration. However, there was no significant change in the biomass and lipid content of T. cutaneum even when the concentration of PEG-200 was as high as 0.5 g/L. Interestingly, although Brij 35 didn’t inhibit the cell growth, it could promote the lipid accumulation of the three yeasts. The cationic surfactants, including DTAB, TTAB and BC had significantly inhibitory effects on the cell growth and lipid accumulation of T. fermentans, T. cutaneum and L. starkeyi, especially when their concentrations reached 0.5 g/L, the growth of the yeasts stopped completely. In the anionic surfactants, SDS had inhibitory effect on the cell growth and lipid accumulation of the three yeasts. Potassium oleate showed no inhibition on cell growth of the three yeasts, and it could promote the lipid accumulation of the three yeasts when its concentration reached 0.1 g/L. Sodium stearate had no significant influence on the cell growth and lipid accumulation of T. fermentans, but 0.1 g/L of which could improve the lipid content of T. cutaneum and L. starkeyi.The culture of L. starkeyi which had the best performance in the flask fermentation was enlarged in a 5 L fermentor. It was found that the biomass, lipid content and lipid yield of L. starkeyi increased with the extension of fermentation and reached the maximals of 29.21 g/L, 42.93% and 12.47 g/L at day 10. The kinetics modelings related to the cell growth, lipid accumulation and glycerol consumption of L. starkeyi in the fermentor were done based on the logistic, Lueeking-Piret, and Luedeking-Piret-like equations. The results showed that the correlation coefficients between the maximum specific growth rate, lipid synthesis and the cell growth were 0.40 and 0.56, respectively.This study is of significance in promoting the application of crude glycerol in the microbial oil fermentation and reducing the production cost of microbial oil.