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Biodiesel is one of the most important renewable energy sources. At present, the main materials for biodiesel production are animal fats and vegetable oils. Although biodiesel has been already used in US, EU and Japan and many other places, the high cost restricts its wide application. Moreover, the use of vegetable oils for biodiesel production would compete with edible oils, thus leading to the soar of food price. Therefore, the key factor for the industrialization and wide application of biodiesel is to find a cheap and sustainable feedstock. Microbial oils are lipids accumulated by oleaginous microorganisms under special conditions. Their fatty acid compositions are similar to that of vegetable oils. Lipid production by microbial fermentation, can not only relieve the shortage of lipid resources from animals and plants, but is expected to provide cheap feedstock for biodiesel production. Presently, the industrial application of microbial oils is hampered by the high cost of nitrogen-limited mediums used in microbial oil production. Trichosporon fermentans has been screened out and proved to be able to accumulate lipid with xylose in our lab previous study and the possibility of production of microbial oils with bagasse hemicellulose hydrolysate was explored in this dissertation, and the effects of related factors on the fermentation were systematically examined. Based on this, response surface methodology (RSM) was employed to optimize the fermentation conditions. To further reduce the production cost and improve the lipid production efficiency, the effect of adding small amount of molasses instead of organic nitrogen into the medium on the cell growth and lipid accumulation of T. fermentans. At the same time, the preliminary scale-up in 1 L bioreactor was also investigated based on shaking flask fed-batch fermentation.Crushed bagasse( particle diameter≤0.5 mm) was hydrolyzed by 1.5% diluted sulfuric acid (w/v) and the hydrolysis conditions were as follows: solid-liquid ratio(w/v) 1:10, temperature 121℃, time 1.5 h. After filtration, the total sugar concentration of resulted hydrolysate was 40.8 g/L, and the main components of which were xylose (30.2 g / L), glucose (5.2 g / L), galactose (1.5 g / L), arabinose (3.9 g / L) plus inhibitors such as acetic acid and furfural etc. The hydrolysate was pretreated by the methods of overliming, concentration and activated carbon adsorption and the total sugar concentration of pretreated hydrolysate was 122.5 g/L including 92.9 g / L xylose, 16.8 g / L glucose, 2.4 g / L galactose and 11.4 g / L arabinose. Meanwhile, inhibitors were almost relieved except acetic acid. the harmful The the harmful substances such as furfural was mostly removed. T. fermentans could grow well with efficient lipid accumulation in the pretreated bagasse hydrolysate. After fermentation for 9 days, the biomass and lipid content reached 34.2 g / L and 39.9%, and the lipid yield was 13.7 g/L, which was higher than the corresponding value with molasses (12.8 g/L) or rice straw hydrolysate (11.5 g/L) as medium. GC analysis showed that the resulted lipid contained myristic acid, palmatic acid, palmitoleic acid, stearic acid, oleic acid and linoleic acid, which was similar to the composition of vegetable oils, indicating it can be used as feedstock for biodiesel production.The results of single factor experiments showed that the optimal fermentation time, carbon to nitrogen ratio (C/N), inoculum concentration, temperature and initial pH were 9 d, 168, 10%, 25℃and 7.5, respectively. Based on this, a 3 levels 4 factors Box-Behnken design was adopted to evaluate the effects of C/N ratio, inoculum concentration, initial pH and fermentation time on the lipid yield of fermentation by T. fermentans. A corresponding statistic mathematical model was established based on the experimental data, and then through the response surface analysis, the optimum fermentation conditions were obtained as follows: fermentation time 9 d, C/N ratio 165, inoculum concentration 11% and initial pH 7.6. Under the optimum conditions, a lipid yield of 15.8 g/L, which is very close to the predicted value of 15.6 g/L was obtained after fermentation for 9 d. The relative error was 1.41%, indicating that the established mathematical model can well simulate the actual fermentation process and is highly significant.To further improve the lipid yield and reduce the production cost, the effect of fed-batch culture in shaking flask on the fermentation was investigated. The lipid yield reached 20.7 g/L after fermentation for 13 d by fed-batch hydrolysate twice. Meanwhile, the effect of medium with small amount of molasses instead of expensive organic nitrogen on cell growth and lipid accumulation of T. fermentans was also examined. A relatively high lipid yield(12.4 g/L)was achieved with the hydrolysate containing 8.5 g/L molasses as substrate. Based on this, the fed-batch culture in 1 L bioreactor with the hydrolysate containing 8.5 g/L molasses as substrate was explored. After feeding hydrolysate three times and fermentation for 180 h, the lipid yield attained 24.6 g/L and the lipid production rate was 0.14 g/ (L?h), which was 112% higher than that of shaking flask fed-batch culture (0.066 g/(L?h)) .This study is of great significance to realize the high-value utilization of biomass resources and reduce the microbial oil production cost as well. It also helps to solve the bottleneck problem of oil raw material which hinders the large-scale production and wide application of biodiesel.
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