| Biodiesel is an important renewable energy, which synthesized by catalytic conversion of oils and short chain alcohols (such as fatty acid methyl ester, FAME/fatty acid ethyl ester, FAEE). Biodiesel have been widely applied in many countries. About14millions ton biodiesel was produced in the world in2008, and the worldwide demand for biodiesel is growing at a fast speed in recent years. Currently, high cost of raw materials and complex processes are main constraints on biodiesel production. Therefore, how to produced biodiesel directly by fermentation and built a new biological routine alternative to petroleum diesel and existing biodiesel production technology are very urgent.In this thesis, engineered yeast strain capable of direct production of long-chain biodiesel was constructed and its fermentation conditions were optimized. Medium-chain fatty acid ethyl esters synthesized by immobilized lipase and directly by engineered bacteria were studied.1. A strain of Saccharomyces cerevisiae was genetically engineered to produce long-chain biodiesel directly. The recombinant plasmid YEp352-PLC was transformed into S.cerevisiae and the transformants were screened by nutritional supplement. The results showed that a42kDa protein was expressed in recombinant S.cerevisiae and the specific activity of the lipase was12.12U/mg. The content of long-chain fatty acids exceeded92%in extracted lipid. After96h fermentation culture, the yield of FAEEs reached3.7mg/g (DCW).2. Response surface method was applied to optimize the fermentation conditions for lipid production by S.cerevisiae. Under the optimized fermentation conditions, a lipid production of14.55%was achieved which was about2-fold of the control.3. Uniform design was used to optimize the cofermentation conditions of lipid and ethanol production by S. cerevisiae. Effects of aeration rate, agitation rate, and glucose feeding on lipid and ethanol production were investigated. Two equations with90%correlation were obtained by nonlinear fitting. By analysis, the stirring speed was observed to exert more influence than other factors on lipid and ethanol production. The optimized fermentation conditions for lipid and ethanol production was achieved by calculation and verified by experiments.4. Fermentation conditions for FAEEs production by engineered S. cerevisiae were optimized. On the basis of the favored fermentation condition for lipid production, a high FAEEs yield of11.4mg/g (DCW) was attained with three-stepwise4%(V/V) ethanol addition.5. A new process for medium-chain biodiesel synthesis with camphor tree seed oil was provided. Microwave assisted extraction was implied to extract camphor tree seed oil and the extraction conditions was optimized by orthogonal experiment. The results showed that the rate of oil extraction exceeded90%and the predominant fatty acid composition were capric acid (53.4%) and lauric acid (38.7%), which content of medium-chain fatty acids reached94%of the total fatty acids. Secondly, the immobilized Candida sp.99-125lipase was used to catalyze camphor tree seed oil for biodiesel synthesis. A FAEEs yield of93.5%was obtained after optimization of the synthetic process conditions, the water content of10%(wt), enzyme loading15%(wt), oil/ethanol molar ratio1:3.2,9stepwise ethanol addition (intervals2.67h), reaction temperature40℃, shaking speed170rpm, reaction time24h.6. An engineered bacteria capable of medium-chain fatty acid production was constructed. ccFatB from Cinnamonum camphora was cloned and transformed into E. coli. By fermentation, the yield of myristic acid was approximately52mg/L, which is10.6%of the total fatty acid content.7. An engineered bacteria capable of medium-chain biodiesel production was constructed. atfA was cloned from Acinetobacter baylyi. A dual-gene expression vector pETDuet-ccFatB-atfA was constructed and transformed into E. coli. FAEEs were intracellularly synthesized and the maximum yield of myristic acid ethyl ester reached2.1mg/L by adding4%(V/V) ethanol, which is10%of the total ester content. |
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