| Geraniol is an acyclic monoterpene alcohol that is widely used in fragrances, cosmetic, food, medicines and other fields. It has an increasing market demand. Isolation from plant and synthesis from petrochemicals are two major ways for geraniol synthesis. However, the productions by both methods are subject to various problems, such as high cost, pollution environment, poor selectivity, and so on. Microbial production of geraniol has become a development trend due to cheap and renewable biomass as the material. This paper first focused on the construction of engineered E. coli which could synthesize geraniol using glucose as the raw material; then in the level of shake flask, fermentation conditions and fermentation medium were optimized to improve the yield of geraniol; finally in 5L fermenter, high yield of geraniol was obtained. Meanwhile fermentation production of 2500 tons of geraniol every year was designed.The main contents and results of the thesis include the following aspects:(1) The metabolic pathways of bio-synthesis of geraniol using glucose as raw material is constructed in E.coli through the expression of MVA pathway and geraniol synthase gene by molecular biology technique. By gas chromatography-mass spectrometry (GC-MS) detection, it was determined that the fermentation product was the geraniol. The quantitative detection method of geraniol was established through the use of gas chromatography (GC) and the quantitative standard cure was built.(2) The optimization of shake flask fermentation conditions and the fermentation medium:The one-variable-at-a-time was used to optimize fermentation conditions. The fermentation conditions optimized were:induction time of OD600nm 1.0, IPTG concentration of 1.0mM, induction temperature of 30℃, pH 6.0 and the speed of 180r/min and they would be used in medium optimization experiment. The response surface method was used to optimize fermentation medium. Firstly, screening the key factors of the medium affecting the yield of geraniol was done by Plackett-Burman design method; then using the steepest ascent method, it could make the concentration of the key factors close to the optimum range; finally by central composite design, the optimal concentration of the key factors and the regression model were determined. The results showed that ferric ammonium citrate, magnesium sulfate and trace element solution were key factors affecting the production of geraniol. The optimal concentration of key factors were:ferric ammonium citrate 0.13g/L, magnesium sulfate 17.6mmol/L and trace elements 16.7ml/L. The optimized production of geraniol was from the initial 50.12mg/L increased to 223.24mg/L, increasing 4.45 times compared to the initial fennentation conditions and medium.(3) The study of 5L fermenter fermentation technology:The fermentation technology of geraniol was established in 5L fermenter level. Because geraniol has antimicrobial activity and volatility, diphasic fennentation was used in 5L fermenter, which meant extractant (isopropyl myristate) was added in the fermentation process. So fermentation and separation were achieved simultaneously, to achieve a stable production of geraniol. After induction for 48h, the OD600nm of bacteria reached about 45, the concentration of geraniol was about 2g/L, and the yield was 3.5%(theoretical yield of 28.5%).(4) Industrial production of bio-geraniol was designed. Diphasic fermentation was applied in production process, and the goal was to produce 2500 tons of geraniol every year(purity>90%), and the yield can reach 18%(theoretical yield of 28.5%). The design included design of process, selection of major equipments and the drawing of PFD. |
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