【作者】 赵静波；

【导师】 吴兆亮；

【作者基本信息】 河北工业大学 ， 化学工艺， 2004， 硕士

【摘要】 D-核糖具有重要的生理功能，在食品和医药工业中的应用越来越广泛，目前其最主要的生产方法是微生物发酵法。针对国内外D-核糖发酵的研究现状及发展趋势，本文提出了评价诱变效果的新指标，并确定了最适诱变剂量；研究了初始碳氮源浓度和各操作条件对D-核糖发酵的影响并对其进行了优化，在此基础上着重研究了摇瓶和发酵罐中的补料工艺；最后建立了D-核糖补料分批发酵的动力学模型。 1．D-核糖生产菌的紫外诱变表征及工艺。确定合适的诱变剂量是D-核糖生产菌紫外诱变育种的一个重要原则，用传统的评价指标无法确定最适诱变剂量。提出新的评价指标——平均正突变幅度，给出了其定义及计算方法，并将其应用于D-核糖生产菌的紫外诱变中，得到在15W紫外灯、距离25cm条件下不同照射时间时的平均正突变幅度，并结合致死率曲线确定20s为最佳紫外照射时间。 2．初始碳氮源浓度对D-核糖发酵的影响及摇瓶补料工艺。研究了初糖浓度对D-核糖发酵的影响，并确定了较为适宜的初糖浓度。针对底物抑制现象，研究了补糖工艺对D-核糖发酵的影响，确定发酵24h后补加50g／L的葡萄糖为较优的补糖工艺，在此工艺条件下D-核糖产量可提高48.3％。研究了氮源和初始氮源浓度对D-核糖发酵的影响，在此基础上对摇瓶中补加氮源工艺进行了研究，确定了较优的补加氮源的工艺为：发酵60h后补加3g／L的硫酸铵，在此工艺条件下D-核糖产量可提高8.2％。 3．操作条件对发酵罐中D-核糖发酵的影响及其优化。通过单因素实验研究操作条件对发酵罐中D-核糖发酵的影响，得到了不同温度、pH、搅拌转速和溶氧下的菌体生长、葡萄糖消耗和D-核糖生成曲线，结果表明搅拌转速对D-核糖发酵影响最大。综合考虑D-核糖产量、转化率和生产强度，确定了较优的操作条件为：通气量0.082m³／h，罐压0.02MPa，培养温度36℃，不控制pH，搅拌转速700r／min。在此条件下，D-核糖产量、转化率和生产强度分别为47.89g／L、0.3193g／g和0.8869 g·L^-1·h^-1。 4．发酵罐中D-核糖的补料分批发酵。研究了补料时间、补料成分、结合两段控制的补料和补料方式对D-核糖发酵的影响，确定了D-核糖补料分批发酵的较优工艺条件为：通气量0.082m³／h，罐压0.02MPa，培养温度36℃，发酵25h前搅拌转速为900r／min，25h后搅拌转速为700r／min，采用间歇递减流加方式，发酵到30h，开始每隔5h流加浓度为800g／L的葡萄糖补料液，共流加3次，每次分别为50mL、40mL和30mL。在此工艺条件下，发酵时间为50h，最终D-核糖产量、残糖浓度、转化率和生产强度分别为61.97g／L、33g／L、0.3146g／g和1.239 g·L^-1·h^-1。 5．D-核糖补料分批发酵的动力学。建立了D-核糖补料分批发酵的菌体生长、产物生成和底物消耗的动力学模型，确定了模型参数，模型计算值与实验值基本吻合，模型能较好的反映发酵过程。模型分析表明，发酵15h以前是菌体大量积累的阶段，葡萄糖的消耗主要用于菌体生长；发酵15h以后是D-核糖大量生成的阶段，葡萄糖的消耗主要用于D-核糖生成。产物生成与菌体生长呈部分偶联关系，但偶联程度很小。更多还原

【Abstract】 D-ribose plays an important physiological role and has more and more extensive usages in foodstuff and medicine fields. Now the most useful way for D-ribose production is microorganism fermentation. Aiming at research actuality and development trend of D-ribose fermentation, in this thesis a new evaluating index for mutation was proposed and the optimal mutagen dosage was determined. Effects of initial carbon source concentration, nitrogen source concentration and operation conditions on D-ribose fermentation were studied. Based on the optimization of these technology conditions feeding process of D-ribose fed-batch fermentation was studied emphatically. At last kinetic model of D-ribose fed-batch fermentation was established.1. Representation and technological process of ultraviolet induced mutation of strain for producing D-ribose. The use of appropriate mutagen dosage is important in UV induced mutation of D-ribose producing strain. But it was hard to find the optimal mutagen dosage with traditional evaluating index. So a new evaluating index-average positive mutation extent was proposed and its definition and calculating method were given. When it was used in UV induced mutation of D-ribose producing strain that was 25cm below a 15w UV lamp, the value of average positive mutation extent of different irradiation time was got. Combining the average positive mutation extent with lethal rate curve 20s was found as the best irradiation time.2. Effects of initial carbon source concentration and nitrogen source concentration on D-ribose fermentation and feeding process in flask. The effects of initial glucose concentration on D-ribose fermentation were studied. Appropriate initial glucose concentration was obtained. In order to avoid substrate inhibition glucose feeding process was studied. Adding 50g/L glucose after 24h fermentation was established as the appropriate method to increase D-ribose concentration. With this method the ultimate D-ribose concentration was 48.3% higher than thai of the comparison group. The effects of nitrogen source and initial nitrogen source concentration on D-ribose fermentation were studied. Based on these researches nitrogen source feeding process in flasks was studied and adding 3g/L ammonium sulphate after 60h fermentation was established as the appropriate method. With this method the ultimate D-ribose concentration was 8.2% higher than that of the comparison group.3. Effects of operation conditions on D-ribose fermentation in fermentor and optimization of operation conditions. Effects of operation conditions on D-ribose fermentation in fermentor were studied by means of single factor experiments. Cell growth, glucose consume and D-ribose formation curve of different temperature, pH, agitation speed and dissolved oxygen were obtained. The results showed that agitation speed has the greatest effect on D-ribose fermentation. By considering D-ribose concentration, yield and productivity comprehensively the optimal operation conditions were obtained: aeration rate 0.082m3/h, fermentor pressure 0.02MPa, culture temperature 36℃, without pH control and agitation speed 700r/min. Under such operation conditions the D-ribose concentration, yield and productivity were 47.89g/L, 0.3193g/g and 0.8869 g.L-1.h-1 respectively.4. D-ribose fed-batch fermentation in fermentor. Effects of feeding time, feeding composition, feeding with two-stage control and feeding model on D-ribose fermentation were studied. Optimal conditions for D-ribose fed-batch fermentation were determined: aeration rate 0.082m3/h, fermentor pressure 0.02MPa, culture temperature 36℃, agitation speed 700r/min before 25h and 900r/min after 25h, feeding 800g/L glucose solution by successive batch feed model, feeding time 30h after fermentation, interval time 5h, amount of feeding 50mL, 40mL and 30mL respectively in 3 times. Under such conditions fermentation time was 50h and D-ribose concentration, residual glucose concentration, yield and productivity were 61.97g/L, 33更多还原