【作者】 马立娟；

【导师】 闻建平；

【作者基本信息】 天津大学 ， 生物化工， 2009， 博士

【摘要】 随着石油资源日益枯竭,工业模式逐渐由石油炼制向生物炼制转变。本文针对甘油生物炼制1,3-二羟基丙酮(DHA)过程中存在的底物投料浓度低、DHA生产效率低及后续提取分离工序复杂、收得率低等问题,对氧化葡萄糖酸杆菌(Gluconobacter oxydans)转化甘油生产DHA进行了产品开发研究。为提高Gluconobacter oxydans (G. oxydans)转化甘油生产DHA的能力,利用He-Ne激光辐照该菌,在21 mW 19~21 min剂量范围内,获得了较高的正突变率。通过甘油和DHA耐受性筛选,最终获得具有高DHA生产能力和稳定遗传特性的正突变株GM51。通过对GM51和出发菌株的性能对比研究,甘油初始浓度为100 g/L时,GM51中甘油脱氢酶活力比出发菌株提高了75.17%;7 L发酵罐中发酵周期缩短了10~15 h,DHA对甘油的得率达到了92%,比出发菌株提高了77.6%,体积生产速率由1.29 g·L-1·h-1提高到了2.29 g·L-1·h-1。在单因素实验结果基础上,采用响应曲面法确定了最佳发酵培养基组成(g/L)为:酵母膏2.39,蛋白胨2.18,甘油100.0,(NH4)2SO4 2.0,MgSO4·7H2O 1.0,K2HPO4·3H2O 2.62,MnSO4·1H2O 0.53,CaCO3 2.5,CaCl2 1.25。培养条件:接种量5%,初始pH5.9,摇床转速210 rpm,培养时间45 h。在最佳条件下进行G. oxydans GM51发酵,DHA的产量为82.81 g/L,比优化前提高了41%。在7 L发酵罐中考察了DHA间歇发酵、分批补料发酵和流加发酵特征。实验表明,分批补料发酵可解决间歇发酵后期营养物质耗竭导致的DHA合成受限问题;连续补料发酵可弥补分批补料发酵引起的发酵液环境参数的瞬间剧烈变化,使DHA产量达到123.8 g/L,较间歇发酵提高约36%。建立了适当的G. oxydans GM51间歇发酵生产DHA过程中菌体生长、底物消耗及产物生成动力学模型,并可较准确的预测发酵过程中细胞生物量、产物和底物的变化趋势。利用天然高分子絮凝剂壳聚糖和活性炭对DHA发酵液进行预处理后,醇沉去除发酵液中蛋白、核酸、多糖等大分子及盐,实验结果显示,经上述工艺后菌体和蛋白的去除率分别达到100%和98.6%,处理前后溶液总电导率下降96.4%,DHA损失8.6%。经乙醇中溶析结晶后DHA的结晶得率为81%。采用以上确定的整个提取分离工艺,所得DHA成品的收得率大于72%,纯度达99.8%,达到了市售商品质量规格要求。更多还原

【Abstract】 With the depletion of petroleum resources, petroleum refining is being replaced by biorefining gradually. To solve the problems during the production of 1,3-dihydroxyacetone (DHA) from glycerol by biorefinery such as low initial substrate concentration, low production efficiency, complicated subsequent extraction process, low DHA yield and so on, the whole production technology of DHA from glycerol by Gluconobacter oxydans has been researched in this work.In order to increase the DHA production capacity of G. oxydans from glycerol, He-Ne laser was used to irradiate G. oxydans and among the dose range of 21 mW 19~21 min, higher positive mutant rate could be obtained. After the tolerance experiment of glycerol and DHA, mutant G. oxydans GM51 with high DHA production capacity and stable heredity was obtained finally. By comparison the performance of mutant GM51 and wild strain, the activity of glycerol dehydrogenase in GM51 was 75.17% higher than that in wild strain when the initial glycerol concentration was 100 g/L. The culture cyclic of mutant GM51 had been shortened by 10~15 hours and the yield of DHA had been increased by 77.6% during the culture in 7 L fermenter. The DHA productivity had been improved from 1.29 g·L-1·h-1 to 2.29 g·L-1·h-1.Based on the experimental results of single factor, response surface methodology was used to determine the optimal fermentation medium composition (g/L): yeast extract 2.39, peptone 2.18, glycerol 100.0, (NH4)2SO4 2.0, MgSO4·7H2O 1.0, K2HPO4·3H2O 2.62, MnSO4·1H2O 0.53, CaCO3 2.5, CaCl2 1.25; inoculum concentration 5% (v/v), initial pH 5.9, rotational speed of the cradle 210 rpm, culture time 45 h. Under the optimal conditions, the yield of DHA was 82.81 g/L by mutant GM51, which was 41% higher than that before optimization.The characteristics of batch, semi-fed-batch and fed-batch fermentation in a 7 L bioreactor were investigated. In the late phase of the batch fermentation, DHA biosynthesis would be repressed due to the exhaustion of nutrition substances. Semi-fed-batch could improve the substrate supply and increase DHA productivity. Fed-batch fermentation could prevent the dramatic transient phenomenon in fermentation environment aroused by semi-fed-batch culture; therefore the DHA yield was further increased by about 36% to 123.8 g/L. A proper kinetic model for cell growth, substrate consumption and DHA formation by mutant GM51 was established and it could predict the variation tendency of biomass, substrate and product correctly.Natural macromolecular flocculant—chitosan was used to remove cell and protein and absorbent charcoal was used to remove the pigment in the fermentation broth. Protein, nucleic acid, polyoses and salt were removed further by alcohol precipitation. The experimental results indicated that 100% cell and 98.6% protein were removed respectively through the above process, and most salt was separated and removed by crystallization with 96.4% decrease of the total conductivity. Meanwhile, DHA loss was 8.6% during the process. After crystallization in alcohol, the crystallization yield of DHA was 81%. By using the above separation technology, the yield of DHA was more than 72% and the purity of DHA was up to 99.8%. And the DHA product produced in this work has been met the quality requirements compared with DHA of commercial grade.更多还原