微生物发酵制备丙酸的过程控制及工艺优化
文献类型：学位论文
| 作者 | 刘寅 |
| 学位类别 | 博士 |
| 答辩日期 | 2010-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 朱建航 ; 咸漠 |
| 关键词 | 丙酸 产酸丙酸杆菌 响应面法 甘油/葡萄糖共发酵 混菌培养 |
| 学位专业 | 环境工程 |
| 中文摘要 | 丙酸是一种重要的精细化工产品和基本化工原料，在食品、饲料、农药、医药等领域应用广泛。生物法制备丙酸具有原料廉价易得、生产条件温和及产物为天然绿色产品等优点，是当前丙酸产业的研究热点。但是，生物法制备丙酸面临受终产物强抑制，丙酸发酵效率低和副产物（特别是乙酸）含量高等问题，导致其生产成本较高，无法规模化生产。鉴于此，本文从发酵过程控制、代谢工程调控及混菌协同作用入手，对 Propionibacterium acidipropionici CGMCC1.2225 丙酸发酵机理和过程优化控制进行了研究，以此解析丙酸合成代谢的调控机制，为丙酸发酵过程优化控制奠定了理论基础。现将主要研究成果归纳如下：研究了培养基成分和环境条件对 P. acidipropionici CGMCC1.2225 发酵合成丙酸过程的影响。通过优化碳源、氮源和碳氮比等营养条件，确定了最佳发酵培养基的组成为（g/L）: 甘油 26.7，葡萄糖 13.3，酵母提取物 10，胰酶大豆肉汤 5，K2HPO4 2.5，KH2PO41.5；同时考察了一些环境因素（如温度、pH 等）对丙酸发酵的影响。最终在 5 L 发酵罐水平上，在最优条件下，丙酸最高产量达到21.2 g/L，比优化前提高了 24.7%。采用Box- Behnken 设计和响应面法（RSM），以P. acidipropioniciCGMCC1.2225 发酵产丙酸的3个关键因素（培养温度、pH和接种量）为自变量，以丙酸产量为响应值，对上述因素的最佳水平范围进行了探讨与优化。实验结果表明，培养温度和pH对丙酸产量有显著性影响，并据此建立了相关的数学模型。得到的工艺参数的优选结果是：培养温度为29.73℃、pH值为6.61、接种量为6.17%（v/v），经过优化，丙酸产量提高了33.1%。基于P. acidipropionici CGMCC1.2225发酵单一碳源葡萄糖或甘油产丙酸过程中生物量及代谢产物变化规律，提出了采用葡萄糖和甘油双底物共发酵产丙酸的新策略，并通过实验证明其可行性。采用共发酵工艺，在最优条件（甘油/葡萄糖摩尔比为4 / 1）下，实现丙酸产量分别比利用单一葡萄糖和单一甘油碳源提高了90.4%和21.0%，丙酸产率0.572 g/g，丙酸生产强度0.152 g/L· h。丙酸产量和生产强度分别提高了20%和21%。考察了五株酵母菌和一株乳酸菌培养物的添加对产酸丙酸杆菌丙酸发酵过程的协同促进作用，从中优选出对丙酸合成有较强促进作用的菌株 Candidarugosa BS-1。补料分批发酵时，混菌发酵的丙酸产量达到了 54.2 g/L，比 P.acidipropionici CGMCC1.2225 纯菌发酵提高了 46.1%；混菌发酵的底物转化率为0.729 g/g，比纯菌发酵提高了 29.5%；混菌发酵的生产强度为 0.151 g/L·h，比纯菌发酵提高了 46.6%。这些结果表明，混合培养时，C. rugosa BS-1 可以增强菌株 P. acidipropionici CGMCC1.2225 对丙酸和乙酸等产物的耐受，而且能促进产酸丙酸杆菌菌体生长和丙酸合成代谢。通过本文研究，利用有关控制丙酸发酵作用机制的基础知识，通过对各种影响 丙酸发 酵的因 素和 发酵条 件进行 优化 ，再结 合甘油/葡萄 糖共发 酵与 P.acidipropionici CGMCC1.2225 和C. rugosa BS-1混菌发酵技术，可以开发出一个更经济的丙酸生物合成工艺过程。 |
| 英文摘要 | Propionic acid, an important fine chemical and chemical intermediate, is widely used in food, feed, pesticide, medicine and other fields. Currently, propionic acid biosynthesis is expected to be a promising option and hot topic due to its renewable and cheap raw sources, the mild production conditions, and the green products. Although there has been great interest in producing propionic acid from biomass via fermentation, the relatively low efficiency of propionic acid fermentation, high by-products concentration and high production costs have presented major barriers for economical applications. In this research, propionic acid fermentation control mechanism and process optimization studies, based on the regulation of metabolic engineering, fermentation process control, and synergistic interactions, were carried out. Main results are summarized as follows:Fermentation conditions such as carbon source and nitrogen source for P. acidipropionici CGMCC1.2225 were optimized. The optimal fermentation medium contained 26.7 g/L glycerol, 13.3 g/L glucose, 10 g/L yeast extract, 5 g/L tryptic soy broth, 2.5 g/L K2HPO4, 1.5 g/L KH2PO4. Several environment factors such as culture pH, temperature, and rotating speed, which affected the propionic acid production by P. acidipropionici, were investigated. The maximum production of propionic acid (21.2 g/L) was obtained when batch fermentation was conducted in a 5 L stirred bioreactor, which increased by 24.7% compared with that before being optimized.Response surface methodology (RSM) based on a three-factor Box-Behnken design of experiments was used to optimize the propionic acid yield. The critical factors selected for the investigation were temperature, pH and inoculum size. The results showed that the effect of culture temperature and pH on the propionic acid yield were significant. The optimized parameters were temperature of 29.73℃, pH of 6.61, and inoculum size of 6.17% (v/v). After optimization, the propionic acid yield increased by 33.1%.A novel glycerol/glucose co-fermentation strategy for propionic acid production, based on biomass and metabolites variation of fermentation process, was put forward and proved by experiments feasibility. Glycerol/glucose co-fermentation produced 21.0% more propionic acid than glycerol alone and 90.4% more propionic acid than glucose under the optimal conditions (4/1, mol/mol). The optimal molar ratio of glycerol/glucose at 4/1 (mol/mol) enhanced this production, with propionic acid yield (0.572 g/g) and productivity (0.152 g/L·h) increased 20% and 21%, respectively, compared with optimal results from sole carbon sources (glycerol).For the mixed culture, five species of yeasts and a lactobacillus strain were examined, and C. rugosa BS-1 was confirmed to enhance propionic acid production. In a fed-batch mixed culture of P. acidipropionici CGMCC1.2225 and C. rugosa BS-1, propionic acid yield (54.2 g/L), productivity (0.151 g/L·h) and substrate conversion efficiency (0.729 g/g) significantly increased (46.1%, 46.6%, and 29.5%, respectively), compared with those in pure cultures. These results indicate that mixed culture of C. rugosa BS-1 and P. acidipropionici CGMCC1.2225 could enhance the acid tolerance of strains, cell growth, and propionic acid biosynthesis.Glycerol/glucose co-fementation, the knowledge of the underlying mechanism in controlling propionic acid fermentation, and the mixed culture of P. acidipropionici and C. rugosa should allow us to develop an economical bioprocess for propionic acid production. |
| 学科主题 | 生物基化学品 |
| 语种 | 中文 |
| 公开日期 | 2011-08-29 |
| 源URL | [http://ir.qibebt.ac.cn//handle/337004/329]  |
| 专题 | 青岛生物能源与过程研究所_材料生物技术研究中心 |
| 推荐引用方式 GB/T 7714 | 刘寅. 微生物发酵制备丙酸的过程控制及工艺优化[D]. 北京. 中国科学院研究生院. 2010. |
入库方式： OAI收割
来源：青岛生物能源与过程研究所
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