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微生物发酵制备丙酸的过程控制及工艺优化

By 刘寅

Abstract

<font size="3"><span style="font-family: 宋体">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</span><span><font face="Calibri"> Propionibacterium acidipropionici CGMCC1.2225 </font></span><span style="font-family: 宋体">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:</span></font><font size="3"><span style="font-family: 宋体">研究了培养基成分和环境条件对</span><span><font face="Calibri"> P. acidipropionici CGMCC1.2225 </font></span><span style="font-family: 宋体">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(</span><span><font face="Calibri">g/L</font></span><span style="font-family: 宋体">)</span><span><font face="Calibri">: </font></span><span style="font-family: 宋体">甘油</span><span><font face="Calibri"> 26.7</font></span><span style="font-family: 宋体">,葡萄糖</span><span><font face="Calibri"> 13.3</font></span><span style="font-family: 宋体">,酵母提取物</span><span><font face="Calibri"> 10</font></span><span style="font-family: 宋体">,胰酶大豆肉汤</span><span><font face="Calibri"> 5</font></span><span style="font-family: 宋体">,</span><span><font face="Calibri">K2HPO4 2.5</font></span><span style="font-family: 宋体">,</span><span><font face="Calibri">KH2PO41.5</font></span><span style="font-family: 宋体">;同时考察了一些环境因素(如温度、</span><span><font face="Calibri">pH </font></span><span style="font-family: 宋体">等)对丙酸发酵的影响。最终在</span><span><font face="Calibri"> 5 L </font></span><span style="font-family: 宋体">发酵罐水平上,在最优条件下,丙酸最高产量达到</span><span><font face="Calibri">21.2 g/L</font></span><span style="font-family: 宋体">,比优化前提高了</span><span><font face="Calibri"> 24.7%</font></span><span style="font-family: 宋体">。</span></font><font size="3"><span style="font-family: 宋体">采用</span><span><font face="Calibri">Box- Behnken </font></span><span style="font-family: 宋体">设计和响应面法(</span><span><font face="Calibri">RSM</font></span><span style="font-family: 宋体">),以</span><span><font face="Calibri">P. acidipropioniciCGMCC1.2225 </font></span><span style="font-family: 宋体">发酵产丙酸的</span><span><font face="Calibri">3</font></span><span style="font-family: 宋体">个关键因素(培养温度、</span><span><font face="Calibri">pH</font></span><span style="font-family: 宋体">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</span><span><font face="Calibri">pH</font></span><span style="font-family: 宋体">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</span><span><font face="Calibri">29.73</font></span><span style="font-family: 宋体">℃、</span><span><font face="Calibri">pH</font></span><span style="font-family: 宋体">值为</span><span><font face="Calibri">6.61</font></span><span style="font-family: 宋体">、接种量为</span><span><font face="Calibri">6.17%</font></span><span style="font-family: 宋体">(</span><span><font face="Calibri">v/v</font></span><span style="font-family: 宋体">),经过优化,丙酸产量提高了</span><span><font face="Calibri">33.1%</font></span><span style="font-family: 宋体">。</span></font><font size="3"><span style="font-family: 宋体">基于</span><span><font face="Calibri">P. acidipropionici CGMCC1.2225</font></span><span style="font-family: 宋体">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</span><span><font face="Calibri">/</font></span><span style="font-family: 宋体">葡萄糖摩尔比为</span><span><font face="Calibri">4 / 1</font></span><span style="font-family: 宋体">)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</span><span><font face="Calibri">90.4%</font></span><span style="font-family: 宋体">和</span><span><font face="Calibri">21.0%</font></span><span style="font-family: 宋体">,丙酸产率</span><span><font face="Calibri">0.572 g/g</font></span><span style="font-family: 宋体">,丙酸生产强度</span><span><font face="Calibri">0.152 g/L</font></span><span style="font-family: 宋体">&middot;</span><span><font face="Calibri"> h</font></span><span style="font-family: 宋体">。丙酸产量和生产强度分别提高了</span><span><font face="Calibri">20%</font></span><span style="font-family: 宋体">和</span><span><font face="Calibri">21%</font></span><span style="font-family: 宋体">。</span></font><font size="3"><span style="font-family: 宋体">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</span><span><font face="Calibri"> Candidarugosa BS-1</font></span><span style="font-family: 宋体">。补料分批发酵时,混菌发酵的丙酸产量达到了</span><span><font face="Calibri"> 54.2 g/L</font></span><span style="font-family: 宋体">,比</span><span><font face="Calibri"> P.acidipropionici CGMCC1.2225 </font></span><span style="font-family: 宋体">纯菌发酵提高了</span><span><font face="Calibri"> 46.1%</font></span><span style="font-family: 宋体">;混菌发酵的底物转化率为</span><span><font face="Calibri">0.729 g/g</font></span><span style="font-family: 宋体">,比纯菌发酵提高了</span><span><font face="Calibri"> 29.5%</font></span><span style="font-family: 宋体">;混菌发酵的生产强度为</span><span><font face="Calibri"> 0.151 g/L</font></span><span style="font-family: 宋体">&middot;</span><span><font face="Calibri">h</font></span><span style="font-family: 宋体">,比纯菌发酵提高了</span><span><font face="Calibri"> 46.6%</font></span><span style="font-family: 宋体">。这些结果表明,混合培养时,</span><span><font face="Calibri">C. rugosa BS-1 </font></span><span style="font-family: 宋体">可以增强菌株</span><span><font face="Calibri"> P. acidipropionici CGMCC1.2225 </font></span><span style="font-family: 宋体">对丙酸和乙酸等产物的耐受,而且能促进产</span></font><font size="3"><span style="font-family: 宋体">酸丙酸杆菌菌体生长和丙酸合成代谢。</span></font><font size="3"><span style="font-family: 宋体">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</span><font face="Calibri"> </font><span style="font-family: 宋体">丙酸发</span><font face="Calibri"> </font><span style="font-family: 宋体">酵的因</span><font face="Calibri"> </font><span style="font-family: 宋体">素和</span><font face="Calibri"> </font><span style="font-family: 宋体">发酵条</span><font face="Calibri"> </font><span style="font-family: 宋体">件进行</span><font face="Calibri"> </font><span style="font-family: 宋体">优化</span><font face="Calibri"> </font><span style="font-family: 宋体">,再结</span><font face="Calibri"> </font><span style="font-family: 宋体">合甘油</span><span><font face="Calibri">/</font></span><span style="font-family: 宋体">葡萄</span><font face="Calibri"> </font><span style="font-family: 宋体">糖共发</span><font face="Calibri"> </font><span style="font-family: 宋体">酵与</span><span><font face="Calibri"> P.acidipropionici CGMCC1.2225 </font></span><span style="font-family: 宋体">和</span><span><font face="Calibri">C. rugosa BS-1</font></span><span style="font-family: 宋体">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。</span></font><span><font size="3"><font face="Calibri">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 </font></font></span><span><font size="3"><font face="Calibri">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:</font></font></span><span><font size="3"><font face="Calibri">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.</font></font></span><font size="3"><span><font face="Calibri">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</font></span><span style="font-family: 宋体">℃</span><span><font face="Calibri">, pH of 6.61, and inoculum size of 6.17% (v/v). After optimization, the propionic acid yield increased by 33.1%.</font></span></font><span><font size="3"><font face="Calibri">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&middot;h) increased 20% and 21%, respectively, compared with optimal results from sole carbon sources (glycerol).</font></font></span><span><font size="3"><font face="Calibri">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&middot;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.</font></font></span><span><font size="3"><font face="Calibri">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.</font></font></span

Topics: 丙酸, 产酸丙酸杆菌, 响应面法, 甘油/葡萄糖共发酵, 混菌培养, 生物材料::生物基化学品, 环境工程
Year: 2010
OAI identifier: oai:ir.qibebt.ac.cn:337004/329
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