| 题名 | 扩张床原位提取丙酸耦合维生素B12发酵的研究 |
| 作者 | 王鹏 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-06-05 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 苏志国 |
| 关键词 | 丙酸 维生素B12 费氏丙酸杆菌 扩张床吸附 产物原位提取 |
| 其他题名 | In-situ Propionic Acid Recovery by Expanded-bed Adsorption Coupled with Vitamin B12 Fermentation |
| 学位专业 | 生物化工 |
| 中文摘要 | 微生物厌氧发酵生产维生素B12(ado-cobalamin, ado-cbl)同时会产生大量丙酸。传统发酵一般是进行细胞分离后提取胞内维生素B12而将含丙酸的发酵清液排放。本文建立起一种新型的以扩张床原位吸附为特征的生物反应过程，实现了丙酸/维生素B12的联合发酵。对费氏丙酸杆菌维生素B12发酵过程的代谢进行了研究。通过带有二极管阵列检测器的HPLC和离子阱质谱的分析，发现了一种维生素B12关键中间产物，确定为腺苷钴啉醇酰胺(ado-cobinamide, ado-cbi)。Ado-cbi在5，6-二甲基苯并咪唑(DMB)的诱导下转化为维生素B12。建立了ado-cbi控制策略，指导发酵过程中DMB的添加，从而优化了厌氧生产维生素B12的发酵工艺。进行了100L放大实验，通过ado-cbi控制策略得到的ado-cbl预测值与测量值之间的偏差可以控制在1%以内。采用离子交换吸附法分离发酵液中的丙酸。通过静态吸附实验，筛选出了一种弱碱性阴离子交换介质IPE-A，该介质在流化床中对发酵液中丙酸的吸附量达到51 mg/g，并且选择性较好，在对发酵液吸附丙酸的过程中受发酵液环境影响较小。对该介质进行热力学初步研究，表明，丙酸在IPE-A介质上的吸附遵循Langmuir吸附等温方程，拟合后线性系数达到0.998。对丙酸抑制发酵的动力学进行了研究。考察了起始培养基中不同浓度丙酸对于细胞生长和丙酸合成的影响，确定了产物原位提取过程中分离单元的切入点为丙酸浓度10 g/L。分批发酵实验对丙酸原位提取过程中设备模式进行了筛选，确定了外部-直接模式的产物原位提取设备可以极大提高丙酸浓度﹑底物转化率以及底物消耗速率。在确定了介质和设备模式的基础上，选用符合外部-直接模式的扩张床设备进行了从费氏丙酸杆菌发酵液中提取丙酸的吸附和解吸工艺研究，优化工艺参数为：发酵液pH 5.0、上料流速为 25 mL/min、吸附时间20 min，发酵液与介质体积比为10:1时介质动态吸附量为58 mg/g；解吸剂NaOH 3 mol/L、流速1.32 mL/min时解吸率达到93.2%。进行了IPE-A介质的生物相容性实验。建立起了一种新型的以扩张床原位吸附为特征的生物反应过程，将该过程应用在丙酸/维生素B12联合发酵中，可以实现半连续方式将发酵液中的丙酸有效提取，从而消除了丙酸对发酵过程的抑制。通过160 小时的补料分批发酵实验，获得了53 g/L的丙酸和43 mg/L的维生素B12，底物转化得率分别达到0.66 g/g 和0.54 mg/g。 |
| 英文摘要 | Fermentation production of vitamin B12 (ado-cobalamin, ado-cbl) produces great amount of propionic acid simultaneously that is the by-product of fermentation. Traditional fermentation always extracts intracellular vitamin B12 and discharges fermentation liquor containing propionic acid. In this work, a novel bioprocess by in situ expanded bed adsorption (EBA) was designed and set up. The aim was for development of a simple and economical production of propionic acid and vitamin B12 in a single process. The anaerobic biosynyhesis route of vitamin B12 by Propionibacterium freudenreichii was investigated. An important intermediate of ado-cbl separated from the cell extract was detected and identified as adenosylcobinamide (ado-cobinamide, ado-cbi) by high-performance liquid chromatography (HPLC) coupled to an ultraviolet diode array detector (UV-DAD) and ESI mass spectrometry analysis. Ado-cbi can be converted to ado-cbl when linked to DMB, which is an essential compound for ado-cbi bioconversion. Ado-cbi is useful not only in determining ado-cbl concentration in the fermentation process but also in serving as an effective compound to guide DMB incorporation for the harvest of the maximum ado-cbl concentration. Accordingly, with scaling up to 100 liters’ fermentation, the experimental results showed that the discrepancy between predictive value and measure value was less than 1% using the developed prediction technique. The adsorption behavior of propionic acid from broth was studied by using ion-exchange method. Through the resin selection experiments, the IPE-A resin showed the best capability among kinds of resins (adsorption capacity reached 51 mg/g in fluidized bed adsorption). The IPE-A resin was chosen to investigate adsorption thermodynamics of propionic acid. The results showed that the Langmuir adsorption isotherm was suitable for describing the adsorption process (R2: 0.998). Relevant product inhibition kinetics was discussed. The results demonstrated that at elevated concentrations, propionic acid inhibited the growth and biotransformation activity of Propionibacterium freudenreichii CICC 10019 and it was essential to keep the aqueous propionic acid concentration below 10 g/L for in situ propropionic acid recovery (ISPR) process. A batch study, in which three ISPR configuration mode designs were compared, was conducted. The comparison indicated that employing an external-direct ISPR mode had significant advantages over other modes in terms of increased concentration and product yield, with a corresponding decrease in substrate consumption. Based on the selected resin and equipment mode, the expanded bed adsorption and desorption process of propionic acid were investigated systematically. Under optimized operation, the dynamic adsorption capacity could reach 58 mg/g when the ratio of broth to resin was 10:1 (volume), pH 5.0, flow velocity 25 mL/min for 20 min. The desorption rate could reach 93.2% when the elution rate was 1.32 mL/min with 3 mol/L NaOH. A new type of in situ product recovery (ISPR) technique of expanded bed adsorption (EBA) was studied to simultaneously produce extracellular propionic acid and intracellular vitamin B12 by Propionibacterium freudenreichii CICC 10019. Resin screening experiments showed that the IPE-A resin had the best biocompatibility and highest adsorption for propionic acid. Through the EBA bioreactor, propionic acid could be recovered efficiently by semi-continuous recirculation of the unfiltered broth, which eliminated the feedback inhibition of propionic acid. Fed-batch fermentation was carried out using the EBA system, resulting in a propionic acid concentration of 53 g/L and vitamin B12 concentration of 43 mg/L at 160 h, which corresponded to product yields of 0.66 g/g and 0.54 mg/g, respectively. |
| 语种 | 中文 |
| 公开日期 | 2013-09-25 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1788]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 王鹏. 扩张床原位提取丙酸耦合维生素B12发酵的研究[D]. 中国科学院研究生院. 2012. |
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