【作者】 陈国；

【导师】 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2006， 博士

【摘要】 生物微胶囊作为最常用的固定化活细胞技术,受人工器官、基因治疗及生物化工等领域发展的推动,引起研究者的广泛兴趣。聚电解质成膜反应以其温和快速的特点成为制备生物微胶囊的最重要方法。本文以纤维素硫酸钠（NaCS）/聚二甲基二烯丙基氯化铵（PDMDAAC）为聚电解质成囊反应的代表,提出了改变胶囊大小及膜特性的方法,研究了聚电解质成膜反应特性与聚电解质溶液特性之间的关系,并应用NaCS/PDMDAAC微胶囊固定化Candida krusei和Klebsiella pneumoniae,最后通过二过程的串联实现了葡萄糖到1,3-丙二醇的转化。 首先,建立了可控制胶囊大小的气流微囊发生器。该装置通过调节气速来控制液滴大小,进而控制胶囊的大小,可有效调节胶囊直径在1.0mm到2.6mm之间,并建立了气速大小与胶囊大小之间的关系式。 其次,在纤维素硫酸钠（NaCS）—聚二甲基二烯丙基氯化铵（PDMDAAC）胶囊体系的基础上,通过引入羧甲基纤维素（CMC）,制备得到一种新型的三组分CMC-NaCS/PDMDAAC胶囊。以开始收缩时间、直径、膜厚和机械强度等宏观特性为胶囊的考察对象,研究了不同反应物浓度、不同反应条件下,制备得到的CMC-NaCS/PDMDAAC胶囊的特性。以强度为衡量标准,CMC-NaCS/PDMDAAC胶囊的较优的反应条件为:35-40g/L NaCS,6-8g/L CMC,60g/L PDMDAAC在25℃下反应30-40分钟。并详细研究了小分子物质(葡萄糖、甘油、酪氨酸和维生素B₁₂)透过胶囊的扩散特性,初步考察了胶囊稳定性及菌的泄漏情况。 第三,在对CMC-NaCS/PDMDAAC胶囊研究的基础上,详细考察了添加其它类型物质（中性小分子、中性聚合物、小分子盐）对NaCS/PDMDAAC体系胶囊的影响,并对影响方式进行了定性的分析。通过测定添加物对聚电解质稀溶液比浓粘度、Zeta电位,以及对聚电解质阴阳离子反应后悬浊液浊度、Zeta电位的影响,将稀溶液特性与制备胶囊过程关联。提出了改善胶囊特性的二种方式:一是添加物参与聚电解质体系的反应（如CMC引入NaCS/PDMDAAC体系）,二是添加物对聚电解质在溶液中的伸展特性要有强烈的影响(如PEG6000和NaCl引入更多还原

【Abstract】 Bioencapsulation as one of cell immobilization techniques, has greatly extended its application in the realm of artificial organ, gene therapy and bioengineering, and arouse lots of attention. Biocapsules are mainly formed by mild reaction between polyanion and polycation. Sodium cellulose sulphate （NaCS） / poly （dimethyldiallylammonium chloride） （PDMDAAC） system as the representative of polyelectrolyte complex capsules, was studied in detail in this dissertation. The methods of making capsules with controllable characteristics such as size, membrane thickness, etc. were investigated. The relationship between characteristics of capsules and properties of dilute polyelectrolytes solution, especially under the condition of other molecules introduced, was elucidated. The capsules were also used to immobilize Candida krusei and Klebsiella pneumoniae. And the reactors with encapsulated C. krusei and K. pneumoniae were stringed to produce 1,3-propanediol （1,3-PD） from glucose by fermentation, in which the cell growth and metabolism were studied carefully.First, a practical device was designed to control the size of capsule. The size of capsule was controlled by the size of drop from nozzle which could be adjusted by velocity of airflow. The diameter of capsule could be controlled between 1.0 and 2.6mm. And a formula was constructed to correlate size of capsule and velocity of air at the nozzle.Second, carboxymethyl cellulose （CMC） was added to NaCS solution to form co-polyanion solution, and then dropped to PDMDAAC solution to form CMC-NaCS/PDMDAAC capsules. The membrane thickness and mechanical intensity of capsules could be adjusted by changing the CMC and NaCS concentration in co-polyanion solution. In terms of mechanical intensity as criterion, the optimum operation conditions for preparation of CMC-NaCS/PDMDAAC capsules were recommended as 35-40g/L NaCS, 6-8g/L CMC, 60g/L PDMDAAC and polymerization for 30-40 min. Diffusion of substances with low molecular weight into capsules was investigated, and diffusion coefficients of glucose, glycerol, tyrosine and vitamin B₁₂ in membrane were calculated by using the developed model. The stability of capsules and cell leakage properties from capsules were also investigated.Third, unionized small molecule, soluble unionized polymer and electrolyte were individually added into NaCS solution to check their effects on NaCS/PDMDAAC capsule formation. Simultaneously the effects of molecules added on reduced viscosity and zeta potential of dilute NaCS solution were investigated in detail. And the turbidity and zeta potential of solution formed by reaction between dilute NaCS and PDMDAAC solution was also studied. By using the lattice model of solution to correlate the properties of dilute polyelectrolyte solution and capsule characteristics, two methods for controlling the polyelectrolyte complex （PEC） membrane properties were proposed. One is the introduced molecule joining the reaction between polyions such as CMC, the other is the introduced molecule heavily impacting the polyelectrolyte molecule extension in solution.Fourth, NaCS/PDMDAAC capsules were used to immobilize osmotolerant Candida krusei to produce glycerol. The effects of osmoregulators such as PEG4000, NaCl and glycerol on cell growth and glycerol production were investigated firstly. Results showed that osmoregulators inhibited the cell growth and improve production of glycerol. The maximum net glycerol concentration of 99g/L, glycerol/glucose mass yield of 55% and glycerol productivity of 22gL"1day"1, were obtained at the initial glycerol concentration of 80g/L as osmoregulator. The cell density in capsules, glucose and glycerol concentration in medium and capsules were carefully measured. Two logarithm growth stages were observed which caused by metabolism pathway transference due to low substrate concentration in capsules. To overcome the problems above, PEG4000 which could not permeate out of capsules was co-entrapped with C. krusei to create a continuous local high osmotic stress environment. By this way, the fermentation time was shortened and the glycerol concentration increased by 25%, compared with common encapsulation. The results were reproduced in air-lift reactor for five batches. The steady glycerol concentration, glycerol/glucose mass yield and glycerol productivity were 65g/L, 35% and 22gL"1day"1, respectively.Fifth, NaCS/PDMDAAC capsules were used to immobilize facultative anaerobe of Klebsiella pneumoniae to produce l,3-propanediol（l,3-PD）. In shake flask, biomass was enriched to 6g/L in microcapsules after 12h compared with 2.5g/L in free culture after 27h. Glycerol consumption was accelerated by immobilization. And1,3-PD/glycerol yield of 0.61mol/mol by immobilized cells was higher than 0.55mol/mol by free cell culture. The concentration of glycerol in capsules kept in a low value due to the transfer limitation caused by membrane of capsule, which could effectively avoid substrate inhibition. The process was operated in a packed-bed reactor provided with medium containing different concentration of glycerol, eg. 40g/L, 60g/L, 80g/L and 120g/L. Results indicated that cell could tolerate higher concentration of glycerol than free cells and higher 1,3-PD/glycerol molar yield was obtained. When medium with glycerol concentration of 120g/L was provided, 1,3-PD concentration, 1,3-PD/glycerol molar yield and 1,3-PD productivity were 63.1g/L, 0.65mol/mol and 5.74gL’’h"1, respectively. In fed batch fermentations, byproducts of ethanol and acetic acid would stop the fermentation, and the fermentation could continue after fresh medium was provided. In continuous fermentations in the packed bed reactor, at low dilution rate 1,3-PD concentration and 1,3-PD/glycerol molar yield was high, and the 1,3-PD productivity was low;and at high dilution rate the results were inverse.Finally, the air-lift reactor with encapsulated Candida krusei and the column packed with encapsulated Klebsiella pneumoniae were successfully strung up to produce 1,3-PD from glucose, and the final 1,3-PD/glucose molar yield was 0.295. The acetic acid and ethanol produced in process affected the growth of Klebsiella pneumoniae and the production of 1,3-PD.更多还原