Formation Characteristics Of NaCS/PDMDAAC Biocapsule And Production Of 1,3-propanediol By Encapsulated Cells

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.