In this study newly designed hollow fiber bioreactors for aerobic whole cell immobilization were introduced. First, the dual hollow fiber bioreactor consisting of outer silicone membrane for oxygen transport and three inner isotropic polypropylene hollow fibers for liquid nutrient transport were designed and its performance was examined by cultivating the aerobic microbes such as $\underline{E}$. $\underline{coli}$, $\underline{Nocardia}$ $\underline{mediterranei}$, $\underline{Aspergillus}$ $\underline{niger}$ and $\underline{Streptomyces}$ $\underline{griseus}$ (chapters 2-6). Second, aerobic hollow fiber bioreactors with internal aeration circuits were newly designed and their performance was examined by cultivating the fungus, $\underline{Aspergillus}$ $\underline{niger}$, producing citric acid.
In chapter 2, the dual hollow fiber bioreactor carried out by simulation. Oxygen penetration depth and volumetric oxygen uptake rate were not much affected by the silicone membrane thickness and a major portion of oxygen transfer resistance existed in the immobilized cell layer.
In chapter 3, the cell density and packing characteristics of $\underline{E}$. $\underline{coli}$ in a dual hollow fiber bioreactor were investigated. The cells have grown forming the layer like animal tissue in a nearly 100% packing density. The dry biomass density was 550 gram/liter of void volume for cell growth, which was the highest among the biomass densities ever reported.
In chapter 4, continuous production of rifamycin B was studied using $\underline{Nocardia}$ $\underline{mediterranei}$ immobilized in a dual hollow fiber bioreactor. In the reactor operation the volumetric productivity based on the volume occupied by the immobilized cells was 108mg/L.h when air was used for aeration and was 143 mg/L.h with pure oxygen. These corresponded to 22 and 30-fold increase over the productivity of the comparable batch system. However, the specific productivity of the cell was 30-40% of that in shake-flask culture. These high volumetric productivities were due to the high cell mass density of 550 gram/L. As the residence time of medium in the reactor increased, pH of the effluent rose to an alkaline region that was off from its optimal condition near neutral pH and the yield and productivity decreased.
In chapter 5, $\underline{Aspergillus}$ $\underline{niger}$ B60 was immobilized in a dual hollow fiber bioreactor to produce citric acid continuously. The fungi proliferated well in the interstitial region formed by a parallel arrangement of three microporous polypropylene hollow fibers contained within a silicone tube. Long-term operation with nitrogen-enriched medium was not possible due to expansion of the silicone tubes by continual cell growth. The fungal growth could be controlled by supplying a nitrogen-deficient medium at the production stage. With pure oxygen aeration and nitrogen-deficient medium, volumetric productivity reached 1.62g/L.h at a residence time of 4.02 h, which corresponded to a 27-fold increase over that of shake-flask fermentation. When the residence time was increased to 20.1 h, citric acid at a concentration of 26 g/L was continuously produced, with a yield of 80-90% and a volumetric productivity of 1.3 g/L. h. This represents a significant improvement in final concentration, yield and the volumetric productivity over the equivalent values of the corresponding batch fermentation, which were 18 g/L 40% and 0.06 g/L.h, respectively.
In chapter 6, a new process using the dual hollow fiber bioreactor system for the whole cell enzyme immobilization was developed. The current method allows $\underline{Streptomyces}$ $\underline{griseus}$ with glucose isomerase activity to proliferate in the reactor to a desired density and convert glucose to fructose with high productivity. After 6 day culture the dry cell mass density was amounted to 140 g/L, based on the space volume available for cell growth. The volumetric productivity of fructose was 22.5 g/L. h.(based on 34% glucose conversion and the inner silicone tube volume), which corresponded to 12-fold increase over that of batch (1.8 g/L.h. based on 44% glucose conversion).
In chapter 7, newly designed hollow fiber bioreactors for three phase biological systems were introduced and strictly aerobic fungus, $\underline{Aspergillus}$ $\underline{niger}$, producing citric acid was cultivated as a model system. This reactors consisted of a bundle mixed with hydrophilic membranes for liquid nutrient transport and hydrophobic membranes for oxygen transport. The aerobic cells were successfully cultivated. However, the reactor was full of the cells and the hollow fiber membranes were compresses and blocked by excessive fungal cell growth. Citric acid was produced with high volumetric productivity compared with that of shake-flask fermentation, but the production rapidly dropped. Rapid decrease of production was thought to be due to the blockage of the hollow fiber membranes, and thus further improvement was required for successful continuous operation.
본 논문은 호기성 미생물 고정화를 위하여 새롭게 고안된 실관 생물 반응기에 관한 연구 결과이다. 첫째로, 외벽은 산소 통과를 위한 실리콘 튜브로 그리고 그 안쪽엔 액체 배지 공급을 위한 3개의 폴리 프로필렌 실관 막으로구성된 이중 실관 반응기에서 $\underline{E}$. $\underline{coli}$, $\underline{Nocardia}$ $\underline{mediterranei}$, $\underline{Aspergillus}$ $\underline{niger}$와 $\underline{Streptomyces}$ $\underline{griseus}$가 배양 되었으며(2-6장), 둘째로, 반응기 내부에 산소 전달관을 지닌 새로운 구조의 반응기가 고안 되었으며, 이 반응기를 이용하여 $\underline{Aspergillus}$ $\underline{niger}$가 배양 되었다.
제 2장에서는 이중 실관 반응기의 구조 및 제작 방법 등이 소개되었고, simulation에 의해 산소 전달을 이론적으로 해석하였다. 산소 전달 깊이와 체적당 산소 전달 속도는 실리콘 막 두께에는 거의 영향을 받지 않았으며 산소전달 저항은 대부분 고정화 세포층에 존재하였다.
제 3장에서는 이중 실관 생물 반응기에 고정화된 $\underline{E}$. $\underline{coli}$의 세포 밀도 및 특성이 연구 되었다. 세포들은 거의 100% 밀집되어 동물 세포에서 처럼 층을 이루면서 자랐다. 세포 농도는 550 g/L로써 지금까지 보고된 세포 농도중 가장 높았다.
제 4장에서는 이중 실관 생물 반응기에 고정화된 $\underline{Nocardia}$ $\underline{mediterranei}$를 이용하여 리파마이신 B의 연속 생산에 대한 연구가 수행 되었다. 체적당 생산성은 공기가 사용되었을 때 108 mg/L.h, 순수 산소가 사용되었을 때가 143 mg/L.h로써, 이것은 각각 회분식 조업에서 보다 22, 30배 높은 값이었다. 그러나 단위 세포당 생산성은 shake-flask때의 30-40% 정도에 해당되었다. 반응기내의 액체 배지의 체류 시간이 길어짐에 따라 effluent의 pH가 알칼리 영역으로 올라 갔으며, 수율과 생산성이 급격히 감소 되었다.
제 5장에서는 이중 실관 생물 반응기에 곰팡이 $\underline{Aspergillus}$ $\underline{niger}$가 고정화 되었으며, 구연산의 연속 생산에 대한 연구가 수행 되었다. 곰팡이는 박테리아 배양과는 달리 실리콘 튜브를 팽창 시키면서까지 활발히 성장하므로 장기적인 조업에는 문제가 있었다. 따라서, 세포 성장후 생산 단계에서 질소원 결핍 배지를 이용하여 세포 성장을 억제시키는 조업 방법을 사용하였다. 순수 산소와 질소원 결핍 배지를 이용하고 액체 배지의 체류시간 4.02 h 에서 생산성은 1.62 g/L.h 로써 shake-flask 발효에서 보다 27배 높았다. 액체배지의 체류 시간을 20.1 h 로 증가 시켰을 때 26 g/L의 구연산이 연속 생산 되었으며 수율은 80-90%, 생산성은 1.3 g/L.h 였다. 이것은 희분식 발효때의 18 g/L, 40%, 0.06 g/L.h에 비해 상당히 향상된 결과이다.
제 6장에서는 이중 실관 생물 반응기를 whole cell enzyme 고정화에 이용하는 새로운 방법이 연구되었다. 이 방법은 포도당 이성화 효소의 활성을 지닌 $\underline{Streptomyces}$ $\underline{griseus}$를 반응기 내에서 고농도로 배양한 후 효소로 이용하는 새로운 방법이다. 6일 배양후 세포 농도 140 g/L를 얻었으며 과당의 생산성은 22.5 g/L.h로써 회분식 조업에서 보다 12배 높았다.
제 7장에서는 새롭게 고안된 삼상 실관 생물 반응기에 대한 연구가 수행되었다. 이 반응기는 친수성, 배수성 실관막을 서로 섞어주고 친수성 실관막으로는 액체 배지를, 배수성 실관막으로는 공기를 공급할 수 있도록 고안 되었다. 호기성 미생물인 $\underline{Aspergillus}$ $\underline{niger}$ 배양 결과, 성공적으로 배양 되었지만 실관막 구조를 변형시키는 등 조업 및 반응기 제작에 있어서 해결해야 할 많은 문제점들이 제시되었다.
