Earticle

The production of red pigment from glucose by fed-batch culture of Monascus anka was investigated. In batch culture using fermentor, 200 rpm of agitation speed, 1vvm of aeration volume, and 10% (v/v) of inoculum size were optimal, respectively. The red pigment production was increased by removal of wall-attached mycelium. In an intermittent feeding fed-batch culture, dry cell weight increased to 30 g/l, adn the red pigment content reached 350 of absorbance at 495nm. In a continuous feeding fed-batch culture, dry cell weight increased to 22g/l, and the red pigment content reached 190 of absorbance at 495nm.

고농도 유전자 재조합 대장균을 이용하여 pyruvate dehydrogenase dehydrogenase complex-E2 특이성 인간 모노클론 항체의 Fab 부분을 효율적으로 생산하기 위해 회분식, 이단 연속식, 반 유가식, two-stage cyclic fed-batch 등 여러 가지 배양 방법이 조사되었다. 먼저 플라스미드 안정성 문제를 극복하기 위해 growth stage와 production stage를 분리하는 two-phase 회분식 배양과 이단 연속식 시스템을 시도하였다. 그 결과 two-phase 회분식 배양보다는 이단 연속식 배양에서의 세포농도와 항체 생산성이 우수하였다. 또한 이단 연속식 배양에서의 세포 성장과 항체 생산성은 용존산소를 제어한 경우가 그렇지 않은 경우보다 월등하게 높았다. 그리고 plasmid 안정성에 있어서 는 실험기간 내에 거의 100%를 유지하여 높은 안정도를 보여주었다. 유가식 공정에 적합한 공급 배지로 변형된 M9 배지가 최적배지로 선정되었고 이 배지 중 최적의 C/N 비율을 조사한 결과 2:3으로 결정되었다. 반 유가식 시스템에서 constant feeding 전략을 사용할 경우 최적 공급속도는 0.6 g/ℓ/hr이었다. 또한 pulse에 의해 공급배지를 공급할 경우에는 총 공급량이 같을 경우 소량으로 자주 공급해 주는 것이 공급배지를 한꺼번에 많은 양을 공급해주는 것 보다 바람직하였다. 여러 가지 feeding 전략을 조사해 본 결과 linear feeding 방법이 가장 효과적이었다. 하지만 linear feeding 방법마저도 고농도 세포배양에 한계가 있었기 때문에 pH-stat 방법을 이용한 two-stage cyclic fed-batch 시스템을 시도하여 54 g/ℓ의 세포 를 얻을 수 있었다. 따라서 이 방법이 일단 생산성 향상을 위한 세포의 고농도 배양에는 조사한 여러 배양 시스템 중에 가장 효율적인 시스템임을 알 수 있었다. 하지만 이 시스템에서 포도당을 낮은 level로 유지할 수 있었으나, 초산의 과도한 축적으로 항체 생산성의 향상은 예상에 비해 크지 않았다.

Several culture systems including batch, two-stage CSTR, semi-fed batch, and two-stage cyclic fed-batch were investigated for the efficient production of the Fab fraction of PDC-E2 specific human monoclonal antibody using high cell density recombinant E. coli. A two-phase batch system and a two-stage continuous system were examined to overcome plasmid instability problems, by separating the growth and the production stages. The cell density and productivity of the two-stage continuous culture was better than that of the two-phase batch fermentation. In the two-stage continuous culture system with DO-stat, the cell growth and the productivity were superior to those of the system without the DO control. Also, almost total plasmid stability was maintained in the two-stage continuous culture system. Modified M9 medium was selected as an optimum feeding medium for the fed-batch process, and the optimum C/N ratio determined to be 2:3. The optimum feeding rate was 0.6 g/ℓ/hr for a constant feeding strategy in semi-fed batch system. When the feeding medium was fed by pulsing, it was observed that more frequent pulsing resulted in improved cell growth. The linear feeding method was the most efficient of the various feeding methods tested. Finally, high cell density culture using a two-stage cyclic fed batch system with pH-stat was tried because the linear feeding method showed limitations in terms of obtaining high cell densities, and a cell density of 54 g/ℓ was achieved. It was concluded that the two-stage cyclic fed batch system was the most efficient system for high cell density culture of the systems tested. However, productivity improvements were lower than expected due to the extremely high accumulations of acetate, although the low levels of residual glucose were maintained.

A thermostable α-L-arabinofuranosidases (α-L-AFase) is an industrially important enzyme for recovery of Larabinose from hemicellulose. The recombinant α-L-AFase from Thermotoga maritima was expressed in Escherichia coli by using a constitutive pHCE or an inducible pRSET vectors. In batch fermentation, the constitutive expression system resulted in slightly faster growth rate (0.78 vs. 0.74/hr) but lower enzyme activity (2,553 vs. 3,723 units/L) than those of the induction system. When fed-batch fermentation was performed, biomass and enzyme activity reached the highest levels of 36 g/L and 9,152 units/L, respectively. The fed batch cultures performed superior results than batch culture in terms of biomass yield (4.62-5.42 folds) and enzyme synthesis (3.39-4.00 folds). In addition, the fed-batch induction strategy at high cell density resulted in the best productivity in cell growth as well as enzyme activity rather than the induction method at low cell density or the constitutive expression.

Cell growth and lipid production of Chlorella minutissima in fed-batch culture was studied. Two carbon resources, such as CO2 and glucose were used to compare autotrophic and heterotrophic cultivate condition. For fed-batch cultivation, the feeding rate of seawater medium containing glucose was decided to be 0.5 ℓ/day of medium containing glucose. Autotrophic with CO2 showed the maximum cell growth of 9 (g-dry wt./L) and total lipid production of 15%. heterotrophic condition with glucose 10 g/ ℓ showed the maximum cell growth of 15 (g-dry wt./L) and total lipid production of 32%. Also, lipid profiles produced by gas chromatography analysis showed that lipid concentration of C17 and C18 composition is each 16%, 38%. This tells that Chlorella minutissima is a suitable bio-resource to produce the biodiesel. Accordingly, this study showed that the fed-batch cultivation with glucose is most effective for the large scale cultivation for biodiesel production with Chlorella minutissima.

곰팡이 균인 Aspergillus terreus로서 고지혈증인 치료제인 lovastatin을 생산하기 위해 탄소원 조절에 의한 유가식 배양을 통해 lovastatin 생산 발효 조건을 확립하고자 하였다. 또한 발효시 곰팡이의 형태구조가 이차대사산물 생산에 중요함으로 유가식 배양에서의 공급배지농도와 공급속도에 따른 형태 구조의 변화가 생산성과 관련이 있는지를 구명하였다. Glucose 용액을 일정하게 또는 증가시켜 공급하는 방법을 실험하였으며 이 때 펠맅의 분포를 비교하였다. Glucose 용액을 일정하게 공급한 경우가 1 mm이하의 펠맅이 더 많이 분포하였으며, lovastatin 생산량도 높았다. Lovastatin 생산에 적절한 펠맅의 크기가 1 mm이었다는 연구 결과와 일치하였다. 또한 배양 방법 중 3일간 회분 배양 후 4일째부터 glucose를 일정하게 공급한 경우에서 높은 lovastatin 생산량을 나타내었다. 회분식 발효에서 lovastatin 생산을 위한 물리적 조건 중 교반 속도는 400 rpm으로 조절하였고 pH에 대한 영향을 조사하였다. 그 결과 pH 5.8로 유지하였을 때 lovastatin 생산량은 pH 7.4의 7 배 이상인 384 mg/L로서 발효 기간 중 pH 조절이 중요하다는 것을 알 수 있었다. 플라스크에서의 유가식 배양과 회분식 배양결과를 토대로 하여 발효조에서 유가식 배양시 pH 5.8, 400 rpm, 초기 glucose 농도를 30 g/L로하여 3일간 회분 배양 후 4일째부터 180 g/L의 glucose 용액을 공급한 결과, 회분식 배양의 lovastatin 생산량보다 1.5배 많은 547 mg/L를 얻었으며, 생산성은 3.25 mg/L․hr였다.

The biosynthesis of Lovastatin, a cholesterol lowering agent formed by the filamentous fungus, cerulenin-resistant Aspergillus terreus mutant was studied in shake flasks and bioreactors. The lovastatin production could be improved by fed-batch under the limited condition of carbon source. The relationship between the fungal morphology and the lovastatin production was also examined during the fed-batch cultures. The fed-batch studies in shake flasks were carried out to find the optimum glucose feeding method, and the pulsed feeding of glucose from 3 days onward at 24 hours intervals was found to be optimal to increase the lovastatin production and reduce the average pellet size. When the pH was controlled at around 5.8 during the whole fermentation period, the lovastatin concentration reached 384 mg/L, which is much higher than the values obtained pH-uncontrolled and pH 7.4. The optimal glucose feeding strategies was found that 30 g/L of glucose was added initially in batch mode, and then fed-batch was conducted by continuous addition of glucose solution(180 g/L) from 72 to 240 hr at a rate of 1.2 mL/hr at 28℃, pH 5.8, 400 rpm, and 1.0 vvm. The lovastatin concentration of 547 mg/L was obtained in 168 hr. It was about 1.5 times higher than the value of the batch fermentation.

E. faecalis RKY1을 사용한 유가식 배양을 통하여 기질저해를 받지 않도록 하여 발효조 내의 젖산 농도를 최대화시키고자 하였다. 유가식 배양에 의한 젖산발효는 생성물인 젖산으로 인한 저해가 심하여 부피생산성이 감소하였지만, 네 가지 형태의 유가식 배양을 실험한 결과 발효 도중에 발효액을 수 회 제거하고 질소원이 제한되지 않은 배지를 수회 공급하는 간헐적인 유가식 배양의 경우에 가장 나은 결과를 얻을 수 있었으며, 이 경우 발효 84시간째 생산된 젖산의 농도는 최대 224.8 g/L까지 증가하였고 젖산에 대한 총괄 수율은 0.84 g/g, 비생산속도는 0.092 g/g․hr, 그리고 부피생산성은 2.7 g/L․hr로 얻을 수 있었다.

Fed-batch cultures of Enterococcus faecalis RKY1 were performed to maximize the L(+)-lactic acid concentration in the bioreactor. The highest lactic acid concentration was obtained at around 225 g/L by intermittent feeding the concentrated glucose media containing 500 g/L of glucose and 15 g/L (or 75 g/L) of yeast extract. However, in all fed-batch cultures, volumetric productivities of lactic acid gradually decreased due to the inhibitory effect of lactic acid produced during the fermentation. The highest value of lactic acid concentration obtained in this work corresponded to around 1.5-fold increase compared with conventional batch fermentation.

균사형성 미생물에 의한 항생물질의 대량생산은 적절한 산소공급이 요구되며, 산소전달속도는 생산성에 영향을 미치는 속도제한단계이다. 그러므로 발효에 이용하기 위해 Streptomyces avermitilis와 같은 균사형성 미생물 배양액에서의 산소전달계수를 측정하였다. 그러나 회분식 배양에서 kLa 측정을 위한 적절한 용존산소 유지가 어려웠으며, 이를 극복하기 위해서 유가식 배양을 도입하였다. 배지의 단계적 공급으로 미생물의 성장속도를 조절할 수 있었고, 낮은 교반속도에서도 적정용존산소의 유지가 가능하였으며, 결과적으로 kLa 측정도 용이하였다. 7 g/L이하의 낮은 균체량에서는 350 rpm의 교반속도가 교반효율과 전단응력을 고려했을 때 가장 적절한 조건임을 알 수 있었다. 그러나 균체가 증가할수록 배양액 점도가 증가하여 혼합이 효과적이지 못한 이유로 더 높은 교반속도가 필요하였다. 통기량의 증가에 의한 kLa 증가율은 건조균체질량이 고 농도 일수록 미미하였다. 그러므로 높은 균체농도에서는 이차대사산물에 영향을 주지 않는 범위에서 교반속도를 증가하여 용존산소 농도를 조절하는 것이 통기속도의 조절보다 더 효율적 일 것으로 판단되었다.

The large-scale production of antibiotics by filamentous mycelial organism requires an adequate supply of dissolved oxygen. In terms of productivity, it means that oxygen transfer is the rate-limiting step. Therefore, the oxygen transfer coefficients(kLa) were determined in a broth involving a filamentous mycelial organism such as Streptomyces avermitilis for use in fermentations. To determine kLa in a stirred vessel, a great deal of effort is required to provide all the cells with a sufficient oxygen supply. To overcome the oxygen limitation in a batch culture, a fed-batch culture was applied to control the growth rate by an intermittent supply of nutrients. Thus, it was possible to maintain a suitable dissolved oxygen concentration at a low agitation rate. The optimal agitation speed was 350 rpm at low cell concentrations (below 7 g/L) by considering the efficiency of agitation and shear stress. The kLa was found to decrease from 64.26 to 29.12 hr-1 when the biomass concentration was increased from 9.82 to 12.09 g/L. In addition, an increase in viscosity was also observed during the growth phase. By comparing the kLa values for the various agitation and aeration rates, it was found that the effect of an increase in kLa by aeration was reduced dramatically at high biomass concentrations. However, this effect was not observed when altering the agitation rate. This suggests that controlling the dissolved oxygen concentration by altering the agitation rate was more efficient than increase the aeration rate.

Fed-batch fermentations were carried out in order to improve the efficiency of hirudin production by recombinant Saccharomyces cerevisiae. A fed-batch fermentation done with the optimized semi-synthetic medium resulted in a maximum hirudin concentration of 342mg/l by keeping a galactose concentrations between 10 and 30g/l which corresponded to a 11.4-fold increase in hirudin concentration compared with the simple bach fermentation done with the same medium. Comparison of the chromatographic pattern of proteins in the growth medium clearly showed that the use of the semi-synthetic medium is more advantageous for separation of hirudin than the case o fusing the complex medium. Continuous cell recycle fermentation done at dilution rate of 0.1h-1 and an inlet galactose concentration of 100g/l yielded a maximum hirudin productivity of 19.1mg hirudin/lh

Bacillus thuringiensis를 생물농약으로 이용하기 위해서는 고농도의 포자형성에 의한 높은 살충성의 δ-endotoxin를 생산하는 것이 중요하다. 따라서 본 연구에서는 B. thuringiensis의 고농도 배양에 의한 높은 포자형성 수율을 얻기 위하여 40％의 용존산 소량과 28℃에서 여러 가지 방법의 유가식 배양법을 검토하였다. 최종 배양액의 glucose 농도가 50 g/L가 되도록 하는 경우의 유가식 배양에서는 continuos fed-batch culture의 linear gradient feeding 법에 의하여 9.37×109 cell/mL의 최대 생존 세포 수와 8.33×109 spore/mL의 최대 포자 수를 얻었으며, 이때의 포자 형성율은 88.9％ 이었다. 최종 배양액의 glucose 농도가 100 g/L가 되도록 하는 경우의 유가식 배양에서는 intermittent fed-batch culture의 pH-stat 법에 의하여 1.38×1010 cell/mL의 최대 생존 세포 수와 1.35×1010 spore/mL의 최대 포자 수를 얻었으며, 이 때의 포자 형성율은 97.8％ 이었다.

Both the production of high spore concentration and high insecticidal activity are required in the production of Bacillus thuringiensis to be used for the bacterial insecticide. In the production of high cell and spore concentrations of B. thuringiensis, the continuous fed-batch culture (CFBC) and intermittent fed-batch culture (IFBC) were investigated at 28℃ by maintaining 40％ dissolved oxygen concentration. When the final glucose concentration was 50 g/L, the maximum viable cell number obtained using the CFBC with linear gradient feeding was 9.37×109 cells/mL and maximum spore concentration was 8.33×109 spores/mL which was approximately 84.4％ yield of spore formation. When the final glucose concentration was 100 g/L, the maximum viable cell and spore concentrations obtained using the IFBC with pH-statb were 1.38×1010 cells/mL and 1.35×1010 spores/mL respectively and the yield of spore formation was approximately 97.8％.

발효조의 냉각수 공급 제어 신호와 냉각수의 유출입 지점의 온도를 온라인으로 측정하여 이 값들로부터 냉각량을 추정할 수 있는 실험식을 얻었다. 회분식 배양을 통하여 냉각량이 온라인으로 측정될 수 있음을 확인하였고 기질의 재투입시 냉각량의 변화를 통하여 유가배양의 제어변수로 활용할 수 있음을 점검하였다. 미리 프로그램된 목표 냉각량의 증가곡선에 따라 냉각량이 증가되면서 세포가 자라도록 유가배양을 실시하였다. 본 연구를 통하여 냉각량의 온라인 측정방법을 제시하였고 이를 유가배양 제어변수로 할용하여 세포의 성장속도를 조절할 수 있음을 보였다.

A laboratory jar fermenter was modified to measure the duration for cooling water supply and the temperatures of the coolant at the inlet and outlet of water jacket. Successful operation of temperature control and on-line measurement was achieved by adjusting optimum parameters of the Proportion-Integral-Derivative temperature controller. The variables measured on-line were used to estimate cooling rates from empirical equations comprised of the time period of cooling water supply and the temperatures of coolant. The measured cooling rate showed a good correlation to the specific growth rate during batch cultivation of E. coli. Cooling rate was measured and applied to programmed cell growth in a fed-batch cultivations. Three fed-batch cultivations were demonstrated by feeding substrate to follow the programmed cooling rates increasing exponentially.

본 연구에서는 음식물쓰레기를 당화시키기 위해 섬유소분해효소를 효율적으로 배양하고자 먼저 refill하는 멘델배지의 농도를 0.5%로, 새로운 배지의 주입시간을 12시간으로 결정하였다. Flask 레벨에서는 fill-and-draw 방법으로 12시간 단위로 연속배양한 결과, amylase 활성은 300시간까지 1.0 U/mL 내외로 유지되었으며, FPase 활성은 156시간까지 0.40 U/mL 이상으로 유지되었다. 이때의 효소생산성은 amylase 3.49 U/L․hr, FPase 1.02 U/L․hr 이었다. 10 L에서는 batch, fed-batch, fill-and-draw 방법으로 효소를 생산한 결과 batch에서 가장 높은 효소생산성을 나타내었으며, 그다음은 fed-batch 이었다. Batch에서의 효소생산성은 amylase 42.30 U/L․hr, FPase 5.60 U/L․hr, fed-batch에서는 각각 23.03, 2.76 U/L․hr 이었다. 그리하여 T. inhamatum KSJ1을 이용한 섬유소분해효소의 연속배양에서 10 L 생물반응기에서 fed-batch 방법이 가장 효율적이었다.

For continuous culture of cellulolytic enzymes production to saccharify food wastes, refill concentration of Mandel's medium for continuous culture was 0.5%, and refill intervals were determined to 12 hours by analysis of COD and total nitrogen concentration after 4-days batch culture in flask level. As a result, amylase and FPase productivities were 3.5 and 1.0 U/L․hr, respectively. In 10 L bioreactor, the batch culture mode was compared with fed-batch, fill-and-draw for continuous production of cellulolytic enzyme. Enzyme productivities were most high at batch culture and followed by fed-batch culture. Amylase and FPase activities were 42.3 and 5.6 U/L․hr at batch culture, and 23.0, 2.8 U/L․hr at fed-batch culture, respectively. As a result, in continuous cultivation of cellulolytic enzymes by T. inhamatum KSJ1, the mode of fed-batch was most effective in 10 L bioreactor.

유가식배양과 세라믹 막을 장착한 생물반응기에서 운전인TCRC 조업을 통해서 Bacillus thuringiensis의 고농도 세포배양을 실시하였다. 유가식 배양에서 B. thuringiensis의 세포 성장은 선형적으로 증가하였고, 이것은 세포성장 모델리의 결과와 잘 맞았다. 낮은 세포 성장속도에도 불구하고 유가식 배양동안에 포자형성은 관찰할 수 없었고, 이것은 연속배양의 결과와는 반대였다. 유가식 조업 후에 회분식 배양으로 바꾸면 300 g/L의 포도당 공급 농도를 사용했을 때 2.7×109 CFU/mL의 포자농도를 얻었다. 생물반응기내에 세라믹 막을 장착한 TCRC 조업에서 포도당 공급 농도의 영향을 결정하였다. 50 g/L의 포도당 농도를 사용했을 때 TCRC 조업에서 82.5 g-cell/L에 해당하는 최대 세포농도 1.8×1010 CFU/mL를 얻었다. TCRC에서 세포성장은 선형적으로 증가하였고 포도당 농도는 제한되었는데 이것은 세포성장은 모델링의 결과와 잘 일치하였다. 1 g/L의 포도당을 공급한 경우 이외에는 TCRC 조업 동안에 포자형성을 관찰할 수 없었다. 50 g/L의 포도당 을 공급한 경우 TCRC조업 후에 회분식 배양으로 전환시키 면 1.2×1010 CFU/mL의 포자농도를 얻었고, 이것은 연구된 여러 배양형태 중에 가장 높은 포자농도이다. 이 때 최적의 포도당 공급속도는 0.55 g glucose/h로 공급하였을 때였다.

High cell density culture of Bacillus thuringiensis was conducted in fed-batch culture and TCRC using a bioreactor incorporating ceramic membrane filter. Cell growth of B. thuringiensis in fed-batch culture increased linearly, which was well matched by the results of cell growth modeling. In spite of the slower growth rate during fed-batch culture, no spore formation was observed, which was contrary to the results of continuous culture. Changing culture mode to batch culture after fed-batch operation induced a 2.7×109 CFU/mL spore concentration using a 300 g/L glucose feed concentration. In TCRC operation incorporating ceramic filter within the bioreactor, the effect of glucose feed concentrations on the cell growth and spore formation of B. thuringiensis was determined. A maximum cell concentration of 1.8×1010 CFU/ml, which corresponds to 82.6 g-cell/L, was obtained in the TCRC using a 50 g/L glucose feed concentration. In the TCRC, cell growth increased linearly and glucose concentration was limited, which agreed well with the results of cell growth modeling. No spore formation was observed except when 1 g/L of glucose was fed. Changing to batch culture induced a 1.2×1010 CFU/mL of spore concentration, which was the highest spore concentration obtained among the various culture modes examined. The optimal glucose feed rate was found to be 0.55 g-glucose/h.

본 연구는 에탄올보다 물리적, 화학적 특성이 뛰어난 부탄올을 투과증발을 이용한 추출 발효를 이용해 효율적으로 생산하기 위한 조건을 찾아내었다. Clostridium acetobutylicum B18 균주를 이용한 부탄올 회분식 발효 결과 최적 포도당 농도는 60 g/L이며 배양 시작 30시간 후에 탄소원 이외의 영양소를 추가로 주입했을 때 부탄올 생산성이 0.112 g/L․h으로 가장 높았다. 그리고 pH가 조절된 실험에서는 pH가 5.5에서 부탄올 생산성이 0.216 g/L․h 로 가장 높았다. 투과증발시스템을 적용하기 위한 공기흐름속도는 2.0 L/min-tubing일 때가 가장 부탄올 투과 효율이 높았다. 투과증발시스템을 이용한 회분식 및 유가식발효에서 포도당 소모 속도가 1.3배 증가하였다. 유가식 배양에서는 영양분의 공급으로 장시간 배양이 가능하였고, 결과적으로 포도당소모를 촉진시킬 수 있었다. 생성되는 부탄올은 투과증발시스템에 의해 제거해줌으로써 균주의 활성을 장시간 유지하여 연속적인 부탄올 생산 가능성을 확인 할 수 있었다.

An extractive fermentation process using pervaporation was studied in a 7 liter fermentor. Pervaporation was performed using a silicone membrane module, and a low-acid-producing strain, Clostridium acetobutylicum B18, was used to produce butanol. In batch culture without pervaporation, pH 5.5 and initial glucose concentration of 60 g/L resulted in the highest butanol productivity (0.216 g/L․h) with butanol yield of 0.261. Butanol flux through the membrane was best at 2.0 L/min-tubing of air flow rate. In batch and fed-batch fermentation glucose consumption rate increased by 1.3 times with pervaporation.

The possibility of application of membrane type immobilized adsorbent to the fed-batch or perfusion culture system with anchorage-independent cells as well as batch system was investigated. The improvement in cell density and cell viability due to the combination of immobilized adsorbent with each culture system was evaluated for the investigation, and the optimum culture system employing immobilized adsorbent system was suggested based on the results. It was observed that the system with immobilized adsorbent showed better cell growth and cell viability than that without immobilized adsorbent in every operation system of batch, fed-batch, and perfusion. In case of batch system, 200% improvement of maximum cell density was observed in the system where ammonium chloride was added on purpose. And 50% improvement of maximum cell density was observed in the fed-batch system where ammonium ion accumulates significantly, while small increase in maximum cell density was observed in the perfusion system where dilution of waste byproducts exists. Especially, the fed-batch system showed the most significant improvement on cell growth because both compensation of nutrient and removal of ammonium ion occurred simultaneously in the system. Therefore a combined system of immobilized adsorbent and fed-batch operation could be suggested as an optimum system with in situ removal of ammonium ion.

제주도 연안에서 분리 동정된 한천분해효소를 생산하는 Agarivorans sp. JA-1의 배양특성을 연구하였다. 균주가 기질로 이용하는 한천은 배지 내의 한천의 농도가 0.2%일때 가장 효율적인 성장을 하였으며 4종의 탄소원에 대해서는 성장에 영향을 미치지 않는 것으로 나타났다. 배양시간에 따른 한천농도는 15시간 이후 모두 분해되었으며 점도의 변화도 이와 유사한 것으로 나타났다. 또한 fed-batch 배양에서 0.8 g 한천을 2회 첨가하였으며 한천을 첨가한 후 agarase생산이 증가되는 것으로 나타났다. 한천올리고당의 성분분석을 한 결과 중합도가 6인 당이 확인되었다.

Characteristics of β-agarase production of Agarivorans sp. JA-1 isolated from north-eastern sea of Jeju marine environment was studied. Optimal cell growth was definite that the medium containing agar is 0.2%. The decreasing pattern of viscosity and agar concentration was same and they reached almost zero after 15 hours. Fed-batch culture was studied to improve agarase productivity by Agarivorans sp. JA-1 in marine broth containing 2.0 g/L agar with intermittent addition of 0.8 g agar two times. The hydrolysis products were identified oligosaccharide of degrees of polymerization 6.

우수한 셀룰로오스 생산 균주인 Acetobacter xylinum BRC5의 교반배양에 의한 셀룰로오스 생산성을 향상시키기 위하여 fed-batch 배양을 하였으며, 기질공급속도, 기질 공급량 및 용존 산소의 영향을 검토하였다. 초기 glucose 양을 변화시켜 회분배양하였을 때 glucose 농도가 10 및 20 g/L인 경우 셀룰로오스 생산량은 각각 2.05와 4.10 g/L이였으며 glucose에 대한 셀룰로오스 수율 (Yp/s)은 0.21이었다. 초기 glucose 농도 40g/L일 때 셀룰로오스 수율을 향상시키기 위해서 초기 glucose 농도 20 g/L에서 회분배양을 시작하여 glucose가 gluconic acid로 완전히 전환된 시점부터 추가적으로 glucose를 공급하여 fed-batch 배양기간에 glucose 공급속도는 셀룰로오스 생산성에 큰 영향을 미쳐 20g/L의 glucose를 2.22 g/L.h의 속도로 9시간 첨가하여 fed-batch 배양한 결과 셀룰로오스 생성량이 10 g/L로 가장 우수하여 초기 glucose 농도 20 g/L로 회분배양하였을 때 비하여 약 2배 증가하였으며, Yp/s도 0.26으로 현저히 향상되었다. 또한 동일조건으로 fed-batch 배양하면서 glucose 공급량을 증가시켜 40g/L의 glucose를 추가적으로 첨가한 경우 셀룰로오스 생산량은 10.7g/L는 거의 증가되지 않았으며, Yp/s가 0.18로 감소하였다. 이는 셀룰로오스 농도가 증가함에 따라 산소 공급이 부족하기 때문이므로 용존산소(DOT)를 2~15% 포화범위에서 조절하여 fed-batch 배양했을 때 DOT를 10% 수준으로 유지하면서 fed-batch 배양기간에 40g/L의 glucose를 추가공급 했을 때 셀룰로오스 생성량은 15.3 g/L로 증가되었고 이때 Yp/s는 0.26로 향상되었다. 이는 DO를 제어하지 않는 경우에 비하여 셀룰로오스 생성량이 1.5배 증가한 결과이다

Glucose fed-batch culture was studied to improve cellulose productivity by Acetobacter xylinum BRC5. When initial glucose concentrations in batch cultures were less than 20 g/L, yield coefficients of cellulose (Yp/s) remained a constant value of 0.21 g cellulose/g glucose. But a low yield coefficient, Yp/s=0.13 was obtained from an initial glucose concentration of 40 g/L. Since initial high glucose concentrations in batch culture resulted in low yields of cellulose, constant fed-batch cultures were carried out. The optimal feed rate for fed-batch culture was 2.22 g glucose/L.h. In constant fed-batch culture without DO control, 10 g/L of cellulose was obtained from 40 g/L of glucose with this feed rate, which was approximately two fold higher than that of the batch culture with the same initial glucose concentration. In DO stat plus fed-batch culture, the highest cellulose productivity could be obtained when dissolved oxygen level was controlled at 10% of air saturation, and cellulose productivity increased about 1.5 times compared with that of the culture without DO control.

natural antibiotic compound from microbial source are promising for future. Batch and fed-batch culture was carried out to enhance the antibiotics production from B.subtilis produce surfactin-like lipopeptide antibiotics. Dextrose was best carbon source for high production in batch culture. For fed-batch, optimal glucose concentration was 1.5 % around and this concentration showed best antibiotics production rate. Cell growth was also highly enhance by glucose feeding. Ammonium was best nitrogen source for antibiotics production for this strain and it concentration was 4% around. Other salts such as magnesium, manganese, ferrous, and potassium ion was also important for growth as well as production.

Since butyric acid has wide applications to chemical, food, and pharmaceutical industries, there has been a great interest in its fermentative production. In particular, butyric acid more recently has been disclosed to be converted to butanol, known as one of the most promising liquid biofuels, via a catalytic reaction with hydrogen. In this study, the kinetics of cell growth, butyric acid formation, and substrate consumption in the batch fermentation of Clostridium tyrobutyricum were investigated. In order to describe cell growth, butyric acid production, and glucose consumption, modified Monod model by adding terms of inhibitions of substrate and product, Luedeking-Piret model, and carbon mass balance were employed, respectively. The parameters used in the models were estimated by m inimizing mean-squared errors between the predictions of the models and the experimental data obtained from a series of batch fermentations. The predictions of the models have showed in good agreements with the fermentation results. Especially, the model-based fed-batch fermentation remarkably increased butyric acid production, and also enhanced the growth of C. tyrobutyricum. The models developed in this study could be used for the development of fermentative chemicals and fuels production processes. This work was supported by Energy Technology Innovation (ETI) Program of the Korean Ministry of Knowledge Economy (MKE).

Nattokinase is a potent fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. The enzymes were discovered from various sources, such as Japanese natto, Korean Chungkookjang soy sauce, and Chinese douchi. Based on its food origin and relatively strong fibrinolytic activity, nattokinase has advantages over other commercially used medicine in preventative and prolonged effects, convenient oral administration, and stability in the gastrointestinal tract. In this study, Bacillus subtilis was cultivated to produce nattokinase. Glucose and NH4Cl were used as carbon and nitrogen sources with peptone supplementation. In flask culture, peptone concentration was fixed at 50 g/L. The highest cell growth (optical density monitored at 600 nm) and nattokinase activity were obtained with 10 g/L of glucose supplementation. At this condition, nattokinase activity was 688 unit/ml. In fed-batch culture of B. subtilis in fermentor, the ratio of peptone and glucose in feeding solution was varied. The highest cell concentration and nattokinase activity were 75 g/L and 15,000 unit/ml, respectively. Nattokinase activity increased with cell growth, showing growthassociated production.