• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2000년도 추계학술발표대회 및 bio-venture fair
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• pp.241-244
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• 2000

CO로부터 수소를 생산할 수 있는 미생물인 R. palustris P4를 반응기에 적용하여 연속적으로 수소를 생산하는 연구를 진행하였다. 고농도 배양을 위해 R. palustris P4를 호기적 조건에서 영양요구성 성장을 시켰고 그 결과 13 g/L정도의 높은 균체 농도를 얻을 수 있었다. CO는 물에 잘 녹지 않는 기상의 기질이므로 반응기내에서 기체의 충분한 체류시간을 제공할 수 있는 반응기인 TBR을 수소생산 단계에 적용하였다. 기체 체류시간이 50분으로 운전되는 조건에서 CO의 분압이 0.4 atm일 때 최대 CO 소모 속도가 16 mmol/L/hr였다. 또한 광합성 미생물 R. rubrum을 적용하여 TBR에서 수소생산을 시도한 Gaddy group의 수소생산속도 3-4 mmol/L/hr에 4-5배 높은 수소생산 속도이다(3).

• KSBB Journal
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• 제10권2호
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• pp.170-175
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• 1995

Methylobacterium organophilum을 이용하여 메탄 올로부터 고점성 다당류의 생산에 대한 질소원 이용의 통력학척 분석을 유가식 배양에서 수행하였다. 발효중 세포는 배지 중에 첨가된 질소원이 고갈된 후에도 계속 성장하였고, 에틸란은 질소원 고갈조건 하에서만 생성되였다 세포 성장은 배양초기에 첨가 한 질소원인 암모니아의 농도에 비례적으로 이루어 졌으나, 에릴란 생산은 첨가한 암모니아 농도가 $0.75g/\ell$ 이상에서는 오히려 현저하게 감소하였다. 초기 암모니아 농도가 높아질수록, 비세포중식 속도, 비산물생성 속도, 비기질소비 속도가 감소하였다. 높 은 암모니아 농도의 발효 속도들에 대한 저해작용을 감소시키기 위해, 질소원을 일정농도(암모니아 농도=$0.15g/\ell$) 이하로 유지시키면서 공급하는 유가식 배양법으로 메틸란의 농도를 $12.5g/\ell$ 까지 증가시킬 수있었다.

• 한국발생생물학회지:발생과생식
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• 제18권3호
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• pp.139-143
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• 2014

Different with other fishes, the guppies (Poecilia reticulata) is ovoviviparity, which retain their fertilized eggs within the follicle throughout gestation. The synchronously growing diplotene oocytes store nutrients in droplets and yolk, before their maturation and fertilization. The lecithotrophic strategy of development entails the provisioning of embryos with resources from the maternal yolk deposit rather than from a placenta, it allows the extracorporeal culture of guppy embryo. Studies on their early development of live bearers like the guppy including lineage tracing and genetic manipulations, have been limited. Therefore, to optimize conditions of embryo in vitro culture, explanted embryos from pregnant females were incubated in embryo medium (L-15 medium, supplemented with 5, 10, 15, 20% fetal bovine serum, respectively). We investigated whether the contents of FBS in vitro culture medium impact the development of embryos, and whether they would hatch in vitro. Our study found that in 5% of FBS of the medium, although embryos developed significantly slower in vitro than in the ovary, it was impossible to exactly quantify the developmental delay in culture, due to the obvious spread in developmental stage within each batch of eggs, and embryos can only be maintained until the early-eyed. And although in culture with 20% FBS the embryos can sustain rapid development of early stage, but cannot be cultured for the entire period of their embryonic development and ultimately died. In the medium with 10% and 15% FBS, the embryos seems well developed, even some can continue to grow after follicle ruptures until it can be fed. We also observed that embryonic in these two culture conditions were significantly different in development speed, in 15% it is faster than 10%. But 10% FBS appears to be more optimizing condition than 15% one on development process of embryos and survival rate to larvae stage.

• KSBB Journal
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• 제15권2호
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• pp.134-138
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• 2000

알콜증류폐액에 빵효모를 배양하는 중에 glucose와 ammo-nium을 자동첨가함으로써 폐액배양액의 균체농도를 높여서 알콜증류폐액의 이용성을 증가시키고자 하였다. 배양중에 glucose를 공급하여 줌으로서 공급하지 않은 경우보다 1.3배 더 높은 균체농도를 얻었으며, glucose와 (NH4)2SO4를 공급하여 줌으로써 5.8배 더 높은 균체농도를 얻었다. Glucose의 경우에는 배양액의 DO를 제어파라미터로 하는 제어방법을 사용하여 자동첨가하고, ammonium의 경우에는 pH조정제로서 NH4OH을 사용함으로서 pH제어기에 의하여 자동으로 공급되도록 배양하였다. 배양결과 증식의 정체없이 배양 22.5시간후에 최대 12.0 g/L의 건조균체량에 도달하였으며, 균의 최대비증식속도는 0.18 hr-1였고, 대당균체수율이 약 0.54g/gdldjTdmamfh glucose의 첨가가 경제적이라고 생각되었다. 증균배양으로 폐액배양액의 COD를 약 22% 더 감소시킬 수 있었다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권8호
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• pp.1144-1148
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• 2001

The implications of incorporating deoiled rice bran (DORB) in diet on the utilization of certain nutrients had been examined through balance studies made with 6- and 10- week-old Japanese quails. The stuffs evaluated in this study had their origin from two different types of processes- a batch extraction (DORB-B) and another continuous process (DORB-C), and each included at 10, 20 or 30% level in an iso-nitrogenous and iso-caloric diet. On chemical analyses, the DORB-B was found to contain crude protein, 19.0; ether extract, 0.79; total ash, 17.05; Ca, 0.11; P, 1.92; glucose, 2.35 and starch 11.22 g/100 g day matter. Corresponding values for DORB-C were 15.02, 1.56, 13.0, 0.40, 2.76, 2.16 and 19.0, respectively. The data on nutrient balances with quails fed diets containing the two-types of DORB suggested no significant (p>0.05) differences in per cent retention of diet dry matter (DM), organic matter (OM), ether extract (EE), gross energy (GE), nitrogen (N), crude fiber (CF), calcium (Ca) or phosphorus (P) content attributable to the extraction process. The age of the birds also appeared to have no significant (p>0.05) effect on nutrient utilization by them. However, the level of inclusion of DORB had a significant (p<0.05) effect on nutrient utilization by quails in that the retention of dietary N, EE, CF, Ca and P was better at lower than at higher levels. It is concluded that the DORB, irrespective of extraction process, can safely be employed upto 20% level in diet of adult Japanese quails without noticeable interference in utilizing the major dietary nutrients.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.257-260
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• 2002

Dissolved oxygen level of cell culture media has a critical effect on cellular metabolism, which governs specific productivity of recombinant proteins and mammalian cell growth However, in the cores of cell aggregates or cell-immobilized beads, oxygen level frequently goes below a critical level. Mammalian cells have a number of genes induced in the lower level of oxygen, and the genes contain a common cis-acting element (-RCGTG-), hypoxia response element (HRE). By binding of hypoxia inducible factor-l (HIF-I) to the HRE, promoters of hypoxia inducible genes are activated, which is a survival mechanism. In this work, to develop a CHO cell capable of producing recombinant proteins in immobilization and high density cell culture efficiently, mammalian expression vectors containing human tissue-type plasminogen activator (t-PA) gene controlled by HRE were constructed and stably transfected into the CHO cells. In $Ba^{2+}$ -alginate immobilization culture, CHO/pCl/dhfr/2HRE-t-PA cells produced 2 folds higher recombinant t-PA activity than CHO/pCl/dhfrlt-PA cells without $CoCl_2$ treatment. Furthermore, in repeated fed batch culture, productivity of t-PA in immobilized CHO/pCI/dhfr/2HRE-t-PA cells was 121 ng/ml/day, total production of 0.968 mg/day at 11 days culture while CHO/pCIIdhfrlt-PA cells was 22.8 ng/ml/day. All these results indicate that HRE is very useful for the enhancement of protein productivity in mammalian cell cultures.

• KSBB Journal
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• 제19권3호
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• pp.192-199
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• 2004

본 연구에서는 글루타치온의 생산 공정을 개발하기 위해 효모의 성장특성, 글루타치온의 생산성 및 공정 모니터링에 관하여 조사하였다. 초기 pH가 4인 경우 40 mg/L 정도의 높은 글루타치온이 생산되었으며 배양온도에 따른 글루타치온의 생산은 3$0^{\circ}C$에서 가장 높게 나타났다. 그리고 최소 배지에 첨가한 시스테인은 배양 12시간에 넣었을 때 글루타치온의 생산성이 높게 나타났다. 생물 반응기를 이용한 회분식 배양에서 기질 농도에 따른 S. cerevisiae 성장 특성 및 글루타치온 생산은 글루코스 농도 20 g/L에서 글루타치온 생산량이 55 mg/L로 가장 높았다. 최소 배양액에 배양 초기에 0.5 % (v/v) 글라이신과 글루탐산을 각각 첨가하고 배양 11시간에 시스테인을 0.5% (v/v) 추가로 첨가한 경우에 글루타치온의 생산량이 많았다. 회분식 배양 후 기질을 첨가하는 유가식 발효 공정에서는 반응기내 글루코스 농도가 0.5 g/L 이하로 유지되도록 글루코스를 계단식으로 공급하였을 때 글루타치온은 약 102 mg/L로 높은 생산량을 나타내었다. 2차원 형광 센서를 이용하여 글루타치온 생산 공정의 온라인 모니터링은 배양액의 배지 조성이나 성장 특성 등 배양기내의 환경 변화에 따라 형광 영역 및 세기가 다르게 나타났으며 실시간 모니터링 된 형광 데이터는 기질 및 생산물 그리고 균체 성장 등의 각종 공정 변수와 좋은 상관성을 보였다. 따라서 2차원 형광 센서에 의한 모니터링은 글루타치온 대량 생산을 위한 실시간 모니터링에 매우 효과적이라 할 수 있다.

• Journal of Microbiology and Biotechnology
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• 제20권11호
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• pp.1529-1533
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• 2010

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 was overexpressed by using the GAL10 promotor in a ${\Delta}ga180$ strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to a mating factor ${\alpha}$ signal sequence for secretory expression. Use of the ${\Delta}ga180$ strain allowed for the galactose-free induction of inulinase expression using a glucose-only medium. Shake-flask cultivation in YPD medium produced 34.6 U/ml of the recombinant inulinase, which was approximately 13-fold higher than that produced by K. marxianus NCYC2887. It was found that the use of the ${\Delta}ga180$ strain improved the expression of inulinase in the recombinant S. cerevisiae in both aerobic and anaerobic conditions by about 2.9- and 1.7-fold, respectively. A 5-l fed-batch fermentation using YPD medium was performed under aerobic condition with glucose feeding, which resulted in the inulinase production of 31.7 U/ml at the $OD_{600}$ of 67. Ethanol fermentation of dried powder of Jerusalem artichoke, an inulin-rich biomass, was also performed using the recombinant S. cerevisiae expressing INU1 and K. marxianus NCYC2887. Fermentation in a 5-l scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and with the pH controlled at 5.0. The temperature was maintained at $30^{\circ}C$ and $37^{\circ}C$, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/l, respectively.

• Journal of Microbiology and Biotechnology
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• 제20권11호
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• pp.1555-1562
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• 2010

Development of the strain and the fermentation process of Hansenula polymorpha was implemented for the production of ${\gamma}$-linolenic acid ($GLA,\;C18:3{\Delta}^{6,9,12}$), an n-6 polyunsaturated fatty acid (PUFA) that has been reported to possess a number of health benefits. The mutated ${\Delta}^6$-desaturase (S213A) gene of Mucor rouxii was expressed in H. polymorpha under the control of the methanol oxidase (MOX) promoter. Without the utilization of methanol, a high-cell-density culture of the yeast recombinant carrying the ${\Delta}^6$-desaturase gene was then achieved by fed-batch fermentation under glycerol-limited conditions. As a result, high levels of the ${\Delta}^6$-desaturated products, octadecadienoic acid ($C18:2{\Delta}^{6,9}$), GLA, and stearidonic acid ($C18:4{\Delta}^{6,9,12,15}$), were accumulated under the derepression conditions. The GLA production was also optimized by adjusting the specific growth rate. The results show that the specific growth rate affected both the lipid content and the fatty acid composition of the GLA-producing recombinant. Among the various specific growth rates tested, the highest GLA concentration of 697 mg/l was obtained in the culture with a specific growth rate of 0.08 /h. Interestingly, the fatty acid profile of the yeast recombinant bearing the Mucor ${\Delta}^6$-desaturase gene was similar to that of blackcurrant oil, with both containing similar proportions of n-3 and n-6 essential fatty acids.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.90-96
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• 2004

A synthetic defined medium, fortified with amino acids, was developed for the stable production of the kringle fragments of human apolipoprotein(a) (apo(a)), rhLK68. Using a complex rich medium containing yeast extract and a high-cell-density fed-batch culture, the expression level of rhLK68 reached 17% of the total cellular protein, which corresponded to $5\;g\;l^{-1}$ of the culture. To replace the complex media with chemically defined media, several amino acids that positively affect cell growth and gene expression were chosen by a statistical method. The various combinations of the selected amino acids were tested for its fortifying effect on a minimal defined medium. When glutamine only was added, the overall expression level of rhLK68 reached 93% of the complex rich medium increasing the specific expression level by 22.4% and decreasing the cell growth by 24%. Moreover, the addition of glutamine resulted in a 2-fold increase in the concentration of rhLK68 in the culture broth, compared with the minimal defined medium. The synthetic defined media developed in this study could be generally applied to high-cell-density cultures of the recombinant Escherichia coli BL21(DE3), especially for the production of therapeutic proteins that require a strict quality control of the culture media and fermentation processes.