Two-Stage Depth Filter Perfusion Culture for Recombinant Antibody Production by Recombinant Chinese Hamster Ovary Cell

이준철,김도연,오덕재,장호남 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.5

A rCHO cell line of DUKX origin 26*-320, producing recombinant antibody against the human platelet, was cultivated in a two-stage depth filter perfusion system (DFPS) for 20 days in order to attain high recombinant antibody concentration. The productivity of the first stage DFPS bioreactor reached 53 times that of the batch culture in a controlled stirred tank reactor and was showed 12.1 mg/L antibody concentration at a perfusion rate of 6.0 d-¹. Glucose concentration in the first DFPS was maintained at 1.5 g/L to avoid cell damage in the perfusion culture. A second stage DFPS system was attached to the first DFPS, which resulted in a low glucose concentration of 0.02 g/L and a high antibody concentration of 23.9 mg/L. The two-stage depth filter perfusion culture yielded 60% higher product concentration than the batch and 49-fold higher produc-tivity of 69.3 mg/L/d in comparison with that (1.4 mg/L/d) in a batch system. Furthermore, antibody concentration of the sec-ond stage was 97% higher than that of the first stage, and the antibody productivities were comparable to that of the first stage. This two-stage DFPS system also showed potential for higher titer production of recombinant antibody and high volumetric productivity for long-term culture of bio-pharmaceutical substances.

Production of Tropane Alkaloids by Two-stage Culture of Scopolia parviflora Nakai Adventitious Root

Kim, Won-Jung,Jung, Hee-Young,Min, Ji-Yun,Chung, Young-Gwan,Lee, Cheol-Ho,Choi, Myung-Suk Plant molecular biology and biotechnology research 2004 Plant molecular biology and biotechnology research Vol.2004 No.-

Scopolia parviflora Nakai, a rare and endangered species, os the sole plant producing tropane alkaloids (TA) among the Korean native species. In order to enhance TA productivity the SP72 root, line was selected by screening 100 of root line, and the optimal culture media for root growth and TA production were investigated with the SP72 roots. Based on the several media, SH and 2B5 medium were determined as growth medium and White and NN medium as production medium. Among the four combinations of two-stage culture, 2BN (2B5 as growth medium plus NN as production medium) showed more enhanced root growth and TA production as compared with production media of White and NN medium and growth media of SH and 2B5 medium, respectively. However, bubble column bioreactor (BCB) cultures applying two-stage culture did not reveal the effective results despite of the each successful operation of two-stage culture in conical flasks and BCB cultures.

Production of Tropane Alkaloids by Two-stage Culture of Scopolia parviflora Nakai Adventitious Root

Won-Jung Kim,Hee-Young Jung,Ji-Yun Min,Young-Gwan Chung,Cheol-Ho Lee,Myung-Suk Choi 韓國藥用作物學會 2004 한국약용작물학회지 Vol.12 No.5

Scopolia parviflora Nakai, a rare and endangered species, is the sole plant producing tropane alkaloids (TA) among the Korean native species. In order to enhance TA productivity the SP72 root line was selected by screening 100 of root line, and the optimal culture media for root growth and TA production were investigated with the SP72 roots. Based on the several media, SH and 2B5 medium were determined as growth medium and White and NN medium as production medium. Among the four combinations of two-stage culture, 2BN (2B5 as growth medium plus NN as production medium) showed more enhanced root growth and TA production as compared with production media of White and NN medium and growth media of SH and 2B5 medium, respectively. However, bubble column bioreactor (BCB) cultures applying two-stage culture did not reveal the effective results despite of the each successful operation of two-stage culture in conical flasks and BCB cultures.

Production of Tropane Alkaloids by Two-stage Culture of Scopolia parviflora Nakai Adventitious Root

Kim, Won-Jung,Jung, Hee-Young,Min, Ji-Yun,Chung, Young-Gwan,Lee, Cheol-Ho,Choi, Myung-Suk The Korean Society of Medicinal Crop Science 2004 韓國藥用作物學會誌 Vol.12 No.5

Scopolia parviflora Nakai, a rare and endangered species, is the sole plant producing tropane alkaloids (TA) among the Korean native species. In order to enhance TA productivity the SP72 root line was selected by screening 100 of root line, and the optimal culture media for root growth and TA production were investigated with the SP72 roots. Based on the several media, SH and 2B5 medium were determined as growth medium and White and NN medium as production medium. Among the four combinations of two-stage culture, 2BN (2B5 as growth medium plus NN as production medium) showed more enhanced root growth and TA production as compared with production media of White and NN medium and growth media of SH and 2B5 medium, respectively. However, bubble column bioreactor (BCB) cultures applying two-stage culture did not reveal the effective results despite of the each successful operation of two-stage culture in conical flasks and BCB cultures.

Production of Tropane Alkaloids by Two-stage Culture of Scopolia parviflora Nakai Adventitious Root

김운정 한국약용작물학회 2004 韓國藥用作物學會誌 Vol.12 No.5

Scopolia parviflora Nakai, a rare and endangered species, is the sole plant producing tropane alkaloids (TA) among the Korean native species. In order to enhance TA productivity the SP72 root line was selected by screening 100 of root line, and the optimal culture media for root growth and TA production were investigated with SP72 roots. Based on the several media, SH and 2B5 medium were determined as growth medium and White and NN medium as production medium. Among the four combinations of two-stage culture, 2BN (2B5 as growth medium Plus NN as production medium) showed more enhanced root growth and TA production as compared with production media of White and NN medium and growth media of SH and 2B5 medium respectively. However, bubble column bioreactor (BCB) cultures applying two-stage culture did not reveal the effective results despite of the each successful operation of two-stage culture in conical flasks and BCB cultures.

Enhancement of Poly-3-hydroxybutyrate (PHB) Productivity by the Two-Stage Supplementation of Carbon Sources and Continuous Feeding of NH₄Cl

여주상,Jae Yeon Park,Sung Ho Yeom,유영제 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.1

Gluconate and glucose were selected as the carbon substrates in the production of poly-3-hydroxybutyrate (PHB). Gluconate was utilized to maximize the specific growth rate during the first stage of cell growth, whereas glucose was used to maximize PHB biosynthesis during the second stage of PHB accumulation. The sequential feeding of gluconate and glucose resulted in a 50% enhancement of PHB productivity as compared to the cultures cultivated on glucose alone. In conjunction with secondary glucose uptake, the presence of a trace amount of ammonium increased the rate of PHB biosynthesis during the stage of PHB accumulation. Via the feeding of 0.03 mmol/h of NH4Cl solution prior to the exhaustion of the initial amount of NH4Cl, PHB productivity was significantly enhanced as compared to the cultures raised on glucose alone. The glucose-grown culture evidenced a higher level of NADPH during the NH4Cl-exausted PHB accumulation stage than was observed in the gluconate-grown culture, which reflects that the reason of higher PHB production observed when glucose was used as a carbon source. NH4Cl feeding following the depletion of initial NH4Cl resulted in elevated levels of both ATP and NADPH, which increased the PHB biosynthesis rate, and also in a decrease in the level of NADH, which reflected the alleviation of the inhibitory effects on the cells caused by nitrogen depletion.

Cultivation of four microalgae for biomass and oil production using a two-stage culture strategy with salt stress

Ra, Chae Hun,Kang, Chang-Han,Kim, Na Kyoung,Lee, Choul-Gyun,Kim, Sung-Koo Elsevier 2015 Renewable energy Vol.80 No.-

<P><B>Abstract</B></P> <P>A two-stage culture strategy was used for maximum biomass production under nutrient-sufficient conditions, followed by cultivation under low-salt stress, to cause the accumulation of oil in the biomass. Controlled conditions of nitrate, salt concentration, and time to exposure to stress were optimized for oil production with four species of microalgae, <I>Isochrysis galbana</I>, <I>Nannochloropsis oculata</I>, <I>Dunaliella salina</I>, and <I>Dunaliella tertiolecta</I>. Using conditions with addition of nitrate to 24.0 mg/L, <I>I. galbana</I> and <I>N. oculata</I> showed higher biomass productions than <I>D. salina</I> and <I>D. tertiolecta</I>. The oil contents of the microalgae increased from 24.0% to 47.0% in <I>I. galbana</I> with 10 psu for 2 days, from 17.0% to 29.0% in <I>N. oculata</I> with 0 psu for 3 days, from 22.0% to 43.0% of <I>D. salina</I> with 10 psu for 1 day, and from 23.0% to 40.0% (w/w) in <I>D. tertiolecta</I> with 0 psu for 2 days as the second stage culture with low-salt stress. Thus, <I>I. galbana</I> could be a suitable candidate microalga for oil production.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A two-stage culture was used for algae biomass production and for oil accumulation. </LI> <LI> Oil accumulation under low-salt stress enhanced overall oil productivity. </LI> <LI> The highest oil content was 47.0% with <I>I. galbana</I> at 10 psu salt stress for 2 days. </LI> <LI> <I>I. galbana</I> could be a suitable candidate microalga for oil production. </LI> <LI> Algae cell growth kinetics was determined by Monod equation. </LI> </UL> </P>

A Novel Two-stage pH Control Strategy for the Production of 5-Aminolevulinic Acid Using Recombinant Streptomyces coelicolor

Diep Ngoc Pham,김창준 한국생물공학회 2021 Biotechnology and Bioprocess Engineering Vol.26 No.4

5-aminolevulinic acid (ALA) has extensive use in photodynamic cancer therapy, tumor diagnosis, and agriculture. In the microbial production of ALA, most efforts have focused on engineering enzymes and the metabolic pathways involved in ALA biosynthesis. The aim of this study was to enhance ALA production using recombinant Streptomyces coelicolor expressing the ALA synthase gene (hem A) of Rhodobacter sphaeroides with a novel two-stage pH control strategy. Batch cultures were performed in production medium at different pH values. Although cells grew well at neutral pH (6.8-7.2), the highest amount of ALA was produced with a long culture time (140 h) at a weakly acidic pH (5.5-6.0). In response, a two-stage pH control strategy was developed in which pH was maintained at 6.8-7.2 for cell growth and then shifted to 5.5-6.0 to promote ALA synthesis, resulting in a significant enhancement in ALA production compared to a one-stage pH control strategy. The titer of ALA was further improved up to 482 mg/L in the two-stage pH culture by supplying more glucose in the medium and shifting the pH during the early phase of cultivation.

Accumulation of Cell Biomass Anthraquinones, Phenolics, and Flavonoids as Affected by Auxin, Cytokinin, and Medium Salt Strength in Cell Suspension Culture of Morinda citrifolia

Kyu-Man Shim(심규만),Eun-Joo Hahn(한은주),Won-Kyung Jeon(전원경),Kee-Yoeup Paek(백기엽) 한국원예학회 2010 원예과학기술지 Vol.28 No.2

노니(Morida citrifolia)의 세포 배양과 2차 대사산물 축적에 적합한 배지 조성을 알기 위해 배지 내 옥신(2,4-D, NAA, IBA: 0, 1.0, 3.0, 5.0, 7.0 and 9.0㎎?L<SUP>-1</SUP>), 싸이토키닌(Kinetin, BA: 0, 0.1, 0.3, 0.5, 0.7, and 0.9㎎?L<SUP>-1</SUP>), 무기물 농도(1/4, 1/2, 1, 3/2, 2MS)를 달리하여 3주간 배양하였다. 세포 생장은 NAA 3.0㎎?L<SUP>-1</SUP>, IBA 5.0 또는 7.0㎎?L<SUP>-1</SUP>에서 양호하였으나 2차대사산물의 축적은 옥신 처리에 의해 억제되었다. 옥신과 싸이토키닌의 혼용 효과를 알기 위해 옥신 실험에서 적합한 배지로 선발된 NAA 3㎎?L<SUP>-1</SUP>을 control 로 하여 kinetin과 BA의 농도를 달리하여 실험한 결과, BA 0.9㎎?L<SUP>-1</SUP>를 혼용하였을 때 2차 대사산물 함량이 증가하였다. 세포 생장과 2차 대사산물 함량에 적합한 MS 배지 내 무기물 농도는 서로 달라 세포 생장은 1MS에서, 2차 대사산물 함량은 2MS에서 가장 높았다. 그러나 2MS 배지의 경우, 세포의 고사율이 1MS에 비해 4배 이상 높아 세포 생장에는 치명적이었다. 이상의 결과로, Morinda citrifolia의 세포 배양의 생산성을 증가시키기 위해서는 세포 증식에 적합한 배지를 조성하여 biomass를 최대한 증가시킨 후 2차 대사산물 축적을 위한 배지로 교환하여 배양하는 2단계 배양(Two-stage culture)이 필요하다는 결론을 얻었다. Morida citrifolia cells were cultured with various medium compositions to determine optimal conditions of auxin, cytokinin and medium salt strength for cell biomass and secondary metabolite accumulation. Cell growth was enhanced by NAA (3.0 ㎎?L<SUP>-1</SUP>) and IBA (5.0 and 7.0 ㎎?L<SUP>-1</SUP>), while the accumulation of secondary metabolites (total anthraquinones, phenolics and flavonoids) was inhibited by auxin. The combination of 3.0 ㎎?L<SUP>-1</SUP> NAA and 0.9 ㎎?L<SUP>-1</SUP> BA accelerated the accumulation of secondary metabolites. The optimal MS medium strength for cell biomass and secondary metabolites differed: Cell growth was maximized at 1 MS, while the secondary metabolite contents reached the highest at 2 MS. The results suggested that a two-stage culture method is required for the cell suspension culture of Morinda citrifolia: Two different culture media should be supplied for the accumulation of cell biomass and secondary metabolites.

A fermentation strategy for production of recombinant protein subjected to plasmid instability

Shu-Jen Chen,Bo-Shun Ke,I-Chung Chiu 한국화학공학회 2008 Korean Journal of Chemical Engineering Vol.25 No.5

The appearance of plasmid-losing cells in a recombinant Escherichia coli culture was observed when the cell mass became doubled after induction, which corresponded to the timing of cell fission. Accordingly, a two-stage fermentation strategy capable of maintaining plasmid stability without selective pressure in a recombinant E. coli culture was proposed. In the first stage (cell growth stage), a high cell density culture was obtained by incubating the cells in the R medium. In the second stage (producing stage), the cells were devoted to producing the recombinant protein by introducing the fresh LB medium supplemented with isopropyl-β-D-thiogalactopyranoside (IPTG). It was necessary to prevent the doubling in the cell mass after induction; otherwise cell fission would occur and generate plasmid-losing cells. The present strategy is expected to be extensively applicable in recombinant E. coli cultures