• Title/Summary/Keyword: high cell density fermentation

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Production of a Platelet Aggregation Inhibitor, Salmosin, by High Cell Density Fermentation of Recombinant Escherichia coli

  • Seo, Myung-Ji;Choi, Hak-Jong;Chung, Kwang-Hoe;Pyun, Yu-Ryang
    • Journal of Microbiology and Biotechnology
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    • v.21 no.10
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    • pp.1053-1056
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    • 2011
  • Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation.

Metabolic Analysis of Poly(3-Hydroxybutyrate) Production by Recombinant Escherichia coli

  • WONG, HENG HO;RICHARD J. VAN WEGEN;JONG-IL CHOI;SANG YUP LEE
    • Journal of Microbiology and Biotechnology
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    • v.9 no.5
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    • pp.593-603
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    • 1999
  • Poly(3-hydroxybutyrate) (PHB) production by fermentation was examined under both restricted- and ample-oxygen supply conditions in a single fed-batch fermentation. Recombinant Escherichia coli transformed with the PHB production plasmid pSYLl07 was grown to reach high cell density (227 g/l dry cell weight) with a high PHB content (78% of dry cell weight), using a glucose-based minimal medium. A simple flux model containing 12 fluxes was developed and applied to the fermentation data. A superior closure (95%) of the carbon mass balance was achieved. When the data were put into use, the results demonstrated a surprisingly large excretion of formate and lactate. Even though periods of severe oxygen limitation coincided with rapid acetate and lactate excretion, PHB productivity and carbon utilization efficiency were not significantly impaired. These results are very positive in reducing oxygen demand in an industrial PHA fermentation without sacrificing its PHA productivity, thereby reducing overall production costs.

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고정상세포분리기의 개발 및 Cyclosporin A 생산을 위한 고정화 연속배양공정에의 적용

  • Lee, Tae-Ho;Park, Sung-Kwan;Chang, Yong-Keun;Chun, Gie-Taek
    • Microbiology and Biotechnology Letters
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    • v.24 no.6
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    • pp.717-725
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    • 1996
  • We have developed an efficient immobilized cell separator for continuous operation of immobilized fungal cell cultures, and applied this separator to actual fermentation process for the production of cyclosporin A (CyA), a powerful immunosuppressant. In the experiments employing highly viscous polymer (carboxymethyl cellulose) solution, the decantor showed good separating performances at high solution viscosites and fast dilution rates. Air duct and cylindrical separator installed inside the decantor turned out to play key roles for the efficient separation of the immobilized cells. By installing the decantor in an immobilized perfusion reactor system (IPRS), continuous immobilized culture was stably carried out even at high dilution rate for a long period, leading to high productivities of free cells and CyA. Almost no immobilized biomass existed in effuluent stream of the IPRS, demonstrating the effectiveness of the decan- tor system for a long-term continuous fermentation. It was noteworthy that we could obtain these results despite of the unfavorable fermentation conditions, i.e., reduced density of the biosupports caused by overgrowth of cells inside the bead particles and existence of high density of suspended fungal cells (10g/l) in the fermentation broth.

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Optimization of Induction Conditions for Bacillus-derived Esterase Production by High-cell Density Fermentation of Recombinant Escherichia coli (재조합 대장균의 고농도 배양과 유도조건 최적화를 통한 Bacillus 유래 esterase의 생산)

  • Kang, Seung-Hoon;Min, Byung-Hyuk;Choi, Hong-Yeol;Kim, Dong-Il
    • Microbiology and Biotechnology Letters
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    • v.45 no.2
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    • pp.149-154
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    • 2017
  • To increase the efficiency of esterase production by Bacillus, high cell-density culture of recombinant Escherichia coli through fed batch fermentation was tested. Cells were cultured to $OD_{600}$ of 76 (35.8 g/l DCW) with dissolved oxygen level controlled to least above 30% air saturation by supplying pure oxygen. Cells were cultured to an $OD_{600}$ of 90 (42.4 g/l DCW) with glucose feeding controlled to at least 1 g/l. However, the cells reached stationary phase at the late stage of culture, despite glucose being supplied. Cells were cultured to an $OD_{600}$ of 185 (87.3 g/l DCW) by supplying additional medium with fortified yeast extract. To increase the productivity of the recombinant protein, cell growth and esterase productivity based on induction time were evaluated. Late exponential phase induction for esterase production in fed batch fermentation resulted in maximum optical density $OD_{600}$ of 190 (89 g/l DCW) and maximum esterase activity of 1745 U/l, corresponding to a 5.8-fold enhancement in esterase production, compared to the early exponential phase induction. In this study, we established fermentation methods for achieving maximum production of Bacillus-derived esterase by optimizing IPTG induction time in high-cell density culture by supplying pure oxygen and a nitrogen source.

Study on the variation of cellular physiology of Escherichia coli during high cell density cultivation using 2-dimensional gel electrophoresis

  • Yun, Sang-Seon;Lee, Sang-Yeop
    • 한국생물공학회:학술대회논문집
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    • 2000.04a
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    • pp.219-222
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    • 2000
  • Physiological changes of Escherichia coli during the fed-batch fermentation process were characterized in this study. Overall cellular protein samples prepared at the different stage of fermentation were separated by 2-dimensional gel electrophoresis (2-DE), and differently expressed 15 proteins, Phosphotransferase enzyme I, GroEL, Trigger factor, ${\beta}$ subunit of ATP synthase, Transcriptional regulator KDGR, Phosphoglycerate mutase 1, Inorganic pyrophosphatase, Serine Hydroxymethyl-transferase, ${\alpha}$ subunit of RNA polymerase, Elongation factor Tu, Elongation factor Ts, Tyrosine-tRNA ligase, DnaK suppressor protein, Transcriptional elongation factor, 30S ribosomal protein S6 were identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). When bacterial cells grow to high cell density, and IPTG-inducible heterologous protein is produced, expression level of overall cellular proteins was decreased. According to their functions in the cell, identified proteins were classified into three groups, proteins involved in transport process, small-molecule metabolism, and synthesis and modification of macromolecules.

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Expression profile analysis of metabolism of Escherichia coli during high cell density cultivation using DNA chip

  • Yun, Seong-Ho;Lee, Sang-Yeop;Im, Geun-Bae
    • 한국생물공학회:학술대회논문집
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    • 2000.04a
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    • pp.600-603
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    • 2000
  • DNA chip containing 207 E. coli genes related to important metabolisms such as (TCA cycle, glycolysis, fermentation and etc) were used to carry out a comprehensive investigation of the change in metabolism and physiology during high cell density culture of E. coli by fed-batch cultivation.

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Fed-batch Fermentation for Production of Nitrile Hydratase by Rhodococcus rhodochrous M33

  • Kim, Bu-Youn;Kim, Jong-Chul;Lee, Hyune-Hwan;Hyun, Hyung-Hwan
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.6 no.1
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    • pp.11-17
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    • 2001
  • To enhance the productivity and activity of nitrile hydratase in Rhodococcus rhodochrous M33, a glucose-limited fed-batch culture was performed. In a fed-batch culture where the glucose was controlled at a limited level and cobalt was supplemented during the fermentation period, the cell mass and total activity of nitrile hydratase both increased 3.3-fold compared to that in the batch fermentation. The productivity of nitrile hydratase also increased 1.9-fold compared to that in the batch fermentation. The specific activity of nitrile hydratase in the whole cell preparation when using a fed-batch culture was 120 units/mg-DCW, which was similar to that in the batch culture.

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Cell separation from high density culture broths of Alcaligenes eutrophus by using Al-based coagulants (Alcaligens eutrohus 고농도 배양액으로부터 알루미늄(Al)계 응집제를 이용한 세포분리)

  • 조경숙;류희욱;정현우;곽종운;장용근
    • KSBB Journal
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    • v.13 no.3
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    • pp.272-278
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    • 1998
  • Cell recovery from high cell density broths of Alcaligenes eutrophus by pretreatment with aluminum-based coagulants such as aluminum sulfate, polyaluminum hydrooxide chloride silicate (PACS), and polyaluminum hydrooxide chloride (Hi-PAX) was carried out. Cells coagulated with coagulants could be successfully recovered above 95-99% by centrifugation or filtration. The optimum initial pH of fermentation broths for cell recovery was in the range of 10 to 12. Optimum coagulants dosage for cell recovery increased with increasing of cell concentrations (21-160 g/L). The optimum coagulant dosages to recover cells with more than 95% cell recovery by centrifugation for the cell concentrations ranged 21-160 g/L were as follows: aluminum sulfate, 416-1708 mg Al/L; PACS, 211-826 mg Al/L; Hi-PAX, 320-960 mg Al/L. At optimum conditions for the coagulation of cells, centrifugal forces for 95% of cell recovery were dependent on the cell concentration. The centrifugal forces at 82 g/L and 160 g/L of cell concentration were only 45${\times}$g and 1600${\times}$g, respectively.

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Linear Correlation between Online Capacitance and Offline Biomass Measurement up to High Cell Densities in Escherichia coli Fermentations in a Pilot-Scale Pressurized Bioreactor

  • Knabben, Ingo;Regestein, Lars;Schauf, Julia;Steinbusch, Sven;Buchs, Jochen
    • Journal of Microbiology and Biotechnology
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    • v.21 no.2
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    • pp.204-211
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    • 2011
  • To yield high concentrations of protein expressed by genetically modified Escherichia coli, it is important that the bacterial strains are cultivated to high cell density in industrial bioprocesses. Since the expressed target protein is mostly accumulated inside the E. coli cells, the cellular product formation can be directly correlated to the bacterial biomass concentration. The typical way to determine this concentration is to sample offline. Such manual sampling, however, wastes time and is not efficient for acquiring direct feedback to control a fedbatch fermentation. An E. coli K12-derived strain was cultivated to high cell density in a pressurized stirred bioreactor on a pilot scale, by detecting biomass concentration online using a capacitance probe. This E. coli strain was grown in pure minimal medium using two carbon sources (glucose and glycerol). By applying exponential feeding profiles corresponding to a constant specific growth rate, the E. coli culture grew under carbon-limited conditions to minimize overflow metabolites. A high linearity was found between capacitance and biomass concentration, whereby up to 85 g/L dry cell weight was measured. To validate the viability of the culture, the oxygen transfer rate (OTR) was determined online, yielding maximum values of 0.69 mol/l/h and 0.98mol/l/h by using glucose and glycerol as carbon sources, respectively. Consequently, online monitoring of biomass using a capacitance probe provides direct and fast information about the viable E. coli biomass generated under aerobic fermentation conditions at elevated headspace pressures.

High Cell Density Cultivation of Pseudomonas oleovorans for the Production of Poly(3-Hydroxyalkanoates)

  • Lee, Sang-Yup
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.1 no.1
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    • pp.51-53
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    • 1996
  • Fed-batch culture of Pseudomonas oleovorans was carried out for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) using octanoate as a carbon source. Octanoate and the salt solution containing ammounium sulfate and magnesium sulfate were intermittently fed in the course of fermentation. Cell mass and PHA concentrations of 42.8 and 16.8g/L, respectively, could be obtained in 40 h. The PHA content and the PHA productivity were 39.2% and 0.42 g PHA/L-h, respectively. The yields of cell mass and PHA were 0.71 g dry cell mass/g octanoate and 0.28g PHA/g octanoate, respectively. Therefore, octanoate can be used for the production of MCL-PHAs to a high concentration with high productivity.

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