• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.2
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• pp.105-111
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• 2002

The biological removal of 2,4,6-trinitrotoluene (TNT) was studied in a bench-scale bioreactor using a bacterial culture of strain OK-5 originally Isolated from soil samples contaminated with TNT. The TNT was completely removed within 4 days of incubation in a 2.5 L bench-scale bioreactor containing a newly developed medium. The TNT was catabolized in the presence of different supplemented carbons. Only minimal growth was observed in the killed controls and cultures that only received TNT during the incubation period. This catabolism was affected by the concentration ratio of the substrate to the biomass. The addition of various nitrogen sources produced a delayed effect for the TNT degradation. Tween 80 enhanced the degradation of TNT under these conditions. Two metabolic intermediates were detected and identified as 2-amino-4, 6-dinitrotoluene and 4-amino-2, 6-dinitrotoluene based on HPLC and GC-MS analyses, respectively. Strain OK-5 was characterized using the BIOLOG system and fatty acid profile produced by a microbial identification system equipped with a Hewlett Packard HP 5890 II gas chromatograph. As such, the bacterium was identified as a Stenotrophomonas species and designated as Stenotrophomonas sp. OK-5.

• 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.

• Journal of Microbiology and Biotechnology
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• v.20 no.8
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• pp.1230-1239
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• 2010

Five bacterial species, capable of degrading the recalcitrant organic compounds (ROCs) diethyleneglycol monomethylether (DGMME), 1-amino-2-propanol (APOL), 1-methyl-2-pyrrolidinone (NMP), diethyleneglycol monoethylether (DGMEE), tetraethyleneglycol (TEG), and tetrahydrothiophene 1,1-dioxide (sulfolane), were isolated from an enrichment culture. Cupriavidus sp. catabolized $93.5{\pm}1.7$ mg/l of TEG, $99.3{\pm}1.2$ mg/l of DGMME, $96.1{\pm}1.6$ mg/l of APOL, and $99.5{\pm}0.5$ mg/l of NMP in 3 days. Acineobacter sp. catabolized 100 mg/l of DGMME, $99.9{\pm}0.1$ mg/l of NMP, and 100 mg/l of DGMEE in 3 days. Pseudomonas sp.3 catabolized $95.7{\pm}1.2$ mg/l of APOL and $99.8{\pm}0.3$ mg/l of NMP. Paracoccus sp. catabolized $98.3{\pm}0.6$ mg/l of DGMME and $98.3{\pm}1.0$ mg/l of DGMEE in 3 days. A maximum $43{\pm}2.0$ mg/l of sulfolane was catabolized by Paracoccus sp. in 3 days. When a mixed culture composed of the five bacterial species was applied to real wastewater containing DGMME, APOL, NMP, DGMEE, or TEG, 92~99% of each individual ROC was catabolized within 3 days. However, at least 9 days were required for the complete mineralization of sulfolane. Bacterial community diversity, analyzed on the basis of the TGGE pattern of 16S rDNA extracted from viable cells, was found to be significantly reduced in a conventional bioreactor after 6 days of incubation. However, biodiversity was maintained after 12 days of incubation in an electrochemical bioreactor. In conclusion, the electrochemical reduction reaction enhanced the diversity of the bacterial community and actively catabolized sulfolane.

• Journal of Plant Biotechnology
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• v.3 no.2
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• pp.107-112
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• 2001

The kinetics of Ti-transformed Salvia miltiorrhiza cell cultures was studied in 250-$m\ell$ shake flasks by using B5 medium with addition of 30 gfL of sucrose. In the cell cultures, the maximum cell mass obtained was 11.5 g DW/L on day 15. The highest amount of phenolic compounds - rosmarinic acid (RA) and lithospermic acid B (LAB) reached 871.3 mg/L (day 15) and 121.3 mg/L (day 13), respectively. The total tanshinone production, i.e., intracellular plus extracellular cryptotanshinone, tanshinone 1, and tanshinone IIA, was 5.3 mg/L on day 13. For the cultivations in 2.4-L stirred bioreactors, the residual sugar level and medium conductivity were a little higher in a small turbine impeller reactor ($T_s$) than those in a large turbine impeller reactor ($T_L$), while a higher cell density was obtained in the $T_L$. For the production of tanshinones and phenolics, better results were obtained in the $T_L$ than in the $T_s$. In the $T_L$, similar or even a little higher production titers of tanshinones and phenolic compounds were achieved compared to those in the flasks. The results suggest that the shake flask results could be successfully scaled up to the $T_L$ reactor. Such a large impeller reactor like $T_L$ may be better than a small impeller one for the large-scale production of the valuable metabolites by the suspension cultures of Ti transformed S.miltiorrhiza cells. This is considered due to the beneficial culture environment in the $T_L$, such as low shear rates as estimated theoretically.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.183-187
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• 2008

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. $VEGF_{165}$ is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human $VEGF_{165}$ $(rhVEGF_{165})$ protein. The production rate of the established CHO cells was over 80mg/l of $rhVEGF_{165}$ protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The $rhVEGF_{165}$ protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified $rhVEGF_{165}$ protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the $rhVEGF_{165}$ protein in CHO cells differed from that in insect cells. The purified $rhVEGF_{165}$ protein in this study was functionally active with a half-maximal effective concentration of 3.8ng/ml and specific activity of $2.5{\times}10^5U/mg$.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1534-1538
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• 2010

Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used a high-purity oxygen-supplying strategy to increase the viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7, was utilized in this study as a host strain in both 5-l and 30-l scale fermentors. To supply high-purity oxygen into a bioreactor, nearly 100% high-purity oxygen from a commercial bomb or higher than 93% oxygen available in situ from a pressure swing adsorption (PSA) oxygen generator was employed. Under the optimal fermentation of H. polymorpha with highpurity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/l and 5.1 g/l in the 5-l fermentor, and 24.8 g/l and 4.5 g/l in the 30-l fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-l and 30-l fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-l fermentor. This study, therefore, proved the positive effect of high-purity oxygen in enhancing viable cell density as well as target recombinant protein production in microbial fermentations.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.35-40
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• 2004

Rotavirus virus-like particle (VLP) composed of VP2, VP6, and VP7 was expressed in the Baculovirus Expression Vector System (BEVS). Sf9 cell, a host of the baculovirus, was cultured from a 0.5-1 spinner flask to the 50-1 bioreactor system. Sf9 cell was maintained at cell density between 3.0E＋05 and 3.0E＋06 cells/ml and grew up to 1.12E＋07 cells/ml in the bioreactor. Growth kinetics was compared under different culture systems and showed similar growth kinetics with 20.1-25.2 h of doubling time. Early exponentially growing cell culture was infected with three recombinant baculoviruses expressing VP2, VP6, and VP7 protein at 1.0, 2.0, and 0.2 moi, respectively. The expression of rotavirus proteins was confirmed by Western blot analysis and its three-layered virus-like structure was observed under an electron microscope. Rotavirus VLP was semipurified and immunized in ICR mice intramuscularly. Rotavirus-specific serum antibody was detected from 2 weeks after the immunization and lasted at least 21 weeks of the post-immunization, indicating its possible use as a vaccine candidate.

• Journal of Plant Biotechnology
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• v.43 no.1
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• pp.110-118
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• 2016

We compared germination efficiency for somatic embryos (SE) of Liriodendron tulipifera using semi-solid (SS), temporary immersion bioreactors (TIB), and continuous immersion bioreactors (CIB) to produce vigorous plants. The bioreactors were designed to be immersed in liquid media with plantlets with an adjustable immersion time. TIB and CIB improved germination rates up to 80.86% and 95.21%, respectively, however, CIB produced more hyperhydric plantlets than TIB. The height of plantlets in TIB was significantly higher than for those in CIB. Fresh weights of plantlets grown in CIB of were significantly lower than for those grown in TIB. The lowest chlorophyll concentration was found in in vitro plantlets from CIB. We examined abnormally developed leaves, stems, and apical zones of in vitro plantlets that were produced in CIB. Among the three types, SS showed the highest stomatal density and the shortest stomatal length in in vitro plantlets. After acclimatization, plants from CIB exhibited the lowest values in biomass, such as height, root collar diameter, leaf fresh weight, leaf length, leaf width, petiole length, petiole diameter, and leaf area. Photosynthesis and transpiration rates of ex vitro plants were not significantly different among the three culture types, but stomatal conductance was higher in TIB than in the SS and CIB. Therefore, the results suggest that TIB is the preferable bioreactor to improve in vitro plantlet regeneration of L. tulipifera. TIB-originated plants showed higher growth rate than SS and CIB after transferring to soil.

• Horticultural Science & Technology
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• v.33 no.6
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• pp.877-882
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• 2015

This study was carried out treatment of polyethylene glycol (PEG) in order to increase of survival rate of acclimated plants of strawberry's in vitro plantlets through bioreactor culture. We used PEG with molecular weight 6000 (PEG 6000) in this study. Concentration of PEG is non-treatment, 5, 10, 15, and $20g{\cdot}L^{-1}$ each bioreactor. $5g{\cdot}L^{-1}$ of PEG was treated at $1^{st}$, $2^{nd}$, $3^{rd}$, $4^{th}$, and $5^{th}$ week during culture. We investigated growth characteristics of in vitro plantlets after 6 weeks cultivation. Growth amount of all PEG treatment decreased as compared to non-treatment. The more concentration increased, the more plant growth decreased. In $5g{\cdot}L^{-1}$ of PEG, shoot length was shorter than non-treatment that shoot length was 7.9 cm and especially fresh weight that is 1.6 g was more decrease than non-treatment. Shoot length was ranged 3.0-3.9 cm at $1^{st}$ week treatment to $4^{th}$ week treatment of $5g{\cdot}L^{-1}$ PEG. The shoot length was not significant by 5.3 cm at $5^{th}$ week treatment. The survival rate was improved 5.4% at the treatment of $4^{th}$ week and was improved 8.7% at the $5^{th}$ week as compared to non-treatment. In order to improve of survival rate of strawberry' in vitro plantlets through bioreactor culture, the method is suitable that adding $5g{\cdot}L^{-1}$ of PEG in the medium and $5^{th}$ week's treatment is suitable.

• 한국생물공학회:학술대회논문집
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• 2000.11a
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• pp.159-162
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• 2000

It has been commonly known that cell growth is inhibited by the lack of dissolved oxygen and mass transfer inhibition of nutrients at stationary phase in fed-batch culture. In this study, Pluronic F-68 and oxygen vectors were added in Angelica gigas Nakai suspension culture in order to enhance cell growth in fed-batch culture. It was observed that the addition of 6%(w/v) Pluronic F-68 promoted cell growth up to 6.1% compared to control and that the use of 4%(v/v) n-hexadecane markedly enhanced cell growth up to 11.4%.