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Shake Flask Culture (shake + flask_culture)
Selected AbstractsModeling and optimization of hairy root growth in fed-batch processBIOTECHNOLOGY PROGRESS, Issue 3 2010Francis Mairet Abstract This article proposes a feeding strategy based on a kinetic model to enhance hairy roots growth. A new approach for modeling hairy root growth is used, considering that there is no nutrient limitation thanks to an appropriate feeding, and the intracellular pools are supposed to be always saturated. Thus, the model describes the specific growth rate from extracellular concentration of the major nutrients and nutrient uptakes depend on biomass growth. An optimized feeding strategy was determined thanks to the model to maintain the major nutrient levels at their optimum assuming optimal initial concentrations. The optimal feed rate is computed in open loop using kinetic model prediction or in closed loop using conductivity measurements to estimate biomass growth. Datura innoxia was chosen as the model culture system. Shake flask cultures were used to calibrate the model. Finally, cultures in bioreactor were performed to validate the model and the control laws. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Influence of nutritional conditions on the mycelial growth and exopolysaccharide production in Paecilomyces sinclairiiLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2002S.W. Kim Aims:,The objective of the study was to optimize the submerged culture conditions for the production of exopolysaccharide from Paecilomyces sinclairii. Methods and Results:,The optimal temperature and initial pH for exopolysaccharide production by Paecilomyces sinclairii in shake flask culture were found to be 30°C and 6·0, respectively. Sucrose (60 g l,1) and corn steep powder (10 g l,1) were the most suitable carbon and nitrogen source for exopolysaccharide production. Conclusions:,Under optimal culture medium, the maximum exopolysaccharide concentration in a 5-l stirred-tank fermenter indicated 7·4 g l,1, which was approximately three times higher than that in basal medium. The maximum specific growth rates (,max) and yield coefficient (YP/S) in the optimal culture medium was 0·16 h,1 and 0·19, respectively. Significance and Impact of the Study:,The optimal culture conditions reported in this article can be widely applied to the processes for submerged cultures of other mushrooms. [source] Sensitivity to Hydrogen Peroxide of Growth and Hyaluronic Acid Production by Streptococcus zooepidemicus ATCC 39920ASIA-PACIFIC JOURNAL OF CHEMICAL ENGINEERING, Issue 5-6 2005M.D. Mashitah Abstract The sensitivity to hydrogen peroxide (H2O2) of growth and hyaluronic acid (HA) production by Streptococcus zooepidemicus ATCC 39920 was studied under various conditions. In sheep blood agar-plates, no detectable zone was observed even when the concentration of H2O2 was increased to 0.15 mM. With brain heart infusion-agar and chemically defined medium-agar plates, a profound zone was detected at 0.015 mM concentration of H2O2. To determine the minimal inhibitory concentration (MIC) of the strain in culture broth, various concentrations of H2O2 (0-200 mM) were maintained in the medium prior to fermentation. The result showed that for higher concentrations of H2O2 in the medium, the greater was the inhibition. Streptococcus is catalase-negative and known to produce H2O2 which may affect growth, HA production and glucose utilization. In order to determine at which growth phase H2O2 had the maximum inhibitory activity, a batch fermentation of S. zooepidemicus was conducted in shake flask culture. It was found that H2O2 production took place during the growth phase, and HA production started after the growth had reach late exponential phase when H2O2 in the culture media was depleted. This indicates that H2O2 produced by the cells did not affect cell growth but influenced HA production. [source] In situ magnetic separation for extracellular protein productionBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Tobias Käppler Abstract A new approach for in situ product removal from bioreactors is presented in which high-gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non-porous bacitracin linked magnetic adsorbents were employed directly in the broth during the fermentation, followed by in situ magnetic separation. Proof of the concept was first demonstrated in shake flask culture, then scaled up and applied during a fed batch cultivation in a 3.7 L bioreactor. It could be demonstrated that growth of B. licheniformis was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we could show that protease yield in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in bio production processes which are often limited due to weak downstream operations. Potential limitations encountered during a bioprocess can be overcome such as product inhibition or degradation. We also discuss the key points where research is needed to implement in situ magnetic separation in industrial production. Biotechnol. Bioeng. 2009;102: 535,545. © 2008 Wiley Periodicals, Inc. [source] Plasmid system for the intracellular production and purification of affinity-tagged proteins in Bacillus megaterium,BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Rebekka Biedendieck Abstract A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His6 -tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry. Biotechnol. Bioeng. 2007;96: 525,537. © 2006 Wiley Periodicals, Inc. [source] Improved Paclitaxel and Baccatin III Production in Suspension Cultures of TaxusmediaBIOTECHNOLOGY PROGRESS, Issue 3 2002Rosa M. Cusidó A cell suspension culture of Taxus media was established from a stable callus line of this species. The growth rate and production of paclitaxel and baccatin III of this cell suspension were significantly increased during the shake flask culture in its respective optimum media for cell growth and product formation, which were selected after assaying 24 different culture media. The highest yields of paclitaxel (2.09 mg L,1) and baccatin III (2.56 mg L,1) in the production medium rose (factors of 7.0 and 3.0, respectively) in the presence of methyljasmonate (220 ,g g,1 FW). When the elicitor was added together with mevalonate (0.38 mM) and N -benzoylglycine (0.2 mM), the increase in the yields of paclitaxel and baccatin III was even higher (factors of 8.3 and 4.0, respectively). Thereafter, a two-stage culture for cell suspension was carried out using a 5,l stirred bioreactor running for 36 days, the first stage being in the cell growth medium until cells entered their stationary growth phase (12 days) and the second stage being in the production medium supplemented with the elicitor and two putative precursors in the concentrations indicated above. Under these conditions, 21.12 mg L,1 of paclitaxel and 56.03 mg L,1 of baccatin III were obtained after 8 days of culture in the production medium. [source] Microbiological and geochemical dynamics in simulated-heap leaching of a polymetallic sulfide oreBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2008Kathryn Wakeman Abstract The evolution of microbial populations involved in simulated-heap leaching of a polymetallic black schist sulfide ore (from the recently-commissioned Talvivaara mine, Finland) was monitored in aerated packed bed column reactors over a period of 40 weeks. The influence of ore particle size (2-6.5 mm and 6.5-12 mm) on changes in composition of the bioleaching microflora and mineral leaching dynamics in columns was investigated and compared to fine-grain (<2 µm) ore that was bioprocessed in shake flask cultures. Both column reactors and shake flasks were inoculated with 24 different species and strains of mineral-oxidizing and other acidophilic micro-organisms, and maintained at 37°C. Mineral oxidation was most rapid in shake flask cultures, with about 80% of both manganese and nickel and 68% of zinc being leached within 6 weeks, though relatively little of the copper present in the ore was solubilised. The microbial consortium that emerged from the original inoculum was relatively simple in shake flasks, and was dominated by the iron-oxidizing autotroph Leptospirillum ferriphilum, with smaller numbers of Acidimicrobium ferrooxidans, Acidithiobacillus caldus and Leptospirillum ferrooxidans. Both metal recovery and (for the most part) total numbers of prokaryotes were greater in the column reactor containing the medium-grain than that containing the coarse-grain ore. The bioleaching communities in the columns displayed temporal changes in composition and differed radically from those in shake flask cultures. While iron-oxidizing chemoautotrophic bacteria were always the most numerically dominant bacteria in the medium-grain column bioreactor, there were major shifts in the most abundant species present, with the type strain of Acidithiobacillus ferrooxidans dominating in the early phase of the experiment and other bacteria (At. ferrooxidans NO37 and L. ferriphilum) dominating from week 4 to week 40. With the coarse-grain column bioreactor, similar transitions in populations of iron-oxidizing chemoautotrophs were observed, though heterotrophic acidophiles were often the most abundant bacteria found in mineral leach liquors. Four bacteria not included in the mixed culture used to inoculate the columns were detected by biomolecular techniques and three of these (all Alicyclobacillus -like Firmicutes) were isolated as pure cultures. The fourth bacterium, identified from a clone library, was related to the Gram-positive sulfate reducer Desulfotomaculum salinum. All four were considered to have been present as endospores on the dried ore, which was not sterilized in the column bioreactors. Two of the Alicyclobacillus -like isolates were found, transiently, in large numbers in mineral leachates. The data support the hypothesis that temporal and spatial heterogeneity in mineral heaps create conditions that favour different mineral-oxidizing microflora, and that it is therefore important that sufficient microbial diversity is present in heaps to optimize metal extraction. Biotechnol. Bioeng. 2008;101: 739,750. © 2008 Wiley Periodicals, Inc. [source] Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell cultureBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2008Aleksei Rozkov Abstract Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h,1) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68,107 g,L,1 wet weight) achieves high volumetric yields of plasmid (95,277 mg,L,1 depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg,1 or less. Biotechnol. Bioeng. 2008;99: 557,566. © 2007 Wiley Periodicals, Inc. [source] Mixed Culture Bioconversion of 16-Dehydropregnenolone Acetate to Androsta-1,4-diene-3,17-dione: Optimization of ParametersBIOTECHNOLOGY PROGRESS, Issue 2 2003Tushar Banerjee Bioconversion of 16-dehydropregnenolone acetate (16-DPA) to androsta-1,4-diene-3,17-dione (ADD), an intermediate for the production of female sex hormones, by mixed culture of Pseudomonasdiminuta MTCC 3361 and Comamonas acidovorans MTCC 3362 is reported. Various physicochemical parameters for the bioconversion of 16-DPA to ADD have been optimized in shake flask cultures. Nutrient broth inoculated with actively growing co-culture proved ideal for bacterial growth and bioconversion. A temperature range of 35,40 °C was most suitable; higher or lower temperatures adversely affected the bioconversion. Dimethylformamide below 2% concentration was the most suitable carrier solvent. Maximum conversion was recorded at 0.5 mg mL,1 16-DPA. A pH of 5.0 yielded a peak conversion of 62 mol % in 120 h incubation period. Addition of 9,-hydroxylase inhibitors failed to prevent further breakdown of ADD to nonsteroidal products. 16-DPA conversion in a 5 L fermenter followed a similar trend. [source] | ||||||||||