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Jochen Büchs - One of the best experts on this subject based on the ideXlab platform.
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online measurement of co2 and total gas production in parallel anaerobic Shake Flask cultivations
Biochemical Engineering Journal, 2020Co-Authors: Andreas Schulte, Garret Munch, Marcel Mann, Robert Dinger, Lars Regestein, Lars Rehmann, Jochen BüchsAbstract:Abstract Online measurements of off-gas streams are often crucial for studying bioconversion processes. However, for anaerobic processes, options for online off-gas analysis are typically restricted to lab-scale bioreactors or larger systems, while gas measurements at smaller scales typically do not discriminate between different gases. In this work, a method for online measurement of CO2 and total gas production in anaerobic fermentations at the Shake Flask scale is described, extending capabilities of a previously reported device developed for aerobic processes to anaerobic bioprocesses. The novel design allows anaerobic fermentations to be performed in multiple parallel vessels, all of which collect online gas signals. The online gas signals are used to calculate the transfer rates, allowing near real-time visualization of the progress of eight fermentations operating in parallel. Conditions such as carbon source depletion, inhibition of growth, and exhaustion of a single carbon source in a dual-substrate fermentation can all be clearly distinguished. The combination of online signals and offline analysis allowed for carbon balances to be performed with high degrees of closure. The new design allows for higher throughput screening of anaerobic bioprocesses, an area lacking in small-scale options with off-gas analysis capabilities.
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validation of the transferability of membrane based fed batch Shake Flask cultivations to stirred tank reactor using three different protease producing bacillus strains
Journal of Bioscience and Bioengineering, 2019Co-Authors: Janina Muller, Tobias Habicher, Anne Hutterott, Nina Musmann, Jochen BüchsAbstract:Most industrial fermentation processes are operated in fed-batch mode to overcome catabolite repression, undesired by-product formation and oxygen limitation. To maintain comparable process conditions during screening of optimal production strains, the implementation of a fed-batch mode at small scale is crucial. In this study, three different protease producing Bacillus species, Bacillus aeolius, B. licheniformis and B. pumilus, were cultivated using the previously described membrane-based fed-batch Shake Flasks. Under carbon-limited conditions, catabolite repression was avoided, so that proteases were produced in all strains. Protease yields of B. aeolius and B. licheniformis increased 1.5-fold relative to batch cultivations. To validate process scalability between Shake Flasks and stirred tank reactors, membrane-based fed-batch Shake Flask cultivations were transferred to laboratory-scale stirred tank reactors with equal feeding rates. Despite inevitable differences between the scales such as pH control, feed supply and feed start, comparable results were achieved. Oxygen transfer rates of B. licheniformis and B. pumilus measured with the respiration activity monitoring system (RAMOS) in Shake Flasks and in stirred tank reactor with an off-gas analyzer were almost identical in both cultivation systems. The protease activities referring to the total consumed glucose were also mostly comparable. A slight decrease from Shake Flask to stirred tank reactor could be observed, which is presumably due to differences in pH control. This study successfully demonstrates the transferability of membrane-based fed-batch Shake Flask cultivations to laboratory-scale stirred tank reactors.
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parallel substrate supply and ph stabilization for optimal screening of e coli with the membrane based fed batch Shake Flask
Microbial Cell Factories, 2018Co-Authors: P. Philip, D. Kern, J. Goldmanns, F Seiler, Andreas Schulte, Tobias Habicher, Jochen BüchsAbstract:Screening in the fed-batch operation mode is essential for biological cultivations facing challenges as oxygen limitation, osmotic inhibition, catabolite repression, substrate inhibition or overflow metabolism. As a screening tool on Shake Flask level, the membrane-based fed-batch Shake Flask was developed. While a controlled supply of a substrate was realized with the in-built membrane tip, the possibilities for replenishing nutrients and stabilizing pH values was not yet exploited. High buffer concentrations were initially used, shifting the medium osmolality out of the biological optimum. As the growth rate is predefined by the glucose release kinetics from the reservoir, the resulting medium acidification can be compensated with a controlled continuous supply of an alkaline compound. The focus of this research is to establish a simultaneous multi-component release of glucose and an alkaline compound from the reservoir to enable cultivations within the optimal physiological range of Escherichia coli. In combination with the Respiratory Activity MOnitoring System, the membrane-based fed-batch Shake Flask enabled the detection of an ammonium limitation. The multi-component release of ammonium carbonate along with glucose from the reservoir resulted not only in the replenishment of the nitrogen source but also in the stabilization of the pH value in the culture medium. A biomass concentration up to 25 g/L was achieved, which is one of the highest values obtained so far to the best of the author’s knowledge with the utilization of a Shake Flask and a defined synthetic medium. Going a step further, the pH stabilization allowed the decrease of the required buffer amount to one-fourth establishing an optimal osmolality range for cultivation. As optimal physiological conditions were implemented with the multi-component release fed-batch cultivation, the supply of 0.2 g glucose in a 10 mL initial culture medium volume with 50 mM MOPS buffer resulted in a twofold higher biomass concentration than in a comparable batch cultivation. The newly introduced multi-component release with the membrane-based fed-batch Shake Flask serves a threefold purpose of replenishing depleted substrates in the culture medium, stabilizing the pH throughout the entire cultivation time and minimizing the necessary amount of buffer to maintain an optimal osmolality range. In comparison to a batch cultivation, these settings enable to achieve higher biomass and product concentrations.
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Three-dimensional (3D) evaluation of liquid distribution in Shake Flask using an optical fluorescence technique
Journal of biological engineering, 2017Co-Authors: Amizon Azizan, Jochen BüchsAbstract:Biotechnological development in Shake Flask necessitates vital engineering parameters e.g. volumetric power input, mixing time, gas liquid mass transfer coefficient, hydromechanical stress and effective shear rate. Determination and optimization of these parameters through experiments are labor-intensive and time-consuming. Computational Fluid Dynamics (CFD) provides the ability to predict and validate these parameters in bioprocess engineering. This work provides ample experimental data which are easily accessible for future validations to represent the hydrodynamics of the fluid flow in the Shake Flask. A non-invasive measuring technique using an optical fluorescence method was developed for Shake Flasks containing a fluorescent solution with a waterlike viscosity at varying filling volume (VL = 15 to 40 mL) and shaking frequency (n = 150 to 450 rpm) at a constant shaking diameter (do = 25 mm). The method detected the leading edge (LB) and tail of the rotating bulk liquid (TB) relative to the direction of the centrifugal acceleration at varying circumferential heights from the base of the Shake Flask. The determined LB and TB points were translated into three-dimensional (3D) circumferential liquid distribution plots. The maximum liquid height (Hmax) of the bulk liquid increased with increasing filling volume and shaking frequency of the shaking Flask, as expected. The toroidal shapes of LB and TB are clearly asymmetrical and the measured TB differed by the elongation of the liquid particularly towards the torus part of the Shake Flask. The 3D liquid distribution data collected at varying filling volume and shaking frequency, comprising of LB and TB values relative to the direction of the centrifugal acceleration are essential for validating future numerical solutions using CFD to predict vital engineering parameters in Shake Flask.
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Systematic evaluation of characteristics of the membrane-based fed-batch Shake Flask
Microbial cell factories, 2017Co-Authors: P. Philip, Cornelia Bähr, Kristina Meier, D. Kern, J. Goldmanns, F. Stockmeier, Jochen BüchsAbstract:The initial part of process development involves extensive screening programs to identify optimal biological systems and cultivation conditions. For a successful scale-up, the operation mode on screening and production scale must be as close as possible. To enable screening under fed-batch conditions, the membrane-based fed-batch Shake Flask was developed. It is a Shake Flask mounted with a central feed reservoir with an integrated rotating membrane tip for a controlled substrate release. Building on the previously provided proof of principle for this tool, this work extends its application by constructive modifications and improved methodology to ensure reproducible performance. The previously limited operation window was expanded by a systematic analysis of reservoir set-up variations for cultivations with the fast-growing organism Escherichia coli. Modifying the initial glucose concentration in the reservoir as well as interchanging the built-in membrane, resulted in glucose release rates and oxygen transfer rate levels during the fed-batch phase varying up to a factor of five. The range of utilizable membranes was extended from dialysis membranes to porous microfiltration membranes with the design of an appropriate membrane tip. The alteration of the membrane area, molecular weight cut-off and liquid volume in the reservoir offered additional parameters to fine-tune the duration of the initial batch phase, the oxygen transfer rate level of the fed-batch phase and the duration of feeding. It was shown that a homogeneous composition of the reservoir without a concentration gradient is ensured up to an initial glucose concentration of 750 g/L. Finally, the experimental validity of fed-batch Shake Flask cultivations was verified with comparable results obtained in a parallel fed-batch cultivation in a laboratory-scale stirred tank reactor. The membrane-based fed-batch Shake Flask is a reliable tool for small-scale screening under fed-batch conditions filling the gap between microtiter plates and scaled-down stirred tank reactors. The implemented reservoir system offers various set-up possibilities, which provide a wide range of process settings for diverse biological systems. As a screening tool, it accurately reflects the cultivation conditions in a fed-batch stirred tank reactor and enables a more efficient bioprocess development.
A Glomme - One of the best experts on this subject based on the ideXlab platform.
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comparison of a miniaturized Shake Flask solubility method with automated potentiometric acid base titrations and calculated solubilities
Journal of Pharmaceutical Sciences, 2005Co-Authors: A Glomme, J Marz, Jennifer B DressmanAbstract:Solubility is one of the most important parameters for lead selection and optimization during drug discovery. Its determination should therefore take place as early as possible in the process. Because of the large numbers of compounds involved and the very low amounts of each compound available in the early development stage, it is highly desirable to measure the solubility with as little compound as possible and to be able to improve the throughput of the methods used. In this work, a miniaturized Shake-Flask method was developed and the solubility results were compared with those measured by semiautomated potentiometric acid/base titrations and computational methods for 21 poorly soluble compounds with solubilities mostly in the range 0.03-30 microg/mL. The potentiometric method is very economical (approximately 100 microg of a poorly soluble compound is needed) and is able to create a pH/solubility profile with one single determination, but is limited to ionizable compounds. The miniaturized Shake-Flask method can be used for all compounds and a wide variety of media. Its precision and throughput proved superior to the potentiometric method for very poorly soluble compounds. Up to 20 compounds a week can be studied with one set-up. Calculated solubility data seem to be sufficient for a first estimate of the solubility, but they cannot currently be used as a substitute for experimental measurements at key decision points in the development process.
Bala Muntari - One of the best experts on this subject based on the ideXlab platform.
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recombinant bromelain production in escherichia coli process optimization in Shake Flask culture by response surface methodology
AMB Express, 2012Co-Authors: Bala Muntari, Azura Amid, Mohammed Saedi Jami, Hamzah Mohd SallehAbstract:Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in Shake Flask culture.
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recombinant bromelain production in escherichia coli process optimization in Shake Flask culture by response surface methodology
AMB Express, 2012Co-Authors: Bala Muntari, Azura Amid, Mohammed Saedi Jami, Hamzah Mohd SallehAbstract:Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in Shake Flask culture.
Hamzah Mohd Salleh - One of the best experts on this subject based on the ideXlab platform.
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recombinant bromelain production in escherichia coli process optimization in Shake Flask culture by response surface methodology
AMB Express, 2012Co-Authors: Bala Muntari, Azura Amid, Mohammed Saedi Jami, Hamzah Mohd SallehAbstract:Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in Shake Flask culture.
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recombinant bromelain production in escherichia coli process optimization in Shake Flask culture by response surface methodology
AMB Express, 2012Co-Authors: Bala Muntari, Azura Amid, Mohammed Saedi Jami, Hamzah Mohd SallehAbstract:Bromelain, a cysteine protease with various therapeutic and industrial applications, was expressed in Escherichia coli, BL21-AI clone, under different cultivation conditions (post-induction temperature, L-arabinose concentration and post-induction period). The optimized conditions by response surface methodology using face centered central composite design were 0.2% (w/v) L-arabinose, 8 hr and 25°C. The analysis of variance coupled with larger value of R2 (0.989) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced bromelain activity of 9.2 U/mg while validation experiments gave bromelain activity of 9.6 ± 0.02 U/mg at 0.15% (w/v) L-arabinose, 8 hr and 27°C. This study had innovatively developed cultivation conditions for better production of recombinant bromelain in Shake Flask culture.
M Z Abdin - One of the best experts on this subject based on the ideXlab platform.
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comparative study of withanolide production and the related transcriptional responses of biosynthetic genes in fungi elicited cell suspension culture of withania somnifera in Shake Flask and bioreactor
Plant Physiology and Biochemistry, 2017Co-Authors: Seema Ahlawat, Parul Saxena, Athar Ali, Shazia Khan, M Z AbdinAbstract:Ashwagandha (Withania somnifera) is one of the most reputed medicinal plants in the traditional medicinal system. In this study, cell suspension culture of W. somnifera was elicited with cell homogenates of fungi (A. alternata, F. solani, V. dahliae and P. indica) in Shake Flask and the major withanolides like withanolide A, withaferin A and withanone were analysed. Simultaneously expression levels of key pathway genes from withanolides biosynthetic pathways were also checked via quantitative PCR in Shake Flask as well as in bioreactor. The results show that highest gene expression of 10.8, 5.8, 4.9, and 3.3 folds were observed with HMGR among all the expressed genes in cell suspension cultures with cell homogenates of 3% P. indica, 5% V. dahliae, 3% A. alternata and 3% F. solani, respectively, in comparison to the control in Shake Flask. Optimized concentration of cell homogenate of P. indica (3% v/v) was added to the growing culture in 5.0-l bioreactor under optimized up-scaling conditions and harvested after 22 days. The genes of MVA, MEP and withanolides biosynthetic pathways like HMGR, SS, SE, CAS, FPPS, DXR and DXS were up-regulated by 12.5, 4.9, 2.18, 4.65, 2.34, 1.89 and 1.4 folds, respectively in bioreactor. The enhancement of biomass (1.13 fold) and withanolides [withanolide A (1.7), withaferin A (1.5), and withanone (1.5) folds] in bioreactor in comparison to Shake Flask was also found to be in line with the up-regulation of genes of withanolide biosynthetic pathways.
