Bacillus subtilis is a non-pathogenic, sporulating, Gram-positive bacteria with pronounced antimicrobial and metabolic activity and great potential for wide application in various fields. The aim of ...this paper was to determine the optimum B. subtilis NCIM 2063 growth conditions and to scale up biomass production from shake flasks to a bioreactor level. The critical growth parameters and their interaction effects were studied using Box?Behnken experimental design and response surface methodology. Developed model equations were statistically significant with good prediction capability. It was found that during shake flask cultivation glucose should be added in concentration up to 5 g L-1 in DSM medium, OTR at 10 mol m-3 h-1 and temperature of 33?C, to achieve the maximum number of viable cells and spores. To scale up the process from shake flasks to the bioreactor level kLa was used as a main criterion. Scale up effect was evaluated by comparing the growth kinetics in the shake flasks and in a laboratory bioreactor. The total number of cells obtained in the bioreactor was 4.57x109 CFU mL-1 which is 1.41 times higher than the number of cells in the shake flasks (3.24x109 CFU mL-1), proving that the scale-up procedure was conducted successfully.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
To overcome catabolite repression, industrial fermentation processes are usually operated in substrate‐limited fed‐batch mode. Therefore, the implementation of such an operating mode at small scale ...is crucial to maintain comparable process conditions. In this study, Bacillus licheniformis, a well‐known producer of proteases, was cultivated with carbon (glucose)‐ and nitrogen (ammonium)‐limited fed‐batch conditions using the previously introduced membrane‐based fed‐batch shake flasks. A repression of protease production by glucose and ammonium was thus avoided and yields increased 1.5‐ and 2.1‐fold relative to batch, respectively. An elevated feeding rate of glucose caused depletion of ammonium, which was recognizable within the oxygen transfer rate (OTR) signal measured with the Respiration Activity MOnitoring System (RAMOS). Ammonium limitation was prevented by feeding ammonium simultaneously with glucose. The OTR signal clearly indicated the initiation of the fed‐batch phase and gave direct feedback on the nutrient release kinetics. Increased feeding rates of glucose and ammonium led to an elevated protease activity without affecting the protease yield (YP/Glu). In addition to YP/Glu, protease yields were determined based on the metabolized amount of oxygen (YP/O2). The results showed that the protease production correlated with the amount of consumed glucose as well as with the amount of consumed oxygen. The membrane‐based fed‐batch shake flask in combination with the RAMOS device is a powerful combination to investigate the effect of substrate‐limited fed‐batch conditions.
Small scale shaken bioreactors operated in fed‐batch mode have become important tools for screening and initial process development. In this study, Habicher and coworkers used a membrane‐based fed‐batch shake flask to realize continuous substrate release with parallel online monitoring of the oxygen transfer rate (OTR). It was demonstrated that defined substrate‐limited conditions allow overcoming catabolite repression in a protease producing Bacillus licheniformis strain.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Processes involving mammalian cell cultures - especially CHO suspension cells - dominate biopharmaceutical manufacturing. These processes are usually developed in small scale orbitally shaken ...cultivation systems, and thoroughly characterizing these cultivation systems is crucial to their application in research and the subsequent scale-up to production processes. With the knowledge of process engineering parameters such as oxygen transfer rate, mixing time, and power input, in combination with the demands set by the biological production system, biomass growth and product yields can be anticipated and even increased. However, the available data sources for orbitally shaken cultivation systems are often incomplete and thus not sufficient enough to generate suitable cultivation requirements. Furthermore, process engineering knowledge is inapplicable if it is not linked to the physiological demands of the cells.
In the current study, a simple yet comprehensive approach for the characterization and design space prediction of orbitally shaken single-use cultivation systems is presented, including the “classical” Erlenmeyer shake flask, the cylindrical TubeSpin bioreactor and the alternately designed Optimum Growth flask. Cultivations were performed inside and outside the design space to validate the defined culture conditions, so that cultivation success (desired specific growth rates and viable cell densities) could be achieved for each cultivation system.
•Process engineering characterization of 5 different orbitally shaken systems.•Determining limitations of CHO suspension cells in orbitally shaken systems.•Application of a design space approach to ensure optimal cultivation parameters.•Process transfer between bioreactors that assure cultivation success.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Purpose
Simultaneous membrane-based feeding and monitoring of the oxygen transfer rate shall be introduced to the newly established perforated ring flask, which consists of a cylindrical glass flask ...with an additional perforated inner glass ring, for rapid bioprocess development.
Methods
A 3D-printed adapter was constructed to enable monitoring of the oxygen transfer rate in the perforated ring flasks.
Escherichia coli
experiments in batch were performed to validate the adapter. Fed-batch experiments with different diffusion rates and feed solutions were performed.
Results
The adapter and the performed experiments allowed a direct comparison of the perforated ring flasks with Erlenmeyer flasks. In batch cultivations, maximum oxygen transfer capacities of 80 mmol L
−1
h
−1
were reached with perforated ring flasks, corresponding to a 3.5 times higher capacity than in Erlenmeyer flasks. Fed-batch experiments with a feed reservoir concentration of 500 g glucose L
−1
were successfully conducted. Based on the oxygen transfer rate, an ammonium limitation could be observed. By adding 40 g ammonium sulfate L
−1
to the feed reservoir, the limitation could be prevented.
Conclusion
The membrane-based feeding, an online monitoring technique, and the perforated ring flask were successfully combined and offer a new and promising tool for screening and process development in biotechnology.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The experimental conditions that affect equilibrium solubility values measured by the classical saturation shake-flask method have been examined, using hydrochlorothiazide as a model compound. ...Modifications in temperature, sedimentation time, composition of aqueous buffer and the technique of separation of solid and liquid phases were all found to influence the equilibrium solubility results strongly. However, variations in the amount of solid excess and stirring time were found to have less influence. In the light of these observations, a new, shorter protocol has been developed for measurements of equilibrium solubility, together with recommendations for good analytical practice. The equilibrium solubilities of five other drugs were measured to verify the new protocol.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Alfamart is a company engaged in retail. Companies involved in the retail sector are certainly inseparable from buying and selling products, and every transaction that occurs will be detailed in the ...invoice exchange. The problem that arises is because Alfamart wants to accommodate the electronic invoice exchange process. Therefore, Alfamart built a B2B TTF application that can accommodate the electronic invoice exchange process and help its accounting management. The application is made using the Research and Development method because it can address urgent needs and has a high validation value. It is built using the Flask framework and is integrated with Google Cloud to overcome application deployment speed problems and be more flexible. In addition, the implementation of Optical Character Recognition using Google Vision is used to validate uploaded invoice files. This study's results are in the form of a B2B TTF application that can make it easier for users to exchange invoices. The results of using Google Vision have a relatively high percentage of 77%. The B2B TTF application uses the Flask framework and is integrated with Google Cloud, which can assist users in the process of exchanging invoices electronically.
Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from ...meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism
Ralstonia eutropha
. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L
-1
of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L
-1
CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production.
Key points
•
Evaluation of shake flask designs for cultivating with hydrophobic raw materials
•
Development of a workflow for microwell plate cultivations with hydrophobic raw materials
•
Production of polyhydroxyalkanoate in small scale experiments from waste animal fat
Graphical Abstract
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CEKLJ, DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
In the present investigation, the production of amylase, cellulase, and protease activity by Bacillus subtilis ATCC 6051a was evaluated on different raw materials (soybean meal, peas, sorghum flour, ...corn) and combined feed (CF). The effect of various fermentation conditions (24-72 h) on enzymatic production through shake-flask culture (Erlenmeyer 100 mL) in optimum conditions (150 rpm, pH 7.0±0.2, 37◦C) was investigated. The inoculum strain presents 1.907 optical density (OD) 600 nm with a concentration of 1.6 x 109 CFU/mL. The screening showed a capacity of amylase, cellulase, and protease strain production. The maximum amylase level was obtained at 72 h when the strain was cultured in CF fermentation medium (13.19±0.15 U/mL), followed by corn (11.72±0.15 U/mL), peas (9.22±0.11 U/mL), soybean meal (7.29±0.19 U/mL) and sorghum (6.31±0.2 U/mL). For production of cellulase by DNS method, the activity was noticed in corn (4.39±0.04 U/mL), sorghum (4.05±0.02 U/mL), peas (2.94±0.06 U/mL), soybean meal (2.87±0.04 U/mL) and CF medium (1.41±0.02 U/mL). Maximum protease activity was observed after 24 h in CF (4.91±0.08 U/mL), with minimum enzyme production in the rest of fermentation media, both at 48 h and 72 h. In conclusion, qualitative analysis revealed that Bacillus subtilis ATCC 6051a could be successfully used for high scale production of amylase, cellulase and to less extend protease, particularly in fermented medium containing CF or corn, and may be considered a potential candidate for supplementation of animal feed.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cultivating cells in shake flasks is a routine operation that is largely unchanged since its inception. A glass or plastic Erlenmeyer vessel with the primary gas exchange taking place across various ...porous plugs is used with media volumes typically ranging from 100 mL to 2 L. Oxygen limitation and carbon dioxide accumulation in the vessel is a major concern for studies involving shake flask cultures. In this study, we enhance mass transfer in a conventional shake flask by replacing the body wall with a permeable membrane. Naturally occurring concentration gradient across the permeable membrane walls facilitates the movement of oxygen and carbon dioxide between the flask and the external environment. The modified flask called the breathable flask, has shown a 40% improvement in mass transfer coefficient (k
a) determined using the static diffusion method. The prokaryotic cell culture studies performed with Escherichia coli showed an improvement of 28%-66% in biomass and 41%-56% in recombinant product yield. The eukaryotic cell culture study performed with Pichia pastoris expressing proinsulin exhibited a 40% improvement in biomass and 115% improvement in protein yield. The study demonstrates a novel approach to addressing the mass transfer limitations in conventional shake flask cultures. The proposed flask amplifies its value by providing a membrane-diffusion-based sensing platform for the integration of low-cost, noninvasive sensing capabilities for real-time monitoring of critical cell culture parameters like dissolved oxygen and dissolved carbon dioxide.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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