Background Fed-batch conditions are advantageous for industrial cultivations as they avoid unfavorable phenomena appearing in batch cultivations. Those are for example the formation of overflow metabolites, catabolite repression, oxygen limitation or inhibition due to elevated osmotic concentrations. For both, the early bioprocess development and the optimization of existing bioprocesses, small-scale reaction vessels are applied to ensure high throughput, low costs and prompt results. However, most conventional small-scale procedures work in batch operation mode, which stands in contrast to fed-batch conditions in large-scale bioprocesses. Extensive expenditure for installations and operation accompany almost all cultivation systems in the market allowing fed-batch conditions in small-scale. An alternative, more cost efficient enzymatic glucose release system is strongly influenced by environmental conditions. To overcome these issues, this study investigates a polymer-based fed-batch system for controlled substrate release in microtiter plates. Results Immobilizing a solid silicone matrix with embedded glucose crystals at the bottom of each well of a microtiter plate is a suitable technique for implementing fed-batch conditions in microtiter plates. The results showed that the glucose release rate depends on the osmotic concentration, the pH and the temperature of the medium. Moreover, the applied nitrogen source proved to influence the glucose release rate. A new developed mathematical tool predicts the glucose release for various media conditions. The two model organisms E. coli and H. polymorpha were cultivated in the fed-batch microtiter plate to investigate the general applicability for microbial systems. Online monitoring of the oxygen transfer rate and offline analysis of substrate, product, biomass and pH confirmed that fed-batch conditions are comparable to large-scale cultivations. Furthermore, due to fed-batch conditions in microtiter plates, product formation could be enhanced by the factor 245 compared to batch cultivations. Conclusions The polymer-based fed-batch microtiter plate represents a sophisticated and cost efficient system to mimic typical industrial fed-batch conditions in small-scale. Thus, a more reliable strain screening and early process development can be performed. A systematical scale-down with low expenditure of work, time and money is possible. Electronic supplementary material The online version of this article (10.1186/s13036-019-0147-6) contains supplementary material, which is available to authorized users.
BackgroundAmong disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen kLa and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal kLa correlation.ResultsA Respiration Activity Monitoring System (RAMOS) was used to measure kLa values in cylindrical disposable shaken bioreactors and Buckingham’s π-Theorem was applied to define a dimensionless equation for kLa. In this way, a scale- and volume-independent kLa correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxygen supply of tobacco BY-2 cell suspension cultures.ConclusionThe resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/− 30%. To our knowledge, this is the first kLa correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.
BackgroundBiotechnological screening processes are performed since more than 8 decades in small scale shaken bioreactors like shake flasks or microtiter plates. One of the major issues of such reactors is the sufficient oxygen supply of suspended microorganisms. Oxygen transfer into the bulk liquid can in general be increased by introducing suitable baffles at the reactor wall. However, a comprehensive and systematic characterization of baffled shaken bioreactors has never been carried out so far. Baffles often differ in number, size and shape. The exact geometry of baffles in glass lab ware like shake flasks is very difficult to reproduce from piece to piece due to the hard to control flow behavior of molten glass during manufacturing. Thus, reproducibility of the maximum oxygen transfer capacity in such baffled shake flasks is hardly given.ResultsAs a first step to systematically elucidate the general effect of different baffle geometries on shaken bioreactor performance, the maximum oxygen transfer capacity (OTRmax) in baffled 48-well microtiter plates as shaken model reactor was characterized. This type of bioreactor made of plastic material was chosen, as the exact geometry of the baffles can be fabricated by highly reproducible laser cutting. As a result, thirty different geometries were investigated regarding their maximum oxygen transfer capacity (OTRmax) and liquid distribution during shaking. The relative perimeter of the cross-section area as new fundamental geometric key parameter is introduced. An empirical correlation for the OTRmax as function of the relative perimeter, shaking frequency and filling volume is derived. For the first time, this correlation allows a systematic description of the maximum oxygen transfer capacity in baffled microtiter plates.ConclusionsCalculated and experimentally determined OTRmax values agree within ± 30% accuracy. Furthermore, undesired out-of-phase operating conditions can be identified by using the relative perimeter as key parameter. Finally, an optimum well geometry characterized by an increased perimeter of 10% compared to the unbaffled round geometry is identified. This study may also assist to comprehensively describe and optimize the baffles of shake flasks in future.
Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument-caused deviations in enzyme kinetics between two MTP-readers, we comprehensively quantified temperature distribution in 96-well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96-well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature-induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development.
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