Validation of computational fluid dynamics of shake flask experiments at moderate viscosity by liquid distributions and volumetric power inputs.

Computational fluid dynamics (CFD) has recently become a pivotal tool in the design and scale-up of bioprocesses. While CFD has been extensively utilized for stirred tank reactors (STRs), there exists a relatively limited body of literature focusing on CFD applications for shake flasks, almost exclusively concentrated on fluids at waterlike viscosity. The importance of CFD model validation cannot be overstated. While techniques to elucidate the internal flow field are necessary for model validation in STRs, the liquid distribution, caused by the orbital shaking motion of shake flasks, can be exploited for model validation. An OpenFOAM CFD model for shake flasks has been established. Calculated liquid distributions were compared to suitable, previously published experimental data. Across a broad range of shaking conditions, at waterlike and moderate viscosity (16.7 mPa∙s), the CFD model's liquid distributions align excellently with the experimental data, in terms of overall shape and position of the liquid relative to the direction of the centrifugal force. Additionally, the CFD model was used to calculate the volumetric power input, based on the energy dissipation. Depending on the shaking conditions, the computed volumetric power inputs range from 0.1 to 7 kW/m3 and differed on average by 0.01 kW/m3 from measured literature data.

Black glucose-releasing silicon elastomer rings for fed-batch operation allow measurement of the oxygen transfer rate from the top and optical signals from the bottom for each well of a microtiter plate.

BACKGROUND: In industrial microbial biotechnology, fed-batch processes are frequently used to avoid undesirable biological phenomena, such as substrate inhibition or overflow metabolism. For targeted process development, fed-batch options for small scale and high throughput are needed. One commercially available fed-batch fermentation system is the FeedPlate®, a microtiter plate (MTP) with a polymer-based controlled release system. Despite standardisation and easy incorporation into existing MTP handling systems, FeedPlates® cannot be used with online monitoring systems that measure optically through the transparent bottom of the plate. One such system that is broadly used in biotechnological laboratories, is the commercial BioLector. To allow for BioLector measurements, while applying the polymer-based feeding technology, positioning of polymer rings instead of polymer disks at the bottom of the well has been proposed. This strategy has a drawback: measurement requires an adjustment of the software settings of the BioLector device. This adjustment modifies the measuring position relative to the wells, so that the light path is no longer blocked by the polymer ring, but, traverses through the inner hole of the ring. This study aimed at overcoming that obstacle and allowing for measurement of fed-batch cultivations using a commercial BioLector without adjustment of the relative measurement position within each well. RESULTS: Different polymer ring heights, colours and positions in the wells were investigated for their influence on maximum oxygen transfer capacity, mixing time and scattered light measurement. Several configurations of black polymer rings were identified that allow measurement in an unmodified, commercial BioLector, comparable to wells without rings. Fed-batch experiments with black polymer rings with two model organisms, E. coli and H. polymorpha, were conducted. The identified ring configurations allowed for successful cultivations, measuring the oxygen transfer rate and dissolved oxygen tension, pH, scattered light and fluorescence. Using the obtained online data, glucose release rates of 0.36 to 0.44 mg/h could be determined. They are comparable to formerly published data of the polymer matrix. CONCLUSION: The final ring configurations allow for measurements of microbial fed-batch cultivations using a commercial BioLector without requiring adjustments of the instrumental measurement setup. Different ring configurations achieve similar glucose release rates. Measurements from above and below the plate are possible and comparable to measurements of wells without polymer rings. This technology enables the generation of a comprehensive process understanding and target-oriented process development for industrial fed-batch processes.