Characterization and simulation of substrate release in a novel 48-well fed-batch microtiter plate

  • Charakterisierung und Simulation der Substratfreisetzung in einer Neuartigen 48-Well Fed-Batch Mikrotiterplatte

Lattermann, Clemens; Büchs, Jochen (Thesis advisor); Pich, Andrij (Thesis advisor)

Aachen (2018, 2019)
Dissertation / PhD Thesis

Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2018


Industrial large-scale bioprocesses are often run in fed-batch operation mode. It provides several advantages compared to the conventional batch mode such as optimal cultivation conditions as well as enhanced product yields. In contrast, small-scale screening is often run in batch mode. However, the physiological conditions between both operation modes strongly vary and suboptimal strains might be selected from the screening process. In the past years, different small-scale fed-batch bioreactors have been developed. A novel screening tool represents the fed-batch microtiter plate, presented in this work. The system enables diffusive substrate release from a hydrogel into a culture well. Within this work, the microtiter plates and substrate release are characterized in detail. In a first step, the diffusion properties of a polyvinyl alcohol (PVA) and polyallylamine (PAAm) hydrogels were analyzed for feeding concentrations up to 700 g/L. For the PVA-glycerol and PAAm-glucose systems, maximum diffusion rates of 0.86 mg/h and 0.58 mg/h were determined, respectively. Moreover, the apparent lag-phase tlag was identified as key parameter regarding the hydrogel stability. In a second step, substrate diffusion through the hydrogel was calculated based on a hydrodynamic diffusion model. Moreover, a simulation model based on Fick’s diffusion laws was developed. Predictions about the diffusive mass flows were made and the local volume change of the hydrogel was calculated. As a result, substrate diffusion could be predicted up to +/- 20 % by means of the simulation model. In addition, water counter diffusion was identified as having considerable impact onto the hydrogel stability. For the PAAm hydrogel, a relative volume loss of 7.5 - 12.5 % per section was calculated as critical volume loss at feeding concentrations above 300 g/L. In addition, a maximum volume loss of 19 % after 0.8 h was observed for a feeding concentration of 700 g/L. To demonstrate the biological application of the novel fed-batch microtiter plate, cultivations experiments with E. coli BL21 (DE3) pRhotHi-2-Ec FbFP were performed. Here, carbon limited fed-batch conditions could be observed. Finally, experiments regarding the temperature distribution across conventional microtiter plates have been performed. A relative deviation of the temperature of 1.5 - 2.3 °C across the microtiter plate was determined.