Advancing automated bioprocess development for industrial platform organisms

Jansen, Roman Pascal; Oldiges, Marco (Thesis advisor); Blank, Lars M. (Thesis advisor); Wiechert, Wolfgang (Thesis advisor)

Aachen (2020, 2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Industrial biotechnology is often hampered through long process development times. Consequently, methods allowing for accelerated process development are of great interest, offering the potential to reduce the time to market. Automation, miniaturization and parallelization have proven to be valuable tools for this, as several integrated microbioreactor (MBR) platforms have been developed, enabling increased throughput for quantitative phenotyping and process development. Nevertheless, these robotic systems still suffer from several limitations when reproducing manual laboratory workflows. Consequently, novel workflows advancing automated process development for various platform organisms are developed in this thesis. MBR systems are well established for yeast and bacterial microorganisms and batch cultivations. However, in industry fed-batch processes are frequently preferred, e.g. to avoid oxygen limitations and substrate excess inhibition. This discrepancy between batch screening and fed-batch process targets might lead to the selection of wrong strains and parameters, due to unsuitable conditions at the screening level. Therefore, novel workflows allowing for microscale fed-batch are in high demand. Two approaches for miniaturized fed-batch were developed and validated for protein producing C. glutamicum as a model organism. A feed-back-regulated enzyme-based slow-release system for fed-batch cultivation in microtiter plates (MTP) was developed, enabling reproducible single-sided pH control as well as constant, linear or exponential feeding for 48 parallel cultivations. Furthermore, a workflow based on microfluidic MTPs was established, where through specific medium and feed solution design, miniaturized fed-batch cultivations as a standalone solution was enabled. Filamentous fungi of the genus Aspergillus are one of the workhorses for industrial production processes. However, so far they have not fully been implemented on the robotic platforms, due to their challenging morphology. Novel workflows allowing the robust and reproducible MBR cultivation of microfilamentous A. niger, mycelial aggregates of A. giganteus and pellets of A. carbonarius were established and validated. With tailor made liquid handling parameters for fungal biomass, automated sample harvest and processing was enabled. Moreover, a workflow allowing automated morphology analysis was developed. With successful implementation of microscale fed-batch workflows and handling of non-standard microorganism, a variety of biological case studies can be performed. Especially when combining these novel workflows new opportunities of quantitative phenotyping are possible, for example, automated morphology analysis from fed-batch cultivation to detect spore formation under carbon limitation. Consequently, automation, miniaturization and paral-lelization remain powerful tools to accelerate bioprocess development.