Strategies and tools to select E. coli fermenterphiles for industrial application

The world faces growing challenges due to humankind’s dependence on fossil resources for energy, fuel, and materials. Biomanufacturing in the past decades has proven its potential to provide alternative, sustainable ways to produce medicines, commodity chemicals, materials, and fuels from renewable resources by cultivating microbial cell factories. New bioprocesses and microbial cell factories are often developed under ideal conditions in the laboratory. However, under industrial cultivation conditions, microbial cell factories are exposed to a heterogeneous bioreactor environment due to mixing gradients, potentially altering microbial performance at scale and resulting in lower-than-expected productivity. Therefore, selecting robust microbial production strains that maintain unaltered production performance under industrial cultivation conditions is one of the strategies that will lead to a successful scale-up. Such strains are also called fermenterphiles. Appropriate scale-down simulations, i.e., experiments that mimic industrial-scale conditions at a laboratory scale, are necessary to understand the microbial response to industrial cultivation conditions and to eventually identify and select fermenterphile strains based on their transcriptional response to environmental changes. This Ph.D. thesis addresses the design and implementation of scale-down experiments. It provides an in-depth analysis of the physiology and transcriptional regulation of the bacterium Escherichia coli under industrial-scale cultivation conditions.

Before investigating scale-specific industrial cultivation challenges, this Ph.D. thesis addresses the transcriptional and physiological state of E. coli in carbon-limited fed-batch cultivations to establish a baseline for the following studies. The transcriptional analysis leads to the design of a suitable medium to perform high-cell-density fed-batch cultivations. Further, the response of E. coli to industrial cultivation conditions with a focus on glucose concentration and dissolved oxygen concentration gradients is investigated in detail experimentally. A compartment modeling approach is applied to predict the mixing gradients in a 90 m3 stirred tank reactor, based on which scale-down experiments relying on the oscillation of glucose feeding and aeration are designed. A new finding reported here is the transcriptional downregulation of the general stress response, which was previously reported to be upregulated in glucose-oscillation scale-down experiments. Furthermore, one of the key conclusions arrived at is that the response of E. coli to industrial cultivation conditions is highly genotype-dependent, which calls for the implementation of high-throughput scale-down experiments to assess the fermenterphile phenotype of different strains on a case-by-case basis. With respect to combined glucose and oxygen oscillation scale-down experiments, this thesis identifies a physiological and transcriptional response distinct from the responses to isolated glucose and oxygen oscillations, highlighting that scale-down experiments must represent large-scale bioreactor phenomena holistically.

Taken together, the results presented in this Ph.D. thesis expand the understanding of E. coli’s physiology and transcriptional regulation when grown under industrially relevant conditions. Future research on developing E. coli fermenterphiles can be conducted based on this knowledge base. Further, the framework of this work may be used to implement scale-down experiments in bioprocess development workflows.