Considerations of Bacterial Robustness and Stability to Improve Bioprocess Design

Harnessing nature’s ingenuity with microorganisms for industrial production is an attractive solution to today’s climate concerns. Nature’s innate diversity allows the production of many value-added chemicals and can be expanded on through genetic engineering. Although the use of microbial cell factories (MCFs) has been extremely successful at lab scale, the numbers of successful bioprocesses remain limited. High cell densities and long cultivation times lead to reductions in productivity over the course of the cultivation through the effects of genetic and expression instability of the strain. This instability leads to population diversification. In this review, we explore the roots of genetic instability in microorganisms, focusing on prokaryotic bioprocesses, and how organisms cope with this instability. We spotlight single-cell detection methods capable of monitoring populations within the bioprocess both in- and on-line. We also examine different approaches to minimizing population diversification, both through strain development and bioprocess engineering. With this review, we highlight the fact that population-averaged metrics overlook the single-cell stresses driving genetic and functional instability, leading to an overestimation of microbial bioprocess robustness. High-throughput single-cell monitoring in industry-like conditions remains essential to identify and select truly stable microbial cell factories and bioprocesses.