Understanding and utilising bacterial growth rate changes at high external osmolarities

Abstract

Bacteria colonized nearly all corners of the globe because of their amazing ability to adapt and evolve. To humans, bacteria represent both an incredible tool, that can be harnessed for food production, medicine, and biotechnology; and a danger that can cause famine and disease. Understanding what drives bacterial growth is therefore of great importance to harness the power of microbes both good and bad. The traditional model bacterium, Escherichia coli, can survive and grow in a variety of different environments, that vary in pH, temperature, osmolarity, and other physical parameters. However, our understanding of how E. coli manages to overcome these adverse conditions to continue growth is incomplete. Determining the physical limits imposed on growth by the external environment holds significant scientific importance, as it helps prevent unwanted bacterial growth and enables increased industrial bacteria production for biotechnology and agriculture applications.

For more than a century, scientists have extensively studied the growth rate of bacteria. When bacteria are allowed to adapt to their environment, they exhibit constant growth. However, the speed at bacteria grow varies significantly and depends on the strain of bacteria as well as the conditions in which it is grown. Unraveling the specific mechanisms that govern growth is challenging due to the complexity of biological systems, such as E. coli. Growth can be viewed as the process in which bacteria transform nutrients from their environment into more cellular material. Growth, therefore emerges from the metabolic flux of nutrient substrates to bacterial products, catalyzed by bacterial enzymes, raising questions about what limits the rate of metabolic flux under specific conditions. How does the cell effectively balance metabolic flux across diverse environmental circumstances to optimize its growth rate? Previous studies have extensively explored the relationship between the nutritional composition of growth media and the rate of growth.

However, nutrient limitation is just one of the many stresses bacteria face in nature. They also encounter other challenges, such as oxygen limitation, extreme temperatures (both low and high), pH variations, and fluctuations in osmolarity, all of whichcan impact their growth rate. This thesis aims to investigate how hyperosmotic stress affects bacterial growth. I demonstrate that increasing the media osmolarity does not affect the growth rate by providing an energy burden, or a proteome burden on the cell. I also show that the growth rate slow down is not caused solely by a decrease in cytoplasmic water concentration. I present evidence that a similar slow down can be achieved by loading the E. coli cytoplasm with compatible solutes that do not cause osmotic stress. Therefore, the decrease in growth rate seems to be independent of osmotic stress. Instead data suggests osmotic stress causes cell-wide changes in metabolic efficiency. I hypothesize that the reduced growth is the result of increased cytoplasmic viscosity decreasing the rate of metabolic flux. Finally I apply knowledge gained during this fundamental scientific investigation to explore whether lactobacillus experience osmotic stress during industrial fermentation and whether strategies used by E. coli can lead to potential improvements of the growth rate and yield. I find that there is minimal osmotic stress during fermentation and that addition of osmotic stress by addition of NaCl reduces yield after culture and does not provide any additional benefits when freezing or freeze-drying bacteria.