Quantitative fermentation of sesquiterpene producing microorganisms

Abstract

Due to the increasing demand for molecules derived from natural resources, coupled with the heterogeneity of production crops, seasonality, and low yields leading to scarcity, the need for optimizing biotechnological processes for producing such molecules has grown. Key challenges in this area include the practical and economic feasibility of these production processes, often based on large-scale cultivation of high-yielding microorganisms (fermentation).
To capture the formed products during the production process, "In Situ Product Recovery" (ISPR) can be applied, for example, by adding an organic solvent during fermentation. ISPR also has the added benefit of preventing the accumulation of hydrophobic products in the culture medium to levels that inhibit the growth and production of the microorganisms. However, the use of organic solvents combined with surface-active compounds (SACs) poses challenges for product recovery after the stabilization of the culture medium.
The research described in this thesis was conducted within the Delft Integrated Recovery Column project (DIRC), which focuses on designing an integrated reactor for the production and recovery of hydrophobic compounds such as alkanes or isoprenoids.
This thesis explores the production of α-santalene and α-humulene, terpenes naturally found in the essential oils of Santalum album and Humulus lupulus, respectively. The production of these compounds in two different Escherichia coli BL21(DE3) strains was studied during fed-batch fermentations using glycerol as a growth-limiting substrate. Significant differences were observed between the two tested strains regarding cell viability and productivity.
Chapter 1 describes why sesquiterpenes (C15H24) are valuable non-polar molecules for various industrial sectors, based on their bioactivity or use as biofuels and fragrances. It also provides an overview of the biosynthetic pathways for terpene production, particularly the biosynthetic route for using glycerol as a carbon source. Finally, it reviews the literature on the advantages and limitations of Escherichia coli for the expression of heterologous products.
Chapter 2 presents the initial results of the biotechnological production of α-santalene, using ISPR (with the addition of dodecane) in 2 L bioreactors. Various process variations (such as different feed rates and induction strategies) were tested in fed-batch culture experiments with a genetically modified Escherichia coli BL21 (DE3) strain known for its high variability in sesquiterpene productivity. Moreover, the values of the black-box parameters (a, ms) from the Herbert-Pirt correlation for the specific substrate consumption rate (qs) were estimated based on the experimental data obtained.
Chapter 3 focuses on the same α-santalene-producing strain studied in Chapter 2, but instead investigates plasmid dynamics during an experiment without dodecane: while this organic solvent improves bioprocess performance, it interferes with qPCR assays.
The biotechnological production of α-humulene, also carried out in 2L bioreactors, is discussed in Chapter 4. This was done using a better-producing E. coli strain that our group gained access to in the final phase of this project. The productivity exceeded expectations compared to the findings described in the previous chapters. The influence of the substrate glycerol feed rate was examined, in combination with ISPR, as well as the impact of the absence of dodecane on specific productivity. Herbert-Pirt parameters were estimated for a fed-batch experiment conducted with a constant glycerol feed rate. Additionally, correlations between specific humulene productivity (qp) and specific growth rate (µ) were presented.
To base the modeling on realistic data, Chapter 5 determined and compared experimental oxygen transfer rates under actual extractive ("overlay") and non-extractive conditions for sesquiterpene fermentation with current insights into coalescing and non-coalescing systems. While the kLa values in extractive fermentation (ISPR) indicate non-coalescing behavior, the kLa of solvent-free fed-batch follows a general correlation for coalescing systems. Combined with the specific productivity results reported for the humulene-producing strain in Chapter 4, Chapter 5 outlines the design of scaled-up production processes, to be carried out in 1 m³ and 10 m³ bioreactors. Using a developed black-box model, the effects of two different glycerol feed strategies were compared: an exponentially increasing feed rate (Exponential Feed, EF) and a controlled feed rate aimed at a constant maximum oxygen consumption rate (Constant Oxygen Consumption Rate, COCR).
Chapter 6 summarizes the key findings and provides recommendations for further research and development.