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.

A crucial challenge during the initial stages of bioprocess development is that tools used to screen microorganisms and optimize cultivation conditions do not represent the environment imposed at industrial scale. Inside an industrial-scale bioreactor, microorganisms are often cultivated under fed-batch conditions, where nutrients are supplied during the culture. Additionally, microorganisms continuously keep crossing zones with low and high concentrations of substrate and dissolved oxygen. However, during initial bioprocess development, growth and productivity of microorganisms are evaluated under batch conditions due to the difficulty of dynamically controlling nutrient and dissolved oxygen concentrations in screening equipment such as micotiter plates. This inconsistency in cultivation conditions often leads to selection of strains that fail to perform at industrial scale. The difficulty in continuously supplying minute amounts of nutrients to microorganisms in microtiter plates and imposing dynamic dissolved oxygen levels throughout the cultivation experiment necessitates an alternative approach. Microfluidic technology holds the potential to address this inconsistency with fidelity by offering high-throughput experimentation and excellent control over the culture microenvironment. The central theme of this Ph.D. project is the design and development of droplet-based microfluidic technology, that enable studying microorganisms under such dynamically controlled cultivation conditions. As such, the outcomes from this Ph.D. project form a foundation step towards narrowing the gap between screening and industrial-scale use, with an eye to keeping the technology sufficiently simple to be adopted by the biotechnology and bioengineering community.

Trichoderma harzianum P49P11 was selected among several other microorganisms at the Biorenewables National Laboratory (LNBR, Brazil) and it was considered a promising strain to produce cellulase. Here in this project, a mathematical model and simulation platforms were developed as potential tools to be used for cellulase maximization using fed-batch mode (Chapter 2). Feeding strategies were simulated to maximize cellulase production, at first, only using cellulose as the substrate, and then using glycerol for cell growth and cellulose for cellulase production. Although the mathematical model and simulation platforms were built up for a wild type strain, these tools help to predict data and they can be adapted for optimized strains.
Chapter 3 evaluates the continuous production of enzymes using different carbon sources under carbon-limited conditions. It was found that glucose has a positive influence on the production of enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (PNPGase). Sucrose and fructose seem to inhibit PNPGase synthesis; however, these substrates could also have a positive influence on the synthesis of other enzymes not evaluated in this project. Cells can uptake glucose without the need to synthesize extracellular enzymes like PNPGase. The increase in the production of PNPGase during the continuous culture using glucose as the carbon source indicates the presence of inducers. It was also discovered in this project that polysaccharides were present in the supernatant of all conditions using glucose, fructose/glucose and sucrose (Chapter 4 and Chapter 5). This suggests that the possible inducers could have come from fragments of the extracellular polysaccharides.
Sugar analysis showed the presence of sugar with the same retention time as gentiobiose in the supernatant of the conditions using glucose as the carbon source, which could be a fragment from polymers released from the cell wall. Gentiobiose could be acting as an inducer of enzymes. In addition, a mechanism was also proposed for continuous PNPGase production under glucose-limited conditions assuming that PNPGase includes beta-glucosidase (Chapter 4).
The carbon sources used under carbon-limited conditions influenced the PNPGase productivity and possibly the whole enzymatic cocktail secreted by the fungus. For this reason, shotgun proteomics and SDS-PAGE analysis were performed for the proteins present in the supernatant of the conditions using glucose, fructose/glucose and sucrose (Chapter 4). The shotgun proteomics analysis suggested that the different carbon sources used provided the production of different extracellular proteins including several uncharacterized proteins, which can also include different enzymes. This brings the possibility of creating a hypothesis that different carbon sources easily assimilated by the cells could lead to the synthesis of different inducers (fragments of extracellular polysaccharides), which could induce the synthesis of different enzymes under carbon-limited conditions.
Extracellular polysaccharides were the by-products discovered in this project during the production of enzymes under carbon-limited conditions. The behaviour of intracellular metabolites (glycolysis, citric acid cycle, pentose phosphate pathway and nucleotides) was evaluated under four different conditions in duplicate during the production of extracellular polysaccharides by Trichoderma harzianum under carbon-limited conditions (Chapter 5). This chapter has provided the first step for the optimization of the production of extracellular polysaccharides and the information about the behaviour of intracellular metabolites using this wild type strain is essential to the development of optimal strains.