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.
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The growing group of health-conscious consumers drives the development and sales in the market of low- or non-alcoholic beverages. In particular, sales in alcohol-free beer (AFB) has drastically gained in recent years, giving the incentive to further advance alcohol-free products. This thesis comprises the development of a new technology to produce an alcohol-free beer with an improved flavour, that is, an AFB with a significantly lower concentration in Strecker aldehydes. Despite the fact that Strecker aldehydes are present at trace concentrations of a few micrograms per litre, these compounds are majorly associated with the characteristic wort offflavour in alcohol-free beer. In the first stage, the origin of wort flavours is elucidated, and furthermore, the impact of the changed production process in comparison to regular beer and its resulting changes in the product is discussed. The most significant pathways of origin are the Maillard reactions and Strecker degradation, however, many other formation pathways have been suggested. While alcohol-free beers produced through regular fermentation and subsequent dealcoholisation are generally low in wort off-flavour, biologically produced AFBs exhibit a rather strong wortiness. This is because yeast’s activity during the fermentation is restricted to prevent alcohol formation, having the side effect that other compounds such as aldehydes are not converted to neutral or characteristic beer flavours. As it is impossible to prevent the formation of wort flavour, selective removal is required to produce an alcohol-free beer with improved flavour profile (balance). Subsequently, technologies applied to remove aldehydes from aqueous streams are investigated. Mechanisms that can be exploited for such removal are various – from separation based on size or volatility (distillation, pervaporation or membrane filtration) through sorption or conversion – past research has nearly studied all removal techniques available for aldehydes. However, most of the available techniques are not selective enough for a complex matrix such as beer, or not suitable for the application in the food industry. Nonetheless, adsorption is identified as the most promising technique to facilitate selective aldehyde removal. As a benchmark system, a dealcoholisation plant (spinning cone column) is studied with special focus on wort flavour removal. While volatile aldehydes such as 3-methylbutanal can relatively easily be reduced, methional, having a high boiling point, is not sufficiently separated from the end-product. Furthermore, due to the impact of heat, and ergo, the continuous formation and evaporation of these flavours, a plateau in their concentration is observed, where no further removal seems possible. This theory is proven by spiking a reactive sugar into the base and forming the mass balance over the system. Hence, in order to achieve a high reduction on high boiling wort flavours, another technology, such as adsorption, is required. To test, whether a suitable adsorbent can be identified, a wide screening experiment covering three adsorbent types (amine-functionalized polymers, hydrophobic resins and zeolites) and a total of 21 materials is performed. ZSM-5 type zeolites prove to have a superior selectivity as well as a relatively high capacity compared to other adsorbents. The choice of the right pore size and hydrophobicity is crucial for the separation success. Adsorption of the compounds of interest is non-competitive within the design space and robust against small changes in the beer matrix. The process is transferred to pilot scale, where it is shown that the zeolite effectively reduces wort off-flavour while maintaining the original character of the beverage. The reduced wort flavour concentration is also maintained over a period of 4 months ageing. Interestingly, the formation rates of aldehydes related to ageing are similar for the treated and reference product, with the exception of trans-2-nonenal, which is manifold lower in the treated AFB. A trained sensory panel confirms the difference in taste for both, the fresh and the aged product. In order to upscale this process to industrial scale, also the dynamics of the adsorption process on granular, binderless ZSM-5 zeolite is studied. The homogenous surface diffusion model is employed to regress the intraparticle diffusivity. The so obtained parameters indicate strong intraparticle mass transfer limitations as well as an inverse correlation of the effective diffusion coefficient to the molecules’ hydrophobicity. To avoid this process bottleneck, material improvements such as smaller crystal size and smaller granules are recommended. Finally, all obtained parameters are translated into a unit operation design. With a simple packed-bed operation of crushed particles, the production costs can be as much as 40 % lower than thermal dealcoholisation unit. Nonetheless, several assumptions still need to be verified, such as the material regenerability as well as other non- process-related factors such as material stability and food safety. Concluding, the newly developed technology for selective wort flavour removal in AFB in combination with restricted fermentation represents a great alternative to conventionally produced AFB. @en