It is common practice in the development of bioprocesses to genetically modify a microorganism and study a large number of resulting mutants in order to select the ones that perform best for use at the industrial scale. At industrial scale, strict nutrient-controlled growth conditions are imposed to control the metabolic activity and growth rate of the microorganism, thereby enhancing the expression of the product of interest. Although it is known that microorganisms that perform best under these strictly controlled conditions are not the same as the ones that perform best under uncontrolled batch conditions, screening, and selection is predominantly performed under batch conditions. Tools that afford high throughput on the one hand and dynamic control over cultivation conditions on the other hand are not yet available. Microbioreactors offer the potential to address this problem, resolving the gap between bioprocess development and industrial scale use. In this review, we highlight the current state-of-the-art of microbioreactors that offer the potential to screen microorganisms under dynamically controlled conditions. We classify them into: (i) microtiter plate-based platforms, (ii) microfluidic chamber-based platforms, and (iii) microfluidic droplet-based platforms. We conclude this review by discussing the opportunities of nutrient-fed microbioreactors in the field of biotechnology.

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A key bottleneck in bioprocess development is that state-of-the-art tools used for screening of cells and optimization of cultivation conditions do not represent the conditions enforced at industrial scale. At industrial scale, cell growth is strictly controlled (“fed-batch”) to optimize the metabolites produced by the cells. In contrast, cell growth is uncontrolled (“batch”) in microwells commonly used for bioprocess development due to the difficulty to continuously supply minute amounts of nutrients to the cells in these wells over the course of the cultivation experiment. This work addresses this bottleneck through the development of a droplet-based fed-batch nanobioreactor. A key challenge addressed in this work is the implementation of the required non-steady droplet operations on chip to establish a semi-continuous nutrient supply, while keeping the chip and its operation as simple as possible. The ability to study micro-organisms under nutrient-controlled fed-batch conditions is demonstrated using the yeast Cyberlindnera (Pichia) jadinii, with the cell growth rate controlled through the glucose concentration. Given the relative ease of operation and the potential to extend its features, the presented nanobioreactor provides a solid platform technology for further development and use in the field of bioprocess development and beyond.

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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. @en

We developed a microfluidic droplet on-demand (DoD) generator that enables the production of droplets with a volume solely governed by the geometry of the generator for a range of operating conditions. The prime reason to develop this novel type of DoD generator is that its robustness in operation enables scale out and operation under non-steady conditions, which are both essential features for the further advancement of droplet-based assays. We first detail the working principle of the DoD generator and study the sensitivity of the volume of the generated droplets with respect to the used fluids and control parameters. We next compare the performance of our DoD generator when scaled out to 8 parallel generators to the performance of a conventional DoD generator in which the droplet volume is not geometry-controlled, showing its superior performance. Further scale out to 64 parallel DoD generators shows that all generators produce droplets with a volume between 91% and 105% of the predesigned volume. We conclude the paper by presenting a simple droplet-based assay in which the DoD generator enables sequential supply of reagent droplets to a droplet stored in the device, illustrating its potential to be used in droplet-based assays for biochemical studies under non-steady operation conditions.

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We report the development of a droplet-based microfluidic device, which enables long term culturing of microorganisms inside water in oil microdroplets under semi-continuous conditions. Firstly, a microdroplet containing yeast cells is trapped on chip. After an initial incubation phase, this cell laden droplet is coalesced with smaller nutrient droplets to enable cultivation and time-lapsing under semi-continuous conditions. We demonstrate a proof-of-principle study on a sequential batch experiment, which lays a sound foundation for screening of microorganisms under semi-continuous conditions.

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We report the continuous production of microcapsules composed of an aqueous core and permeable hydrogel shell, made stable by the controlled photo-cross-linking of the shell of an all-aqueous double emulsion. While most previous work on water-based emulsions focused on active droplet formation, here double emulsion droplets were spontaneously generated at a three-dimensional flow-focusing junction through the break-up of a double jet formed by immiscible aqueous solutions of polyethylene glycol and cross-linkable dextrans. The capsules obtained with this lipid-free, organic-solvent-free, and surfactant-free approach displayed excellent stability under a variety of harsh conditions (3 <pH <13, high salinity). Drying and rehydration experiments demonstrate the permeability of the shell, which may enable molecular-weight-dependent release and uptake of polar solutes.

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