Fed‐Batch Droplet Nanobioreactor for Controlled Growth of Cyberlindnera (Pichia) jadinii: A Proof‐Of‐Concept Demonstration

Abstract

Improvement of the microorganism's product yield on feedstock is possible by targeted genetic modification, which requires a solid understanding of the species metabolism and genetics. Alternatively, genetic modifications can be introduced randomly such that the chance that a resulting mutant performs better than the original one is small. Consequently, many mutants need to be created and subsequently screened in order to identify the best performing ones. [1] Given the large number of experiments, it is common practice to grow and study all the different mutants in individual microliter-sized wells on microtiter plates. While this allows for parallel screening in an automated fashion using pipetting robots and plate readers, conventional microtiter plates lack the ability to feed nutrients and control pH by base or acid addition. It is indeed challenging to feed liquids at flow rates in the nanoliter per hour range to all the individual wells of a microtiter plate and to do so accurately. Therefore, screening of mutants is commonly performed under batch conditions with all feedstock present from the start and no further control over feedstock concentration and pH. In contrast, more than 80% of the processes in industrial biotechnology are operated under so called fed-batch conditions with control over feedstock concentration and pH. [2,3] This incompatibility between the physiological conditions during screening and industrial operation not only leads to selection of false positives which fail to generate competitive yields at industrial scale, but also fails to identify the best mutants for fed-batch conditions. [4][5][6] The effectiveness of screening hence greatly benefits from a technology that enables growing and studying a large number of microorganisms under precisely controlled conditions representative of industrial bioreactors. Besides screening of mutants, optimization of process conditions, a second important aspect in bioprocess development, also benefits from such a technology. [7] Although there has been progress in recent years, there remains both need and opportunity to cost-effectively and with fidelity miniaturize fermentation to screen under fed-batch conditions.Several strategies have been developed to study microorganisms under controlled growth conditions. One strategy is to 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 ...

Microbioreactors for nutrient‐controlled microbial cultures: Bridging the gap between bioprocess development and industrial use

“…A recent example of a droplet‐based fed‐batch microbioreactor that does allow the repeated and controlled supply of nutrients to a cell‐containing droplet immobilized in a chamber was demonstrated in the lab of the authors. [ 47 ] The design of the geometry in which the nutrient droplets are produced allows a robust periodic on‐demand supply of droplets in a device free of valves, just through the use of a commercially available pressure pump, [ 48 ] see Figure 6G. The authors demonstrated the cultivation of cells under fed‐batch conditions, with the growth rate of the cells inside the immobilized cell‐containing droplet controlled by the concentration of the nutrient‐containing droplets.…”

“…Adapted from Tomasi et al [46] (G) Fed-batch droplet-based microbioreactor comprising of droplet-on-demand junctions (left) for the controlled supply of nutrient droplets to a cell-containing droplet immobilized in a trap (right). Adapted from Totlani et al [47,48] (H) Channel-free approach in which cell containing droplets are bio-printed in a yield stress fluid. This approach has the potential to repeatedly supply reagents to the cell-containing droplets and study cells under controlled conditions.…”

“…Adapted from Totlani et al. [ 47,48 ] (H) Channel‐free approach in which cell containing droplets are bio‐printed in a yield stress fluid. This approach has the potential to repeatedly supply reagents to the cell‐containing droplets and study cells under controlled conditions.…”