Engineering picoliter droplets for ultra-high throughput microbial biotechnology: microfluidic strategies for culturing and screening the microverse and exploit its biosynthetic potential

Abstract

From the microbiologists perspective, microfluidic droplets deliver a paradigm-changing experimental approach, providing a powerful platform in which the biochemical and physiological parameters of microbial samples can be explored with millions of droplets per experiment. The aim of this thesis was to develop technological and experimental enhancements to establish microfluidic droplet assays for the investigation and exploitation of microbial diversity. These developments include: A system for microbial cultivation in millions of picoliter enabling an enhanced and uniform oxygenation and control of pH for the microorganisms in all the droplets simultaneously. A method to take a single image of each droplet and linked the image acquisition to analysis and microfluidic actuation, enabling real-time image-based droplet sorting for the first time. With custom image analysis algorithms it was possible to detect and quantify microbes proliferating in droplets while also keeping quality descriptors monitored over the course of experimentation. A droplet population encoding strategy based on the encapsulation and detection of colored polymer microbeads was developed in order to test multiple conditions simultaneously. A strategy for facile production of multi-structured 3D microfluidic chips, making customized chip production accessible in non-specialized labs. An opto-electronic strategy to be able to simultaneously detect multiple fluorescent channels using a single sensor. A system enabling automated transfer and deposition of single sorted droplets onto Petri dishes or into wells of microplates; for the recovery of microorganisms isolated in single droplets. These developments and applications provide ground work for a broad application of droplet microfluidics in microbiology and biotechnology; contributing to further progress in the field, with both academic and industrial perspectives.