Capsule Formation Mechanisms in Interfacially Initiated Macroporous Hydrogels to Tailor Microstructures for the Encapsulation of Living Bacteria

Bacteria are used in a range of sectors, such as wastewater treatment, bioremediation, or as soil additives. For these applications, live bacteria are encapsulated to protect them from mechanical damage and desiccation. Unlike other types of cargo, bacteria are not always required to be released because when encapsulated, they can interface with their environment and fulfill their roles via molecular transport through the capsule walls. The aims of encapsulation are then shifted away from delaying release to making capsules that are mechanically robust while permitting sufficient diffusion to support the metabolic activity of the bacteria. Here, we produced covalent hydrogel capsules from a water-in-oil (W/O) emulsion of aqueous poly(ethylene glycol) diacrylate (PEGDA) in hexadecane containing a UV-radical initiator. Upon initiation, PEGDA polymerization begins at the W/O interface to produce hydrogel capsules. We discovered three classes of capsule microstructures with differing levels of macroporosity that could be tailored by changing the polymerization conditions. Systematic investigations showed how the UV energy input and the PEGDA macromonomer concentration can be used to selectively create honeycomb, sponge like, or dense spherical capsules. To explain the sponge-like structure, we propose a capsule formation mechanism based on diffusion-limited aggregation of PEGDA microbeads. The structures resemble random-walk simulations of sticky beads and, furthermore, satisfy the theoretical volume fractions required for percolation. We successfully encapsulated live Mycobacterium smegmatis within the sponge structures, demonstrating biocompatibility. Importantly, the internal hydrogel microstructure allows the growth of bacteria. This mechanistic understanding is paramount for designing robust covalent capsules while optimizing porosity within hydrogel structures.