Characterization of Single and Dual-Consortium Microbial Systems in 3D-Printed Multi-Material Hydrogels

Engineered Living Materials (ELMs) capture the efficiency of cellular systems within polymeric matrices to provide bioreactor designs for production of commodities such as biofuels, vaccines, and the classic Seattle IPA. With extrusion-based 3D printing, we can print multi-material ELMs that afford controlled spatial organization of cell populations. With F127-bisurethane methacrylate (F127-BUM), an easily accessible triblock copolymer, we can synthesize bioinks with proper yield strain for extrusion-based printing while also gaining robust biocompatibility and free diffusion of metabolites across the material. We maintain a basic understanding of F127-BUM structure but have limited knowledge of how the network interacts with cells. While these hydrogel constructs display robust metabolic function, prior studies have shown cells escaping into the surrounding media despite complete encapsulation, suggesting random migration through the material. However, in studies that culture two microbial species in adjacent F127 gels, higher-order spatial organization is observed as cell populations consisting of one species do not migrate into gel areas occupied by another cell species. In our studies, we propose an alternative 3D-printed core-shell cube structure that consists of a cell-laden inner core surrounded by a cell-free outer shell. This construct affords a model to study cell behavior of single and dual-cell consortiums in F127-BUM hydrogels as we seek to improve cell containment and preserve metabolic function in 3D printed hydrogel designs for bioproduction.<br/><br/>Using the core-shell cube structure, we demonstrated robust multi-kingdom functionality with Green Fluorescence Protein (GFP)-secreting <i>E. coli </i>and betaxanthin-producing <i>S. cerevisiae</i>. After six days, the multi-material constructs showed continuous GFP and betaxanthin production and diffusion into the media<b>.</b> Optical fluorescence microscopy showed GFP E. coli distribution focused on the core-shell interface while S. cerevisiae distribution is uniform throughout the core. While visual spatial separation between the core and the shell elements was maintained, cell escape after 48 h was observed using SEM as a result of microscopic inconsistencies in the printing process and cross-contamination of the extrusion nozzles during printing. By altering flow speed and the print design g-code, we improved cell containment.<br/><br/>To investigate impact of form factor on microbial behavior, we proposed a rectangular prism design with improved surface area-to-volume ratio to investigate the impact of geometric parameters on bioproduction and diffusion. We saw improvement in the GFP-system but observed little difference in our betaxanthin system. Additionally, we tested the performance of both core-shell constructs in co-culture systems by placing gels containing one cell population into free media containing a different microbial species. With the gel system, viability was maintained in both cell species for 6 d.<br/><br/>Following the single consortia models, we expanded the core-shell design to contain a dual-microbial consortium. Inspired by symbiosis, this multi-kingdom hydrogel design contained a metabolically constitutive relationship consisting of engineered <i>E. </i><i>coli</i> and <i>S. cerevisiae </i><i>strains.</i><i> S. cerevisiae </i>only produces betaxanthin in presence of L-DOPA.<i> </i><br/><br/>During a six-day period, final betaxanthin production remained low as a result of the inner core consortia having limited access to oxygen as the outer shell consortia blocks nutrient access during overgrowth. As a result, we will adopt designs such as coaxial fibers or thinner dual-material biofilms to avoid limitations in oxygen and nutrient diffusion.<br/><br/>Overall, our investigation of multi-material F127-BUM hydrogels and their ability to afford a wide range of microbial systems and design possibilities establishes a necessary model to study the intricate relationship between hydrogel structure and cell behavior for bioproduction applications.