Engineered living materials (ELMs) are a class of hybrid materials that include engineered microbes encapsulated by a polymer matrix. The biotic and abiotic components define the ELMs design space and can be altered to improve performance and function. While current synthetic materials in the field display robust biocompatibility with both native and engineered living systems, we have a limited understanding of how to leverage three-dimensional (3D) form factors to spatially organize and control microbial dynamics within the material. Motivated by this knowledge gap, we employed extrusion-based 3D printing to fabricate multi-kingdom hydrogel constructs for the encapsulation of both single and multi-kingdom microbial systems. Core-shell cubic constructs enabled the spatial organization of a constitutive multi-kingdom system of levodopa (L-DOPA)-producingE. coliand betaxanthins (BXN)-producingS. cerevisiae. This spatial organization in 3D materials can introduce precise control over bioproduction, bioprotection, and biocontainment features that are critical to the efficacy of current ELMs. The relative spatial organization of the organisms, as well as the surface area-to-volume ratio were investigated to determine how these design elements impact microbial behavior (metabolite production, growth, expression, and cell distribution) over time. We demonstrated that F127-bis-urethane methacrylate (F127-BUM) core-shell geometries enable the hierarchical 3D printing of multi-kingdom constructs, offering customizable control over bioproduction, bioprotection, and biocontainment. With the optimization of these core-shell structures for continuous bioproduction, these ELMs could be deployed as compact and sustainable bioreactors in remote environments.
Keywords: additive manufacturing; bioproduction; engineered living materials; microbial co-culture; multi-kingdom; multi-material hydrogels.
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