Embedding modified microorganisms in polymeric and composite matrices, thereby generating genetically programmable Engineered Living Materials (ELM), is a field of high potential that gained tremendous momentum with the advent of the Synthetic Biology era. Among the numerous methods for designing and manufacturing ELM, 3D bioprinting stands out due to its remarkable ability to precisely control both the structure and integrity of the fabricated constructs and the spatial distribution of cells within them. While bioprinting of engineered microbial cells has been reported for a number of bacterial species, its application for their dormant life stages, so-called spores, is still in its infancy, despite their advantages, being robust, metabolically inactive and long-lived. Endospores of Bacillus subtilis, in particular, hold great potential for ELM, since they provide a second layer to implement engineered functionalities that can be genetically programmed into the DNA of the microbial cells. By translationally fusing a gene-of-interest to a gene encoding a suitable anchor protein, a target protein can be immobilized and hence displayed on the spore envelope during the natural differentiation cycle forming so called SporoBeads.

In our study, we combined 3D bioprinting with functionalized SporoBeads to create a dynamic and adaptable ELM. SporoBeads were printed using various alginate-based bioinks, and the printing process was optimized with respect to spore density, scaffold stability, and controllability of cell growth. By applying growth medium and sporulation medium to the printed scaffolds, we successfully transitioned the printed spores from their dormant phase into an active vegetative state, and subsequently reverted these vegetative cells back into the spore stage. This process increases the concentration of SporoBeads and regenerates the immobilized proteins on the spore surface. Through the attachment of a fluorescent protein to the surface of the SporoBeads and the labeling of a vegetative gene with a separate fluorescent marker, we demonstrated our capacity to regulate the life cycle stages of B. subtilis within the bioprinted scaffold. This ongoing investigation establishes a foundation for developing a range of ELMs with renewable enzymatic functions, leveraging the robust and versatile nature of SporoBeads.