Micro-biogeography greatly matters for competition: Continuous-chaotic bioprinting of spatially-controlled bacterial microcosms

Abstract

Cells do not work alone but instead function as collaborative micro-societies. Consequently, the spatial distribution of different bacterial strains (micro-biogeography) in a shared volumetric space, and their degree of intimacy, greatly influences their societal behavior. Current microbiological techniques are commonly focused on the culture of single bacterial strains or well-mixed bacterial communities and fail to reproduce the micro-biogeography of polybacterial societies. Moreover, the production of micro-spatially controlled architectures at high resolution is currently challenging. In this contribution, we use chaotic flows induced by a printhead containing a static mixer to bioprint fine-scale bacterial microcosms. This straightforward approach (i.e., continuous-chaotic bioprinting) allows us to fabricate hydrogel constructs with 4 to 64 intercalated layers of two bacterial strains. These multi-layered constructs were used to analyze how the spatial distributions of bacteria affect their social behavior and/or survival abilities. We demonstrate that these biological microsystems engage in either cooperation or competition, depending on the degree of shared interface between their different bacterial communities. Remarkably, the extent of inhibition in predator-prey scenarios increased when bacteria were in greater intimacy. Furthermore, two E. coli strains showed competitive behavior in well-mixed microenvironments, whereas stable coexistence prevailed for longer durations in spatially structured consortia. Finally, we demonstrate, for the first time, the simultaneous extrusion of four inks using chaotic printing. This development enables creating higher complexity scenarios, such as four-bacteria microcosms or physically isolated consortia, which may find applications in basic or applied science. We envision that chaotic bioprinting will contribute to the development of a greater complexity of polybacterial microsystems, tissue-microbiota models, and biomanufactured materials.