Floatony formation in liquid environments: liquid drawing-based fabrication of three-dimensional microbial structures.

Abstract

The spatial organization of microorganisms plays a pivotal role in regulating microbial physiology, community behavior, and ecological interactions. However, reconstructing such three-dimensional (3D) microbial architectures<i>in vitro</i>remains a major challenge because conventional culture systems rely on solid or gel-based matrices that restrict microbial motility and molecular diffusion. Here, we introduce 'floatony', a liquid-based strategy for the fabrication of spatially defined microbial colonies using liquid drawing technology. This approach enables the formation and retention of 3D microbial assemblies entirely within a liquid environment, without solidification or crosslinking. Using<i>E. coli</i>as a model organism, we examined how the rheological properties of the supporting liquid matrix influenced the stability of drawn structures of microbial assemblies. Although the optimal conditions depend on the molecular architecture of thickening agents, we identified an empirical design criterion-tan<i>δ</i>< 1.8-under which 3D structures of microbial assemblies were stably retained while maintaining low viscosity (∼10^-1Pa·s) conducive to efficient molecular diffusion. Enzymatic activity assays confirmed that<i>E. coli</i>maintained functional enzyme activity within the supporting liquid matrix, and that the diffusion of low-molecular-weight reaction products was preserved. Furthermore, complex two-dimensional and 3D structures of microbial assemblies were successfully fabricated and visualized in a liquid, including floating 3D structures, as confirmed by fluorescence imaging. This liquid drawing-based approach provides a new experimental framework for reconstructing and studying spatially organized microbial systems, offering opportunities for investigating microbial interactions and developing engineered living materials beyond conventional solid-supported platforms.