Characterizing spatiotemporal microbial colony distributions in printed PEG-DA hydrogel films

Biofilms are surface-attached microbial communities that play vital roles in natural ecosystems and contribute to persistent problems in medicine and industry. These communities exhibit heterogeneous chemical, physical, and physiological properties, which are governed by reciprocal structure-function relationships. Linking structure to function is crucial for understanding biofilm physiology but remains challenging due to the structural complexity of naturally formed biofilms. Bioprinting offers exquisite control over biofilm structure and holds potential for systematically exploring these relationships; however, the microscale colony distributions that emerge within hydrogel-based print resins remain unexplored. To address this, we use light-based bioprinting to create single-layer hydrogel films containing homogeneously dispersed<i>Pseudomonas fluorescens</i>bacteria and characterize the spatiotemporal distribution of colonies that develop within these films. We systematically vary the concentration of bacteria over nearly three orders of magnitude, track colony growth using microscopy, and quantify structural features with image analysis. We observe empirical relationships between initial cell concentration and key structural features: colony size, colony volume, total biovolume, and characteristic gradient length scale. This knowledge can be used to print microbial communities with well-defined features, is readily applicable to more complex three-dimensional shapes, and provides a tool for advancing our understanding of microbial communities.