Biofabrication of microstructured bacterial ecosystems using chaotic bioprinting: advancing <i>in vitro</i> research for microbial engineering

Mixed microbial communities are essential for various ecosystems, with bacteria often exhibiting unique behaviors in structured environments. However, replicating these interactions<i>in vitro</i>remains challenging, as traditional microbiology techniques based on well-mixed cultures fail to capture the spatial organization of natural communities. Chaotic 3D printing offers a versatile, high-throughput method for fabricating hydrogel constructs with multilayered microstructure in which different bacterial strains can coexist, closely mimicking the partial segregation seen in natural microbial ecosystems. Using a Kenics static mixer printing nozzle, we bioprinted a bacterial consortium consisting of<i>Lactobacillus rhamnosus, Bifidobacterium bifidum</i>, and<i>Escherichia coli</i>as a simplified model for human gut microbiota. Chaotic bioprinting enabled the creation of microstructured cocultures with distinct niches, allowing all bacterial strains to coexist (without being scrambled) and reach a population equilibrium. We characterized the cocultures through fluorescence microscopy, colony counting, and quantitative polymerase chain reactions. Our results demonstrate that the microarchitecture of the printed fibers significantly influences bacterial growth dynamics. Stratified arrangements enhanced coculture viability and balance over 72 h compared to well-mixed and suspension conditions. Chaotic printing also allows the rational arrangement of strict anaerobic bacteria, such as<i>B. bifidum</i>, by positioning them in construct layers that are more susceptible to hypoxia. Chaotic bioprinting presents a powerful tool for engineering microbial ecosystems with precise spatial control in the range of tens of micrometers. This approach promises to advance our understanding of microbial interactions and has potential biomedical applications in antibiotic testing, microbiota research, bioremediation, and synthetic biology.