Microbial tale: using chaotic bioprinting to create a structured multi-strain probiotic

Abstract

Probiotic therapies offer great potential for addressing gut dysbiosis, but current approaches are limited by low strain diversity, high production costs, and the challenges of culturing strict anaerobes. To overcome these limitations, this work introduces a novel strategy combining based on continuous chaotic bioprinting to create structured (micro-architected) co-cultures of probiotic bacteria. Using a Kenics static mixer–based printhead, we fabricated alginate hydrogel filaments with an internal multilayered microarchitecture containing four probiotic strains: Bifidobacterium bifidum, Bacteroides fragilis, Lactobacillus rhamnosus, and Streptococcus thermophilus. The spatial arrangement of the multilayered architecture was designed to promote cooperative interactions, particularly by embedding strict anaerobes between facultative anaerobes to create self-sustaining hypoxic niches. The printed constructs were characterized over 72 hours using fluorescence microscopy, colony-forming unit counts, LIVE/DEAD assays, and qPCR. Results showed that structured co-cultures exhibited higher viability, enhanced growth, and more balanced population dynamics than the monocultures of each bacterial strain and unstructured (scrambled) co-cultures. This study demonstrates that chaotic bioprinting enables precise spatial control over microbial ecosystems, allowing the rational design of microbial communities with tailored interactions. The approach presents a powerful and scalable platform for next-generation probiotic production and opens new opportunities for engineered microbiomes, synthetic biology, and living material design.