Scalable Flow Reactors for Stable Biofilm Formation and Continuous Whole-Cell Catalysis

Stable and productive biofilms are highly attractive for continuous whole-cell biocatalysis, yet their controlled formation and long-term operation remain challenging. Here, we introduce a modular and scalable microfluidic reactor platform that exploits flow-induced deposition to generate robust biofilms directly from suspended cells. Starting from a validated pillar reactor design, we systematically scale reactor geometries and apply computational flow simulations to identify microscale hydrodynamic features that govern cell attachment, streamer formation, and biofilm stability. Fluorescence microscopy reveals the emergence of stratified biofilm architectures enriched in extracellular DNA that persist under continuous flow. Enzymatic depletion of extracellular DNA selectively delays early streamer formation, highlighting its critical role during initial matrix assembly. Under continuous flow, biofilm growth and shear-induced detachment establish a dynamic steady state that maintains a self-sustaining catalytic matrix, enabling long-term continuous whole-cell biocatalysis in an upscaled reactor where the deposited biofilms remain catalytically active and structurally stable for at least 28 days. Together, these results establish flow-structured microreactors as a generalizable strategy for hydrodynamically controlled biofilm engineering, linking microscale flow fields to biofilm architecture, long-term stability, and catalytic function, and providing a foundation for scalable continuous whole-cell biocatalysis.