Influence of mechanical properties of agarose hydrogels on bacterial growth and secretory activity

Engineered living materials (ELMs) rely on the ability to control cell behavior in material systems. ELMs containing bacteria secreting beneficial molecules are being developed for therapeutic purposes. Using commensal strains embedded in physically cross-linked agarose hydrogels, we systematically investigate how gel rigidity and initial bacterial density affect the morphology of bacterial colonies and their secretory function. Although often considered independently, these parameters jointly define the microscale environment experienced by embedded cells, influencing nutrient access, mechanical interactions, and potential cell-to-cell communication. We show that matrix rigidity effectively tunes aggregate morphology, modulating their shape and compactness, without compromising bacterial growth or secretion. In parallel, initial bacterial density determines the biomass accumulation dynamics and spatial distribution of aggregates, which in turn influence the onset and temporal profile of secretory activity, without altering its final magnitude. This decoupling between structural organization and secretory output opens new possibilities for engineering ELMs with tailored architectures and prolonged secretory and release activity.