Hydrodynamic modulation of bacterial biofilm formation via cellular motility

Bacterial biofilms are complex, surface-associated communities whose formation is tightly regulated by environmental cues such as nutrient availability, substrate stiffness, and pH. Although the role of motility in biofilm development has been widely acknowledged, its interaction with physical and chemical environmental factors under conditions of limited nutrients remains poorly understood. In this study, we systematically investigate how varying nutrient concentrations, agar stiffness, and pH modulate the biofilm formation dynamics of Escherichia coli and Bacillus subtilis. Using a combination of time-resolved macroscopic imaging, profilometry, particle image velocimetry-based motility mapping, and microscopy, we demonstrate that nutrient-rich environments (Luria Bertani broth) and soft substrates (low agar concentrations) synergistically promote rapid and extensive biofilm expansion. Importantly, we report a novel quantitative characterization of biofilm front propagation, revealing an early burst in cellular activity followed by a pronounced slowdown indicative of maturation. A striking finding is that alkaline pH (pH 9) significantly enhances biofilm growth even in minimal media, whereas acidic pH (pH 5) suppresses biofilm formation across conditions. Microscopic analysis reveals that Escherichia coli and Bacillus subtilis biofilms exhibit nutrient-dependent morphological heterogeneity, including the emergence of dormant persister-like cells under stress. This highlights phenotypic diversification as a key survival strategy modulated by environmental cues and genetic interactions. Together, these results advance our understanding of how mechanical and chemical cues jointly regulate biofilm physiology and offer new insights for designing context-sensitive biofilm control strategies in clinical, industrial, and environmental settings.