Species-Specific Flow Interactions and Environmental Cues Shape Biofilm Dynamics

Biofilm formation is a complex, multiscale process regulated by both biological and physicochemical factors. While bacterial motility and environmental cues such as nutrient availability and substrate stiffness are known to influence biofilm development, the role of species-specific hydrodynamic interactions in shaping early biofilm organization remains poorly understood. In this study, we systematically investigate how pairwise bacterial interactions and environmental parameters jointly modulate biofilm initiation, structure, and dynamics. Using Escherichia coli, GFP-expressing E. coli, and Bacillus subtilis as model systems, we examine three configurations, single-species, same-species, and multi-species, under varying nutrient conditions and agar stiffness. Time-resolved imaging, profilometry, particle image velocimetry-based flow mapping, and microscopy reveal that pairwise hydrodynamic interactions generate species-dependent flow fields that critically determine spatial organization and aggregate growth. Single-species systems produce uniform flow patterns promoting clustering and microcolony initiation, whereas same-species mixtures enhance alignment and coordinated motion, accelerating biofilm expansion. In contrast, multi-species systems exhibit heterogeneous and competing flow fields driven by differences in motility and surface adhesion, resulting in dynamic and unstable architectures. Environmental factors further modulate these hydrodynamic effects: nutrient-rich and soft substrates promote rapid biofilm propagation and structural maturation. These findings demonstrate that species identity, hydrodynamic interactions, and environmental cues collectively govern biofilm architecture, mechanics, and stability, providing an integrated biophysical framework for understanding and controlling biofilm formation across bacterial systems.