Surface-attached bacterial biofilms are self-replicating active liquid crystals and the dominant form of bacterial life on earth ( 1 – 4 ). In conventional liquid crystals and solid-state materials, the interaction potentials between the molecules that comprise the system determine the material properties. However, for growth-active biofilms it is unclear whether potential-based descriptions can account for the experimentally observed morphologies, and which potentials would be relevant. Here, we overcame previous limitations of single-cell imaging techniques ( 5 , 6 ) to reconstruct and track all individual cells inside growing three-dimensional (3D) biofilms with up to 10,000 individuals. Based on these data, we identify, constrain, and provide a microscopic basis for an effective cell-cell interaction potential, which captures and predicts the growth dynamics, emergent architecture, and local liquid crystalline order of Vibrio cholerae biofilms. Furthermore, we show how external fluid flows control the microscopic structure and 3D morphology of biofilms. Our analysis implies that local cellular order and global biofilm architecture in these active bacterial communities can arise from mechanical cell-cell interactions, which cells can modulate by regulating the production of particular matrix components. These results establish an experimentally validated foundation for improved continuum theories of active matter and thereby contribute to solving the important problem of controlling biofilm growth.

SignificanceMost living systems, from individual cells to tissues and swarms, display collective self-organization on length scales that are much larger than those of the individual units that drive this organization. A fundamental challenge is to understand how properties of microscopic components determine macroscopic, multicellular biological function. Our study connects intracellular physiology to macroscale collective behaviors during multicellular development, spanning five orders of magnitude in length and six orders of magnitude in time, using bacterial swarming as a model system. This work is enabled by a high-throughput adaptive microscopy technique, which we combined with genetics, machine learning, and mathematical modeling to reveal the phase diagram of bacterial swarming and that cell–cell interactions within each swarming phase are dominated by mechanical interactions.

SINGLE CELL AGENT-BASED SIMULATIONS Model descriptionOur single cell model is based on the agent-based framework described in [1], with modifications to include the effect of the flow on the cells and cell-cell polar adhesion. Cells are modeled as ellipsoids of half-length l and half-width r; each cell is described by its position x, orientationn and effective local viscosity µ . The dynamics of the cells are approximated as over-damped, as cells live at low Reynolds number Re ≈ 10 −4 [1]. Denoting the identity matrix by I and the dynamic viscosity of water by µ w , the over-damped translational and orientational dynamics for a single cell are

Bacterial biofilms are matrix-bound multicellular communities. Biofilms represent a major form of microbial life on Earth and serve as a model active nematic system, in which activity results from growth of the rod-shaped bacterial cells. In their natural environments, from human organs to industrial pipelines, biofilms have evolved to grow robustly under significant fluid shear. Despite intense practical and theoretical interest, it is unclear how strong fluid flow alters the local and global architectures of biofilms. Here, we combine highly time-resolved single-cell live imaging with 3D multi-scale modeling to investigate the effects of flow on the dynamics of all individual cells in growing biofilms. Our experiments and cell-based simulations reveal that, in the initial stages of development, the flow induces a downstream gradient in cell orientation, causing asymmetrical droplet-like biofilm shapes. In the later stages, when the majority of cells are sheltered from the flow by the surrounding extracellular matrix, buckling-induced cell verticalization in the biofilm core restores radially symmetric biofilm growth, in agreement with predictions from a 3D continuum model.

Controlling and suppressing bacterial accumulation at solid surfaces is essential for preventing biofilm formation and biofouling. Whereas various chemical surface treatments are known to reduce cell accumulation and attachment, the role of complex surface geometries remains less well understood. Here, we report experiments and simulations that explore the effects of locally varying boundary curvature on the scattering and accumulation dynamics of swimming Escherichia coli bacteria in quasi-two-dimensional microfluidic channels. Our experimental and numerical results show that a concave periodic boundary geometry can decrease the average cell concentration at the boundary by more than 50% relative to a flat surface.