Spatial self-organization of confined bacterial suspensions

Lab studies of bacteria usually focus on cells in spatially extended, nutrient-replete settings, such as in liquid cultures and on agar surfaces. By contrast, many biological and environmental settings—ranging from mucus in the body to ocean sediments and the soil beneath our feet—feature multicellular bacterial populations that are confined to tight spots where essential metabolic substrates (e.g., oxygen) are scarce. What influence does such confinement have on a bacterial population? Here, we address this question by studying suspensions of motile Escherichia coli confined to quasi two-dimensional (2D) droplets. We find that when the droplet size and cell concentration are both large enough, the initially uniform suspension spatially self-organizes into a concentrated, immotile inner "core" that coexists with a more dilute, highly motile surrounding "shell." By simultaneously measuring cell concentration, oxygen concentration, and motility-generated fluid flow, we show that this behavior arises from the interplay between oxygen transport through the droplet from its boundary, uptake by the cells, and corresponding changes in their motility in response to oxygen variations. Furthermore, we use biophysical theory and simulations to quantitatively describe this interplay. Our work thus sheds light on the rich collective behaviors that emerge for bacterial populations in confined environments, with implications for understanding ecological niches and engineering artificial systems.

A.M.-C. acknowledges support from the Princeton Center for Theoretical Science, the Princeton Center for the Physics of Biological Function, and the Human Frontier Science Program through the grant LT000035/2021-C. S.S.D. acknowledges support from NSF Grants CBET-1941716, DMR-2011750, and EF-2124863 as well as the Camille Dreyfus Teacher-Scholar and Pew Biomedical Scholars Programs, the Eric and Wendy Schmidt Transformative Technology Fund, and the Princeton Catalysis Initiative. We thank Henry Mattingly for assistance with the preparation of the oxygen probe dye; Daniel Amchin, Enkeleida Lushi, and Ned Wingreen for thoughtful discussions; and Daniel Amchin, Rhea Braun, and Nadine Ziegler for assistance with a preliminary version of the experiments at the inception of this project.

All movies and analysis codes are available at Zenodo (https://doi.org/10.5281/zenodo.14894704) (118). The code used to solve the coupled reaction–diffusion equations describing bacteria and oxygen dynamics in the manuscript are available at https://github.com/amcalv/2025-Code-Spatial-self-organization-of-confined-bacterial-suspensions (119). All other data are included in the manuscript and/or supporting information.