Non-genetic heterogeneity drives pattern formation and provides survival advantages in bacterial biofilms

Biofilms are a dominant lifeform of bacteria in which individual bacterial cells form surface-associated aggregates embedded in a self-produced polymeric matrix. Biofilm structure and biological activity make it a complex form of active matter, with implications in both health and industrial settings. While the classical picture of biofilm life cycle stresses synchronous and collective decisions via chemical communications, non-genetic cell-to-cell variation, or phenotypic heterogeneity, also plays a significant role in biofilm development. Using fluorescent biosensors and single-cell time-lapse microscopy, we measure heterogeneity in a key signaling molecule that regulates biofilm formation in Vibrio cholerae. We find high levels of heterogeneity in growing biofilms, as well as a concomitant pattern formation linked to bimodal phenotypes. These patterns emerge from physical interactions mediated by biofilm matrix, as we demonstrate through genetic mutations, lineage tracing, and numerical simulations. We further show that non-genetic heterogeneity provides survival advantages to biofilm-forming bacteria in fluctuating environments via bet-hedging and optimizing colonization.