Multiscale X-ray study of Bacillus subtilis biofilms reveals interlinked structural hierarchy and elemental heterogeneity

Significance Biofilms are multicellular, soft microbial communities that are able to colonize synthetic surfaces as well as living organisms. To survive sudden environmental changes and efficiently share their common resources, cells in a biofilm divide into subgroups with distinct functions, leading to phenotypic heterogeneity. Here, by studying intact biofilms by synchrotron X-ray diffraction and fluorescence, we revealed correlations between biofilm macroscopic, architectural heterogeneity and the spatiotemporal distribution of extracellular matrix, spores, water, and metal ions. Our findings demonstrate that biofilm heterogeneity is not only affected by local genetic expression and cellular differentiation but also by passive effects resulting from the physicochemical properties of the molecules secreted by the cells, leading to differential distribution of nutrients that propagate through macroscopic length scales.