Oxygen Limitation within a Bacterial Aggregate

A. K. Wessel, T. Arshad, M. Fitzpatrick, J. L. Connell, R. Bonnecaze, J. B. Shear, and M. Whiteley

Cells within biofilms exhibit physiological heterogeneity, in part because of chemical gradients existing within these spatially structured communities. Previous work has examined how chemical gradients develop in large biofilms containing >108 cells. However, many bacterial communities in nature are composed of small, densely packed aggregates of cells (≤105 bacteria). Using a gelatin-based three-dimensional (3D) printing strategy, we confined the bacterium Pseudomonas aeruginosa within picoliter-sized 3D “microtraps” that are permeable to nutrients, waste products, and other bioactive small molecules. We show that as a single bacterium grows into a maximally dense (1012 cells ml−1) clonal population, a localized depletion of oxygen develops when it reaches a critical aggregate size of ~55 pl. Collectively, these data demonstrate that chemical and phenotypic heterogeneity exists on the micrometer scale within small aggregate populations.