Multiscale mechanics of granular biofilms

Biofilms produce and maintain extracellular polymeric substances (EPS) essential for their form and function. While biofilms are commonly lamellar and frequently targets of removal, granular biofilms are increasingly incorporated into water treatment strategies. In both cases, the EPS (mainly consisting of proteins, polysaccharides, and extracellular DNA) is largely responsible for their persistence. Unlike many granular biofilms, which are formed in engineered industrial bioreactors, the "pink berry" consortia is a naturally-occurring and robust granular biofilm of photosynthetic bacteria, found only in intertidal pools of salt marshes around Woods Hole, Massachusetts (USA). The pink berry biofilms unique ecological niche has sparked over three decades of study, yet their mechanical properties are completely unknown. Here, we characterized the structural and mechanical landscape of pink berry granules to determine the extent to which microscale heterogeneity influences macroscale material properties. We performed microindentation measurements on intact granules and nanoindentation measurements on thin sections. We report that intact pink berry granules exhibited low reduced elastic moduli (E*pink berry {approx} 0.5-10 kPa) and fast stress relaxation times ({tau}1/2 {approx} seconds), consistent with previous investigations of soft and viscoelastic biofilms. Nanomechanical measurements of thin pink berry sections revealed two mechanically-distinct domains: a very soft extracellular polymeric substance (EPS) matrix surrounding stiffer microcolonies of purple sulfur bacteria (PSB). Light sheet fluorescence microscopy revealed the spatial organization and distribution of cell-dense PSB microcolonies (34 vol.%) within EPS matrix (66 vol.%), suggesting the nanomechanical behavior of EPS dominates macroscale pink berry mechanics. Our multiscale experimental approach combining mechanics and imaging may be broadly applicable to investigations of complex soft materials, from synthetic hydrogel composites to biologically heterogeneous spheroids, organoids, and tissues.