Intra-colony channel morphology in Escherichia coli biofilms is governed by nutrient availability and substrate stiffness

Nutrient-transporting channels are found throughout mature Escherichia coli biofilms, however the influence of environmental conditions on intra-colony channel formation is poorly understood. We report the effect of different substrate nutrient concentrations and agar stiffness on the structure and distribution of intra-colony channels in mature E. coli colony biofilms using fluorescence mesoscopy and quantitative image analysis. Intra-colony channel width was observed to increase non-linearly with radial distance from the centre of the biofilm and channels were, on average, 50% wider at the centre of carbon-limited biofilms compared to nitrogen-limited biofilms. Channel density also differed in colonies grown on rich and minimal medium substrates, with the former creating a network of tightly packed channels and the latter leading to well-separated, wider channels with easily identifiable edges. We conclude that intra-colony channel morphology in E. coli biofilms is influenced by both substrate composition and nutrient availability.