Inoculum-dependent instability of a biofilm pellicle is rescued by non-biofilm formers

Various microbes are known to colonize the air-liquid interface of an unshaken culture by forming a pellicle-like biofilm. Occupying the spatial niche at an air-liquid interface is beneficial for aerobic microbes to secure access to oxygen and thus has a big impact on ecology and evolution. Although genetic factors that drive pellicle formation, such as extracellular matrices and fimbriae, have been extensively investigated, no clear understanding of the spatio-temporal dynamics has been achieved. In this work, combining experimental and computational approaches, we studied the physical principle of bacterial colonization at an air-liquid interface and its ecological and evolutionary implications. First, we experimentally showed that pellicle formation tended to fail when starting from a low inoculum. This inoculum effect was caused by the detachment of floating microcolonies from the air-liquid interface due to physical instability. To overcome the instability, collective colonization was critical to rapidly cover the space prior to the accumulation of biomass. We further tested the role of non-biofilm formers, which had been regarded as cheaters because they produced less extracellular metrices. Strikingly, the non-biofilm formers rescued the inoculum-dependent collapse of a pellicle by providing physical support. Our results characterize the universal dynamics of pellicle formation and highlight a novel physical contribution of cheaters that do not produce common goods.