Bacillus subtilis is a common model system for studying biofilm formation. In the study, we have linked the development of a floating biofilm at the water-air interface with the distribution and viability of individual B. subtilis cells in the water column. Using IRS and CLSM methods, we have correlated the morphological changes with the mechanical properties of the growing floating biofilm. Six key events in biofilm development accompanied by a large change in morphological and viscoelastic behaviour were identified. The results show that the development of the floating biofilm is a multifaceted response to the changing environment facilitated by bacterial growth, resulting in population redistribution within the model system, reduction of suitable habitat to the water-air interface, cell development, and morphogenesis. The collapse of the floating biofilm, when the biofilm loses its ability to bear mechanical loads, correlates with the process of endospore release and the system entry into a state of rest. By growing genetically heterogeneous floating biofilms, we have determined the influence of relatedness between strains on the formation and development of mixed floating biofilms. We have shown that in combinations of less related strains, the dominant bacterial strain can completely suppress the growth and development of the less related strain. By growing less related strains in a microfluidic device we have shown that spatially dispersed aggregates of different strains are formed until the microchamber is filled, then the dominant strain gradually reduces the density of the cell population of the less related strain and eliminates it. By changing the initial ratio, we have shown that the outcome of the interaction is strongly dependent on the initial ratio of competing strains.
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