The surface morphology of mature biofilms is heterogeneous and can be divided into concentric rings wrinkles (I), labyrinthine networks wrinkles (II), radial ridges wrinkles (III), and branches wrinkles (IV), according to surface wrinkle structure and distribution characteristics. Due to the wrinkle structures, channels are formed between the biofilm and substrate and transport nutrients, water, metabolic products, etc. We find that expansion rate variations of biofilms growing on substrates with high and low agar concentrations (1.5, 2.0, 2.5 wt.%) are not in the same phase. In the first 3 days' growth, the interaction stress between biofilm and each agar substrate increases, which makes the biofilm expansion rate decreases before wrinkle pattern IV (branches) comes up. After 3 days, in the later growth stage after wrinkle pattern IV appears, the biofilm has larger expansion rate growing on 2.0 wt.% agar concentration, which has the larger wrinkle distance in wrinkle pattern IV reducing energy consumption. Our study shows that the stiff substrate does not always inhibit the biofilm expansion, although it does in the earlier stage; after that, mature biofilms acquire larger expansion rate by adjusting the growth mode through the wrinkle evolution even in nutrient extremely depletion.
Keywords: Bacillus subtilis (MTC817) biofilm; expansion rate; substrates stiffness; wrinkle morphology.