Characterization of biofilm surfaces: Morphology, roughness, friction coefficient, spring constant, hydrophobicity and charge

Five stages are involved in biofilm formation in an aqueous environment: initial reversible attachment (Stage 1), irreversible attachment (Stage 2), microcolony formation (Stage 3), biofilm maturation (Stage 4) and steady state growth (Stage 5). To determine the effect of nano-scale biofilm surface roughness on hydrodynamic conditions above the biofilm surface, the HBL thickness of two pure culture biofilms, Pseudomonas aeruginosa and Nitrosomonas europaea, (sampled at Stages 3 and 5) was evaluated using micro-particle image velocimetry (μ-PIV) for various free stream velocities within the laminar regime. The results indicate that the rougher biofilm surface (N. europaea biofilm) significantly increased the effect of HBL thickness. No effect was observed for changes in biofilm roughness on the coefficient of friction. Atomic force microscopy (AFM) has been widely used to evaluate microbial surfaces properties due to its ability to quantify surface stiffness and interactive forces under in-situ conditions and to produce high resolution three-dimensional images. The physiological structure of the biofilm surface (morphology, roughness, spring constant, hydrophobicity and charge properties) was evaluated using AFM with CH3/COOH/NH2 functionalized tips at different biofilm growth stages. The results indicate that the pure culture Pseudomonas aeruginosa biofilm underwent morphology changes during biofilm growth. The surface roughness reached a maximum level in Stage 3, decreased in Stage 4 due to the accumulation of EPS, and stabilized in Stage 5. Surface stiffness increased during Stages 3 to 4 and then reached a plateau. Biofilm surface physicochemical properties (hydrophobicity and charge properties) were significantly altered in Stage 4 but were relatively stable in both Stages 3 and 5. These results indicate that the composition, structure, and chemical properties of a biofilm is highly dependent on the growth stage of the biological matrix