Quantitative analysis of biofilm thickness variability.

The thickness variability of biofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the binary population combination of these two species was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and applying image analysis to construct thickness profiles along linear transects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell attached to the surface (<5 microm) and as thick as 1000 microm. Week-old biofilms of three different species compositions displayed distinct structural features as indicated by their mean thicknesses and by a roughness coefficient. Monopopulation biofilms of P. aeruginosa (29 microm mean thickness) or K. pneumoniae (100 microm mean thickness) were thinner than the binary population biofilm (400 microm mean thickness). A roughness coefficient developed in this investigation corroborated the qualitative visual characterization of P. aeruginosa biofilms as relatively uniformly thick (mean roughness coefficient 0.15), K. pneumoniae biofilms as patchy (mean roughness coefficient 1.14), and the binary population biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum completely, significant areas of essentially bare substratum were apparent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilms may be due to the fact that this organism is nonmotile. A spatial correlation analysis of the thickness data indicated that thickness measurements were still correlated even when separated by distances that exceeded the mean biofilm thickness. Cell aggregates, some of them hundreds of microns in size, were observed in the effluent of K. pneumoniae and binary population biofilm reactors. Measurements of thickness variability and other observations reported in this article provide a quantitative basis for analysis of microscale structural heterogeneity of biofilms.