An aerobic biofilm was characterized using confocal scanning laser microscopy (CSLM), O2 micro-electrodes, particle tracking and microinjection of fluorescent dyes. The biofilms were found to consist of microbial clusters of cells and Extra-cellular Polymeric Substance (EPS) separated by interstitial voids. The cell clusters were ca 300 μm and the voids were ca 100 μm wide. The voids were open channels connected with the bulk fluid. Fluorescein micro-injection showed that liquid could flow through the voids, but was always stagnant in the cell clusters. Consequently, in voids both diffusion and convection may contribute to mass transfer, while in cell clusters transport is determined by diffusion only. Particle tracking with CSLM showed that flow velocity inside the biofilm was proportional to the bulk flow velocity. The importance of convective mass transport in biofilms was demonstrated by oxygen distribution measurements. At high flow velocities of the bulk liquid, the mass boundary layer followed the irregular biofilm surface. At lower velocities the mass boundary layer was parallel to the substratum. Mass transfer from voids to cell clusters increased with flow velocity, as result from vonvective mass transport from the bulk to the voids. Convective transport was insignificant at low flow velocities, but at high flow velocities it increased the total mass transport by 200-250%. The local diffusion coefficients in biofilms were measured using microinjection of fluorescent dyes and quantitative analysis of the subsequent plume formation using CSLM. The diffusion coefficient of small, non-binding molecules in cell clusters is close to that in water. Very large molecules were impeded in their diffusion through the biofilm matrix. It was calculated that the cell cluster matrix had the characteristics of a gel network with pore diameters of 80 nm.

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