LOCAL MACROMOLECULE DIFFUSION-COEFFICIENTS IN STRUCTURALLY NONUNIFORMBACTERIAL BIOFILMS USING FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING (FRAP)

Pure culture Pseudomonas putida biofilms were cultivated under control led conditions to a desired overall biofilm thickness, then employed w ithin classical half-cell diffusion chambers to estimate, from transie nt solute concentrations, the effective diffusion coefficient for seve ral macromolecules of increasing molecular weight and molecular comple xity. Results of traditional half-cell studies were found to be errone ous due to the existence of microscopic water channels or crevasses th at perforate the polysaccharidic gel matrix of the biofilm, sometimes completely to the supporting substratum. Thus, half-cell devices measu re a composite transfer coefficient that may overestimate the true, lo cal flux of solutes in the biofilm polysaccharide gel matrix. An alter native analytical technique was refined to determine the local diffusi on coefficients on a micro-scale to avoid the errors created by the bi ofilm architectural irregularities. This technique is based upon the F luorescence Return After Photobleaching (FRAP), which allows image ana lysis observation of the transport of fluorescently labeled macromolec ules as they migrate into a micro-scale photobleached zone. The techni que can be computerized and allows one to map the local diffusion coef ficients of various solute molecules at different horizontal planes an d depths in a biofilm. These mappings also indirectly indicate the dis tribution of water channels in the biofilm, which was corroborated ind ependently by direct microscopic observation of the settling of fluore scently-labeled latex spheres within the biofilm. Fluorescence return after photobleaching results indicate a significant reduction in the s olute transport coefficients in biofilm polymer gel vs. the same value in water, with the reduction being dependent on solute molecule size and shape. (C) 1998 John Wiley & Sons, Inc.