Local macromolecule diffusion coefficients in structurally non-uniform bacterial biofilms using fluorescence recovery after photobleaching (FRAP).

Pure culture Pseudomonas putida biofilms were cultivated under controlled conditions to a desired overall biofilm thickness, then employed within classical half-cell diffusion chambers to estimate, from transient solute concentrations, the effective diffusion coefficient for several macromolecules of increasing molecular weight and molecular complexity. Results of traditional half-cell studies were found to be erroneous due to the existence of microscopic water channels or crevasses that perforate the polysaccharidic gel matrix of the biofilm, sometimes completely to the supporting substratum. Thus, half-cell devices measure a composite transfer coefficient that may overestimate the true, local flux of solutes in the biofilm polysaccharide gel matrix. An alternative analytical technique was refined to determine the local diffusion coefficients on a micro-scale to avoid the errors created by the biofilm architectural irregularities. This technique is based upon the Fluorescence Return After Photobleaching (FRAP), which allows image analysis observation of the transport of fluorescently labeled macromolecules as they migrate into a micro-scale photobleached zone. The technique can be computerized and allows one to map the local diffusion coefficients of various solute molecules at different horizontal planes and depths in a biofilm. These mappings also indirectly indicate the distribution of water channels in the biofilm, which was corroborated independently by direct microscopic observation of the settling of fluorescently-labeled latex spheres within the biofilm. Fluorescence return after photobleaching results indicate a significant reduction in the solute transport coefficients in biofilm polymer gel vs. the same value in water, with the reduction being dependent on solute molecule size and shape.