Local mass transfer coefficients in bacterial biofilms using fluorescence recovery after photobleaching (frap)

Abstract

Pure culture Pseudomonas putida biofilms were cultivated under controlled conditions to desired overall biofilm thicknesses. Said biofilms were placed within half-cell diffusion chambers to estimate, from transient solute concentrations in each chamber, the effective diffusion coefficient for severed macromolecules of increasing molecular weight and molecular complexity. Results of the 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 an average transfer coefficient that overestimates the true, local flux of solutes in the biofilm alginate gel matrix. An alternative analytical technique was refined to determine the local diffusion coefficients on a micro-scale in order to avoid the errors created by the water channels. This technique is based upon the Fluorescence Return After Photobleaching (FRAP) which allows image analysis observation of the transport of fluorescently labeled molecules as they migrate into a micro-scale photobleached zone. The technique allowed us to map the local diffusion coefficients of various solute molecules at different horizontal planes and depths in a biofilm. These maps also indirectly indicate the distribution of water channels in the biofilm. FRAP results illustrate a significant reduction in macromolecule transport coefficients in biofilm polymer gel versus the same value in water, with the reduction being dependent on solute molecule size and shape.