Modified enzyme activity assay to determine biofilm biomass

An assay of potential exoproteolytic enzyme activity was modified to quantitatively measure the biomass of attached biofilm. The assay utilized the nonfluorescent compound L-leucine-beta-naphthylamide (LLbetaN) that becomes fluorescent when bacterial exoenzymes break the peptide bond, releasing the fluorochrome beta-naphthylamine. Fluorescence development was measured by pumping the liquid phase of a biofilm sample through a fluorescence detector and recording the detector output using a personal computer. A significant linear relationship was shown to exist between the rate of fluorescence development and the biofilm's biomass as carbon, determined using total direct cell counts, measured cell volumes and an existing relationship between cell volume and cell carbon. The technique was used to measure biofilm biomass for experiments where iron oxides were the substratum. Biofilm biomass measurements made using heterotrophic plate counts (HPCs) on R2A medium were shown to correlate well to biomass measurements made using the modified enzyme assay. The technique was shown to be sufficiently sensitive to measure biomass on samples containing little biofilm biomass, such as those exposed to free chlorine. While granular and porous media were used for the experiments presented, small biofilm coupons could easily be used to measure biofilm biomass, expanding the number of possible applications for the enzyme assay technique.