Relevance of polymeric matrix enzymes during biofilm formation

Extracellular polymeric substances (EPS) contribute to biofilm stability and adhesion properties. The EPS matrix might also be a site for free extracellular enzyme activity; however, little is known about participation of enzyme activity in EPS during biofilm formation. In this study, we analyzed the activities of beta-glucosidase, leu-aminopeptidase, and beta-glucosaminidase during the colonization of artificial substrata (glass tiles) in a stream distinguishing enzyme activity in EPS matrix (matrix-enzymes) and total biofilm extracellular enzyme activity. The 1-h incubation of a biofilm suspension and cation-exchange resin followed by centrifugation seems appropriate to extract the matrix fraction (supernatant) and measure matrix enzymes (including free and linked to EPS) in freshwater biofilms, although there is a methodological limitation for using a biofilm suspension instead of an undisrupted biofilm. Total biofilm activities and matrix-enzyme activities showed similar capabilities to decompose organic matter compounds, with a greater capacity for peptide decomposition (leu-aminopeptidase) than for polysaccharides (beta-glucosidase), and a low decomposition of chitin and peptidoglycan (beta-glucosaminidase). Matrix-enzyme activity increased with colonization time, but more slowly than that of total enzyme activity. At the beginning of the colonization experiment (days 1-4) matrix enzymes accounted for 65-81% of total biofilm enzyme activity. Higher proportion of polysaccharides in EPS versus total biofilm, and higher matrix-enzyme activities per microgram of polysaccharides in the EPS were measured during the first 1-3 days of biofilm formation, indicating a high rate of enzyme release into the matrix during this period. Relative contribution of matrix-enzyme activities decreased as biofilm matures, but was maintained at 13-37% of total enzyme activity at the 42- to 49-day-old biofilm. These enzymes, retained and conserved in the EPS, may contribute to community metabolism. When analyzing extracellular enzymes in biofilms, the contribution of matrix enzymes must be considered, especially for young biofilms.