Microbial enzymatic activity measurements by fl uorogenic substrates: Evidence of inducible enzymes in oligotrophic Mediterranean areas

Background: In aquatic environments, organic polymers such as proteins, polysaccharides and organic phosphates are cleaved and up taken by microorganisms through the expression of specifi c enzymes such as Leucine Aminopeptidase (LAP), beta Glucosidase (GLU) and Alkaline Phosphatase (AP), respectively. Microbial enzymatic activities are a fundamental step in the organic matter utilization and turnover into simple monomers. The context and purpose of the study: An experiment was carried out by combining the enzymatic assay using fluorogenic substrates with microscopical counts obtained using the viability marker 5-cyano-2,3 ditolyl-tetrazolium chloride (CTC). The main objective was to verify whether the enzyme activity played by microbial cells depends of the cell viability or rather whether the hydrolytic activity is directly stimulated by the availability of organic substrates for microbial metabolism. Results: This study provides evidence that in oligotrophic waters the fluorescence signal often decreases after incubation with the substrate analogue used for the enzymatic assay. For such samples, the extension of the incubation period from 2 to 5 hours has allowed the detection of a positive fl uorescence signal. Simultaneous counts of the abundance of CTC+ cells revealed that only a fraction close to 30% of the total bacterioplankton was actively respiring, suggesting that the observed increase was related to the presence of inducible enzymes rather than of actively metabolising cells. Main findings: Since the synthesis of hydrolytic enzymes by the microbial community can be induced by the presence of the organic substrates, adapting the incubation period to the trophic condition of the examined area is required for accurate enzymatic measurements, especially for oligotrophic environments. Conclusions: This is the fi rst contribution to link the enzyme activity rates with the viability properties (in terms of actively metabolising components) of bacterioplankton inhabiting pelagic Mediterranean waters. Brief summary: Evidence of inducible enzymes in oligotrophic Mediterranean areas was provided by the increase of fl uorescence recorded after incubation up to 5 hours with specifi c fl uorogenic substrates, in concomitance with the detection of a low fraction of actively respiring cells. While microbial cells were quite active for AP synthesis, LAP and GLU were mostly inducible enzymes, activated by the addition of their specifi c polymeric organic substrate. Any potential implications: In the examined oligotrophic waters the enzymatic activities seem to be stimulated mostly by the availability of metabolisable organic substrates.