Spatial patterns of ectoenzymatic kinetics in relation to biogeochemical properties in the Mediterranean Sea and the concentration of the fluorogenic substrate used

Abstract. Ectoenzymatic activity, prokaryotic heterotrophic abundances and production were determined in the Mediterranean Sea. Sampling was carried out in the sub-surface, the deep chlorophyll maximum layer (DCM), the core of the Levantine intermediate waters and in the deeper part of the mesopelagic layers. Michaelis–Menten kinetics were assessed using a large range of concentrations of fluorogenic substrates (0.025 to 50  µ M). As a consequence, Km (Michaelis–Menten half-saturation constant) and Vm (maximum hydrolysis velocity) parameters were determined for both low- and high-affinity enzymes for alkaline phosphatase, aminopeptidase (LAP) and β -glucosidase ( β GLU). Based on the constant derived from the high-LAP-affinity enzyme (0.025–1  µ M substrate concentration range), in situ hydrolysis of N proteins contributed 48 %  ±  30 % to the heterotrophic bacterial nitrogen demand within the epipelagic layers and 180 %  ±  154 % in the Levantine intermediate waters and the upper part of the mesopelagic layers. The LAP hydrolysis rate was higher than bacterial N demand only within the deeper layer and only when considering the high-affinity enzyme. Based on a 10 % bacterial growth efficiency, the cumulative hydrolysis rates of C proteins and C polysaccharides contributed on average 2.5 %  ±  1.3  % to the heterotrophic bacterial carbon demand in the epipelagic layers sampled (sub-surface and DCM). This study clearly reveals potential biases in current and past interpretations of the kinetic parameters for the three enzymes tested based on the fluorogenic-substrate concentration used. In particular, the LAP  / β GLU enzymatic ratios and some of the depth-related trends differed between the use of high and low concentrations of fluorogenic substrates.