Seasonal study of extracellular phosphatase expression in the phytoplankton of a eutrophic reservoir

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Many phosphorus-deficient algae and cyanobacteria produce extracellular, mostly cell-attached, phosphatases, presumably to make ambient organically bound phosphorus available. The distribution of phosphatase activity among natural phytoplankton populations and its ecological significance is largely unknown. Bulk extracellular phosphatase activity of plankton (using a standard fluorometric assay) and expression of phosphatases at the level of single phytoplankton cells were studied in the eutrophic Římov reservoir during three consecutive seasons. The new enzyme labelled fluorescence (ELF) technique was modified by introducing (i) fixation with HgCl2 to preserve fragile species and (ii) use of polycarbonate filters to concentrate the phytoplankton. After enzymatic hydrolysis of artificial substrate (ELF®97 phosphate), the fluorescent product (ELF®97 alcohol, ELFA) formed insoluble precipitates at the site of phosphatase activity. Inhibition experiments suggested that both the standard assay and the ELF technique detected the same group of phosphatases. Weak ELFA formation and/or stability at pH > 8 might prevent sufficient detection of alkaline phosphatases using the ELF technique. ELFA labelling was detected in most algal classes, except for Euglenophyceae and the majority of Cryptophyceae and Chrysophyceae. Surprisingly, phosphatase activity was almost absent in the dominant populations. No ELFA labelling occurred in the phytoplankton in early spring 2000 and during the clear-water phases in all sampling years. Species of Cyanobacteria, Chlorophyceae and Conjugatophyceae showed phosphatase activity mainly in summer and at the beginning of autumn, while one species of Chrysophyceae (Synura petersenii) and three diatoms (Aulacoseira italica, Cyclotella spp., and Stephanodiscus hantzschii) produced phosphatases in spring. Several green algae (Ankyra ancora, Ankyra judayi, Coelastrum pseudomicroporum, Eudorina elegans, and Pediastrum spp.) were ELFA-labelled whenever present in the phytoplankton. Some species produced the ectoenzyme only in one season, such as Aphanizomenon flos-aquae (Cyanobacteria), Elakatothrix genevensis (Chlorophyceae), or Cryptomonas spp. On the other hand, one third of the 56 species studied never expressed any ELFA labelling. Large variability of phosphatase production was found not only among different algal species, but also within the population of one species. Not all cells of the population were equally ELFA-labelled and also the level of ELFA fluorescence was different. In particular cases, ELFA labelling on algal cells could be produced by bacterial rather than algal ectoenzymes. In comparison to standard methods, the ELF method provided more specific information about the variability of phosphatase activity (i.e. phosphorus stress) within the whole phytoplankton community as well as within one species populations.