ELF(R)97 phosphate (ELFP) is a phosphatase substrate which produces ELF(R)97 alcohol (ELFA), a fluorescent water-insoluble product, upon hydrolysis. We studied the kinetics of ELFA precipitation in freshwater samples at levels of total plankton and single phytoplankton cells, and tested the suitability of ELFP for measurement of surface-bound algal extracellular phosphatases. Samples from acidic Plesné Lake (pH approximately 5; high phosphatase activity) and eutrophic Rímov reservoir (pH approximately 7-10; moderate phosphatase activity) were incubated with ELFP for 5-300 min, fixed with HgCl2 and filtered through polycarbonate filters. Relative fluorescence of filter-retained ELFA precipitates was quantified with image analysis. Time-courses of ELFA formation exhibited lag periods followed by finite periods of linear increase. In Plesné Lake, lag-times were shorter (1-18 min) and rates of increase in ELFA fluorescence higher (by approximately 2 orders of magnitude) than in Rímov reservoir (lag-times 30-200 min). Similar patterns of ELFA formation kinetics were also observed in Plesné Lake samples in cuvette spectrofluorometer measurements (which failed in Rímov reservoir). Linear regression of seasonal data on rates of increase in ELFA fluorescence from image cytometry and spectrofluorometry (r2 = 0.65, n = 10) allowed for calibration of image cytometry in terms of amount of cell-associated ELFA. Preliminary measurements of extracellular phosphatase activities of several algae resulted in rates (10-2260 fmol cell-1 h-1) which are comparable to data reported in the literature for algal cultures.

A tropical planktic filamentous cyanobacterium, tentatively identified as Lyngbya robusta, recently increased in abundance in Lake Atitlán, Guatemala, and since 2008 annual water-blooms occurred. This was one from the first known cases of L. robusta water-blooms worldwide. A polyphasic evaluation of L. robusta using 16S rRNA gene sequencing, cytomorphological markers, and ecological characteristics was made. This species had several unique features. It produced aerotopes that were irregularly spaced in cells; cyanotoxins were not found, and it fixed nitrogen in spite of the lack of heterocytes. It contained a high amount of carotenoids, which caused an unusual brown color of the macroscopic scum on the water level. Molecular phylogenetic analyses using the 16S rRNA gene showed that L. robusta, together with few other planktic species, formed a clade, separated from typical Lyngbya species. The main diacritical markers of this clade were the planktic type of life and formation of gas vesicles in cells. Based upon molecular, morphological and ecological data, a new genus Limnoraphis was proposed with four species.

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 Ř ı´mov reservoir during three consecutive seasons. The new enzyme labelled fluorescence (ELF) technique was modified by introducing (i) fixation with HgCl 2 to preserve fragile species and (ii) use of polycarbonate filters to concentrate the phytoplankton. After enzymatic hydrolysis of artificial substrate (ELF 1 97 phosphate), the fluorescent product (ELF 1 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 4 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 clearwater 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.

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