AME 37:159-169 (2004)  -  doi:10.3354/ame037159

Flow cytometric detection of phosphatase activity combined with 13C-CO2 tracer-based growth rate assessment in phytoplankton populations from a shallow lake

Marco Dignum1,2,*, Hans L. Hoogveld1, Virgilio Floris1, Herman J. Gons1, Hans C. P. Matthijs2, Roel Pel1

1Netherlands Institute of Ecology (NIOO-KNAW), Centre for Limnology, Rijksstraatweg 6, 3631 AC Nieuwersluis, The Netherlands
2University of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics, Department of Aquatic Microbiology, Nieuwe Achtergracht 127, 1018 WS Amsterdam, The Netherlands

ABSTRACT: To determine nutrient availability and growth rates of phytoplankton on a population level, improvement of discriminative power in fluorescence-activated cell sorting is required. We have combined fluorescence of the endogenous photosynthetic pigments chlorophyll a and phycocyanin with a phosphate deficiency (P-deficiency) related stain (enzyme-labelled fluorescence, ELF-97) that yields green fluorescent precipitates at the site of phosphatase activity, referred to as ELF alcohol (ELFA) fluorescence, to sort phytoplankton from Lake Loosdrecht (The Netherlands). Stable isotope labelling with 13C-enriched CO2 enabled assessment of specific growth rates of sorted populations by pyrolytic methylation-gas chromatography and in-line compound specific isotope-ratio mass spectrometry. The dominant population in the lake, the filamentous cyanobacterium Limnothrix sp., was growing under P-deficiency in spring, but the availability of phosphate increased in summer. Continuous flow of phosphate-rich medium into a laboratory-scale enclosure of lake water resulted in washout of the cells with ELFA fluorescence, and increased growth rates. In addition, this study revealed population heterogeneity within the cluster of phycocyanin-containing cyanobacteria. ELFA fluorescence thus reflects the level of P-deficiency of freshwater cyanobacteria and micro-algae, but is modulated by metabolic activity of cells within the population.


KEY WORDS: Alkaline phosphatase · Enzyme-labelled fluorescence · Filamentous cyanobacteria · Fluorescence-activated cell sorting · Population-specific growth rate · Phytoplankton · Stable isotope labelling


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