Extraction methods for phycocyanin determination in freshwater filamentous cyanobacteria and their application in a shallow lake

Abstract

Phycocyanin (PC) is one of the water-soluble accessory pigments of cyanobacteria species, and its concentration in aquatic systems is used to estimate the presence and relative abundance of blue-green algae. PC concentration and the PC/Chl-a ratio of four N2-fixing filamentous cyanobacteria strains (Cylindrospermopsis raciborskii, Anabaena spiroides, Aphanizomenon flos-aquae and Aphanizomenon issatschenkoi) common to Lake Balaton (Hungary) were determined using repeated freezing and thawing. A strong linear correlation was found between the extracted PC and Chl-a concentrations for all strains at high Chl-a concentrations (almost stable PC/Chl-a ratio in the range of 20−100 µg l−1 Chl-a). Extraction of PC and Chl-a from samples with low biomass of cyanobacteria (less than 20 µg l−1 Chl-a) proved to be unreliable using the standard protocol of freeze–thaw cycles (coefficients of variation exceeding 10–15%). In order to find an extraction method that is robust in fresh waters characterized by low algae biomass (e.g. Lake Balaton), the effectiveness of four extraction methods (repeated freeze–thaw method and homogenization with mortar and pestle, Ultrasonic, and Polytron homogenizer) were compared using C. raciborskii. It was found that the efficiency of extraction of phycocyanin was highest when a single freeze–thaw cycle was followed by sonication (25% additional yield compared with using the freeze–thaw method alone). Applying this combined method to surface water samples of Lake Balaton, a strong correlation was found between PC concentration and cyanobacterial biomass (R 2 = 0.9436), whilst the repeated freezing–thawing method found no detectable PC content. Here we show that the combined sonication/freeze–thaw method could be suitable for measuring filamentous cyanobacteria PC content, even at low concentrations; as well as for the estimation of cyanobacterial contribution to total biomass in fresh waters.