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

摘要

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-alpha ratioof four N_2-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-alpha concentrations for all strains at high Chl-alpha concentrations (almost stable PC/Chl-alpha ratio in the range of20-100 ug 1~(-1) Chl-alpha). Extraction of PC and Chl-alpha fromsamples with low biomass of cyanobacteria (less than 20 ug 1~(-1) Chl-alpha) 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 homo-genization 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.