Spectrophotometric Quantification of Pigments in Anabaena Flos-Aquae: Investigation of Cell Disruption Methods

Chlorophyll a and phycobiliproteins are ubiquitous cyanobacterial secondary products of vast scientific and technological interest and their accurate measurement is necessary in many application fields. In this work, three different cell disruption methods of freeze-thawing, ball milling, and ultrasonication were extensively investigated for the extraction and spectrophotometric determination of Chlorophyll a and phycobiliproteins from the filamentous cyanobacterium Anabaena flos-aquae. By studying the effect of the duration of the last two treatments, a total of five different procedures were explored. While all methods were found to return statistically equivalent results for Chlorophyll a under the test conditions (4.078±0.256 to 4.706±0.201 µg/mL), ultrasonication and ball milling for longer durations outperformed others for phycobiliproteins extraction (with phycocyanin concentrations of 19.485 and 19.557 µg/mL, respectively). In addition, the interference of the overlapping Chlorophyll a peak during the phycobiliproteins quantification was analyzed and ultrasonication for extended duration was shown to offer not only a reasonably high yield but a low Chlorophyll a interference as well. Furthermore, common equation sets for the calculation of phycobiliprotein concentration from the spectral data were compared and potential discrepancies were highlighted. This study is believed to provide insights for future research regarding the extraction and spectrophotometric determination of cyanobacterial pigments.