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STABILITY OF CHLOROPHYLL AND PHYCOCYANINE EXTRACTED FROM CYANOBACTERIA WITH OCTANOL:WATER

Abstract

Chlorophyll and phycocyanine are photosynthetic pigments used as surrogates for the monitoring of algae and cyanobacteria, respectively, in environmental water bodies. Cyanobacteria are an important contributor to harmful algal blooms, a growing threat to water bodies worldwide. Currently, these pigments are extracted and measured separately, as the hydrophobic chlorophyll is extracted with organic solvents, while the hydrophilic phycocyanine is extracted with an aqueous-based methodology. A significant problem with these technologies, especially chlorophyll extraction, is the stability of the pigments. It is recommended that chlorophyll be extracted and measured within at least 24 hours, and sooner is better. Simultaneous extraction of chlorophyll and phycocyanine from the cyanobacterium Microcystis aeruginosa has been reported previously. This is a biphasic extraction system where cells are lysed by the presence of octanol, and the cell contents are released into either the upper hydrophobic octanol phase or the lower aqueous phase. Here we show that under the conditions of extraction both chlorophyll (in the octanol phase) and phycocyanine are stable, as determined by absorbance spectra, for at least 3 days while stored separately in the dark at room temperature. In addition, since the earlier report of this methodology, miniaturization of the procedure has been made in order to accommodate high through-put monitoring. Miniaturization included vigorous homogenization of samples in a 2 mL centrifuge tube with a Mini Bead-Beater and reading 0.1 mL aliquots of each phase placed separately into wells of a 384-well microplate. Absorbance spectra were read by a BioTek H1 Synergy plate reader. However, the broader applicability of this methodology to other cyanobacterial species as well as to algal species other than cyanobacteria remains to be determined. This work is supported by the East Georgia State College Baccalaureate Biology Program.

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