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Simultaneous Extraction and Measurement of Chlorophyll and Phycocyanin from Cyanobacteria; Modification of Phycocyanin denaturation by Octanol

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 new methodology under investigation involves extraction with a water-octanol biphasic system that concentrates chlorophyll into an upper octanol layer and the phycocyanin into an aqueous layer. A significant problem with octanol is its denaturation of phycocyanin with extended extraction times. This report shows the quantification of this denaturation under different conditions: 1) the concentration of the material extracted; 2) the efficiency of extraction; and 3) the introduction of agents into the octanol-water biphasic extraction system. Preliminary results show that 3M fructose in the aqueous layer, replacing the pure water in the water-octanol system, significantly reduces the denaturation effect of the octanol. However, it may reduce the efficiency of extraction. 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 (BioSpec Products) 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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