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, 26 (5), 2065-2074

Quantitative Analysis of Extracted Phycobilin Pigments in Cyanobacteria-An Assessment of Spectrophotometric and Spectrofluorometric Methods

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Quantitative Analysis of Extracted Phycobilin Pigments in Cyanobacteria-An Assessment of Spectrophotometric and Spectrofluorometric Methods

Monika Sobiechowska-Sasim et al. J Appl Phycol.

Abstract

Phycobilins are an important group of pigments that through complementary chromatic adaptation optimize the light-harvesting process in phytoplankton cells, exhibiting great potential as cyanobacteria species biomarkers. In their extracted form, concentrations of these water-soluble molecules are not easily determined using the chromatographic methods well suited to solvent-soluble pigments. Insights regarding the quantitative spectroscopic analysis of extracted phycobilins also remain limited. Here, we present an in-depth study of two methods that utilize the spectral properties of phycobilins in aqueous extracts. The technical work was carried out using high-purity standards of phycocyanin, phycoerythrin, and allophycocyanin. Calibration parameters for the spectrofluorometer and spectrophotometer were established. This analysis indicated the possibility of detecting pigments in concentrations ranging from 0.001 to 10 μg cm-3. Fluorescence data revealed a reproducibility of 95 %. The differences in detection limits between the two methods enable the presence of phycobilins to be investigated and their amounts to be monitored from oligotrophic to eutrophic aquatic environments.

Keywords: Absorbance; Calibration; Environmental monitoring; Fluorescence; Phycobilin concentration.

Figures

Fig. 1
Fig. 1
Examples of absorption (a) and fluorescence (b) spectra of phycobilin pigment standards obtained for known pigment standard concentrations. Fluorescence emission spectra of PC obtained for excitation at 590 nm, APC (600 nm), and PE (530 nm) at a lamp intensity set to 800. The marked wavelengths correspond to the maximum values of a absorbance and b emission
Fig. 2
Fig. 2
Calibration curves of phycobiliprotein standards: phycocyanin (diamonds), allophycocyanin (squares), and phycoerythrin (triangles) obtained for a spectroscopic measurements (Spectrophotometer UV/Vis, Hitachi U-2800) and bd spectrofluorometric measurements, Fluorescence Spectrophotometer (Cary Eclipse, Varian, Agilent Technologies) for different lamp intensity settings: b 600 V, c 800 V, and d 1000 V. The characteristic wavelengths for which instruments gave the illustrated responses are specified in Table 3
Fig. 3
Fig. 3
Relative standard deviation (RSD) calculated for absorbance and fluorescence data for selected dilution series of phycobilin standards
Fig. 4
Fig. 4
Series of fluorescence emission values obtained at λem = 644 nm for a standard solution of PC (concentration 0.1 μg cm−3). The fluorescence spectrometer was set to different incident light intensities (λex = 590 nm): 600, 800, and 1000 V. Solid line mean value for a sequence, dotted line range of divergence from the mean value
Fig. 5
Fig. 5
Average temporal changes in a absorbance and b fluorescence response values (lamp settings 600 V) for PC (n = 3). Fresh means measured 2 h after the solutions for extraction were prepared
Fig. 6
Fig. 6
Emission spectra obtained for selected monoculture extracts. a Microcystis aeruginosa. b Synechococcus sp.
Fig. 7
Fig. 7
Phycocyanin concentration (μg cm−3) and relative PC/chl a ratios determined for cyanobacteria monoculture extracts. Comparison of data assessed using two analytical techniques: spectrophotometry (blue) and spectrofluorometry (red)
Fig. 8
Fig. 8
Emission spectra obtained for a PE-rich cyanobacteria monoculture

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