Instrumental considerations for deriving spectral photosynthetic absorption coefficients from total phytoplankton absorption

Instrumental considerations for deriving spectral photosynthetic absorptioncoefficients from total phytoplankton absorptionMary E. Culver1, Richard F. Davis2 and Mary Jane Perry11School of Oceanography, WB- 10, University of Washington, Seattle, WA 981952College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, OR 97331ABSTRACTThe phytoplankton absorption coefficient is primarily due to absorption by photosynthetic andphotoprotective pigments. Absorption by photosynthetic pigments can be separated from that by otherpigments by the former's ability to induce chlorophyll a (chl a) fluorescence. Chl a fluoresces atwavelengths between 660 and 760 nm with a distinat peak at 683 nm and a broad shoulder near 730 nm.The use of Basic Blue 3 as a quantum counter in spectrofluorimetry allows measurement of chi afluorescence at 730 nm and hence, determination of phytoplankton fluorescence excitation spectra between380 and 700 nm. Extension of measurements beyond 600 nm, the limit of the conventional quantumcounter rhodamine B, allows direct comparison of the total absorption spectrum and the fluorescenceexcitation spectrum between 380 and 700 nm. Errors in the measurement of both types of spectra must beidentified and minimized for reliable interpretation of either spectrum. Factors of primary importanceinclude instrument-specific correction spectra for spectrofluorimeter configurations, accuracy in theabsorption measurement, and sample handling. Accurate separation of spectral phytoplankton absorptioncoefficients into photoprotective and photosynthetic components will reduce the variability and errors inestimates of primary production based on optical models and will provide information on thephotoadaptive state of the cells.2. INTRODUCTIONThe phytoplankton absorption coefficient is an inherent optical property, and its spectral shape andmagnitude affects the diffuse attenuation coefficient, reflectance, and bio-optically modeled estimates ofprimary productivity. Particle-specific,1 chemical,2 and statistical3'4 methods that separate totalphytoplankton absorption from particle absorption have been developed. Two classes of pigments areresponsible for phytoplankton absorption: photosynthetic and photoprotective pigments. Thephytoplankton absorption coefficient measured spectrophotometrically is suitable for models of lightattenuation but overestimates absorption when used in productivity models, particularly in surface waters.The fluorescence excitation spectrum has been proposed as an alternative to improve estimates ofphotosynthetic phytoplankton absorption for modeling primary production.5To generate a fluorescence excitation spectrum, a whole water sample is illuminated across thevisible wavelengths while the emission of chlorophyll a (chl a) at 730 nm is monitored. This wavelengthis a secondary emission peak for Photosystem II (P511) chi a. The excitation spectrum represents thepigments involved in the transfer of energy from light-harvesting photosynthetic pigments to P511 chi areaction centers.6 Absorption near 675 nm is dominated by chl a with minimal contributions byphotoprotective or residual detritus pigments. The fluorescence excitation spectrum is normalized to theabsorption at 675 nm allowing quantification of pigment absorption associated with PSII and, therefore,with photosynthesis.5' 7,8Quantification of a fluorescence excitation spectrum depends on the use of a quantum counter tomonitor variations in the intensity of the excitation irradiance during the course of a measurement.Previously, rhodamine B was used, and the excitation wavelengths were limited to those less than 600 nmbecause absorption by rhodamine B is minimal beyond this wavelength. The quantum counter 2,7-Bis(diethylamino)pheno-azoxonium chloride (Basic Blue 3) is effective for excitation wavelengths ranging