MEASUREMENT OF PHYTOPLANKTON PHOTOSYNTHESIS RATE USING A PUMP-AND-PROBE FLUOROMETER

T.K. Antal, P.S. Venediktov, D.N. Matorin, M. Ostrowska*, B. Wozniak*, A.B. Rubin

Moscow Lomonosov State University, Department of Biology

*Institute of Oceanology PAS, Sopot, Poland

 

ABSTRACT

In this work we have studied the possibility to determine the rate of phytoplankton photosynthesis in situ using a submersible pump-and-probe fluorometer in water areas differing in their trophic level, as well as climatic and hydrophysical characteristics. A biophysical model was used to describe the relationship between photosynthesis, underwater radiation, and the intensity of phytoplankton fluorescence excited by an artificial light source. Fluorescence intensity was used as a measure of light absorption by phytoplankton and for assessment of the efficiency of photochemical energy conversion in photosynthetic reaction centers. Parameters of the model that could not be measured experimentally were determined by calibrating fluorescence and radiation data against the primary production measured in the Baltic Sea by radioactive carbon method. It was shown that standard deviation of these parameters in situ did not exceed 20%, and the use of their mean values to estimate phytoplankton photosynthetic rate showed a good correlation between the calculated and measured data on primary production in the Baltic (r=0.89), Norwegian (r=0.77) and South-China (r=0.76) Seas.

 

INTRODUCTION

Photosynthesis of microalgae can be measured as the rate of radioactive carbon assimilation (Steemann Nielsen,1952) or as an increase in the concentration of soluble oxygen in a sample (Williams, 1982; Langdon, 1984). These methods are rather labor-consuming, and their application involves numerous artifacts due to prolonged isolation of phytoplankton in bottles (Eppley, 1980), difference between net and gross photosynthesis (Bender et al., 1987), and metal toxicity (Fitzwater et al., 1982). The application of chlorophyll fluorescence methods avoids these problems and allows gross photosynthesis of microalgae to be continuously measured in real time without affecting their physiological state (Kolber et al., 1990; Green et al., 1992). The relationship between chlorophyll a (C_a) fluorescence and photosynthesis is described in a number of biophysical models of the primary processes of photosynthesis (Weis and Berry, 1987; Genty et al.,1989; Kiefer and Reynolds, 1992). The model of carbon assimilation V_c (mM C m^-3 s^-1) by phytoplankton, which was used in our work, is based on light dependence of photosynthesis (Jassby and Platt, 1976), which can be described by a coefficient of solar radiation absorption by photosynthetic pigments in suspension of microalga (a_PSP)_s (m^-1) averaged over the spectral range 400-700 nm, where PSP stands for photosynthetic pigments (Dubinsky et al., 1986), and the efficiency of the conversion of absorbed energy in photosynthetic reactions, f (mM C mE^-1). According this assumption the photosynthesis rate is equal to:

 

V_c(I) = (a_PSP)_S*f(I)*I (1)

 

where I is the total radiation (mE m^-2 s^-1).

The value of f is proportional to the relative number of functionally active (Ѓ), open (q_P) reaction centers PS II in algal cells, to the efficiency of photochemical conversion of light energy in open reaction centers (f_RC, mM electron mE^-1), and to the efficiency of electron transfer from H₂O to CO₂ (f_e, mM C (mM electron)^-1):

 

V_c(I) = (a_PSP)_S*Ѓ*q_P(I)* f_RC *f_e *I (2)

 

The parameters a_PSP and f*f_RC were determined by measuring fluorescence parameters Fo and F_v/F_m by pump-and-probe method (Mauzerall, 1972; Kolber et al., 1990) in phytoplankton adapted to ambient light. Parameters that could not be measured directly (by pump-and-probe method) were determined by substituting photosynthetic rate measured by radiocarbon method for V_c in formula (2) or by measuring light absorption by algae - a_psp. In this work, we investigated variation of these indirectly measured parameters in the Baltic Sea. The possibility of application of the mean values of these parameters to determine the primary production of microalgae in the Baltic, Norwegian, and South China Seas was also studied.

 

METHODS

 

Structure of the model

Determination of (a_PSP)_S, f, and f_RC from phytoplankton fluorescence characteristics

The intensity of fluorescence excited by an artificial light source, with open reaction centers (RC) in algae, can be found from the equation:

 

F₀ = G*I_fl*(a_PSP)_fl*f_Fo (3)

 

where I_fl is the intensity of exciting flash (in our fluorometer, I_fl(l) was nearly uniformly distributed over spectral range 400-550 nm), a constant; (a_PSP)_fl is the coefficient of exciting flash absorption by PSP of PS II in algal suspension, averaged over spectral range 400-550 nm; f_Fo is the quantum yield of fluorescence in cells with open RC; G is a coefficient defined by geometric characteristics and sensitivity of the fluorescence light sensor, a constant.

Taking into account that (G*I_fl)^-1 = const, the coefficient of solar radiation absorption by PS II of microalgae can be related to fluorescence intensity as follows:

 

(a_PSP)s = const*