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
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.
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 (Ca) 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 Vc (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 (aPSP)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:
Vc(I) = (aPSP)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 (¦), open (qP) reaction centers PS II in algal cells, to the efficiency of photochemical conversion of light energy in open reaction centers (fRC, mM electron mE-1), and to the efficiency of electron transfer from H2O to CO2 (fe, mM C (mM electron)-1):
Vc(I) = (aPSP)S*¦*qP(I)* fRC *fe *I (2)
The parameters aPSP and f*fRC were determined by measuring fluorescence parameters Fo and Fv/Fm 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 Vc in formula (2) or by measuring light absorption by algae - apsp. 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.
Structure of the model
Determination of (aPSP)S, f, and fRC 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*Ifl*(aPSP)fl*fFo (3)
where Ifl is the intensity of exciting flash (in our fluorometer, Ifl(l) was nearly uniformly distributed over spectral range 400-550 nm), a constant; (aPSP)fl is the coefficient of exciting flash absorption by PSP of PS II in algal suspension, averaged over spectral range 400-550 nm; fFo 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*Ifl)-1 = const, the coefficient of solar radiation absorption by PS II of microalgae can be related to fluorescence intensity as follows:
(aPSP)s = const*fFo-1*E*F0 = k(fFo, E)*F0 (4)
where E = (aPSP)S/(aPSP)fl; k is a proportionality coefficient, which depends on fFo and E.
The photochemical efficiency of open reaction center of PS II can be determined from the ratio of fluorescence parameters: fRC » (Fm-F0)/Fm=Fv/Fm (Klughammer, 1992). It was shown that the decrease in the Fv/Fm ratio corresponds to the decrease in fraction of functioning PS II reaction centers (¦) (Kolber 1988, 90), a process which is induced by excessive irradiation (Vasiliev et al., 1994; Long et al., 1994) (photoinhibition) and/or limitation of phytoplankton growth by mineral nutrients (Green et al., 1992; Falkowski et al., 1989). Thus, parameters fRC and ¦ are proportional to the relative yield of variable fluorescence of chlorophyll in microalgae adapted to natural radiation, so we assume:
Determination of parameters qp and fe
It is known that photochemical conversion of light energy in PS II takes place only in open reaction centers. The relative concentration of open centers qp can be found from the model of light dependent transition of reaction centers between the open and closed states. We used model proposed by Kiefer and Mitchell, (1983):
qp(I) = I1/2/(I+I1/2) (6)
where I1/2 is light intensity, at which half of the RC are in closed state.
The value of fe was estimated from the following considerations. To reduce one molecule of CO2, 4 electrons should be transferred from H2O, so, theoretical fe may be as high as 0.25, however, a fraction of electron flow is used for nitrate and sulfate reduction (Dubinsky et al., 1986; Laws, 1991), for cyclic electron transport around PS I (Slovacek et al., 1980; Myers, 1987) and PS II (Falkowski et al., 1986a), as well as for O2 reduction (Chemeris, 96). Comparison of fe with the maximum quantum yield of carbon fixation allows to assume that fe is approximately constant (Kiefer et al., 1989; Morel, 1991) and is not over 0,16 for natural phytoplankton (Bannister and Wiedmann, 1984). Thus, we assume that fe =0,16.
By substituting 4, 5, 6, in 2 and introducing the coefficient 6.9 = 12*10-3 (mgC (mM C)-1) *3600 (s h-1)*fe, the equation for vertical profile of algae photosynthesis rate (mgC m-3 h-1) can be written as follows:
Vc(z) = 6.9*k(z)*F0(z)*Fv/Fm(z)*I1/2/(I(z)+I1/2) *I(z) (7)
where z is depth (m).
Estimation of k and I1/2.
The unknown parameters k and I1/2 were found by comparing the primary production of phytoplankton Pc (mgC m-3 time-1) measured by radiocarbon method with fluorescence and irradiation data according to the formula:
Pc(z) = 6.9*(km*F0(z)*Fv/Fm(z)* I1/2m/(I(z)+I1/2m) *I(z)*Dt)i (8)
where n is the number of fluorescence and radiation profiles measured for the period of bottle exposure at a station; Dt is the time period between these measurements (h); km and I1/2m are values of parameters k and I1/2, respectively, averaged in the water column. They were calculated by approximating the Pc versus z dependence with equation 8 by the method of least squares.
Parameter k was also estimated under laboratory conditions by calibrating Fo against the coefficient of exciting flash absorption with suspension of microalgal cells afl (m-1) taken at a natural concentration (Ca=0.1-10 mg m-3). Spectral distribution of light absorption by cells was similar to fluorescence excitation spectra, hence, afl = (aPSP)fl. Parameter k was determined from formula (4) for E = 1. The value of afl was measured with a laboratory instrument. Light from a KGM 150/24 halogen lamp of a slide projector passed through SZS22 glass filter and a dark chamber 0.2 m in length filled with sample, and the output quantum flux density was measured with a laboratory made quantum sensor. Calculations were made from formula: afl = 5*(In,c-In)/In,c, where In is the intensity of light passed through suspension of microalgae with concentration n; In,c is the same for suspension of the algal cells bleached by illumination in the presence of 1 mM hydroxylamine.
For laboratory experiments, sea algae were grown on Goldberg medium prepared with artificial sea water in bottles at constant temperature in light (Lanskaya, 1971).
The vertical distribution of radiation, fluorescence, primary production of phytoplankton, and chlorophyll concentration were measured in the Bay of Nhatrang of the South China Sea (12o09’-12o18’N, 109o12’-109o20’E) and during cruises in the Baltic (13o10’-25o15’N, 53o25’-58o10’E) and Norwegian Seas (64o15’-70o20’N, 4o40’W-4o30’E):
1. June-July 1993 - the cruise of the r/v "Humbolt", according to the program "Plankton", presented are data of measurements at 7 stations near the Southern and Eastern coasts of the Baltic Sea;
2, 3, 4, 5 - May 1993, September 1993, May 1994 and September 1995, respectively, - the cruises of r/v Oceania, Institute of Oceanology of the Polish AS, presented are data of measurements at 16 stations in central and coast waters of the Baltic Sea;
6. June-July 1997 - cruise of r/v A. Petrov of VNIIRO of the Russian AS, presented are data of the measurements at 13 stations in central deep-aquatic areas of the Norwegian Sea;
7. March 1998 - 8 measurements were made at 8 stations in the Bay of Nhatrang of the South China Sea.
Vertical profiles of in situ fluorescence were registered with a "PrimProd" submersible pump-and-probe fluorometer designed at Biophysical Department of the Faculty of Biology of Lomonosov Moscow State University. The instrument also recorded quantum flux density in PAR region (mE m-2 s-1), temperature, and depth. The fluorometer generates sequential pump and probe flashes at a frequency of 2 Hz. The saturating (pump) flash of 1 J/0.01 ms power per duration was given 1 s after the first probe flash (0.01J/0.01 ms), and the second probe flash follows after pump flash via 50 ms. The impulses were generated by an SSh-20 (MELZ, Russia) xenon lamp. The flashes are isolated from the sample by the light blue-green filter SZS-22. The spectrum of the fluorescence excitation is distributed practically evenly within the range of wavelengths from 400 to 520 nm.
During probe submersion, external water gets passively into an open dark chamber in which fluorescence of phytoplankton cells, adapted to underwater radiation is measured within 0.5 s. The probe submersion rate was 0.3-0.5 m s-1, which allowed for resolution depth profiles.
First probing flash measures F0, fluorescence intensity with open centers of PS II. Subsequent saturating flash converts most of RC in closed state, and the second probing flash, which is given within 50 ms, a time comparable to the time reaction center turnover, measures fluorescence, which corresponds to level I1 of fluorescence saturation (Schreiber et al., 1995). Fm is calculated according to the formula: Fm = 1.4*I1, where 1.4 = Fm,DCMU/I1 is the ratio of the maximum fluorescence obtained in the presence of DCMU, an inhibitor of electron transport in PS II, to fluorescence yield measured by the PrimProd.
Fluorescence signal is recorded by photomultiplier-68 after passing through a KS-17 cut-off glass filter.
The recorded signals of fluorescence as well as underwater irradiation, temperature and pressure (depth) are transmitted in real time via a cable-rope connected to a personal computer.
The primary production of phytoplankton was measured in the Baltic Sea by radiocarbon method at 5-10 horizons down to a depth of 30 m using a routine method (Steemann Nielsen, 1952), and its modification in the Norwegian Sea (Sorokin, 1960) and by the oxygen method in the Bay of Nhatrang and Norvegian Sea (Vinberg, 1969). During measurement by radiocarbon method, bottles were exposed for 6 hours during the 1st cruise, for 4 hours during the 2nd, 3d, 4th cruises, for 2 hours during the 5th cruise, and for 6 hours during the 6th cruise.
The content of chlorophyll a was determined by a standard spectrophotometric method (Bender et al., 1987).
To determine the rate of phytoplankton photosynthesis according to formula 7, it is necessary to estimate unknown quantities k(fFo, E) and I1/2 and their variability in the regions studied. As follows from experiments (Dera, 1995; Ernst et al., 1986; Wozniak et al., 1997), photosynthetic parameters fFo and I1/2 change under stress action of abiotic factors. In natural phytoplankton, according to Ostrowska et al. (2000a and b) the parameter fFo did not significantly depend on environmental factors while parameter I1/2 changes mainly with temperature of water body (Antoine and Morel, 1996; Dera, 1995; Morel, 1991; Wozniak et al., 1997). We suppose that they are nearly constant in regions with similar temperature (Wozniak et al., 1992). The mean values of k and I1/2 in the water column: km and I1/2m accordingly, were calculated (formula 8) at 23 stations of the Baltic Sea in the central and coastal areas (from the Bay of Riga to Pomorsky Bay), where the average concentration of Ca in water column varied from 0.7 to 10 mg m-3. The data are given in Table 1.
Variation of km between stations of Baltic sea
The mean value of this parameter was 5.6*10-5 (standard deviation SD = +-17%) at stations of the Baltic Sea.
The k variation can be related to the factor E = (aPSP)S/(aPSP)fl (see formula 4), which is induced by the differences in blue light and solar radiation absorption by sea algae. The value of E depends, mainly, on taxonomic composition of microalgae and phisiological condition. For example, E calculated in vivo from absorption spectra (as shown in Fig. 1A) for three taxonomic groups: diatomea Phaeodactylum tricornutum, yellow-green Nephrochloris salina, and green Platimonas virdis, grown under optimum conditions and at low irradiation, elevated temperatures, or nitrogen deficiency, varied from 0.6 to 0.75. For a samples of natural phytoplankton from Baltic sea, E = 0.74. Since spectral distribution of light absorbed by these cell cultures corresponded to fluorescence excitation spectra, it can be supposed that a(l) = aPSP(l). The experimental value of E was <1, due is to the fact that, usually, the absorption coefficient of sea algae for blue light is much higher than their absorption coefficient averaged over the PAR region: (aPSP)fl > (aPSP)S. Thus, it can be expected that, in the first approximation, in the upper water layers, where irradiation spectrum is close to that of solar radiation, the values E for natural phytoplankton should vary from 0.6 to 0.75.
Changes in the spectrum of underwater irradiation with depth are accompanied by changes in E: E(z) = E(0)*j(z), where E(0) = (aPSP)S/(aPSP)fl is E value at water surface and j(d) is depth dependence of E. In clear water, attenuation of red light with depth must leads to an increase in E(z) from 0.6-0.7 at the surface to 1 at 20 m and at greater depths, where spectra of probing flash and underwater radiation are similar (Fig. 1B). Thus, it can be expected that mean values of E in water column, which affect km, should be higher than 0.7 and vary to a lesser extent than at the surface.
For such mixed waters as Baltic, taxonomic composition of phytoplankton within the euphotic zone can be assumed to be uniform, and at low irradiation level (I(0)<I1/2), fFo(z) is a constant, therefore, E(z) = const*Ca(z)/F0(z). We estimated E at a depth of 1 m from 11 measurements according to formula: E(1) =Ca(1)*Fo(20)*E(20)/(Ca(20)*Fo(1)), where E(20) = 1. The values obtained varied from 0.8 to 1.1, which exceeds E(0) values obtained from analysis of absorption spectra of algal cultures. This may be attributed to significant extinction of red light at the depth of 1 m (Fig. 1B (curve 2)). Thus, we can assume the average value of E in the water column is near to 1 and does not influence on the km variability.
As seen from the histogram of km distribution, which is shown in Fig. 2A, the standard deviation of this parameter was mainly due to variation in k values in the range of k > 7*10-5. Figure 3 shows the dependence of km on I(0) for stations with distinct surface inhibition of Pc and phytoplankton fluorescence. As seen from Fig. 3, a weak positive correlation between km and surface irradiation was observed only for km > 7*10-5, which were measured at stations 11, 13, 14, 16 and 22.
Vertical profiles of Ca were uniform at stations where km > 7*10-5, and F0 decreased in 2-4 times in surface water. Taking into account that E only slightly changes with depth, our data indicate a light-dependent decrease in the fFo in the upper layers under intense irradiation, which was the cause for overestimation of calculated km values at these stations. It should also be noted that 4 of the 5 stations were investigated at different time in the same area of the Baltic Sea - the Pomorsky Bay (Oder mouth). The recalculation of km at these stations, taking into account vertical distribution of F0, corrected for Ca, resulted in a reduction of the standard deviation of this parameter by 17 to 9%, as compared to that calculated previously. This indicates a rather considerable contribution of light-dependent changes in fFo to dispersion of km. At other 17 stations, where the noon depression of fluorescence was also recorded, same reduction in both Fo and Ca was observed in surface water. Thus, the vertical profiles of F0 at most stations demonstrated distinct depth dependence of microalgae concentration and their absorption capacity, but not fFo, which agrees with the assumption that fFo is about constant in natural phytoplankton. The low level of fFo, which is not typical for the studied area as a whole, could be related to characteristics phytoplankton physiological state in the Pomorsky Bay.
Therefore, the variation of km at 23 stations of the Baltic Sea was mainly due to light-dependent decrease fFo at 5 stations.
Variation of I1/2 at Baltic sea stations.
Column 6 of Table 1 gives averaged over water column I1/2 values (I1/2m) in the Baltic Sea, as calculated according to formula 8. The maximum and minimum values of this parameter made up 98 and 190, respectively, the mean value at all stations was 137 mE m-2 s-1, the standard deviation was 22%, indicating a greater variation of this parameter, as compared to km (Fig. 2B). I1/2m did not correlate with daily changes in solar radiation (see also Antal et al., 1999), however, I1/2m tended to decrease with chlorophyll concentration (Fig. 4A).
Below presented is the result of polynomial regression of the dependence of I1/2m on the average content of chlorophyll a in the water column (Cam):
I1/2m = 171-14.7*Cam+0.8*(Cam)2 (9)
Comparison of Figs. 2B and 4B shows that the degree of I1/2m variation decreases from 22% to 16%, when the standard deviation of this parameter is calculated with respect to values of I1/2m, which had been determined from formula 9, but not with respect to mean value at all stations. Thus, the variation of I1/2m at stations in Baltic sea was partly associated with an error in determining this parameter as well as with variation in the content of chlorophyll a at the stations, which indicates a rather wide variation rage for this parameter, depending on the trophicity of water studied.
Primary production of phytoplankton, Pcc.
Primary production of phytoplankton, Pcc, was calculated by substituting fluorescence, underwater irradiation, km = 5.4*10-5, and I1/2m, determined from formula 9, to the right part of formula 8 and by integrating Pcc(z) over depth. In addition, the effect of light-dependent decrease in fFo was taken into account and appropriate correction of F0 profiles for Ca at 5 stations was made. Pcc calculated in this way correlated well with production measured by the direct method, the coefficient of correlation r = 0.94 and standard deviation was +-25% (Fig. 5). When Pcc was calculated without taking into account the light dependent decrease in fFo by substituting value I1/2m = 137 to formula 8, it slightly less correlated with the measured production: r = 0,89. Both results indicate quite a high accuracy of estimation of the rate of phytoplankton photosynthesis by the suggested fluorescence method, but the best result was obtained when the data on vertical distribution of chlorophyll a were used.
This method of determining the primary production of phytoplankton showed good results at 23 stations of the Baltic Sea in coastal and central waters in spring, summer, and autumn in different years. It seems likely that it can successfully be applied for estimation of productivity in the Baltic Sea.
We also investigated the possibility of determining Pcc in other climatic zones, which differ from the Baltic Sea in trophicity and hydrophysical characteristics. Pcc was calculated in central mesotrophic stratified waters of the Norwegian Sea, where Ca, averaged over water column, varied between stations from 0.20 to 0.49 mg m-3, and in coastal oligo- mesotrophic stratified waters of the South China Sea (the Bay of Nhatrang), where chlorophyll content varied from 0.025 to 0.25 mg m-3. When calculating, we substituted parameters km and I1/2m in formula 8 with the average values for the Baltic Sea: 5.4*10-5 and 137, respectively. The calculated and measured primary production correlated with each other slightly lower than in the Baltic Sea: r = 0.77 (radiocarbon method) and 0.70 (oxygen method) in the Norwegian Sea and 0.76 in the Bay of Nhatrang (oxygen method). The comparison of F0 and Ca profiles showed that there was no drastic changes in fFo, thus, the lower correlation, as compared to the Baltic Sea, may be related to variations in I1/2 and to a low accuracy of direct measurement of Pc in these regions: samples were collected from only two horizons and the samples were incubated on ship board. Furthermore, Pc measured by oxygen method correlated with Pcc only qualitatively exceeding it threefold on the average (see Sapozhnikov et. al., 2000). As described above, the fluorometer probe was calibrated against radiocarbon methods, which gives lower values, as compared to those obtained with oxygen method, due to differences in calculation (Naletova and Sapozhnikov, 1995) and measurement methods (Koblents-Michke and Vedernikov, 1977).
Measurement of parameter k by direct calibration of fluorescence data in terms of light absorption by microalgae allows for independent estimation of Vc in microalgae and its comparison with the data obtained by direct measurements. We measured F0 as a function of absorption afl under laboratory conditions in green (Chlorella vulgaris), diatomic (Thalassiosera west), and yellow-green (Nephrochloris salina) algae (data not shown). The dependencies were linear at Ca < 10 mg m-3. Values of k, as determined at E =1 by non-linear regression of this dependency, only slightly varied within the range 8-9*10-5. When the decrease in E under natural conditions in surface water (see above) was taken into account, the upper and lowest limits of k values were equal to 6.4*10-5 and 9*10-5, respectively, which is slightly above of the radiocarbon data, which are within the range 4,32 - 6,20*10-5 (without taking into account the values k > 7*10-5, see Table 1). Photosynthetic rate calculated with the use of the average value k=7.7*10-5, which was determined by this method, is about in 1.5 times higher than radiocarbon data, but in two times lower than the data obtained by oxygen method.
Thus, primary production determined by the pump-and-probe fluorometer correlated well with that measured by direct methods at stations in the Baltic, Norwegian, and South China Seas, which allows application of this method, taking into account changes in fFo in various aquatic regions.
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