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Measurement of phytoplankton photosynthesis rate using a pump-and-probe fluorometer*

OCEANOLOGIA, 43 (3), 2001. pp. 291 ­ 313. 2001, by Institute of Oceanology PAS.
KEYWORDS

Phytoplankton Primary production Plant luminescence Fluorometric method

Taras K. Antal 2 Pavel S. Venediktov 2 Dimitrii N. Matorin 2 Miroslawa Ostrowska 1 Bogdan Woniak 1 Andrei B. Rubin 2
1

Institute of Oceanology, Polish Academy of Sciences, PowstacÑw Warszawy 55, PL­81­712 Sopot, Poland; e-mail: ostra@iopan.gda.pl
2

Department of Biophysics, Faculty of Biology, Moscow State University, Moscow, 119899 Russia
Manuscript received 21 June 2001, reviewed 26 July 2001, accepted 1 August 2001.

Abstract In this work we have studied the possibility of determining 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 in climatic and hydrophysical characteristics. A biophysical model was used to describe the relationship between
* The study results published in this paper will be presented at the `Third Workshop on Luminescence and Photosynthesis of Marine Phytoplankton', Sopot­Sulczyno, 8­12 October 2001, sponsored by the Polish State Committee for Scientific Research and organised by the Marine Physics Department of the Institute of Oceanology PAS in Sopot, the Environmental Physics Department of the Pomeranian Pedagogical University in Slupsk and the Department of Biophysics of the Lomonsov University in Moscow.


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photosynthesis, underwater irradiance, 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 assessing the efficiency of photochemical energy conversion at photosynthetic reaction centers. Parameters of the model that could not be measured experimentally were determined by calibrating fluorescence and irradiance data against the primary production measured in the Baltic Sea with the radioactive carbon method. It was shown that the standard deviation of these parameters in situ did not exceed 20%, and the use of their mean values to estimate the 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.

1. Introduction
Photosynthesis of phytoplankton can b e measured as the rate of radioactive carb on assimilation (Steemann-Nielsen 1952) or as the increase in the concentration of soluble oxygen in a sample (Williams & Jenkinson 1982, Langdon 1984). These methods are rather lab or-consuming, and their application involves numerous artifacts owing to the prolonged isolation of phytoplankton in b ottles (Eppley 1980), the difference b etween 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 phytoplankton to b e measured continuously in real time without their physiological state b eing affected (Kolb er et al. 1990, Green et al. 1992). The relationship b etween chlorophyll a (Ca ) fluorescence and photosynthesis is describ ed in a numb er of biophysical models of the primary processes of photosynthesis (Weis & Berry 1987, Genty et al. 1989, Kiefer & Reynolds 1992). The aim of our work was to elab orate the methodology of determining the rate of photosynthesis in situ using theoretically justified biophysical models. The model of carb on assimilation Pc [µM C m-3 s-1 ] by phytoplankton used in our work is based on the light dep endence of photosynthesis (Jassby & Platt 1976) and can b e describ ed by the following product: ¯ Pc = apl
, P SP

(E )E,

(1)

where:1 apl, P S P [m-1] ­ mean coefficient of solar irradiance absorption by phyto¯ plankton photosynthetic pigments (PSP) in the 400­700 nm sp ectral range (PAR) (after Dubinsky et al. 1986),
For the reader's convenience, we append a list of symbols denoting the physical quantities used in the text. The nomenclature and denotations are in line with the conventions employed in the subject literature.
1


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(E ) [µM C µE-1 ] ­ efficiency (quantum yield) of the conversion of absorb ed energy in photosynthetic reactions, E [µE m-2 s-1 ] ­ total irradiance in the PAR range. The value of is prop ortional to the relative numb er of functionally active (f ) and op en (qp ) PS I I reaction centers in algal cells, to the efficiency of photochemical conversion of light energy in op en reaction centers (RC ), [µM electron µE-1 ], and to the efficiency of electron transfer from H2 O to CO2 (e ), [µM C (µM electron)-1 ]: Pc = apl ¯
, P SP

f qp (E )

RC

e E.

(2)

Some parameters of equation (2), like apl, P S P or f ,can b e determined by ¯ measuring fluorescence parameters F0 and F /Fm by the pump-and-probe method (Mauzerall 1972, Kolb er et al. 1990) in phytoplankton adapted to ambient light; alternatively, by substituting the photosynthetic rate measured by the radiocarb on method for Pc in formula (2), or by measuring light absorption by algae. In this work, we investigated the variation of these indirectly measured parameters in the Baltic Sea. The p ossibility of applying the mean values of these parameters to determine the primary production of phytoplankton in the Baltic, Norwegian, and South China Seas was also studied.

2. Methods
2.1. Determination of apl, P S P , f , and ¯ fluorescence characteristics
RC

from phytoplankton

The intensity of fluorescence excited by an artificial light source, with op en reaction centers (RC) in algae, can b e found from the equation ¯ F0 = G Ifl apl where: Ifl
, P S P, f l

F 0 ,

(3)

apl ¯

, P S P, f l

[m-1

F G

0

­ total intensity of the exciting flash (in our fluorometer, Ifl () was nearly uniformly distributed over the 400­550 nm sp ectral range), (constant), ] ­ coefficient of exciting flash absorption by PSP, averaged over the 400­550 nm sp ectral range, ­ quantum yield of fluorescence in cells with op en RC, ­ coefficient defined by geometric characteristics and the sensitivity of the fluorescence light sensor, (constant).


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Taking into account the fact that (G Ifl )-1 = const, the coefficient of solar irradiance absorption by algae can b e related to the fluorescence intensity as follows: apl ¯
, P SP

= const

-1 F0

AF0 = k(F0 , A) F0 ,

(4)

where: ¯ A = apl, P S P /apl,P S P,f l , ¯ k(F0 , A) ­ a prop ortionality coefficient, which can b e determined by intercalibration ­ see b elow. The photochemical efficiency of an op en PS I I reaction center can b e determined from the ratio of fluorescence parameters RC (Fm - F0 )/Fm = F /Fm (Klughammer 1992). It was shown that the decrease in the F /Fm ratio corresp onds to part of the decrease in the fraction of functioning PS I I reaction centers (f ) (Kolb er et al. 1988, 1990), a process induced by excessive irradiation (Long et al. 1994, Vassiliev et al. 1994) (photoinhibition) and/or limitation of phytoplankton growth by mineral nutrients (Falkowski et al. 1989, Green et al. 1992). Thus, parameters RC and f are prop ortional to the relative yield of variable fluorescence of chlorophyll in phytoplankton adapted to natural radiation. We therefore assume that f
RC

= F /Fm .
e

(5)

2.2. Determination of qp and

It is known that photochemical conversion of light energy in PS I I takes place only in op en reaction centers. The relative concentration of op en centers qp can b e found from the model of the light-dep endent transition of reaction centers b etween the op en and closed states. We used the model expressed by the Michaelis-Menten equation, which was prop osed among others by Kiefer & Mitchell (1983): qp (E ) = E1/2 (E + E1/2 )-1 , (6) where E1/2 is the light irradiation, at which half of the RC are in the closed state ­ see b elow. The value of e was estimated from the following considerations. To reduce one molecule of CO2 , 4 electrons should b e transferred from H2 O. Theoretically, therefore, e may b e as high as 0.25; however, a certain fraction of the electron flow is consumed for nitrate and sulfate, for cyclic electron transp ort around PS I and PS I I, as well as for O2 reduction (Slovacek et al. 1980, Dubinsky et al. 1986, Falkowski et al. 1986, Myers 1987, Laws 1991). Comparison of e with the maximum quantum yield of carb on fixation leads to the assumption that e is approximately


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constant (Kiefer et al. 1989, Morel 1991) and is not over 0.16 for natural phytoplankton (Bannister & Weidemann 1984). Hence, we assume that e = 0.16. By substituting 4, 5, 6, in eq. (2) and introducing the coefficient 6.9 = 12 â 10-3 [mg C (µM C)-1 ] 3600 [s h-1] e , the equation for the vertical profile of the algae photosynthesis rate [mg C m-3 h-1 ] can b e written as Pc (z ) = 6.9 k(z ) F0 (z ) F /Fm (z ) where z is depth [m]. 2.3. Estimation of k and E1/2 The unknown parameters k and E1/2 were found by comparing the primary production of phytoplankton P c [mg C m-3 time-1 ] measured by the radiocarb on method, and fluorescence and irradiance measurements, according to the formula
n

E1/2 E (z ), E (z )+ E1/2

(7)

P c (z ) = 6.9
i=1

(km F0 (z )F /Fm (z )

E1/2 m E (z )t)i , E (z )+ E1/2 m

(8)

where n is the numb er of fluorescence and irradiation profiles measured for the p eriod of b ottle exp osure at a station; t [h] is the time p eriod b etween these measurements; km and E1/2 m are the resp ective values of the parameters k and E1/2 averaged in the water column. They were calculated by approximating the P c versus z dep endence with eq. (8) by the least squares method. Parameter k was also estimated under lab oratory conditions by calibrating F0 against the coefficient of exciting flash absorption by phytoplankton taken at a natural concentration (Ca = 0.1­10 mg m-3 ). Parameter k was determined from formula (4) for A =1. The valueof apl, P S P, f l was measured ¯ with a lab oratory instrument. Light from the KGM 150/24 halogen lamp of a slide pro jector passed through an SZS22 glass filter and a 0.2-m-long dark chamb er containing the sample, and the output quantum flux density was measured with a lab oratory-made quantum sensor. Calculations were done using the formula apl, P S P, f l [m-1 ] = 1/0.2 (In, c - In )/In, c = 5 ¯ (In, c - In )/In, c , where In is the intensity of light passed through a susp ension of algae of concentration n; In, c is the same for a susp ension of algal cells bleached by illumination in the presence of 1 mM hydroxylamine. For lab oratory exp eriments, marine algae were grown on Goldb erg medium prepared with artificial sea water in flasks at constant temp erature in light (Lanskaya 1971).


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2.4. Data recording 2.4.1. Material The vertical distribution of irradiance, fluorescence, primary production of phytoplankton, and chlorophyll concentration were measured in the Bay of Nhatrang in the South China Sea (12 09 ­12 18 N, 109 12 ­109 20 E) and during cruises in the Baltic (13 10 ­25 15 N, 53 25 ­58 10 E) and Norwegian Seas (64 15 ­70 20 N, 4 40 W­4 30 E): (1) June­July 1993 ­ the cruise of the r/v `Humb oldt', according to the `Plankton' program; measurements at 7 stations near the southern and eastern coasts of the Baltic Sea. (2) May 1993 ­ the cruise of r/v `Oceania', Institute of Oceanology PAS; measurements at 4 stations in central and coastal waters of the Baltic Sea. (3) Septemb er 1993 ­ the cruise of r/v `Oceania', Institute of Oceanology PAS; measurements at 3 stations in central and coastal waters of the Baltic Sea. (4) May 1994 ­ the cruise of r/v `Oceania', Institute of Oceanology PAS; measurements at 3 stations in central and coastal waters of the Baltic Sea. (5) Septemb er 1995 ­ the cruise of r/v `Oceania', Institute of Oceanology PAS; measurements at 6 stations in central and coastal waters of the Baltic Sea. (6) June­July 1997 ­ cruise of r/v `A. Petrov', VNIRO of the RAS; measurements at 13 stations in the deeps of the central Norwegian Sea. (7) March 1998 ­ measurements at 8 stations in the Bay of Nhatrang in the South China Sea. 2.4.2. Measurements Vertical profiles of in situ fluorescence were recorded with a `Prim Prod' submersible pump-and-probe fluorometer designed at the Biophysical Department of the Faculty of Biology of Lomonosov Moscow State University. The instrument also recorded irradiance in the PAR region [µEm-2 s-1 ], temp erature, and depth. The fluorometer generates sequential pump and prob e flashes at a frequency of 2 Hz. The saturating (pump) flash of 1 J/0.01 ms p ower p er duration was given 1 s after the first prob e flash (0.01 J/0.01 ms), and the second prob e flash follows the pump flash after 50 µs. The impulses were generated by an SSh­20 (MELZ, Russia) xenon


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lamp. The flashes are isolated from the sample by a light blue-green filter SZS­22. The sp ectrum of the fluorescence excitation is distributed practically evenly within the range of wavelengths from 400 to 520 nm. During prob e submersion, external water passively enters an op en dark chamb er in which the fluorescence of phytoplankton cells adapted to underwater radiation is measured every 0.5 s. The prob e submersion rate was 0.3­0.5 m s-1 , which allowed for resolution depth profiles. The first probing flash measures F0 , the fluorescence intensity with op en PS I I centers. The subsequent saturating flash converts most of the RC to the closed state, and the second probing flash, which follows within 50 µs, a time comparable to the reaction center turnover time, measures the fluorescence, which corresp onds to the I 1 level of fluorescence saturation (Schreib er et al. 1995). Fm is calculated according to the formula Fm = 1.4 â I 1, where Fm, DCMU /I 1 = 1.4 is the ratio of the maximum fluorescence obtained in the presence of DCMU, an inhibitor of electron transp ort in PS I I, to the fluorescence yield measured by the PrimProd. After passing through a KS­17 cut-off glass filter, the fluorescence signal is recorded by photomultiplier-68. The recorded fluorescence signals as well as the underwater irradiance, temp erature and pressure (depth) are transmitted in real time via a cable-rop e connected to a p ersonal computer. The primary production of phytoplankton was measured in the Baltic Sea with the radiocarb on method at 5­10 horizons down to a depth of 30 m using a routine method (Steemann-Nielsen 1952), a modification of it in the Norwegian Sea (Sorokin 1960), and by the oxygen method in the Bay of Nhatrang and Norwegian Sea (Vinb erg 1969). During measurement with the radiocarb on method, b ottles were exp osed for 6 hours during the 1st cruise, for 4 hours during the 2nd, 3rd and 4th cruises, for 2 hours during the 5th cruise, and for 6 hours during the 6th cruise. The chlorophyll a content was determined with a standard sp ectrophotometric method (Bender et al. 1987).

3. Results
To determine the rate of phytoplankton photosynthesis according to formula 7, it is necessary to estimate the unknown quantities k(F0 , A) and E1/2 and their variability in the regions studied. As exp eriments have shown (Ernst et al. 1986, Dera 1995, Woniak et al. 1997), photosynthetic parameters F0 and E1/2 vary under the stress action of abiotic factors. In natural phytoplankton, according to Ostrowska et al. (2000a and b), the parameter F0 does not dep end significantly on environmental factors (with the exception of the fluorescence photoinhibition that is p ossible


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Table 1. Values of km and E1/2 m calculated at given time intervals from 51 profiles of phytoplankton production, fluorescence, and underwater irradiation at 23 stations in the Baltic Sea. The cruise and station numbers, dates and areas of measurements are also given Cruise Station Date Area km â 105 [relative units]
3­6 6­9 9­12 12­15

E1/2 m [µE m-2 s

-1

]

3­6 6­9 9­12 12­15

1

2

3

4

5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23



23.06.1993 26.06.1993 28.06.1993 30.06.1993 01.07.1993 09.07.1993 10.07.1993 13.05.1993 14.05.1993 15.05.1993 10.05.1993 28.09.1993 29.09.1993 30.09.1993 09.05.1994 11.05.1994 13.05.1994 08.09.1995 09.09.1995 10.09.1995 12.09.1995 13.09.1995 14.09.1995

5 5 4 3 3 2 2 1 1 4 5 5 5 5 1 5 6 3 3 1 1 1 1

5 5 5 5 4 5

­ .6 .2 .6 .0 .7 .0 ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­

0 8 0 0 2 0

4.64 4.92 5.48 5.32 5.52 4.60 4.80 ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ 5.40 ­ 5.60 ­

5.48 4.8 6.20 4.32 ­ 5.88 4.44 4.92 5.68 5.80 8.44 5.48 7.24 7.96 5.60 8.60 5.28 6.08 5.32 5.48 5.88 5.76 5.12



­ 5.60 5.20 4.44 ­ 5.60 5.00 ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ 5.48 ­ 6.20 ­ 7.44 ­



­ 134 170 176 113 188 170 ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­

155 164 134 116 100 95 134 ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ 148 ­ 180 ­

167 135 144 107 ­ 134 140 124 118 178 145 164 104 110 125 115 140 98 145 190 133 165 131



­ 161 128 124 ­ 139 155 ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ 127 ­ 121 ­ 138 ­

Areas: 1 ­ central waters, 2 ­ the Gulf of Riga, 3 ­ the Lithuanian coast, 4 ­ the Gulf of Gdask, 5 ­ the Pomeranian Bay, 6 ­ the coastal waters between 4 and 5; the results were averaged over several measurements.

in phytoplankton from overexp osed shallow waters under intense natural irradiance), while parameter E1/2 can change to some degree with the trophic typ e of water and does change mainly with the temp erature of the water b ody (Morel 1991, Dera 1995, Antoine & Morel 1996, Woniak et al. 1997). We presume that it is nearly constant in regions with similar temp erature (Woniak et al. 1992). The mean values of k and E1/2 in the water column ­ km and E1/2 m ­ were calculated (formula 8) at 23 stations


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299

in the central and coastal areas of the Baltic Sea (from the Gulf of Riga to the Pomeranian Bay), where the average concentration of Ca in the water column varies from 0.7 to 10 mg m-3 . The data are given in Table 1. 3.1. Variation of km at the Baltic Sea stations The mean value of this parameter at the Baltic Sea stations was 5.6 â 10-5 (standard deviation SD = ± 17%). The km variation can b e related to the factor A (see formula 4), which is induced by the differences in blue light and solar radiation absorption by marine phytoplankton. The value of A dep ends mainly on the taxonomic comp osition of the algae and their physiological condition. For example, A calculated in vivo from absorption sp ectra (as shown in Fig. 1a) for three taxonomic groups ­ diatomea Phaeodactylum tricornutum, yellow-green algae Nephrochloris salina, and green algae Platymonas virdis ­ grown under optimum conditions and at low irradiation, elevated temp eratures, or nitrogen deficiency, varied from 0.6 to 0.75. For samples of natural phytoplankton from the Baltic Sea, A = 0.74. The exp erimental value of A was <1 due to the fact that the absorption coefficient of marine algae for blue light is usually much higher than their absorption coefficient averaged ¯ over the PAR region: apl, P S P, f l > apl, P S P . Thus, it can b e exp ected that, to ¯ a first approximation, the values A for natural phytoplankton should vary from 0.6 to 0.75 in the upp er water layers, where the irradiance sp ectrum is close to that of solar radiation.
0.10

excitation irradiance I [arbitrary units] relative underwater irradiance E(l) / E(lmax) [relative units]

a
0.08 0.06 0.04 0.02 0.00 400 500 600 700
Ñ apl, PSP fl Ñ apl, PSP

b
1.2 1.0 0.8 0.6 0.4 0.2 0.0 400 500 20 m 600 5m 1m

absorption [mß1]

700

wavelength l [nm]

wavelength l [nm]

Fig. 1. Exemplary spectrum of phytoplankton light absorption coefficient from the central Baltic Sea. Values of absorption coefficients are averaged over the 400­550 nm (¯pl, P S P, f l ) and 400­700 nm (¯pl, P S P ) spectral regions (a). Spectrum a a of the light used to excite chlorophyll fluorescence in the PrimProd fluorometer (solid line), and spectral distributions of underwater irradiance in the sea at different depths (dashed lines ­ data by M. Ostrowska and R. Hapter) (b)


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Changes in the sp ectrum of underwater irradiance with depth are accom¯ panied by changes in A : A(z )= A(0) â T (z ), where A(0) = apl, P S P /apl, P S P, f l ¯ is the value of A at the water surface and T (z ) is the depth dep endence of A. In clear water, the attenuation of red light with depth must lead to an increase in A(z ) from 0.6­0.7 at the surface to 1 at 20 m and greater depths, where the sp ectra of the probing flash and underwater irradiance are similar (Fig. 1b). Thus, it can b e exp ected that mean values of A in the water column, which affect km , should b e higher than 0.7 and vary to a lesser extent than at the surface. As a first approximation we can assume that A = 1 and does not influence the value of km (see section 4).
a
15 SD = ±18% 15 SD = ±21%

b

frequency [%]

frequency [%]
4.0 4.8 5.6 6.4 7.2 8.0 km ´105 [relative unit] 8.8

10

10

5

5

0

0

100

120 E1/2

m

140 160 180 [mEin mß2 sß1]

Fig. 2. Histograms of km (a) and E1/2 Sea stations

m

(b) distribution, determined for the Baltic

As can b e seen from the histogram of km distribution (Fig. 2a), the high standard deviations of this parameter were due mainly to the occurrence of km values in the range km > 7 â 10-5 . Figure 3 shows the dep endence of km on E (0) for stations with a distinct surface inhibition of P c and phytoplankton fluorescence. As can b e seen from Fig. 3, there is a weak p ositive correlation b etween km and surface irradiance only for km > 7 â 10-5 , which were measured at stations 11, 13, 14, 16 and 22. Vertical profiles of Ca were uniform at stations where km > 7 â 10-5 , and F0 decreased 2­4 times in surface water. Taking into account the fact that A changes only slightly with depth, our data indicate a light-dep endent decrease in F0 in the upp er layers under intense irradiance, which is why


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301

parameter km ´ 10ß5 [relative units]

7.2

4.8

2.4 km > 7 ´ 10 km < 7 ´ 10 0 1000 1500 2000
ß2 ß1 ß5 ß5

surface irradiance E(0) [mEin m s ]

Fig. 3. Dependence of km on surface irradiance E (0) for the Baltic Sea stations. Dashed lines show the standard deviation band

the calculated km values at these stations were overestimated. It should also b e noted that 4 of the `5-area' stations were investigated at different times in the same area of the Baltic Sea ­ the Pomeranian Bay (Oder mouth). The recalculation of km at these stations to take into account the vertical distribution of F0 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-dep endent changes in F0 to the disp ersion of km . At the other 18 stations, where the noon depression of fluorescence was also recorded, b oth F0 and Ca were reduced in surface water. The vertical profiles of F0 at most stations thus demonstrated the distinct depth dep endence of phytoplankton concentration and its absorption capacity, but not of F0 , which confirms the assumption that F0 is roughly constant in natural phytoplankton. The low level of F0 , which is not typical of the study area as a whole, could b e related to the characteristic physiological state of the phytoplankton in the Pomeranian Bay. Therefore, the variation of km at 23 stations of the Baltic Sea was due mainly to light-dep endent inhibition F0 at 5 stations. 3.2. Variation of E1/2 at Baltic Sea stations Column 6 of Table 1 gives E1/2 values (E1/2 m ) averaged over the water column in the Baltic Sea calculated according to formula 8. The minimal and maximal values of this parameter are 98 and 190, resp ectively; the mean


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value for all stations was 137 µE m-2 s-1 , and the standard deviation was 22%, which indicates a greater variation in this parameter in comparison to km (Fig. 2b). E1/2 m did not correlate with daily changes in solar irradiance (see also Antal et al. 1999); however, E1/2 m did tend to decrease with chlorophyll concentration (Fig. 4a).
a
200 150 100 50 0 1 C
am

b
E1/2 m = 171ß14.7 Cam+ 0.8 C
2 am

15

SD = ±16%

[mEin mß2 sß1]

frequency [%]
10 [mg m ]
ß3

10

E1/2

5

m

0

0.5 E
1/2 m, c

1 /E
1/2 m, m

2

Fig. 4. The dependence of E1/2 m on the chlorophyll a Cam content averaged over the water column and polynomial regression of this dependence (a); histogram of distribution of the ratio of E1/2 m, c /E1/2 m, m (index c ­ calculated, using polynomial regression values of E1/2 m , index m ­ observed values of this parameter) (b)

The result of the p olynomial regression of the dep endence of E1/2 the average content of chlorophyll a in the water column (Cam ) is E1/2
m

m

on (9)

= 171 - 14.7 Cam +0.8(Cam )2 ,

Comparison of Figs. 2b and 4b shows that the degree of E1/2 m variation decreases from 22% to 16% when the standard deviation of this parameter is calculated with resp ect to values of E1/2 m determined from formula 9, but not with resp ect to the mean value for all stations. Thus, the variation of E1/2 m at stations in the Baltic Sea was partly due to an error in determining this parameter, as well as to the variation in chlorophyll a content at the stations. This indicates that there is some range of variation for this parameter, dep ending on the trophicity of the investigated waters. 3.3. Primary production of phytoplankton, PPc The primary production of phytoplankton, PPc , was calculated substituting fluorescence, underwater irradiance, and parameters = 5.4 â 10-5 , and E1/2 m , determined from formula 9, in the right-h side of formula 8 and by integrating Pc (z ) over depth. The effect of by km and the


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303

light-dep endent decrease in F0 was also taken into account. PPc calculated in this way is well correlated with the production measured directly; the coefficient of correlation r = 0.94 and the standard deviation is ± 25% (Fig. 5). When PPc was calculated by substituting the value of E1/2 m = 137 in formula 8 without the light-dep endent decrease in F0 b eing taken into account, it was slightly less well correlated with the measured production: r = 0.89. However, b oth results indicate that the suggested fluorescence method yields a fairly accurate estimate of the rate of phytoplankton photosynthesis.
a
calculated primary production [mg C mß3 timeß1]
100 r = 0.94 6

b
SD = ±25%

frequency [%]
10 measured primary production [mg C mß3 timeß1] 100

4 2 0

10

0.5

1 ratio calculated / measured primary production

2

Fig. 5. Dependence of the calculated primary production of phytoplankton on the measured value averaged over the water column at Baltic Sea stations (a), histogram of the distribution ratio of the calculated productions to the measured values (b)

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 b e applied successfully to the estimation of productivity in the Baltic Sea. We also investigated the p ossibility of determining PPc in other climatic zones, which differ from the Baltic Sea in trophicity and hydrophysical characteristics. PPc was calculated in central mesotrophic stratified waters of the Norwegian Sea, where Ca , averaged over the water column, varied b etween stations from 0.20 to 0.49 mg m-3 , and in the oligo-mesotrophic, stratified, coastal waters of the South China Sea (the Bay of Nhatrang), where the chlorophyll content varied from 0.025 to 0.25 mg m-3 . In the calculations, we substituted the parameters km and E1/2 m in formula 8 with the average values for the Baltic Sea: 5.4 â 10-5 and 137,


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resp ectively. The calculated and measured primary production are slightly less well correlated with each other than in the Baltic Sea: r = 0.77 (radiocarb on method) and 0.70 (oxygen method) in the Norwegian Sea and 0.76 in the Bay of Nhatrang (oxygen method). Comparison of F0 and Ca profiles showed that there were no abrupt changes in F0 . Thus, the lower correlation, as compared to the Baltic Sea, may b e related to variations in E1/2 and to the low accuracy of direct measurement of P c in these regions: samples were collected from only two horizons and the samples were incubated on b oard ship. Furthermore, Pc measured by the oxygen method was only qualitatively correlated with PPc , exceeding it threefold on average (see Sap ozhnikov et al. 2000). As describ ed ab ove, the fluorometer prob e was calibrated against radiocarb on methods, which gives lower values as compared to those obtained with the oxygen method, owing to differences in the methods of calculation (Naletova & Sap ozhnikov 1995) and measurement (Koblentz-Mischke & Vedernikov 1977). Measurement of parameter k by direct calibration of fluorescence data in terms of light absorption by phytoplankton allows for an indep endent estimation of PPc in phytoplankton and its comparison with the data obtained by direct measurements. We measured F0 as a function of absorption under lab oratory conditions in green (Chlorel la vulgaris), diatomic (Thalassiosira west.), and yellow-green (N. salina) algae (data not shown). The dep endencies were linear at Ca < 10 mg m-3 . Values of k, as determined at A = 1 by the non-linear regression of this dep endency, varied only slightly within the range 8­9 â 10-5 . When the decrease of A under natural conditions in surface water (see ab ove) was taken into account, the upp er and lower limits of k were equal to 6.4 â 10-5 and 9 â 10-5 , resp ectively, which is slightly ab ove the radiocarb on data values, which lie within the range 4.32­6.20 â 10-5 (without taking into account the values k > 7 â 10-5 , see Table 1). The photosynthetic rate calculated with the use of the average value k = 7.7 â 10-5 , which was determined by this method, is ab out 1.5 times higher than the radiocarb on data result, but only half as great as the result obtained using the oxygen method data.

4. Conclusion and final remark
Primary production determined by the pump-and-prob e fluorometer correlated well with that measured by direct methods at stations in the Baltic, Norwegian, and South China Seas: application of this method takes into account changes in F0 in various marine areas. Hence, b ecause of its efficiency and sp eed, the method presented in this pap er could b e used as an alternative to the traditional method.


Measurement of phytoplankton photosynthesis rate ...

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Using the PrimProd fluorescence method for estimating primary production, one has to b ear in mind inconveniences and simplifications that are the cause of certain inaccuracies, in particular, the following: · Firstly, this method requires calibration by traditional measurements and the determination of the constant km (related to the sp ecific absorption coefficient) and the constant E1/2 (related to the shap e of the light-photosynthesis curve) in equation (8). This is why this method is not universal. · Secondly, when using this method we assume that the fluorescence F0 is the measure of the mean coefficient of the total photosynthetic pigment light absorption. While this assumption is correct, it should b e realized that this mean absorption coefficient, apl, P S P, f l , is in ¯ reality the mean absorption coefficient with the weight of the sp ectrum of the exciting light:
apl,
P SP

()I ()d

apl ¯

, P S P, f l fl

= const â F0 where F0 =

fl



I ()d


,

(10)

is the quantum yield of fluorescence, and range of the exciting light. On the other hand, to determine the energy absorb have to use a similar absorption coefficient apl, P S P ~ weight of the sp ectrum scalar irradiance in the sea apl ~ 1 = I
700 nm

where

is the sp ectral ed by pigments we averaged with the I ():



, P SP

700 nm

, P SP

apl

() I ()d,

I =
400 nm

I ()d. (11)

400 nm

~ Unfortunately, these absorption coefficients (apl, P S P, f l and apl, P S P ) ¯ are not prop ortional. In reality, as a result of changes in I () ¯ sp ectra with depth, the ratio apl, P S P /apl, P S P, f l is also strongly ~ depth-dep endent. Moreover, this ratio dep ends on the trophicity. Let us denote this ratio as Amod : Amod = apl, P S P ~ . apl, P S P, f l ¯

As we can see in Fig. 6, the parameter Amod differs for different trophic typ es of sea (we assume the surface chlorophyll a concentration Ca (0) to b e the trophic index of the sea) and also varies with depth. These changes are significant, esp ecially in regions of high and low trophicity. This illustrates the imprecision of the assumption in eq. (8) that parameter km is constant for all depths in the sea. Therefore, the assumption of a constant value of km for all depths in eq. (8) can b e the source of significant errors.


306
a
0 2

T. K. Antal, P. S. Venediktov, D. N. Matorin et al.
E2 E3

b
E4 E5 E6 0 50 E4 E3 E2 E1 P M O3 O2 O1 0.5 1 A
mod

E5

E6

optical depth t

4 6 8 E1 10 0.5 M O1

depth z [m]
2 2.5

100 150 200 250 300

O2

P 1

O3 1.5

1.5

2

2.5

Amod [dimensionless]

[dimensionless]

Fig. 6. Computed (using the model according to Ostrowska 2000) depth profiles of the ratio Amod = apl, P S P /apl, P S P, f l for optical (a) and real (b) ~ ¯ depths for different trophic types of sea. Surface chlorophyll a concentrations Ca (0) [mg tot. chl a m-3 ] were assumed to represent the water trophic type index (according to Woniak et al. 1992): O1 ­ Ca (0) = 0.035; O2 ­ Ca (0) = 0.07; O3 ­ Ca (0) = 0.15; M ­ Ca (0) = 0.35; P ­ Ca (0) = 0.7; E1 ­ Ca (0) = 1.5; E2 ­ Ca(0) = 3.5; E3 ­ Ca (0) = 7; E4 ­ Ca (0) = 15; E5 ­ Ca (0) = 35; E4 ­ Ca (0) = 70

· Thirdly, qp is a function of temp erature in the sea. The initially determined qp as a function of the absorb ed energy PU RPS P (part of the Photosynthetically Usable Radiation due to photosynthetic pigments (p er unit of chlorophyll a mass)) and temp erature is given by the equation (Ostrowska 2000) qp =
KP U RPS P (temp) PU RPS P 1 - exp - PU RPS P KP U RPS P (temp)

,

where KP U RPS P (temp) (the so-called `saturation irradiance') dep ends on temp erature according the Arrhenius law: KP U RPS P (temp) = KP U RPS P, 0

Q10

temp/10 C

.

The values of KP U RPS P, 0 (`saturation irradiance' at 0 C) and Q10 (a factor describing the increase in saturation irradiance caused by the temp erature increase (temp = 10 C)) should b e determined empirically. To a first approximation (according to Ostrowska 2000): KP U RPS P, 0 = 8.39 â 10-7 [Ein s-1 (mg tot. chl a)-1 ], Q10 = 1.9 [dimensionless].


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The authors intend to take into consideration all these remarks and modifications in their forthcoming pap ers.

References
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Ostrowska M., Ma jchrowski R., Matorin D. N., Woniak B., 2000a, Variability of the specific fluorescence of chlorophyl l in the ocean. Part 1. Theory of classical `in situ' chlorophyl l fluorometry, Oceanologia, 42 (2), 203­219. Ostrowska M., Matorin D. N., Ficek D., 2000b, Variability of the specific fluorescence of chlorophyl l in the ocean. Part 2. Fluorometric method of chlorophyl l a determination, Oceanologia, 42 (2), 221­229. Sapozhnikov V. V., Gorunova V. S., Levenko B. A., Dulov L. E., Antal T. K., Matorin D. N., 2000, Comparison of primary production determination in Norway Sea by different methods, Engl. Okeanologiya, 40 (2), 234­240. Schreiber U., Hormann H., Neubauer C., Klughammer C., 1995, Assessment of photosystem II photochemical quantum yield by chlorophyl l fluorescence quenching analysis, Plant Physiol., 22, 209­220. Slovacek R. E., Crowther D., Hind J., 1980, Relative activities of linear and cyclic electron flows during chloroplast CO2 -fixation, Biochim. Biophys. Acta, 592, 495­505. Sorokin Y. I., 1960, Method for measurement of primary production in the sea with 14 C, Proc. All-Union Soc. Hydrobiol., 10, 235­254, (in Russian). Steemann-Nielsen E., 1952, The use of radio-active carbon 14 C for measuring organic production in the sea, J. Cons. Int. Explor. Mer., 18 (3), 117­140. Vassiliev I. R., Prasil O., Wyman K. D., Kolber Z., Hanson A. K., Prentice J. E., Falkowski P. G., 1994, Inhibition of PS II photochemistry by PAR and UV radiation in natural phytoplankton communities, Photosynth. Res., 42, 61­64. Vinberg G. G., 1969, Primary production of water bodies, Russ. Acad. Sci., Minsk, 348 pp., (in Russian). Weis E., Berry J. T., 1987, Quantum efficiency of photosystem II in relation to energy-dependent quenching of chlorophyl l fluorescence, Biochim. Biophys. Acta, 894, 198­208. Williams P. J., Jenkinson N. W., 1982, A transportable microprocessor control led Winkler titration suitable for field and shipboard use, Limnol. Oceanogr., 27, 576­584. Woniak B., Dera J., Koblentz-Mishke O. J., 1992, Bio-optical relationships for estimating primary production in the Ocean, Oceanologia, 33 (1), 5­38. Woniak B., Dera J., Ma jchrowski R., Ficek D., Koblentz-Mishke O. J., Darecki M., 1997, `IO PAS initial model' of marine primary production for remote sensing applications, Oceanologia, 39 (4), 377­395.


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Appendix
List of symbols and abbreviations denoting the physical quantities Symbol apl, ¯
P SP

Denotes mean coefficient of solar irradiance absorption by phytoplankton photosynthetic pigments (PSP) in the 400­700 nm spectral range (PAR) mean coefficient of exciting flash absorption by PSP, averaged over the 400­550 nm spectral range mean absorption coefficient averaged with the weight of the spectrum scalar irradiance in the sea ratio of the mean absorption coefficient: mean in PAR range and averaged over the 400­550 nm spectral range

Units m-1

apl,P ¯

S P,f l

m-1

apl, ~

P SP

m-1

A

dimensionless

Amo

d

ratio of the mean absorption coefficient averaged with the weight of the spectrum scalar irradiance in the sea and averaged over the 400­550 nm spectral range value of A at the water surface sum of chlorophylls a + pheo, or total chlorophyll (chl a + divinyl chl a) concentrations averaged content of chlorophyll a in the water column an inhibitor of electron transport in PS II total irradiance in PAR range light irradiation at which half of the RC are in the closed state

dimensionless

A(0) Ca

dimensionless mg tot. chl a m-3

Cam

mg tot. chl a m-3

DCMU E E1/2

µE m-2 s µE m-2 s µE m-2 s

-1 -1

E1/2

m

value of parameter E1/2 averaged in the water column

-1


Measurement of phytoplankton photosynthesis rate ...

311

Appendix
List of symbols and abbreviations (continued) Symbol f Denotes relative number of functionally active PSII reaction centers in vivo phytoplankton fluorescence yield induced by a weak probe flash in the dark, under ambient light, and following a saturating flash, all measured in a light-adapted state F Fm,
DCMU

Units dimensionless

F0 , Fm

conv. units

variable fluorescence = Fm - F0 fluorescence measured after adding inhibitor DCMU coefficient defined by geometric characteristics and sensitivity of the fluorescence light sensor, (constant)

conv. units conv. units

G

Ifl

total intensity of fluorescence excitation light intensity of light passed through a suspension of algae of concentration n

quanta m-2 s quanta m-2 s

-1

In

-1

In,

c

intensity of light passed through a suspension of the algal cells of concentration n bleached by illumination in the presence of 1 mM hydroxylamine
,A

quanta m-2 s

-1

k (F0 km

)

proportionality coefficient value of parameter k averaged in the water column

dimensionless dimensionless


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T. K. Antal, P. S. Venediktov, D. N. Matorin et al.

Appendix
List of symbols and abbreviations (continued) Symbol
KP U RPS P (temp)

Denotes photosynthesis saturation PU RPS P energy `saturation irradiance' (e.g. photosynthesis saturation PU RPSP energy) at 0 C number of fluorescence and radiation profiles measured for the period of bottle exposure at a station carbon assimilation by phytoplankton primary production of phytoplankton primary production determined using the described method averaged in the water column photosynthetically available radiation

Units Ein (mg tot. chl a)-1 s
-1

KP U RPS

P, 0

Ein (mg tot. chl a)-1 s

-1

n

Pc

µM C m-3 s

-1

P

c

mg C m-3 time

-1

PPc

mg C m-3 time

-1

PAR

PU R



photosynthetically usable radiation (per unit of chlorophyll a mass)
P

Ein (mg tot. chl a)-1 s

-1

PU RPS

part of PU R due to photosynthetic pigments photosynthetic pigments photosystem 2

Ein (mg tot. chl a)-1 s

-1

PSP PS II


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313

Appendix
List of symbols and abbreviations (continued) Symbol Trophic type symbols: O M P E Q
10

Denotes

Units

oligotrophic mesotrophic intermediate eutrophic factor describing the increase in saturation irradiance caused by a temperature increase temp = 10 C relative number of functionally open reaction centers PS II in algal cells correlation coefficient reaction center depth dependence of A depth in the sea efficiency (quantum yield) of the conversion of absorbed energy in photosynthetic reactions efficiency of electron transfer from H2 O to CO2 quantum yield of fluorescence quantum yield of fluorescence in cells with open RC efficiency of photochemical conversion of light energy in open reaction centers spectral range of exciting light dimensionless dimensionless
-1

qp

dimensionless

r RC T (z ) z (E )

dimensionless

dimensionless m µM C µE
-1

e

fl F0

RC

µM electron µE



nm