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Chlorophyll fluorimetry as a metho d for studying light absorption by photosynthetic pigments in marine algae*

OCEANOLOGIA, 46 (4), 2004. pp. 519 ­ 531.
C

2004, by Institute of Oceanology PAS.
KEYWORDS

Fluorescence Photosynthetic pigments Light absorption Phytoplankton

Dmitrii N. Matorin1 Taras K. Antal1 Miroslawa Ostrowska2 Andrei B. Rubin1 Dariusz Ficek3 Roman Majchrowski3 Department of Biophysics, Faculty of Biology, Moscow State University, Moscow, 119899 Russia; e-mail: matorin@biophys.msu.ru Institute of Oceanology, Polish Academy of Sciences, PowstacÑw Warszawy 55, PL­81­712 Sopot, Poland
3 Pomeranian Pedagogical University, Arciszewskiego 22b, PL­76­200 Slupsk, Poland 2 1

Manuscript received 18 June 2004, reviewed 14 October 2004, accepted 5 November 2004.

Abstract Using laboratory cultures of algae and natural phytoplankton populations from Nhatrang Bay (South China Sea), the relationship between the chlorophyll fluorescence F0 , the chlorophyll a concentration Ca and light absorption capacities of algae cells was studied. It is shown that the ratio F0 /Ca depends mainly on the
* This work was carried out within the framework of IOPAN's statutory research, and also as part of pro ject PZB­KBN 056/PO4/2001/3 of the Institute of Physics, Pomeranian Pedagogical Academy, Slupsk. The complete text of the pap er is available at http:/ /www.iopan.gda.pl/oceanologia/


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species composition of the algae population; hence, the concentration Ca can be measured with the fluorescence metho d with acceptable accuracy only when the species composition of algae populations varies over a rather narrow range. The fluorescence F0 can, however, be a go o d index of the total absorption capacities of different phytoplankton species, because the intensity of F0 depends on the sum total of light absorbed by all photosynthetic pigments in a plant cell. Thus, the fluorescence F0 measures not only the concentration of chlorophyll a, but that of all photosynthetic pigment concentrations.

Introduction
Characterised by a high sensitivity and allowing rapid non-invasive assessment of several characteristics of phytoplankton, the methods of measuring chlorophyll fluorescence have b ecome very p opular in recent times (Matorin & Venediktov 1990, Falkowski & Raven 1997, Fadeev et al. 1999, Ostrowska et al. 2000a, b, Ficek et al. 2000, Ostrowska 2001, Woniak et al. 2002). It was demonstrated some time ago that fluorescence intensity is correlated with phytoplankton concentration as measured by routine methods, for example, from the cell concentration or biomass (Karabashev 1987, Sirenko et al. 1988). However, the most frequently used characteristic of the phytoplankton content is the chlorophyll a concentration Ca (Vinb erg 1969, Foy 1987), which has long b een determined in vitro by measuring fluorescence in pigment extracts (Yentsch & Menzel 1963, Lorenzen 1966). Measurements of chlorophyll fluorescence, F0 , excited artificially by weak light, are now a common way of determining chlorophyll concentration in situ. Many authors have shown that F0 correlates well with the chlorophyll concentration in natural water b odies. For example, Vedernikov et al. (1990) showed that, in the Black Sea, the correlation coefficients b etween these characteristics were equal to 0.7­0.85. Such a high correlation is not normally the rule, however. This may b e due to the fact that the value of F0 is prop ortional to the total content of photosynthetic pigments (carotenoids, chlorophylls a, b, c, etc.), whereas the fraction of chlorophyll a in pigments varies over a wide range, dep ending b oth on the taxonomic affiliation of the algae (Cullen 1982, Soo Hoo et al. 1986, Matorin & Venediktov 1990) and on such environmental conditions as irradiance, concentration of mineral nutrients, and p ollutants (Kolb er et al. 1988). We have b een able to determine chlorophyll a concentrations much more accurately by using not only F0 measurements but also our own physical models of light absorption and fluorescence in different natural plant communities (Woniak et al. 1999, 2000, 2003, Ma jchrowski et al. 2000, Ostrowska et al. 2000a, b, Ma jchrowski 2001). Our method takes into consideration the fact that F0 reflects the total concentration of photosynthetic pigments. Treated as a characteristic of light absorption by algae, it can then b e used to determine


Chlorophyll fluorimetry as a method for studying light absorption . . .

521

the photosynthetic production of phytoplankton (Antal et al. 1999, Ficek 2001, Ostrowska 2001, Woniak et al. 2002). The aim of the present work is to investigate and present the relationships b etween fluorescence F0 , chlorophyll a concentration Ca and the light absorption capacities of cell susp ensions using lab oratory cultures of algae and natural phytoplankton. Theoretical background The fluorescence F0 , excited by an artificial light source, has b een measured in dark-adapted algae in which the PS I I reaction centres are op en, so that absorb ed light energy can b e converted into chemical b ond energy with maximum efficiency (Klughammer 1992). The intensity of F0 can b e calculated from the equation F0 = G Ifl apl, P S P, f l F0 , where 1 Ifl ­ total intensity of exciting flash (in our fluorimeter, Ifl () was nearly uniformly distributed over the 400­550 nm sp ectral range), (constant); apl, P S P, f l [m-1 ] ­ coefficient of absorption of the exciting flash by photosynthetic pigments, averaged over the 400­550 nm sp ectral range; F0 ­ quantum yield of fluorescence in cells with op en RC; G ­ coefficient defined by the geometric characteristics and the sensitivity of the fluorescence light sensor, (constant). 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: 1 apl, P S P = const -0 AF0 , F where A = apl
, P SP

/apl

, P S P, f l

To simplify the analysis, we have assumed that A 1, b ecause the sp ectrum of the exciting light in our fluorimeter is similar to that of natural light in the sea. Thus, apl, P S P apl, P S P, f l . In reality, parameter A can vary for different trophic typ es of sea and also with depth. This problem is discussed in Antal et al. (2001); see also Ostrowska et al. (2000a, 2001).

Material and Methods
The exp eriments were carried out with three cultures of algae: the green algae Chlorel la pyrenoidosa, Platymonas viridis, and Ankistrodesmus sp.,
1

See the app endix for the list of symb ols used in the text.


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D. N. Matorin, T. K. Antal, M. Ostrowska, A. B. Rubin et al.

the yellow-green alga Nephrochloris salina, the diatom Thalassiosira weissflogii, and natural phytoplankton from Nhatrang Bay (Vietnam). Ch. pyrenoidosa (thermophilic strain CALU-175 from the collection of the Biology Institute, St. Petersburg State University, Russia) was grown in 1/5 Tamiya medium (pH 6.8) (Tamiya et al. 1961) at 32 C, under 60 µE m-2 s-1 background irradiation, and with moisturised air b eing bubbled through it. The culture was maintained in a log growth phase by daily dilution with fresh medium to maintain a cell density of ab out 1­10 â 105 cells ml-1 . The marine algae P. viridis, Ankistrodesmus sp., Th. weissflogii and N. salina were grown at 20 C under indirect daylight with a maximum light flux density of 120 µE m-2 s-1 in an artificial medium describ ed in Lanskaya (1971). The mean effective absorption coefficient for blue light (sp ectrum similar to the sp ectrum of the flash-exciting F0 in the fluorimeter) for cells in the algal susp ension, apl, blue , were measured with a lab oratory apparatus. Light ~ from a KGM 150/24 halogen slide-pro jector lamp was passed through a bluegreen filter SZS-22 with < 600 nm and a dark chamb er l =0.2 m in length filled with the sample, and the output quantum flux density was measured with a lab oratory-made quantum sensor. The calculations were p erformed according to the formula apl ~
, blue -2

= (Inc - In )/(l Inc ) or apl ~
-1

, blue

= (ln Inc - ln In )/l,

] ­ the intensity of light passed through the algal susp ension of concentration n ; -2 s-1 ] ­ the intensity of light passed through the susp ension of In,c [µE m algal cells bleached by illumination in the presence of 1 mM hydroxylamine. The chlorophyll a concentration Ca in the algae samples was measured on a HITACHI-557-SPECTR (Japan) sp ectrophotometer using a routine method (Parsons & Strickland 1963). Measurements of phytoplankton fluorescence were carried out over the whole area of Nhatrang Bay (Vietnam) from 23rd March to 5th April 1998. At each station, vertical profiles of fluorescence F0 were measured in situ with a submersible fluorimeter. Functioning on the principle of the pumpand-prob e method (Mauzerall 1972, Kolb er et al. 1990), this fluorimeter was designed at the Faculty of Biology of the Moscow State University for the real-time measurement of initial and maximum fluorescences (F0 , and Fm ), as well as underwater irradiation, temp erature and pressure (depth) (Matorin et al. 1996, Antal et al. 1999). The fluorimeter was also used to measure the fluorescence of algae under lab oratory conditions, s

where In [µE m


Chlorophyll fluorimetry as a method for studying light absorption . . .

523

the fluorescence-exciting impulses b eing generated by an SSh-20 (MELZ, Russia) xenon lamp. The flashes were isolated from the sample by the bluegreen filter SZS-22. The sp ectrum of the fluorescence excitation light was distributed practically uniformly within the range of wavelengths from 400 to 520 nm. The fluorescence signal was recorded by a photomultiplier-68 after having passed through a KS-17 cut-off glass filter ( > 680 nm).

Results and discussion
Fig. 1 shows the dep endence of F0 on Ca , measured under lab oratory conditions with a susp ension of Ch. pyrenoidosa cells. It is evident that this dep endence is nearly linear over the range of naturally occurring phytoplankton concentrations (Ca < 16 mg m-3 ). Some deviation from linearity was observed only near the limits of the sensitivity ranges when the sensitivity of the sensors was changed. As follows from the curve, the F0 signal can b e automatically converted into chlorophyll a concentration units in the course of in situ measurements. However, this method yields only an approximate value of the chlorophyll concentration and can b e used, for example, for a quick assessment of the trophic status of water b odies; for more accurate estimates of Ca content, one should take into account the fact that the F0 /Ca ratio is a function of the sp ecies comp osition of the microalgae. Fig. 2 shows F0 as a function of Ca in four sp ecies of marine algae from three different taxa grown under lab oratory conditions. Here we see that in the yellow-green (N. salina ) and diatomaceous (Th. weissflogii )
4

fluorescence F0 [r.u.â103]

3

2

1

0

0

4

8

12

16

chlorophyll a concentration Ca [mg m­3]

Fig. 1. Dependence of F0 yield on chlorophyll a concentration in a suspension of Chlorel la vulgaris cells. Vertical lines show the points at which the fluorescence sensor sensitivity was altered


524

D. N. Matorin, T. K. Antal, M. Ostrowska, A. B. Rubin et al.
1

fluorescence F0 [r.u.â103]

2.0 1.5 1.0 0.5 0.0

2 3

4

0

2

4

6

8

chlorophyll a concentration Ca [mg m­3]

Fig. 2. F0 vs Ca in suspensions of marine algae from three different taxa: diatom Thalassiosira weissflogii (1), yellow-green Nephrochloris salina (2) green Ankistrodesmus sp. and Platymonas viridis (3 and 4 respectively). The algae were grown under laboratory conditions

algae, in which chlorophyll comprises the least part of the light-harvesting pigments, the intensity of F0 p er unit of Ca concentration was ab out 2­3 times higher than in green alga, in which chlorophyll is the ma jor lightharvesting pigment. The F0 /Ca ratio in these algae is correlated with the pigment index (the a430 /a663 ratio in a 90% acetone extract), which characterises the contribution of carotenoids to the total light absorption (Margalef 1963, Matorin et al. 1997). The ratio F0 /Ca is also different in natural phytoplankton in the same water b ody. For example, deep-sea and shore stations in Nhatrang Bay (Fig. 3) and in the Baltic Sea (data not shown) differed significantly in their F0 dep endence on chlorophyll a concentration. These results show that the light-harvesting complexes of algal cells from shore regions were richer in chlorophyll a than algae from deep-water regions. This may b e related to a shift in the ratio of dominant algal sp ecies and to the high levels of mineral nutrients in shore waters, and also to photo-acclimation to the changing light environment. Previously, we showed that a deficiency in the main mineral nutrients (nitrogen, phosphorus) in the culture medium led to a smaller contribution of chlorophyll a to the light-harvesting complex (Matorin et al. 1997). Earlier, F0 was shown to correlate more closely with another phytoplankton characteristic ­ the light absorption capacity of the photosynthetic pigments in PS I I centres (Ostrowska et al. 2000a, Antal et al. 2001). In principle, the intensity of F0 is prop ortional to apl, P S P ,


Chlorophyll fluorimetry as a method for studying light absorption . . .
3.6 3.6

525

a
fluorescence F0 [r.u.â103]
2.4 2.4

b

1.2

1.2

0.0 0.0

0.1

0.2

0.3

0.0 0.0

0.1

0.2

0.3

chlorophyll a concentration Ca [mg m­3]

chlorophyll a concentration Ca [mg m­3]

Fig. 3. Dependences of F0 on Ca in deep water (a) and coastal (b) stations of Nhatrang Bay and the results of the linear regression of these dependences. The values of F0 and Ca are averaged over the water column

the constant distribution light in the 2001). Thes
5 4 3 2 1 0

of prop ortionality b eing dep endent on F0 , if of the light exciting F0 is similar to that of sea, i.e. A = 1 (see Ostrowska et al. 2000a, e assumptions were confirmed by studies of the
3 21

the sp ectral the natural Antal et al. relationship

fluorescence F0 [r.u.â103]

0.0

0.1

0.2

0.3

0.4

mean effective absorption coefficient for blue light ~ apl, blue [m-1]

Fig. 4. Dependences of F0 on apl, blue in suspensions of marine algae from ~ three different taxa: diatom Thalassiosira weissflogii (square), green Chlorel la pyrenoidosa (circle) and yellow-green Nephrochloris salina (triangle). The results of the linear regression of these dependences are marked 1, 2 and 3 respectively. The algae were grown under laboratory conditions


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D. N. Matorin, T. K. Antal, M. Ostrowska, A. B. Rubin et al.

b etween F0 and the absorption coefficient of blue light, apl, blue in cell ~ susp ensions of various groups of marine algae. Exp erimental relatinships ~ of F0 as a function of apl, blue were measured for green (Ch. pyrenoidosa ), diatomaceous (Th. weissflogii ) and yellow-green (N. salina ) algae over the range of natural concentrations (Fig. 4). The algae were grown under optimum conditions (for example, at a low irradiance level): we may assume, therefore, that the values of F0 in these sp ecies of algae are similar. ~ Determined for each group from the linear regression method, the F0 /apl, blue ratios were almost identical, whereas the F0 /Ca ratios varied greatly among ~ the algal taxa (see Fig. 2). Small differences in the F0 /apl, blue ratios may b e due to the difference b etween the light absorption capacity of whole cells and that of the PS I I photosynthetic pigments, i.e. apl, blue = apl, P S P, f l . ~ The so-called packaging effect could also b e the reason for such differences. This problem has b een analysed in detail in our previous pap ers (Woniak et al. 1999, 2000). As noted ab ove, the relationship b etween apl, P S P and F0 dep ends on F0 and factor A, which can vary. Previous studies of natural phytoplankton p opulations have shown that in some p olluted areas, near the surface, the quantum yield of F0 was suppressed (Antal et al. 2001); nevertheless, this was observed rather rarely, and the value F0 can b e assumed constant (Ostrowska et al. 2000a). We also showed that the sp ectrum of underwater irradiation in natural water b odies from a depth of 5 m to the lower limit of the euphotic zone is very close to that of the exciting F0 light of the
excitation irradiance I [arbitrary units] relative underwater irradiance E(l)/E(lmax)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 400 5m 20 m 500 20 m 5m 1m 1m

600

700

wavelength l [nm]

Fig. 5. Spectrum of the light used to excite chlorophyll fluorescence in the PrimPro d fluorimeter (solid line), and the spectral distribution of underwater irradiance in the sea at different depths (after Antal et al. 2001)


Chlorophyll fluorimetry as a method for studying light absorption . . .

527

fluorimeter (400­600 nm), i.e. A 1 (see Fig. 5). Hence, estimates of the light absorption capacity by measuring F0 are probably correct over most of the photosynthetic zone. However, this assumption may b e not true for highly eutrophic waters (Ca > 10 mg m-3 ), in which the fraction of blue light (< 500) decreases at a depth of 10 m and more b ecause it is absorb ed by yellow substances, and green light with its sp ectral maximum at 550 nm is dominant (Dera 1995).

Conclusions
We analysed the relationships b etween fluorescence F0 and chlorophyll a concentration as well as b etween fluorescence F0 and total absorption earlier for a comprehensive empirical in situ database of different natural plant communities in the World Ocean (Fig. 6) (Ostrowska et al. 2000a, b). The correlation coefficients for these empirical data are r =0.916 for F0 = f (Ca ) and r =0.942 for F0 = f (apl, P S P ). Evidently, the general tendency in algae from different seas is that the fluorescence F0 is more closely related to light absorption capacities than to the chlorophyll concentration. The results of the analyses presented in this work for lab oratory cultures and natural phytoplankton p opulations confirm our earlier results.
10000 10000

a
fluorescence F0 [r.u.]
1000 1000

b

100

100

10 r = 0.916 1 0.001 0.01 0.1 1 10 100

10 r = 0.942 1 0.0001 0.001 0.01 0.1 1

chlorophyll a concentration Ca [mg m­3]

light absorption coefficient apl, PSP, fl [m-1]

Fig. 6. The relationship between fluorescence F0 and chlorophyll a concentration (a) and fluorescence F0 and the light absorption co efficient apl,P S P,f l (b), for data from different regions of the World Ocean (after Ostrowska et al. 2000b)

Our data show that measurement of F0 for in situ estimation of chlorophyll a content should b e p erformed after fluorescence calibration in a particular area, i.e. when the spatial and temp oral variations in the sp ecies comp osition of phytoplankton are not significant. The fraction


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D. N. Matorin, T. K. Antal, M. Ostrowska, A. B. Rubin et al.

of chlorophyll a in the total photosynthetic pigments in algal cells can b e estimated by calibrating F0 on Ca , thus enabling the dominant algal groups in the region to b e characterised. It is more correct to use F0 for estimating the light absorption capacity by phytoplankton cells b ecause the fluorescence b etter reflects this characteristic. The light absorption capacity is a more appropriate measure of phytoplankton abundance than Ca , b ecause it is prop ortional to the concentration of the total photosynthetic pigments. In addition, the light absorption capacity can b e used to estimate photosynthetic production in phytoplankton (Antal et al. 2001, Ostrowska 2001).

Acknowledgements
We would like to thank Dr. Ho Hai Sam (Oceanography Institute, Nha Trang , Vietnam) for the data on chlorophyll concentration, and the Russian-Vietnamese Trop center for the opp ortunity to conduct these studies in Vietnam. This work was carried out in accordance with the World Ocean Program (Russia).

References
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Appendix
List of the main symb ols and abbreviations used in this pap er
Symbol a ~ a
pl, blue pl, P S P, f l

Denotes

Units

a

pl, P S P

A

Ca E () F0 ,Fm

G

In In,
c

I () Ifl PAR PSP PS II RC Fo

mean effective absorption co efficient for m-1 blue light mean co efficient of exciting flash absorption m-1 by PSP, averaged over the 400­550 nm spectral range mean co efficient of light absorption by PSP, m-1 averaged over the 400­700 nm spectral range (PAR) ratio of the mean absorption co efficient: dimensionless mean in PAR range and averaged over the 400­550 nm spectral range sum of chlorophylls a + pheo, or total mg tot.chl a m-3 chlorophyll (chl a + divinyl chl a ) concentrations spectral scalar irradiance Ein m-2 s-1 nm-1 in vivo phytoplankton fluorescence yield relative units induced by a weak probe flash in the dark (initial), and following a saturating flash (maximum), measured in a light-adapted state co efficient defined by geometric relative units characteristics and sensitivity of the fluorescence light sensor (constant) intensity of light passed through µEin m-2 s-1 a suspension of algae of concentration n intensity of light passed through a suspension µEin m-2 s-1 of algae of concentration n bleached by illumination in the presence of 1 mM hydroxylamine spectrum of light excitation ­ depends quanta m-2 nm-1 s on the light source used by the instrument total intensity of exciting flash (constant) quanta m-2 s-1 photosynthetically available radiation photosynthetic pigments photosystem II reaction centre quantum yield of fluorescence in cells dimensionless with open RC light wavelength nm

-1