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Oxygen evolution and photosynthetic energy storage during the cell cycle of green alga Scenedesmus armatus characterized by photoacoustic spectroscopy
J. Plant Physiol. 158. 1061 – 1067 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
Oxygen evolution and photosynthetic energy storage during the cell cycle of green alga Scenedesmus armatus characterized by photoacoustic spectroscopy Janusz Szurkowski1 *, Agnieszka Ba´scik-Remisiewicz2, Krystyna Matusiak2, Zbigniew Tukaj2 1
Institute of Experimental Physics, University of Gdansk, ´ Wita Stwosza 57, 80-952 Gdansk, ´ Poland
2
Department of Plant Physiology, University of Gdansk, ´ Al. Marszałka J. Piłsudskiego 46, 81-378 Gdynia, Poland
Received August 25, 2000 · Accepted February 19, 2001
Summary The photosynthetic activity of Scenedesmus armatus, synchronized by light/dark regime of 14/10 hours, was characterized using the photoacoustic (PA) spectroscopy technique. Frequency-dependent analysis of the PA signal was carried out to separate its photobaric and photothermal components. The photochemical energy storage (ES) measured at a high frequency of 180 Hz and the efficiency of oxygen evolution measured at a low frequency of 4 Hz were found to have different runs during the light phase (14 h) of the cell cycle. ES changed from 27% for young autospores illuminated for 0.5 h to 35 % for mother cells starting to release the next generation of autospores. The efficiency of oxygen evolution rapidly increased, reaching the maximum value in 3 – 4 h, and next gradually declined with time during the cell cycle. The time of the photothermal component of the PA signal creation measured in a PA cell changed from 1.63 to 1.87 ms. The coefficient of oxygen diffusion through the cell wall was 7.84 · 10 –7 cm2 s –1. In view of a very good correlation between the PT signal and the cell volume during the cell cycle, the PA technique can also be employed to monitor the growth of cells or biomass production. Key words: cell cycle – energy storage – photoacoustic spectroscopy – oxygen evolution – Scenedesmus Abbreviations: Ath amplitude of photothermal signals. – Aox amplitude of photobaric signals. – ES energy storage. – PA photoacoustic. – PS photosystem
Introduction Light-induced, synchronous cultures of chlorococcalean green algae Scenedesmus are frequently used to study the * E-mail corresponding author: [email protected]
cell cycle. Depending on culture conditions, these autotrophic organisms form unicells or multicellular coenobia, are haploidal during their cell cycle, and divide by the multiple fission. Different strains and mutants of S. obliquus and S. quadricauda are usually investigated, and photosynthesis is one of the processes to which particular attention has been 0176-1617/01/158/08-1061 $ 15.00/0
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paid (see review of Krupinska and Humbeck 1994). Each Scenedesmus cell contains a single chloroplast dividing before the cell cycle termination. Due to its partial genetic autonomy, it has its own cycle divided into characteristic phases (Zachleder et al. 1995). Both changes in the fine structure of the chloroplast (Nilshammer and Walles 1974) and molecular organization of the photosynthetic apparatus during the cell cycle (Senger and Krupinska 1986) are quite well known. Therefore, synchronized cultures of Scenedesmus, relatively simple to obtain, are a convenient tool for studying biochemical and biophysical processes within chloroplasts and metabolic relationships occurring during the cell cycle. The light energy absorbed by chloroplastic pigments is in part stored in photosynthetic products and in part dissipated in the form of heat and fluorescence. The overall photosynthetic activity of algae is measured as the rate of the oxygen evolution and photoassimilatory fixation of inorganic carbon, both appearing to fluctuate strongly during the cell cycle. A Clark-type oxygen electrode is most frequently used for the determination of photosynthetic production oxygen and C14 incorporation into the carbohydrate as a measure of c-assimilation. In addition to these two most popular methods, chlorophyll fluorescence has also been used for many years as a noninvasive indicator for the assessment of in vivo photosynthesis (Schreiber et al. 1993). A series of fluorescence parameters provide useful information on both photophysical processes occurring in photosystems and photosynthetic electron transfer between them, recently applied to characterization of photosynthetic systems of S. obliquus, (Strasser et al. 1999) and S. quadricauda (Kaftan and al. 1999). Some other information can be obtained from measurements of the thermal dissipation carried out using a photoacoustic technique, applied also to study the photosynthesis of both intact algae (Herbert et al. 1990, Cha and Mauzerall 1992) and those exposed to abiotic stress (Tukaj and Szurkowski 1993, Szurkowski and Tukaj 1995). The PA effect is the production of pressure modulations within and around a sample when it absorbs intensity-modulated light. In the case of photosynthetic samples in a gas volume, thermal expansion of the gas surrounding the sample and photosynthetic oxygen evolution are the major contributors to the acoustic wave. Since heat diffuses more rapidly from chloroplasts to the extracellular gas than does oxygen, the photothermal and oxygen (photobaric) components of the photoacoustic signal can be separated (Poulet et al. 1983). In the photosynthetic system, the photothermal component of the PA signal represents conversion of the absorbed light energy to heat. The photothermal part of the photoacoustic signal is reduced by a fraction equal to the part of the absorbed energy stored by the photosynthetic process as chemical energy. By measuring heat emission in the presence or absence of a nonmodulated, saturating light background, the photosynthetic ES is evaluated. The oxygen component of the PA signal consists largely of oxygen evolution at photosystem (PS) II. Two O2-consuming processes such as
photorespiration and respiration are considered as too slow to be recorded by the PA-spectroscopy (Charland et al. 1992). Moreover, when photosynthesis is at a low level, it is possible that the Mehler type O2 uptake in PS I may be modulated by and contributed to the photobaric signal (see review of Malkin and Canaani 1994). In the present paper, we have tried to apply photoacoustic spectroscopy for characterization of the photosynthetic system of Scenedesmus armatus grown in cultures synchronized by the light/dark regime. In vivo measurements of the cell volume, the efficiency of photosynthetic oxygen evolution, and photochemical energy storage are described. The time of photothermal signal creation in a photoacousic cell and the coefficient of oxygen diffusion through the cell wall are calculated. Heterogeneity of PS II is also considered and discussed in relation to results obtained by other authors. All measurements were carried out during the light phase (0.5 – 14 h) of the cell cycle, and the term «cell cycle» in this work refers to this period only.
Materials and Methods Organism and cultures The green microalga Scenedesmus armatus used in this study, isolated from the phytoplankton of the southern Baltic, was obtained from culture collection of the Polish Academy of Sciences’ Institute of Oceanology (Sopot), where it is assigned as the B1-76 strain. Algae from bacterial slants were transferred to liquid BBM medium (Nichols and Bold 1965) and incubated for a few days in precultures to adapt them to the conditions of the batch culture. Suspensions of cells were cultured in plate-parallel vessels of a 600 mL volume, which were immersed in a water bath at a constant temperature of 30 ˚C. Cultures were illuminated from one side by 10 fluorescent tubes, cool white (Philips TLD 36W/94), with an irradiance at the surface of the culture vessels of 60 W · m – 2. The cultures were aerated with atmospheric air enriched with CO2 (2 %, v/v), and sterilized by passing through a filter (Sartorius 2000; 0.2 µm PTFE). Algae were synchronized by alternating light and dark periods (14 : 10 h). The lengths of the light periods of the cell cycle were chosen so that the light was switched off when the first daughter cells of the population were released. Ten hours of the dark period were sufficient to terminate the daughter cell release. At least 3 light/dark cycles were needed for sufficient synchronization. After each dark phase, the cultures were diluted at the beginning of the light periods to a constant density of about 1.5 × 106 cells per 1cm3. Cell numbers were determined by counting in the Bürker chamber using standard procedure. Cells dimensions were measured under a microscope, and cell volume was calculated assuming the Scenedesmus armatus cell to be a rotational ellipsoid.
Samples preparation and phtoacoustic measurements The samples were prepared using a modified version of the method described by Carpentier et al. (1983). The algae were deposited on a Sartorius SM 11306 membrane filter (0.45 µm pore size). A 15 mm ring was cut out from the filter 3 – 4 min after filtration and the sample was
Photosynthesis in the cell cycle of Scenedesmus then introduced into the photoacoustic cell. The volume of the culture was filtered in such a manner that about 106 cells were deposited on the sample, covering the filter-ring with a monolayer of cells. In the studies presented, an experimental set-up based on a lockin amplifier SR 850 was used, which not only measured the signal but also controlled the operation of the laser diode that was a source of the modulated light (Szurkowski and Wartewig 1999). A laser diode (type SDL-73311) emitted modulated light with a wavelength of 680 nm. The laser beam was provided through a fiber wire to the measuring cell constructed by the authors. Nonmodulated white background light from a fiber optic illuminator (PL-800) was focused onto the sample through a fiber-optic light guide. The photoacoustic signal from a microphone is phase-sensitive detected using a lock-in amplifier (type SR 850). An internal generator of the amplifier was also used for operating the laser diode controller (LPC 400). The entire data collection and further processing was performed on a PC working in the GPIB standard. The introduction of the laser diode and electronic light modulation system, compared to its mechanical analogue, allowed a higher signal-to-noise ratio to be achieved. The light modulation frequency varied from 4 to about 200 Hz. The photoacoustic signal of the sample was analyzed as a vectorial summation of photothermal and photobaric contributions, according to the method of Poulet et al. (1983). The amplitude of the oxygen signal (Aox) was evaluated using the following equation: Aox =
√A
2 th
+ A2 – 2 A th A cos (ϕ)
(1)
where Ath and A are the amplitude of photothermal and total signals, respectively, in the absence of background light, and ϕ – is the phase of the total signal versus the photothermal one. Ath was calculated as follows: Ath = K · ATth
(2)
where K is the ratio of the acoustic signal in the absence of background light to the signal in the presence of background light at 180 Hz modulation frequency, and AthT is the signal in the presence of background light at the given frequency. It should be noted that the above equations are based on the assumption that practically no change in the phase angle occurs in the photothermal signal when the background light is applied. This was verified experimentally in the high-frequency limit for photoacoustic measurements (Poulet et al. 1983), and also at low frequency by direct photothermal radiometry measurements (Kanstad et al. 1983).
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reactions are not saturated, and a reduced photoacoustic signal is observed as a result of the storage of a substantial part of the absorbed light as photosynthesis products. All measurements were carried out at room temperature with a measuring light wavelength of 680 nm. The modulated light intensity was equal to 25 µmol photons m – 2 s –1 and the nonmodulated background actinic light of 2500 µmol photons m – 2 s –1 were used.
Results Under growth conditions and with the medium composition used in this work, Scenedesmus armatus forms only unicells. In synchronized populations of this strain, three sequences of reproductive processes such as DNA replication, nuclear division, and protoplast fission are triggered, resulting in the formation of 8 daughter cells (Tukaj et al. 1996). Hence, presumably, an 8-fold increase in the cell volume from about 200 µm3 (young autospores) to 1600 µm3 (mother cells) was observed (Fig. 2), and a new generation of autospores started to release at the end of the light period of the cycle (13–14 h) and terminated during next 10 h in the dark. The photoacoustic (PA) signal gathered from the photosynthetic material evolving oxygen contains at least two contributions. Both can be separated by frequency-dependent measurements (Bults at al. 1981). At a low frequency, the PA signal comprises both modulated heat and oxygen evolution, whereas at high frequencies it represents only heat emission. The photothermal signal amplitude versus the time of culture of Scenedesmus armatus in light period is depicted in Figure 1. The relation is assumed to be linear and the correlation coefficient is 0.99. A similar relation has already been evaluated as useful in monitoring the number of cells in a fermentation by Schmidt and Beckmann (1998). According to the Rosencwaig-Gersho theory (1976), the amplitude of the photothermal signal is proportional to the optical density of the sam-
Photoacoustic energy storage measurements Energy storage was calculated and expressed as a percentage: A bc – A A bc
· 100
(3)
where Abc is the photoacoustic signal produced by the modulated light plus the nonmodulated background light, and A is the photoacoustic signal in the modulated light alone. The application of strong background light results in the saturation of photochemical reactions in the sample, increasing the conversion of the absorbed modulated light to heat to nearly 100 %, and producing a maximum photoacoustic signal proportional to the absorption of the modulated light by the sample. In the presence of the modulated light alone, photochemical
Figure 1. Photothermal signal amplitude changes during the light period of Scenedesmus cell cycle. PA signal was gathered at 4 Hz and derived from 106 cells deposited on membrane filter. R is the correlation coefficient.
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Figure 2. Relationship between the photothermal signal amplitude and cell volume. The samples collected from the same culture were divided into 2 parts and immediately analyzed in spectrometer and under microscope. Linear dimensions of at least 20 randomly chosen cells were measured to calculate the cell volume. R is the correlation coefficient.
ple. Since the total number of algae in the sample was the same (about 106 cells on the filter ring), in the subsequent measuring runs during the cell cycle, the increase in the photothermal signal amplitude corresponds to the increase of the signal coming from one cell, i.e. to the linear increase of the cell biomass during its growth. From the dependence of the photothermal signal amplitude on the cell volume (Fig. 2), it is clear that the biomass growth during the cell cycle is related to the increase of the cell volume. Thus, the photothermal signal amplitude can be a good measure of the culture biomass and dimensions of the cells growing therein. A natural limitation of this technique when applied to a water culture is the effect of light scattering by particles and gas bubbles on the measurements. However, even in a water culture, the photoacoustic method is much less affected than the turbidity method (Schmidt and Backmann 1998). The photobaric signal amplitude allowed the estimation of both the rate and yield of oxygen evolution during the cell cycle (Fig. 3). The rate of oxygen evolution per cell during the cell cycle grows linearly with the time of culture (Fig. 3 a). As opposed to the above, a quite different run was observed when the ratios of photobaric to photothermal signal amplitude were calculated (Fig. 3 b). This corresponds to the yield of oxygen evolution per unit of biomass. During the first 3 – 4 h of the 14 h light period, the O2 evolution rapidly increased. After 4 h, the yield steadily decreased, reaching its minimum at the 9th h of the cell cycle. Despite a different method, a similar dependence has already been observed for a light-saturated rate of O2 evolution of synchronously grown S. quadricauda (Kaftan et al. 1999) and S. obliquus (Strasser et al.
1999), measured using the Clark electrode and fluorescence methods, respectively. These authors explain the increase in the steady-state O2 evolution rate at the beginning of the light period by the increased rate of the electron flow subsequent to PS II, and by the decline observed toward the end of the light period due to limited PS II activity. Figure 4 shows that the energy storage (ES) calculated from Eq. 3. ranged from 27 % for young autospores illuminated for 0.5 h to 35 % for mother cells at the end of the cell cycle. At the beginning of the cell cycle, as in the case of oxygen evolution efficiency, the ES increase was fast up to 3 – 4 h, then it became slower, reaching an almost constant value in the middle of the cell cycle (33 %). The results are consistent with those reported by Cha and Mauzerall (1992), who used the pulsed time-resolved photoacoustics and showed that the overall thermal efficiency (energy storage) in asynchronously growing Chlorella cells was 30 %, the mean value we obtained. Just before the dark period, a value of about 35 % was measured, also obtained theoretically (Clayton 1980). The application of a two-phase amplifier allowed the measurement of the characteristic time of the photothermal signal creation. For signal analyses at modulation frequencies of about 200 Hz (where only the photothermal signal is dealt with), the phase-modulation method was employed. It is
Figure 3. Changes in the photobaric signal amplitude (a) and the ratio of photobaric to photothermal signal (b) during the light period of the cell cycle. Photobaric signal gathered at 4 Hz relates to oxygen evolution, and the ratios correspond to oxygen evolution yield per biomass unit.
Photosynthesis in the cell cycle of Scenedesmus
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widely used in fluorescence decay time studies (Lakowicz 1983). According to Lakowicz, by using a fluorometer with a single modulation frequency (f), it is possible to determine the characteristic decay time (τ), assuming a one-exponent decay from the equation: ϕ = arctg(2 πfτ)
(4)
Since in our measurements the photothermal signal is studied instead of fluorescence signals, the same mathematical approach can be applied. A variability of the time characterizing the photothermal signal creation, measured at the modulation frequency 180 Hz, is presented in Figure 5. The time increases monotonically from (1.63 ± 0.03) ms to (1.87 ± 0.03) ms during the light cycle. As known from fluorescence studies of higher plant chloroplasts, at least two components, differing significantly in their decay times and related to PS II heterogenity, can be distinguished (Melis et. al. 1988). Both subpopulations of PS II display equal fluorescence quantum yields (Kaftan et al. 1999). Despite the population balance change between them, no decrease of the energy storage at the end of the light cell cycle can be seen in Figure 4. Measurements carried out during the cell cycle of the green alga Scenedesmus quadricauda by Kaftan et al. (1999) yielded the inactive PS II of approximately 3 % of the total PS II at the beginning of the cell cycle, and about 15 % in the dividing cells. Similar results were also obtained for green alga Scenedesmus obliquus (Strasser et al. 1999). Here, we report a study on the oxygen-evolution (photobaric) signal as a function of the frequency of modulated light. The relative damping of the photobaric signal, compared to the photothermal one, can be explained by the diffusion of oxygen from the chloroplasts through the cell wall to the gaseous phase, and by the electron-transport reactions occurring in the time between the photochemical act and water
Figure 4. Changes of energy storage of Scenedesmus cells during the cell cycle. The intensity of the measuring light (680 nm) was 25 µmol photons m – 2 s –1, the background actinic light was 2500 µmol photons m – 2 s –1, and modulation frequency was 180 Hz.
Figure 5. Time, which characterises the generation of the photoacoustic signal measured at 180 Hz in a PA cell, during the cell cycle.
Figure 6. Logarithm of the ratio of the photobaric (Aox) to photothermal (Ath) signal amplitude, as a function of square root of the modulation frequency for algae from the 13th hour of the cell cycle. Linear dependence is the best-fit approximation to the experimental data in the form: y = ax + b (R = – 0.997), which yields the diffusion coefficient Dox = 7.84*10 –7 cm2 s –1.
splitting (Poulet et al. 1983). It can be shown that for lower frequencies the principal role in the attenuation of the photobaric signal is played by the diffusional process, whereas at higher modulation frequencies ( ≥ 200 Hz), the signal is damped at the source by a kinetic factor due to the chain of electrontransport reactions between the photochemical reaction and the step of oxygen formation (Bults et al. 1982). The logarithm of Aox/Ath as a function of the square root of the modulation frequency is plotted in Figure 6. Since the amplitudes of the modulated oxygen concentration field and temperature field are attenuated exponentially at the cell 1 boundary, the plot of ln(Aox/Ath) versus (f) ⁄2 tends to be a straight line in the low frequency range, from the slope of
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which Dox can be computed using the formula proposed by Poulet et al. (1983); ln
A ox A th
=–
√π f 冢
1
–
1
√D √D ox
th
冣
l,
(5)
where; Dth – heat diffusion coefficient (diffusivity), Dox – oxygen diffusion coefficient in the aqueous medium of the cell, l – distance from the source. When reasonable values are assumed for l and Dth (Dth = 1.44 · 10 – 3 cm2 s –1), the term it is comparable to
√
1 can usually be neglected if D th
√
1 and omitted in Eq. 5. Hence, a simD ox
plified equation is obtained with a corresponding slope ( – √π
1
√D
l ). Deviations from this line at higher frequencies ox
(see Fig. 6, the bottom right angle) can be estimated using the attenuation factor attributed to the reaction steps that lead to oxygen evolution. Assuming a mean diffusion length of 10 – 4 cm and thermal diffusivity as in pure water (1.44 · 10 – 3 cm2 s –1 – handbook data), the measured value of coefficient oxygen diffusion through the wall is 7.84 · 10 –7 cm2 s –1. This value is by two orders of magnitude smaller than that of Dox in pure water, and is quite reasonable considering the much higher density and viscosity of the cell wall. It is by an order of magnitude lower than the value obtained for tobacco leaves by Poulet et al. (1983). However, in comparison to their condition, our sample is much more uniform and better defined, one being thick cell layer of green alga Scenedesmus armatus at the same developmental stage. The results presented above were obtained after a 13-hour light period of synchronous growth. However, the corresponding slope during the whole light period is almost constant, varying not more than 5 %.
Discussion The usefulness of the photoacoustic (PA) spectroscopy method in characterization of the cell and its photosynthetic system has been demonstrated for synchronized cultures of green alga Scenedesmus armatus. The data obtained shows that the method allows determination of the changes in several key parameters in alga during its cell cycle, such as biomass production (Fig. 1), cell volume increase (Fig. 2), photosynthetic oxygen evolution (Fig. 3), and photochemical energy storage (Fig. 4). It also allows the heterogenity of PS II (Fig. 5) to be detected. The portable set-up used in the photoacoustic measurements presented, in which the laser diode was a source of modulated light, appeared to be a very useful tool for photoacoustic measurements without antivibration insulation almost
everywhere electric power was available. As a result, the time between the alga culture sampling and the measurement in the photoacoustic cell was limited to the few minutes required by cells to accommodate to new conditions in the measuring cell. The changes in the key physiological parameters of synchronously growing Scenedesmus armatus cells obtained via the PA method are in agreement with data obtained by other authors for Scenedesmus quadricauda (Kaftan et al. 1999) and Scenedesmus obliquus (Strasser et al. 1999). Despite different methods used (S. quadricauda – the Clark-type electrode, S. obliquus – fluorescence, S. armatus – photoacoustics), all the strains exhibit a similar trend in a steady-state of O2 evolution rate with a maximum in the 4th hour of the cell cycle. It is known that there are two PS II forms having the same fluorescence yield, and, consequently, the same efficiency of absorbed energy deactivation and equal share in the photoacoustic signal response. An increase in the PA signal characteristic time from 1.63 to 1.87 ms (Fig. 5) seems to indicate changes in the heterogeneity of PS II. The inactive form of PS II displays longer deactivation time compared to the active one. The measurements of the photoacoustic signal vs modulation frequency allowed determination of the value of diffusion coefficient for oxygen migration through the cell wall (7.84 · 10 –7 cm2 s –1) (Fig. 6). This value, lower by two orders of magnitude than that of Dox in pure water, is quite reasonable in view of much higher density and viscosity of the cells wall. In conclusion, the photoacoustic method proves to be a source of valuable information on the photosynthetic apparatus of Scenedesmus armatus during the cell cycle. A part of this information is not accessible by other methods (e.g., energy storage, diffusion parameters etc.). Short measurement time allows continuous monitoring of alga developmental stages. Acknowledgements. This work was supported by the University of Gdansk ´ under Grants no. BW/5200-5-0306-0 and no. BW/1110-50129-0.
References Bults G, Horwitz BA, Malkin S, Cahen D (1982) Photoacoustic measurements of photosynthetic activities in whole leaves photochemistry and gas exchange. Biochim Biophys Acta 679: 452 – 465 Carpentier R, La Rue B, Leblanc RM (1983) Photoacoustic spectroscopy of Anacystis nidulans. I. Effect of sample thickness on the photoacoustic signal. Arch Biochem Biophys 222: 403 – 410 Cha Y, Mauzerall DC (1992) Energy storage of linear and cyclic electron flows in photosynthesis. Plant Physiol 100: 1869–1877 Charland M, Veeranjaneyulu K, Charlebois D, Leblanc RM (1992) Photoacuostic signal generation in leaves: are O2-consuming processes involved. Biochim Biophys Acta 1098: 261– 265
Photosynthesis in the cell cycle of Scenedesmus Clayton RC (1980) Photosynthesis: Physical Mechanisms and Chemical Patterns. Cambridge University Press, Cambridge Herbert SK, Fork DC, Malkin S (1990) Photoacoustic measurements in vivo of energy storage by cyclic electron flow in algae and higher plants. Plant Physiol 94: 926 – 934 Kaftan D, Meszaros T, Whitmarsh J, Nedbal L (1999) Characterization of photosystem II activity and heterogeneity during the cell cycle of the green alga Scenedesmus quadricauda. Plant Physiol 120: 433 – 441 Kanstad SO, Cahen D, Malkin S (1983) Simultaneous detection of photosynthetic energy storage and oxygen evolution in leaves by photothermal radiometry and photoacoustics. Biochim Biophys Acta 722: 182–189 Krupinska K, Humbeck K (1994) Light-induced synchronous cultures, an excellent tool to study the cell cycle of unicellular green algae. J Photochem Photobiol Biology 26: 217– 231 Lakowicz JR (1983) Principles of fluorescence spectroscopy. Plenum, New York Malkin S (1998) Attenuation of the photobaric-photoacoustic signal in leaves by oxygen-consuming processes. Israel J Chem 38: 261– 268 Malkin S, Canaani O (1994) The use and characteristics of the photoacoustic method in the study of photosynthesis. Annu Rev Plant Physiol Mol Biol 45: 493 – 526 Melis A, Guenther GE, Morrissey PJ, Ghirardi (1988) Photosystem II heterogeneity in chloroplasts. In: Lichtenthaler HK (ed) Applications of Chlorophyll Fluorescence. Kluwer Academic Publishers, Dordrecht, pp 33 – 43 Nichols WH, Bold HC (1965) Trichosarcina polymorpha Gen. et Sp. Nov. J Phycol 1: 34 – 38 Nilshammer N, Walles B (1974) Electron microscope studies on cell differentiation in synchronized cultures of the green alga Scenedesmus. Protoplasma 79: 317– 332 Poulet P, Cahen D, Malkin S (1983) Photoacoustic detection of photosynthetic oxygen evolution from leaves. Quantitative analysis by phase and amplitude measurements. Biochim Biophys Acta 724: 433 – 446
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Rosencwaig A, Gersho A (1976) Theory of the photoacoustic effect with solids. J Appl Phys 47: 64 – 68 Schmidt K, Beckmann D (1998) Biomass monitoring using the photoacoustic effect. Sensors and Actuators B 51: 261– 267 Schreiber U, Bilger W, Neubauer C (1993) Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. In: Schultz ED, Caldwell MM (eds) Ecophysiology of Photosynthesis. Ecological Studies, vol 100. Springer-Verlag, Berlin, pp 49–70 Senger H, Krupinska K (1986) Changes in molecular organization of thylakoid membrans during the cell cycle of Scenedesmus obliquus. Plant Cell Physiol 27(6): 1127–1139 Strasser BJ, Dau H, Heinze I, Senger H (1999) Comparison of light induced and cell cycle dependent changes in the photosynthetic apparatus: A fluorescence induction study on the green alga Scenedesmus obliquus. Photosynth Res 60: 217– 227 Szurkowski J, Tukaj Z (1995) Characterization by photoacoustic spectroscopy of the photosynthetic Scenedesmus armatus system affected by fuel oil contamination. Arch Environ Contam Toxicol 29: 406 – 410 Szurkowski J, Wartewig S (1999) Application of photoacoustic spectroscopy to studies of thin olive oil layers on water. Instrum Scien Technol 27: 311– 317 Tukaj Z, Szurkowski J (1993) Photoacoustic spectra affected by fuel oil in the chlorococcal alga Scenedesmus armatus. Acta Physiol Plant 15: 219 – 226 Tukaj Z, Kubinova A, Zachleder V (1996) The effect of irradiance on the growth and reproductive processes during the cell cycle in the alga Scenedesmus armatus (Chlorophyta). J Phycol 32 (4): 624 – 631 Zachleder V, Kawano S, Kuroiwa T (1995) The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cycle of the alga Scenedesmus quadricauda. Protoplasma 188: 245 – 251
Oxygen evolution and photosynthetic energy storage during the cell cycle of green alga Scenedesmus armatus characterized by photoacoustic spectroscopy Janusz Szurkowski1 *, Agnieszka Ba´scik-Remisiewicz2, Krystyna Matusiak2, Zbigniew Tukaj2 1
Institute of Experimental Physics, University of Gdansk, ´ Wita Stwosza 57, 80-952 Gdansk, ´ Poland
2
Department of Plant Physiology, University of Gdansk, ´ Al. Marszałka J. Piłsudskiego 46, 81-378 Gdynia, Poland
Received August 25, 2000 · Accepted February 19, 2001
Summary The photosynthetic activity of Scenedesmus armatus, synchronized by light/dark regime of 14/10 hours, was characterized using the photoacoustic (PA) spectroscopy technique. Frequency-dependent analysis of the PA signal was carried out to separate its photobaric and photothermal components. The photochemical energy storage (ES) measured at a high frequency of 180 Hz and the efficiency of oxygen evolution measured at a low frequency of 4 Hz were found to have different runs during the light phase (14 h) of the cell cycle. ES changed from 27% for young autospores illuminated for 0.5 h to 35 % for mother cells starting to release the next generation of autospores. The efficiency of oxygen evolution rapidly increased, reaching the maximum value in 3 – 4 h, and next gradually declined with time during the cell cycle. The time of the photothermal component of the PA signal creation measured in a PA cell changed from 1.63 to 1.87 ms. The coefficient of oxygen diffusion through the cell wall was 7.84 · 10 –7 cm2 s –1. In view of a very good correlation between the PT signal and the cell volume during the cell cycle, the PA technique can also be employed to monitor the growth of cells or biomass production. Key words: cell cycle – energy storage – photoacoustic spectroscopy – oxygen evolution – Scenedesmus Abbreviations: Ath amplitude of photothermal signals. – Aox amplitude of photobaric signals. – ES energy storage. – PA photoacoustic. – PS photosystem
Introduction Light-induced, synchronous cultures of chlorococcalean green algae Scenedesmus are frequently used to study the * E-mail corresponding author: [email protected]
cell cycle. Depending on culture conditions, these autotrophic organisms form unicells or multicellular coenobia, are haploidal during their cell cycle, and divide by the multiple fission. Different strains and mutants of S. obliquus and S. quadricauda are usually investigated, and photosynthesis is one of the processes to which particular attention has been 0176-1617/01/158/08-1061 $ 15.00/0
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paid (see review of Krupinska and Humbeck 1994). Each Scenedesmus cell contains a single chloroplast dividing before the cell cycle termination. Due to its partial genetic autonomy, it has its own cycle divided into characteristic phases (Zachleder et al. 1995). Both changes in the fine structure of the chloroplast (Nilshammer and Walles 1974) and molecular organization of the photosynthetic apparatus during the cell cycle (Senger and Krupinska 1986) are quite well known. Therefore, synchronized cultures of Scenedesmus, relatively simple to obtain, are a convenient tool for studying biochemical and biophysical processes within chloroplasts and metabolic relationships occurring during the cell cycle. The light energy absorbed by chloroplastic pigments is in part stored in photosynthetic products and in part dissipated in the form of heat and fluorescence. The overall photosynthetic activity of algae is measured as the rate of the oxygen evolution and photoassimilatory fixation of inorganic carbon, both appearing to fluctuate strongly during the cell cycle. A Clark-type oxygen electrode is most frequently used for the determination of photosynthetic production oxygen and C14 incorporation into the carbohydrate as a measure of c-assimilation. In addition to these two most popular methods, chlorophyll fluorescence has also been used for many years as a noninvasive indicator for the assessment of in vivo photosynthesis (Schreiber et al. 1993). A series of fluorescence parameters provide useful information on both photophysical processes occurring in photosystems and photosynthetic electron transfer between them, recently applied to characterization of photosynthetic systems of S. obliquus, (Strasser et al. 1999) and S. quadricauda (Kaftan and al. 1999). Some other information can be obtained from measurements of the thermal dissipation carried out using a photoacoustic technique, applied also to study the photosynthesis of both intact algae (Herbert et al. 1990, Cha and Mauzerall 1992) and those exposed to abiotic stress (Tukaj and Szurkowski 1993, Szurkowski and Tukaj 1995). The PA effect is the production of pressure modulations within and around a sample when it absorbs intensity-modulated light. In the case of photosynthetic samples in a gas volume, thermal expansion of the gas surrounding the sample and photosynthetic oxygen evolution are the major contributors to the acoustic wave. Since heat diffuses more rapidly from chloroplasts to the extracellular gas than does oxygen, the photothermal and oxygen (photobaric) components of the photoacoustic signal can be separated (Poulet et al. 1983). In the photosynthetic system, the photothermal component of the PA signal represents conversion of the absorbed light energy to heat. The photothermal part of the photoacoustic signal is reduced by a fraction equal to the part of the absorbed energy stored by the photosynthetic process as chemical energy. By measuring heat emission in the presence or absence of a nonmodulated, saturating light background, the photosynthetic ES is evaluated. The oxygen component of the PA signal consists largely of oxygen evolution at photosystem (PS) II. Two O2-consuming processes such as
photorespiration and respiration are considered as too slow to be recorded by the PA-spectroscopy (Charland et al. 1992). Moreover, when photosynthesis is at a low level, it is possible that the Mehler type O2 uptake in PS I may be modulated by and contributed to the photobaric signal (see review of Malkin and Canaani 1994). In the present paper, we have tried to apply photoacoustic spectroscopy for characterization of the photosynthetic system of Scenedesmus armatus grown in cultures synchronized by the light/dark regime. In vivo measurements of the cell volume, the efficiency of photosynthetic oxygen evolution, and photochemical energy storage are described. The time of photothermal signal creation in a photoacousic cell and the coefficient of oxygen diffusion through the cell wall are calculated. Heterogeneity of PS II is also considered and discussed in relation to results obtained by other authors. All measurements were carried out during the light phase (0.5 – 14 h) of the cell cycle, and the term «cell cycle» in this work refers to this period only.
Materials and Methods Organism and cultures The green microalga Scenedesmus armatus used in this study, isolated from the phytoplankton of the southern Baltic, was obtained from culture collection of the Polish Academy of Sciences’ Institute of Oceanology (Sopot), where it is assigned as the B1-76 strain. Algae from bacterial slants were transferred to liquid BBM medium (Nichols and Bold 1965) and incubated for a few days in precultures to adapt them to the conditions of the batch culture. Suspensions of cells were cultured in plate-parallel vessels of a 600 mL volume, which were immersed in a water bath at a constant temperature of 30 ˚C. Cultures were illuminated from one side by 10 fluorescent tubes, cool white (Philips TLD 36W/94), with an irradiance at the surface of the culture vessels of 60 W · m – 2. The cultures were aerated with atmospheric air enriched with CO2 (2 %, v/v), and sterilized by passing through a filter (Sartorius 2000; 0.2 µm PTFE). Algae were synchronized by alternating light and dark periods (14 : 10 h). The lengths of the light periods of the cell cycle were chosen so that the light was switched off when the first daughter cells of the population were released. Ten hours of the dark period were sufficient to terminate the daughter cell release. At least 3 light/dark cycles were needed for sufficient synchronization. After each dark phase, the cultures were diluted at the beginning of the light periods to a constant density of about 1.5 × 106 cells per 1cm3. Cell numbers were determined by counting in the Bürker chamber using standard procedure. Cells dimensions were measured under a microscope, and cell volume was calculated assuming the Scenedesmus armatus cell to be a rotational ellipsoid.
Samples preparation and phtoacoustic measurements The samples were prepared using a modified version of the method described by Carpentier et al. (1983). The algae were deposited on a Sartorius SM 11306 membrane filter (0.45 µm pore size). A 15 mm ring was cut out from the filter 3 – 4 min after filtration and the sample was
Photosynthesis in the cell cycle of Scenedesmus then introduced into the photoacoustic cell. The volume of the culture was filtered in such a manner that about 106 cells were deposited on the sample, covering the filter-ring with a monolayer of cells. In the studies presented, an experimental set-up based on a lockin amplifier SR 850 was used, which not only measured the signal but also controlled the operation of the laser diode that was a source of the modulated light (Szurkowski and Wartewig 1999). A laser diode (type SDL-73311) emitted modulated light with a wavelength of 680 nm. The laser beam was provided through a fiber wire to the measuring cell constructed by the authors. Nonmodulated white background light from a fiber optic illuminator (PL-800) was focused onto the sample through a fiber-optic light guide. The photoacoustic signal from a microphone is phase-sensitive detected using a lock-in amplifier (type SR 850). An internal generator of the amplifier was also used for operating the laser diode controller (LPC 400). The entire data collection and further processing was performed on a PC working in the GPIB standard. The introduction of the laser diode and electronic light modulation system, compared to its mechanical analogue, allowed a higher signal-to-noise ratio to be achieved. The light modulation frequency varied from 4 to about 200 Hz. The photoacoustic signal of the sample was analyzed as a vectorial summation of photothermal and photobaric contributions, according to the method of Poulet et al. (1983). The amplitude of the oxygen signal (Aox) was evaluated using the following equation: Aox =
√A
2 th
+ A2 – 2 A th A cos (ϕ)
(1)
where Ath and A are the amplitude of photothermal and total signals, respectively, in the absence of background light, and ϕ – is the phase of the total signal versus the photothermal one. Ath was calculated as follows: Ath = K · ATth
(2)
where K is the ratio of the acoustic signal in the absence of background light to the signal in the presence of background light at 180 Hz modulation frequency, and AthT is the signal in the presence of background light at the given frequency. It should be noted that the above equations are based on the assumption that practically no change in the phase angle occurs in the photothermal signal when the background light is applied. This was verified experimentally in the high-frequency limit for photoacoustic measurements (Poulet et al. 1983), and also at low frequency by direct photothermal radiometry measurements (Kanstad et al. 1983).
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reactions are not saturated, and a reduced photoacoustic signal is observed as a result of the storage of a substantial part of the absorbed light as photosynthesis products. All measurements were carried out at room temperature with a measuring light wavelength of 680 nm. The modulated light intensity was equal to 25 µmol photons m – 2 s –1 and the nonmodulated background actinic light of 2500 µmol photons m – 2 s –1 were used.
Results Under growth conditions and with the medium composition used in this work, Scenedesmus armatus forms only unicells. In synchronized populations of this strain, three sequences of reproductive processes such as DNA replication, nuclear division, and protoplast fission are triggered, resulting in the formation of 8 daughter cells (Tukaj et al. 1996). Hence, presumably, an 8-fold increase in the cell volume from about 200 µm3 (young autospores) to 1600 µm3 (mother cells) was observed (Fig. 2), and a new generation of autospores started to release at the end of the light period of the cycle (13–14 h) and terminated during next 10 h in the dark. The photoacoustic (PA) signal gathered from the photosynthetic material evolving oxygen contains at least two contributions. Both can be separated by frequency-dependent measurements (Bults at al. 1981). At a low frequency, the PA signal comprises both modulated heat and oxygen evolution, whereas at high frequencies it represents only heat emission. The photothermal signal amplitude versus the time of culture of Scenedesmus armatus in light period is depicted in Figure 1. The relation is assumed to be linear and the correlation coefficient is 0.99. A similar relation has already been evaluated as useful in monitoring the number of cells in a fermentation by Schmidt and Beckmann (1998). According to the Rosencwaig-Gersho theory (1976), the amplitude of the photothermal signal is proportional to the optical density of the sam-
Photoacoustic energy storage measurements Energy storage was calculated and expressed as a percentage: A bc – A A bc
· 100
(3)
where Abc is the photoacoustic signal produced by the modulated light plus the nonmodulated background light, and A is the photoacoustic signal in the modulated light alone. The application of strong background light results in the saturation of photochemical reactions in the sample, increasing the conversion of the absorbed modulated light to heat to nearly 100 %, and producing a maximum photoacoustic signal proportional to the absorption of the modulated light by the sample. In the presence of the modulated light alone, photochemical
Figure 1. Photothermal signal amplitude changes during the light period of Scenedesmus cell cycle. PA signal was gathered at 4 Hz and derived from 106 cells deposited on membrane filter. R is the correlation coefficient.
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Figure 2. Relationship between the photothermal signal amplitude and cell volume. The samples collected from the same culture were divided into 2 parts and immediately analyzed in spectrometer and under microscope. Linear dimensions of at least 20 randomly chosen cells were measured to calculate the cell volume. R is the correlation coefficient.
ple. Since the total number of algae in the sample was the same (about 106 cells on the filter ring), in the subsequent measuring runs during the cell cycle, the increase in the photothermal signal amplitude corresponds to the increase of the signal coming from one cell, i.e. to the linear increase of the cell biomass during its growth. From the dependence of the photothermal signal amplitude on the cell volume (Fig. 2), it is clear that the biomass growth during the cell cycle is related to the increase of the cell volume. Thus, the photothermal signal amplitude can be a good measure of the culture biomass and dimensions of the cells growing therein. A natural limitation of this technique when applied to a water culture is the effect of light scattering by particles and gas bubbles on the measurements. However, even in a water culture, the photoacoustic method is much less affected than the turbidity method (Schmidt and Backmann 1998). The photobaric signal amplitude allowed the estimation of both the rate and yield of oxygen evolution during the cell cycle (Fig. 3). The rate of oxygen evolution per cell during the cell cycle grows linearly with the time of culture (Fig. 3 a). As opposed to the above, a quite different run was observed when the ratios of photobaric to photothermal signal amplitude were calculated (Fig. 3 b). This corresponds to the yield of oxygen evolution per unit of biomass. During the first 3 – 4 h of the 14 h light period, the O2 evolution rapidly increased. After 4 h, the yield steadily decreased, reaching its minimum at the 9th h of the cell cycle. Despite a different method, a similar dependence has already been observed for a light-saturated rate of O2 evolution of synchronously grown S. quadricauda (Kaftan et al. 1999) and S. obliquus (Strasser et al.
1999), measured using the Clark electrode and fluorescence methods, respectively. These authors explain the increase in the steady-state O2 evolution rate at the beginning of the light period by the increased rate of the electron flow subsequent to PS II, and by the decline observed toward the end of the light period due to limited PS II activity. Figure 4 shows that the energy storage (ES) calculated from Eq. 3. ranged from 27 % for young autospores illuminated for 0.5 h to 35 % for mother cells at the end of the cell cycle. At the beginning of the cell cycle, as in the case of oxygen evolution efficiency, the ES increase was fast up to 3 – 4 h, then it became slower, reaching an almost constant value in the middle of the cell cycle (33 %). The results are consistent with those reported by Cha and Mauzerall (1992), who used the pulsed time-resolved photoacoustics and showed that the overall thermal efficiency (energy storage) in asynchronously growing Chlorella cells was 30 %, the mean value we obtained. Just before the dark period, a value of about 35 % was measured, also obtained theoretically (Clayton 1980). The application of a two-phase amplifier allowed the measurement of the characteristic time of the photothermal signal creation. For signal analyses at modulation frequencies of about 200 Hz (where only the photothermal signal is dealt with), the phase-modulation method was employed. It is
Figure 3. Changes in the photobaric signal amplitude (a) and the ratio of photobaric to photothermal signal (b) during the light period of the cell cycle. Photobaric signal gathered at 4 Hz relates to oxygen evolution, and the ratios correspond to oxygen evolution yield per biomass unit.
Photosynthesis in the cell cycle of Scenedesmus
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widely used in fluorescence decay time studies (Lakowicz 1983). According to Lakowicz, by using a fluorometer with a single modulation frequency (f), it is possible to determine the characteristic decay time (τ), assuming a one-exponent decay from the equation: ϕ = arctg(2 πfτ)
(4)
Since in our measurements the photothermal signal is studied instead of fluorescence signals, the same mathematical approach can be applied. A variability of the time characterizing the photothermal signal creation, measured at the modulation frequency 180 Hz, is presented in Figure 5. The time increases monotonically from (1.63 ± 0.03) ms to (1.87 ± 0.03) ms during the light cycle. As known from fluorescence studies of higher plant chloroplasts, at least two components, differing significantly in their decay times and related to PS II heterogenity, can be distinguished (Melis et. al. 1988). Both subpopulations of PS II display equal fluorescence quantum yields (Kaftan et al. 1999). Despite the population balance change between them, no decrease of the energy storage at the end of the light cell cycle can be seen in Figure 4. Measurements carried out during the cell cycle of the green alga Scenedesmus quadricauda by Kaftan et al. (1999) yielded the inactive PS II of approximately 3 % of the total PS II at the beginning of the cell cycle, and about 15 % in the dividing cells. Similar results were also obtained for green alga Scenedesmus obliquus (Strasser et al. 1999). Here, we report a study on the oxygen-evolution (photobaric) signal as a function of the frequency of modulated light. The relative damping of the photobaric signal, compared to the photothermal one, can be explained by the diffusion of oxygen from the chloroplasts through the cell wall to the gaseous phase, and by the electron-transport reactions occurring in the time between the photochemical act and water
Figure 4. Changes of energy storage of Scenedesmus cells during the cell cycle. The intensity of the measuring light (680 nm) was 25 µmol photons m – 2 s –1, the background actinic light was 2500 µmol photons m – 2 s –1, and modulation frequency was 180 Hz.
Figure 5. Time, which characterises the generation of the photoacoustic signal measured at 180 Hz in a PA cell, during the cell cycle.
Figure 6. Logarithm of the ratio of the photobaric (Aox) to photothermal (Ath) signal amplitude, as a function of square root of the modulation frequency for algae from the 13th hour of the cell cycle. Linear dependence is the best-fit approximation to the experimental data in the form: y = ax + b (R = – 0.997), which yields the diffusion coefficient Dox = 7.84*10 –7 cm2 s –1.
splitting (Poulet et al. 1983). It can be shown that for lower frequencies the principal role in the attenuation of the photobaric signal is played by the diffusional process, whereas at higher modulation frequencies ( ≥ 200 Hz), the signal is damped at the source by a kinetic factor due to the chain of electrontransport reactions between the photochemical reaction and the step of oxygen formation (Bults et al. 1982). The logarithm of Aox/Ath as a function of the square root of the modulation frequency is plotted in Figure 6. Since the amplitudes of the modulated oxygen concentration field and temperature field are attenuated exponentially at the cell 1 boundary, the plot of ln(Aox/Ath) versus (f) ⁄2 tends to be a straight line in the low frequency range, from the slope of
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which Dox can be computed using the formula proposed by Poulet et al. (1983); ln
A ox A th
=–
√π f 冢
1
–
1
√D √D ox
th
冣
l,
(5)
where; Dth – heat diffusion coefficient (diffusivity), Dox – oxygen diffusion coefficient in the aqueous medium of the cell, l – distance from the source. When reasonable values are assumed for l and Dth (Dth = 1.44 · 10 – 3 cm2 s –1), the term it is comparable to
√
1 can usually be neglected if D th
√
1 and omitted in Eq. 5. Hence, a simD ox
plified equation is obtained with a corresponding slope ( – √π
1
√D
l ). Deviations from this line at higher frequencies ox
(see Fig. 6, the bottom right angle) can be estimated using the attenuation factor attributed to the reaction steps that lead to oxygen evolution. Assuming a mean diffusion length of 10 – 4 cm and thermal diffusivity as in pure water (1.44 · 10 – 3 cm2 s –1 – handbook data), the measured value of coefficient oxygen diffusion through the wall is 7.84 · 10 –7 cm2 s –1. This value is by two orders of magnitude smaller than that of Dox in pure water, and is quite reasonable considering the much higher density and viscosity of the cell wall. It is by an order of magnitude lower than the value obtained for tobacco leaves by Poulet et al. (1983). However, in comparison to their condition, our sample is much more uniform and better defined, one being thick cell layer of green alga Scenedesmus armatus at the same developmental stage. The results presented above were obtained after a 13-hour light period of synchronous growth. However, the corresponding slope during the whole light period is almost constant, varying not more than 5 %.
Discussion The usefulness of the photoacoustic (PA) spectroscopy method in characterization of the cell and its photosynthetic system has been demonstrated for synchronized cultures of green alga Scenedesmus armatus. The data obtained shows that the method allows determination of the changes in several key parameters in alga during its cell cycle, such as biomass production (Fig. 1), cell volume increase (Fig. 2), photosynthetic oxygen evolution (Fig. 3), and photochemical energy storage (Fig. 4). It also allows the heterogenity of PS II (Fig. 5) to be detected. The portable set-up used in the photoacoustic measurements presented, in which the laser diode was a source of modulated light, appeared to be a very useful tool for photoacoustic measurements without antivibration insulation almost
everywhere electric power was available. As a result, the time between the alga culture sampling and the measurement in the photoacoustic cell was limited to the few minutes required by cells to accommodate to new conditions in the measuring cell. The changes in the key physiological parameters of synchronously growing Scenedesmus armatus cells obtained via the PA method are in agreement with data obtained by other authors for Scenedesmus quadricauda (Kaftan et al. 1999) and Scenedesmus obliquus (Strasser et al. 1999). Despite different methods used (S. quadricauda – the Clark-type electrode, S. obliquus – fluorescence, S. armatus – photoacoustics), all the strains exhibit a similar trend in a steady-state of O2 evolution rate with a maximum in the 4th hour of the cell cycle. It is known that there are two PS II forms having the same fluorescence yield, and, consequently, the same efficiency of absorbed energy deactivation and equal share in the photoacoustic signal response. An increase in the PA signal characteristic time from 1.63 to 1.87 ms (Fig. 5) seems to indicate changes in the heterogeneity of PS II. The inactive form of PS II displays longer deactivation time compared to the active one. The measurements of the photoacoustic signal vs modulation frequency allowed determination of the value of diffusion coefficient for oxygen migration through the cell wall (7.84 · 10 –7 cm2 s –1) (Fig. 6). This value, lower by two orders of magnitude than that of Dox in pure water, is quite reasonable in view of much higher density and viscosity of the cells wall. In conclusion, the photoacoustic method proves to be a source of valuable information on the photosynthetic apparatus of Scenedesmus armatus during the cell cycle. A part of this information is not accessible by other methods (e.g., energy storage, diffusion parameters etc.). Short measurement time allows continuous monitoring of alga developmental stages. Acknowledgements. This work was supported by the University of Gdansk ´ under Grants no. BW/5200-5-0306-0 and no. BW/1110-50129-0.
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Photosynthesis in the cell cycle of Scenedesmus Clayton RC (1980) Photosynthesis: Physical Mechanisms and Chemical Patterns. Cambridge University Press, Cambridge Herbert SK, Fork DC, Malkin S (1990) Photoacoustic measurements in vivo of energy storage by cyclic electron flow in algae and higher plants. Plant Physiol 94: 926 – 934 Kaftan D, Meszaros T, Whitmarsh J, Nedbal L (1999) Characterization of photosystem II activity and heterogeneity during the cell cycle of the green alga Scenedesmus quadricauda. Plant Physiol 120: 433 – 441 Kanstad SO, Cahen D, Malkin S (1983) Simultaneous detection of photosynthetic energy storage and oxygen evolution in leaves by photothermal radiometry and photoacoustics. Biochim Biophys Acta 722: 182–189 Krupinska K, Humbeck K (1994) Light-induced synchronous cultures, an excellent tool to study the cell cycle of unicellular green algae. J Photochem Photobiol Biology 26: 217– 231 Lakowicz JR (1983) Principles of fluorescence spectroscopy. Plenum, New York Malkin S (1998) Attenuation of the photobaric-photoacoustic signal in leaves by oxygen-consuming processes. Israel J Chem 38: 261– 268 Malkin S, Canaani O (1994) The use and characteristics of the photoacoustic method in the study of photosynthesis. Annu Rev Plant Physiol Mol Biol 45: 493 – 526 Melis A, Guenther GE, Morrissey PJ, Ghirardi (1988) Photosystem II heterogeneity in chloroplasts. In: Lichtenthaler HK (ed) Applications of Chlorophyll Fluorescence. Kluwer Academic Publishers, Dordrecht, pp 33 – 43 Nichols WH, Bold HC (1965) Trichosarcina polymorpha Gen. et Sp. Nov. J Phycol 1: 34 – 38 Nilshammer N, Walles B (1974) Electron microscope studies on cell differentiation in synchronized cultures of the green alga Scenedesmus. Protoplasma 79: 317– 332 Poulet P, Cahen D, Malkin S (1983) Photoacoustic detection of photosynthetic oxygen evolution from leaves. Quantitative analysis by phase and amplitude measurements. Biochim Biophys Acta 724: 433 – 446
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Rosencwaig A, Gersho A (1976) Theory of the photoacoustic effect with solids. J Appl Phys 47: 64 – 68 Schmidt K, Beckmann D (1998) Biomass monitoring using the photoacoustic effect. Sensors and Actuators B 51: 261– 267 Schreiber U, Bilger W, Neubauer C (1993) Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. In: Schultz ED, Caldwell MM (eds) Ecophysiology of Photosynthesis. Ecological Studies, vol 100. Springer-Verlag, Berlin, pp 49–70 Senger H, Krupinska K (1986) Changes in molecular organization of thylakoid membrans during the cell cycle of Scenedesmus obliquus. Plant Cell Physiol 27(6): 1127–1139 Strasser BJ, Dau H, Heinze I, Senger H (1999) Comparison of light induced and cell cycle dependent changes in the photosynthetic apparatus: A fluorescence induction study on the green alga Scenedesmus obliquus. Photosynth Res 60: 217– 227 Szurkowski J, Tukaj Z (1995) Characterization by photoacoustic spectroscopy of the photosynthetic Scenedesmus armatus system affected by fuel oil contamination. Arch Environ Contam Toxicol 29: 406 – 410 Szurkowski J, Wartewig S (1999) Application of photoacoustic spectroscopy to studies of thin olive oil layers on water. Instrum Scien Technol 27: 311– 317 Tukaj Z, Szurkowski J (1993) Photoacoustic spectra affected by fuel oil in the chlorococcal alga Scenedesmus armatus. Acta Physiol Plant 15: 219 – 226 Tukaj Z, Kubinova A, Zachleder V (1996) The effect of irradiance on the growth and reproductive processes during the cell cycle in the alga Scenedesmus armatus (Chlorophyta). J Phycol 32 (4): 624 – 631 Zachleder V, Kawano S, Kuroiwa T (1995) The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cycle of the alga Scenedesmus quadricauda. Protoplasma 188: 245 – 251