The effect of dynamic light regimes on Chlorella I. Pigments and cross sections

Transcription

1 Hydroiologia 238: 1-8, T. Berman, H. J. Gons & L. R. Mur (eds), The Daily Growth Cycle of Phytoplankton. O 1992 Kluwer cademic Pulishers. Printed in Belgium. 1 The effect of dynamic light regimes on Chlorella I. Pigments and cross sections Bernd M.. Kroon,' Mikel Latasa, 2 Bas W. Ielings & Luuc R. Mur 1 1 University of msterdam, Nieuwe chtergracht 12, 118 WS msterdam, The Netherlands; 2Instituto de Ciencas del Mar, Paseo Nacional, s/n, 83 Barcelona, Spain Key words: dynamic light regime, chlorophyll a, chlorophyll, optical, in vivo asorption, cross sections stract The patterns of diurnal variations in pigmentation and optical cross-section were compared for two cyclostat cultures of Chlorella pyrenoidosa, where the dynamics of the photoperiod differed. Populations were light-limited, nutrient rich and growing on an 8:16 light-dark (LD) cycle. One light regime was an 8 h sine function of the light period (sinusoidal culture), while the second had an 1 h sine function super-imposed on the 8 hour sine function (oscillating sinusoidal culture). Hourly samples were taken throughout a 12 h period including the light period. Determinations were made of chlorophyll (Chl) a and aundance, in vivo asorption spectra, cell numer and volume and used to derive oth cell-specific (acell) and optical chlorophyll specific (chl) cross sections, as well as the asorption efficiency, Q, of the cells. The results indicate that C. pyrenoidosa is capale of adapting to dynamics in light intensity within an 8 h photoperiod. The sinusoidal culture showed a constant decrease in the Chl a/ ratio of 28 % while the total Chl content per cell increased slightly and achl and Q remained constant, suggesting coordinated changes in reaction centers and light harvesting complexes. Over the oscillating photoperiod, however, the second culture displayed a diurnal variation in Chl a/ ratio, a 2 % increase in achl and an apparent oscillation in Q. These oservations suggest that an oscillating photoperiod promoted the capaility of Chl molecules to collect light and that the fractional area of all Chl molecules exposed to the photon flux is inversely related to the photon flux. Introduction In natural environments the intensity of the light regime experienced y phytoplankton is influenced y its position in the water column and the surface irradiance, which varies as a sine function with time of day (cf. Kirk, 1983). t any given depth, the underwater light climate will oscillate in phase with surface irradiance, and the magnitude of the diurnal periodicity in photosynthetically availale radiation (PR) will dampen out with depth (cf. Kirk, 1983). If phytoplankton are confined to a narrow depth range, as is the case in well stratified water columns, then the diurnal pattern of PR will resemle a sine curve. However, if phytoplankton are moved through a significantly large change in optical depth, as is the case when vertical mixing dominates or phytoplankton ride the pycnoclines of strong internal waves, then the sine function of the photoperiod

2 2 will have additional oscillations imposed upon it. The time scale of these light intensity fluctuations can e smaller than the actual growth rates and small compared to reported adaptation rate constants of pigmentation and asorption properties (Pr6zelin & Matlick, 198; Marra, 198; Post etal., 1985; Falkowski, 1984; Pr6zelin etal., 1991). The light fluctuations can range from darkness to full sunlight, the extremes of which have a severe inhiitory effect on photosynthesis. In oth a sinusoidal and an oscillating sinusoidal light regime the range of irradiances is likely too large to assume that optimal photosynthetic performance can e maintained throughout the photoperiod without some flexiility in the composition and/or function of the components of the photosynthetic machinery determining light asorption and radiation utilization efficiencies. general limitation in the efforts to predict in situ primary productivity is the lack of accurate measures quantifying variations in the optical and physiological state of phytoplankton throughout the entire photoperiod. Thus the predictions are often ased on discrete proxy measures of the photosynthetic characteristics. More often optical parameters are used to predict primary productivity (e.g. Smith etal., 1989; Kroon etal., 1989). The optical parameters primarily determine the rate of excitons arriving at the photosystems, given a certain photon flux, and thus largely determine the primary production rate. Changing the optical parameters would have the effect of feed forward control with respect to photosynthesis. Knowing the time dependent changes of the optical properties with regard to the actual light regime, would greatly improve the predictive capaility of optical models. The aim of the present study was to determine the pattern of diurnal variation and adaptaility of Chlorella pyrenoidosa in pigment composition, optical and cellular cross sections to light regimes with a dynamic light supply during the photoperiod. The applied light regimes simulated a stratified system, where the light intensity is a sine function, and a mixed system, where the light intensity is an oscillating function of which the envelope was a sine function. The outcome of such studies might serve to etter predict primary productivity using optical parameters. Materials and methods Samples of two continuous cultures of Chlorella pyrenoidosa differing in light regime were used to examine pigmentation, in vivo asorption spectra, cell volume, physical and optical cross sections during the light period. One culture was grown on 8:16 h. light:dark (LD) cycles, where irradiance was varied as a sine function of time equal to the length of the photoperiod (sinusoidal culture; total light dose 1.3 mol m - 2 d- 1). The second culture differed from the first only in that a second oscillation with a period of 1 h. was superimposed on the 8 h. sine function (oscillating sinusoidal culture; total light dose molm - 2 d-). During the oscillating light regime, darkness reoccurred once every hour. Both cultures were grown on BG11 medium and were operated at a dilution rate of. d- 1 and had a volume of 23 ml. The culture unit as well as an example of an oscillating photoperiod are descried in Kroon et al. (1992). Chlorophyll (Chl) a and were extracted for 3 minutes in 1% methanol and concentrations determined spectrophotometrically. In vivo asorption spectra (range 4-5 nm;.35 nm resolution) were recorded with an minco DW2 spectrophotometer with the cuvette placed directly in front of the photomultiplier. The asorption data were converted from logarithm ase 1 to ase e and the average asorption value etween 3 and 5 nm was sutracted from the spectra to correct for residual scattering (Duysens, 1956). The spectral averaged asorption coefficient () for photosynthetically availale radiation (PR) was calculated assuming a light source with a constant spectral composition. Cell numer was determined with a Coulter Counter Model ZM after appropriate dilution of the sample in Isoton. The averaged cell diameter was calculated from the size distriution. The Chl-specific (achl) and cell-specific (ocell)

3 3 optical cross sections were calculated as the ratio of and measurements of Chl (a + ) concentration and cell density respectively. The asorption efficiency (Q) of the cells was calculated as the ratio of cell and physical cell cross section. Results Pigments The culture exposed to the sinusoidal light regime showed small variations in Chl (a + ) content during the first half of the light period (Fig. 1). The sudden increase (late in the photoperiod) was an artifact, caused y the formation of visile cell aggregates. s a result, the numer of cells determined with the Coulter Counter underestimated the true cell density. The Chl a/ molar ratio decreased 28 % during the light period from 4.3 to 3.1, with the largest part of the decline evident prior to the time of cell aggregation (Fig. 2a). Both Chl (a + ) content and ratio of the oscillating sinusoidal culture contrast with the sinusoidal culture. In the former, the chlorophyll content was aout half compared to the sinusoidal population and increased aout 1% during the light period (Fig. l). The Chl a/ ratio decreased 9 % during the first 3 hours of the light period and ( 5. _. 3.5 J = "1-2. co.o 5. B 3.5, q n, Fig. 2a, The molar ratio of chlorophyll a and chlorophyll as a function of local time for the culture receiving the sinusoidal (2a) and the oscillating (2) light regime. The shaded area and vertical ar indicate the dark period. Starting at the time indicated y ' the data were influenced y cell aggregation. returned to the initial value (3.3) during the last three light hours (Fig. 2). The overall values of the Chl a/ ratios of oth cultures were comparale. Cell density and volume Cell density (Fig. 3) decreased due to continuous dilution of the culture, and cell volume (Fig. 4) increased during the light period of oth cultures. Both phenomena indicated partial synchronization of cell division. Whereas the decrease in cell _, I. = I.5 _ B 6 W - 2. a -Y I-Ia (9 3.25, U " io w 53. a, (. (I ōc =... = oc Oil Fig. Ia, Cell chlorophyll (a + ) concentration (1-'5 mol per cell) as a function of local time for the culture receiving the sinusoidal (la) and the oscillating (l) light regime. The shaded area and vertical ar indicate the dark period. Note the difference in ordinate scaling. Starting at the time indicated y ' the data were influenced y cell aggregation. Fig. 3a, Cell numer [ 16 cell ml- '] as a function of local time for the culture receiving the sinusoidal (3a) and the oscillating (3) light regime. The shaded area and vertical ar indicate the dark period. Note the difference in ordinate scaling. Starting at the time indicated y" the data were influenced y cell aggregation.

4 4 density of the sinusoidal culture occurred almost during the entire light period, the major decrease in cell density of the oscillating culture was restricted to the second half of the light period (Fig. 3a,). The latter culture contained a three fold higher cell density. The cell volume of the oscillating culture increased two fold, mainly etween 11: and 15:, which is in contrast to the sinusoidal culture where the volume increase occurred almost throughout the entire light period (Fig. 4a,). Cross sections The Chl-specific optical cross section (achl) is a direct measure of the capacity of chlorophyll molecules to collect photons. The achl of the sinusoidal culture remained constant during the light period at aout 4 #m 2 /cell (Fig. 5a). The culture receiving the oscillating light regime showed an increase of 2% in achl during the light period, especially from 12: onward (Fig. 5). The cellular optical cross section, cell, is a function of the area of a single cell which participates in light asorption; ecell does not represent a physical area of a discrete particle. The value of cell is determined y the cumulative statistical chance of all cellular components (mainly pigments) to asor photons from the radiant flux incident on one cell. The quotient of ocell and the -a W Q) Q, U _ t v =-- -a z Qm -a ) U, Fig. 4a, Cell volume [#im 3 per cell] as a function of local time for the culture receiving the sinusoidal (4a) and the oscillating (4) light regime. The shaded are and vertical ar indicate the dark period. physical cellular cross section (PCC) is the asorption efficiency (Q) of the cross sectional area of a cell in asoring light (Kirk, 195; Morel & Bricaud, 1981). The value of cell for the sinusoidal culture increased during the photoperiod. Due to cell aggregation, the values from 15: onward are uncertain. s a result cell does not clearly covary with the cellular physical cross section, although a trend does exist efore clumping of cells occurred (Fig. 6a). If true, Q would e constant during the sinusoidal light period. Figure a shows that Q was constant until the cells aggregated. pplying an oscillating light regime causes the increase in acell to occur out of phase and earlier than the increase in physical cell cross section, PCC, (Fig. 6). In contrast to the sinusoidal culture, Q is inversely related to the hourly integrated light dose (Fig. ). During the morning hours when the hourly light dose is increasing, Q decreases. The change is reversed during the second half of the light period. In vivo asorption Whether changes in spectrally-averaged cross section are driven y changes in the concentration of one or more pigments can e revealed y examining in vivo asorption difference spectra. Figure 8 shows the wavelength dependent difference spectra of acell, determined as 13: minus 9: (M) and as 1: minus 13: spectra (PM). The M difference spectrum of the oscillating culture is slightly negative, while the PM difference spectra of oth cultures are positive. The PM increase in wavelength dependent cell resemles a normal in vivo asorption spectrum. The ratio of this afternoon difference spectrum with the asorption spectrum of 13: was constant over the wavelength range (data not shown). These facts suggest that the increase in cell is due to a alanced increase of all pigments present. The positive sinusoidal M spectrum shows a roadening of the main Chl (a + ) asorption peak etween 65 and 68 nm, indicating a relative higher increase in Chl compared to Chl a.

5 -h ' 5 N 6 4 I k,,\- V Z i c' 6 4 :i I B j 2 Fig. 5a, Chlorophyll-specific optical cross sections (achl [m 2 mmol Chl]) as a function of local time for the culture receiving the sinusoidal (a) and the oscillating (5) light regime. The shaded area and vertical ar indicate the dark period. ZU 1 Z o 1 B.... I ii 5 Z l -- Fig. 6a, Optical (acell, continuous line) and physical (PCC, roken line) cell cross sections [ym 2 per cell] as a function of local time for the culture receiving the sinusoidal (6a) and the oscillating (6) lighl regime. The shaded areai and vertical aar indicate the dark period. Starting at the time indicated y ' the data were influenced y-cell aggregation. Discussion and conclusions The dynamics of Chi a and content and ratio significantly differed etween the sinusoidal and oscillating culture. s Chl is only present in light harvesting complexes of photosystems I and II (e.g. Fujita et al., 1989), we interpret the differences in Chi (content and ratio) etween the two cultures to reflect dissimilar changes in the size and numer of the photosynthetic units (PSU's) during the light period. linear regression of Chl /cell vs Chl a/cell of the sinusoidal culture had a slope of.396 and an x-axis intercept of.136 (r 2 =.9). The values for the oscillating culture were.28 and -.14 with r 2 =.3, respectively. The values of the regression analyses of the sinusoidal culture were comparale to the values (respectively.38 and.8 with r 2 =.985) reported y Ley (1986) using Chlorella vulgaris grown in atch cultures under a range of light intensities. He argued that this linear relationship implied that increases in cell Chl were achieved y the addition of Chl a and Chl in fixed molar ratios. ccording to the model proposed y Ley (1986), this means that the changes in total cell Chl of the sinusoidal culture are the result of co-

6 6 o.4 n,1 V B... i Fig. a, The asorption efficiency (Q) as a function of local time for the culture receiving the sinusoidal (a) and the oscillating () light regime. The shaded area and vertical ar indicate the dark period. Starting at the time indicated y ' the data were influenced y cell aggregation. ) o - t I a) C a) U.vu Wavelength [nm] - tn Wavelength [nm] Fig. 8a, Difference spectra etween wavelength dependent acell spectra etween 13: minus 9: (.M.) and etween 1: minus 13: (P.M.) of the sinusoidal (a) and oscillating () culture. The difference is given in relative units ([-]). Note the difference in ordinate scaling. ordinated changes in oth reaction centers and light harvesting complexes. We suggest that the dynamics of Chl (a + ) content during a sinusoidal photoperiod (see Fig. la) were the result of variation in the numer of PSU's. The Chl a/cell vs Chl /cell regression results of the oscillating sinusoidal culture indicate more complex changes in reaction centers and light harvesting complexes. Cells adapted to high light intensities generally have lower Chl levels. The cause of such an adaptation to high light is hypothesized y Falkowski (198) to e the result of changes in the glutamine/glutamate levels which are regulated y the TP supply. The twofold lower Chl levels of the oscillating culture can only e accounted for y the 1% higher total irradiance it received, if the comined action of the photosystems would e iassed towards TP synthesis. This effect could have occurred, if newly formed Chl was preferentially incorporated in the light harvesting complexes of photosystem I. Flow cytometric analysis (Hans Balfoort, pers. comm.) indicated that the percentage of iomass in clusters increased from 3% at 14: to 98% at the end of the sinusoidal photoperiod. The cell clumping reversed during the night period (data not shown) and might reflect a physiological response to the highest light intensities of the sinusoidal photoperiod. We suggest that during maximum light intensities polymers are excreted and

7 cells start to aggregate. Cells in clusters will e protected from photodamage due to increased self shading. The cell numer of the sinusoidal culture decreased aout 5% whereas this decrease only was 8% for the oscillating culture. ssuming the cells of oth cultures to divide into 4 autospores, the results indicate a higher synchronization of cell division in the sinusoidal culture. The lack or low energy supply during part of the oscillating light regime might have provoked these differences (Donnan et al., 1985). The value of achl is reported to e constant (Osorne & Raven, 1986) or to vary with changes in size, shape and refractive index of the cells (Spinrad & Yentsch, 198; see also Nelson & Pr6zelin, 199). nother argument for changes in achl is suggested to e variaility in stacking of the chlorophyll molecules in the thylakoid memranes (Falkowski et al., 1985). To minimize the package effect from 15: onwards as cells ecame clustered, we sonicated the sample unit cell aggregates were no longer visile. However, the flow cytometric results (data not shown) indicated that 3% of the iomass was in aggregated form etween 9: and 14:. We did not try to correct for the apparent package effect, ecause of easily made overestimation (Mauzerall & GreenBaum, 1989). s a result the values given for achl in the sinusoidal culture may underestimate the actual values. With increasing cell Chl, the sinusoidal culture showed a constant value for achl, while the value of achl for the oscillating culture increased during the photoperiod (Fig. 5). Higher values of achl might cause the pigments to e more susceptile to photodamage. Bearing in mind the small increase in chlorophyll content (Fig. l), the results suggest a change in physical arrangement of the chlorophyll molecules such that the light asoring capacity of the Chl molecules increased. We oserved a remarkale difference in the time course of Q during oth light periods. In a sinusoidal photoperiod, a constant fractional area of the cell (constant Q) is capale of asoring light, irrespective of a prominent increase in cell size and dynamic light supply (Fig. a). dditional experiments with a 12 h sinusoidal photoperiod did indicate that acell correlated with the PCC (r 2 =.95, n = 8), especially during the first 8 h. of the light period (Kroon, unpul. data). For the oscillating sinusoidal photoperiod, the late day increase of Q coincided with the increase in cell Chl content (Fig. l). The effect of the dynamics in oth achl and Q is that, while the capacity of the chlorophyll molecules to asor light is monotonically increasing during the light period, the fractional cell area exposed to the radiant flux is lowest when the light intensity reaches its daily maximum. s the asorption efficiency Q is lowered during high irradiance, the chance of the pigments ecoming photodamaged decreases. The results presented indicate differences in the time course and asolute values of pigmentation, cell division cycle, physical and optical cross sections of Chlorella pyrenoidosa during the light period when the dynamics of the light supply is varied. By changing the dynamics of the light dose in a photoperiod, the maximum light intensities occurring will also differ. The effect of the highest irradiance during an oscillating photoperiod should e investigated in order to improve the reliaility of optical models for phytoplankton productivity. cknowledgements The results presented formed a part of all measurements performed during the Vth GP workshop. The experiments could only have een done due to the cooperative spirit during the workshop. The authors thank B. B. Pr6zelin for valuale comments on the original manuscript. References Donnan, L.,. P. Carvill, T. J. Gilliland & P. C. L. John, The cell cycles of Chlamydomonas and Chlorella. New Phytol. 99: 1-4. Duysens, L. N. M., The flattening of the asorption spectrum of suspensions, as compared to that of solutes. Biochim. Biophys. cta 19: Falkowski, P. G., 198. Light-shade adaptation in marine

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