Journal of Applied Phycology 12: 417–426, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
417
Changes in chlorophyll fluorescence quenching and pigment composition in the green alga Chlorococcum sp. grown under nitrogen deficiency and salinity stress J. Masoj´ıdek1, G. Torzillo2∗ , J. Kopeck´y1 , M. Kobl´ıžek1,3, L. Nidiaci2 , J. Komenda1 , A. Lukavsk´a1 & A. Sacchi2 1 Department
of Autotrophic Microorganisms, Institute of Microbiology, Academy of Sciences, 379 81 Tˇreboˇn, Czech Republic 2 Centro di Studio dei Microrganismi Autotrofi del CNR, Piazzale delle Cascine 27, 50144 Florence, Italy 3 Faculty of Biology, University of South Bohemia, 370 05 Cesk´ ˇ e Budˇejovice, Czech Republic (∗ Author for correspondence; e-mail
[email protected]) Received 1 November 1999; revised 16 April 2000; accepted 16 April 2000
Key words: astaxanthin, Chlorococcum sp., fluorescence, nitrogen deficiency, photobioreactor, secondary carotenoid, salinity, xanthophyll cycle
Abstract Changes in the in vivo chlorophyll fluorescence quenching, photosynthesis and pigment composition were followed in the green alga Chlorococcum sp. during exposure of the culture to nitrogen deficiency and salinity stress with the aims to study the interrelations between changes in physiological and photochemical parameters and xanthophyll-cycle pigments content during adaptation to stress, and to evaluate the capacity of this green alga to produce secondary carotenoids in tubular photobioreactors. Exposure of Chlorococcum to nitrogen deficiency, 0.2 M NaCl and high irradiance outdoors caused a strong depression of the photosynthetic activity and of photochemical quantum yield of PSII (Fv /Fm ). These changes were accompanied by an increase of the non-photochemical quenching coefficient (NPQ), of the amount of xanthophyll-cycle pigments and of the carotenoid/chlorophyll ratio. As a result of exposure to stress conditions, cell division completely stopped, although an increase in the biomass dry weight could be detected due to an increase in the cell size. These processes were followed, with a certain delay (15–20 h), by massive appearance of secondary carotenoids that reached the maximum (about 50% total carotenoids) after 2–3 days of cultivation. The results show that despite of the lower carotenoid content (2 mg g−1 dry wt) as compared with Haematococcus, Chlorococcum can be a potentially interesting strain for secondary carotenoid production because of its higher growth rate. 0
Abbreviations: Fo , Fv , Fm – minimum, variable and maximum fluorescence in dark-adapted cultures; F, Fm – steady-state and maximum fluorescence in light-adapted cultures; Fv /Fm – maximum photochemical quantum 0 yield of photosystem II; NPQ – Stern-Volmer quenching, (Fm /Fm )-1.
Introduction Photosynthetic organisms must adapt to unfavourable conditions in their environment to optimise and preserve the function of the photosynthetic apparatus. Such an adaptation becomes crucial under conditions
where the absorbed light greatly exceeds their photosynthetic capacity. Photosynthetic organisms have developed photoadaptive and photoprotective mechanisms at the level ranging from the whole plant, the leaf or cell, to the photosynthetic membranes (Björkman & Demmig-Adams, 1994); biochemical
418 changes in the content and composition of cell pigments (chlorophylls, carotenoids) allow them to downregulate energy transduction. Carotenoids are widely found in plants and animals (Goodwin, 1976; 1980). In photosynthetic organisms the carotenoids serve at least two important functions in photosynthesis, namely light harvesting and photoprotection (Burnet, 1976; Mathis & Schenck, 1982; Schroeder & Johnson, 1993). Under physiological conditions, microalgal cells possess carotenoids normally found in the chloroplast of higher plants, namely neoxanthin, violaxanthin, lutein, zeaxanthin and β-carotene, also referred to as primary carotenoids. Plants and some green algae exhibit rapid, light-dependent, reversible interconversions of violaxanthin to zeaxanthin via antheraxanthin (xanthophyll cycle). Zeaxanthin formation has often been directly related to the rapidly reversible component of non-photochemical quenching in higher plants (e.g. Björkman, 1987; DemmigAdams, 1990; Gilmore, 1997). However, in spite of these data indicating the existence of the zeaxanthindependent non-photochemical quenching, a number of reports have been published showing poor correlation between light-induced zeaxanthin accumulation and quenching of variable chlorophyll fluorescence in higher plants (Jahns & Krause, 1994; Schindler & Lichtenthaler, 1994; Tardy & Havaux, 1996) and microalgae (Niyogi et al., 1997; Casper-Lindley & Björkman, 1998; Masojídek et al., 1999). Secondary carotenoids (carboxylated xanthophylls), such as astaxanthin and canthaxanthin are produced by certain algae, fungi and crustaceans. The secondary carotenoids almost exclusively accumulate under stress conditions (e.g. nutrient starvation, salinity, temperature extremes in synergism with high irradiance) and this process is species specific. The physiological function of secondary carotenoids has not been clarified yet. However, it is generally believed that they function as passive photoprotectants (i.e. as a filter) reducing the amount of light which can reach the light-harvesting pigment complexes of PSII (Bidigare et al., 1993; Zlotnik et al., 1993; Hagen et al., 1994) The secondary carotenoid astaxanthin can be accumulated in some algae, such as Haematococcus (Droop, 1955), Euglena and Acetabularia (Czeczuga, 1974, 1986), Chlorella (Rise et al., 1994) and Chlorococcum (Brown et al., 1967; Zhang et al., 1997). Haematococcus has received the most interest due to its high astaxanthin content of up to 5% of dry wt (Borowitzka et al., 1991; Lee & Soh, 1991; Boussiba & Vonshak, 1991, 1992). However, its cultivation on
an industrial scale may be difficult for several reasons: (i) difficulty in achieving high productivity in outdoor cultures due to its slow growth rate (about 0.03 h−1 ); (ii) relatively low biomass concentration (about 1 g L−1 ) (Chaumont & Thepenier, 1995); (iii) optimal temperature for growth between 24–28 ◦ C (Lu et al., 1994), which may reduce the economic advantage of using a closed system due to high cost for culture cooling; (iv) susceptibility to contamination and predation in open cultures. The awareness of such limitations prompts the search for alternative secondary carotenoid producing microalgal strains with higher temperature tolerance and faster growth rate. Recently the green alga Chlorococcum has been proposed as another candidate for astaxanthin production (Zhang et al., 1997); however there is little information on the physiological and photochemical changes during the transition of the cells to the stressed conditions which cause accumulation of secondary carotenoids. Knowledge of such changes may help in finding the type of stress which best stimulates secondary carotenoid synthesis. The aims of the present study were to examine the interrelations between changes in physiological and photochemical parameters and xanthophyll-cycle pigments content during adaptation of the unicellular alga Chlorococcum sp. to stress conditions (N-deficiency + salinity, under high irradiance) and to evaluate its capacity to produce secondary carotenoids in outdoor culture. Materials and methods Organism and culture conditions The microalga Chlorococcum sp. was isolated locally and identified by G. Torzillo. Laboratory cultures were grown photoautotrophically in BG11 medium (Rippka et al., 1979) at 30 ◦ C under continuous illumination (150 µmol photon m−2 s−1 ). Photosynthetically active radiation (PAR) was provided by four fluorescent lights and measured with a Li-185B quantum sensor (Li-Cor, USA). The experiments were performed outdoors in horizontal 50 L tubular photobioreactors made of 10 parallel Pyrex glass tubes (length 2 m, i.d.= 48.5 mm) (Bocci et al., 1987). The photobioreactor was placed in a stainless steel basin containing water controlled of 32◦ C ± 1◦ C. The pH of the culture was controlled at 7.0 ± 0.1; the dissolved oxygen concentration was kept at about 20 mg L−1 by automatic
419 addition of nitrogen; the circulation speed of the culture was 0.46 m s−1 , corresponding to a Reynolds number of about 11000 (whole turbulent flow). Most of the measurements were made between 0900 and 1700 h. Previous laboratory experiments had shown that nitrogen deficiency, increased salinity (0.1–0.3 M NaCl) and their combination, under high irradiance (800–1000 µmol photon m−2 s−1 ) caused an inhibition of the photochemical activities. This process was reflected in a reduction of the growth and an increase in the production of secondary carotenoids after 2 to 3 days of cultivation, particularly in the culture exposed to the combination of nitrogen deficiency and salinity stress. According to these results, we decided to perform similar experiments in tubular photobioreactors outdoors in summer. In stressed cultures (secondary carotenoid inductive conditions), the algae were harvested by centrifugation and resuspended in a nitrogen-free medium (BG110); 0.2 M NaCl was added to the culture 30 min before the start of the experiment. At the beginning of the second day the control culture was diluted to approximately the same cell density as the stressed one using fresh BG11 medium (50% v/v) in order to keep the cell densities of the cultures comparable. Analytical procedures Pigment content (Chl a/b, total carotenoids) was determined spectrophotometrically in 80% acetone (Lichtenthaler & Wellburn, 1983). The amounts of individual carotenoids were assessed by HPLC according to the procedure of Gilmore & Yamamoto (1991). Dry weight was determined in duplicates in 25-mL samples using 3 µm cellulose nitrate filters (Sartorius, Germany). Cell number was determined by counting triplicate samples in a Bürker haemacytometer.
mL) taken from photobioreactors at time intervals during the day. Minimum fluorescence Fo was measured by modulated light (< 0.3 µmol photon m−2 s−1 ) from a light-emitting diode (peak wavelength at 655 nm, 1600 Hz). A single, high-intensity flash (5500 µmol photon m−2 s−1 , 0.8 s in duration) was applied to raise fluorescence yield to the maximum value Fm (the maximum fluorescence yield in the dark-adapted state). Actinic light intensity was provided by a halogen lamp (FL-103, Walz). The steady-state F level was recorded after 90–180 s of illumination. Then, a satur0 ating pulse was applied to reach the Fm level, i.e. the maximum fluorescence yield in the light. Non-photochemical quenching coefficient was measured under a constant PFD of 2000 µmol m−2 s−1 and calculated as the Stern-Volmer quenching coeffi0 cient NPQ = Fm /Fm -1 (relative change in the rate constant of total non-photochemical energy dissipation) (Bilger & Björkman, 1990; Gilmore & Yamamoto, 1991). The effective absorption cross-section of open PSII reaction centres σ PSII (rate of PSII closure) was calculated from the induction curve on 5-min dark adapted samples measured by PAM 2000 (H. Walz, Germany) under 100 µmol m−2 s−1 of red light in the presence of 1 × 10−5 M 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The fluorescence induction curve was then fitted by cumulative one-hit Poisson function F = Fm − (Fm − Fo ) × exp(−σPSII · t), where F is the fluorescence measured during the induction, Fo is the minimal fluorescence, Fm is the maximal level of fluorescence reached in saturation, t is the time and σ PSII is the effective absorption cross-section.
Fluorescence and oxygen measurement
Results
Both chlorophyll fluorescence and oxygen evolution were measured in a stirred cuvette (model DW2, Hansatech, King’s Lynn, England) connected to an O2 electrode control box and chart recorder. The fibreoptic light guide of a pulse-amplitude-modulation fluorimeter (PAM 101-103 coupled with the emitterdetector unit ED-101US, H. Walz, Germany) was placed in one of the four transparent ports of the cuvette. The fluorescence nomenclature follows van Kooten & Snel (1990). The Fv /Fm ratio was determined in dark-adapted (10–15 min) culture samples (0.5
The experiments were carried out in tubular photobioreactors, outdoors in summer when the light intensity reached 2000 µmol photon m−2 s−1 . In order to increase the amount of light received by single cells and thus better to stimulate carotenoid synthesis, the initial chlorophyll concentration of the culture was set at 3 mg Chl L−1 in both control (standard BG11) and stressed cultures (BG110 plus 0.2 M NaCl). Although the major changes in physiological and photochemical parameters occurred during the first 2 days, the experiments were continued for several days in order to
420
Figure 1. Time course of the Fv /Fm ratio of control (open symbols) and in stressed, N-deficient + 0.2 M NaCl, cultures (closed symbols) of Chlorococcum sp. grown in photobioreactors under ambient irradiance (dashed line).
reach the maximum secondary carotenoid content in the stressed cultures. The exposure of diluted cultures to high irradiance during the day led to a decrease of the Fv /Fm ratio in both control and stressed cultures (Figure 1). During the first day of the experiment, in the control culture, Fv /Fm decreased from 0.72 to 0.41 between 0900 and 1300 h when the irradiance reached 1950 µmol m−2 s−1 . Thereafter, in the afternoon, the Fv /Fm ratio slowly recovered to 0.65 by 1700 h. In the stressed culture the Fv /Fm ratio was more depressed, from the morning value of 0.72 to 0.28 at 1300; during the afternoon it recovered only to 0.40. It is worth noting that the greatest decrease of Fv /Fm in this culture occurred during the first hour of exposure to light. During the second day, the reduction of the Fv /Fm ratio in both cultures was less dramatic than on the first day, however, the starting Fv /Fm value of the stressed culture was significantly lower (0.57) than the control culture (0.76). Generally, the Fv /Fm ratio of the stressed culture during the experiment was about one third lower than that of the control culture. The time course of Stern-Volmer quenching NPQ measured during the first two days of cultivation revealed significant differences between control and stressed cultures (Figure 2). Similar to the initial Fv /Fm changes, the NPQ values showed significant differences between the two cultures after 30 min sunlight exposure; the stressed culture reached a much
Figure 2. Changes in the Stern-Volmer non-photochemical quenching (NPQ) in control (open symbols) and in stressed, N-deficient + 0.2 M NaCl, cultures (closed symbols) of Chlorococcum sp. grown outdoors in photobioreactors. Measurements made at 2000 µmol photon m−2 s−1 .
Figure 3. Changes in the effective absorption cross-section of open PSII reaction centres in control (open symbols) and in stressed, N-deficient + 0.2 M NaCl, cultures (closed symbols) of Chlorococcum sp. grown outdoors in photobioreactors. Bars represent SE; n=3 replicates.
421 Table 1. Changes of the carotenoid content and carotenoid/chlorophyll a+b (Car/Chl) ratio in control and stressed cultures of Chlorococcum sp. during the first and second day of outdoor cultivation Day
Time of day (h)
Control culture Car content Car/Chl (mg L−1 )
Stressed culture Car content Car/Chl (mg L−1 )
1 1 2 2
9 17 8 17
0.72 1.5 1.41 4.85
0.77 0.85 0.94 1.50
0.24 0.28 0.25 0.25
0.24 0.35 0.40 0.52
higher level of NPQ compared to the control one (1.5 vs. 0.5). During the first day, the mean value of the NPQ of the stressed culture was 50% higher than that calculated for the control culture, and this increased to 60% on the second day. The measurements of the effective absorption cross-section of open PSII reaction centres σ PSII showed only minor differences between the two cultures during the first day, except for the last measurement at 1700 h (Figure 3). During the second day the value of σ PSII of the stressed culture remained high as at the end of the first day, but it was substantially higher than in the control culture (by about 50–70%). Pigment content, determined spectrophotometrically, revealed a lower Car/Chl ratio in the control culture at the end of the first day compared with the stressed culture (Table 1). At 0900 h on the first day this ratio was 0.24 in both cultures, while at 1700 h it changed to 0.28 in the control culture and to 0.35 in the stressed one. During the second day the Car/Chl ratio stayed unchanged in the control culture but it further increased from 0.40 to 0.52 in the stressed one. Typical examples of HPLC elution profiles of control (first day, 1100 h) and stressed cultures (after 3 days of cultivation) are shown in Figure 4. When the data were normalised to the same Chl a concentration, it was evident that the stressed culture contained significantly higher amounts of carotenoids than the control one. The secondary carotenoids astaxanthin (λmax about 482 nm), canthaxanthin (λmax about 472 nm) and astaxanthin esters (diester and monoester) were identified according to their absorption spectra measured using a diode-array detector (Jeffrey et al., 1997) and to their retention time. No secondary carotenoids were found in the control culture, even after 3 days of cultivation under high solar irradiance.
Figure 4. Two examples of HPLC elution profiles of control (sampled on the first day at 1100 h, solid line) and the culture grown under N-deficiency in the presence of 0.2 M NaCl (dashed line, sampled after 3 days of cultivation). The sequence of pigments according to the increasing retention time is: neoxanthin, violaxanthin/astaxanthin, unknown compound, antheraxanthin, lutein, zeaxanthin, canthaxanthin, chlorophyll a, chlorophyll b, astaxanthin esters and β-carotene. Data are normalised to the chlorophyll a content.
Figure 5. Changes in the content of astaxanthin, lutein and canthaxanthin in control (open symbols) and in stressed, N-deficient + 0.2 M NaCl, cultures (closed symbols) of Chlorococcum sp. grown outdoors in photobioreactors. The amount of xanthophylls is expressed as µg pigment per mg Chl.
422 In the stressed culture some traces of free astaxanthin and canthaxanthin as well as a partial increase of the lutein content were already seen at 1700 h of the first day (Figure 5). After two days of cultivation the secondary carotenoids increased several times in parallel with the 70% decrease of the lutein content. After 3 days of cultivation the content of lutein, astaxanthin (including the free form and its mono- and diester) and canthaxanthin became stable and did not change substantially during further cultivation, reaching values of 48.8 µg mg−1 Chl, 55.4 µg mg−1 Chl and 31.2 µg mg−1 Chl, respectively. After three days of cultivation under sunlight, the final content of secondary carotenoids in the stressed culture reached about 2 mg g−1 dry wt. Further extension of the cultivation period did not result in an increase in secondary carotenoids accumulation. The changes in the content of the xanthophyll cycle pigments during the first 2 days of cultivation are shown in Figure 6. It can be observed that (i) the extent of changes was much greater during the first day and (ii) the pool size of the xanthophyll cycle pigments (calculated in µg per mg Chl) was larger in the stressed culture than in the control one. The maximum content of zeaxanthin (54 µg mg−1 Chl) in the control culture was found at 1100 h on the first day (panel A in Figure 6) while the maximum of zeaxanthin in the stressed culture (90 µg mg−1 Chl) appeared at 1500 h, 4 h later (panel B in Figure 6). In the stressed culture the decrease of the violaxanthin content occurred mainly during the first 2 h of cultivation when about 75% of violaxanthin converted to antheraxanthin and zeaxanthin. The antiparallel increase of the zeaxanthin content continued for another 4 h (panel B in Figure 6) since a de novo synthesis of xanthophylls probably occurred during this period as their total amount increased by about 50%. The extent of the xanthophyll cycle changes was much smaller during the second day in both cultures. The lower amount of xanthophylls in the control culture showed its better light acclimation as compared with the stressed one (Figure 6). Comparing the extent of NPQ and the content of zeaxanthin during the first and second day in the stressed culture, we observed significant differences. It is interesting to note that, contrary to what is usually observed in higher plants (Demmig-Adams, 1990), the maximum levels of zeaxanthin and NPQ did not match. Indeed, on the first day the maximum value of NPQ was observed at 1300 h while the highest amount of zeaxanthin was found at 1500 h (compare Figures 2 and 6).
Figure 6. Changes in the content of violaxanthin (circles), anteraxanthin (squares) and zeaxanthin (triangles) in control (A) and stressed (N-deficient + 0.2 M NaCl) cultures (B), of Chlorococcum sp. grown outdoors in tubular photobioreactors.
The time course of the maximum photosynthetic activity (oxygen evolution) of the cultures is reported in Figure 7. During the first day of cultivation, the photosynthetic activity of the control and stressed cultures followed an opposite pattern. In the control culture, the photosynthetic activity increased by about 30% during the day, while in the stressed one it decreased up to one third of the morning value. During the second
423
Figure 7. Changes in photosynthetic oxygen evolution (PSOE) in control (open symbols) and in stressed, N-deficient + 0.2 M NaCl, cultures (closed symbols) of Chlorococcum sp. grown outdoors in photobioreactors. Measurements were done at 2000 µmol m−2 s−1 .
day, a further increase in photosynthetic activity was observed in the control culture while in the stressed one it remained strongly depressed to about one fifth of the value measured at the start of the experiment. Growth parameters (Chl, cell number, dry weight), are reported in Figure 8. During the first day the Chl content increased by 60% in the control culture, and decreased slightly in the stressed one. On the following day, the Chl content of the control culture grew 3.5-fold while it was almost constant in the stressed culture (Figure 8A). The number of cells in the control culture almost doubled during the first day, whereas in the stressed culture, the cell number did not change as significantly indicating an inhibition of the cell division as a result of the stress conditions (Figure 8B). At the beginning of the second day, the control culture was diluted by 50% (v/v) with fresh medium in order to keep the amount of light received by cells in both cultures comparable. In spite of such a dilution, the cell number in the control culture did not change significantly from the evening value indicating that a massive cell division had taken place during the night (Figure 8B). During the second day, the cell number increased by only 10% in the control culture while it remained almost constant in the stressed culture. Changes in dry weight in both control and stressed cultures are reported in Figure 8C. During the first day an increase in dry weight of about 65% and 30% oc-
Figure 8. Changes in total chlorophyll content (panel A), cell number (panel B) and dry weight (panel C) in control (open symbols) and in stressed, N-deficient + 0.2 M NaCl, cultures (closed symbols) of Chlorococcum sp. grown outdoors in photobioreactors.
424 Table 2. Growth rates of control and stressed cultures of Chlorococcum sp. during the first and second day of outdoor cultivation. ∗ calculated on dry wt basis; ∗∗ calculated on cell number basis Day
Control culture µ (h−1 )∗ µ (h−1 )∗∗
Stressed culture µ (h−1 )∗ µ (h−1 )∗∗
1 2
0.134 0.139
0.056 0.051
0.070 0.018
0.024 0.0
curred in control and stressed culture, respectively. On the second day, the dry weight increased 3.5-fold in the control culture and 1.6-fold in the stressed one. Microscopic observation showed an increase in the cell size during the day in both cultures, accompanied with change of the cell shape from ellispoid to spherical. Comparison of the average specific growth rates calculated on the basis of dry weight or cell number, showed striking differences in both cultures (Table 2). For example, during the first day in the control culture, specific growth rate calculated on dry weight basis was about 2-fold higher than that calculated on cell number basis, this is because of the significant increase in the size of cells which occurred during the day. In the stressed culture, cells number slightly increased during the first day while it did not change significantly on the second day, so that growth rates normalised on cell basis were close to zero. However, due to an increase in cell size, growth rates on dry weight basis reached values of 0.056 and 0.051 h−1 during the first and the second day, respectively.
Discussion Exposure of Chlorococcum cells to N-deficiency, 0.2 M NaCl and high irradiance outdoors caused an inhibition of cell division and a strong depression of photosynthetic activity. However, cells continued to increase in size resulting in a 35% increase in dry weight during the first day. Secondary carotenoids (astaxanthin and canthaxanthin) started to accumulate in the cells about one day after the exposure to sunlight. In agreement with the results of Zhang et al. (1997) astaxanthin was accumulated in free and ester forms (mono- and diester). When traces of secondary carotenoids appeared in the stressed culture by the late afternoon of the first day, the content of xanthophyll cycle pigments was about twice as high than in the control culture. This phenomenon was accompanied
by a depression in the photosynthetic activity to about one third of the morning value. Contrary to what was expected, effective absorption cross-section of PSII reaction centres resulted higher in the stressed culture than in the control one; we expected this parameter to be smaller in the stressed culture due to its higher NPQ than in the control one. We hypothesise that inactive PSII reaction centres can still collect excitons and spill them over to active centres, thus increasing the effective antenna size. This phenomenon, although not as pronounced, has also been reported in other microalgae (Scenedesmus, Chlorella) under high irradiance (Masojídek et al., 1999). During the second day of cultivation the synthesis of astaxanthin and canthaxanthin reached its highest rate; their increase was lower during the third and fourth day. The most striking differences between the control and the stressed culture was observed through the measurement of the photosynthetic oxygen evolution. In the stressed culture, the start of secondary carotenoids accumulation was preceded by a strong reduction of the photosynthetic oxygen evolution. Another important feature of the stressed culture was almost no change of the cell number while the dry weight doubled during two days of cultivation. In the process of adaptation of Chlorococcum to stress, we tried to find out whether the xanthophyll cycle plays some role in dissipating excess light energy. Our results from fluorescence and xanthophyll cycle measurements have shown that in this microalgal strain whose light-harvesting and xanthophyll-cycle pigments are similar to higher plants, there is not a direct relationship between light-induced zeaxanthin formation and NPQ. Indeed, changes in the value of NPQ and in zeaxanthin content did not match. In the stressed culture, zeaxanthin accumulation was much greater during the first day compared to the second day, but NPQ was higher during the second day (compare Figures 2 and 6A). The discrepancy was even more striking in the control, where on the second day the maximum amount of zeaxanthin reached only about 10% of the maximum value of the first day, but the average value of NPQ was only 5 to 10% lower. Similar conclusions were reached by Masojídek et al. (1999) with Scenedesmus and Chlorella where zeaxanthin-dependent quenching was found to contribute only to a limited extent to overall non-photochemical quenching. Our studies indicate that Chlorococcum can be easily grown at 32◦ C with high biomass growth rate (0.13 h−1 ) in standard medium, while under stressed
425 conditions (N-deficiency and increased salinity) its growth rate decreases by one half. Both growth rate and optimal temperature are significantly higher (µ = 0.03 h−1 , t = 24–28 ◦ C) than those reported for Haematococcus pluvialis (Lu et al., 1994; Ding & Lee, 1994). These results are in agreement with those obtained by Zhang et al. (1997) who suggested the suitability of Chlorococcum for outdoor cultivation at moderate and warmer climate zones and the possibility of developing astaxanthin production in a closed system. In outdoor cultures, a concentration of secondary carotenoids (astaxanthin, its esters and canthaxanthin) of about 2 mg g−1 dry wt was obtained after 3 days of cultivation, corresponding to a yield of about 1 mg L−1 in the 50-litre photobioreactor. These values are comparable with the yeast Phaffia but much lower than those obtained with Haematococcus (4–5% dry weight). A further increase in the secondary carotenoid content in Chlorococcum sp. could be obtained by optimising the stress conditions that better stimulate the synthesis of carotenoids. However, it must be pointed out that the almost 4-fold higher biomass growth rate attainable with Chlorococcum (0.139 h−1 ), may partially compensate for its lower astaxanthin content. Our results represent a step in the search for algal strains other than Haematococcus suitable for secondary carotenoid production and in the development of a cultivation technology in a large-scale photobioreactor.
Acknowledgements This paper is dedicated to Professor David O. Hall – friend, colleague and mentor. Research carried out in the framework of the Bilateral Agreement between the National Research Council of Italy and the Czech Academy of Sciences and in part supported by the project No. 206/96/1222 of the Grant Agency of the Czech Republic. We thank Ms. Jana Hofhanzlová, Ms. Anna Mati and Mr. Edoardo Pinzani for their able assistance during experiments.
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