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. Masojı́dek1, G. Torzillo2∗ , J. Kopecký1 , M. Koblı́žek1,3, L. Nidiaci2 , J. Komenda1 ,
A. Lukavská1 & A. Sacchi2
1 Department
of Autotrophic Microorganisms, Institute of Microbiology, Academy of Sciences, 379 81 Třeboň,
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 České Budějovice, 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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