Hydrobiologia 238: 149-157, 1992.
T. Berman, H. J. Gons & L. R. Mur (eds), The Daily Growth Cycle of Phytoplankton.
© 1992 Kluwer Academic Publishers. Printed in Belgium.
Variations in biochemical parameters of Heterocapsa sp. and
Olisthodiscus luteus grown in 12:12 light:dark cycles
II. Changes in pigment composition
Mikel Latasa, Elisa Berdalet & Marta Estrada
Institut Ciencies del Mar, Passeig nacional s/n, 08039 Barcelona, Spain
Key words: Heterocapsa sp., Olisthodiscus luteus, cycles, pigments, HPLC
Photosynthetic pigment composition was studied in batch cultures of Heterocapsa sp. and Olisthodiscus
luteus growing exponentially in a 12:12 light:dark cycle. Both species divided in the dark. The synthe-
sis of pigments was continuous for both species. However for chlorophyll c and peridinin, in Heterocapsa
sp., and chlorophyll c and fucoxanthin, in 0. luteus, (pigments belonging to light harvesting complexes)
the synthesis was significantly higher during the light period. Concentrations per total cell volume (TCV)
of chlorophyll a, chlorophyll c, peridinin and diadinoxanthin in Heterocapsa sp., and chlorophyll a,
chlorophyll c, fucoxanthin and violaxanthin in 0. luteus, showed a maximum at the onset of light and
decreased during the light period. The values of the chlorophyll a:chlorophyll c, chlorophyll a:peridinin
and chlorophyll a:fucoxanthin ratios are compared with data reported in the literature.
In oceanography, measurements of the concen-
tration of photosynthetic pigments, particularly
chlorophyll a, have been routinely used as esti-
mators of phytoplankton biomass. In addition,
photosynthetic pigment composition has been
used as chemotaxonomical marker (Jeffrey, 1974;
Gieskes & Kraay, 1983; Smith et al., 1987).
However, the concentration of different pigments
in the phytoplankton cells, and also the ratios
between these concentrations, are subjected to
variability in response to external factors such as
irradiance, nutrient availability and temperature.
This variability complicates the use of pigment
determination in biomass and taxonomical stud-
ies, but has provided a basis for employing pig-
ment composition as an indicator of the physio-
logical state of populations (Margalef, 1960, 1963;
Manny, 1969; Hallegraeff, 1976). In addition to
the effect of external factors, changes in the cel-
lular contents of pigment may be linked with the
cell division cycle, which, in photosynthetic or-
ganisms, exhibits typically a more or less tight
coupling to the light/dark cycle (see Sournia,
1974, for review). In spite of the importance of
investigating all sources of pigment variability,
both to understand the functions of some of these
pigments and to use them as taxonomical or
physiological markers, there are few experimental
works dealing with aspects such as rhythmical
fluctuations. Our study focused on the 12:12
light:dark periodicity and the alignment with the
division cycle, of the major pigment components
of two species, Heterocapsa sp. (Dinophyceae)
and Olisthodiscus luteus (Raphidophyceae) grow-
ing in batch culture. A parallel study of the fluc-
tuations in RNA and DNA in the same cultures
is reported in Berdalet et al. (1992).
Material and methods
A unialgal culture of Heterocapsa sp. (sensu Mor-
rill & Loeblich III, 1981), isolated by E. Berdalet
from the Barcelona Harbour and another of
0. luteus (provided by the Plymouth Marine Lab-
oratory) were grown in 4 1 Pyrex flasks filled with
3 1 of f/2 culture medium without silicate (f/2-Si)
(Guillard, 1975), and kept at a constant temper-
ature of 18 + 1 C. A quantum flux density of
150 pE m-2 s - 1 on a 12:12 light:dark (L:D) cycle
was provided by cool white fluorescent lamps.
Inocula were taken from exponentially growing
cultures adapted to the same environmental con-
ditions. Samples for the experiment were taken at
3 hours intervals, for 48 hours, during the expo-
nential phase of the cultures.
Cell number and the distribution of particle
size in 32 volume classes were determined in vivo
using a Multisizer Coulter Counter with a 140 #jm
0 aperture tube. Additional countings using the
inverse microscope technique (Utermohl, 1958)
were made to check the Multisizer Coulter re-
sults. We observed that high cell concentration
(> 5 % coincidence) resulted 'in an underestima-
tion in the number of particles recorded by the
Multisizer Coulter, although the volume/particle
ratio remained almost constant. According to
that, we applied microscopic countings to calcu-
late cell number and total cell volume (TCV) in
Heterocapsa sp.. In 0. luteus, the coincidence level
was always less than 5%, because this culture
never reached a density as high as Heterocapsa sp.
(Figs. 2a and 3a), and microscopic results agreed
with Multisizer Coulter countings.
HPLC analysis of pigment composition was
based on the method of Mantoura and Llewellyn
(1983), with minor modifications. Samples were
filtered through Whatman GF/C filters which
were subsequently ground with a teflon-glass
grinder (Braun-Melsungen), in 90 % acetone. The
chromatographic system consisted of a LKB 2152
controller, a LKB 2150 pump, a 7125 Rheodyne
injection valve and a ODS-2 column (5# m
spherical packing, 150 mm length, 4.6 mm inter-
nal diameter). Absorbance was measured at
440 nm (LKB, 2151 variable wavelength moni-
tor), and fluorescence detection (Perkin-Elmer
Em = 600-800 nm) was used to aid in the iden-
tification of chloropigments. Peak areas were
measured (Shimadzu C-R3A integrator) on the
For analysis, a 200 l mixture of 2:1 pigment
extract in 90% acetone and PIC (the ion-pairing
solution, see below) was injected into the chro-
matographic system. A linear gradient was run
80:10:10) to 100% eluent B (methanol:acetone
70:30) in 8 min, and elution was continued
with 100% B until 20min. Flow rate was
1.3 ml min- . The ion-pairing solution consisted
of 1.5 g of tetrabutylammonium acetate, and 7.7 g
of ammonium acetate in 100 ml of distilled water,
with pH corrected to 7.1. Organic solvents were
HPLC grade, and water was purified by a Milli-
Q system (Millipore Corp.).
To identify pigments, fractions were collected
as they eluted from the column. Reversed-phase
separation cartridges (C-8) were used to transfer
pigments from the eluent to ethanol. Spectra were
recorded in a Hitachi 150-20 spectrophotometer.
Extinction coefficients from Foppen (1971),
Mantoura & Llewellyn (1983) Wright & Shearer
(1984) and Rowan (1989) were employed to cal-
culate concentration of standards. Typical chro-
matograms are shown in Fig. 1.
To estimate variability due to sampling and
analytical error (which we will call thereafter ex-
perimental error) for pigment analysis, 4 and 6
samples were taken respectively from exponen-
tially growing cultures of Heterocapsa sp. and
0. luteus. Cell number and total cell volume
(TCV) were estimated from an additional sample
for each species. The resulting coefficients of vari-
ation are given in brackets in Table 2 and 3.
In both species studied, we considered that
volume was proportional to biomass because of
the nonexistence of gas vacuoles. Thus, our de-
terminations of pigment concentration per TCV
could be taken as estimates of concentration per
Overall growth rates (doublings/day, Kd) for
the cultures were calculated between 0 and 24
Ex = 450 nm,
10 15 20
Fig. 1. Typcial absorbance chromatograms of both species:
(A) Heterocapsa sp. and (B) O. luteus. Peak identities:
(1) Chlorophyll c, (2) Fucoxanthin, (3) Violaxanthin, (4) Chlo-
rophyll a, (5) B-carotene, (6) Peridinin, (7) Diadinoxanthin.
hours (first day), and between 24 and 48 hours
(second day), according to the formula (Guillard,
Kd = K/ln2, where K = (InNi - InNO)/tl - to).
Raw data were smoothed calculating sliding av-
erage value of two consecutive points. All param-
eters were treated in the same way.
In both species, cellular division occurred during
the dark period, as can be deduced from the de-
crease in average cell volume and the increase in
cell number (Figs 2 and 3). Cellular growth took
place during the light period, as reflected in the
increase in average cell volume and in TCV per
unit volume of culture. The average size of Het-
erocapsa sp. was smaller the second day, and the
growth rate varied from 0.97 to 1.10 doublings/
day, between the first and the second day. In
0. luteus the change in growth rate during the
same period was also negligible (from 1.01 to 0.96
Pigment synthesis seemed to be continuous
throughout the L:D cycle (Fig. 4). Chlorophyll c
and peridinin, in Heterocapsa sp., and chloro-
24 36 48
Fig. 2. Heterocapsa sp., changes in (a) cell number and total cell volume (TCV), (b) mean cell volume. Shadow areas indicate dark
Fig. 3. 0. luteus, variations in (a) cell number and total cell volume (TCV), (b) mean cell volume. Shadow areas indicate dark
Fig. 4. Pigment concentration in the cultures. (a) Heterocapsa sp., (b) 0. luteus. Dark periods are indicated by shadow areas.
phyll c and fucoxanthin, in 0. luteus, presented
light:dark fluctuation with a production maximum
during the light time; no clear periodicity could be
observed in chlorophyll a and the other pigments
Concentrations per TCV of chlorophyll a,
chlorophyll c, peridinin and diadinoxanthin in
Heterocapsa sp., and chlorophyll a, chlorophyll c,
fucoxanthin and violaxanthin in 0. luteus, showed
a maximum at the onset of light and decreased
during the light period (Fig. 5). This accumula-
tion started in the middle of the dark period in
Heterocapsa sp. and near the end of the darkness
in 0. luteus. All pigments increased slightly their
concentrations per cell volume from the begin-
ning to the end of the experiment (Fig. 5).
The similarity of the concentration increase
patterns of fucoxanthin and chlorophyll c in
0. luteus and peridinin and chlorophyll c in Het-
erocapsa sp. (Table 1) could be explained by their
association in the Light Harvesting Complexes
(LHC) (Pr6zelin & Haxo, 1976; Friedman & Al-
berte, 1984). We have not calculated the incre-
ment of ,-carotene due to the low concentrations
Table 1. Percentage increase of each pigment in the light and
dark periods. These values were obtained from the following
expression: [(PrPo)/Po] x 100, where Pf and Po mean respec-
tively, pigment concentration referred to culture volume at the end
and the beginning of the period.
found during the experiment, which bordered the
Table 2 presents the average concentration per
TCV of the major pigments, for all samples com-
Fig. 5. Changes in pigment concentration related to total cell volume (TCV). (a) Heterocapsa sp., (b) 0. luteus. Dark periods are
indicated by shadow areas.
Table 2. Averaged concentration of pigments referred to Total
Cell Volume (TCV) for samples taken between the first and
the second maximum in average cell volume (n = 8, a 'gener-
ation time'). An estimation of the experimental error (the co-
efficient of variation, see text) associated to experimental con-
ditions is presented in brackets.
prised between the first and the second peak in
cell volume (which we will consider as a 'gener-
ation time'). The coefficients of variation ranged
from 4.5 to 14.5, except for f-carotene in Hetero-
capsa sp., whose concentration
a coefficient of variation of 35.3. The average
values of the ratios chlorophyll a:other pigments
are given in Table 3. In Heterocapsa sp. the
Table 3. Chlorophyll a: pigment ratios in both species. Molar
and weight relationship are presented. (n = 8, comprising a
'generation time' for each species). The error (coefficient of
variation) associated to experimental conditions is shown in
mol/mol w/w C.V. %
Chl a/chl c
chlorophyll a:/-carotene ratio showed also a
higher variability than other ratios (38.6 against a
range between 5.4 and 11.6).
The ratios in which violaxanthin is included are
also shown. They may be interesting for chemo-
taxonomical purposes, because, although neither
fucoxanthin nor violaxanthin are markers of a
single algal group, they appear together only in
Chrysophyceae (Rowan, 1989). 0. luteus used to
be included in the Chrysophyceae class due to
xanthophyll content (Gibbs et al., 1980), but re-
cent studies have placed this species among the
Raphidophyceae (Vesk & Moestrup, 1987).
The most striking finding of this experiment is
that pigment synthesis occurred during the light
and dark periods in both species (Fig. 4).This
contrasts with the results of most experiments
reported in the literature in which the production
of chlorophyll stopped at dark time in species
belonging to a wide variety of groups, such as
diatoms (Owens etal., 1980), Prasinophyceae
(Kohata & Watanabe, 1988), Raphydophyceae
(Kohata & Watanabe, 1989) or Chlorophyceae
(see references in Meeks, 1974). However, Guer-
rero et al. (1988) had reported chlorophyll a syn-
thesis in darkness for algal cultures of Isochrysis
galbana Parke, Platymonas spp., Dunaliella marina
and Pavlova lutheri, growing in exponential phase
in LD light regime and subsequently changed to
continuous darkness. Similar phenomena was
observed by Welschmeyer & Lorenzen (1985) in
Thalasiossira pseudonana and Dytilum brightwelli
and by Yentsch (1965) in Phaeodactylum tricor-
nutum. In Gonyaulaxpolyedra which has a marked
light-dark rhythmicity, pigments synthesis is con-
tinuous (Prezelin & Sweeney, 1977). A priori,
there should be no reason to preclude chlorophyll
production in the dark, since algal species grow-
ing heterotrophically in darkness are able to syn-
thesize chlorophyll a (Meeks, 1974). Guerrero
et al. (1988), attributed cell growth during dark-
ness to the effect of the illumination used at the
sampling time. However, during our experiment
the amount of light reaching the cultures at each
sampling time did not exceed 1 E m -2s - dur-
ing 10 min. Given that our cultures were adapted
to 150 IE m- 2s - 1, it is unlikely that an irradi-
ance of 1 E m- 2s-1 affected a biochemical
process, such as pigment synthesis, in a quanti-
tatively important way. Thus, we assume a pro-
duction of pigments by the cells throughout the
light:dark period, although at variable rates
The increase in concentration per TCV after
cell division resulted in maximum pigment con-
centrations at the onset of light. This pattern may
have the advantage of allowing the cells to start
photosynthetic activity with an optimum pigment
pool. Some sort of anticipation has been reported
also by Putt & Prezelin (1985), in relation to pho-
tosynthetic activity in a natural population.
As discussed in Berdalet et al. (this volume),
DNA and RNA concentrations per TCV were
highest at the end of the dark period. In addition,
the RNA/DNA ratio indicated a preferential syn-
thesis of RNA during darkness. High values of
RNA (mostly ribosomic RNA) are coupled to
active periods of proteins synthesis (Dortch et al.,
1983), including proteins associated to pigments.
Thus it is likely our results evidenced a relation-
ship between increase in RNA concentration and
synthesis of pigments.
In both species, the pigment/volume ratio was
slightly higher the second day. Among the many
factors affecting pigment concentration in cells,
the best studied are the irradiance level, nutrient
availability and temperature (see review in Meeks,
1974). In our experiment, temperature remained
constant and nutrients were never limiting, but
the irradiance received by the cells could have
decreased due to self-shading, and this could have
caused an increase in pigment concentration
(Steeman Nielsen & Jorgensen, 1968). For Het-
erocapsa sp., the smaller average cell size during
the second day may be a partial explanation for
the increment of the ratio in this specie. Kohata
& Watanabe (1988) had noticed also that self-
shading induced an increase of pigment concen-
tration and a decrease of cell size.
The ratios given by Jeffrey etal. (1975) for
chlorophyll a:peridinin and chlorophyll a:chloro-
phyll c averaged 2.24 + 0.66 and 3.49 + 0.79
(mean + standard deviation, w:w), respectively,
for 22 species of dinoflagellates. These values
are slightly higher than those we found for
Heterocapsa sp. (Table 3). The higher illumina-
tion in our experiment (150 ME m- 2s-1 versus
1000 W cm
-2-around 50 gE m-2 s - '-in
frey et al., 1975) could have caused a lower chlo-
rophyll a:chlorophyll c ratio. Concerning the
chlorophyll a:peridinin ratio, Pr6zelin (1976) ob-
served that, with decreasing irradiance in Gleno-
dinium sp., chlorophyll a increased in relation to
chlorophyll c but decreased in relation to peridi-
nin. Thus, it appears that the lower chlorophyl-
l a:peridinin ratio in our experiment cannot be
attributed to the irradiance difference.
In 0. luteus, the chlorophyll a:chlorophyllc,
chlorophyll a:fucoxanthin and chlorophyll a:vio-
laxanthin ratios were similar or slightly higher
than the values of Kohata & Watanabe (1988) for
Chattonella antiqua grown under irradiance of
565 Em- 2s-1
(although the authors do not
give values of those ratios, we have calculated
them from their Figs. 5 and 8). According to some
authors (Loeblich & Fine, 1977) C. antiqua is a
species very closely related to 0. luteus.
Apart from the floristic difference, it is
likely that pigment composition may change
150 E m-2 s- 1, than
565 E m- 2s - 1, since 50 E m-2s - 1 is proba-
bly limiting to photosynthesis, while both 150 and
565 iE m-2s- 1 are likely to be saturating but
not high enough to have inhibitory effects. The
results of Falkowski & Owens (1980) and
Falkowski et al. (1981) seem to support this idea.
Nevertheless there are too few works dealing with
these ratios to allow a generalized description of
the patterns of light adaptation.
Assessment of the ranges of variability of pig-
ment content per TCV and of selected pigment
ratios in different phytoplankton groups is neces-
sary in order to allow direct estimation of the
contribution to total volume (and biomass) of
these groups. Data generally available in the lit-
erature refer pigment composition to chloro-
50 /E m- 2 -1
phyll a (Jeffrey et al., 1975; Vesk & Jeffrey, 1977;
Falkowski et al., 1981; Gieskes & Kraay, 1983;
Gallagher et al., 1984; Burkill et al., 1987), but the
high variability of the chlorophyll a:volume (or
cell) (Foy, 1987) ratio complicates extrapolation
to phytoplankton composition in terms of biom-
ass, which is often the variable of interest.
The fluctuations of pigment content per TCV
and pigment ratios due to light:dark cycles (Ta-
bles 2 and 3) were small when compared with
variability due to environmental factors such as
irradiance or nutrient availability (Senger & Fleis-
chhacker, 1978; Manny, 1969; Watson & Os-
borne, 1979; Latasa, unpublished data). How-
ever, assessing diel periodicity is important for
understanding physiological processes in the phy-
toplankton cell, and represents a first step for an
accurate estimate of the source of variability af-
fecting phytoplankton pigments in natural and
experimental conditions. An improved knowledge
of the effect of internal and external factors on
pigment concentrations is necessary for a reliable
use of pigments as indicators of biomass and as
chemotaxonomic or physiological markers.
The authors are grateful to E. Saiz for the com-
ments on the laboratory work. We also thank Dr
P. J. Wangersky who reviewed the manuscript.
This research was supported by FPI grants from
the Ministerio de Educaci6n y Ciencia de Espafia
and funds from Programa Fronts MAR-880252-
CYCIT and CSIC.
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