Journal of Biotechnology 89 (2001) 65 – 71

www.elsevier.com/locate/jbiotec

Two-stage cultures for the production of Astaxanthin from

Haematococcus plu6ialis
Jaime Fábregas *, Ana Otero, Ana Maseda, Adolfo Domı́nguez
Laboratorio de Microbiologı́a, Facultad de Farmacia, Uni6ersidad de Santiago, Santiago 15782, Spain

Received 24 November 2000; received in revised form 23 April 2001; accepted 18 May 2001

Abstract

A two-stage culture system was established for the production of astaxanthin from Haematococcus plu6ialis. In a
first stage green vegetative cells were produced in semicontinuous cultures maintained with daily renewal rates
between 10 and 40%. The steady-state cell density decreased with increasing renewal rates. Highest cell productivity,
64 ×106 cells l − 1 day − 1 was obtained with a daily renewal rate of 20%. In a second stage the harvested cultures were
submitted to high light (240 mmol photon m − 2 s − 1) under batch conditions for 15 days in order to stimulate the
transition to the aplanospore stage and the accumulation of astaxanthin. No decrease in cell density was recorded
during the induction period in any of the cultures. Cultures obtained at high renewal rates continued growing during
the induction period and no astaxanthin was accumulated until all nitrogen in the media had been consumed. The
final concentration of astaxanthin was inversely correlated to the growth rate at which first-stage cultures were
maintained. Optimal renewal rate for maximal astaxanthin production depended on the duration of the induction
period. After a 12-day induction period the highest astaxanthin production, 5.8 mg l − 1 of semi-continuous culture
day – 1, was obtained with cultures maintained at a renewal rate of 20%. When the induction period was increased to
15 days maximal astaxanthin productivity, 9.6 mg l − 1 of semi-continuous culture day – 1, was obtained from cultures
maintained at a renewal rate of 40% despite the much lower astaxanthin concentration achieved in these cultures.
Results demonstrate the feasibility of semi-continuous cultivation of H. plu6ialis for the two-stage production of
astaxanthin. © 2001 Published by Elsevier Science B.V.

Keywords: Haematococcus; Astaxanthin; Semi-continuous cultures; Two-stage cultures

1. Introduction technological importance with applications in the

The ketocarotenoid astaxanthin (3,3%dihydroxy-
b,b-carotene-4,4%-dione) is a pigment of bio-
* Corresponding author. Tel.: + 34-981-563-100x14944; fax:
+34-981-592-210.
E-mail address: fabregas@usc.es (J. Fábregas).
nutraceutical, pharmaceutical and food industries.
The freshwater microalga Haematococcus plu6ialis
is one of the best microbial sources of astaxan-
thin, that is accumulated in the transition between
green vegetative cells and red aplanospores as the
result of stress conditions (Margalith, 1999). Since
different culture conditions are required for the

0168-1656/01/$ - see front matter © 2001 Published by Elsevier Science B.V.

PII: S0168-1656(01)00289-9
66 J. Fábregas et al. / Journal of Biotechnology 89 (2001) 65–71
production of green vegetative cells of Haemato-
coccus and the accumulation of astaxanthin in the
red aplanospores, a two-stage production process
has been proposed (Kobayashi et al., 1991;
Harker et al., 1996; Olaizola, 2000). In previous
works batch cultures of H. plu6ialis were allowed
to achieve stationary phase and the accumulation
of astaxanthin was further induced by the addi-
tion of NaCl (Harker et al., 1996) or acetate
(Kobayashi et al., 1991). Another factors that halt
cell division such as nitrogen deprivation, high
light, high temperature and oxidative stress
(Borowitzka et al., 1991; Kobayashi et al., 1993;
Fan et al., 1994) can be also applied to stimulate
the accumulation of astaxanthin during the induc-
tion phase. Key factors for the efficiency of the
process are the continuous production of green
vegetative cells and the application of a separate
induction process under conditions that could
avoid cell death. The development of an opti-
mised culture media has improved the production
of H. plu6ialis vegetative cells in semi-continuous
cultures (Fábregas et al., 2000). These cultures
were maintained under circadian light/dark condi-
tions and culture media was renewed every 24 h,
which resulted in a continuous production of
green vegetative cells at constant growth rates and
of constant physiological characteristics when
considered on a daily basis (Fábregas et al., 1996,
2000). In the present work the productivity of
semi-continuous cultures of H. plu6ialis main-
tained with different renewal rates is studied and
the efficiency of the induction of the accumulation
of astaxanthin in batch conditions under high
light depending on the physiological conditions of
the vegetative cells obtained semi-continuously
reported.
2. Materials and methods
H. plu6ialis (CCAP 37/4) was grown in OHM
medium (Fábregas et al., 2000): KNO3 0.41 g l − 1,
Na2HPO4 0.03 g l − 1, MgSO4 · 7H2O 0.246 g l − 1,
CaCl2 · 2H2O 0.11 g l − 1, Fe(III)citrate · H2O 2.62
mg l − 1, CoCl2 · 6H2O 0.011 mg l − 1,
CuSO4 · 5H2O 0.012 mg l , Cr2O3 0.075 mg l − 1,
−1
MnCl2 · 4H2O 0.98 mg l − 1, Na2MoO4 · 2H2O 0.12
mg l − 1, SeO2 0.005 mg l − 1, biotin 25 mg l − 1,
thiamine 17.5 mg l − 1 and B12 15 mg l − 1. Semi-
continuous cultures were carried out in 70-ml
tubes (diameter 30 mm) arranged in a rack that
was internally illuminated at a light intensity of 40
mmol photon m − 2 s − 1 provided by cool white
fluorescent lamps and a circadian dark/light cycle
(12 h:12 h), at 25 °C and aerated intermittently
for 10 s every 10 min at a rate of 250 ml min − 1
(Fábregas et al., 2000). Aeration was supple-
mented with CO2 in order to maintain the pH
between 7.2 and 7.8. Cultures were inoculated at a
cell density of 105 cell ml − 1 with cells from an
exponentially growing culture and allowed to
grow for 3 days before the beginning of the
semicontinuous regime. A percentage of the vol-
ume of the culture (renewal rate) was harvested
daily and replaced with fresh culture media. Four
renewal rates were tested: 10, 20, 30 and 40%.
This resulted in a continuous production of vege-
tative green cells at a constant growth rate when
considered on a 24-h cycle basis. Previous experi-
ments demonstrated that under the tested condi-
tions higher renewal rates were unstable
(Fábregas et al., 2000). The renewal of the cul-
tures was carried out during the first hour of the
light cycle. Three replicates were set for each of
the culture conditions. Cell density was monitored
daily in the harvested cultures by microscope
counting using an improved Neubauer
haematocytometer.
Once all cultures had achieved the steady state,
the semi-continuous regime was ceased and light
intensity was increased to 240 mmol photon m − 2
s − 1 for 15 days in order to induce the transition
to the aplanospore stage and accumulation of
astaxanthin. All other culture conditions were
kept as for the semi-continuous period and no
nutrients were added. During the induction pe-
riod, samples were taken every 3 days for pigment
analysis, cell counts and NO− 3 quantification. For
pigment analyses, 2-ml samples were centrifuged
at 3100 rpm and pellets stored at − 20 °C until
analysis. Cell pigments were extracted with ace-
tone–methanol (1:2 v/v), dried under nitrogen
flux and re-suspended in acetone. All processes
were conducted in the dark. Pigments were
analysed by reverse-phase high-performance liq-
 J. Fábregas et al. / Journal of Biotechnology 89 (2001) 65–71
uid chromatography (HPLC) as described by
Kraay et al. (1992). Chlorophyll a, b and ß-
carotene were identified and quantified by com-
parison with commercial standards (Sigma).
Lutein standard was obtained from Fluka. The
standard for astaxanthin was provided by Hoff-
mann-LaRoche. The 3D-absorption spectra were
determined with HPLC3D Chemstation (Hewlett
Packard) for the identification of the astaxanthin
esters (Grung et al., 1992) which was confirmed
by saponification of the isolated peaks (Renstrøm
et al., 1981). The HPLC used was a Hewlett
Packcard series 1050, with reverse phase column
Waters Nova-Pack® C-18, 3 mm and 3.9×150
mm equipped with an UV-Vis diode array detec-
tor. Chlorophyll a was quantified at 436 nm,
astaxanthin at 474 nm and all other carotenoids
and chlorophyll b were quantified at 460 nm.
Nitrate was quantified spectrophotometrically in
the supernatant of cultures using the standard
method (Cleresci et al., 1989).
3. Results
All cultures reached the steady state after 2– 5
days of the beginning of the semi-continuous
regime. Cultures were maintained for at least 8
days without significant changes in the steady-
state cell density. The steady-state cell decreased
with increasing renewal rate (Fig. 1). Maximal
steady-state cell density, 5.72×105 cells ml − 1 was
obtained with a daily renewal of 10% of the
volume of the culture. This cell density was only
slightly lower than stationary phase cell density
obtained in 14-days batch cultures with the same
nutrient concentration 6.25×105 cells ml − 1 (Fáb-
regas et al., 1998). Cells of cultures maintained
with a renewal rate of 40% received a very high
effective light intensity due to the low steady-state
density, 0.79×105 cells ml − 1, but no
aplanospores or palmella cells were detected.
-) in semicontinuous cultures of H. plu6ialis. Cultures were
Cell productivity calculated as the number of
cells produced per litre of culture per day is
dependent of both steady-state cell density and
the percentage of culture that is harvested daily
(renewal rate). Despite highest steady-state cell
density was achieved with a renewal rate of 10%,
67
maximal cell productivity, 64.6× 106 cells l − 1
day − 1 was obtained with a renewal rate of 20%
(Fig. 1).
No significant decrease in cell density was
recorded when cells were transferred to the high-
light batch condition in any of the renewal rates
tested (Fig. 2). Little changes in cell density were
observed in cultures that were originated at re-
newal rates 10 and 20%. Cultures from renewal
rates 30 and 40% continued growing for 6 and 12
days, respectively, reaching cell densities around
4× 105 cells ml − 1 (Fig. 2). Unlike cell density, the
chlorophyll a concentration increased during the
induction period for all cultures, including those
obtained at renewal rates 10 and 20% during the
first 6 days of induction, remaining almost stable
thereafter (Fig. 3). The concentration of chloro-
phyll a reached values between 10 and 14 mg ml − 1
for all cultures after 15 days of high light treat-
ment (Fig. 3). Lutein and b-carotene concentra-
tion increased during the induction period in a
manner similar to that of chlorophyll a.
The rate of accumulation of astaxanthin during
the induction phase was dependent on the renewal
rate applied in the semi-continuous culture, and
Fig. 1. Steady-state cell density (--) and cell productivity
(-
maintained with daily renewal rates between 10 and 40% of
the volume of the cultures. The productivity of astaxanthin
when the outflow of the green semicontinuous cultures were
transferred for 6 days (open bars), 12 days (dashed bars) or 15
days (crossed bars) to batch conditions under high light (240
mmol photon m − 2 s − 1) is also shown. Vertical bars represent
S.D. (n =3).
68 J. Fábregas et al. / Journal of Biotechnology 89 (2001) 65–71

tinuous culture per day, both, the duration of

Fig. 2. Evolution of cell density in the outflow of semicontinu-
ous cultures of H. plu6ialis transferred to batch conditions
under high light (240 mmol photon m − 2 s − 1) during 15 days
for the induction of astaxanthin accumulation. Cultures were
produced semicontinuously at renewal rates 10% (-
-), 20% (Fig. 1).
(--), 30% (--) and 40% (--). Vertical bars represent S.D.
(n= 3).
the induction period and the renewal rate
strongly affected the productivity results. The
highest astaxanthin concentration was achieved
in the outflow of cultures maintained at a re-
newal rate of 10%, but total astaxanthin produc-
tivity on a ‘per volume of culture basis’ was low
since only a 10% of the cultures was harvested
daily and made available for the induction
phase. Moreover, astaxanthin productivity was
strongly dependent on the duration of the induc-
tion period (Fig. 1). When short induction peri-
ods are applied (6 days) highest productivity, 2.5
mg l − 1 day − 1, was achieved in cultures ob-
tained at the lowest renewal rate while almost
no astaxanthin could be obtained in cultures
maintained with high renewal rates. After 12
days of induction maximal astaxanthin produc-
tivity, 5.8 mg l − 1 day − 1, was obtained with a
renewal rate of 20% (Fig. 1). Induction periods
of 15 days produced maximal productivities of
9.6 mg l − 1 day − 1 with a renewal rate of 40%

therefore on the growth rate at which cells were

growing (Fig. 4). The lower the renewal rate, the
earliest the beginning of the accumulation of as-
taxanthin (Fig. 4). The beginning of the accumu-
lation of astaxanthin was correlated with the
amount of nitrate remaining in the culture me-
dia, no accumulation being detected before all
the nitrate had been consumed. The astaxathin
was first detected in cultures obtained at a re-
newal rate of 10% after 3 days of being trans-
ferred to the high light condition and increased
up to 49 mg ml − 1 on day 15 (Fig. 4). When cells
are obtained at a renewal rate of 40% astaxan-
thin was detected only after 9 days of exposure
to the high light, once cell growth has ceased Fig. 3. Evolution of chlorophyll a concentration in the outflow
and all nitrogen had been depleted from the cul- of semicontinuous cultures transferred to batch conditions
ture media (Fig. 4). under high light (240 mmol photon m − 2 s − 1) during 15 days
for the induction of astaxanthin accumulation. Cultures were
When astaxanthin productivity is calculated produced semicontinuously at renewal rates 10% (-
-), 20%
on the basis of the astaxanthin produced at the (--), 30% (--) and 40% (--). Vertical bars represent S.D.
end of the induction period per litre of semicon- (n =3).
 J. Fábregas et al. / Journal of Biotechnology 89 (2001) 65–71
Fig. 4. Astaxanthin (closed symbols) and nitrate (open sym-
bols) concentration during the induction period in two-stage
cultures of H. plu6ialis. Cultures were produced semicontinu-
ously at renewal rates 10% (-
-), 20% (--), 30% (--) and OHM even in those maintained with very high
40% (--) and transferred to batch conditions under high light
(240 mmol photon m − 2 s − 1) for the induction of astaxanthin
accumulation. Vertical bars represent S.D. (n=3).
4. Discussion
Most experimental cultivation of H. plu6ialis
has been done in batch cultures due to the
difficulties of maintaining continuous cultures of
this slow-growing species. In batch cultures the
optimal Haematococcus media (OHM) produced
a cell density of 6.25× 105 cells ml − 1 after 14
days of culture with no astaxanthin being accu-
mulated (Fábregas et al., 1998). Much lower sta-
tionary phase cell densities were obtained in
autotrophic cultures after 20 days (2.5× 105 cells
ml − 1, Harker et al., 1996) or in mixotrophic
conditions after 12 days (4.2× 105 cells ml − 1,
Tripathi et al., 1999). Most importantly, OHM
can sustain high steady-state cell densities in semi-
continuous cultures. Despite the final optimisa-
tion process for the semicontinuous production of
H. plu6ialis cells with OHM had been carried out
for a renewal rate of 20%, the steady state ob-
tained with OHM at a renewal rate of 40%,
0.79 ×105 cells ml − 1, is 3 times higher than with
the non optimized media (Fábregas et al., 2000).
Maximal cell productivity, 64.6×106 cells l − 1
day − 1 was obtained with a renewal rate of 20%
69
(Fig. 1). This productivity is higher than the max-
imal productivity obtained in a 25 000 l outdoor
photobioreactor, 52×106 cells l − 1 day − 1
(Olaizola, 2000), although the light available in
the outdoor system was much higher. The concen-
tration of OHM used was selected on the basis of
minimum nitrate available at the end of a day
cycle with a renewal rate of 20% at 40 mmol
photon m − 2 s − 1 (Fábregas et al., 2000). An
increase in the concentration of the optimized
media, together with an increase in the effective
light available would improve the productivity of
vegetative cells with OHM (Fábregas et al., 2000).
Although the presence of astaxanthin in actively
growing cultures has been described (Lee and
Soh, 1991; Chaumont and Thépenier, 1995; Ha-
gen et al., 2001) no astaxanthin was observed in
the semicontinuous cultures maintained with
renewal rate. Results demonstrate the importance
of media formulation for the achievement of max-
imal microalgal productivities.
When the harvested cultures were transferred to
high light condition cell division continued in
cultures obtained at renewal rates 30 and 40%
until all nitrogen had been consumed despite the
low cell density of cultures obtained at renewal
rate 40% (Fig. 4). Arrested cell division and astax-
anthin accumulation has been reported in cultures
of H. plu6ialis maintained in continuous high-light
condition even in the presence of nitrate (Fan et
al., 1998). Continuous illumination rather than
light/dark illumination cycles have been demon-
strated to be more favourable for astaxanthin
formation in H. plu6ialis, light quantity being
more important than light intensity (Kobayashi et
al., 1992). The use of continuous light instead of
light/dark cycles may represent an additional
source of stress and could be used to accelerate
the process of astaxanthin accumulation.
Chlorophyll a concentration increased during
the induction period while cultures were still
growing and stabilised thereafter. Several authors
have found an important decrease in chlorophyll
cell content during the formation of aplanospores
(Harker et al., 1996; Grünewald et al., 1997)
although other authors could not find such dra-
matic decrease (Zlotnik et al., 1993). In very
70 J. Fábregas et al. / Journal of Biotechnology 89 (2001) 65–71
diluted cultures submitted to high light in the
absence of nitrogen an important decrease in
chlorophyll content during aplanospore formation
has been also recorded in our cultures (non pub-
lished data). Therefore the evolution of chloro-
phyll cell content seems to be strongly dependent
on the experimental conditions and on the rela-
tionship between cell density and light intensity,
and independent of astaxanthin formation, ques-
tioning the photo-protective role of astaxanthin
during the formation of aplanospores.
The accumulation of astaxanthin was strongly
dependent on the growth rate applied in the semi-
continuous cultures. The renewal rate applied
strongly affected the amount of nitrogen avail-
able. Only the semi-continuous cultures main-
tained with a renewal rate of 10% consumed all
the nitrogen added daily (Fig. 4). Astaxanthin was
accumulated only when cell division was stopped
and all nitrogen in the culture media had been
consumed (Fig. 4). The steady-state cell density of
cultures maintained at a renewal rate of 40% was
very low (0.79× 105 cells ml − 1). The transfer of
these cultures to high light condition during the
induction period resulted in a very high light per
cell but no astaxanthin was accumulated during 9
days, with a strong correlation between astaxan-
thin formation and nitrogen depletion in the cul-
ture media (Fig. 4). Although the role of
astaxanthin as a passive photoprotectant can not
be completely ruled out (Yong and Lee, 1991;
Hagen et al., 1994), the strong correlation be-
tween nitrogen depletion and astaxanthin forma-
tion regardless the effective light intensity
reaching the cells corroborates previous results
that supported the idea of astaxanthin synthesis
being driven by nutrient depletion (Droop, 1955;
Borowitzka et al., 1991; Fábregas et al., 1998) as
far as the photosynthetic apparatus is still active.
As demonstrated by our results, the establish-
ment of optimal nutrient concentration in the
semicontinuous cultures allowing minimal nitro-
gen concentration in the culture media at the
beginning of the induction period is essential in
order to achieve maximal productivities of astax-
anthin in short induction periods. The most con-
venient renewal rate applied in the
semicontinuous cultures in order to maximize as-
taxanthin productivity depends on the length of
the induction period. While for short induction
periods lower renewal rates are preferred, longer
induction periods produce higher productivities in
cultures maintained with high renewal rates. In
fact, when the induction period is prolonged to 15
days a maximal productivity of 9.6 mg l − 1 day − 1
was obtained with a renewal rate of 40%, but the
volume of culture to be handled during the induc-
tion period and harvest is much higher with high
renewal rates, certainly affecting production costs.
The light intensity applied during the induction
period, 240 mmol photon m − 2 s − 1, is much lower
than the full sun-light intensity that can be ob-
tained in outdoor cultures, which would surely
improve the pigment yield and shorten the induc-
tion period. A combination of factors could be
applied during the second stage of the process in
order to shorten the induction period. The addi-
tion of NaCl to induce carotenogenesis produced
undesirable cell death even when added sequen-
tially, resulting in lower productivies (Harker et
al., 1996). Total carotenoid reached a concentra-
tion of 40 mg ml − 1 after 40 days of NaCl induc-
tion (Harker et al., 1996), a concentration lower
than that obtained after 12 days of high-light
induction (Fig. 4). The addition of acetate and the
ferrous ion can also increase the rate of astaxan-
thin accumulation but cell death has also been
reported (Kobayashi et al., 1993). In batch cul-
tures in which a modified medium with high
content of iron, acetate and continuous high light
were used for the induction maximal carotenoid
concentration achieved was 25 mg ml − 1 after 6
days of induction (Kobayashi et al., 1991). After
20 days, 64 mg ml − 1 of astaxanthin were obtained
in a feed-batch culture in the presence of acetate
(Zhang et al., 1999).
Results demonstrate the feasibility of the estab-
lishment a two-stage production system of astax-
anthin from H. plu6ialis with a continuous
production of vegetative cells during the first
stage. Correct culture parameter selection during
the production of vegetative cells, including nutri-
ent concentration and renewal rate, is essential in
order to achieve maximal astaxanthin productivi-
ties during the induction period.
 J. Fábregas et al. / Journal of Biotechnology 89 (2001) 65–71
Acknowledgements
This work was supported by a project from
Xunta de Galicia (XUGA20317B97). We are
grateful to F. Hoffman-La Roche Ltd. for kindly
providing Astaxanthin and Zeaxanthin standards.
References
Borowitzka, M.A., Huisman, J.M., Osborn, A., 1991. Culture
of the astaxanthin-producing green alga Haematococcus
plu6ialis. 1. Effects of nutrients on growth and cell type. J.
Appl. Phycol. 3, 295 – 304.
Cleresci, L.S., Greenberg, A.E., Trussell, R.R., 1989. Standard
Methods for the Examination of Water and Wastewater,
17th Edition. American Public Health Association, Wash-
ington DC.
Chaumont, D., Thépenier, C., 1995. Carotenoid content in
growing cells of Haematococcus plu6ialis during a sunlight
cycle. J. Appl. Phycol. 7, 529 –537.
Droop, M.R., 1955. Carotenogenesis in Haematococcus plu6i-
alis. Nature 175, 42.
Fábregas, J., Patiño, M., Morales, E.D., Cordero, B., Otero,
A., 1996. Optimal renewal rate and nutrient concentration
for the production of the marine microalga Phaeodactylum
tricornutum in semicontinuous cultures. Appl. Environ.
Microbiol. 62, 266 – 268.
Fábregas, J., Domı́nguez, A., Garcia-Álvarez, D., Lamela, T.,
Otero, A., 1998. Induction of astaxanthin accumulation by
nitrogen and magnesium deficiencies in Haematococcus
plu6ialis. Biotech. Lett. 20, 623 –626.
Fábregas, J., Domı́nguez, A., Regueiro, M., Maseda, A.,
Otero, A., 2000. Optimization of culture medium for the
continuous cultivation of the microalga Haematococcus
plu6ialis. Appl. Microbiol. Biotechnol. 53, 530 –535.
Fan, L., Vonshak, A., Boussiba, S., 1994. Effect of tempera-
ture and irradiance on growth of Haematococcus plu6ialis
(Chlorophyceae). J. Phycol. 30, 829 – 833.
Fan, L., Vonshak, A., Zarka, A., Boussiba, S., 1998. Does
astaxanthin protect Haematococcus against light damage?
Z. Naturforsch. 53c, 93 –100.
Grünewald, K., Hagen, C., Braune, W., 1997. Secondary
carotenoid accumulation in flagellates of the green alga
Haematococcus lacustris. Eur. J. Phycol. 32, 387 – 392.
Grung, M., D‘Souza, F.M.L., Borowitzka, M., Liaaen-Jensen,
S., 1992. Algal carotenoids 51. Secondary carotenoids 2.
Haematococcus plu6ialis aplanospores as a source of (3S,
3%S)-astaxanthin esters. J. Appl. Phycol. 4, 165 –171.
Hagen, C., Braune, W., Björn, L.O., 1994. Functional aspects
of secondary carotenoids in Haematococcus lacustris
71
(Volvocales). III Action as a ‘sunshade’. J. Phycol. 30,
241 – 248.
Hagen, C., Grünewald, K., Xyländer, M., Rothe, E., 2001.
Effect of cultivation parameters on growth and pigment
biosynthesis in flagellated cells of Haematococcus plu6ialis.
J. Appl. Phycol. 13, 79 – 87.
Harker, M., Tsavalos, A.J., Young, A.J., 1996. Autotrophic
growth and carotenoid production of Haematococcus plu-
6ialis in a 30 liter air-lift photobioreactor. J. Ferm. Bioeng.
82, 113 – 118.
Kobayashi, M., Kakizono, T., Nishio, N., Nagai, S., 1992.
Effects of light intensity, light quality, and illumination
cycle on astaxanthin formation in a green alga, Haemato-
coccus plu6ialis. J. Ferm. Bioeng. 74, 61 – 63.
Kobayashi, M., Kakizono, T., Nagai, S., 1993. Enhanced
carotenoid biosynthesis by oxidative stress in acetate-in-
duced cysts cells of a green unicellular alga, Haematococcus
plu6ialis. Appl. Environ. Microbiol. 59, 867 – 873.
Kobyashi, M., Kakizono, T., Nagai, S., 1991. Astaxanthin
production by a green alga, Haematococcus plu6ialis ac-
companied with morphological changes in acetate media.
J. Ferm. Bioeng. 71, 335 – 339.
Kraay, G.W., Zapata, M., Veldhuis, M.J.W., 1992. Separation
of chlorophylls c-1, c-2, and c-3 of marine phytoplankton
by reversed-phase-C18-high-performance liquid chro-
matography. J. Phycol. 28, 708 – 712.
Lee, Y.-K., Soh, C.W., 1991. Accumulation of astaxanthin in
Haematococcus lacustris (Chlorophyta). J. Phycol. 27,
575 – 577.
Margalith, P.Z., 1999. Production of ketocarotenoids by mi-
croalgae. Appl. Microbiol. Biotechnol. 51, 431 – 438.
Olaizola, M., 2000. Commercial production of astaxanthin
from Haematococcus plu6ialis using 25000-liter outdoor
photobioreactors. J. Appl. Phycol. 12, 499 – 506.
Renstrøm, B., Borch, G., Skulberg, O.M., Liaaen-Jensen, S.,
1981. Optical purity of (3S,3’S)-astaxanthin from Haema-
tococcus plu6ialis. Phytochemistry 20, 2561 – 2564.
Tripathi, U., Sarada, R., Ramachandra Rao, S., Ravishankar,
G.A., 1999. Production of astaxanthin in Haematococcus
plu6ialis cultured in various media. Biores. Technol. 68,
197 – 199.
Yong, Y.Y.R., Lee, Y.K., 1991. Do carotenoids play a photo-
protective role in the cytoplasm of Haematococcus lacustris
(Chlorophyta)? Phycologia 30, 257 – 261.
Zhang, X.W., Gong, X.-D., Chen, F., 1999. Kinetic models
for astaxanthin production by high cell density
mixotrophic culture of the microalga Haematococcus plu6i-
alis. J. Ind. Microb. Biotechnol. 23, 691 – 696.
Zlotnik (Shmerler), I., Sukenik, A., Dubinsky, Z., 1993. Phys-
iological and photosynthetic changes during the formation
of red aplanospores in the Chlorophyte Haematococcus
plu6ialis. J. Phycol. 29, 463 – 469.