Annals of RSCB Vol.

XV, Issue 2

ASTAXANTHIN PRODUCTION FROM A NEW STRAIN OF

HAEMATOCOCCUS PLUVIALIS GROWN IN BATCH CULTURE
N. Dragoş1, 2, V. Bercea1, Adriana Bica1, B. Drugă1, Ana Nicoară1,
C. Coman1, 2
1
INSTITUTE OF BIOLOGICAL RESEARCH, CLUJ-NAPOCA, ROMANIA
2
DEPARTMENT OF BIOLOGY, BABEŞ-BOLYAI UNIVERSITY, CLUJ-NAPOCA,
ROMANIA

Summary
Astaxanthin is the most common red-colored pigment of many freshwater and
marine organisms with important applications in aquaculture and human health.
Haematococcus pluvialis, a green unicellular freshwater microalga, is able to
accumulate large quantities of astaxanthin in inducible stress conditions. In this paper
we report a preliminary evaluation of the growth characteristics, pigment composition
and astaxanthin content of a new isolate of Haematococcus pluvialis green alga
(Chlorophyta). The strain AICB 223 was isolated by micromanipulation of a single
cell and grown in a batch system with CO2 supplying. The separation of carotenoids
was performed by thin-layer chromatography, and the components were identified
and quantified by spectrophotometric method, based on the absorption coefficients.
The astaxanthin synthesis was induced by changing the light intensity (increased
irradiance coupled with continuous illumination). The astaxanthin content in the algal
biomass (red stage, with aplanospores) was approximately of 5.7 mg · g-1 per dry
biomass. In light stress conditions, the biomass composition was significantly
changed, the increase of the astaxanthin content by approximately 10 times being
accompanied by the decrease of protein amount and increase of the carbohydrates
content.
Keywords: Haematococcus, astaxanthin, light stress, red aplanospores.
ndragos@hasdeu.ubbcluj.ro

Introduction (Norsker et. al., 2010). In this context, one

Carbon dioxide, the greenhouse gas
which together with other gases causes the
global warming is an essential nutrient
involved in the growth and reproduction of
the algae, both in their natural environment
and in culture. The exploitation of the
microalgae potential in fixing the residual
carbon dioxide is tightly related to the
development of the commercial application
of the biomass (Hung et al., 2010). The
ratio between the production cost of the
algal biomass and the market price of the
by-products obtained from it is crucial in
the development of these applications
353
of the most provocative biological
challenge with respect to the research of
microalgae potential is the identification
and bringing into cultures of the taxa/strains
with optimal features for a given
application (Greenwell et. al., 2010)
Astaxanthin, a symmetric keto-
carotenoid, (3,3’-dihydroxy-β,β’-carotene-
4,4’-dione) is the most common red-colored
pigment of many freshwater and marine
organisms, the salmon or trout “pink flesh”
color being good examples in this respect
(Breithaupt, 2007). Because the aquatic
organisms are not capable of carotenoids de
novo synthesis, this xanthophyll and others
Annals of RSCB
which were also identified in their bodies
(e.g. canthaxanthin and lutein) originate in
the diet, microorganisms being the main
source of them (Johnson and An, 1991). In
addition to the astaxanthin applications in
aquaculture (Bjerkeng, 2008), its benefits
for the human health were ascertained also,
due to its properties: pro-vitamin A, more
effective antioxidant that other carotenoids,
decelerate age-related macular
degeneration, immunomodulatory effects,
etc (Jyonouchi et al., 1991; Shimidzu et al.,
1996; Lorenz and Cysewsky, 2000; Naguib,
2000; Dufossé et al., 2005; Breithaupt,
2007). Therefore, the astaxanthin was uses
in pharmaceutics, cosmetics and as food
additives with a recent great success on the
market (Dufossé et al., 2005).
Haematococcus pluvialis is a green
unicellular freshwater microalga capable to
accumulate large quantities of astaxanthin
(0.2-2% from dry biomass) (Lorenz and
Cysewski, 2000; Cysewski and Lorenz,
2004; Dufossé et al., 2005). Even if other
astaxanthin producing green algae were
recently identified (Coelastrella striolata –
Abe et al., 2007; Monoraphidium sp. –
Fujii et al., 2009; Scenedesmus obliquus –
Quin et al., 2008; Chlorella zofingiensis –
Ip and Chen, 2005), H. pluvialis alongside
with the heterobasidomycetous yeast
Xanthophyllomyces dendrorhous (formerly
Phaffia rhodozyma), are the best microbial
source (Johnson and An, 1991; Dufossé et
al., 2005). Numerous attempts were made
in finding the best way to cultivate this
alga, in selecting the optimal strains and in
increasing the astaxanthin contents within
the cells (Boussiba et al., 1991, 1999; Lee
and Soh, 1991; Harker et al., 1995; Hata et
al., 1995, among others) and in making
more feasible the implementation of the
production process. The selection strategies
were focused on isolation of wild and
mutant astaxanthin highly producing strains
and/or with optimal growth conditions
(Tjahjono et al., 1994; Tripathi et al., 2001;
Chen et al., 2003; Wang et al., 2005;
Kamath et al., 2008). We report in this
paper, a preliminary evaluation of the
354
Vol. XV, Issue 2
growth characteristics, pigment
composition and astaxanthin content of a
newly isolate of Haematococcus pluvialis
green alga.
Material and Methods
Strain and culture conditions.
Haematococcus pluvialis AICB 223 was
isolated by micromanipulation of a single
cell under a light microscope. One sample
of freshwater phytoplankton, collected from
a fishpond near Cefa (Bihor District,
Romania) was used in this procedure. The
strain was deposited in the Culture
Collection of Algae and Cyanobacteria of
the Institute of Biological Research (AICB)
from Cluj-Napoca (Romania). The
isolation, the stock culture and the growth
experiments were carried out in Bold Basal
Medium (BBM) (Dragoş et al., 1997). H.
pluvialis AICB 223 was grown in batch
system using an Applikon bioreactor with
controlled temperature of 25±1oC and
continuous stirring (150-200 rpm),
containing 800 ml algal suspension. White
fluorescent light was used in a dark: light
cycle of 8:16 hours with a light intensity of
315 µmolphoton·m-2·s-1. After 10 days, the
culture was continuously exposed to light,
using a double irradiance (630 µmol
photon·m-2·s-1) to enhance the
carotenogenesis. The carbon dioxide (12%
in air) was supplied with a 0.036 l/min/l
flow within the algal suspension. The
growth curve was generated based on daily
spectrophotometer measurements (600 nm)
of optical density, following Sorokin`s
protocol (1973).
Light microscopy. The morphological
features of H. pluvialis AICB 223 cells
were observed from samples collected from
1 to 3 days, using an Olympus BX41
microscope equipped with digital camera.
The examination was conducted based on
the species diagnosis (Ettl, 1983).
Pigment extraction and analysis. The algal
samples, collected 17 days after the
initiation of the growth process, were
filtered on silica gel and the retained
material was grind with glass beads using
Annals of RSCB
acetone as solvent. The separation of the
chemical components was performed by
thin-layer chromatography on Merck
kieselgel 60 plates (Hager and Bertenrath,
1966). The pigments were identified and
quantified following spectrophotometric
method, based on the absorption
coefficients and the highest value of the
wave length (Arnon, 1949; Davies, 1965).
The pigment kinetics in the algal culture
was evaluated from samples (20 ml)
collected from 1 to 3 days. After the
acetone extraction the concentration of the
pigments (chlorophyll and carotenoids) was
calculated based on the absorption
coefficients in VIS domain.
Fig. 1. Light microphotographs of H. pluvialis
AICB 223 aplanospores. The green
aplanospores (a) have been found in a lower
irradiance (315 µmolphoton·m-2· s-1, light:dark
cycle of 16:8 h). The increase of irradiance (630
µmolphoton·m-2·s-1) associated with continuous
illumination produced mature red aplanospores
(b). Scale bar=100µm.
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Vol. XV, Issue 2
Protein and carbohydrate measurement.
The protein content of the algal biomass
was estimated by measuring the total
content of the organic nitrogen (Kjeldhal
method). The measurement was based on
dry weight samples which were mineralized
for 2.5 hours in concentrated sulfuric acid
using a Gerhardt apparatus. The distillation
was done with an adjustable Vapodest 30s
(Gerhardt) device for 6.5 min. The
carbohydrates content was
spectrophotometrically measured from algal
samples following the protocol described
by Dubois et al. (1956). The analysis results
were related to dry weight which was
measured gravimetrically after Sorokins`s
method (1973).
Results and Discussion
Morphology. The culture was derived from
an inoculum with typical characteristics of
motile stage, with biflagellate cells. The
growth in bioreactor, with mechanical
steering, has favored the occurrence of
more or less mature aplanospores (Fig. 1a).
This stage becomes dominant together with
the evolution of growth. The aplanospores
color turns gradually into red, because of
high accumulation of carotenoids in
chloroplasts, and especially outside of
them, in lipid globules (astaxanthin). The
red aplanospores are known as
“haematocysts”. This stage may appear
under stress conditions induced by light,
high temperature, increase of salinity,
nutritional limitation, change of carbon
source etc (Kobayashi et al., 1991; Hagen
et al., 2000; Wang et al., 2003; Garcia-
Malea et al., 2005; Ranjbar et al., 2008).
The morphological analysis of cells by light
microscopy has shown, without a doubt,
that during the second stage of the culture,
the cells have been undergone through light
stress (630 µmol photon·m-2·s-1), thus the
majority of the cells being haematocysts
(Fig. 1b).
Annals of RSCB
5 16
4.5
4
3.5
D log2 OD
3 10
2.5
2
1.5
1
0.5
0 0
0 2 4 6 8 10
∆ log2 OD Chl. a Carotenoids
Fig. 2. Growth curve of the AICB 223 batch
culture and the kinetics of chlorophyll and total
carotenoids in different conditions of light. The
changing in irradiance is marked by different
plotting symbols of the OD measurements.
Effect of irradiance on growth kinetics.
Strain AICB 223 was grown in batch
system for 17 days, the growth curve being
plotted based on daily measurements of the
optical density (Fig. 2). During the first
stage of growth (10 days), the batch culture
was carried out in a light:dark cycle of 16:8
hours under a 315 µmolphoton·m-2·s-1
intensity. During the second phase (7 days)
the illumination was modified to induce
carotenogenesis. Thus, the level of
irradiance was doubled (630 µmol
photon·m-2·s-1), under continuous
illumination, the other growth conditions
remaining unchanged. The growth was
rapid, apparently without a lag phase (Fig.
2), the exponential growth being of 5 days,
with a 1.7 days doubling time. Both during
the exponential and stationary growth, the
concentration in chlorophylls and total
carotenoids increased in a similar way and
the algal suspension remained green. After
a short increase of the optical density (2
days), the doubling of irradiance caused the
culture entering in the stationary phase and
then in a slow decline. The high irradiance
has caused the red color of the suspension
and a dramatic increase of caretonoids level
in cell suspension, this not being associated
to a similar increase in chlorophylls (Fig.
2). Thus, our experiment indicated that the
growth of the AICB 223 strain was active at
a lower irradiance and that an increased
Vol. XV, Issue 2
level of irradiance induced the
carotenogenesis accompanied by a dramatic
Chl. a - carotenoids (mg/l)
14
12 change of suspension color. Similar
observations were also reported for other H.
8
pluvialis strains. Thus, Wang et al. (2003)
6
4
observed that carotenogenesis at high levels
2
of irradiance is followed by the increase of
the carotenoids/chlorophylls ratio. The rise
12 14 16 18 of astaxanthin levels shows an
days
accommodation response to light stress
which enables cells to maintain the
structural and functional integrity of PSII.
The ability of H. pluvialis to accumulate
astaxanthin is an adaptation to habitats with
high irradiance (Hagen et al., 1994) in
addition to formation of aplanospores with
thick, rigid cell walls (Montsant et al.,
2001; Hagen et al., 2002).
Pigment content in the biomass. Thin layer
chromatography on kieselgel plates of
acetone extracts was performed on algal
suspension samples collected after
carotenogenesis induction by light (red
stage). To compare, a similar extract was
analyzed from the inoculum (green stage).
The method allowed a good separation of
chlorophylls, carotene and xanthophylls
(Fig. 3). Nine pigments were
chromatographically identified in visible
domain (as distinct lanes – fig. 3) (Fig. 4): 2
chlorophylls, the carotene and 6
xanthophylls. Their content in the biomass
(as dry weight) is presented in Tab. 1.
Fig. 3. Chromatograms on kieselgel plates. A –
green stage (inoculum), B – red stage. a –
carotene, b-e – astaxanthin, f – pheophytin and
other degraded xanthophylls, g-h – chlorophylls
a and b, i – lutein, j – violaxanthin, k –
neoxanthin.
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Annals of RSCB Vol. XV, Issue 2

In the chromatographic plates the Biomass composition in relation to

astaxanthin was identified in 3-4 lanes (Fig.
3). An explanation could be based on the
existence of multiple forms of astaxanthin
in Haematococcus cells. Kobayashi et al.
(1991) and Miao et al. (2006) showed that
the free form of astaxanthin is accompanied
by monoesteric and diesteric forms, the
esteric fractions being dominant. The
astaxanthin synthesis was induced in a
relatively short period of time by increasing
the irradiance. Thus, the red stage of AICB
223 strain contained in its biomass a
quantity of astaxanthin of aprox. 10 times
higher than in the green stage. Zhang et al.
(2009) reported a maximum content of 27.9
mg/g of dry mass in Haematococcus. In the
biomass of the AICB 223 strain, in the
described conditions (which are not yet
optimal) we observed a content of aprox.
5.7 mg/g of dry weight. These results show
that AICB 223 can be an astaxanthin highly
producing strain if its performances could
be improved throughout optimization of
growth in the green stage and induction of
astaxanthin synthesis by stress factors.
astaxanthin content. The protein and
carbohydrate contents of biomass in green
and red stages were investigated in order to
find significant changes in the cellular
metabolism associated with the astaxanthin
synthesis, due to the light stress. The major
observation is the radical change in the
cellular metabolism, the astaxanthin
synthesis being associated with a significant
decrease of the protein content (estimated
from the organic nitrogen by Kjeldahl
method) and an increase of the
carbohydrate content (Fig. 5).
It is likely, that the reducing of the
photosynthetic capacity in stress conditions
would determine the protein bulk synthesis
to become energetically unsustainable.
Kobayashi et al. (1997) noticed the
formation and maturation of the
aplanospores which was accompanied by
the rapidly increasing degradation of the
cellular proteins, process related to an
enhanced carotenogenesis.

Table 1. Pigment composition of H. pluvialis

AICB 223 biomass (mg/g dry weight)
Pigment Green stage Red
stage
Chlorophyll a 10.053 4.027
Chlorophyll b 7.829 1.665
Astaxanthin 0.572 5.753
Carotene (α + β) 0.295 0.542
Zeaxanthin 0.352 0.317
Lutein 1.554 1.471
Lactucaxanthin - 0.258
Violaxanthin 0.274 0.241
Neoxanthin 0,338 0.512
Carotenoids/ 0.189 1.598
chlorophylls

357
Annals of RSCB Vol. XV, Issue 2

Fig. 4. The visible absorption spectra of the major pigments identified in H. pluvialis AICB 223
biomass.

70 7
% protein - carbohydrate

60 6
mg astaxanthin/g d.w.

It is acknowledged that the cells of 50 5

the green algae produce large quantities of 40 4
intraplastidial starch when stress conditions 30 3
are applied (Hu, 2004). Thus, the 20 2
astaxanthin synthesis induced by stress is 10 1
associated with a strong decrease in protein
0 0
synthesis and with considerable AICB 223 - green AICB 223 - red
accumulations of intraplastidial starch
Protein Carbohydrate Astaxanthin
quantities.

Fig. 5. Protein and carbohydrate contents of H.

pluvialis AICB 223 in relation to astaxanthin
level, in different light conditions of the batch
culture.

358
Annals of RSCB
Conclusions
Strain AICB 223, Haematococcus
pluvialis, which was isolated by
micromanipulation of a single cell is
capable of astaxanthin synthesis in batch
system when CO2 is supplied. The
astaxanthin synthesis was induced by
changing the light intensity (increased
irradiance coupled with continuous
illumination). The astaxanthin content in
the algal biomass (red stage with
haematocysts) was approximately of 5.7 mg
· g-1 per dry biomass. The astaxanthin
synthesis was accompanied by an overall
increase in the carotenogenesis. Beside
astaxanthin, 5 more xanthophylls were
identified in the algal biomass. In
conditions of light stress the biomass
content is significantly changed, the
increasing of the astaxanthin content by
approximately 10 times being followed by
the reduction of the protein content and an
increase of the carbohydrate components.
The results certainly indicate that in
Haematococcus cultures, the astaxanthin
production through CO2 fixation must be
addressed in 2 directions: the optimization
of the growth of the biomass (acquiring the
dense cultures in green, vegetative stage)
and properly inducement of the astaxanthin
synthesis (fast process, induced by stress
factors) with the formation of haematocysts
in order to increase the commercial value of
biomass.
Acknowledgements
Financial support to this study was
provided by ANCS (Autoritatea Nationala
pentru Cercetare Stiintifica – Romania),
PNII Project No 22-085/2008
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Vol. XV, Issue 2
Tripathi, U., Venkateshwaran, G., Sarada, R.,
Ravishankar, G.A.: Studies on
Haematococcus pluvialis for improved
production of astaxanthin by mutagenesis.
World J. Microbiol. Biotechnol., 17, 143–
148, 2001.
Wang, B., Zarka, A., Trebst, A., Boussiba, S.:
Astaxanthin accumulation in Haematococcus
pluvialis (Chlorophyceae) as an active
photoprotective process under high
irradiance. J. Phycol., 39, 1116–1124, 2003.
Wang, S.-B., Chen, F., Sommerfeld, M., Hu,
Q.: Isolation and proteomic alalysis of cell
wall-deficient Haematococcus pluvialis
mutants. Proteomics, 5, 4839–4851, 2005.
Zhang, B.Z., Geng, Y.H., Li, Z.K., Hu, H.J., Li,
Y. G.: Production of astaxanthin from
Haematococcus in open pond by two-stage
growth one-step process. Aquaculture, 295,
275–281, 2009.