Journal of Biotechnology 125 (2006) 540546

A novel double-layered photobioreactor for

simultaneous Haematococcus pluvialis cell
growth and astaxanthin accumulation
In Soo Suh, Hyun-Na Joo, Choul-Gyun Lee
Institute of Industrial Biotechnology, Department of Biological Engineering,
Inha University, 253 Yonghyun-Dong, Nam-Gu, Incheon 402-751, Republic of Korea

Received 19 October 2005; received in revised form 8 March 2006; accepted 21 March 2006

Abstract

This study proposes a novel double-region photobioreactor to simplify the commercial two-stage process of astaxanthin pro-
duction by the cultivation of Haematococcus pluvialis. The feasibility of the double-region photobioreactor has been investigated
and found to achieve high biomass yield in the inner core region and simultaneous astaxanthin accumulation in the outer jacket
region. Among many environmental factors, light condition and nitrate level were manipulated for selective cell growth and
astaxanthin production. In the outer jacket region, efficient astaxanthin production was accomplished by excessive irradiation
(770 20 E m2 s1 ) and nitrate starvation, resulting in a dramatic increase of astaxanthin productivity (357 mg l1 ). Mean-
while, attenuated light energy (40 3 E m2 s1 ) and sufficient nitrates were supplied to the vegetative cells in the inner core
region, which continued to grow to a high cell concentration of 4.0 105 cells ml1 . The sequential batch run was performed by
utilizing the high-density vegetative cells as inoculum for the next batch run. The cultivation results exhibited similar trends as
the previous run, reaching high cell density (4.3 105 cells ml1 ) in the inner core region and high astaxanthin content (5.79%
on a dry weight basis) in the outer jacket region. The present study indicates that the double-region photobioreactor and its
method of operation possess a good potential for commercial production of astaxanthin by H. pluvialis.
2006 Elsevier B.V. All rights reserved.

Keywords: Double-region photobioreactor; Haematococcus pluvialis; Astaxanthin; Inner core region; Outer jacket region

1. Introduction aquatic environments. Their biotechnological poten-

Through their photosynthetic activities, microalgae
are the major primary producers of organic matter in
Corresponding author. Tel.: +82 32 860 7518;
fax: +82 32 872 4046.
E-mail address: leecg@inha.ac.kr (C.-G. Lee).
tial has been widely studied for the production of new
bioproducts and possible environmental applications
over the last several decades. Specifically, there has
been considerable interest in the production of many
clinically and medically important biochemicals from
algae; most of the biochemicals of interest are not
obtainable by chemical synthesis.

0168-1656/$ see front matter 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2006.03.027
 I.S. Suh et al. / Journal of Biotechnology 125 (2006) 540546
A green alga, Haematococcus pluvialis, has recently
received increased interest as one of the best producers
of astaxanthin (3,3 -dihydroxy-,-carotene-4,4 -
dione), which holds high antioxidant activity approx-
imately 10 times greater than other carotenoids, such
as zeaxanthin, lutein, canthaxanthin, and -carotene,
and over 500 times greater than -tocopherol. Using
its potential effects of cancer prevention, immune
response enhancement, and a free radial quenching, the
use of this red ketocarotenoid has expanded in applica-
tion from a preferred pigment in feeds for farmed fish
to a vitamin source for the poultry industry; a colorant,
a natural preservative, and a food additive in the food
industry; a superior antioxidant in the cosmetic indus-
try; an antiaging reagent as a precursor of Vitamin A;
an anticancer agent through singlet oxygen quenching;
and an immunomodulator in the pharmaceutical
industry (Lorenz and Cysewski, 2000; Guerin et al.,
2003).
To date, many biotechnologists have advanced
large-scale high-density cultivation techniques for the
commercial production of astaxanthin from H. pluvi-
alis because of the high levels of astaxanthin accu-
mulation in the cells. However, the commercial use
of H. pluvialis cells has been restricted due to low
growth rates, ease of contamination, its thick cell wall,
and difficulties in mass cultivation. Because the cul-
ture requirements for astaxanthin accumulation are
different from those for cell growth, the commercial
processes for astaxanthin production have been oper-
ated as a two-stage H. pluvialis cultivation process
(Boussiba et al., 2000; Lorenz and Cysewski, 2000;
Olaizola, 2000). The first stage is conducted by main-
taining the environmental conditions to achieve high
H. pluvialis growth rates in the vegetative form. The
cells at the stationary growth phase are transferred to
the second reddening stage and the astaxanthin accu-
mulation in the cells is then induced by environmen-
tal stresses, including light intensity, nutrient levels,
metal ions, oxidative stress, salt stress, temperature,
and pH (Lee and Zhang, 1999). Among these environ-
mental stresses, the irradiance level has the greatest
effect on the morphological changes of H. pluvialis
cells and their carotenoid formation. The two-stage
processes currently employed by most manufacturers
require long cultivation times and high production costs
for complex equipment and vast plots of land. The use
of a two-stage batch production process for astaxanthin
541
production has been fully discussed in the previous lit-
erature (Fabregas et al., 2001; Zhang and Lee, 2001;
Hata et al., 2001).
In order to simplify the conventional two-stage pro-
cess, this study introduced a novel double-region pho-
tobioreactor for the production of astaxanthin from H.
pluvialis. In the cylindrical double-region photobiore-
actor, the vegetative cell growth was accomplished in
the inner core region while simultaneous astaxanthin
accumulation was achieved in the outer jacket region.
The excessive irradiation to the outer surface of the
reactor enhanced the accumulation of astaxanthin in
the cells. While penetrating the outer jacket region, the
supplied light energy was decreased by mutual shading
of the red cells in the outer jacket region. The attenuated
light energy was used for the growth of green cells in the
inner core region. In this study, the cylindrical double-
region photobioreactor was designed as a model sys-
tem; the technical feasibility of the double-region pho-
tobioreactor has been demonstrated to simultaneously
accomplish vegetative cell growth and astaxanthin pro-
duction.
2. Materials and methods
2.1. Strain and culture conditions
The unicellular green alga, H. pluvialis UTEX 16
(CCAP 34/1b, H. lacustris), was obtained from the
University of Texas Culture Collection (Austin, TX,
U.S.A.) and photoautotrophically cultivated in fortified
Bolds Basal Medium (FBBM) (Park et al., 2001; Choi
et al., 2003). After the initial pH was adjusted to 6.5,
the cells grown on the agar were inoculated into 120 ml
sterilized medium in a 250 ml Erlenmeyer flask. The
seed cultures were incubated at 25 C under continuous
shaking (175 rpm) and irradiated at 40 2 E m2 s1
with white fluorescent lamps (Park and Lee, 2001).
2.2. Photobioreactor construction
A novel air-lift photobioreactor was designed and
constructed as shown in Fig. 1. The cylindrical air-
lift photobioreactor (1 l working volume) consisted of
two concentric, cylindrical tubes made of Pyrex glass.
Their bottom parts were modified into a cone-shape to
reduce cell sedimentation. The outer tube (inner diam-
542 I.S. Suh et al. / Journal of Biotechnology 125 (2006) 540546

Fig. 1. Photograph and schematic diagrams of a double-layered photobioreactor: (a) photograph, (b) side view, and (c) top view of the photo-
bioreactor: (1) inner-core region, (2) outer-jacket region, and (3) fluorescent lamp; (i) inoculation port, (ii) sampling port, (iii) medium feeding
port, (iv) gas outlet port, and (v) gas inlet port.

eter = 7.5 cm) had two necks, and both of the necks
had a silicone stopper where four ports were equipped
for inoculation, fresh medium inlet, gas outlet, and
sampling. In the case of the inner tube (inner diame-
ter = 5.1 cm), these ports were installed at a top head
cover. The cells in both regions were mixed by the
motion of the rising gases, which were introduced
through the gas-inlet port at the end of the respec-
tive tapered jacket. For irradiation, fluorescent lamps
(FL20SSD/18, OSRAM Korea Co., Korea) were fixed
on a frame and positioned symmetrically at the same
distance from each other. The experiments were carried
out under continuous irradiation; the light condition
inside the photobioreactor was regulated by changing
the number and positions of the fluorescent lamps (Choi
et al., 2003).
2.3. Cell cultivation methods
It has been recognized that culture requirements for
H. pluvialis cell growth and astaxanthin induction are
different. The basal operating conditions of environ-
mental factors were determined in our previous works
(Park et al., 2001; Park and Lee, 2001). The broth tem-
perature in both regions was maintained at 25 C, and
the gas flow rate of 5% CO2 enriched air was main-
tained at 100 ml min1 (0.2 vvm). Two regions in the
photobioreactor were inoculated by the cells at a period
of exponential growth; these cells were pre-incubated
under seed-culture conditions.
The selective vegetative cell growth and aplanos-
pore formation were accomplished by regulating the
light conditions and nitrate levels of the inner core and
outer jacket regions, respectively, inside the double-
region photobioreactor. Light energy is essential for
phototropic growth of algae, which is of particular con-
cern for industrial applications. The high light energy
needed for astaxanthin induction was supplied to the
outer surface of the photobioreactor. The attenuated
light energy by mutual shading effects during the pen-
etration of the outer jacket region was transferred to the
inner core region and utilized for vegetative cell growth
in that region.
Since a nitrogen source is an essential nutrient for
high-density culture, nitrate was intermittently fed into
the cell growth region to maintain the nitrate level of the
fresh medium. For astaxanthin accumulation, a condi-
tion of nitrate starvation was used as a stress condition
 I.S. Suh et al. / Journal of Biotechnology 125 (2006) 540546
in order to accelerate astaxanthin accumulation in H.
pluvialis cells.
2.4. Analytical methods
The cell number, average cell size, cell size distri-
bution, and cell fresh weight were measured using a
Coulter Counter (model Z2, Beckman Coulter, Inc.,
Fullerton, CA, U.S.A.) after the culture samples were
diluted with an electrolyte solution, Isoton II (Beck-
man Coulter, Inc.). The data from the Coulter Counter
were converted by AccuComp software (ver. 2.01,
Beckman Coulter, Inc.) and exported to the Microsoft
Excel program (ver. 2002, Microsoft Co.). Cell dry
weight was determined from a 20 ml sample. Each
sample was centrifuged at 2000 rpm for 10 min and
washed twice with distilled water. Samples were then
dried overnight at 90 C prior to the weighing of the
crucibles.
Following acetone extraction and dilution to the
proper concentrations, the total chlorophyll, and astax-
anthin concentrations were analyzed by a spectropho-
tometer (model HP8453B, Hewlett Packard, Wald-
bronn, Germany) (Park and Lee, 2001). Nitrate concen-
tration was also analyzed by a spectrophotometer after
the centrifuged sample was treated with HCl accord-
ing to the standard method (APHA et al., 1998). The
photon flux density was measured using a quantum sen-
sor (model LI-190SA, LI-COR, Lincoln, NE, U.S.A.)
equipped with a Datalogger (model LI-1400, LI-COR).
The light intensity at the surface of the photobioreactor
was determined by averaging the values of light inten-
sity measured at 16 points (every /8 radian).
3. Results and discussion
3.1. Feasibility test of a double-region
photobioreactor
In a preliminary experiment, a novel double-region
photobioreactor was introduced for batch cultivation
of H. pluvialis. Two regions, the inner core and outer
jacket, were inoculated with cells from an exponential
growth period at a cell density of 1.2 104 cells ml1 .
The cells were then photoautotrophically cultivated
with nitrate feeding and a light energy supply of
60 5 E m2 s1 at the outer surface of the photo-
543
bioreactor, which corresponded to 40 3 E m2 s1
at the surface of the inner jacket of the photobioreac-
tor.
Fig. 2 presents the changes in cell number, cell
fresh weight, total chlorophyll, and astaxanthin con-
centration in the inner core and outer jacket regions.
During early growth stage of the batch run, the cell
number in both regions increased exponentially, corre-
sponding with high chlorophyll synthesis rates, while
the astaxanthin concentration increased only slightly.
However, as the cell growth entered into the linear
phase, a difference in the growth patterns of the inner
core and outer jacket regions was observed. The cell
growth in the inner core region became impeded by an
insufficient light energy supply due to the mutual shad-
ing effects by the biomass in the outer jacket region.
Thus, the maximal cell number (4.0 105 cells ml1 )
in the inner core region was 36.5% lower than that
(5.5 105 cells ml1 ) in the outer jacket region.
After the onset of the stationary phase of algal
growth in the inner core region, the induction of
carotenogenesis in the outer jacket region was con-
ducted by exposure to a high level of irradiation
(770 20 E m2 s1 ). Meanwhile, the cells in the
inner core region received decreased light energy
(<15 E m2 s1 ) due to the intensified mutual shading
effects in the outer jacket region. The nitrate-depleted
condition in the outer jacket region was maintained in
order to accelerate astaxanthin accumulation, while the
concentrated nitrate solution was fed into the inner core
region. The cultivation results indicate that the cells in
the inner core region were continuously maintained in a
small, green vegetative state without astaxanthin accu-
mulation in the cells. However, the cell growth in the
outer jacket region ceased, and large, red (astaxanthin-
rich), immobile aplanospores were formed which con-
tinued to increase in astaxanthin concentration. The
highest astaxanthin concentration obtained in this reac-
tor was measured to be 357 mg l1 (4.95% on a dry
weight basis) in the outer jacket region, which was
about nine times higher than the levels obtained in inner
core region.
This investigation allows a preliminary evaluation
of the applicability of a double-region photobioreac-
tor for the cultivation of H. pluvialis. Furthermore, the
patterns of cell growth and astaxanthin accumulation
indicate that the inner core and outer jacket regions of
the photobioreactor can be used for selective vegetative
544 I.S. Suh et al. / Journal of Biotechnology 125 (2006) 540546

Fig. 2. Time course profiles of (a) cell number, (b) cell fresh weight, (c) total chlorophyll concentration, and (d) astaxanthin concentration in
the inner-core region (
) and outer-jacket region () of the photobioreactor. At 12th day, the light condition and nitrate level were changed for
astaxanthin accumulation in the outer-jacket region.

cell growth and astaxanthin accumulation through the
regulation of the light condition and nutrient level.
3.2. Simultaneous cell growth and astaxanthin
production
In order to make this batch cultivation viable, it is
desirable to operate the double-region photobioreactor
systematically. When one batch run was completed,
the aplanospore cells in the outer jacket region were
harvested for further down-stream processing. In this
study, the vegetative cells in the inner core region could
be utilized as an inoculum for the next cultivation. The
high-density vegetative cells obtained from the previ-
ous batch run were resuspended with fresh medium at
2.1 105 and 1.2 104 cells ml1 , which were trans-
ferred to the outer jacket and inner core regions, respec-
tively, at a clean bench. Different culture conditions
in the inner core and outer jacket regions were then
manipulated for vegetative cell growth and astaxan-
thin accumulation, respectively. Excessive light energy
(770 20 E m2 s1 ) was supplied to the outer sur-
face of the photobioreactor. While penetrating the outer
jacket region, the supplied light energy was attenu-
ated to 40 3 E m2 s1 at the surface of the inner
jacket of the photobioreactor. In order to promote selec-
tive cell growth and astaxanthin induction, the optimal
nitrate concentration was established by nitrate feeding
to the inner core region and simultaneous nitrate star-
vation in the outer jacket region of the photobioreactor.
Growth and pigment levels of cells during the batch
run are shown in Fig. 3. In the inner core region, the cell
 I.S. Suh et al. / Journal of Biotechnology 125 (2006) 540546 545

Fig. 3. Time course profiles of (a) cell number, (b) cell fresh weight, (c) total chlorophyll concentration, and (d) astaxanthin concentration in
the inner-core region (
) and outer-jacket region () of the photobioreactor.

number and cell fresh weight increased continuously to
4.3 105 cells ml1 and 3.2 g l1 , respectively. Since
the inoculum had been prepared according to a dif-
ferent procedure, the growth rate was relatively low
compared with the previous culture, shown in Fig. 2.
Additionally, the lower fresh weight in the inner core
was caused by the small cell size, which indicates that
the cells in the inner core region were maintained in
the vegetative form. The profiles in Fig. 3(c) and (d)
exhibit the expected increase in total chlorophyll and
the maintenance of a low astaxanthin level during the
full course of the batch run. Meanwhile, the culture in
the outer jacket region was observed to be in the transi-
tion from green vegetative cells to red aplanospores.
Although the rate of cell division was very low at
the beginning of the culture, the cell numbers actually
declined after all of the nitrogen had been consumed.
However, an increase of the cell fresh weight was
observed because the cells had encysted and increased
in size. A reduction in the level of total chlorophyll
in the cells was accompanied by an increase in the
astaxanthin concentration, up to 226 mg l1 , which cor-
responded to the 5.79% astaxanthin content on a dry
weight basis.
From these results, it is evident that conventional
two-stage batch processes can be replaced by a process,
which uses a double-region photobioreactor to allow
simultaneous growth of vegetative cells and astaxan-
thin production. Furthermore, the use of the double-
region photobioreactor enabled us to minimize energy
546 I.S. Suh et al. / Journal of Biotechnology 125 (2006) 540546
loss for irradiation, reduce equipment cost and cultiva-
tion time, and simplify the process configuration. This
study has also demonstrated that the use of vegetative
cells in the inner core region as inoculum for the next
batch allows sequential batch operation of the double-
region photobioreactor.
4. Conclusion
In this paper, a novel double-region photobioreac-
tor was established and applied for astaxanthin pro-
duction from the green microalga, H. pluvialis. The
experimental results indicate that it was practicable to
use the double-region photobioreactor for astaxanthin
production in the outer jacket region with simultane-
ous vegetative cell growth in the inner core region.
In order to achieve higher productivity of the two
regions of the photobioreactor, further optimization
study is required to determine the most favorable cul-
ture conditions for the growth of vegetative cells and
the induction of astaxanthin accumulation. We are cur-
rently investigating a strategy for up-scaling this pro-
cess and for (semi-) continuous operation of the double-
region photobioreactor. The present study provides
an alternative biotechnological strategy to develop a
cost-efficient bioprocess for astaxanthin production by
H. pluvialis.
Acknowledgement
This study was supported by Inha University
Research Fund, for which the authors are grateful.
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