COMMUNICATION TO THE EDITOR

On the Relative Efficiency of Two- vs. One-stage

Production of Astaxanthin by the Green Alga
Haematococcus pluvialis
Claude Aflalo, Yuval Meshulam, Aliza Zarka, Sammy Boussiba
Microalgal Biotechnology Laboratory, The Blaustein Institutes for Desert Research,
Ben-Gurion University, Sede-Boker Campus 84990, Israel; telephone: 972-8-6596817;
fax: 972-8-6596742; e-mail: aflaloc@bgumail.bgu.ac.il
Received 2 December 2006; revised 21 January 2007; accepted 12 February 2007
Published online 22 February 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21391

coccus pluvialis is cultured in large scale facilities for the

ABSTRACT: Haematococcus pluvialis under stress condi-
tions overproduces the valuable red ketocarotenoid astax-
anthin. Two proposed strategies for commercial production
are under current analysis. One separates in time the pro-
duction of biomass (optimal growth, green stage) and
pigment (permanent stress, red stage), while the other uses
an approach based on continuous culture under limiting
stress at steady state. The productivities, efficiencies and
yields for the pigment accumulation in each case have been
compared and analyzed in terms of the algal basic physiol-
ogy. The two-stage system indoors yields a richer astax-
anthin product (4% of dry biomass) with a final astaxanthin
productivity of 11.5 mg L
1 day
1, is more readily upscal-
able and amenable to outdoors production. Furthermore,
each stage can be optimized for green biomass growth and
red pigment accumulation by adjusting independently the
respective ratio of effective irradiance to cell density. We
conclude that the two-stage system performs better (by a
factor of 2.5–5) than the one-stage system, and the former is
best fit in an efficient mass production setup.
Biotechnol. Bioeng. 2007;98: 300–305.
ß 2007 Wiley Periodicals, Inc.
KEYWORDS: Haematococcus pluvialis; growth yield; astax-
anthin productivity; carotenoid; one-stage; two-stage
Introduction
Microalgae can be elicited to overproduce secondary
metabolites under special circumstances, often related to
as stress conditions (Zhao et al., 2005). These compounds,
existing at low concentration in cells growing under
favorable conditions, are needed to counter or accommo-
date biotic or abiotic environmental stresses, such as salinity,
nutrient deprivation, exposure to prohibitive light intensity,
drought, etc. (Boussiba, 2000). The green alga Haemato-
production of the valuable ketocarotenoid astaxanthin
(Borowitzka et al., 1991) accumulated in response to stress
into lipid globules (Zhekisheva et al., 2005) that can traffic in
the cytosol between around the nucleus and the cell
periphery. The pigment can be used for either coloring
salmonids flesh (Foss et al., 1987) or in the nutraceutical
market as a potent antioxidant (Guerin et al., 2003).
We have proposed that efficient astaxanthin production
by Haematococcus can be achieved in a two-stage process
(Boussiba et al., 1992), first producing green biomass under
optimal growth conditions (‘green’ stage) and next exposing
the algae to adverse environmental conditions to induce the
accumulation of astaxanthin (‘red’ stage). This sequential
production system (Boussiba et al., 1997) is routinely used
by other groups for the production of astaxanthin by the
green alga (Choi et al., 2002; Cifuentes et al., 2003; Cordero
et al., 1996; Fabregas et al., 2001; Hata et al., 2001;
Lababpour et al., 2004; Olaizola, 2000; Sarada et al., 2002;
Tripathi et al., 1999).
The feasibility of a one step astaxanthin production
system has been reported (Lee and Soh, 1991). In such a
recently revised system, the pigment astaxanthin is produced
under a high irradiance and limiting nitrogen regime,
allowing cells to divide continuously in asynchronic cultures
(Del Rio et al., 2005). In this elaborated study, productivity
data have been presented in support to the claims that this
new technology has the highest production rate of
astaxanthin ever reported in the literature, and is superior
in terms of efficiency to the two-stage system. This latter
conclusion by Del Rio et al. (2005) stands in contradiction
with the consensus stating that secondary metabolites pro-
duction occurs preferentially in cells with low proliferation

Correspondence to: Claude Aflalo

300 Biotechnology and Bioengineering, Vol. 98, No. 1, September 1, 2007 ß 2007 Wiley Periodicals, Inc.
activity (Boussiba, 2000; Glombitza et al., 2004; Wang et al.,
2003; Zhao et al., 2005).
This communication sets the record straight by doc-
umenting a higher productivity of astaxanthin in the two-
stage system, refuting the claims in favor of a single-step
process. The comparison between both systems is discus-
sed in light of basic physiological aspects of astaxanthin
accumulation in Haematococcus, as well as their relevance
in resolving biotechnological issues encountered in the
commercial production of this valuable ketocarotenoid.
Materials and Methods
Haematococcus pluvialis Flotow 1844 em. Wille K-0084 was
obtained from the Scandinavia Culture Center for Algae
and Protozoa (SCCAP) at the University of Copenhagen,
Denmark.
Indoors Growth Conditions
Cells were cultivated in 4 cm wide, 500 mL glass columns
immersed in a thermostated (25 8C) water bath in modified
BG-11 medium (Boussiba and Vonshak 1991). The cultures
were stirred by bubbling (0.3 L min
1) using a mixture of
1.5% CO2 in air. Continuous illumination was provided by
external cool-white fluorescent lamps. Cultures of green
cells were grown for 5 days under incident light intensity of
75 mE m
2 s
1 (normal light). These actively growing cells
were then resuspended in fresh medium to a cell concen-
tration of 200 106 cells/L (or 200 Mc/L, corresponding to
about 0.3 g/L in dry biomass) and grown under normal or
various stress conditions, including high salinity (0.8%
NaCl), high irradiance (350 mE m
2 s
1) and/or nutrient
deprivation. Nitrogen, phosphorus, or sulfur deficiency was
achieved by iso-osmotic exchange of NaNO3, K-phosphate,
or MgSO4 in the complete medium by NaCl, KCl, or MgCl2,
respectively.
Outdoors Cultures
Two types of photobioreactors were used: a 5 cm wide, 500 L
flat vertical panel type (Richmond and Cheng-Wu, 2001)
and a 5 cm wide, 2000 L horizontal tubular type (Richmond
et al., 1993) for the green (4 days cycle) and red stage (6 days
cycle), respectively. Both the reactors were cooled (24–268C)
and mixed by either air bubbling (300 L min
1) or using
a diaphragm pump (50 cm s
1), respectively. CO2 was
supplied in both cases as needed to maintain a constant
pH (6.4–7.0). The outdoors cultures were carried out during
summer (14 light h/day) at peak solar light intensity of
2000 mE m
2 s
1.
Measurement of Growth Variables
Cell concentration, biomass dry weight and chlorophyll or
total carotenoid (Tcar) content in hot DMSO extracts were
Aflalo et al.: Best Production of Astaxanthin by a Two-Stage System
determined as described previously (Boussiba and Vonshak,
1991). The variability between duplicate measurements was
routinely less than 5%. In addition to the quantization,
the cells morphology was routinely monitored under the
microscope. Increased cell carotenoid content was corre-
lated with the appearance of large, red cysts (Kobayashi
et al., 2001). Total extracted carotenoids were fractionated
and analyzed by HPLC (Boussiba et al., 1999); after 5 days of
nutrient deprivation, astaxanthin esters represented more
than 90% of total carotenoids. Similar results were obtained
under high light irradiance at days 2 and 3, beyond which
the culture became acclimated to the stress, and continued
with ‘green’ growth.
All indoors experiments presented in this work are
representative of at least three independent replicates per-
formed with different batches of algae, and resulted in
consistent behavior with some inter-batch variability.
Results and Discussion
During the first (green) stage, Haematococcus cells are
cultured in complete medium under standard conditions,
avoiding high light irradiance. In the second (red) stage, the
green cultures are basically exposed to stress conditions by
increasing the average cell exposure to light (by diluting the
culture, increasing the incident light intensity, and/or
reducing the light path of the culture) and/or nutrient(s)
deprivation.
Figure 1. Growth kinetics indoors under increasing light intensity. A low density
culture was illuminated at light intensity of 25 mE m
2 s
1. Every 7 days the medium
was replaced and light intensity was increased as indicated. The closed symbols
represent values measured after 14 days with no changes of medium or light intensity.
Chl: chlorophyll.
301
Biotechnology and Bioengineering. DOI 10.1002/bit
Figure 2. Growth kinetics of high-density cultures indoors under two light
intensities. Actively growing cultures were harvested, resuspended at 2  106
cells/mL (or 2 Mc/mL) in fresh mBG-11 medium, and illuminated at normal
(75 mE m
2 s
1; open symbols) or high (500 mE m
2 s
1; closed symbols) light
intensity. Average green biomass productivities of 0.63 and 1.83 g L
1 day
1 (corre-
sponding to growth rates of 0.19 and 0.40 day
1) can be calculated for normal and high
irradiance, respectively.

Production of Green Biomass—The Green Stage

Several studies have described the conditions for the pro-
duction of vegetative green Haematococcus cells biomass; in
most cases the growth rate was in the range 0.2–0.4 day
1,
with reported biomass productivity of up to 0.55 g L
1
day
1 (Garcia-Malea-Lopez et al., 2006). The average
exposure of the cells to light is a major factor affecting
Haematococcus growth rate in indoors cultures (Choi et al., Figure 3. Growth and carotenoids accumulation in outdoors cultures. The
2003; Richmond et al., 2003). This concept is illustrated in conditions are as described in Materials and Methods section. A: Green stage culture.
The biomass in the culture was monitored, partially harvested and periodically diluted
Figure 1. A constant growth rate could be maintained by with fresh medium as indicated by broken lines. The calculated average green
adjusting the light intensity to cell density, while keeping biomass productivity was 0.37  0.05 g L
1 day
1 (corresponding to growth rate of
the nutrients at saturating levels, yielding a high overall 0.28 day
1). B: Red stage. Green biomass resuspended in medium lacking nitrate, was
monitored. Total carotenoids content (%Tcar) is expressed as percentage of the
productivity (about 0.8 g L
1 day
1). Implementing this biomass dry weight. Astaxanthin content was 1.0 and 102 mg L
1 at days 0 and 6,
strategy (light controlling growth rate) and using a higher respectively, yielding a calculated productivity of 16.8 mg L
1 day
1, and a
cell density as inoculum, one can even achieve higher specific rate of formation of 72.1 mg g
1 day
1, on the basis of a biomass productivity
of 0.23 g L
1 day
1.
productivities, up to 2 g L
1 day
1 (Fig. 2), the highest ever
reported for this alga. Furthermore, the resulting green
biomass, having been exposed to relatively high light
intensity (compared to both standard conditions, and those

Table I. Astaxanthin accumulation indoors under different stress conditionsa

Stress condition Total carotenoid productivity (mg L
1 day
1) Astaxanthin:Tcar final ratio Tcar final content (% DW)
None (full medium, normal light) 2.0–3.0 0.01–0.05 0.6–1.2
No sulfate 6.6–7.0 0.80–0.90 3.0–3.5
No phosphate 7.2–7.6 0.80–0.90 3.0–3.5
No nitrate 10–12 0.93–0.96 3.5–4.0
High lightb 12–15 0.75–0.80 3.0–3.5
High light, no nitrate 20–25 0.94–0.97 4.0–4.5
High salinity 5.0–5.3 0.80–0.90 3.0–3.5
a
Values are observed ranges of 5 days indoors cultures grown in airlift columns as described in Materials and Methods, representative of 3–8 independent
experiments for each condition. Tcar: total carotenoids; DW: dry weight.
b
High light intensity (350 mE m
2 s
1) in the presence of saturating nutrients represents a transient stress, inducing a peak response between day 2 and 3,
whose values are reported.

302 Biotechnology and Bioengineering, Vol. 98, No. 1, September 1, 2007

DOI 10.1002/bit
Table II. Comparison of biomass and astaxanthin yield under different conditionsa
Conditions
Indoors (continuous light) Biomass productivity (g L
1 day
1)
Tcar productivity (mg L
1 day
1)
Final astaxanthin content (% DW)
Outdoors (day:night cycle) Biomass productivity (g L
1 day
1)
Tcar productivity (mg L
1 day
1)
Final astaxanthin content (% DW)
a
The two-stage standard conditions indoors are as described in Materials and Methods section. The one-stage conditions and values are from Del Rio et al.
continuous cultures at high light intensity and limiting nitrogen source (Del Rio et al., 2005).
b
The total carotenoids (Tcar) in the green stage include only about 5% astaxanthin.
c
Calculated as the time-weighted average of both stages productivities.
d
The reported Tcar productivity refers to astaxanthin specifically.
e
Calculated value corrected for ‘unproductive’ dark hours (10-day
1 in summer), including over 90% astaxanthin.
in Fig. 1), contains larger cells commited to partial stress,
and are thus better prepared to respond efficiently in
astaxanthin production during a subsequent stage under full
stress conditions. Similarly in outdoor cultures grown using
higher (although discontinuous) irradiance (Fig. 3A), an
average productivity of about 0.37 g L
1 day
1 could be
maintained for a month during the summer period.
Production of Red Biomass—The Red Stage
The green biomass obtained from these cultures can then be
exposed to several environmental stresses to trigger the
production of the secondary metabolite astaxanthin as an
antioxidative response to the stress-induced formation of
reactive oxygen species (Kobayashi, 2000). Total carotenoids
productivity obtained indoors under different stresses vary
between 5 and 25 mg L
1 day
1 depending on the stress
imposed (Table I); in all cases, a high astaxanthin content is
prominent. The highest rate is obtained with exposure of the
green biomass to a combination of high light and nitrogen
deprivation. In such a culture outdoors (tubular reactor of
2000 L), the average carotenoid productivity for the red
stage is 12–17 mg L
1 day
1. Representative changes in
such reactor of the parameters measured are depicted in
Figure 3B. In this process, the cells stop dividing, increase
in size and mass, transform into cysts with a hard cell wall,
and accumulate astaxanthin up to 4% of the dry biomass
(Boussiba et al., 1997).
Comparing Both Systems and Some
Biotechnological Considerations
The two-stage (batch-based) and one-stage (steady-state at
limiting Nitrogen) culture systems being different in
essence, they will be compared on the basis of normalized
rates of pigment accumulation, relevant to commercial scale
production. These are the volumetric (weight L
1 day
1)
and specific (weight g biomass
1 day
1) astaxanthin pro-
Aflalo et al.: Best Production of Astaxanthin by a Two-Stage System
Two-stage
One-stage
Green stage Red stage Green þ red Steady state
0.50 0.21 0.36c 0.9
2.1b 20.8 11.5c 5.6d
0.05 4.0 4.0 0.8
0.37 0.21 0.27c, 0.46e NA
3.1b 14.8 10.1c, 17.3e NA
0.1 3.8 3.8 NA
ductivities, providing information on scalable effective rates
and inherent algal capacity for production, respectively.
Astaxanthin Productivity
The highest rate obtained in the one-stage system was
5.6 mg L
1 day
1 at steady state, while for the two-stage
system the maximal rate obtained was 25 mg L
1 day
1, at
the red stage (Table I). Taking into account the period
required for the production of the green biomass (4 days,
Fig. 2) the overall rate indoors would be about 13.9 mg L
1
day
1, which is still over twice faster than that reported for
the one-stage continuous production (Del Rio et al., 2005).
Finally, the two-stage approach is readily upscaled and
implemented outdoors, yielding an astaxanthin productivity
of about 8–10 mg L
1 day
1 over a 10 (4 green þ 6 red stage)
days cycle, or up to 17 mg L
1 day
1 for the light hours only,
more than three times that of the continuous one-stage
system (Table II). The specific rates (calculated per overall
biomass produced during the light hours: 0.46 g L
1 day
1)
for astaxanthin accumulation reveals an even larger
difference between the two-stage and one-stage systems
(37.4 vs. 6.2 mg g
1 day
1, respectively).
Carotenoids Profile of the Product
The maximal astaxanthin content obtained in the one-stage
system is about 0.8% of dry weight (Del Rio et al., 2005).
However this type of cells, although enriched in astaxanthin,
actually contains also other carotenoids, such as lutein and
b-carotene, estimated as about 65, 20, and 5% of total
carotenoids, respectively (Torzillo et al., 2003). One will
need to separate these pigments to obtain clean astaxanthin.
In comparison, red cells in the two-stage system accumulate
up to over 4% astaxanthin (Table I, Fig. 3). This type of cells
contains nearly pure astaxanthin, more than 95% of the total
carotenoid fraction (Boussiba et al., 1997). Another issue
rising from the low pigment content in the biomass relates
to the major commercial application of the carotenoid
production; as an additive to fish feed, a relatively large
303
Biotechnology and Bioengineering. DOI 10.1002/bit
amount of this biomass should be added to affect pigmen-
tation as compared to the 4% enriched pigment biomass.
Type of Cells Produced
In a continuous system at steady state, cells are in a division
cycle most of the time; this implies that the cells in the
culture are either in flagellated or palmelloid forms, devoid
of a hard cell wall (Del Rio et al., 2005). This creates a major
problem in up-scaling the system. The soft cells are very
sensitive to light and mechanical breakage as reported
previously (Gudin and Chaumont, 1991). In the two-stage
system, green cells are usually harvested at the end of the
growth cycle as mature palmelloids with hard cell wall
(Montsant et al., 2001). These cells exposed to further stress
under relatively harsh outdoors conditions turn into cysts
after a day or two, soon enabling them to resist mechanical
breakage and counter the oxidative burst developing under
high light and other stresses (Kobayashi, 2000; Kobayashi
et al., 2001). Finally, the harvest of the resulting large and
heavy cysts is much more easily done (by gravitational
decantation) than that of the heterogeneous cells obtained at
steady state in the one-stage system.
Haematococcus Cell Wall
In our view, a stiff cell wall represents a major advantage in
the large-scale development of microalgal biotechnology.
The cell wall hampers digestion by invading predators in the
culture, conveys resistance to high light and UV irradiation
in particular and to mechanical breakage as well. This
natural ‘‘suitcase’’ represents a major asset of the alga
exposed to harsh outdoors environmental conditions. These
cell characteristics do not apply to cells obtained in the one-
stage system, and there is no real biotechnological advantage
to vigorously growing cells for production of the secondary
carotenoid astaxanthin.
Pigment Extraction
An apparent drawback of the two-stage system is the
extraction of astaxanthin from heavily walled cysts, as
compared to the one-stage mode involving softer cells. It
would seem that milder treatments for extraction should be
sufficient for the later case. However, since the steady-state
culture is not synchronous and high astaxanthin content is
generally correlated with the presence of cysts, most the
pigment in that culture would be associated with the heavily
walled cells fraction. Thus, a mild extraction procedure,
while technically advantageous, is likely to turn counter-
productive in terms of the yield for extraction of astaxan-
thin. In fact, efficient breakage technology is available today
at relatively low-cost, so that the extraction procedure from
cysts (in both systems) does not represent a real production
bottle-neck.
304 Biotechnology and Bioengineering, Vol. 98, No. 1, September 1, 2007
Concluding remarks
Although the one-step production system is interesting to
look into the physiology of production of a secondary
metabolite in a continuous mode, it has several drawbacks in
terms of its relevance to mass production of astaxanthin, the
more prominent, beside low astaxanthin content, being its
unsuitability to outdoors setting (e.g., requirement of
continuous illumination). The two-stage production system
has been for the first time extensively documented in terms
of productivities and efficiency parameters in this commu-
nication. On the rational level, its experimental flexibility
and potential for further optimisation based on physiolo-
gical and production process arguments makes it in our
opinion the system of choice for large scale, commercial
production of astaxanthin from the green alga Haemato-
coccus pluvialis.
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