Journal of Biotechnology 123 (2006) 329342

Comparative analysis of the outdoor culture of Haematococcus

pluvialis in tubular and bubble column photobioreactors
M.C. Garca-Malea L opez
a
, E. Del Ro S anchez
b
, J.L. Casas L opez
a
,
F.G. Aci en Fern andez
a
, J.M. Fern andez Sevilla
a
, J. Rivas
b
,
M.G. Guerrero
b
, E. Molina Grima
a,
a
Department of Chemical Engineering, University of Almera, Ca nada San Urbano S/N, Almera 04071, Spain
b
Inst. Bioqumica Vegetal y Fotosntesis, University Sevilla-CSIC, E-41092-Sevilla, Spain
Received 19 July 2005; received in revised form 27 October 2005; accepted 23 November 2005
Abstract
The present paper makes a comparative analysis of the outdoor culture of H. pluvialis in a tubular photobioreactor and a
bubble column. Both reactors had the same volume and were operated in the same way, thus allowing the inuence of the reactor
design to be analyzed. Due to the large changes in cell morphology and biochemical composition of H. pluvialis during outdoor
culture, a new, faster methodology has been developed for their evaluation. Characterisation of the cultures is carried out on a
macroscopic scale using a colorimetric method that allows the simultaneous determination of biomass concentration, and the
chlorophyll, carotenoid and astaxanthin content of the biomass. On the microscopic scale, a method was developed based on
the computer analysis of digital microscopic images. This method allows the quantication of cell population, average cell size
and population homogeneity. The accuracy of the methods was veried during the operation of outdoor photobioreactors on a
pilot plant scale. Data from the reactors showed tubular reactors to be more suitable for the production of H. pluvialis biomass
and/or astaxanthin, due to their higher light availability. In the tubular photobioreactor biomass concentrations of 7.0 g/L (d.wt.)
were reached after 16 days, with an overall biomass productivity of 0.41 g/Lday. In the bubble column photobioreactor, on
the other hand, the maximum biomass concentration reached was 1.4 g/L, with an overall biomass productivity of 0.06 g/Lday.
The maximum daily biomass productivity, 0.55 g/Lday, was reached in the tubular photobioreactor for an average irradiance
inside the culture of 130 E/m
2
s. In addition, the carotenoid content of biomass from tubular photobioreactor increased up to
2.0%d.wt., whereas that of the biomass from the bubble column remained roughly constant at values of 0.5%d.wt. It should
be noted that in the tubular photobioreactor under conditions of nitrate saturation, there was an accumulation of carotenoids
due to the high irradiance in this reactor, their content in the biomass increasing from 0.5 to 1.0%d.wt. However, carotenoid
accumulation mainly took place when nitrate concentration in the medium was below 5.0 mM, conditions which were only
observed in the tubular photobioreactor. A similar behaviour was observed for astaxanthin, with maximum values of 1.1 and

Corresponding author. Tel.: +34 950 015032; fax: +34 950 015484.
E-mail address: emolina@ual.es (E.M. Grima).
0168-1656/$ see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2005.11.010
330 M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342
0.2%d.wt. measured in the tubular and bubble column photobioreactors, respectively. Fromthese data astaxanthin productivities
of 4.4 and 0.12 mg/Lday were calculated for the tubular and the bubble column photobioreactors. Accumulation of carotenoids
was also accompanied by an increase in cell size from 20 to 35 m, which was only observed in the tubular photobioreactors.
Thus it may be concluded that the methodology developed in the present study allows the monitoring of H. pluvialis cultures
characterized by fast variations of cell morphology and biochemical composition, especially in outdoor conditions, and that
tubular photobioreactors are preferable to bubble columns for the production of biomass and/or astaxanthin.
2005 Elsevier B.V. All rights reserved.
Keywords: Haematococcus; Photobioreactors; Astaxanthin; Digital image; Colorimeter; Outdoor
1. Introduction
Microalgae are a potential source of biomass or spe-
cic products such as lipids, pigments, antioxidants,
etc. One of the most recent processes based on microal-
gae is the production of astaxanthin fromHaematococ-
cus pluvialis. Astaxanthin is a high-value carotenoid
pigment with important applications in the nutraceuti-
cal, cosmetics, food and feed industries (Guerin et al.,
2003). The major market for astaxanthin is as a pigmen-
tation source in aquaculture, primarily in salmon and
trout (Guerin et al., 2003). In this sense, the microalgae
Haematococcus pluvialis is the richest source of natu-
ral astaxanthin, and it is nowcultivated on an industrial
scale (Olaizola and Huntley, 2003).
Astaxanthin sells for US $2500 kg with an annual
worldwide market estimated at US $200 million
(Lorenz and Cysewski, 2000). Although 95% of this
market consumes synthetically derived astaxanthin,
consumer demand for natural products makes the syn-
thetic pigments much less desirable and provides an
opportunity for the production of natural astaxanthin
by H. pluvialis. This strain contains 1.53.0% astaxan-
thin and has gained acceptance in aquaculture and other
markets as a concentrated formof natural astaxanthin
(Lorenz and Cysewski, 2000; Olaizola and Huntley,
2003). In this sense, natural astaxanthin from H. plu-
vialis is currently produced in a two-step process. In
the rst step green vegetative cells are produced under
controlled culture conditions, frequently indoors, using
stirred tank or bubble columns. In the second step,
green cells are exposed to stress conditions (high irra-
diance, nitrate and/or phosphate deprivation, high tem-
perature) to induce accumulation of astaxanthin, using
open raceways or tubular photobioreactors. Flat panel,
bubble columns and tubular photobioreactors have
been extensively proposed as outdoor closed photo-
bioreactors for the industrial production of microalgae
(Tredici and Materassi, 1992; Richmond et al., 1993;
Molina et al., 1994; Aci en et al., 1998; Garca et al.,
1999). However, only bubble columns and tubular pho-
tobioreactors have proven capable of scaling up to high
volumes. Although the outdoor production of H. plu-
vialis in tubular photobioreactors has been referenced
(Olaizola, 2000; Chaumont and Thepenier, 1995), no
data from bubble column has been referenced in spite
of the highly adequate light prole inside this type of
reactor (Garca et al., 1999). In addition, Chaumont
and Thepenier (1995) have reported the rapid variation
of biomass concentration and pigment content in day-
light using tubular photobioreactors, with carotenoid
content increasing from 0.6 to 1.4%d.wt. in 4 h, from
7:00 to 11:00 h.
Due to the fast and great variation of Haematococ-
cus cells during outdoor culture, a fast methodology
to characterize the cultures at macroscopic and micro-
scopic scale is necessary. Microscopic characterisation,
i.e. cell population and size distribution, is usually per-
formed either by direct observation, or using automated
devices such as haemocytometers or more sophisti-
cated cell counters (Harker et al., 1996; Tripathi et
al., 1999). However, apart from cell counters, the other
methods do not quantify cell size and homogeneity,
and the use of cell counters for Haematococcus cells is
very problematic due to variations in cell size between
different cell morphotypes, frequently more than 10-
fold. Macroscopic culture characterisation is usually
performed as the dry weight measurement of biomass
concentrationandthe determinationof pigment content
byspectrophotometryHPLC(Del Campoet al., 2000).
Dry weight measurements are tedious and results are
not obtained until at least 12 h after sampling. The
spectrophotometric measurement of pigment content
requires a time-consuming extraction process of the
pigments using adequate solvents, which must also
ensure effective cell wall breakage. In the case of
M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342 331
H. pluvialis, efcient extraction of pigments is only
properly achieved using mechanical procedures of dis-
ruption (grounding with alumina) or chemical methods
(DMS) (Del Campo et al., 2000). In addition, the astax-
anthin content of the biomass must nally be quantied
by HPLC. In short, the measurement of biomass pig-
ment content is both a time and labour consuming
method, taking a minimum of 24 h.
In the present paper a comparative analysis on the
performance of H. pluvialis cultures carried out in bub-
ble columns and tubular photobioreactors is performed.
The objective is to determine the best reactor to be used
for the outdoor production of astaxanthin, as well as to
identify the main variables governing the behaviour of
the cultures. For this, and due to fast changes in cell
morphology and biochemical composition of this strain
when accumulation of astaxanthin takes place, two fast
methods for the microscopic and macroscopic charac-
terization of H. pluvialis cultures are developed. The
microscopic characterization is performed by a com-
puter analysis of digital images of the cultures obtained
with a microscope. The macroscopic characterization
is carried out by measuring colour changes directly on
H. pluvialis cultures, and the method is capable of mea-
suring biomass concentration and pigment content. The
development of fast characterization methods as well
as determining the most adequate photobioreactor and
the main variables governing the behaviour of outdoor
cultures of H. pluvialis is the rst step towards opti-
mising the outdoor production of astaxanthin from H.
pluvialis.
2. Materials and methods
2.1. Micro-organism and culture conditions
The microalga Haematococcus pluvialis (strain
CCAP 34/8) was from the Culture Collection of Algae
and Protozoa of the Centre for Hydrology and Ecol-
ogy (Ambleside, UK), and was grown in an inor-
ganic medium free of acetate (Garcia-Malea et al.,
2005). Cultures for the calibration of fast character-
ization methods were grown in 2 L photobioreactors
in laboratory conditions. The photobioreactors were
bubbled with air at 1.0 v/v/min, with the temperature
maintained at 20

C by passing thermostated water

through a jacket, and the pH maintained at 8.0 by on
demand injection of CO
2
. Batch cultures were car-
ried out at different irradiances, nitrate concentrations,
and elapsed culture times, to obtain different morpho-
logical types of cells. The irradiance ranged from 50
to 2000 Em
2
s
1
, whereas the nitrate concentration
ranged from 3.0 to 15.0 mM, and the elapsed culture
time ranged from 6 to 12 days.
Outdoor cultures were performed in two types of
reactors: an airlift tubular reactor and a bubble column
reactor (Fig. 1). In both reactors, the culture temper-
ature was controlled at 20

C by using heat exchang-

ers. The pH was controlled by injection of dry CO
2
,
whereas air was bubbled to circulate the liquid and
remove dissolved oxygen. The reactors were operated
in batch mode. The tubular airlift reactor had a volume
of 55 L, and consisted of a 98 m polymethylmethacry-
late tube of 0.03 m diameter arranged horizontally
inside a water pool, with a degasser at a height of 3.5 m.
Air was supplied at the beginning of the riser. The bub-
ble column had a culture volume of 55 L, and consisted
of a 2.0 m high vertical tube of 0.20 m diameter. Air
was supplied at the bottom of the column. In both pho-
tobioreactors temperature, pH and dissolved oxygen
probes were installed and connected to a data acqui-
sition system for on-line registration of measurement
and control. The instantaneous photon-ux density of
the photosynthetically active radiation (PAR) on a hori-
zontal surface and inside the water pool were measured
on-line using two quantum scalar irradiance meters
(LI-190 SA, Licor Instruments, Lincoln, NE, USA)
connected to a data acquisition card. Measured values
were integrated to determine the mean solar irradiance
during the daylight period. The mean irradiance on the
surface of the bubble column was calculated by multi-
plying the irradiance on the horizontal surface by 0.48.
This factor was previously determined by experimen-
tal measurements (Garca et al., 1999). The average
irradiance inside the reactors was calculated as a func-
tion of irradiance on the reactor surface, I
o
, the tube
diameter, p, the extinction coefcient of the biomass,
K
a
, and the biomass concentration, C
b
, as (Aci en et al.,
1998),
I
av
=
I
o
K
a
pC
b
(1 exp(K
a
pC
b
)) (1)
where the extinction coefcient of the biomass, K
a
, was
considered to be constant and equal to 0.1 m
2
/g.
332 M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342
Fig. 1. Scheme of outdoor photobioreactors used: (A) airlift tubular photobioreactor and (B) bubble column.
2.2. Analytical methods
Classic analytical methods were used for reference.
The number of cells in the cultures was determined in
a Neubauer counting chamber by direct observation on
an Olympus CH20 microscope. The biomass concen-
tration was determined by dry weight measurements.
For this purpose, 50 mL of culture was ltered through
a 0.45 mWhatmann lter, washed with 0.1NHCl and
oven-dried at 80

C for 24 h. Chlorophyll content was

determined by the method of Mackinney (1941), after
disruption of cells by sonication for 5 min and extrac-
tion with methanol at 40

C for 1 h. Total carotenoid

content was determined spectrophotometrically by the
method of Davies (1976), after disruption of cells by
sonication for 5 min and extraction with acetone for
3 h.
For pigment analysis by HPLCcells were ground in
a mortar with alumina and the extracts prepared in pure
acetone. To quantify astaxanthin, saponication was
performed, as follows. Acetone extracts were evapo-
rated under nitrogen gas and the residue re-dissolved
in pure ethyl ether. The same volume of KOHin MeOH
(2%, w/v) was added. The stirred mixture was allowed
to react for 15 min at 0

C in darkness under nitrogen

gas. To stop the reaction and to remove excess alkali,
10%NaCl was added. After separation of phases, ether
was evaporated under nitrogen gas and the remaining
pigment re-dissolved in pure acetone. Pigments were
analysed by the chromatographic method of Mnguez-
Mosquera et al. (1992), as modied by Del Campo
et al. (2000). Standards of astaxanthin, cantaxanthin
and -carotene were kindly provided by Hoffman-La
Roche, Switzerland. Violaxanthin was a gift from Dr.
L. Lubi an, Instituto de Ciencias Marinas, CSIC, Cadiz,
Spain. Lutein was fromSigma Chemical Co., St Louis,
MO, USA.
2.3. Fast characterization methods
For microscopic characterization of the cultures dig-
ital images of the samples were obtained by using
a CMOS camera (Evolution LC Color from Media
Cybernetics) mounted on the same microscope (Olym-
pus CH20). Images were analyzed by software using
Image-Pro Plus 4.5.1 from Media Cybernetics. This
type of software allows us to identify each cell in the
photo and to quantify the number of events, their mean
size and the standard deviation of sizes (Sabri et al.,
1997).
For macroscopic characterization of the cultures
a colorimetric method was used. The colour of the
M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342 333
Table 1
Cell density, biomass concentration and pigments content of biomass from cultures used for the calibration of the fast characterization methods
proposed
Culture Cell number (cell/mL) C
b
(g/L) Chlorophyll (%d.wt.) Carotenoid (%d.wt.) Astaxanthin (%d.wt.) Cell type
1 459000 0.25 5.00 1.00 0.20 Flagellated
2 819000 2.50 1.50 2.00 1.80 Cysts
3 1138500 0.63 4.52 0.24 0.00 Flagellated
4 3937500 1.79 5.90 0.50 0.13 Flagellated
5 2142000 1.88 3.84 0.72 0.43 Palmeloids
6 1206000 3.33 1.12 1.79 1.64 Cysts
7 3244500 1.79 5.90 0.50 0.13 Flagellated
8 1521000 1.33 4.00 1.02 0.61 Palmeloids
Determinations were performed by classical methods.
samples was determined using a CM-3500d Minolta
spectrophotometercolorimeter with Spectramagic 3.6
software (Minolta, Germany). For these measurements
a special glass cuvette (3 cm wide, 5 cm high, 1 cm
deep) was lled with 12 mL of sample and colour
parameters were immediately obtained, in addition to
transmittance at wavelengths from 400 to 700 nm. In
the agricultural and food industries the most popular
numerical colour-space system is the L*a*b*, which
is also referred to as the CIE-LAB system, originally
dened by the CIE in 1976 (CIE, 1986). In this colour
space, L* indicates the clearness, whereas a* and b*
are the chromaticity coordinates. The parameter a*
measures the red and green characteristics, whereas b*
measures the yellow and blue characteristics. Another
frequently used colour-space system is the L*C*h*.
Measurements were carried out with both colour-space
systems.
2.4. Statistical analysis
Statistical analysis of data was carried out using
Statgraphics version 7.0 software. Analysis of variance
and correlations between the variables were also cal-
culated using this software.
3. Results
Before carrying out the experiments in outdoor pho-
tobioreactors, the development of necessary character-
ization methods was needed. In this sense, two methods
were developed for the microscopic and macroscopic
fast characterization of the cultures. For this, eight
different batch cultures performed at laboratory scale
under different growth conditions were used. Data from
these cultures, measuredbyclassical methods, are sum-
marized in Table 1. The biomass concentration of the
cultures ranged from 0.25 to 3.33 g/L, whereas the
chlorophyll and carotenoid content ranged from 1.12
to 5.90%d.wt., and from 0.24 to 2.00%d.wt., respec-
tively. The cell density of the cultures ranged from
0.46 10
6
to 3.94 10
6
cell/mL, and cell morphol-
ogy varied from agellated cells to palmeloid cells,
and cysts.
For the calibration of the microscopic fast character-
ization method proposed, samples of cultures three to
six were used. Fromeach culture, aliquots were diluted
at ve levels, and four repetitions of each dilution were
measured, a total of 80 samples being quantied by
both the classical Neubauer counting chamber method
and the proposed digital image-analysis method. Anal-
ysis of the digital images of the samples allowed us to
determine not only the cell number, but also the aver-
age cell size as diameter and standard deviation of cell
size. Analysis of variance (Table 2) showed that the
culture sample was the major signicant variable, the
results yielded by the different images taken for each
sample showing no signicant difference. On the other
hand, the dilution ratio inuenced the results only at
high values of this variable. The analysis of the results
carried out at different dilution ratios showed that to
avoid the inuence of this variable the measurements
by digital image analysis must be performed at cell
densities higher than 10
5
cell/mL. Fig. 2 shows the
agreement of the classical Neubauer chamber counting
methodandmicroscopic fast characterizationbydigital
image analysis, verifying the adequacy of the proposed
methodology. Thus, characterization of overall batch
cultures by the proposed digital image analysis method
334 M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342
Table 2
Statistical analysis of data obtained from microscopic characterization of the cultures (n =80)
Cell number (cell/mL) Cell size (m) Standard deviation size (m)
F-ratio Signicant level F-ratio Signicant level F-ratio Signicant level
Culture 64.732 0.0000 53.132 0.0000 7.257 0.0003
Dilution 3.226 0.0174 3.669 0.0091 0.572 0.6838
Replica 0.107 0.9558 0.360 0.7817 0.012 0.9981
Inuence of independent variables (culture, dilution and replica) on the measured values of microscopic characterization. Signicant variables
are shadowed.
Fig. 2. Correlation between cell number measured by classical
Neubauer chamber counting method and digital image analysis
method, both in cells/mL.
is shown in Fig. 3. It can be observed that besides cell
density, the proposed methodology also allows us to
determine mean cell size and standard deviation of cell
size inside the culture. Data showed that both the cell
Fig. 3. Measurements obtained from digital image analysis of sam-
ples from batch cultures performed on different culture conditions.
size and standard deviation of cell size were lowest for
agellated cells (cultures 1, 3, 4 and 7) and highest
for cysts (cultures 2 and 6), cell size ranging from19 to
29 mand standard deviation ranging from5 to 12 m,
respectively.
Regarding the macroscopic fast characterization of
the cultures a colorimetric method was developed.
Colour measurements allow one to determine the
colour coordinates (L*, a*, b*, C*, h*) of each sam-
ple instantaneously, directly over the fresh culture. For
the evaluation of the colorimetric method samples of
all the cultures were used. Aliquots from each culture
were diluted at ve levels, and three repetitions of each
dilution were measured, making a total of 120 samples
quantied. Analysis of variance (Table 3) showed that
the culture was the only signicant variable; neither
culture dilution nor replica were signicant. In addi-
tion, colour coordinates L*, a* and C* showed higher
signicance levels, and were therefore selected as cor-
relation variables. In this sense, correlation analysis
of data (Table 4) showed how biomass concentration
correlated with the clearness index L*, whereas the
red coordinate a* correlated with the carotenoid and
astaxanthin concentration in the culture, and green
coordinate C* correlated with the chlorophyll con-
centration in the culture. Mathematical relationship
and tting between experimental and calculated values
(Fig. 4) proved that the macroscopic fast characteri-
zation method allows a fast quantication of culture
biomass and pigment concentrations in only a few sec-
onds, and directly using the fresh culture.
Once the fast characterisation methods were devel-
oped and veried, the behaviour of outdoor cultures of
H. pluvialis in pilot scale photobioreactors was stud-
ied. The photobioreactors used had the same culture
volume, 55 L, but very different designs. The bubble
column photobioreactor is characterised by a tube of
M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342 335
Table 3
Statistical analysis of data obtained from macroscopic characterization of the cultures (n =120)
L a* b* C h
F-ratio Signicant
level
F-ratio Signicant
level
F-ratio Signicant
level
F-ratio Signicant
level
F-ratio Signicant
level
Culture 105 0.0003 68.2 0.0356 32.2 0.0553 78.6 0.0235 16.3 0.0665
Dilution 6.53 0.0173 3.21 0.1456 1.66 0.2658 4.65 0.2256 1.23 0.2685
Replica 0.10 0.9668 0.23 0.9865 0.35 0.9859 0.16 0.9987 0.46 0.9866
Inuence of independent variables (culture, dilution and replica) on the measured values of macroscopic characterization. Signicant variables
are shadowed.
Table 4
Statistical analysis of data obtained from macroscopic characterization of the cultures
Parameter C
b
(g/L) Chlorophylls (mg/L) Carotenoids (mg/L) Astaxanthin (mg/L)
Coefcient
correlation
F-ratio Coefcient
correlation
F-ratio Coefcient
correlation
F-ratio Coefcient
correlation
F-ratio
L* 0.9345 227 0.7185 35 0.7120 2 0.6257 15
a* 0.7319 38 0.0935 1 0.9646 321 0.9618 295
C* 0.4881 10 0.9472 288 0.0087 1 0.1260 1
Correlation between spectrophotometric-colour parameters and measured variables of the samples. Shadow data corresponded to the best
correlation factor determined.
Fig. 4. Correlation between selected parameters and measured variables of the samples: (A) biomass concentration vs. L*; (B) chlorophylls
concentration vs. C*; (C) carotenoids concentration vs. a*; (D) astaxanthin concentration vs. a*.
336 M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342
Fig. 5. Experimental data from the discontinuous culture of H. pluvialis in the airlift tubular photobioreactor. The biomass concentration,
pigments content, cell density, cell size and deviation of cell size were determined according to fast characterization methods proposed.
large diameter (0.20 m) arranged vertically and with a
low surface/volume ratio (22 m
2
/m
3
). In contrast, the
airlift tubular photobioreactor is characterised by a tube
of small diameter (0.03 m) placed horizontally and with
a high surface/volume ratio (188 m
2
/m
3
). Both pho-
tobioreactors were inoculated with 10% of the same
inoculum, and the cultures were then operated in batch
mode for 16 days. Data from the airlift tubular photo-
bioreactor and the bubble column photobioreactor are
shown in Figs. 5 and 6, respectively.
Fig. 5 shows how, due to the immersion of the exter-
nal loop of the photobioreactor in a 15 cm deep water
pool panted white, the irradiance impinging on the
reactor surface was higher than that measured on a hor-
izontal plane, with mean values in the daylight period
of 1600 E/m
2
s. In spite of the high irradiance, the
culture was not photoinhibited, as conrmed by val-
ues of chlorophyll uorescence above 0.6 regardless
of the mean daily irradiance on the reactor surface.
In this way, the biomass concentration increased up
to 7.0 g/L after 16 days, including the lag phase. The
maximum growth rate during the exponential phase
was 0.040 h
1
, whereas the overall biomass produc-
tivity was 0.41 g/Lday. Concerning the pigment con-
tent, accumulation of carotenoids took place during
the rst 9 days, the carotenoid content increasing from
0.5 to 1.0%d.wt., due to high irradiance on the reac-
tor surface. However, the major accumulation took
place when the nitrate concentration decreased below
5 mM, when maximumcarotenoid values of 2.0%d.wt.
were recorded. In contrast, the chlorophyll content
was high under nitrate saturation conditions, with a
mean value of 1.8%d.wt. but decreasing to values
of 0.5%d.wt. when accumulation of carotenoids took
place due to nitrate limitation (after day 9). Concerning
the microscopic characterisation of the cultures, data
showed that the cell density increased in similar fash-
ion to biomass concentration up to 4.5 10
6
cell/mL
M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342 337
Fig. 6. Experimental data from the discontinuous culture of H. pluvialis in the bubble column photobioreactor. The biomass concentration,
pigments content, cell density, cell size and deviation of cell size were determined according to fast characterization methods proposed.
on day 9, whereas the cell size and standard devi-
ation of cell size remained constant with values of
21 and 5 m, respectively. However, under conditions
of nitrate limitation, cell division diminished and cell
density remained constant in spite of higher biomass
concentration. This was due to encystment of the cells,
which became red, the cell size and standard devia-
tion of cell size increasing to values of 32 and 9 m,
respectively.
A different behaviour was observed from data of
the bubble column photobioreactor (Fig. 6). Due to the
vertical arrangement the irradiance on the reactor sur-
face was lower than measured on a horizontal surface;
maximum mean values of 425 E/m
2
s were recorded
in daylight. The lowirradiance protected the culture by
preventing photoinhibition, with values of uorescence
of chlorophylls above 0.6 whatever the irradiance, but
it also reduced the growth and biomass concentration
of the culture. Thus, a maximum biomass concentra-
tion of 1.4 g/Lday and a maximum growth rate on
the exponential phase of 0.021 h
1
were recorded.
The overall biomass productivity of the culture was
low, 0.06 g/Lday. This imposed a low nitrate uptake
rate, and so the nitrate concentration of the culture
slowly decreased from the initial value of 20.0 mM
to a nal value of 16 mM, meaning that the culture
was always nitrate saturated. This fact, along with
the low irradiance on the reactor surface explains that
the chlorophyll and carotenoid content of the biomass
remained roughly constant, with mean values of 1.7
and 0.5%d.wt., respectively. Cell size and standard
deviation of cell size remained constant, with val-
ues of 18 and 4 m. The cell density increased from
0.5 10
6
to 2.5 10
6
cells/mL as the biomass con-
centration increased, but cells remained as small green
agellated vegetative cells.
338 M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342
Fig. 7. Variation of astaxanthin content of the biomass during the
discontinuous culture of H. pluvialis on tubular photobioreactor and
bubble column used. Data measured according to fast characteriza-
tion method proposed.
Differences between the reactors also determine
differences in the astaxanthin accumulation of the
biomass. Reactor shape is the only factor that explains
why the astaxanthin content of the biomass from the
bubble column photobioreactor only increases slightly
from 0.10 to 0.25%d.wt., as opposed to an increase
from 0.10 to 1.1%d.wt. in the airlift tubular photo-
bioreactor (Fig. 7). The higher astaxanthin content in
the biomass from the tubular photobioreactor is also
related, as in the case of total carotenoids, to the higher
irradiance and the depletion of nitrate in this reactor. It
is also important to note that the major accumulation
of astaxanthin took place in the tubular photobioreactor
when nitrate was depleted.
To quantify the inuence of design of the reactor in
the light availability and the behaviour of the cultures,
the average irradiance to which the cells were exposed
inside the culture was calculated. Fig. 8 shows how
the biomass productivity increased with the average
irradiance up to 130 E/m
2
s, and then decreased due
to the low biomass concentration inside the cultures.
However, the average irradiance was 10 times higher in
the tubular photobioreactor than in the bubble column,
due to the different arrangement and tube diameter of
both photobioreactors. In this sense, the low average
irradiance inside the bubble column, with maximum
values of 70 E/m
2
s, determines that the growth rate
was much lower than the specic maximum growth
rate, and that no accumulation of astaxanthin takes
place in this reactor. In contrast, the high average irra-
Fig. 8. Inuence of the average irradiance inside the culture on the
daily biomass productivity of H. pluvialis cultures on discontinuous
mode, in both tubular and bubble column photobioreactors.
diance inside the tubular photobioreactor, with values
of up to 600 E/m
2
s, indicates that this reactor can be
used to maximise the production of both biomass and
astaxanthin.
Finally, in order to verify the data obtained by using
the fast characterisation methods, a comparison was
performed with data measured using classic methods.
Fast microscopic characterisation was demonstrated
to reproduce the experimental data measured using
the Neubauer chamber counting method, as conrmed
with a statistical procedure analogous to Fig. 2 (not
shown). Concerning the macroscopic characterisation,
Fig. 9 summarises the agreement of measurements
of biomass concentration and pigment content carried
Fig. 9. Fitting between experimental values of biomass concentra-
tion and pigments content measured by classical methods and using
fast method proposed.
M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342 339
out using classical methods and using the proposed
fast method. It is clear that the proposed methodology
allows us to quantify the biomass concentration and
pigment content of the biomass, and although some
errors were over 15%, residues are unscrewed, and the
methodology is, therefore, useful for determining the
behaviour of the cultures.
4. Discussion
In order to produce natural astaxanthin from H.
pluvialis at competitive cost the production must
take place in outdoor conditions and using optimally
designed processes. The core of the production process
is the photobioreactor to be used, and it is therefore
necessary to determine the inuence of the reactor
geometry on the yield of the process. In addition,
outdoor cultures of H. pluvialis are characterised
by fast variations of biochemical composition and
cell morphology in response to variations of culture
conditions, such as irradiance or nitrate concentration.
The availability of fast characterisation methods
can therefore contribute to the optimisation of this
process.
In this sense, the production of astaxanthin from
H. pluvialis cultures is currently based on a two-step
process (Olaizola and Huntley, 2003), in which the
biochemical composition, morphology and wall of the
cells undergo considerable changes. Thus, the determi-
nation of the culture status is not complete if the usual
practice of a single microscopic or macroscopic quan-
tication is carried out (Harker et al., 1996; Tripathi
et al., 1999; Gong and Chen, 1998). Moreover, the
methods for the macroscopic characterisation of the
cultures vary greatly from one cell type to another,
mainly because the strengthened cell wall membrane
that appears in certain cell forms requires the use of
special cell wall disruption systems such as grounding
alumina (Del Campo et al., 2000). To further compli-
cate matters, the changes in cell morphology are fast
and can take place all of a sudden induced by different
stress factors such as temperature, light, nutrients, etc
(Boussiba and Vonshak, 1991; Borowitza et al., 1991;
Tjahjono et al., 1994; Harker et al., 1996; Cordero et al.,
1996; Sarada et al., 2002). Thus, for the optimal man-
agement of these processes and for the enhancement or
improvement of new processes, a fast analysis method
of cell status and composition are very convenient. In
this sense, the use of digital image software and col-
orimeters for the microscopic and macroscopic char-
acterisation of the cultures has been proved adequate
in the present work. Digital image analysis software
is nowadays more accessible and commonplace in the
laboratory, though more scarce on an industrial scale.
Colorimeters are usually used in the quality control of
processes related with paints, ink, etc., but they have
also been used extensively in food technology for meat
(Wulf and Wise, 1999), coffee (Ortola et al., 1998),
tomato (Arias et al., 2000), etc.
Regarding the microscopic characterisation, the
cycle and reason why the different cell types mod-
ify the others is not clear (Kobayashi et al., 1997).
A simplied representation of the population consist-
ing of three basic cell types (agellated, palmeloid and
cyst) is usually accepted, although transitions and vari-
ations of these types are also referenced (Kobayashi
et al., 1997). However, most researchers only make a
qualitative characterisation of the cultures indicating
only the cell number and predominant cell morphol-
ogy. The microscopic method developed in this work
makes this characterisation quantitative, measuring the
cell number and also the average cell size and its stan-
dard deviation, providing a more accurate description
of homogeneity of the cell population. This is pre-
sented in Fig. 3, where the agellated cells are shown
to have a mean diameter of 20 m, while palmeloid
cells are larger, with a diameter of 25 m, and cysts
are larger than 30 m. Regarding the homogeneity of
the population, it can also be observed that the agel-
lated cells corresponding to fast growing young cells
are more homogeneous, whereas when the culture is
stressed cells of slow growth (palmeloids cells and
cysts) appear and the homogeneity of the population
decreases because some agellated and transition cell
types remain. Fromthese data the great variation of cell
weight between different cell types can also be quanti-
ed. They vary from500 pg/cell for agellated cells, to
1000 pg/cell for palmeloid cells, and even 3000 pg/cell
for cysts; a direct general relationship between cell
number and dry weight measurements cannot therefore
be established.
The development of fast characterisation methods
allows us to analyse the behaviour of H. pluvialis cul-
tures in outdoor conditions. The experiments carried
out highlight the importance of irradiance and nitrate
340 M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342
concentration on the growth and astaxanthin accumula-
tion of H. pluvialis. The irradiance or light availability
is a function of location and design of the reactor. The
use of small tube diameter horizontal photobioreac-
tors enhances the irradiance on the reactor surface,
thereby increasing the growth rate and the biomass
and astaxanthin productivity of the reactor. Thus, the
mean daily irradiance on the tubular photobioreactor
was 2.8 times higher than in the bubble column, and
the maximum daily biomass productivity in the tubu-
lar photobioreactor was 0.55 g/Lday, as opposed to
0.12 g/Lday measured in the bubble column. The bub-
ble column was strongly light limited, the maximum
biomass productivity in this reactor was measured at
the maximum average irradiance of 70 E/m
2
s. On
the other hand, the tubular photobioreactor was light
saturated, and the maximum biomass productivity of
0.55 g/Lday was measured at an optimal average irra-
diance of 130 E/m
2
s. Thus, the biomass yield in
light was higher in the bubble column, 2.15 g/E, than
in the tubular photobioreactor, 0.61 g/E, although the
biomass productivity was higher in the latter. The
productivity measured was higher than those refer-
enced for the production of biomass under autotrophic
conditions, 0.065 g/Lday (F abregas et al., 2001), het-
erotrophic conditions, 0.17 g/Lday (Hata et al., 2001),
mixotrophic conditions, 0.20 g/Lday (Zhang et al.,
1999), and also higher than had been reached for this
step on an industrial scale, 0.052 g/Lday (Olaizola,
2000). Similar biomass productivityof 0.58 g/Ldayhas
only been referenced in the laboratory under high irra-
diance conditions (Garca-Malea et al., in press). As
regards astaxanthin, productivity in the tubular pho-
tobioreactor was 4.5 mg/Lday, i.e. higher than those
reported by Lee and Ding (1995) (0.1 mg/Lday); Lee
and Soh (1991) (3.5 mg/Lday); and Olaizola (2000)
(2.2 mg/Lday), obtained under different stress condi-
tions, but lower than the 5.6 mg/Lday achieved under
continuous culture (Del Ro et al., 2005). The higher
light availability also favoured the accumulation of
carotenoids/astaxanthin in the biomass fromthe tubular
photobioreactor. Under nitrate saturation conditions,
carotenoids were accumulated due to high irradiance
on the reactor surface, but the major accumulation
took place when the nitrate concentration decreased
below 5 mM. Stimulation by nitrogen starvation of
astaxanthin accumulation in Haematococcus cells has
received considerable attention (Borowitza et al., 1991;
Boussiba et al., 1999; Boussiba and Vonshak, 1991;
F abregas et al., 2001; Lee and Soh, 1991; Orosa et
al., 2005). Moreover, some studies on the inuence
of other stress factors such as light, acetate, dissolved
oxygen, pH, salinity, etc. (Kobayashi et al., 1992;
Orosa et al., 2001) concluded that they might indi-
rectly affect nitrogen consumption by the cells. The
data reported in the present work support the cumu-
lative character of both factors in the induction of
astaxanthin accumulation as has recently been reported
(Del Ro et al., 2005). Moreover, synthesis of astax-
anthin in Haematococcus is accompanied by morpho-
logical and biochemical changes (Boussiba, 2000).
Our results show that cell weight increases six-fold
from agellated cells to astaxanthin-rich cyst cells.
This value is similar to previously reported four-fold
increases in cell size from small green motile cells
to large red resting haematocysts, induced by severe
stress conditions (Boussiba and Vonshak, 1991), and
larger than the mere 40% increase reported under
continuous operation (Del Ro et al., 2005). These
large differences between batch and continuous oper-
ation demonstrate the inuence of culture conditions
and the importance of a complete characterisation of
the cultures on both microscopic and macroscopic
scale.
In short, this work demonstrates that the two anal-
ysis methods proposed allow in-depth characterisation
of Haematococcus pluvialis cultures whatever the mor-
phology of the cells. This characterisation is fast and
provides both the macroscopic and microscopic data of
the culture. Fast characterisation methods allow analy-
sis of the behaviour of fast changing outdoor cultures
of H. pluvialis. Tubular photobioreactors are shown
to be preferable to bubble columns for the outdoor
production of biomass and/or astaxanthin. For the pro-
duction of biomass the nitrate concentration must be
kept higher than 5 mM, producing agellated cells of
20 m diameter. For the massive accumulation of
astaxanthin the nitrate concentration must be lower
than5 mM. Under nitrate depletionconditions cell divi-
sion stops and astaxanthin content increases, and rest-
ing cells as large as 30 m diameter are obtained. The
continuous production of astaxanthin under lownitrate
concentrations, ranging from 2 to 5 mM has recently
been referenced (Del Ro et al., 2005), and the tubular
photobioreactor is the most adequate photobioreactor
to perform this operation in outdoor conditions.
M.C.G.-M. L opez et al. / Journal of Biotechnology 123 (2006) 329342 341
Acknowledgements
This research was supported by the Ministerio
de Ciencia y Tecnologa (PPQ 2001-3822-C02-02;
PPQ2001-3822-C02-01) and Junta de Andaluca, Plan
Andaluz de Investigaci on III (CVI 173, CVI 263).
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