Research Article

Received: 9 August 2010 Revised: 29 September 2010 Accepted: 1 October 2010 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.2532

Comparison of different cultivation modes

and light intensities using mono-cultures
and co-cultures of Haematococcus pluvialis
and Chlorella zofingiensis
Suphi S. Oncel,∗ Esra Imamoglu, Emre Gunerken and Fazilet Vardar Sukan

Abstract
BACKGROUND: Recent studies indicate that microalgal cultivation using organic carbon sources has the potential to provide
high yields. Haematococcus pluvialis and Chlorella zofingiensis, two important carotenoid producers, were selected for co-culture
cultivations to utilize the unique advantages of both organisms. A co-culture production process was investigated in terms of
the effects of organic carbon source, co-cultivation method, and light intensity on carotenoid production.

RESULTS: The addition of 5 g L−1 glucose resulted in a growth rate of 0.60 day−1 for H. pluvialis and 0.59 day−1 for C. zofingiensis,
which were higher than those for other carbon sources tested and the control group. Incremental increase of light intensity
instead of direct increase to 170 µE m−2 s− prevented cell loss in both cultures. Co-cultivation based on cell numbers (60%
H. pluvialis and 40% C. zofingiensis) prevented population domination of one microalgae over the other. The biomass production
rate of the co-culture was higher (0.61 g L−1 day−1 ) in glucose-enriched medium. The total carotenoid content of the co-culture
in the control culture was higher (0.83 mg total carotenoids g−1 cell) than that obtained in glucose-enriched medium (0.54 mg
total carotenoids g−1 cell) but not as high as the amounts reached in mono-cultures.

CONCLUSION: Total carotenoid content of the mono-cultures gave higher yields in standard bold basal medium (BBM).
Preliminary high performance liquid chromatography (HPLC) studies indicated a variation in the amounts of astaxanthin
isomers produced. Further studies are in progress to determine the effects of carbon-enriched media and co-cultivation on the
type of isomers and caretenoids produced.

c 2010 Society of Chemical Industry

Keywords: algae; bioprocesses; biomass; co-culture

INTRODUCTION
Microalgae are unique and valuable microorganisms containing
chlorophyll and other photosynthesis-related pigments such as
carotenoids, which enable them to absorb and utilize CO2 as
principal carbon source in the growth process.1 The red keto-
carotenoid astaxanthin surpasses the antioxidant activity of other
carotenoids such as β-carotene, zeaxanthin, and vitamin E. Thus,
the pharmaceutical and cosmetic industries are increasingly inter-
ested in astaxanthin since it can, for instance, protect against the
damaging effects of ultraviolet radiation and chemically induced
cancers, and also enhance the immune system. Owing to the high
market price of synthetic astaxanthin, efforts have been made
to use microorganisms such as the green algae Haematococcus
pluvialis, Chlorococcum sp., Chlorella vulgaris or the heteroba-
sidiomycete yeasts Xanthophyllomyces dendrorhous (anamorph:
Phaffia rhodozyma) as a natural source of the product.2,3,4,5,6
Compared with other astaxanthin-producing microorganisms,
Haematococcus pluvialis and Chlorella zofingiensis have the
potential to accumulate large amounts of astaxanthin because
they possess sequestering systems for storing astaxanthin in lipid
bodies.7,8 H. pluvialis has been used commercially to produce
natural astaxanthin, whereas C. zofingiensis has only recently
attracted attention due to its high growth rate under various
conditions with minimal negative environmental effects.9,10
Furthermore, C. zofingiensis can grow and produce astaxanthin
without light when exogenous glucose is supplemented.11,12
High carbon to nitrogen ratios have been suggested to induce
astaxanthin biosynthesis because nitrogen limitation in the
presence of excess organic carbon sources such as acetate and
glucose has proven effective in enhancing astaxanthin production
in mixotrophic cultures.11,13
Mixed cultures of microorganisms are common in ecosystems.
In recent years, exploration of mixed cultures has become critical to
many key biochemical processes. Mixed cultures have been used in
various applications, such as complementary bio-transformations,
multi-step bio-transformations, in situ enzyme regeneration, in situ
oxygen generation, waste degradation and waste remediation.
∗ Correspondence to: Suphi S. Oncel, Ege University, Faculty of Engineering,
Department of Bioengineering, Izmir, 35100, Turkey.
E-mail: suphi.oncel@ege.edu.tr
Ege University, Faculty of Engineering, Department of Bioengineering, Izmir,
35100, Turkey

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 www.soci.org
Co-cultures are similar to mixed cultures but there is a unique
difference in cultivation. In co-cultures the quantity and type
of organisms in the culture are all defined at inoculation
whereas in naturally occurring mixed cultures, different organisms,
depending on culture conditions, may become dominant during
the cultivation period.
The main objectives of this work were: (a) determination of the
optimum organic carbon ratio; (b) selection of the carbon source;
(c) determination of the co-cultivation method based on biomass
and cell-count data; (d) to investigate the effect of gradually
increasing light intensity on mono-cultures of H. pluvialis and C.
zofingiensis in organic carbon enriched culture media; and (e) to
investigate the effect of light intensity and organic carbon source
on the co-cultivation of H. pluvialis and C. zofingiensis to produce
higher carotenoid yields.
MATERIALS AND METHODS
Seed culture conditions
Haematococcus pluvialis and Chlorella zofingiensis obtained from
Ege University Bioengineering Department Microalgae Culture
Collection (EGE-MACC) were grown photoautotraphically in bold
basal medium (BBM).1 Cultures were grown in 250 mL Erlenmayer
flasks containing a culture medium of 100 mL at 100 rpm. The
temperature was maintained at 25 ± 1 ◦ C and the cultures were
illuminated continuously with cool white fluorescent lamps at
30 µE m−2 s−1 intensity. All light intensities in the experiments
were measured with a LI-250 quantum photometer (Lambda
Instrument Corp., USA) at the culture surface.
After cells reached their mid-logarithmic phases (determined
spectrophotometrically), they were transferred to bubble column
photobioreactors (PBRs) of 5 cm diameter and 500 mL culture
volume. Cultures were aerated with 1 vvm CO2 (1% v/v) enriched
air mixture under the same culture conditions as the Erlenmeyer
cultivations. Cells in the bubble column PBRs were transferred
according to the planned experiments using a 10% inoculation
ratio.
Batch cultivations for the determination of the organic carbon
source ratio
Cultures were grown in 250 mL Erlenmayer flasks with 100 mL
culture volume. Glucose was used as organic carbon source.
Glucose was added to BBM media at ratios of 3, 5 and 10 g L−1 .
Cultures were grown photomixotrophically under 30 µE m−2 s−1
illumination, at 25 ± 1 ◦ C with 100 rpm orbital shaking. Results of
cell count, biomass and carotenoid yields obtained were compared
with those of the control group cultivated using standard BBM.
Batch cultivations for selection of the organic carbon source
Subsequent to the determination of the glucose ratio, glycerol
and xylose were tested as alternative carbon sources. The ratios
of the alternative carbon sources were calculated based on equal
numbers of molecular carbons using the optimum glucose ratio
identified in previous experiments. Cultures were cultivated in
500 mL bubble PBRs under 30 µE m−2 s−1 light intensity and at
25 ± 1 ◦ C, aerated using 1 vvm air. The control group was not
supplemented by an organic carbon source.
Batch cultivations for the selection of optimum co-cultivation
ratio
Bubble column PBR cultures were transferred to 500 mL Er-
lenmayer flasks with 300 mL working volume containing BBM.
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J Chem Technol Biotechnol (2010)
SS Oncel et al.
Cultures were grown phototrophically, under 30 µE m−2 s−1 light
intensity, at 25 ± 1 ◦ C and at 100 rpm orbital shaking. The co-
cultivation ratio was determined using two different parameters,
namely biomass content and number of cells.
Experiments based on biomass content were carried out using
seed cultures with a final biomass concentration of 2 g L−1 at
logarithmic phase of growth from both cultures and the co-
cultures were inoculated at C. zofingiensis : H. Pluvialis (C : H) ratios
of 20 : 80, 30 : 70, 40 : 60 and 50 : 50.
Similarly, experiments based on cell number were carried out
using seed cultures with a final cell concentration of 4 ± 0.5 cell
L−1 and the co-cultures were inoculated mixed at ratios of 40 : 60,
50 : 50 and 60 : 40 C : H.
Batch cultuvations of H. pluvialis and C. zofingiensis under
incremental increase of light intensity, using organic carbon
enriched culture media
Cultures of H. pluvialis and C. zofingiensis were grown in standard
BBM in 500 mL bubble column PBRs with an aeration rate of 1
vvm under a light intensity of 30 µE m−2 s−1 at 25 ± 1 ◦ C. The
light intensity of the cultures was increased stepwise to 60 µE
m−2 s−1 , 85 µE m−2 s−1 , and 115 µE m−2 s−1 every 2 days, reaching
a maximum intensity of 170 µE m−2 s−1 on the 12th day to trigger
light stress. Glucose was added to standard BBM as organic carbon
supplement at a ratio of 5 g L−1 ).
Co-culture of H. pluvialis and C. zofingiensis under incremental
increase of light intensity, using organic carbon enriched
culture media
Co-culture experiments were inoculated using 60% H. pluvialis
to 40% C. zofingiensis (cell number approach), to 500 mL bubble
column PBRs at aeration rates of 1 vvm under a light intensity of
30 µE m−2 s−1 at 25 ± 1 ◦ C. Glucose was added to standard BBM
at a ratio of 5 g L−1 . Light intensity was increased stepwise up to
170 µE m−2 s−1 . Results obtained were compared with those of
the standard BBM culture as control.
Analytical methods
Dry weight: Daily samples, 5 mL for dry biomass determination
and 1 mL for cell count and turbidity measurements, were taken
from the cultures. Fresh medium was added to the culture to
compensate for the volume loss following sample removal. Dry cell
weight was determined by filtering the aliquots on pre-weighed
GF/C filter paper (Whatman, UK). The filtered cells were dried at
105 ◦ C until constant weight was obtained and were cooled to
room temperature in a desiccator before weighing.
Cell density: Cell growth was monitored by measuring the optical
density at 560 nm with a UV–Vis spectrophotometer (Jenway
6400; Staffordshire, UK).
Cell count: Each microalgal cell was counted under the microscope
with a Neubauer hemocytometer (Wertheim, Germany).
Cell desruption: 5 mL of culture broth was harvested by centrifuga-
tion at 2000g, for 5 min; the pellet obtained was frozen at −20 ◦ C
and then lyophilized. 2 mg freeze-dried biomass was placed in a
2 mL eppendorph containing 1 mL acetone and was sonicated for
cell disruption to release intracellular biological molecules, using a
sonicator (Bandelin Electronic UW 2070, Berlin, Germany) for 30 s
at 0.1 s intervals. The dismembrator had a maximum power out-
put of 180 W and was operated at a constant frequency of 20 kHz.
The horn was placed at the center of the tube. Sonication was
Micro-algal ultivation using mono-cultures and co-cultures www.soci.org

Table 1. Change in growth rate and biomass productivities of the processes

Growth rate (day−1 ) Biomass production rate (g L−1 day−1 )

Glucose Haematococcus Chlorella Haematococcus Chlorella

concentration pluvialis zofingiensis pluvialis zofingiensis

Control 0.22 0.22 0.22 0.16

3% 0.38 0.37 0.57 0.49
5% 0.40 0.39 0.69 0.72
10% 0.42 0.39 0.69 0.69

Haematococcus Chlorella Haematococcus Chlorella

Carbon sources pluvialis zofingiensis pluvialis zofingiensis

Control 0.36 0.31 0.13 0.11

Glycerol 0.47 0.55 0.42 0.42
Glucose 0.60 0.59 0.53 0.43
Xylose 0.48 0.44 0.28 0.36

Increasing light Haematococcus Chlorella Haematococcus Chlorella

intensity pluvialis zofingiensis pluvialis zofingiensis

Control 0.43 0.44 0.29 0.27

Glucose 0.73 0.60 0.56 0.49

carried out in an ice-water bath to prevent undesired temperature
increases affecting product quality.
Total carotenoid content: Determined using a modified method
from Mendes-Pinto et al.14 A suspension containing 2 mg biomass
and 1 mL acetone (Carlo Erba (Italy) %99) was agitated for 3–4 min
in a vortex and was left for 16 h at room temperature. The cell debris
was removed by centrifugation prior to analysis. The absorbance
of the combined extracts was measured at 480 nm.
RESULTS AND DISCUSSION
Effects of carbon source concentration
Although previous studies have shown that full and efficient
glucose metabolism is very important for algal cultivation,15 the
effects of different carbon concentrations on microalgal growth
have not been studied. Since glucose, as a low cost carbon
source, is a widely used monosaccharide it was selected as carbon
supplement for the experiments. BBM was prepared by adding
different concentrations of glucose (3, 5 and 10 g L−1 ) and the
cultures of both microalgae were cultivated for 11 days. During
cultivation of H. pluvialis, the addition of glucose showed a net
increase in biomass production rate when compared with the
control group having zero glucose (Table 1). A positive correlation
between C-metabolism and cell production was observed. As
expected the best result was obtained with the BBM supplemented
with 10 g L−1 glucose, since the mixotropic metabolism yields
faster cell growth and maintenance. On the other hand, the
5 g L−1 concentration had a better performance than the 3 g L−1
concentration. Based on these results, 5 g L−1 glucose supplement
was selected for the other experiments, since it yielded high growth
rates, approaching that of the 10 g L−1 concentration values, while
possessing lower contamination risks.
Similar results were obtained with the C. zofingiensis cultures.
C. zofingiensis, reached a maximum biomass production rate of
0.72 g L−1 day−1 with 5 g L−1 addition of glucose medium, which
was 4% (g L−1 ) higher than in 10 g L−1 glucose concentration
(Table 1). Thus, a similar trend in favour of glucose addition was
seen with 5 g L−1 glucose concentration yielding optimum values
in both biomass and growth rates.
The results obtained were parallel to the findings of Liang et al.,16
where low dose additions were reported to be more effective than
high doses for C. vulgaris biomass production.
Effects of various carbon sources
Subsequent to experiments with glucose supplement, other
alternative carbon sources were tested on the basis of equivalent
moles of carbon using bubble column PBRs. 5 g L−1 glucose
equivalent concentrations of glycerol and xylose showed again
a net increase in both microalgae compared with results using
standard BBM. In C. zofingiensis cultivation, xylose with five carbon
atoms had a lower effect on production than the other carbon
sources, while glycerol-added cultures showed similar behaviour
to glucose-added cultures. Similar biomass production rate values
were obtained with H. pluvialis cultures for all carbon sources
(Table 1). The difference in growth rate and biomass production
rates observed between Erlenmeyer and PBR cultivations is
due to the more homogeneous growth in PBRs. However, a
faster change in the color of the cells was observed for both
glucose- and glycerol-added cultures, compared with xylose-
added media and the control culture. The color change was related
to the triggering of carotenoids, presenting an advantage of
carbon supplementation. In heterotrophic production, the amount
of astaxanthin accumulation can be increased by metabolic
stress. Ip and Chen11 reported that C. zofingiensis synthesized
higher concentrations of astaxanthin in the dark. Growth and
carotenogenesis on glucose is also affected by light intensity. The
maximum biomass productivity of C. vulgaris grown on 1% glucose
was 0.254 g L−1 day−1 with light and was 0.151 g L−1 day−1 in the
dark over 6 days.16 It was observed that light-stimulated growth
yielded higher biomass and lipid productivities.
Selection of co-culture inoculum ratios
The microalgae cultures were mixed to form a co-culture in two
different ways. The first approach based on cell biomass ratio

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 www.soci.org SS Oncel et al.

900 30 30
C6
800
C8 25 H5

Cell Count (nx10^4/ml)

H2
25
700
Cell count (nx10^4/ml)

H4 C5
600 20
20
500
15
400
15

300 10
10
200
5
100
5
0 0
0 1 2 3 4 5 6 7 8 9 10
0
0 2 4 6 8 10 12
Time (day)
Time (day)
Figure 1. Cell count (×104 cell mL−1 ) variation of the co-cultivation in the
ratios of H. pluvialis (20% and 40%) to C. zofingiensis (80% and 60%) under Figure 3. Cell count (×104 cell mL−1 ) variation of the co-cultivation in the
light intensity of 30 µE m−2 s−1 using biomass approach. ratios of 50% H. pluvialis to 50% C. zofingiensis under light intensity of 30 µE
m−2 s−1 using cell numbers approach.

900 30 30
C2 H6
800
C4 25
H6 25 C4

Cell Count (nx10^4/ml)

700
Cell count (nx10^4/ml)

H8
600 20
20
500
15
400 15
300 10

200
10
5
100
5
0 0
0 1 2 3 4 5 6 7 8 9 10
Time (day)
0
0 2 4 6 8 10 12
Figure 2. Cell count (×104 cell mL−1 ) variation of the co-cultivation in the Time (day)
ratios of H. pluvialis (60% and 80%) to C. zofingiensis (40% and 20%) under
light intensity of 30 µE m−2 s−1 using biomass approach. Figure 4. Cell count (×104 cell mL−1 ) variation of the co-cultivation in the
ratios of 60% H. pluvialis to 40% C. zofingiensis under light intensity of 30 µE
m−2 s−1 using cell numbers approach.

aimed to prevent the domination of larger Haematococcus cells

with higher mass over the smaller Chlorella cells with lower mass.
The change in cell numbers was monitored during production by
cell counts of daily samples under the microscope. The cells were
mixed at ratios of 20% to 80% (g g−1 ) individually to give a total
biomass of 2 g L−1 at the start of the culture. Chlorella cells are
smaller than Haematococcus cells, resulting in smaller cell biomass.
Thus, C. zofingiensis dominated the co-culture from the start of
production, due to the larger number of cells and this domination
existed at all ratios (Figs 1 and 2). Therefore this approach was not
suitable for the initiation of co-cultivation.
The second approach was based on number of cells, aiming to
maintain a constant cell count ratio to prevent the domination of
one microalgae over the other. A cell count of 5 × 104 cells mL−1
was selected for Haematococcus as the inoculation concentration
for the co-culture. Based on the biomass experiments, the mixing
ratio was changed to 50–70%. During 20 days of cultivation, slight
domination of Chlorella cells over Haematococcus was observed
(Figs 3 and 4). When the ratios of microalgae were examined
separately the trend was clearer. The growth rates of Chlorella were
higher than Haematococcus. This may be attributed to the stress
imposed on Chlorella due to co-existence with a high biomass
opponent having the same number of cells. However, despite
this disadvantage Chlorella reached higher cell numbers but with
higher oscillations. When Haematococcus cells were taken into the
consideration, the change in cell number was more steady.
These findings were valid at all other ratios tested (Figs 4 and 5).
When the morphological changes in the cells were investigated
under the microscope, Chlorella cells were observed to increase
their biomass in parallel with the changes in Haematococcus cells.
The cell numbers started to decrease after the sixth day. This may
be due to increasing flocculation tendency and/or cystic formation
affecting accurate cell count measurements.
Although all the ratios showed more or less similar behaviour,
the numbers of cells of both microalgae were more or less equal
at a co-cultivation ratio of 60% Haematococcus to 40% Chlorella.
Therefore this ratio was selected for further experiments.
Effect of gradually increased light intensity
on the mono-cultures of H. pluvialis and C. zofingiensis
in organic carbon enriched culture media
Since previous findings17 indicated that growth on carbon-
enriched culture media is also affected by light intensity,
experiments were conducted to investigate the effects of changing
light intensities. The effect of a single step increase of light intensity
to 170 µE m−2 s−1 at the start of cultivation caused high stress in
both microalgae cultures, resulting in high cell loss after a few

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25 The addition of an organic carbon source not only prevents

H7
night biomass loss, but helps to achieve continuous cell growth
during light/dark cycles. The amount of organic carbon source
Cell Count (nx10^4/ml)

20
C3 added should be limited to that quantity which can be completely
metabolized during the night. Incomplete consumption of the
15
added organic carbon source during the night leads to mixotrophic
growth the following day, causing a decrease in the cellular content
10 of photosynthetic metabolites.19
In our studies, mono-culture of H. pluvialis exhibited similar
5 results. Glucose-enriched BBM had a superior effect on growth
compared with the control. The maximum cell number obtained
0 with H. pluvialis was about 33 × 104 cells mL−1 on day 14 (Fig. 6).
0 2 4 6 8 10 12 The increase in H. pluvialis population was lower than that of
Time (day) C. zofingiensis. The biomass production rate of H. pluvialis was
0.56 g L−1 day−1 compared with C. zofingiensis (0.49 g L−1 day−1 )
Figure 5. Cell count (×104 cell mL−1 ) variation
of the co-cultivation in the in glucose-enriched medium (Table 1). Therefore, glucose was
ratios of 70% H. pluvialis to 30% C. zofingiensis under light intensity of 30 µE
observed to give better results when used as an additional organic
m−2 s−1 using cell numbers approach.
carbon source.
The stepwise increase of light intensity affected the cells first
200 45
in cell numbers and then in pigment content, as observed from
colour change in the cells. The first 6 days of cultivation could
180 Cc 40
Cg be considered as an adaptation stage for both strains. This
160 35
Hc period exhibited a distinct increase in biomass indicating speedy
Cell count (nx10^4/ml)

140 Hg
120
100
80
60
40
20
0 0
0 1 2 3 4 5 6 7 8 9
Time (day)
Figure 6. Cell count (×104
cell mL−1 ) variation of the mono-cultivations
of H. pluvialis and C. zofingiensis in control culture medium (c) and in
glucose enriched culture medium (g) under the stepwise increment of
light intensity.
days. Therefore light intensity was increased gradually to prevent
this problem.
The light intensity was increased incrementally every 2 days to
obtain a constant increase in cell number during mono-cultures
of H. pluvialis and C. zofingiensis using organic carbon enriched
culture media in bubble column PBRs. In the mono-culture of C.
zofingiensis no significant change in cell number was observed
during the first 6 days. As shown in Fig. 6, the carbon-enriched
BBM were more effective than the control. After the sixth day, an
increase in cell numbers was observed reaching a maximum value
of 151 × 104 cells mL−1 in glucose-enriched BBM compared with
that of the control group (130 × 104 cells mL−1 ). The biomass
production rate of C. zofingiensis in glucose-enriched medium was
0.49 g L−1 day−1 .
At high light intensities, heterotrophic growth is inhibited and
aerobic respiration in the cell is reduced, whereas at low light
intensities, the demands of growth and respiration are satisfied
by glucose supplementation.17 The specific glucose consumption
rate increases when reducing the light intensity. When the organic
substrate is completely consumed, an autotrophic phase follows
subsequent to an adaptation period. The results of other workers18
also show that it is advisable to add glucose only to light-limited
cultures.
30 utilization of carbon sources. After the sixth day, results similar
25 to constant light intensity experiments were observed. At this
stage, the cells gained more weight, divided continuously and did
20
not flocculate. The constant light intensity did not create suitable
15
conditions for the cultures to continue growth, but the stepwise
10
increase of light intensity helped the cultures to compensate for
5 extra carbon requirements.
The variation in total carotenoid concentration of the cultures
10 11 12 13 14 was also investigated. The total carotenoid value found in the
control BBM was higher than that in organic carbon enriched media
during the production of both microalgae under the stepwise
increase of light intensity regime. The carotenoid content of
H. pluvialis cells reached 1.50 mg total carotenoids g−1 cell while
C. zofingiensis cells reached 0.65 mg total carotenoids g−1 cell in
control cultures. On the other hand, the values were about 1 mg
total carotenoids g−1 cell and 0.3 mg total carotenoids g−1 cell for
glucose cultures of H. pluvialis and C. zofingiensis (Fig. 8).
As reported by Boussiba and Vonshak,20 astaxanthin (a
ketocarotenoid pigment) production by H. pluvialis can be induced
by modifying the environmental conditions. These researchers
obtained the best results with a light intensity of 170 µE m−2 s−1 ,
phosphate starvation and salt stress (NaCl 0.8%). It is suggested
that environmental or nutritional stresses, which interfere with
cell division, trigger the accumulation of astaxanthin. It has been
proposed that secondary metabolites might serve as storage in
order to protect the cell from biotic or abiotic stresses.11 When
the formation of cystic cells is desirable, both nutrient deficiency
and high light intensities were applied exploiting the resulting
strong synergistic effect.21 In a light-independent cultivation
system, compositions of organic compounds in the medium,
especially carbon and nitrogen may be the main factors affecting
carotenogenesis in the algal cells. Glucose might be essential for
providing the carbon skeleton for the formation of secondary
carotenoids including astaxanthin, in the carotenoid biosynthetic
pathway.11
These results seem to contradict our expectations to see a
positive effect of glucose addition on the total carotenoid amount.
Other workers have reported10,11,21 that during stress conditions
total carotenoids content of the cells, which reflects the net

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200 45
180 Cc* 40
Cg*
160 35
Hc*
Cell count (nx10^4/ml)
140 Hg* 30
120
25
100
20
80
15
60
40 10
5
20
0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (day)
4 −1
Figure 7. Cell count (×10 cell mL ) variation of the co-cultivations of H.
pluvialis and C. zofingiensis in control culture medium (c∗ ) and glucose-
enriched culture medium (g∗ ) under the stepwise increment of light
intensity.
change in the carotenoid level, may remain the same, while the
individual components (such as astaxanthin or other primary
and secondary carotenoids) may increase or decrease. Glucose,
a source of energy and a metabolism regulator, affects the level
of chlorophylls and carotenoids differently. Although the cell
dry weight concentration increases when glucose concentration
increases, the lutein, β-carotene, chlorophylls a and b contents
increase or decrease depending on the species and other culture
conditions, when glucose is added to the culture medium. This is
reported to be due to transcriptional activation.22,23,24
It is also reported that singular disturbances in the culture
medium such as addition of organic substrates or application of
high light intensities or severe nutrient deficiencies, during the
induction period, may result in immature cyst cells. Mature cyst
cells are obtained when both nutrient and light effects are applied
jointly because of their strong interaction.11,21,25 The presence of
organic substrates in the culture under stress conditions may have
caused the low total carotenoid accumulation.
Effect of gradually increased light intensity in co-culture
of H. pluvialis and C. zofingiensis in organic carbon enriched
culture media
During co-cultivation, the cells were mixed in ratios of 60%
H. pluvialis to 40% C. zofingiensis in 500 mL bubble column
PBRs. During the first 8 days, the cell numbers of H. pluvialis
were higher than those of C. zofingiensis, but subsequently the
growth rate of H. pluvialis was reduced and the cell numbers of
C. zofingiensis started to increase (Fig. 7). The cell count values
indicated that the results were in favour of glucose-enriched
media (Fig. 7). When compared with the control group (Fig. 7)
the cell count change of the co-culture exhibited a similar trend
for all experiments using glucose supplementation and standard
BBM. When the growth of mono-cultures and co-culture of H.
pluvialis and C. zofingiensis in glucose-enriched culture medium
were compared, the cell concentration was approximately 35%
higher in the co-culture. The biomass production rate of the co-
culture was 0.27 g L−1 day−1 under photoautotrophic conditions
and 0.61 g L−1 day−1 with enriched glucose medium.
H. pluvialis exhibits low growth rates and low final cell densities
under optimal growth conditions in the co-culture of H. pluvialis
and C. zofingiensis.25 In contrast to H. pluvialis, C. zofingiensis grows
very rapidly (approximately three times as fast as H. pluvialis,
wileyonlinelibrary.com/jctb c 2010 Society of Chemical Industry

J Chem Technol Biotechnol (2010)
SS Oncel et al.
1.8
Hc
mg total carotenoids / g cell
1.6
Cc
1.4
Hg
1.2
1 Cg
0.8 H+C (c)
0.6 H+C (g)
0.4
0.2
0
0 2 4 6 8 10 12 14 16
Time (day)
Figure 8. Total carotenoid (mg total carotenoids g−1 cell) variations of
the mono and co-culture of H. pluvialis and C. zofingiensis in control and
organic carbon enriched culture media under the stepwise increment of
light intensity.
reaching specific growth rates of 0.043 h−1 ) and accumulates
significant amounts of secondary carotenoids in the dark. This
feature makes C. zofingiensis a potential candidate for the
production of astaxanthin on a large scale.10,11,26
Supplied with glucose as sole carbon and energy source, C.
zofingiensis can grow and produce astaxanthin without light.11
Other advantages of C. zofingiensis include its ease of culturing
under various growth conditions, and resulting minimal negative
environmental influence.9,16 H. pluvialis cannot grow efficiently in
dark heterotrophic culture, and consequently, the production of
astaxanthin by H. pluvialis has to be through a photosynthetic
process, and often requires extremely high irradiance (e.g. 950 µE
m−2 s−1 ), which may not be economically justified.27,28,29 Other
problems such as mutual shading of cells, photolimitation
and/or photoinhibition also exist in photosynthetic culture
systems.30,31,32
In this study, total carotenoid content of the co-culture of
H. pluvialis and C. zofingiensis was similar to that of the mono-
cultures. The total carotenoid content of the co-culture in control
BBM culture was higher (0.83 mg total carotenoids g−1 cell) than
that obtained in organic carbon enriched BBM (0.54 mg total
carotenoids g−1 cell) but not as high as in the mono-cultures
(1.5 mg total carotenoids g−1 cell) (Fig. 8). An overall analysis of
the values showed that mono-culture of H. pluvialis reached higher
values of total carotenoid content.
Heterotrophic C. zofingiensis accumulates significantly less
astaxanthin (1 mg g−1 or 10 mg L−1 ) than phototrophic H. pluvialis
(40 mg g−1 or 35 mg L−1 ) although the former may reach higher
biomass values (10 g L−1 ).7,11,12 As stated previously, glucose
addition affects the production of chlorophylls and carotenoids
differently.30,31,32
In co-culture, interaction of the two cultures affects the response,
resulting in a higher loss in total carotenoids compared with that
in the mono-cultures.
CONCLUSION
When the organic carbon enriched BBM was considered, glucose
addition was found to have a positive effect on the growth of both
mono-cultures and co-culture of H. pluvialis and C. zofingiensis,
compared with a control group. The effect of a single step
Micro-algal ultivation using mono-cultures and co-cultures
increase of light intensity to 170 µE m−2 s−1 caused high stress,
resulting in high cell loss after a few days for both H. pluvialis and
C. zofingiensis, whereas stepwise increase of light intensity affected
the cell numbers and their carotenoid content subsequently. The
total carotenoid contents of the mono-cultures of both microalgae
gave higher yields in standard BBM. However, preliminary HPLC
studies indicated a variation in the amounts of astaxanthin isomers
produced. The objective of this study was to investigate the effect
of various co-culture conditions.
Further studies are in progress to determine the individual
effects of culture conditions during different growth phases and
the effects of carbon-enriched media on the type of isomers and
caretenoids produced.
ACKNOWLEDGEMENTS
The authors would like to thank The Scientific and Technological
Research Council of Turkey; TUBITAK, for their financial support.
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