Process Biochemistry 43 (2008) 1288–1292
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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Sugar-based growth, astaxanthin accumulation and carotenogenic transcription
of heterotrophic Chlorella zofingiensis (Chlorophyta)
Ni Sun a,b,1, Yan Wang a,c,1, Yan-Tao Li b, Jun-Chao Huang b,*, Feng Chen b,**
a
South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, PR China
School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, PR China
c
Yantai Institute of Coastal Zone Research for Sustainable Development, Chinese Academy of Sciences, Yantai, PR China
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 5 December 2007
Received in revised form 8 July 2008
Accepted 18 July 2008
The green microalga Chlorella zofingiensis can grow and produce the ketocarotenoid astaxanthin in the
dark with glucose as sole carbon and energy source. In the present study, we reported that glucose,
mannose, fructose, sucrose, galactose and lactose could differentially support the cell growth and
astaxanthin biosynthesis. Of the sugars surveyed, glucose and mannose were the best carbon sources for
the algal growth in the dark, as indicated by the relatively high specific growth rates (ca. 0.03 h1) and
high cell dry densities (ca. 10 g l1). Furthermore, the algal cells cultured with glucose and mannose
accumulated the highest amounts of astaxanthin (ca. 1 mg g1), indicating a correlation between cell
growth and astaxanthin formation. In addition, various sugars differentially regulated the transcription
of three carotenogenic genes encoding phytoene desaturase (PDS), b-carotene ketolase (BKT), and bcarotene hydroxylase (CHYb) respectively. By using glucose fed-batch fermentation, high cell dry weight
concentration (ca. 53 g l1) and high astaxanthin production (ca. 32 mg l1) were obtained, which are
much higher than those ever reported in the alga. The present study suggests that C. zofingiensis is
suitable for the production of natural astaxanthin on a massive scale.
ß 2008 Elsevier Ltd. All rights reserved.
Keywords:
Astaxanthin
b-carotene ketolase
b-carotene hydroxylase
Chlorella zofingiensis
Heterotrophic
Sugar
1. Introduction
Green microalgae have already served as a major natural source
of valuable carotenoids [1,2]. The ketocarotenoid astaxanthin has
attracted much attention due to its strong quenching ability of
singlet oxygen, involvement in cancer prevention, and enhancement of immune response [3,4]. Accumulating evidence has shown
that astaxanthin is beneficial to human health by preventing
degenerative diseases [4]. Potential production of astaxanthin
from microorganisms has been a subject of intensive investigation
in recent years [5–10].
Compared with other astaxanthin-producing microorganisms,
some green algae, such as Haematococcus pluvialis and Chlorella
zofingiensis, have the potential to accumulate high amounts of
astaxanthin because they possess sequestering systems to storing
* Corresponding author. Fax: +852 2299 0311.
** Corresponding author. Tel.: +852 2299 0310.
E-mail addresses: [email protected] (J.-C. Huang), [email protected]
(F. Chen).
1
These authors contributed equally.
1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2008.07.014
up astaxanthin in lipid bodies [7,11]. H. pluvialis has been
commercially used for producing natural astaxanthin, whereas C.
zofingiensis has only recently attracted much attention due to its
ease of growing under various conditions with high growth rate,
and minimal negative environmental influence [8,9]. Furthermore, C. zofingiensis can grow and produce astaxanthin without
light when exogenous glucose is supplemented [10]. Thus, darkgrown C. zofingiensis might be more economical for commercial
production of astaxanthin than any light-dependent cultivation
systems, because the well-developed and economical fermentation systems can be used to cultivate the algal cells on a large
scale. However, heterotrophic C. zofingiensis accumulates much
less astaxanthin (ca. 1 mg g1 or 10 mg l1) than phototrophic H.
pluvialis (ca. 40 mg g1 or 35 mg l1) although the former one
may reach much higher biomass (ca. 10 g l1) [7,10]. As
the organic carbon source is the most important factor for
dark-grown C. zofingiensis, in the present study, we investigated
the effect of various sugars on the biosynthesis of astaxanthin and
the transcription of three carotenogenic genes including the
phytoene desaturase (PDS) [12], b-carotene ketolase [13], bcarotene hydroxylase (Genbank accession EU016205) in the
dark-grown alga.
N. Sun et al. / Process Biochemistry 43 (2008) 1288–1292
2. Materials and methods
2.1. Microalga and medium
Chlorella zofingiensis ATCC 30412 was obtained from the American Type Culture
Collection (ATCC, Rockville, USA). This alga was maintained at 4 8C on an agar slant
containing the modified Bristol’s medium CZ-M1 [10]. Briefly, 10 ml of CZ-M1 broth
was inoculated with cells from slants and the alga was grown aerobically in flasks at
27 1 8C for 4 days with orbital shaking at 150 rpm and illuminated with continual
florescence light at 60 mmol m2 s1 measured at the surface of the flask. The cells
were then inoculated at 5% (v/v) into a 200-ml Erlenmeyer flask containing 36 ml of the
medium, and were grown for 4 days and used as seed for batch culture. All media were
adjusted to pH 6.5 prior to autoclaving at 121 8C for 20 min.
2.2. Batch culture
In shake flask batch culture, Erlenmeyer flasks (250 ml), each containing 90 ml
medium supplemented with various carbon sources (glucose, fructose, galactose,
mannose, lactose, and sucrose) were inoculated with 10% (v/v) of exponentially
growing inoculum and then incubated at 25 8C in an orbital shaker at 150 rpm in the
dark.
2.3. Fed-batch fermentation
A two-stage fed-batch culture was carried out in a 3.7-l fermenter (Bioengineering AG, Wald, Switzerland). The working volume of the culture was 2.0 l. The
cultivation conditions in the fermenter were controlled as follows: pH 6.6;
temperature 25 8C; agitation 480 rpm; and dissolved oxygen concentration 50%
saturation. During fed-batch cultivation, the sterilized stock nutrient solution was
fed into the fermenter at intervals so that glucose concentration in the cultures was
kept at 5–20 g l1.
2.4. Analytical methods
For cell dry biomass (DW, g l1) determinations, 5-ml aliquots of the cell culture
were filtered through a pre-dried Whatman GF/C paper, washed three times, and
the filters containing cells were dried until constant weight. Specific growth rate (m,
h1) was calculated from the DW, during the logarithmic phase of growth, using the
equation m = (ln X2 ln X1)/(t2 – t1), where X2 and X1 represent DW values at times
t2 and t1, respectively.
For pigment analysis, cell pellets were obtained by centrifuging the culture
samples at 3000 rpm at 4 8C for 3 min and dried in a DW3 freeze-drier (Heto Dry
Winner, Denmark). The freeze-dried cells (0.01 g) were ground with nitrogen and
extracted with acetone until the cells became colorless. After centrifugation at
14,000 rpm at 4 8C for 5 min, the supernatant was collected and evaporated under
nitrogen gas and the residue was redissolved in 1 ml acetone which was filtered
through a 0.22-mm Millipore organic membrane prior to HPLC analysis. The HPLC
system was equipped with a Waters 2695 separations module and a Waters 2996
photodiode array detector. Twenty microliters of each extract was separated by
HPLC on a Waters Spherisorb1 5 mm ODS2 4.6 250 mm analytical column
(Waters, Milford, MA, USA) according to the method described by Ref. [14].
Pigments were eluted at a flow rate of 1.2 ml min1 with a linear gradient from
100% solvent A (acetonitrile:methanol:0.1 M Tris–HCl, pH 8.0 [84:2:14]) to 100%
solvent B (methanol:ethyl acetate [68:32]) for 15 min, followed by 20 min of
solvent B. The absorption spectra of the pigments were shown between 300 and
700 nm. Peaks were measured at a wavelength of 450 nm. The concentration of
individual carotenoids was determined using standard curves of standard pigments
at known concentrations.
Sugar concentration (g l1) was determined according to the method of Miller
[15].
1289
TATG-30 and reverse, 50 -CCATTTCCCACATATTGCACCT-30 ) and CHYb (forward, 50 GCCAGCCATGAAACGTGTG-30 and reverse, 50 -GTTCCTTCCAGTTATGTAC ACA-30 ). C.
zofingiensis actin (act) primers (50 -TGCCGAGCGTGAAATTGTGA G-30 and reverse, 50 CGTGAATGCCAGCAGCCTCCA-30 ) [13] were used to demonstrate equal amounts of
templates and loading. The GenBank accession numbers for PDS, BKT, and CHYb
were EF621405, AY772713, and EU016205, respectively. Amplification of the cDNA
was done by conventional PCR [94 8C for 2 min followed by 24 cycles (for PDS, BKT
and actin genes) or 26 cycles (for CHYb gene) of 94 8C for 15 s, 58 8C for 20 s, 72 8C for
30 s]. PCR products were separated on a 2% agarose gel and stained with ethidium
bromide for photography (Biorad, USA). The relative transcript levels of the specific
genes were determined based on gel visualization.
3. Results and discussion
3.1. Heterotrophic growth and astaxanthin synthesis of C. zofingiensis
with various carbon sources
Since C. zofingiensis can grow well heterotrophically with
glucose as sole carbon and energy source, we investigated if this
alga is capable of metabolizing other organic carbon sources (e.g.
oligosaccharides) for heterotrophic cultivation to achieve high cell
concentrations. Four monosaccharides (glucose, fructose, galactose, and mannose) and two disaccharides (lactose and sucrose)
were surveyed and the results are presented in Fig. 1. C. zofingiensis
could use all the sugars for heterotrophic growth but their specific
growth rates and dry biomass concentrations were rather different
(Fig. 1). Glucose and mannose were the best carbon sources for
heterotrophic growth of C. zofingiensis which exhibited high
specific growth rates (0.028 h1/0.029 h1) and high dry biomass
concentrations (10.63 g l1/10.17 g l1). Fructose was also a good
carbon source for the heterotrophic growth of C. zofingiensis, with
which the algal cells demonstrated a slightly lower specific growth
rate (0.027 h1) and dry biomass (9.44 g l1) than that with
glucose or mannose. Sucrose and galactose led to low specific
growth rates (0.018 h1/0.012 h1) and low dry biomass
(5.46 g l1/2.17 g l1). Lactose was the poorest carbon source
assimilated by the algal cells, as indicated by the very low specific
growth rate (0.009 h1) and high sugar residual in the medium
(more than 40 g l1, data not shown). The dry biomass from lactose
containing culture was only 1.42 g l1, 87% lower than that from
glucose-supplemented culture.
Some green algae can grow heterotrophically, making use of
sugars, amino acids and organic acids [17]. The uptake of sugars by
the unicellular alga Chlorella was shown inducible [18,19]. When
the alga shifts from carbon autotrophy to heterotrophy, hexose
transport activity increases more than 200 fold [18]. In response to
hexoses, Chlorella cells can induce monosaccharide-H+ symport
catalyzing the energy-dependent transport of D-glucose and
several other hexoses across the plasmalemma [20,21]. Our study
2.5. RNA isolation
RNA techniques were followed according to the standard method described in
Ref. [16]. RNA was isolated from aliquots of about 108 cells cultured with various
sugars (50 g l1) for 2 days. Cells were collected by centrifugation and powdered
under liquid nitrogen using a mortar and pestle. RNA was then isolated using TRI
Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the
manufacturer’s instructions. The concentration of total RNA was determined
spectrophotometrically at 260 nm.
2.6. RT-PCR assay
Total RNA (1 mg) extracted from different samples was reverse transcribed to
cDNA by using a SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad,
CA, USA) for reverse transcription PCR (RT-PCR) primed with oligo(dT) according to
the manufacturer’s instructions. PCR amplification was carried out using specific
primers of PDS (forward, 50 -GATGAATGTATTTGCTGAACTGGGC-30 and reverse, 50 GGCCAGTGCCTTAGCCATAGCG-30 ), BKT (forward, 50 -GTGCTCAAAGTGGGGTGG-
Fig. 1. Heterotrophic growth of C. zofingiensis at 50 g l1 of various sugars. Cell dry
weight concentration (&) and specific growth rate (*). Data are mean values S.D.
of three independent measurements.
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N. Sun et al. / Process Biochemistry 43 (2008) 1288–1292
Fig. 3. Transcript level of b-carotene ketolase (BKT), b-carotene hydroxylase (CHYb)
and phytoene desaturase (PDS) in heterotrophic C. zofingiensis cultured with
50 g l1 of various sugars. The expression of actin gene was used as control.
Fig. 2. Astaxanthin content (&) and yield (&) of C. zofingiensis at 50 g l1 of various
sugars. Data are mean values S.D. of three independent measurements.
showed that C. zofingiensis cells could transport and assimilate
various exogenous sugars with quite different efficiency.
Interestingly, the astaxanthin production from cultures with
different carbon sources (Fig. 2) demonstrated a correlation to the
cell growth. Cultures with glucose or mannose not only grew
fastest leading to the highest dry biomass, but also produced the
highest quantities of astaxanthin (1 mg g1 or 10 mg l1). In
contrast, lactose is a poor carbon source for the heterotrophic
growth of C. zofinginesis, with which the algal cells grew slowly and
only produced a trace amount of astaxanthin (0.06 mg g1).
Cultures with medium containing fructose, sucrose or galactose
exhibited a decreasing trend in cell growth rates, dry biomass as
well as astaxanthin production. These results revealed that the
biosynthesis of astaxanthin in C. zofingiensis might be associated
with the cell growth. In the red yeast Phaffia rhodozyma, the cell
respiration rate was shown to correlate positively with the
astaxanthin production rate [22]. Heterotrophic C. zofingiensis
may share a similar regulation of astaxanthin synthesis as the
yeast.
3.2. Differential transcription of BKT, CHYb, and PDS genes in C.
zofingiensis cells grown with various sugars
We recently found that glucose induced the expression of the
BKT that catalyzed the formation of both canthaxanthin from bcarotene as well as astaxanthin from zeaxanthin in C. zofingiensis
[13]. Since various sugars resulted in different astaxanthin yields
by the alga, we investigated whether the sugars differentially
regulated the related carotenogenic genes. RT-PCR approach was
used to detect the transcription of b-carotene ketolase (BKT), bcarotene hydroxylase (CHYb), and PDS in cells cultured with
various sugars. The results are shown in Fig. 3. The various sugars
differentially regulated the transcription of the genes. Glucose and
mannose, which supported the best growth and astaxanthin
production of the algal cells, also resulted in the highest transcript
levels of all the genes. Frutose, galatose, and sucrose only
moderately enhanced the transcription of the genes; while lactose
which was poorly metabolized by the cells did not up-regulate the
transcription of the carotenogenic genes. Glucose was reported to
induce expression of carotenogenic genes in the cyanobacterium
Synecocystis sp. PCC 6803 [23]. The glucose effects on the
expression of genes are suggested to result from the glucosesensing and signaling with the hexokinase as the key sensor in a
variety of yeasts, mammals and higher plants [24–27], or from
changes in the cytosolic redox state or electron flow of respiratory
electron transport [24,28,29]. A correlation between biomass,
astaxanthin production, and the transcripts of BKT and CHYb genes
was evident in the cells treated with the sugars. Our results
suggested that the uptake and/or the metabolism of sugars might
mediate the transcription of the carotenogenic genes. By using
glucose analogs and specific inhibitors of respiratory electron
transport, it is possible to reveal how the cells regulate
transcription of the carotenogenic genes in response of sugars.
In the present study, C. zofingiensis was shown to differentially
utilize not only different monosaccharides but also different
disaccharides (Fig. 1). Furthermore, different carbon sources not
only supported differential cell growth, but also differential
astaxanthin production in the cells (Fig. 2). As glucose and
mannose benefit cell growth as well as astaxanthin production, it is
possible to enhance the astaxanthin production of C. zofingiensis
via optimizing the culture conditions (e.g. glucose concentration in
medium) or/and culture approaches (e.g. fed-batch fermentation).
3.3. Fed-batch fermentation enhanced the production of astaxanthin
by C. zofingiensis
We previously showed that the highest specific growth rate of
C. zofingiensis was obtained when 20 g l1 glucose was involved in
the medium; whereas the highest cell dry weight and astaxanthin
content were obtained when 50 g l1 glucose was used [10]. We
therefore designed different culturing systems to investigate the
role of carbon source on the cell growth and astaxanthin
production. Batch culture with 50 g l1 glucose was used as
control. In fed-batch cultures, 20 g l1 glucose was initially used.
When the glucose concentration in medium dropped to around
5 g l1 (monitored by sugar detection each day), additional glucose
or medium with glucose was fed to the cultures to reach the initial
glucose concentration (20 g l1). Two times of feeding were carried
out with a total of 50 g l1 glucose used by the fed-batch culture.
The biomass and astaxanthin production of the culture are
summarized in Table 1. The fed-batch culture (glucose/medium
with glucose) obtained much higher biomass concentration
(17.8 g l1/20 g l1) than the batch culture (10.3 g l1). However,
the astaxanthin contents of the fed-batch culture (0.69 mg g1/
0.1 mg g1) were much lower than that of the batch culture
Table 1
Biomass and astaxanthin production by C. zofingiensis in different culture systems
Culture system (glucose concentration (g l1))
Biomass (g l1)
Astaxanthin content (mg g1)
Astaxanthin yield (mg l1)
Batch culture (50)
Glucose fed-batch culture (20 + 13 + 17)
Medium and glucose fed-batch culture (20 + 13 + 17)
10.3 0.18
17.8 0.28
20.0 0.29
1.02 0.05
0.69 0.06
0.10 0.01
10.51 0.2
12.60 0.3
2.00 0.3
N. Sun et al. / Process Biochemistry 43 (2008) 1288–1292
(1.02 mg g1). One reason for the low content of astaxanthin may
be the low dissolved oxygen concentration in the flask cultures at
high cell density, as revealed in the case of red yeast P. rhodozyma
[22]. To answer this question, a 3.7-l fermenter, which could
maintain high dissolved oxygen concentration, was used coupled
with a fed-batch procedure that contained two stages of feeding:
three times of medium with glucose feeding followed by four times
of glucose feeding. The feeding time, time course of cell growth and
astaxanthin production are shown in Fig. 4.
The fed-batch fermentation greatly increased the cell dry
weight concentration from 10.3 g l1 to 51.8 g l1 and astaxanthin
yield from 10.5 mg l1 to 32.4 mg l1 (Fig. 4 and Table 1). During
the first stage of medium and glucose feeding, the cells grew fast
but the astaxanthin content was less than 0.2 g g1 (data not
shown). A high C/N ratio was found to facilitate astaxanthin
formation by the alga [10]. The low content of astaxanthin in cells
fed with medium with glucose may be due to the relatively low C/N
ratio. This explanation was supported by the fact that astaxanthin
accumulation was greatly enhanced when glucose was fed to the
cultures (Fig. 4). Photoautotrophic C. zofingiensis accumulated
astaxanthin up to 1.5 mg g1 or 15 mg l1 when induced with high
light [30]. In contrast, dark-grown C. zofingiensis could accumulate
astaxanthin up to 32.4 mg l1 (this study), which may be further
enhanced by employment of reactive oxygen species as described
in our previous study [31,32] or by optimization of fermentative
medium [33]. Thus, dark-grown C. zofingiensis may be potentially
employed for commercial production of astaxanthin on a large
scale.
Fig. 4. Biomass, glucose consumption and astaxanthin production in a two-stage
fed-batch fermentation of C. zofingiensis in a 3.7-l fermenter. (*) Cell dry weight
concentration; (&) glucose concentration; (~) astaxanthin content; (column)
astaxanthin yield; ( ) medium and glucose feeding and ( ) glucose feeding.
1291
In conclusion, C. zofingiensis can uptake and metabolize a
variety of sugars for heterotrophic growth. Glucose and mannose
are the most suitable carbon source for heterotrophic growth of the
alga. Carbon source and nutrient are two important factors
influencing the growth and astaxanthin production by C.
zofingiensis. The astaxanthin-producing capacity by C. zofingiensis
could be greatly enhanced by using a fed-batch fermentation
strategy.
Acknowledgement
The work described in this paper was fully supported by a grant
from the Research Grants Council of the Hong Kong Special
Administrative Region, China (Project No. HKU 7623/05M).
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