Aquacult Int

DOI 10.1007/s10499-015-9927-2

Evaluation of optimal conditions for cultivation

of marine Chlorella sp. as potential sources of lipids,
exopolymeric substances and pigments

Benjamas Cheirsilp1 Yohanis Irenius Mandik1,2

Poonsuk Prasertsan1

Received: 15 October 2014 / Accepted: 20 June 2015

Springer International Publishing Switzerland 2015

Abstract Marine microalgae are used in aquaculture as a suspension to feed young fishes
and shrimps. Marine Chlorella sp. is one of the most attractive marine microalgae because
in addition to pigments it can accumulate lipids at a high content ([30 %) and release
exopolymeric substances (EPSs) into the culture medium. This study aimed to evaluate the
optimal cultivation of marine Chlorella sp. as potential sources of lipids, EPSs and pig-
ments. Among the culturing regimes tested, mixotrophic cultivation mode produced the
highest yields of biomass, lipids and EPSs. Several factors affecting mixotrophic culti-
vation were optimized. An increase in light intensity up to 65 lmol m-2 s-1 and carbon
dioxide up to 10 % v/v in air enhanced both biomass and product formation. An increase in
glucose concentration up to 1 % w/v enhanced biomass and EPS yields but decreased lipid
and chlorophyll contents. A semi-continuous cultivation under optimal conditions pro-
duced microalgal biomass of 2.76 g L-1 with a high lipid content of 44.9 % and EPS of
1.46 g L-1. This study has also shown that the microalgal lipid and EPS have potential to
be used as biodiesel feedstocks and bioflocculants, respectively. The concomitant pro-
duction of these valuable products together with the microalgal biomass would be a
potential way to offset the production cost and contribute greatly to developing industri-
alized microalgae cultivation.

Keywords Biodiesel feedstocks  Chlorophylls  Culturing regimes  Mixotrophic

cultivation

& Benjamas Cheirsilp

benjamas.che@psu.ac.th
1
Department of Industrial Biotechnology, Faculty of Agro-Industry,
Prince of Songkla University, Hat-Yai 90112, Thailand
2
Department of Chemistry, Faculty of Mathematics and Natural Sciences, University
of Cenderawasih, Jalan Kamp Walker, Jayapura 99358, Indonesia

123
 Aquacult Int

Introduction

Many species of microalgae can produce specific products such as lipids, sterols, peptides,
polysaccharides, enzymes, pigments, antioxidants and toxins (Hossain et al. 2008). Among
these specific products of microalgae, the microalgal lipids have received increasing
attention as alternative sources for biodiesel feedstocks due to their availability and better
ratios of saturated, monounsaturated and polyunsaturated fatty acids enabling better
combustion in thermal engines (Chisti 2007; Mata et al. 2010). In some microalgae, the
lipid content can reach 75 % w/w based on their dry biomass (Mata et al. 2010). Recently,
microalgal exopolymeric substances (EPSs) have been evaluated for their biological
functions (Suarez et al. 2005) and effects on the flocculation efficiency (Wu et al. 2012). In
the aspect of physiology, the presence of an EPS layer surrounding the cells may be
involved in their protection by reducing the penetration of toxic ions through the cell
surface (Goo et al. 2013). In addition to lipids and EPSs, microalgae also produce pigment
chlorophylls for capturing light energy from the sun. The chlorophylls can be used as
natural coloring agents in food, cosmetics and pharmaceutical products. The potential
health benefits of consumption of high chlorophyll diets have been reported. These include
protective properties in the prevention of coronary heart disease, certain cancers such as
skin, colon and liver cancers, diabetes and cataracts (Ferruzzi and Blakeslee 2007; Ma and
Dolphin 1999).
The optimum production of these specific microalgal products can be achieved by
selecting the most suitable microalgae for each product and/or manipulating chemical and
environmental factors such as culture medium, cultivation mode, pH, temperature, light
intensity, carbon dioxide availability, salts and other nutrients (de Oliveira et al. 1999;
Lee 2004; Hossain et al. 2008; Danesi et al. 2011; Fagiri et al. 2013). Up to now, the
commonly used modes for cultivation of microalgae are photoautotrophic, heterotrophic
and mixotrophic modes. In the photoautotrophic cultivation mode, the cells harvest light
energy and assimilate CO2 for their photosynthesis. In the heterotrophic cultivation mode,
the cells assimilate organic carbon sources in the absence of light. This mode can be
performed in conventional microbial bioreactors. The mixotrophic cultivation mode is
another strategy where CO2 and organic carbon sources are simultaneously assimilated
and both photosynthetic and heterotrophic metabolisms operate concurrently (Lee 2004).
Most studies on microalgae have been dominated by the selection of the cultivation
conditions that lead to the highest yield of lipids in the shortest time (Martnez et al.
1997; Liang et al. 2009; Heredia-Arroyo et al. 2010; Cheirsilp and Torpee, 2012; Girard
et al. 2014). However, knowledge on the production of other valuable products in the
different cultivation modes and the optimal condition for each product remains largely
unknown.
Marine Chlorella sp. is one of the most attractive microalgae for lipid production
because it can accumulate lipids to a high content ([30 %) (Cheirsilp and Torpee 2012).
However, the cultivation of this microalga for only one product is not economically
feasible. Therefore, the concomitant production of this microalgal biomass with several
valuable products has drawn attention. The present study aimed to evaluate the possible
concomitant production of lipids, exopolymeric substances and pigments together with the
marine Chlorella sp. biomass. The conditions for maximizing both biomass and product
formation were determined. The compositions and application of the microalgal lipids and
exopolymeric substances were also evaluated.

123
Aquacult Int

Materials and methods

Materials

A marine Chlorella sp. was obtained from the National Institute of Coastal Aquaculture,
Thailand. The media used in this study were the Bolds basal medium (BBM medium)
(Sterin 1973) containing 0.25 g NaNO3, 0.075 g MgSO47H2O, 0.025 g CaCl22H2O,
0.175 g KH2PO4, 0.075 g K2HPO4, 0.025 g NaCl, 0.00498 g FeSO47H2O, 0.01 g Na2-
EDTA, 8.05 mg H3BO3, 1.81 mg MnCl24H2O, 0.222 mg ZnSO47H2O, 0.079 mg
CuSO45H2O, 0.390 mg NaMoO45H2O and 0.0494 mg Co(NO3)26H2O per liter, pH 7.3
and the BG11 medium (Tansakul et al. 2005) containing 1.5 g NaNO3, 0.04 g K2HPO4-
3H2O, 0.2 g KH2PO43H2O, 0.0005 g EDTA, 0.005 g Fe ammonium citrate, 0.005 g citric
acid, 0.02 g Na2CO3 and 1 mL of trace metal solution per liter, pH 7.3. The trace metal
solution contained 2.85 g H3BO3, 1.8 g MnCl24H2O, 0.02 g ZnSO47H2O, 0.08 g
CuSO45H2O, 0.08 g CoCl26H2O and 0.05 g Na2MoO42H2O per liter.

Microalgal cultures

The microalga was precultured in 1 L of BG11 medium with the pH adjusted to 7.3 in a 1-L
flask. Cultures were incubated at 30 C and aerated with an air flow rate of 0.5 volume of air
per volume of medium per minute (vvm) under a 50 lmol m-2 s-1 light intensity with a
16:8 h light/dark cycle for 5 days. The culture medium was filtered through a Whatman No.
40 filter paper and then diluted with the medium to obtain proper dilution with an optical
density (OD) at 660 nm = 2.0. This was used as a starter culture. The batch cultivation of the
microalgae was performed by inoculating 10 % of the starter culture into 400 mL medium
with the pH adjusted to 7.3 in a 500-mL flask. The microalgae were cultivated in various
media including BG11, BBM, BG11 added with a twofold increase in the nitrogen and
phosphorus sources and under photoautotrophic, heterotrophic and mixotrophic cultivation
modes. For photoautotrophic cultivation, the cultures were illuminated with a 50 lmol m-2
s-1 light intensity with a 16:8 h light/dark cycle. For mixotrophic and heterotrophic culti-
vation, glucose was used as an organic carbon source at a concentration of 0.2 % w/v with and
without light illumination, respectively. The effects of salt stress on biomass and product
formation by marine Chlorella sp. were investigated by varying the concentration of sodium
chloride added to the BG11 medium at 0, 0.5 and 1.0 % under photoautotrophic cultivation
mode. The effects of light intensity (5065 lmol m-2 s-1), CO2 concentration (520 % v/v
in air) and initial glucose concentration (0.21.0 % w/v) on the biomass and product for-
mation of marine Chlorella sp. were investigated in the BG11 medium and under mixotrophic
cultivation mode. The initial pH was set at 7.3. All the cultures were incubated at 30 C and
aerated at a flow rate of 0.5 vvm for 5 days. The biomass and amounts of lipid, EPS and
chlorophylls were determined. All experiments were carried out in triplicate.
The microalgae cultivation was scaled up in a 3-L stirred-tank photobioreactor with a
working volume of 2 L. The experiment was conducted under semi-continuous mode for
192 h. The pH of the culture was feedback-controlled at 7.3 by the injection of CO2 in
response to signals from a pH sensor. Aliquots of the culture were manually sampled once
a day. The biomass and amounts of product were measured. The semi-continuous regime
was established by removal of part of the cell suspension (1 L) followed by replacement
with fresh medium every day. The medium were autoclaved before use. The photobiore-
actor was first sterilized using a 2 % (v/v) chlorine solution.

123
 Aquacult Int

Flocculation of microalgal biomass

The initial OD and pH of the microalgal suspension were adjusted to be the same at 1.8 and
10.0, respectively, before the flocculation test. Ten milliliters of this suspension was then
placed in a 20-mL glass and left to settle for 60 min without agitation. Subsequently, the
OD of the supernatant taken from the top half of the height of the clarified layer by
micropipette was measured using a spectrophotometer (LIBRA-S22, Biochrom, UK). The
flocculation efficiency was defined as the ratio of the mass of cells recovered to the total
mass of cells (Wu et al. 2012) and calculated as:
Flocculation efficiency % 1  A1 V1 =A0 V0  100
where A1 is the OD of the supernatant from the top half the height of the clarified layer
after flocculation and A0 is the initial OD of the microalgal suspension before flocculation;
V0 is the volume of microalgal suspension before flocculation, and V1 is the volume of
microalgal supernatant after flocculation; the flocculation time was 60 min.

Analytical methods

Biomass was harvested by centrifugation of the culture sample at relative centrifugal force of
40009g for 15 min. The pellets were then washed twice with distilled water and dried at 60 C
in a hot air oven until a constant weight was obtained. Extraction of lipids from the biomass was
performed according to the modified procedure of Folch et al. (1957). Lipids were extracted
with a mixture of chloroform/methanol (2:1, v/v) for 1 h. The extracted lipids were centrifuged
to obtain a clear supernatant, and the solvent was removed by evaporation under vacuum and
weighed. The lipid-free biomass was calculated by subtracting the lipid weight from the
biomass weight. The chlorophyll was extracted with acetone from the freshly harvested
microalgal cells. The amount of chlorophyll in the extractant was determined by measuring the
absorbance at 645 nm (A645) and 663 nm (A663) with a spectrophotometer (LIBRA-S22,
Biochrom, UK) and then calculated using the following equation (Becker 1994):
Chlorophyll mg=L 8:02  A663 nm 20:21  A645 :

The chlorophyll contents in the microalgal cells (mg/g) were calculated by dividing the
concentration of the chlorophylls (mg/L) by the cell dry weight (g/L).
The method for converting the extracted lipids to fatty acid methyl esters (FAMEs)
involved hydrolysis of the lipids followed by esterification (Jham et al. 1982). The fatty
acid compositions of the FAME were analyzed using a HP6850 Gas Chromatograph
equipped with a cross-linked capillary FFAP column (length 30 m, 0.32 mm I.D, 0.25 lm
film thickness) and a flame ionization detector. Operating condition were as follows: an
inlet temperature was 290 C; the initial oven temperature of 210 C was held for 12 min,
then ramped to 250 C at 20 C/min, held for 8 min, and the detector temperature was
300 C. Fatty acids were identified by comparing their retention times with known pure
standards. The amounts of reducing sugar were estimated by the dinitrosalicylic acid
(DNS) method using a glucose standard calibration curve (Miller 1959).
Exopolymeric substances were precipitated from the supernatant by addition of an equal
volume of cold ethanol (-20 C) followed by centrifugation. The precipitate was re-
dissolved in distilled water. To remove any remaining insoluble materials, the solution was
centrifuged and the clear supernatant was again precipitated in the same way. The resulting
precipitate was re-dissolved in distilled water. The exopolymeric substances were then

123
Aquacult Int

measured colorimetrically by the anthrone method using a standard curve of glucose

(Yeesang et al. 2008). The sugar composition of the exopolymeric substances was deter-
mined using high-performance liquid chromatography (HPLC, Agilent Technologies 1200
series) after acid hydrolysis. The sugar concentration was evaluated by using a calibration
curve generated from HPLC-grade sugars.
The statistical significance of the results was evaluated by one-way ANOVA (analysis
of variance) and Duncans multiple range tests (P \ 0.05) using the SPSS version 13.0
software (SPSS: An IBM Company, Illinois, USA).

Results and discussion

Growth and product formation of marine Chlorella sp. in various media

Since the BBM and BG11 media have been commonly used for Chlorella cultivation
(Buckwalter et al. 2013; Frumento et al. 2013; Liu et al. 2014; Lizzul et al. 2014; Wang
et al. 2014), the growth and product formation of marine Chlorella sp. in these two media
were compared (Fig. 1a). These two media differed mainly in the presence of sodium
carbonate, the concentrations of nitrogen source and the types of buffering agents. The
BG11 medium that contained sodium carbonate and a much higher concentration of
sodium nitrate than the BBM medium gave better growth, more lipids and more EPS. The
chlorophyll content per gram dry cells in the BG11 medium was also significantly higher
than that in the BBM medium (P \ 0.05). During the cultivation, the pH in both media
increased from initial pH of 7.3 up to 8.28.6. Normally, CO2 dissolves in the water in the
form of bicarbonate (HCO3-), and when CO2 is consumed by the microalgae, the OH- is
formed, and the pH becomes more alkaline (Richmond 1986).
To study the effect of nitrogen source and phosphorus source concentrations, the
nitrogen source and phosphorus source in the BG11 medium each was twofold increased
(Fig. 1a, BG11 ? 2N and BG11 ? 2P). With increasing nitrogen source concentration, the
biomass yield slightly increased but the product formation decreased (P \ 0.05). In case of

(A) (B)
1.2 60 1.2 60
Lipid Lipid
Lipid content (%), Chlorophylls

Lipid content (%), Chlorophylls

Lipid-free biomass Lipid-free biomass

Lipid, Biomass, EPS (g/L)
Lipid, Biomass, EPS (g/L)

1.0 EPS 50 1.0 EPS 50

Chlorophylls Chlorophylls A
Lipid content Lipid content
0.8 Final pH 40 0.8 Final pH A 40
(mg/g), pH

A
(mg/g), pH

A A B
A a 30
0.6 30 0.6 b
a b
b b b
0.4 b 20 0.4 20
a ab a
a a
0.2B b b Ab B b 0.2 A b 10
B 10 A b A b b
c b a b a b b
0.0 0 0.0 0
BBM BG11 BG11+2N BG11+2P NaCl 0% NaCl 0.5% NaCl 1.0%
NaCl concentration

Fig. 1 a Biomass and product formation by marine Chlorella sp. grown photoautotrophically in various
media. BG11 ? 2N, the BG11 medium with double the concentration of nitrogen source; BG11 ? 2P, the
BG11 medium with double the concentration of phosphorus source. b Effect of NaCl concentration on the
biomass and product formation by marine Chlorella sp. photoautotrophically cultivated in BG11 medium.
Data are the mean values of triplicate experiments. Different small letters on the bars and different capital
letters on the symbols indicate significant differences between treatments (P \ 0.05)

123
 Aquacult Int

increasing phosphorus source concentration, there was no significant effect on the biomass
and lipid yield, but EPS and chlorophyll content significantly decreased (P \ 0.05). These
results indicated that the original BG11 medium was most suitable for the cultivation of
this marine Chlorella sp. with high biomass and product formation yield.
Different microalgae differ in their adaptability to salinity and their tolerance extent. It
has been reported that salt stress affects the basic processes of photosynthesis, and their
specific metabolic adjustments allow for the maintenance of photosynthetic performance
under unfavorable conditions. Some species of microalgae may produce some metabolites
to protect themselves from salt injury and also to balance the changes of the surrounding
osmotic stress (Richmond 1986). In this study, the effects of salt stress on biomass and
product formation by marine Chlorella sp. were investigated by varying the concentration
of sodium chloride added to the medium (Fig. 1b). When sodium chloride was added, the
biomass decreased from 0.39 to 0.25 g L-1. The lipid and EPS yields also decreased from
0.12 and 0.19 to 0.09 and 0.10 g L-1, respectively (P \ 0.05). However, the lipid content
increased from 30 % up to 37 % (P \ 0.05). Several studies have shown that photosystem
II (PSII) is a major target of increased Na?, due mainly to the degradation of the D1
protein of the reaction center (RCII) and also due to alterations of the water oxidation
complex (Sudhir and Murthy 2004). Similar results have been reported by Qin (2005) who
found that at high salt concentration microalgae failed to grow and the amount of inter-
cellular particles and cell divisions were reduced. However, the chlorophyll content
slightly increased when sodium chloride was added. This could be due to the positive
effect of sodium chloride on the processes of pigment synthesis.

Growth and product formation of marine Chlorella sp. in various cultivation

modes

The growth and product formation of marine Chlorella sp. were studied in photoau-
totrophic, heterotrophic and mixotrophic cultivation modes (Fig. 2a). In this study, 0.2 %
w/v of glucose was used as an organic carbon source for heterotrophic and mixotrophic
cultures. Among the three cultivation modes tested, the mixotrophic cultivation mode gave

(A) (B)
2.5 60 2.5 60
Lipid content (%), Chlorophylls

Lipid
Lipid content (%), Chlorophylls

Lipid
Lipid-free biomass Lipid-free biomass
Lipid, Biomass, EPS (g/L)
Lipid, Biomass, EPS (g/L)

2.0 EPS 50 EPS

2.0 Chlorophylls a 50
Chlorophylls a
Lipid content Lipid content b a
(mg/g), pH

40 Final pH a a 40
(mg/g), pH

Final pH b b
1.5 A a 1.5 a
b
AB a 30 30
a B b Ab c A
1.0 1.0 AB
B
20 20
a b a a a
0.5 b A 0.5A A
A c Ab b 10 A A 10
c
b
b 0
0.0 0 0.0
Photoautotrophic Heterotrophic Mixotrophic 50 65 80 100
2 1
Light intensity (molm s )

Fig. 2 a Effect of the cultivation mode on biomass and product formation by marine Chlorella sp.
cultivated in BG11 medium. b Effect of the light intensity on the biomass and product formation by marine
Chlorella sp. cultivated in BG11 medium under mixotrophic cultivation mode. Data are the mean values of
triplicate experiments. Different small letters on the bars and different capital letters on the symbols indicate
significant differences between treatments (P \ 0.05)

123
Aquacult Int

the highest biomass of 1.41 g L-1. This biomass was about 3 times higher than that
obtained from photoautotrophic culture (0.39 g L-1). The biomass obtained from hetero-
trophic culture (0.50 g L-1) was also slightly higher than that from photoautotrophic
culture. These results indicate that the marine Chlorella sp. may possess an inducible
hexose transport system (Haass and Tanner 1974) that allows it to utilize glucose for the
growth and produce the biomass. Therefore, the microalgal growth is not strictly dependent
on photosynthesis and much higher biomass can be produced from the consumed glucose.
Interestingly, the biomass from mixotrophic culture was also higher than the sum of that
from photoautotrophic and heterotrophic cultures. This may result from the positive effect
of glucose on the photoautotrophic growth and/or the positive effect of light on the glucose
metabolism (Cheirsilp and Torpee 2012).
Under photoautotrophic cultivation mode without the addition of glucose, the marine
Chlorella sp. accumulated lipid content at the highest level of 30 %. With the addition of
glucose, the marine Chlorella sp. accumulated lipids at lower levels of 26 and 21 % under
mixotrophic and heterotrophic cultivation modes, respectively. These results are consistent
with the results of Girard et al. (2014) who found that Scenedesmus obliquus cultivated on
cheese whey permeate under mixotrophic condition gave higher biomass but lower lipid
content compared to those under photoautotropic condition. Liang et al. (2009) also found
that the lipid content of C. vulgaris under photoautotrophic condition was higher than that
under mixotrophic and heterotrophic conditions. However, in the study of Heredia-Arroyo
et al. (2010), they found that the lipid contents of C. protothecoides under different culture
conditions were not significantly different. From these results, it seems that the lipid
accumulation behavior in different culture modes was strain dependent. Although the
photoautotrophic cultivation mode is the suitable mode for attaining the biomass with high
lipid content, this mode gave very low amount of biomass and this led to a lower amount of
lipids per liter (0.12 g L-1) compared to the mixotrophic cultivation mode (0.37 g L-1).
In addition to lipid, the EPS formation by marine Chlorella sp. was also highest under
mixotrophic cultivation. With the addition of glucose, the microalgae produced EPS of
0.36 and 1.14 g L-1 in the absence and presence of light, respectively, which were twofold
and sixfold of that produced under photoautotropic cultivation. There have been few
reports on EPS production by microalgae. The production of exopolysaccharides, one type
of EPS, by a marine microalga Chroomonas sp. was only 0.053 g L-1 (Bermudez et al.
2004), while Rhodella violacea, Porphyridium cruentum and Botryococcus braunii pro-
duced higher concentrations of EPS at 0.594, 0.95 and 1.6 g L-1, respectively (You and
Barnett 2004; Dayananda et al. 2007; Villay et al. 2013). However, there was no available
information on EPS production in various cultivation modes. This study is then the first
study that found that the mixotrophic cultivation is the most suitable mode for EPS pro-
duction by the microalgae.
The chlorophyll content of the marine Chlorella sp. under photoautotrophic condition
was much higher than that under heterotrophic condition. The decrease in the chlorophyll
content could also be observed from the color of the algal cells which changed from dark
green to yellowish during heterotrophic cultivation. The results are consistent with those of
Heredia-Arroyo et al. (2010) who found that the color of photoautotrophic culture broth
was green, while that of heterotrophic culture was yellow, due to different chlorophyll
contents in the cells. Although the chlorophyll content in the mixotrophic culture was
slightly lower than that in the photoautotrophic culture, the microalgae grew better and
produced much higher yields of biomass and products.

123
 Aquacult Int

Optimization of biomass and product formation under mixotrophic condition

The results from the previous section showed that the marine Chlorella sp. grew well and
produced high yields of biomass and products under mixotrophic condition. Therefore, the
mixotrophic condition was then selected for further optimization of biomass and product
formation by this microalga. Because the mixotrophic culture is the combined culture of
photoautotrophic and heterotrophic growth, it is influenced by factors for both growth
conditions. The light intensity and carbon dioxide concentration are the factors for pho-
toautotrophic culture, while the availability of glucose is the factor for heterotrophic culture.
It was then necessary to optimize these three parameters in the mixotrophic culture.

Effect of light intensity

The effects of light intensity on the biomass and product formation of marine Chlorella sp.
are shown in Fig. 2b. The biomass, lipids and EPS yields of the marine Chlorella sp.
increased when the light intensity increased from 50 to 65 lmol m-2 s-1. A further
increase in the light intensity had no further effect. Therefore, the optimal level of light
intensity for supporting biomass, lipid and EPS yields was chosen as 65 lmol m-2 s-1.
However, the optimal light intensity for the chlorophyll content was higher at
80 lmol m-2 s-1. It was possible that with attenuated light the cells respond by synthe-
sizing more chlorophyll. However, at a higher light intensity of 100 lmol m-2 s-1, there
was no further increase in the chlorophyll content. This indicated that the saturation light
intensity for this marine Chlorella sp. would be at 80 lmol m-2 s-1. It should be noted
that the lipid content decreased with an increase in the light intensity. Since high light
intensities enhanced the growth of microalgae, the microalgae might use their synthesized
energy to divide themselves rather than to accumulate it in lipid form. This result was in
contrast to that of the previous reports which had shown that a high light intensity favored
for higher lipid content rather than more biomass (Metzger and Largeau 1999; Tansakul
et al. 2005). However, as increasing cell concentrations during cultivation diminish light
penetration, bright cultures driven under turbidostat mode may be needed to determine the
optimal light intensity for this microalgae.

Effect of CO2 concentration

To investigate the effect of CO2 concentration on the biomass and product formation of
marine Chlorella sp., the CO2 content in air was varied at 5 % (v/v), 10 % (v/v) and 20 %
(v/v) and the air flow rate was 0.5 vvm. The results are compared with those using normal
air (0.03 % CO2) (Fig. 3a). With increasing the CO2 content in air, the microalgae grew
better and produced higher lipid. The biomass increased from 1.41 g L-1 in the culture
aerated with normal air up to 2.19 and 3.10 g L-1 in the cultures aerated with CO2 at 5 and
10 %, respectively. This enhancement was likely due to the enrichment of available CO2 as
a carbon source. The lipid production increased with an increase in the CO2 content and
reached the maximum lipid yield of 0.96 g L-1 at 10 % CO2. At 20 % CO2, there was no
further enhancement of biomass and product formed. This could be due to the limitation of
light penetration through the high cell density of the biomass. It should be noted that the
lipid content was less affected by the CO2 concentration. Similar results have been reported
by Chiu et al. (2008) who found that the lipid content of Chlorella sp. did not respond to
the CO2 concentration. In the present study, the chlorophyll content increased with an

123
Aquacult Int

(A) (B)
6.0 60 6.0 60
Lipid Lipid

Lipid content (%), Chlorophylls

Lipid content (%), Chlorophylls

Lipid-free biomass Lipid-free biomass

Lipid, Biomass, EPS (g/L)

Lipid, Biomass, EPS (g/L)

5.0 EPS 50 5.0 EPS 50

Chlorophylls a Chlorophylls
ab
Lipid content Lipid content

(mg/g), pH
4.0 40 4.0 40

(mg/g), pH
Final pH c Final pH
a
A
3.0 A d a a 30 3.0 A 30
A a a
AB B b a
2.0 b a b a a 20 2.0 b 20
B b
c c b c B
1.0A
c b 10 b c c
B C 1.0A A A d 10
C A
c d c b
0.0 0 0.0 0
CO2 0.03% CO 2 5% CO2 10% CO2 20% 0.2% 0.4% 0.8% 1.0%
Glucose concentration

Fig. 3 a Effect of the CO2 concentration on the biomass and product formation by marine Chlorella sp.
cultivated in BG11 medium with 0.2 % w/v added glucose and under mixotrophic cultivation mode.
b Effect of glucose concentration on the biomass and product formation by marine Chlorella sp. cultivated
in BG11 medium under mixotrophic cultivation mode and aerated with normal air (0.03 % CO2). Data are
the mean values of triplicate experiments. Different small letters on the bars and different capital letters on
the symbols indicate significant differences between treatments (P \ 0.05)

increasing CO2 concentration up to 10 % and the green color of the cells was also getting
darker. It should be noted that the pH in the cultures aerated with 10 and 20 % CO2
remained steady at 7.37.6 (initial pH), while the pH in the cultures aerated with air and
5 % CO2 slightly increased up to 8.18.6. This indicated that the dissolved CO2 generated
by aeration with 1020 % CO2 could compensate for its consumption by the microalgae.

Effect of glucose concentration

To evaluate the effect of glucose concentration on the biomass and product formation of
marine Chlorella sp., the culture was aerated with normal air (0.03 % CO2) and the initial
glucose concentration was varied from 0.2 to 1.0 % w/v (Fig. 3b). The biomass yield was
much improved when the initial glucose concentration was increased from 0.2 to 1.0 %
w/v, while the lipids and EPS formation were less improved. The maximum biomass
obtained at 1.0 % glucose was higher than that obtained at 10 % CO2. However, the lipids
obtained at 1.0 % glucose (Fig. 3b) was lower than that obtained at 10 % CO2 (Fig. 3a).
The lipid content decreased with increasing glucose concentration. It should be noted
that the biomass with a low content of lipids might reduce the efficiency of lipid extraction
and increase the downstream processing cost. Therefore, a biomass with a high content of
lipids was more desirable. The chlorophyll content also decreased with increasing glucose
concentration. This could be due to the increased ratio of the biomass derived from
heterotrophic metabolism of glucose. At the relatively low initial glucose concentration of
0.2 % w/v, no glucose was detected in the medium after 2 days of cultivation. At higher
initial glucose concentrations (0.41.0 % w/v), glucose gradually decreased over time and
was almost completely depleted at the end of cultivation.

Cultivation in photobioreactor with semi-continuous regime

To produce large amounts of microalgal biomass and other valuable products, the mixo-
trophic cultivation of marine Chlorella sp. was scaled up in a 3-L stirred-tank photo-
bioreactor with a working volume of 2 L. The process started under a normal batch regime

123
 Aquacult Int

Lipid content (%), Chlorophylls (mg/g), pH

Fig. 4 Mixotrophic cultivation 6.0 60
of marine Chlorella sp. scaled up Dry cell weight Lipid
in a 3-L stirred-tank EPS Lipid content
photobioreactor using semi- 5.0 Chlorophylls pH 50
continuous regime. Half the

Lipid, Biomass, EPS (g/L)

medium was replaced with fresh 4.0 40
medium at 48, 72, 96, 120 and
144 h. Data are the mean values
of triplicate experiments 3.0 30

2.0 20

1.0 10

0.0 0
0 24 48 72 96 120 144 168 192
Time (h)

until the end of the log phase (48 h of cultivation) and was then switched to run under a
semi-continuous regime for a further 144 h. Half the cell suspension (1 L) was replaced
with fresh medium, and the glucose was supplied to keep the concentration at 0.2 % w/v
every day during the semi-continuous regime. CO2 was supplied at a concentration of
10 % (v/v). Since excessive amounts of CO2 would drop the pH to unsuitable level that
would result in low biomass productivity, CO2 feeding was also feedback-controlled in
response to signals from a pH sensor if necessary. Through this strategy, the CO2 could be
supplied at an appropriate level without a drop in the pH. The biomass and product
formation are shown in Fig. 4.
The cell growth in the batch culture reached the highest value of 2.68 g L-1 at 48 h. The
maximum lipid productivity in the stirred-tank photobioreactor was 12.5 mg L-1d-1 during
2448 h. This was almost twice that in a flask supplemented with 10 % CO2 (7.39 mg
L-1d-1 during the same period). This was probably due to the adequate supply of CO2 using
the pH-stat control system. To continuously produce high cell density of the biomass with
high lipid content, the semi-continuous mode was attempted. An advantage of this type of
cultivation is that the supplementation with fresh medium could provide additional nitrogen
and organic carbon sources that could be efficiently used for cell growth and product for-
mation. As shown in Fig. 4, the microalgae grew vigorously during three cycles of the
medium replacement with the biomass concentration reaching up to 2.482.76 g L-1 in
every cycle, but the microalgae grew slowly and reached lower level of biomass
(1.922.02 g L-1) during the fourth and fifth cycles. Afterwards no obvious growth was
observed. Up to and during 192 h of cultivation, the lipid accumulation continually
increased. The lipid content reached its maximum level of 44.9 % at 168 h. The glucose was
depleted during each cycle. The microalgae also produced EPS with a high productivity until
the 5th cycle. The EPS concentrations reached during the fourth and fifth cycles (1.441.46 g
L-1) were even higher than those in the first, second and third cycles (1.101.14 g L-1).
However, the chlorophyll content decreased along with the fermentation time.

The compositions of lipids and EPS and their application

The lipids extracted from marine Chlorella sp. were converted to fatty acid methyl ester
(FAME). Its major constituents were the two long-chain saturated fatty acids of palmitic

123
Aquacult Int

acid (C16:0) (54.7 %) and stearic acid (C18:0) (15.2 %), and its minor constituents were
unsaturated fatty acids of oleic acid (C18:1) (10.9 %), linoleic acid (C18:2) (3.86 %) and
linolenic acid (C18:3) (1.90 %). Two important chemical properties of biodiesel attributed
to the fatty acid profiles, iodine value (IV) and high heating value (HHV), of the microalgal
lipids were predicted following the theoretical calculation taking into consideration five
fatty acids, namely C16:0, C18:0, C18:1, C18:2 and C18:3 (Gopinath et al. 2009). The
predicted IV (g I2/100 g oil) of the microalgal lipids is 47.3 which follows the European
biodiesel standards (\130), and the predicted HHV is 39.9 which is close to those of
biodiesel derived from plant oils (3941) (Gopinath et al. 2009). In addition, it is also
known that the fatty acid profiles impact on the cetane number (CN) of the biodiesel.
Considering the percentage distribution of fatty acids and the empirical equation (Kris-
nangkura 1986), the microalgal lipids could produce biodiesel with the CN values in the
range of 5764. These values are higher than the minimal requirement for CN values in the
biodiesel standards ASTMD 6751 (USA), DIN 51606 (Germany) and EN 14214 (European
Organization), those have been set at 47, 49 and 51, respectively. Considering these
chemical properties, the microalgal lipids in this study have high potential to be used as
biodiesel feedstocks.
The major monosaccharide of EPS after dilute-acid hydrolysis was glucose. Since it has
been reported that the exopolysaccharides released by the microalgae could influence the
flocculation efficiency (Wu et al. 2012), its application as a bioflocculant was evaluated.
Microalgal cells having small sizes that ranged between 5 and 50 lm always formed stable
suspensions in the medium due to their negative surface charge. Their recovery from the
medium then becomes a critical step that can account for about 2030 % of the total
production costs of microalgal biomass (Gudin and Therpenier 1986). Among several
recovery methods including centrifugation, filtration and flocculation, flocculation is the
most cost-effective and convenient process that allows for a rapid recovery from a large
culture medium volume. Another one is to cultivate dense cells which will settle rapidly
once water motion is stopped and harvested by simple pumping (Shelef et al. 1984). Many
chemicals have been used as flocculants such as inorganic multivalent metal salts and
organic polymer (Duan and Gregory 2003; Vandamme et al. 2010).
In this study, the self-flocculation of the microalgal biomass cultivated in different
modes was compared (Fig. 5a). It was of interest that the flocculation efficiency of the
mixotrophic culture was the highest at 13.3 %. As the initial OD and pH were adjusted to
be the same at 1.8 and 10.0, respectively, before the flocculation test, the higher floccu-
lation efficiency in the mixotrophic culture was likely due to the high concentration of the
EPS (420 mg L-1). Several studies on microalgal harvesting have found that microalgal
extracellular organic matter could interfere with flocculation efficiency (Chen et al. 2009;
Wu et al. 2012). To further evaluate the effect of EPS on the flocculation efficiency, the
EPS produced during the mixotrophic cultivation was recovered. This recovered EPS was
then added into the microalgal suspension from the photoautotrophic culture that originally
contained EPS at 152 mg L-1 (Fig. 5b). Without the addition of EPS, there was no obvious
flocculation of the cells that occurred during the 60-min flocculation time. The flocculation
efficiency gradually increased with an increasing concentration of applied EPS. The
flocculation of the cells most likely required a minimum concentration of applied
EPS [ 300 mg L-1. The highest flocculation efficiency of 22 % was obtained at 500 mg
L-1 of the applied EPS. These results have shown that the EPS could be used as a
bioflocculant for harvesting the cells in the photoautotrophic culture, and it would be also
applied for the harvesting process of other species of microalgae.

123
 Aquacult Int

(A) (B)
30 30
Photoautotrophic (EPS:152 mg/L) Flocculation condition:
-photoautotrophic culture a
Heterotrophic (EPS:257 mg/L)
Flocculation efficiency (%)

25

Flocculation efficiency (%)

25 -initial EPS 152 mg/L
Mixotrophic (EPS:420 mg/L) ab
-time 60 min
20 20

15 15 c

10 10

5 5 d
d d d

0 0
0 10 20 30 40 50 60 0 50 100 200 300 400 500
Time (min) Applied EPS concentration (mg/L)

Fig. 5 a Flocculation efficiency of marine Chlorella sp. cultivated in BG11 medium under various
cultivation modes. The initial OD and pH were adjusted to be the same at 1.8 and 10.0, respectively, before
the flocculation test. b Effect of applied EPS concentration on flocculation efficiency of marine Chlorella sp.
cultivated in BG11 medium under photoautotrophic mode. Data are the mean values of triplicate
experiments. Different letters on the bars indicate significant differences between treatments (P \ 0.05)

Conclusions

A mixotrophic culture of marine Chlorella sp. produced highest yields of biomass, lipids
and exopolymeric substances. Several factors affecting mixotrophic cultivation were
optimized. A semi-continuous cultivation with intermittent medium replacement and
glucose additions was an effective method to maximize the biomass yield with high lipid
content and also the amount of exopolymeric substances. The lipids and exopolymeric
substances produced by marine Chlorella sp. have high potential to be used as biodiesel
feedstocks and bioflocculant, respectively. The concomitant production of these valuable
products together with the microalgal biomass would be a potential way to offset the
production cost and contribute greatly to developing industrialized microalgae cultivation.

Acknowledgments The authors are grateful to the financial support by Prince of Songkla University in the
fiscal year of 2012 under Grant AGR550135S. The first and third authors are also supported by Thailand
Research Fund under Grant No. RTA5780002. Thanks are also given to Dr. Brian Hodgson Faculty of
Pharmaceutical Science, Prince of Songkla University, for assistance with the English.

References
Becker EW (1994) Microalgae: biotechnology and microbiology. Cambridge University Press, New York
Bermudez J, Rosales N, Loreto C, Briceno B, Morales E (2004) Exopolysaccharide, pigment and protein
production by the marine microalga Chroomonas sp. in semicontinuous cultures. World J Microbiol
Biotechnol 20:179183
Buckwalter P, Embaye T, Gormly S, Trent JD (2013) Dewatering microalgae by forward osmosis.
Desalination 312:1922
Cheirsilp B, Torpee S (2012) Enhanced growth and lipid production of microalgae under mixotrophic
culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour
Technol 110:510516

123
Aquacult Int

Chen L, Li P, Liu Z, Jiao Q (2009) The released polysaccharide of the cyanobacterium Aphanothece
halophytica inhibits flocculation of the microalga with ferric chloride. J Appl Phycol 21:327331
Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25:294306
Chiu SY, Kao CY, Chen CH, Kuan TC, Ong SC, Lin CS (2008) Reduction of CO2 by a high-density culture
of Chlorella sp. in a semicontinuous photoreactor. Bioresour Technol 99:33893396
Danesi EDG, Rangel-Yagui CO, Sato S, de Carvalho JCM (2011) Growth and content of Spirulina platensis
biomass chlorophyll cultivated at different values of light intensity and temperature using different
nitrogen sources. Brazil J Microbiol 42(1):362373
Dayananda C, Sarada R, Rani MU, Shamala TR, Ravishankar GA (2007) Autotrophic cultivation of
Botryococcus braunii for the production of hydrocarbons and exopolysaccharides in various media.
Biomass Bioenergy 31:8793
de Oliveira MACL, Monteiro MPC, Robbs PG, Leite SGF (1999) Growth and chemical composition of
Spirulina maxima and Spirulina platensis biomass at different temperatures. Aquacult Int 7(4):261275
Duan J, Gregory J (2003) Coagulation by hydrolysing metal salts. Adv Colloid Interface Sci
100102:475502
Fagiri YMA, Salleh A, El-Nagerabi SAF (2013) Influence of chemical and environmental factors on the
growth performance of Spirulina platensis strain SZ100. J Algal Biomass Utilization 4(2):715
Ferruzzi MG, Blakeslee J (2007) Digestion, absorption, and cancer preventative activity of dietary
chlorophyll derivatives. Nutr Res 27(1):112
Folch J, Lees M, Stanley GHS (1957) A simple method for the isolation and purification of total lipides from
animal tissues. J Biol Chem 226:497509
Frumento D, Casazza AA, Al Arni S, Converti A (2013) Cultivation of Chlorella vulgaris in tubular
photobioreactors: a lipid source for biodiesel production. Biochem Eng J 81:120125
Girard JM, Roy ML, Hafsa MB, Gagnon J, Faucheux N, Heitz M, Tremblay R, Deschenes JS (2014)
Mixotrophic cultivation of green microalgae Scenedesmus obliquus on cheese whey permeate for
biodiesel production. Algal Res 5:241248
Goo BG, Baek G, Choi DJ, Park YI, Synytsya A, Bleha R, Seong DH, Lee CG, Park JK (2013) Charac-
terization of a renewable extracellular polysaccharide from defatted microalgae Dunaliella tertiolecta.
Bioresour Technol 129:343350
Gopinath A, Puhan S, Nagarajan G (2009) Theoretical modeling of iodine value and saponification value of
biodiesel fuels from their fatty acid composition. Renew Energy 34:18061811
Gudin C, Therpenier C (1986) Bioconversion of solar energy into organic chemicals by microalgae. Adv
Biotechnol Process 6:73110
Haass D, Tanner W (1974) Regulation of hexose transport in Chlorella vulgaris. Plant Physiol 53:291302
Heredia-Arroyo T, Wei W, Hu B (2010) Oil accumulation via heterotrophic/mixotrophic Chlorella pro-
tothecoides. Appl Biochem Biotechnol 162:19781995
Hossain ABM, Salleh A, Boyce AN, Chowdhurry P, Naqiuddin M (2008) Biodiesel fuel production from
algae as renewable energy. Am J Biochem Biotechnol 4:250254
Jham GN, Teles FFF, Campos LG (1982) Use of aqueous HCl/MeOH as esterification reagent for analysis of
fatty acids derived from soybean lipids. J Am Oil Chem Soc 59:132133
Krisnangkura K (1986) A simple method for estimation of Cetane index of vegetable oil methyl esters. J Am
Oil Chem Soc 63:552553
Lee YK (2004) Algal nutrition. Heterotrophic carbon nutrition. In: Richmond A (ed) Handbook of
microalgal culture. Biotechnology and applied phycology. Blackwell Scientific Publications Ltd,
Oxford, p 116
Liang YN, Sarkany N, Cui Y (2009) Biomass and lipid productivities of Chlorella vulgaris under auto-
trophic, heterotrophic and mixotrophic growth conditions. Biotechnol Lett 31:10431049
Liu J, Tao Y, Wu J, Zhu Y, Gao B, Tang Y, Li A, Zhang C, Zhang Y (2014) Effective flocculation of target
microalgae with self-flocculating microalgae induced by pH decrease. Bioresour Technol 167:367375
Lizzul AM, Hellier P, Purton S, Baganz F, Ladommatos N, Campos L (2014) Combined remediation and
lipid production using Chlorella sorokiniana grown on wastewater and exhaust gases. Bioresour
Technol 151:1218
Ma L, Dolphin D (1999) The metabolites of dietary chlorophylls. Phytochem 50:195202
Martnez ME, Camacho F, Jimenez JM, Espnola JB (1997) Influence of light intensity on the kinetic and
yield parameters of Chlorella pyrenoidosa mixotrophic growth. Process Biochem 32:9398
Mata TM, Martins AA, Caetano NS (2010) Microalgae for biodiesel production and other applications, a
review. Renew Sust Energy Rev 14:217232
Metzger P, Largeau C (1999) Chemicals of Botryococcus braunii. In: Cohen Z (ed) Chemicals from
microalgae. Taylor and Francis, London, pp 205260

123
 Aquacult Int

Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem
31:426429
Qin J (2005) Bio-hydrocarbons from algae, impacts of temperature, light and salinity on algae growth. A
report for the rural industries research and development corporation. Australia
Richmond A (1986) Handbook of microalgal mass culture. CRC Press Inc., Boca Raton
Shelef G, Sukenik A, Green M (1984) Microalgae harvesting and processing: a literature review. Technion
Research and Development Foundation Ltd., Haifa
Sterin JR (1973) Handbook of phycological methods, culture methods and growth measurements. Cam-
bridge University Press, Cambridge
Suarez ER, Kralovec JA, Noseda MD, Ewart HS, Barrow CJ, Lumsden MD, Grindley TB (2005) Isolation,
characterization and structural determination of a unique type of arabinogalactan from an immunos-
timulatory extract of Chlorella pyrenoidosa. Carbohydr Res 340:14891498
Sudhir P, Murthy SDS (2004) Effect of salt stress on basic processes of phytosynthesis. Photosynthetica
42:481486
Tansakul P, Savaddiraksa Y, Prasertsan P, Tongurai C (2005) Cultivation of the hydrocarbon-rich alga,
Botyococcus braunii in secondary treated effluent from a sea food processing plant. Thai J Agri Sci
38(12):7176
Vandamme D, Foubert I, Meesschaert B, Muylaert K (2010) Flocculation of microalgae using cationic
starch. J Appl Phycol 22:525530
Villay A, Laroche C, Roriz D, Alaoui HE, Delbac F, Michaud P (2013) Optimisation of culture parameters
for exopolysaccharides production by the microalga Rhodella violacea. Bioresour Technol 146:
732735
Wang SK, Hu YR, Wang F, Stiles AR, Liu CZ (2014) Scale-up cultivation of Chlorella ellipsoidea from
indoor to outdoor in bubble column bioreactors. Bioresour Technol 156:117122
Wu Z, Zhu Y, Huang W, Zhang C, Li T, Zhang Y, Li A (2012) Evaluation of flocculation induced by pH
increase for harvesting microalgae and reuse of flocculated medium. Bioresour Technol 110:496502
Yeesang C, Chanthachum S, Cheirsilp B (2008) Sago starch as a low-cost carbon source for exopolysac-
charide production by Lactobacillus kefiranofaciens. World J Microbiol Biotechnol 24(7):11951201
You T, Barnett SM (2004) Effect of light quality on production of extracellular polysaccharides and growth
rate of Porphyridium cruentum. Biochem Eng J 19:251258

123