Biofuels

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Biodiesel production from Nannochloropsis oculata

cultured at stressful carbon dioxide concentration
and light illumination

Jun Zhang, Wen-Yi Tsai, Chao-Hung Hsu & Ching-An Peng

To cite this article: Jun Zhang, Wen-Yi Tsai, Chao-Hung Hsu & Ching-An Peng (2020): Biodiesel
production from Nannochloropsis�oculata cultured at stressful carbon dioxide concentration and
light illumination, Biofuels, DOI: 10.1080/17597269.2020.1787699

To link to this article: https://doi.org/10.1080/17597269.2020.1787699

Published online: 03 Jul 2020.

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BIOFUELS
https://doi.org/10.1080/17597269.2020.1787699

Biodiesel production from Nannochloropsis oculata cultured at stressful

carbon dioxide concentration and light illumination
Jun Zhanga†, Wen-Yi Tsaib†, Chao-Hung Hsuc and Ching-An Penga
a
Department of Biological Engineering, University of Idaho, Moscow, ID, USA; bDepartment of Chemical Engineering, National Taiwan
University, Taipei, Taiwan; cALG Power Technology Corporation, Pingtung, Taiwan

ABSTRACT ARTICLE HISTORY

Nannochloropsis oculata is a promising source of biodiesel due to the production of cellular lipid Received 5 February 2020
which can be converted to biodiesel through transesterification reaction. In this study, N. oculata Accepted 21 June 2020
cells were cultured under a medium light intensity and high CO2 concentrations to improve cell
KEYWORDS
growth and cellular lipid content. With an appropriate initial cell concentration, a 1.5-fold increase in
Nannochloropsis oculata;
cell number and a 4-fold increase in lipid content were achieved when N. oculata was cultured at photoinhibition; CO2 stress;
a medium light intensity of 300 mEm
2s
1 and 10-15% CO2 compared to cells cultured at a low light biodiesel; lipid
intensity of 30 mEm
2s
1 and 0.037% CO2. The cultivation of N. oculata was further scaled up in a
photobioreactor. When cultured with medium light intensity and high CO2 concentration, N. oculata
can obtain higher cell concentrations and lipid amounts. The microalgal biodiesel analyzed by gas
chromatography revealed that high yield was achieved by culturing N. oculata under the combin-
ation of medium light illumination and high CO2 concentration. When cultured in a scaled-up photo-
bioreactor under stressful illumination and high CO2 concentrations, N. oculata not only showed
improved cell concentrations and lipid content, but also showed increased CO2 depletion compared
to cells cultured under low light and low CO2.

Introduction engines. In the 1980s, a chemical modification of natural

The majority of the global energy needs are currently sup-
plied through petrochemical sources, coal and natural gas.
These sources are finite and unsustainable at the current
rates of usage. Moreover, the production and burning of
fossil fuel energy is the main contributor to the accumula-
tion of carbon dioxide (CO2) in the atmosphere. CO2 is rec-
ognized as a major component of the atmospheric
pollutants adding to the “greenhouse effect,” described as
the trapping of heat in the Earth’s atmosphere by gases
capable of absorbing radiation. In order to slow global
warming, limiting the CO2 discharged by current industries
and vehicles is a main goal around the world. Many meth-
ods are employed to reduce CO2, one of which is biological
treatment, i.e. using photosynthetic organisms including
green plants, algae, and photosynthetic bacteria to trap
CO2 by photosynthesis. Microalgae have been found to be
particularly efficient as photosynthesizers. Additionally,
microalgae can be cultured in small containers with a rela-
tively fast growth rate, which makes them a suitable candi-
date as a CO2 sink [1].
The concept of using microalgae as a source of fuel is
not new. However, due to the diminishing sources of pet-
roleum and more significantly, the emerging concern about
global warming that is associated with burning fossil fuels,
there is renewed interest in research and development of
industrial scale production. Non-polar lipids in microalgae,
like those in plant and animal oils, are mainly triacylglycer-
ols (TAGs) which are too viscous to be used in diesel
oils was introduced that helped to bring the viscosity of
microalgal oils to a level similar to that of petroleum diesel.
By reacting microalgal TAGs with alcohols via transesterifi-
cation, one can create fatty acid methyl ester (FAME)
known as biodiesel [2]. Producing biodiesel from microal-
gae has been considered as an efficient way to make bio-
diesel fuel compared to other oilseed crops. The main
advantages of deriving biodiesel from microalgae oil
include reduced emissions of air toxins and carcinogens,
high growth rates, high areal yield, algae can be harvested
daily, highly biodegradable and renewable. For these rea-
sons, algae are able to produce more oil per unit area com-
pared to oil seed crops [1]. There have been many studies
looking at biodiesel production from microalgae [3–7].
Nannochloropsis oculata is a marine unicellular alga
belonging to the Eustigmatophyceae class. It has a spher-
ical shape with a diameter of about 2–4 mm without flagel-
lum or eyespots. The color of the cell is green, but as it
ages it begins to turn yellow. Due to the fact that it has
been confirmed as one of the photoautotrophic producers
of eicosapentaenoic acid (EPA) [8], N. oculata has been
widely used as feed for zooplankton in fish hatcheries and
a source of x-3 polyunsaturated fatty acid for human
use [9].
Carbon/nitrogen (C/N) ratio has been altered in different
algae types to achieve high growth rate or high lipid con-
tent accumulation [10, 11]. CO2 is a commonly used carbon
source for the aeration of algae cultures and manipulation
of relative C/N ratio. N. oculata has previously been studied

CONTACT Ching-An Peng capeng@uidaho.edu Department of Biological Engineering, University of Idaho, 875 Perimeter Drive, Moscow, 83844-0904
ID, USA
†
Equal contribution.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
2 J. ZHANG ET AL.
with regards to CO2 utilization [12–14]. Hoshida et al. reported
that the maximum EPA production was obtained when 2%
CO2 was applied 12 h prior to the end of the exponential
growth phase [15]. Hsueh et al. studied the carbon bio-fix-
ation by photosynthesis of N. oculata [16]; they reported that
the maximum growth rate was obtained at 5% and 8% CO2.
At 10% CO2 concentration, algal growth was inhibited.
The CO2 concentration normally discharged from facto-
ries and power plants is typically at concentrations of
10–15% [17]. Consequently, such high CO2 concentrations
would result in growth inhibition for most microalgae. The
objective of this study is to design a N. oculata culturing
system using stressful light illumination and CO2 concentra-
tions to overcome the photoinhibition problem and thus
achieve improved cellular growth and lipid content.
Stressful light intensity, which also can lead to photoinhibi-
tion of microalgal growth, was tested along with high CO2
concentrations and pertinent initial cell concentrations to
achieve both high biomass yield and lipid content in N.
oculata microalgae.
Materials and methods
Microalgae and medium
N. oculata was supplied from the Tung-Kang Fisher Research
Institute (Pingtung, Taiwan). Seawater (3.5% salinity) was
obtained from the Institute of Fisheries Science (National
Taiwan University, Taiwan). Modified Walne medium consisted
of 100 mg sodium nitrate (NaNO3), 20 mg monosodium phos-
phate (NaH2PO4), 45 mg disodium ethylenediaminetetraace-
tate dihydrate (Na2EDTA), 33.6 mg boric acid (H3BO3), 0.4 mg
manganese(II) chloride (MnCl2), 1.3 mg iron(III) chloride hexa-
hydrate (FeCl3 . 6H2O), 0.044 mg zinc borate (B2O6Zn3),
0.02 mg cobalt(II) chloride hexahydrate (CoCl26H2O), 0.009 mg
sodium molybdate dihydrate (Na2MoO42H2O), 0.02 mg cop-
per sulfate pentahydrate (CuSO45H2O), 0.2 mg vitamin B12,
and 0.01 mg vitamin B1 per liter [18]. N. oculata was inocu-
lated into a 250-mL flask containing 150 mL sterilized sea-
water enriched with modified Walne medium under room
temperature (27 ± 2  C) and light intensity of 30 mEm
2s
1
with air-pump aeration rate of 10 mL min
1. Light was pro-
vided continuously from above with cool-white fluorescent
lights, and was measured at the bottom center of the flask
by using a Quantum Meter (QMSW-SS; Apogee Instruments,
Logan, UT, USA).
Effect of light intensity and CO2 concentration on
algal growth
Stock cultures of N. oculata were grown in 200-mL Roux-type
flat culture bottles containing 160 mL medium and illuminated
at a light intensity of 30 mEm
2s
1 at room temperature with
air-pump aeration. Experimental cultures were inoculated from
stock cultures which were in the late exponential phase of
growth to a starting concentration of 1  107 cells mL
1.
In order to investigate the effects of light intensity on the
growth of N. oculata, light intensities of 25, 50, 100, 200, 300,
and 400 mEm
2s
1 were tested, respectively. It should be
noted that the stressful illumination used in this study (i.e. 
300 mEm
2s
1) is in the range of medium light intensity,
compared to high solar intensity around 1500–2000
mEm
2s
1 in equatorial desert regions. CO2 concentration was
fixed at 0.037% (normal air). Second, the effect of CO2 con-
centration on the growth of N. oculata was also studied. The
CO2 concentrations were supplied at 5, 10, and 15% CO2
mixed with air, and normal air (0.037% CO2) was used as a
control set. Light intensity was fixed at 25 mEm
2s
1. The CO2
concentrations were measured using an Infra-Red CO2
Monitor (D-500, Edinburgh Instruments, Livingston, UK).
Microalgal cultures were aerated and stirred by bubbling
from the bottom of the flask. Finally, the combined effect of
stressful medium light intensity and high CO2 concentration
on microalgal culture was also examined. All experiments
were performed with at least triplicate samples.
Photobioreactor
The cultivation of N. oculata was further scaled up in a photo-
bioreactor, which consisted of a series of five 1.5-L bottles
with 1.4 L of medium in each photobioreactor. Aeration was
provided via an air-stone located near the bottom of the bio-
reactor. The air and CO2 flow rates were set at 10 mL min
1
with a flowmeter. The experimental conditions were as fol-
lows: 30 mEm
2s
1 and 0.037% CO2 (A), 30 mEm
2s
1 and
10% CO2 (B), 300 mEm
2s
1 and 0.037% CO2 (C), 300
mEm
2s
1 and 10% CO2 (D), with an initial cell concentration
of 107 cells mL
1. The cultivation temperature was controlled
at ambient temperature. The bioreactors were illuminated
continuously from the side, and intensity was measured at
the nearest point between the bottle and the light source.
The effluent gases were collected daily with air sampling
bags (Restek, Bellefonte, PA, USA) and then measured with an
Infra-Red CO2 Monitor (D-500).
Cell concentration and specific growth rate
The number of cells were counted by using a microscope
(Eclipse TE2000-U, Nikon, USA) with a hemocytometer
(Neubauer improved bright-line, Marienfeld, Germany). Dry
weight of microalgal cells was measured after centrifuga-
tion and freeze-drying. Specific growth rate (l, d
1) was
calculated as follows:
l ¼ ln ðCf
Ci Þ=Dt
where Cf and Ci are the final and initial cell concentrations,
respectively, and Dt is the cultivation time in days.
Cellular lipid determination
To examine the cellular lipid content, Nile red (9-diethyla-
mino-5H-benzo(a)phenoxazine-5-one, Acros, USA) was used
as previously reported [19–21], with some modifications.
Microalgal cells were collected by centrifugation and re-
suspended with deionized water to obtain 2 mL of 2  107
cells mL
1. The cell suspension was stained with 2-mL Nile
red solution (1 mg mL
1 in acetone). The fluorescent inten-
sity was obtained within the region 500-700 nm with
475 nm as the excitation wavelength with a Vis-NIR
Fluorescent Spectrometer (FP-6300, Jasco, Japan). Since N.
oculata cells are auto-fluorescent, background fluorescence
levels were determined before cells were stained. The rela-
tive fluorescent intensity of Nile red was calculated as
(fluorescent intensity of Nile red stained) minus (auto-

Figure 1. (a) Growth curves rate of N. oculata cultured with normal air and under light intensities of (i-vi) 25, 50, 100, 200, 300 and 400 mEm
2s
1. (b) Specific
growth rate of N. oculata cultured with normal air and under light intensities of (i-vi) 25, 50, 100, 200, 300 and 400 mEm
2s
1. (c) Growth curves values of N.
oculata cultured at different CO2 concentrations under light intensity of 30 mEm
2s
1. (d) Variation of pH values of N. oculata cultured at different CO2 concen-
trations under light intensity of 30 mEm
2s
1. Data represented mean ± standard deviation (n ¼ 3).
fluorescent intensity). N. oculata cells cultured at various con-
ditions were imaged by a light microscope (Eclipse TE2000-U,
Nikon, USA). The size distribution of N. oculata cells were
determined by static light scattering method using a Particle
Size Analyzer (LS 230, Beckman Coulter, USA).
Lipid extraction
The microalgal cells were collected by centrifugation at
9000 rpm for 5 min, and then washed twice with deionized
water. The cells were dried using a freeze dryer, then pulv-
erized with a mortar and pestle. The samples were sus-
pended with methanol/chloroform solution (2:1, v/v), and
extracted with a Soxhlet extractor for 6 h. Chloroform and
1% sodium chloride (NaCl) were added into the mixture to
obtain a ratio of methanol, chloroform, and water of 2:2:1.
The mixture was centrifuged at 9,000 rpm for 10 min, and
the lower layer containing organic lipid was removed and
saved. Finally, the chloroform was removed under vacuum
in a rotary evaporator at 80  C (N-1000, EYELA, Japan) leav-
ing behind the extracted lipids.
FAME production
Approximately 0.2 g microalgal lipid was placed in a tube, dis-
solved with 1 mL n-hexane, and 1 mL internal standard (hep-
tadecanoic acid methyl ester C17:0, 0.08 g in 10 mL n-hexane)
was added, followed by 1 mL 1 N methanolic sodium hydrox-
ide (NaOH) solution, then filled with nitrogen (N2) and the
tube was closed tightly. The tube was vortexed for 30 s and
then saponification was allowed to proceed at 80  C for
15 min. After the tube cooled down, 1 mL 14% methanolic
BIOFUELS 3
boron trifluoride (BF3) solution was added, refilled with N2
and vortexed for 30 s, followed by esterification at 110  C for
15 min. While the tube cooled down, 1 mL n-hexane was
added, the solution was vortexed for 1 min. 6 mL of saturated
NaCl solution was added to the tube, slowly shook, and then
laid down in order to allow the solution to separate into two
phases. The lower layer was collected and dehydrated by
adding anhydrous sodium sulphate and then passing the
solution through a 0.45 mm polytetrafluoroethylene (PTFE)
membrane to obtain the FAME for further analysis.
Gas chromatography
The composition of the FAME was analyzed by gas chroma-
tography (GC) using a column of SP-2380 with dimensions of
60 m  0.25 mm  0.25 mm and a flame ionization detector
(FID). Approximately 1 mL of sample was injected into the col-
umn. The parameters for the oven temperature program
were as follows: starting temperature was 150  C for 5 min,
ramped by 2  C min
1 to 250  C, and then maintained for
15 min. The injector and detector were maintained at 220
and 250  C, respectively. N2 in a split ratio of 100:1 was used
as the carrier gas with a flow rate of 12 cm/sec. The retention
time of the components of FAME was compared with
Supelco 37 component FAME mix standard.
Results and discussion
Effect of light intensity and CO2 concentration on
microalgal growth
The effect of light intensity on the growth of N. oculata
was investigated in batch cultures incubated with an
4 J. ZHANG ET AL.

Figure 2. (a) Growth curves of N. oculata cultured with an initial cell concentration of 106 cells mL
1 and LL: 30 mEm
2s
1, ML: 300 mEm
2s
1, LC: 0.037% CO2, HC:
10% CO2. (b) Growth curves of N. oculata cultured with an initial cell concentration of 107 cells mL
1 and LL: 30 mEm
2s
1, ML: 300 mEm
2s
1, LC: 0.037% CO2, HC:
10% CO2. (c). Growth curves of N. oculata cultured with an initial cell concentration of 107 cells mL
1 and LL: 30 mEm
2s
1, ML: 300 mEm
2s
1, LC: 0.037% CO2,
HC: 15% CO2. (d) Growth curves of N. oculata cultured with an initial cell concentration of 108 cells mL
1 and LL: 30 mEm
2s
1, ML: 300 mEm
2s
1, LC: 0.037% CO2,
HC: 15% CO2. (e) Representative images of N. oculata culture with initial cell concentration of 108 cells mL
1 in Roux-type culture bottles. Cells were cultured for 12 days
with conditions LL: 30 mEm
2s
1, ML: 300 mEm
2s
1, LC: 0.037% CO2, HC: 15% CO2. Irradiance stress induces accumulation of EPA. These changes are reflected in the
yellow-orange coloration of the ML-acclimated (grown) cells. Data represented mean ± standard deviation (n ¼ 3).

irradiance ranging from 30 to 400 mEm
2s
1. Cell concen- cultivation. The maximum cell concentration was obtained
tration was determined by sampling from the cultivation while cultured at 100 mEm
2s
1 with an initial cell concen-
vessel daily. Figure 1(a) shows the growth curves of N. ocu- tration of 107 cells mL
1, increasing to 4.76 ± 1.13  107
lata under different light intensities over 10 days of cells mL
1 by day 10. The specific growth rate (Figure 1(b))
 BIOFUELS 5

Figure 3. (a) Representative pH variation during cultivation of N. oculata with an initial cell concentration of 107 cells mL
1 and LL: 30 mEm
2s
1, ML: 300
mEm
2s
1, LC: 0.037% CO2, HC: 10% CO2. (b) Representative pH variation during cultivation of N. oculata with an initial cell concentration of 108 cells mL
1
and LL: 30 mEm
2s
1, ML: 300 mEm
2s
1, LC: 0.037% CO2, HC: 15% CO2. (c) Representative relative fluorescent intensity of Nile red stained cells during cultiva-
tion of N. oculata cultured at an initial cell concentration of 107 cells mL
1 and LL: 30 mEm
2s
1, ML: 300 mEm
2s
1, LC: 0.037% CO2, HC: 10% CO2. and (d)
Representative relative fluorescent intensity of Nile red stained cells during cultivation of N. oculata cultured at an initial cell concentration of 108 cells mL
1
and LL: 30 mEm
2s
1, ML: 300 mEm
2s
1, LC: 0.037% CO2, HC: 15% CO2.

was observed to increase with increasing light intensity from
25 to 100 mEm
2s
1, with a maximum specific growth rate
measured at 100 mEm
2s
1 of 0.21 d
1. When the illumin-
ation supplied was over 100 mEm
2s
1, the specific growth
rate decreased, a process called photoinhibition [22].
The growth of N. oculata under different CO2 concentra-
tions, with an initial cell concentration of 107 cells mL
1, is
shown in Figure 1(c). After 8 days culturing, the cell con-
centrations measured were 4.55  107, 7.28  107,
3.69  107 cells mL
1 when aerated with 0.037, 5, 10% CO2,
respectively. The specific growth rates were 0.189 d
1,
0.248 d
1, 0.163 d
1, respectively (data not shown). The
growth of N. oculata when supplied with 15% CO2 was
completely inhibited. The pH values of the culture media
are shown in Figure 1(d). It was observed that while cul-
tured with ambient air (containing 0.037% CO2), the pH
increased dramatically within 2 days. As the microalgae
grow, the CO2 in the culture medium is consumed which
results in the decrease of Hþ ions, thus increasing the pH
of the culture medium [23]. In contrast, as CO2-enriched
gas is supplied, the pH value of the cultured medium is
inversely proportional to CO2 concentration. It is surmised
that when the pH is too low, it may be responsible for the
observed inhibition of algal growth at higher CO2 concen-
trations [16]. In this study, we observed that N. oculata cells
grew well at pH values above 6.5, and cell growth was
inhibited when the pH was less than 6.
Combination of light and CO2
It was evident from this study that a light intensity of 300
mEm
2s
1 resulted in significant photoinhibition of N.
oculata. Furthermore, CO2 concentrations over 10% also
inhibited cell growth. With this in mind, attempts to utilize
CO2 discharged from factories and power plants (which is
typically > 15%) for the growth of N. oculata would be
problematic. However, interesting results were observed
when high CO2 concentrations and photoinhibitive light
intensities were combined.
When beginning with an initial cell concentration of 106
cells mL
1, a maximum cell concentration of was obtained
when N. oculata was cultured at 30 mEm
2s
1 and 0.037%
CO2 (Figure 2(a)). In contrast, when a high CO2 concentra-
tion of 10% or a medium light intensity of 300 mEm
2s
1
were used, the growth of N. oculata was inhibited. When
medium light intensity and high CO2 concentration were
supplied simultaneously, the growth rate was also poor.
When the initial cell concentration was increased to 107
cells mL
1, medium light intensity or high CO2 concentra-
tion were also inhibitory (Figure 2(b)). However, compared
to an initial cell concentration of 106 cells mL
1, increased
cell growth was observed with a higher initial cell concen-
tration of 107 cells mL
1 when cultured at 30 mEm
2s
1
and 10% CO2. This suggests that a higher initial cell con-
centration can allow cells to endure high CO2 concentra-
tions, which is consistent with previous findings [16].
However, after 5 days of growth, the cell concentration
under 10% CO2 was lower than that of cultures supplied
with ambient air (0.037% CO2) when the light intensity was
kept constant at 30 mEm
2s
1. In contrast, when inhibitive
light intensity and high CO2 concentration were supplied
simultaneously, the cell concentration reached 90  106 cell
mL
1, 2 times the concentration reached when supplying
low light intensity and atmospheric CO2. Since medium
6 J. ZHANG ET AL.
Figure 4. Cell size distribution of N. oculata cultured at (A) 30 mEm
2s
1 þ 0.037% CO2; (B) 30 mEm
2s
1 þ 10% CO2; (C) 300 mEm
2s
1 þ 0.037% CO2; (D)
300 mEm
2s
1 þ 10% CO2 with an initial cell concentration of 107 cells mL
1.
light intensity inhibits algal growth, association with 10%
CO2 might reach a balance between the medium light and
high CO2 enhancing the photosynthetic rate, thus allowing
CO2 to be quickly depleted. The variation of pH values dur-
ing the cultivation period are shown in Figure 3(a). As
seen, the pH was measured at 5.8–6.2 when cultured at 30
mEm
2s
1 and 10% CO2. The pH increased slightly to
6.2–6.5 when both medium light intensity and high CO2
concentrations were used. A pH of 6.5 is suitable for N.
oculata growth and indicates that, when supplied by 300
mEm
2s
1 and 10% CO2, more CO2 has been photosynthe-
sized and consumed by the cells. As CO2 is depleted, the
pH of the medium increased to a level which would not
inhibit microalgal growth.
The above experiment was repeated substituting 15%
CO2 for 10% CO2. When 15% CO2 was supplied, medium
light intensity associated with high CO2 concentration led
to growth inhibition, in contrast to that of weak light and
normal air (0.037% CO2) when cultured at an initial cell
density of 107 cells mL
1 (Figure 2(c)). This result is similar
to the results seen when the culture was supplied with
10% CO2 at an initial cell concentration of 106 cells mL
1
(Figure 2(a)). Having observed increased tolerance to CO2
concentration with an increase in the starting cell density
when using 10% CO2, we increased the inoculation cell
concentration to 108 cells mL
1 and ran the test again
with 15% CO2. As Figure 2(d) shows, when culturing under
medium light intensity or high CO2 concentration separ-
ately, microalgal growth was inhibited. However, when
combining these two inhibitory conditions, i.e. 300
mEm
2s
1 and 15% CO2, a high specific growth rate was
obtained in N. oculata culture. An almost 1.5-fold increase
in cells growth was obtained when combining medium
light intensity and high CO2 concentration in comparison
to using either separately. The pH value at medium light
intensity was higher than at weak light intensity when sup-
plied with 15% CO2 (Figure 3(b)), which was similar to the
pH when the culture was supplied with 10% CO2 combined
with medium light intensity at an initial cell concentration
of 107 cells mL
1. It should be noted that continuous bub-
bling a fixed percent of CO2 is not an economic approach
to acidify the culture medium. An improvement way is to
supply CO2 as needed into the airstream using a pH con-
troller to prevent over-acidifying the medium and avoid
the wasteful usage of CO2.
Lipid content
The lipid content was measured by using the Nile Red
method and fluorescent intensity was measured before
and after cells were stained. The relative fluorescent inten-
sity of Nile Red dye was obtained after subtraction of the
auto-fluorescence of microalgal cells from the gross read-
ing at 575 nm. For both the initial cell concentrations of
107 cells mL
1 and 108 cells mL
1, the relative fluorescent
intensity was highest when culturing with both medium
light intensity and high CO2 concentration, and the second
highest is with weak light intensity and high CO2
 BIOFUELS 7

Figure 5. (a) Representative growth curves of N. oculata cultured in a photobioreactor with (A) 30 mEm
2s
1 and 0.037% CO2; (B) 30 mEm
2s
1 and 10% CO2;
(C) 300 mEm
2s
1 and 0.037% CO2; (D) 300 mEm
2s
1 and 10% CO2. (b and c) GC histogram of FAME from N. oculata cultured at (a) 30 mEm
2s
1 and
0.037% CO2 and (b) 300 mEm
2s
1 and 10% CO2. (d) Representative effluent CO2 concentration from photobioreactor with N. oculata cultured at (B) 30
mEm
2s
1 and 10% CO2 and (D) 300 mEm
2s
1 and 10% CO2.

concentration over a culture of 12 days (Figure 3(c) and CO2 concentration is combined with medium light inten-
(d)). Higher lipid content can be obtained when the cul- sity, the lipid content is 4-fold higher than when combined
tures are stressed by higher CO2 concentrations, consistent with weak light, most likely because cells are additionally
with results observed by others [24]. Moreover, when high stressed and going into survival mode. Growing N. oculata
8 J. ZHANG ET AL.
Table 1. Composition of FAME produced from N. oculata cultured in a pho-
tobioreactor under (A) 30 lEm
2s
1 þ 0.037% CO2 and (D) 300 lEm
2s
1
þ 10% CO2.
Content (%)
Number FAME A D
1 Methyl myristate (C14:0) 5.26
2 Methyl palmitate (C16:0) 34.42
3 Methyl palmitoleate (C16:1) 24.63
4 Methyl heptadecanoate (C17:0)
5 Methyl stearate (C18:0) 0.88
6 Methyl oleate (C18:1) 12.03
7 Methyl linoleate (C18:2) 3.20
8 Methyl homogamma linolenate (C20:3) 2.92
Saturated FAME 40.56
Saturated FAME 42.78
Others 16.66
Total lipid 14.74
under medium light intensity and high CO2 concentration
can potentially not only increase the biomass, but also
advance the lipid content when beginning with an optimal
initial cell concentration. The diameter of N. oculata cells
became larger when cultured at high CO2 concentrations,
especially when associated with medium light intensity
regardless of the initial cell concentration used (data now
shown). Increased cell size might be due to the higher lipid
accumulation in the microalgal cells, as suggested by the
Nile Red staining data. Figure 4 shows the cell size distribu-
tion analysis of N. oculata. When cultured at medium light
intensity and high CO2 concentration, the particle size
increased. This result is in accordance with the fluorescent
intensity of stained cells.
Scaled-up photobioreactor
Microalgal cultivation was scaled up in a 7.5-L photobior-
eactor (i.e. five of 1.5-L bottles connected in series) with
the light intensity and CO2 concentration being 30, 300
mEm
2s
1 and 0.037, 10%, respectively. Figure 5(a) shows
the growth curves of N. oculata cultured with the four con-
ditions. The highest cell concentration was obtained while
N. oculata was cultured at 300 mEm
2s
1 and 10% CO2,
which agreed with previous results. The dry weight after
culturing for 10 days was measured after centrifugation and
freeze-drying. The dry weight of N. oculata was 0.24, 0.21,
0.18 and 0.71 g L
1 for conditions A–D, respectively. The
dry weight of microalgae under medium light intensity and
high CO2 concentration was almost 3-fold higher than that
of low light and low CO2. However, the cell concentration
was only 2.24-fold higher, indicating that this was because
the particle size was larger when N. oculata was cultured at
medium light intensity and high CO2 concentration and
thus, the dry weight per cell was heavier. Table 1 shows
the composition of FAME produced under low light illumin-
ation and CO2 concentration and medium light illumination
and CO2 concentration (i.e. condition A and D, respect-
ively), the lipid content was about 15% for condition A and
about 25% for condition D. This result of higher lipid con-
tent per cell, obtained from the scaled-up photobioreactor
under the stress conditions of medium light intensity and
high CO2 concentration, was in line with the Roux-type flat
bottle culture utilizing Nile Red dye for examination of lipid
contents at four different conditions. In addition, the poly-
saturated FAME also increased while cultured at medium
light intensity and high CO2 concentration, reaching 50%
of total FAME, in contrast to normal light intensity and CO2
concentration (about 40%). Previous studies showed that
increased levels of CO2 favored the total lipid content, but
decreased the amounts of polyunsaturated fatty acids [24].
Polysaturated FAME is more stable, as polyunsaturated is
5.31
43.07 easily oxidized [1]. GC histograms of condition A and D are
21.06 shown in Figure 5(b and c), respectively.
The effluent CO2 concentration discharged from the
1.75
19.41 scaled-up photobioreactor under 10% CO2 combined with
4.98 low and medium light conditions are shown in Figure 5(d).
1.75
50.13 The outlet CO2 concentration was measured by collecting
47.20 gases from the photobioreactor with an air sampling bag
2.67 for 1 h, and then measured with a CO2 sensor. The effluent
24.83
CO2 concentration of condition B (30 mEm
2s
1 and 10%
CO2) was measured at 8.5% after one day. This might be
due to the CO2 being dissolved in seawater or depleted by
the cells. It increased to nearly 10% day to day, indicating
CO2 was barely consumed by the cells. In contrast, under
condition D (300 mEm
2s
1 and 10% CO2), the outlet CO2
concentration at day 1 was measured at 6%, and continued
at a similar level to day 10, indicating that CO2 was
depleted consistently by the cells. The amount of CO2
removal in the cultures of condition B and D were 0.278 g
d
1 and 1.152 g d
1, respectively.
N. oculata grown in a scaled-up 7.5-L photobioreactor
under 300 mEm
2s
1 and 10% CO2 showed similar results,
with the total dry weight and biodiesel productivity as
5.31 g and 1.32 g, respectively. In contrast, the dry weight
and lipid content were 1.81 g and 0.267 g, respectively,
under 30 mEm
2s
1 and 0.037% CO2. When N. oculata cul-
tured in a photobioreactor at 300 mEm
2s
1 and 10% CO2
and an initial cell concentration of 107 cells mL
1, N. ocu-
lata not only could obtain high cell concentrations and
lipid content, but also depleted more CO2, reaching a CO2
removal of 1.152 g d
1.
Conclusions
In this study, stressful CO2 concentration and light intensity
were used in combination to generate high biomass and
lipid content in the microglae N. oculata. We observed that
the growth and lipid accumulation in N. oculata under
high CO2 concentration and medium light intensity were
far better than cells grown under a low CO2 concentration
and low light intensity. N. oculata grown in a scaled-up
photobioreactor under 300 mEm
2s
1 and 10% CO2, N. ocu-
lata showed increased total dry weight and biodiesel prod-
uctivity compared to cells cultured under 30 mEm
2s
1 and
0.037% CO2. This method can potentially be used in bio-
diesel production from N. oculata microalgae.
Acknowledgments
The GC analysis was provided by Dr. Chan-Chiung Liu of Department
of Food Science, National Pingtung University of Science and
Technology, Pingtung, Taiwan.
Disclosure statement
No potential conflict of interest was reported by the authors.

ORCID
Ching-An Peng http://orcid.org/0000-0003-0389-7311
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