Journal of Microbiology (2019) Vol. 57, No. 2, pp.
101–106
DOI 10.1007/s12275-019-8420-4
Blue-Red LED wavelength shifting strategy for enhancing beta-carotene
production from halotolerant microalga, Dunaliella salina
Sang-Il Han1†, Sok Kim1†, Changsu Lee2,
and Yoon-E Choi1*
1
Division of Environmental Science and Ecological Engineering, Korea
University, Seoul 02841, Republic of Korea
2
Microbiology and Functionality Research Group, World Institute of
Kimchi, Gwangju 61755, Republic of Korea
(Received Aug 2, 2018 / Revised Sep 17, 2018 / Accepted Sep 28, 2018)
In the present study, to improve the photosynthetic beta-
carotene productivity of Dunaliella salina, a blue-red LED
wavelength-shifting system (B-R system) was investigated.
Dunaliella salina under the B-R system showed enhanced
density and beta-carotene productivity compared to D. salina
cultivated under single light-emitting diode light wavelengths
(blue, white, and red light-emitting diode). Additionally, we
developed blue light-adapted D. salina (ALE-D. salina) using
an adaptive laboratory evolution (ALE) approach. In com-
bination with the B-R system applied to ALE-D. salina (ALE
B-R system), the beta-carotene concentration (33.94 ± 0.52
μM) was enhanced by 19.7% compared to that observed for
the non-ALE-treated wild-type of D. salina (intact D. salina)
under the B-R system (28.34 ± 0.24 μM).
Keywords: Dunaliella salina, beta-carotene, light-emitting
diode, wavelength shift, adaptive laboratory evolution
Introduction
Microalgae, which are microscopic unicellular organisms
widely distributed in freshwater and seawater, can convert
solar energy into chemical energy via photosynthesis (Priy-
adarshani and Rath, 2012). Microalgae are highly efficient
producers of biomass because they have higher photosyn-
thetic efficiency than land plants and undergo vigorous cell
division (6–12-h cycle) under ideal growth conditions (Sayre,
2010). Moreover, microalgae contain various bioactive com-
pounds that can be utilized for numerous commercial uses
(Priyadarshani and Rath, 2012). Thus, microalgae have great
potential in many aspects of bio-based manufacturing, such
as the production of biofuels and bio-factories for producing
valuable pharmaceuticals, food additives, and cosmetics
(Mata et al., 2010; Takaichi, 2011; Vílchez et al., 2011).
Among microalgae-derived bioactive compounds, carote-
†
These authors contributed equally to this work.
*For correspondence. E-mail: yechoi@korea.ac.kr; Tel.: +82-2-3290-3042
Copyright G2019, The Microbiological Society of Korea
eISSN 1976-3794
pISSN 1225-8873
noids including beta-carotene are the most important. Al-
though carotenoids are essential for human and animal nu-
trition, carotenoids cannot be synthesized in these organisms
de novo. The natural antioxidant beta-carotene is among the
most important microalgae-derived carotenoids. Beta-caro-
tene has been reported to help protect human and animal
skin against photoaging through its antioxidant activity (Dar-
vin et al., 2011). Additionally, beta-carotene is a major source
of vitamin A, which is indispensable for retina functions
and affects many tissue types by regulating gene expression
(Von Lintig et al., 2005).
Natural beta-carotene can be biosynthesized by plants, al-
gae, and marine organisms. Among various the natural sour-
ces of beta-carotene, the unicellular halotolerant microalga
Dunaliella salina is well-known as the richest source (Lamers
et al., 2012). D. salina can accumulate beta-carotene to as
much as 10–14% of the cellular dry weight (Ribeiro et al., 2011)
under certain extreme environmental conditions, such as
high light intensity (Kró et al., 1997), oxidative stress (Shaish
et al., 1993), high salinity (Borowitzka et al., 1990), and extreme
temperatures (Ben‐Amotz, 1996; Kró et al., 1997).
Of these environmental factors affecting the biosynthesis
of beta-carotene, light conditions are the most crucial factor
(Lamers et al., 2010). Because light is the basic energy source
for microalgae including D. salina, the growth and meta-
bolite biosynthesis of D. salina is limited by insufficient light
or inhibited by overexposure (Tang et al., 2011). Additionally,
the wavelength of light can significantly affect the growth
and biosynthesis efficiency of microalga such as D. salina (Fu
et al., 2013). Therefore, selecting an appropriate light source
and conditions is very important for producing biomaterials
using microalgae including D. salina.
The light-emitting diode (LED) is the preferred light source
for microalgae cultivation because it is easy to apply a specific
wavelength and exhibits low power consumption over a long
duration compared to fluorescent lamps (Zhao et al., 2011).
Thus, various studies have applied LEDs as light sources in
photo-bioreactors for microalgal cultivation. For example,
according to Fu et al. (2013), a red and blue LED illuminated
photo-bioreactor was used to evaluate beta-carotene produc-
tion from D. salina. Additionally, Xi et al. (2016) reported
that appropriate wavelength shift during the life-cycle of Hae-
matococcus pluvialis greatly increased carotenoid (astaxan-
thin) productivity of H. pluvialis (Xi et al., 2016). However,
few studies have examined applying suitable wavelengths of
LEDs for beta-carotene production from D. salina.
In the present study, we investigated the impacts of various
LEDs (blue, red, and white) on the cultivation of D. salina
and evaluated the beta-carotene productivity of D. salina.
Based on the growth and beta-carotene synthesis profiles of
102 Han et al.
D. salina depending on the wavelength of LED light, a blue-
red wavelength shifting system (B-R system) was developed
and investigated to enhance beta-carotene production by
D. salina. To further enhance beta-carotene production by
D. salina under the B-R system conditions, a blue light-
adapted D. salina strain (ALE D. salina) was obtained us-
ing an adaptive laboratory evolution (ALE) approach. The
beta-carotene productivity of ALE D. salina was evaluated
under the B-R system and compared to that of wild-type D.
salina. Here, we report the use of an appropriate wavelength
shift of LEDs and further enhancement of beta-carotene
production following ‘adaptive laboratory evolution’.
Materials and Methods
Strain and materials
The halotolerant green alga Dunaliella salina CCAP 19/18
was obtained from the Culture Collection of Algae and
Protozoa. All cultures were performed in f/2 medium com-
prised of NaNO3 75 mg; NaH2PO4·9H2O 5 mg; Na2SiO3·
9H2O 30 mg; FeCl3·6H2O 3.15 mg; Na2EDTA·2H2O 4.36 mg;
CuSO4·5H2O 9.8 μg; Na2MoO4·2H2O 6.3 μg; ZnSO4·7H2O
22.0 μg; CoCl2·6H2O 10.0 μg; MnCl2·4H2O 180.0 μg; vitamin
B12 0.5 μg; Biotin 0.5 μg; thiamine·HCl 100 μg; per 950 ml of
filtered sea water (Guillard and Ryther, 1962; Guillard, 1975).
All reagents related to cultivation were purchased as culture-
grade from Sigma-Aldrich.
Cultivation conditions
Inocula were prepared from 5-day-old cultures cultivated
in fresh f/2 medium. The initial cell density was adjusted to
approximately 1 × 105 cells/ml. During all cultivations, fil-
ter-sterilized air was supplied at a flow rate of 100 ml/min.
Five-day cultures were inoculated into 150 ml f/2 medium
in 250 ml round flasks at 25 ± 1°C. Continuous 24-h illu-
mination was set to 50 ± 2 μmol/m2·sec using either red (wa-
velength 630–665 nm) or blue (wavelength 430–465 nm)
LEDs. For wavelength-shifting experiments, the cells were
cultivated under the same conditions under blue LEDs, and
then one group of samples (three replicates) was shifted into
red LEDs on the sixth day. Cell growth was measured by
determining the cell density.
Adaptive laboratory evolution (ALE)
The strategy based on ALE was employed to increase the
beta-carotene productivity of the cells. Cells were cultivated
under the same conditions as described above and allowed
to adapt to blue light. Before ALE, only the blue LED (50 ±
2 μmol/m2·sec) was used as the light source. Cultivation
was repeatedly performed over a 5-day cycle. For each new
cycle, the culture was diluted to the same cell density (appro-
ximately 1 × 105 cells/ml) by removing a certain portion of the
culture every 5 days and supplementing the same volume of
fresh medium (Fu et al., 2013). The ALE experiment was
sustained for 5 cycles for 25 days. After the five cycles, cell
density and beta-carotene content were measured.
Cell density and beta-carotene analysis
The number of cells was determined by direct counting with
a hemocytometer under an OPTINITY microscope KB-500
(Korea Lab Tech Corp.). Beta-carotene was extracted from
harvested algal pellets using 80% acetone. Briefly, a 1-ml sam-
ple was removed from the flask, centrifuged at 13,000 rpm
for 10 min, and washed twice with distilled water. After ob-
taining algal pellets, the cells were disrupted by bead-beat-
ing using both 0.6 μm and 1,180 μm acid-washed beads for
90 sec. Next, 1 ml of 80% acetone was added to extract the
pigments. After removing the cell debris by centrifugation at
13,000 rpm for 10 min, absorbance was measured at 412, 431,
460, and 480 nm with a UV-spectrophotometer (GENESYS
10S UV Vis, Thermo Scientific) The concentration of beta-
carotene (μM) was calculated using the following equation
reported previously (Eijckelhoff and Dekker, 1997).
Beta-carotene (μM) = -0.430 × A412 + 0.251 × A431
-4.376 × A460 + 13.216 × A480 (1)
where A412, A431, A460, and A480 indicate the measured UV
absorbance at 412, 431, 460, and 480 nm, respectively.
Results and Discussion
Effect of LED wavelengths on beta-carotene productivity of
D. salina
In the present study, the beta-carotene productivity of D.
salina cultivated in f/2 medium under different LED wave-
lengths such as white, blue, and red lights was compared.
Interestingly, D. salina showed different cell numbers de-
pending on the wavelength used (Fig. 1). Of the different
applied LED lights, red LED (660 nm) light was the most
efficient for D. salina growth compared to white and blue
light (450 nm). In contrast, the blue LED (450 nm) led to
decreased cell numbers compared to the other cultivation
conditions under either the white or red wavelength (Fig. 1A).
After 11 days of cultivation, 167.0 ± 11.4 × 104 cells/ml and
223.3 ± 18.7 × 104 cells/ml for D. salina densities were ob-
served under blue and white light, respectively. Under red
light, D. salina showed 1.45- and 1.08-fold higher cell density
(241.8 ± 20.5 × 104 cells/ml) compared to those under blue
and white light wavelengths, respectively. Under red light,
not only the cell density of D. salina, but also the highest
levels of beta-carotene were observed. As shown in Fig. 1B,
the extent of biosynthesized beta-carotene in D. salina under
red light was 25.21 ± 0.14 μM after 11 days of cultivation.
However, in the case of the white and blue light, beta-car-
otene was produced at 20.01 ± 2.33 and 18.85 ± 4.01 μM,
respectively. This may be because of the light absorbance
properties of D. salina. Phototropic microorganisms includ-
ing green microalga such as D. salina contain chlorophyll-a
and chlorophyll-b for photosynthesis. According to Chappelle
et al. (1992), these chlorophylls display light absorbance at
specific wavelengths (chlorophyll-a: 580, 630, and 670 nm
and chlorophyll-b: 460 and 650 nm). The red-LED wavelength
(630–665 nm) can be absorbed to both chlorophyll-a and -b,
whereas blue-LED wavelength (430–465 nm) can be mainly
 Wavelength shifting for enhancement of beta-carotene production from Dunaliella salina 103

(A)

(B)
Fig. 2. Beta-carotene accumulation by D. salina cells cultivated under
blue, white, and red LED light.

absorbed to chlorophyll-b. Therefore, the red LED light showed

Fig. 1. Effect of LED wavelengths on (A) cell density and (B) beta-carotene
synthesis of D. salina.
better efficiency for biomass growth and beta-carotene pro-
ductivity. Although the highest amount of beta-carotene was
obtained under red LED, the accumulated amounts of beta-
carotene in terms of the cellular basis of D. salina were
similar, regardless of the different light wavelengths used
(Fig. 2). The amounts of beta-carotene accumulated under
blue, white, and red lights were 0.0113 ± 0.0024, 0.009 ±
0.0017, and 0.0104 ± 0.0006 pmol/cell, respectively. Based
on these results, the difference in beta-carotene production
of D. sailna under various light wavelengths was solely de-
pendent on the growth efficiency of D. sailna depending
on different light wavelengths.

(A) (B) Fig. 3. Dunaliella salina cell growth

profiles under (A) blue, (B) white,
(C) red, and (D) B-R systems (blue
to red wavelength shift).

(C) (D)
104 Han et al.
Table 1. Parameters obtained from linear equation
Light condition
0–6 days
4 4
arate (× 10 cells/ml day) bconst (× 10 cells/ml)
Blue 21.14 ± 2.00 -10.83 ± 7.81
White 17.92 ± 1.50 -7.10 ± 5.83
Red 15.79 ± 1.10 -7.07 ± 4.29
Blue-Red 20.29 ± 1.37 -3.73 ± 5.32
Wavelength shifting effect on D. salina cell growth and beta-
carotene productivity
Next, to improve beta-carotene production by D. salina, we
used a light wavelength shifting strategy. According to Xi et
al. (2016), by appropriately exploiting algal growth proper-
ties at specific wavelengths, high-value bio-compounds, such
as astaxanthin produced by H. pluvialis, was enhanced us-
ing a similar LED wavelength shifting strategy (red-blue light
shifting). Because cell density was a crucial factor affecting
the beta-carotene productivity of D. salina applied photo-
bioreactor, based on previously obtained results, growth pro-
files under consistent red, white, and blue LED wavelengths
were carefully evaluated during the time-course of cultiva-
tions. Figure 3 represents the densities of D. salina cells cul-
tivated under these light condition for 11 days. As shown in
Fig. 3, overall, D. salina cell densities were increased during
the cultivation period until 10 days, regardless of the different
illuminations of specific wavelengths, and a reached pla-
teau of stationary phase after 10 days of cultivation. Under
white LED light as a control wavelength, cell density of D.
salina was significantly increased after 6 days of cultivation
time. In the case of blue light, until 6 days of cultivation time,
the cell density of D. salina was continuously increased but
the cell growth rate was decreased after 6 days of cultivation.
The deceleration of growth rate of D. salina under blue LED
might be occurred by stress due to high-level photon energy
of blue LED wavelength (Helena et al., 2016). Under red
LED wavelength, although D. salina cell growth profile was
observed similar to that of white LED, red LED wavelength
led to the most efficient D. salina growth compared to the
other wavelengths. Since red LED wavelength can be ab-
sorbed to main antenna pigments, chlorophyll-a and chlor-
ophyll-b, it might be more effective for photo system of D.
salina than other light wavelengths (Koc et al., 2013). In ad-
dition, according to Guenther et al. (1988), it has been re-
ported that the amount of chlorophyll-a, which acts uniquely
as primary electron donor of the reaction center for photo-
systems is higher than chlorophyll-b in the D. salina. There-
fore, the D. salina could be efficiently triggered by the red
LED wavelength compared to blue LED wavelength. To fur-
ther compare these remarkable patterns of D. salina growth
under red, white, and blue LED light, the following linear
equation was applied to the obtained experimental data.
y = aratextime + bconst
where y is the D. salina cell density (× 104 cells/ml) at a
specific cultivation time (xtime). The arate (× 104 cells/ml·day)
can be regarded as the D. salina growth rate. The bconst is
Parameters
6–11 days
2 4 4 2
R arate (× 10 cells/ml day) bconst (× 10 cells/ml) R
0.965 14.23 ± 1.98 15.80 ± 16.10 0.945
0.973 32.98 ± 2.15 -108.90 ± 17.48 0.987
0.981 40.48 ± 3.37 -168.50 ± 27.39 0.979
0.982 38.70 ± 2.25 -124.75 ± 18.31 0.990
y-intercept of the linear equation. The values obtained from
the linear equations are summarized in Table 1. In the early
stage of D. salina cultivation (0–6 days), the estimated growth
rates of D. salina under white and red LED light conditions
4
were 17.92 ± 1.50 and 15.79 ± 1.10 × 10 cells/ml·day, res-
pectively. During the same period, the growth rate under
blue LED showed the highest value at 21.14 ± 2.00 × 104
cells/ml·day. In the late stage of cultivation (after 6 days),
the D. salina growth rate under blue illumination was de-
creased to 14.23 ± 1.98 × 104 cells/ml·day. In contrast, the D.
salina growth rates of white and red illuminations in the
late stage period were increased compared to those in the
early stage. The growth rates grown under white and red il-
luminations in the late stage were recorded as 32.98 ± 2.15
× 104 and 40.48 ± 3.37 × 104 cells/ml·day, respectively.
Based on these results, we expected that the wavelength shift
strategy can be used for a blue to red wavelength shift (denoted
as the ‘B-R system’) to enhance the growth of D. salina, which
may result in higher beta-carotene production. Under the
B-R system, blue LED light was first used to illuminate D.
salina for 6 days (early stage). After the early stage, the illu-
minating light was shifted to red LED light. According to re-
sults (Fig. 3D), in the early stage, the growth rate of D. salina
cultivated under the B-R system (B-R D. salina) showed a
similar cell growth rate (arate, B-R, early: 20.29 ± 1.37 × 104 cells/
ml·day) as D. salina under the blue wavelength. Additionally,
the D. salina growth rate after 6 days (arate, B-R, late) under the
B-R system was 38.70 ± 2.25 × 104 cells/ml·day. This is sim-
(2)
Fig. 4. Beta-carotene production by D. salina under different illumination
wavelengths with or without adaptive laboratory evolution (ALE) approach.
Single red, continuous red illumination; B-R system, blue to red wavelength
shift; ALE B-R system, adaptive laboratory evolution under the wavelength
shift from blue to red wavelength.
 Wavelength shifting for enhancement of beta-carotene production from Dunaliella salina
ilar to the growth rate of D. salina grown under red illumi-
nation for the same period. Because of the increased cell
growth efficiency in the early stage under the B-R system,
4
D. salina cell density (272.75 ± 18.41 × 10 cells/ml) was in-
creased by 12.8% compared to that of D. salina cultivated
under red illumination (241.75 ± 20.55 × 104 cells/ml) on the
final day of cultivation. Additionally, as cell density increased,
beta-carotene production under the B-R system correspon-
dingly increased. As shown in Fig. 4, beta-carotene produc-
tion by D. salina cultivated under the B-R system was higher
than that cultivated consistently under red illumination (de-
noted as ‘singe red’). In the final stage (11 days of cultivation),
beta-carotene production under the B-R system (28.34 ±
0.24 μM) was increased by 12.5% higher (similar to the in-
creased rate of D. salina cell growth under B-R system) than
that of D. salina cultivated under red illumination (25.21 ±
0.14 μM).
Enhancement of beta-carotene production by ALE
Since D. salina showed the highest growth rate under blue
and red LED light in the early and later growth stages, re-
spectively, we could propose the B-R system, where D. salina
growth efficiency could be significantly enhanced by taking
advantages of specific growth effect of particular wavelength
at different time points. However, although blue LED light
was efficient for growing D. salina under the B-R system,
the blue wavelength eventually caused cellular damage to
D. salina because of the high photon energy reflected by the
short-wavelength property. We predicted that adaptation
to the blue wavelength would overcome this problem. ALE
is a well-established tool for producing evolved biological
strains capable of adapting to certain environmental con-
ditions such as carbon sources, energy sources, and stresses
(Dragosits and Mattanovich, 2013; Fu et al., 2013). Previously,
Fu et al. (2012) successfully applied the ALE method to im-
prove biomass productivity of Chlorella vulgaris in light-
emitting diode-based photobioreactor. Additionally, accord-
Fig. 5. Growth of intact D. salina (dark cycles) or blue-adapted D. salina
with ALE (ALE-D. salina) (white cycles). Both strains became subjects of
wavelength shift strategies of blue to red wavelength shift. Early (0–6 days)
and late cell growth period (after 6 days) was illuminated under blue and
red light, respectively.
105
ing to Reyes et al. (2014), a hyper-producer strain for beta-
carotene production was developed using the ALE method
through hydrogen peroxide shock using Saccharomyces cere-
visiae. Thus, we employed the ALE method to develop blue
light-adapted D. salina (ALE-D. salina) to further enhance
the beta-carotene productivity of D. salina in the wavelength
shifting B-R system.
As shown in Fig. 5, the cell growth profiles of either intact
or ALE subjected D. salina were compared under the B-R
system. ALE-D. salina showed a higher cell density com-
pared to intact D. salina, particularly during the early culti-
vation stage under blue light. The cell densities of ALE-D.
4
salina and intact D. salina were 222.5 ± 8.49 × 10 and 114.5
4
± 5.66 × 10 cells/ml, respectively, at the end of the early stage
(6 days). After shifting the light wavelength to red light, the
cell densities of ALE-D. salina and intact D. salina were in-
creased as the cultivation time increased. ALE-D. salina re-
ached the stationary phase at 8 days after inoculation, allow-
ing for more production of beta-carotene, while intact D.
salina reached the stationary phase at a later time point (10
days after inoculation) (Figs. 4 and 5). Additionally, ALE-D.
salina showed 14.6% higher cell density (312.25 ± 26.52 ×
104 cells/ml) compared to intact D. salina (272.42 ± 8.49 ×
104 cells/ml) at 11 days of cultivation (Fig. 5). This result in-
dicates that ALE-D. salina was successfully adapted to blue
wavelength illumination via the ALE method. Because of
the increased cell density of D. salina after ALE, beta-car-
otene productivity was enhanced by 19.7% in ALE-D. salina
(similar to increased rate of ALE-D. salina density compared
to intact D. salina under the B-R system) (Fig. 4). Beta-car-
otene production rates by intact D. salina and ALE-D. salina
were 28.34 ± 1.01 and 33.94 ± 0.52 μM, respectively (Fig. 4).
Additionally, beta-carotene production by ALE-D. salina un-
der the B-R system was 34.7% higher than that of D. salina
under simple consistent illumination of red LED light, al-
though this condition showed the highest beta-carotene pro-
ductivity among the consistent single wavelength illumina-
tions.
Conclusion
In this study, we compared the beta-carotene productivity
of D. salina under blue, white, and red LED light illumina-
tion. Depending on the light wavelength, growth and beta-
carotene productivity of D. salina showed significant dif-
ferences. For example, under red light, D. salina showed the
highest beta-carotene productivity (25.21 ± 0.14 μM) and
4
cell density (241.75 ± 20.55 × 10 cells/ml). In contrast, blue
illumination was more effective during the early growth of
D. salina compared to the other light wavelengths. Based on
the growth properties of D. salina under blue and red LED
lights, we developed a wavelength shift strategy from blue
to red illumination (B-R system), which enhanced D. salina
cell density and beta-carotene production by 12.8% and
12.5%, respectively, compared to those of D. salina under
single red illumination. We also performed an ALE appro-
ach to obtain blue light-adapted D. salina (ALE-D. salina)
and further enhanced beta-carotene production (33.94 ±
0.52 μM) was achieved compared to that by non-ALE treated
106 Han et al.
D. salina (intact D. salina) under the B-R system (28.34 ±
0.24 μM). Our data clearly indicate that the appropriate
manipulation of wavelength in conjunction with adaptation
can increase the production of secondary metabolites in mi-
croalgae.
Acknowledgements
This work was supported by the Government of South Korea
through the National Research Foundation of Korea (NRF-
2016R1D1A1B03932773). This research was also supported
by the Marine Biotechnology Program of the Korea Institute
of Marine Science and Technology Promotion (KIMST)
funded by the Ministry of Oceans and Fisheries (MOF) (No.
20170488).
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