Bioresource Technology 159 (2014) 240–248

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Manipulation of light wavelength at appropriate growth stage

to enhance biomass productivity and fatty acid methyl ester yield
using Chlorella vulgaris
Dae Geun Kim a, Changsu Lee a, Seung-Moon Park b, Yoon-E Choi c,d,⇑
a
Department of Bioprocess Engineering, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do 561-756, Republic of Korea
b
Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan-si, Jeollabuk-do 570-752, Republic of Korea
c
LED Agri-bio Fusion Technology Research Center, Chonbuk National University, 79 Gobong-ro, Iksan-si, Jeollabuk-do 570-752, Republic of Korea
d
Department of Bioactive Material Sciences, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do 561-756, Republic of Korea

h i g h l i g h t s

 LEDs are suitable light source of microalgal cultivations.

 Blue light LED illumination led to significantly increased cell size.
 Red light LED illumination led to small-sized cell with active divisions.
 Innovative process with wavelength shift increased biomass and FAME.

a r t i c l e i n f o a b s t r a c t

Article history: LEDs light offer several advantages over the conventional lamps, thereby being considered as the optimal
Received 20 December 2013 light sources for microalgal cultivation. In this study, various light-emitting diodes (LEDs) especially red
Received in revised form 21 February 2014 and blue color with different light wavelengths were employed to explore the effects of light source on
Accepted 22 February 2014
phototrophic cultivation of Chlorella vulgaris. Blue light illumination led to significantly increased cell
Available online 3 March 2014
size, whereas red light resulted in small-sized cell with active divisions. Based on the discovery of the
effect of light wavelengths on microalgal biology, we then applied appropriate wavelength at different
Keywords:
growth stages; blue light was illuminated first and then shifted to red light. By doing so, biomass and lipid
LED
Wavelength shift
productivity of C. vulgaris could be significantly increased, compared to that in the control. These results
Chlorella vulgaris will shed light on a novel approach using LED light for microalgal biotechnology.
Microalgal cultivation Ó 2014 Elsevier Ltd. All rights reserved.
Biodiesel

1. Introduction Microalga could also offer several additional advantages not

Due to the higher price of petroleum as well as environmental
concerns regarding to the steep rise of carbon dioxide as a green-
house gas, alternative bio-fuels have been received considerable
attention worldwide (Hill et al., 2006). Among the various biomass
sources, microalgae are considered to be one of the most promising
feedstocks for biodiesel, due to their rapid growth and high lipid
content. For these reasons, the development of biodiesel originated
from microalgae has become a hot topic in recent years (Chisti,
2007).
⇑ Corresponding author at: LED Agri-bio Fusion Technology Research Center,
Chonbuk National University, 79 Gobong-ro, Iksan-si, Jeollabuk-do 570-752,
Republic of Korea. Tel.: +82 63 850 0773.
E-mail address: yechoi@jbnu.ac.kr (Yoon-E Choi).
only limited to microalgal biodiesel but also include a wealthy of
the other benefits. Firstly, microalgae could biologically capture
and fix carbon dioxide during the process of photosynthesis. Sec-
ondly, an enormous variety of compounds with high values includ-
ing antioxidants and potential medications could be also produced
from microalgal biomass. Finally, microalgae could utilized as envi-
ronmental agents, since microalga are capable of rapid uptake of
nitrogen and phosphate in the wastewater. Therefore, microalgal
biomass hold a great promise for future biotechnological applica-
tions and recently it has become reality with intensive effort and
investment around the world.
In order to utilize microalgal biomass for above-mentioned pur-
poses, it must be essential to obtain sufficient microalgal biomass.
Therefore, microalgal cultivation is one of the most important
processes for the use of microalgae to produce any substances of

http://dx.doi.org/10.1016/j.biortech.2014.02.078
0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
 D.G. Kim et al. / Bioresource Technology 159 (2014) 240–248
interest, particularly for biodiesel. Moreover, it has been well ac-
cepted that the cultivation process alone claims high production
cost, restricting further industrialization or commercialization
using microalgae. So far, most of research for the production of bio-
diesel using microalgal biomass was limited in the small scale.
However, in order to achieve economic feasibility, efficient mass
cultivation strategy with high lipid productivity remains a key
challenge to microalgal biotechnology (Uduman et al., 2010).
As with photoautotrophic organisms, microalgae carry out the
photosynthesis as a main metabolism for the acquisition of organic
materials by using energy from light sources such as sunlight or
artificial light. Therefore, multiple lines of evidences suggested that
light is the most significant factor governing the entire process of
mciroalgal cultivation. Based on different approaches to obtain
light source, the strategy for microalgal cultivations could be
broadly categorized into open-pond or photobioreactor. While
open-pond system utilize free sunlight, microalgal cultivation in
the closed photobioreactor with artificial light source have the
indisputable advantages in that biomass productivity could be sig-
nificantly increased, particularly for value added products, com-
pared to those in open pond system. However, again, the supply
of artificial light causes the most expensive cost to operate the
photobioreactor. Moreover, light energy penetrated into microalgal
suspensions was significantly decreased along with increase of
light path-length at high microalgal cells concentrations, which
acts as a bottleneck for microalgal cultivation. Therefore, it will
be crucial to developing efficient process to maximize the utiliza-
tion of light energy, thereby improving economic feasibility in mic-
roalgal cultivation process.
Recently, light-emitting diodes (LED) has been emerged as a
replacement of traditional artificial light source. Compared to the
conventional tubular fluorescence lamps, the recent developed
LEDs light make significant advances in narrowing a specific wave-
length with low power consumption. Additionally, LEDs are suit-
able light source of photobioreactor (PBR) for indoor mass
cultivation due to their small chip size and long duration (Zhao
et al., 2011). Due to these advantages, now, the applications of
LEDs are actively extended to the field of microalgal cultivation
(Wang et al., 2007). Because LEDs could provide a particular wave-
length to illuminate microalgal culture, it will be pertinent to select
LEDs for the purpose of adequate manipulation of microalgal culti-
vation. In accordance with this notion, a number of studies have
addressed that there are optimum wavelengths for each of micro-
algal species, though contradictory results have been obtained
about the influence of specific wavelength of LED on microalgal
growth. Whereas a red light was the most effective for Botryococcus
braunii (Baba et al., 2012), a blue light led to the best biomass pro-
ductivity for Nannochloropsis sp. (Das et al., 2011). Similar contra-
dictory results regarding specific wavelength for microalgal
cultivation were obtained previously (Baba et al., 2012; Lee and
Palsson, 1994). Hence, we still have limited reports regarding
LED applications into the field of microalgal cultivations.
Furthermore, although some researchers attempted to utilize
LEDs for microalgal growth, there has been no research to investi-
gate and consider biological responses of microalgal cells upon the
specific wavelength of LED illuminations, including the cellular
physiology or gene expressions. To date, very few studies have only
focused on the installment of simple LED illuminations for micro-
algal growth without knowing detailed cellular mechanisms upon
specific LED wavelengths. The knowledge on these will become of
great interest to the microalgal bioengineers and could be directly
applied into microalgal biotechnology, such as mass cultivation in
the photobioreactors.
Therefore, in this study, we examined the effect of specific
wavelength of LEDs on microalgal biology including growth, cell
physiology, fatty acid biosynthesis, and gene expressions using
241
Chlorella vulgaris. Indeed, it turned out that red and blue wave-
length has a specific influence on C. vulgaris biology. Considering
the effect of LED wavelengths in particular, we then tried to manip-
ulate red or blue wavelength illumination at appropriate stage of
microalgal cell growth. These results demonstrated that adequate
manipulations of LED wavelengths during the microalgal cell
growth could lead to significant increase in biomass and lipid
productivity. Here, we report a novel discovery of specific influence
of LED wavelengths on microalgal biology and propose the putative
process based on LED wavelength shift for the enhanced produc-
tion of biomass as well as lipid using C. vulgaris.
2. Methods
2.1. Strain and culture condition
C. vulgaris was obtained from the UTEX culture collection.
Inoculums were regularly prepared using modified JM medium
(Thompson et al., 1988) in 500 mL Erlenmeyer flasks at
25 ± 0.5 °C under cool-white fluorescent lights (approximately
50 lmol m2 s1). For the all experiments, cells were cultivated
in 1000 mL Erlenmeyer flasks containing 600 mL JM medium. The
initial cell density was adjusted to be 106 cells mL1 and tempera-
ture was maintained at 26 ± 0.5 °C. Continuous illumination was
supplied at an average light intensity of 100 lmol m2 s1 under
either monochromatic blue (kmax = 430–465 nm) or red (630–
665 nm) light. To ensure sufficient aeration, cultures were bubbled
with sterilized-filtered air at a flow rate of 100 mL min1.
2.2. Analysis
The UV/vis spectrophotometer (T60 U, korea) was used for mea-
suring both optical density and the level of ROS staining.
To analyze chlorophylls, cells were centrifuged, washed twice
with de-ionized (DI) water, and the pellet was dried for 24 h. Then,
equal amount of dried cells was re-suspended in 100% MeOH at
50 °C for 50 min. After centrifugation at 12,000g for 5 min, super-
natant was collected and absorbance was measured at 650 and
665 nm, respectively. The content of chlorophyll a, chlorophyll b,
and the total chlorophyll content (mg/L) was calculated using the
following equations:
Chlorophyll a ¼ 16:5  A665  8:3  A650
Chlorophyll b ¼ 33:8  A650  12:5  A665
Chlorophyllða þ bÞ ¼ 25:5  A650 þ 4:0  A665
2.3. Growth measurements
Algal growth was measured with optical density at 660 nm with
a UV/vis spectrophotometer (T60 U, korea). The number of cells
was determined by direct counting with a haemocytometer
(Hausser Scientific, Horsham, PA) under an OPTINITY microscope
(KB-500, korea). The dry cell weight was measured by filtering
the algal suspension through a pre-dried and pre-weighed,
0.45 lm cellulose nitrate membrane filter (Whatman, USA) and
drying in an oven at 80 °C for 24 h. The specific growth rate (l)
was calculated based on the following equation.
l ¼ ðlnX 1  lnX 0 =t1  t0 Þ
where X1 and X0 were the biomass concentration (g L1) at time t1
and t0, respectively.
242 D.G. Kim et al. / Bioresource Technology 159 (2014) 240–248
The size of Chlorella was measured with OPTINITY microscope
camera (C30, Korea) from more than one hundred microalgal cells
randomly selected.
2.4. Transcription analysis
Microalgal cultivations were performed for 3 days under either
white, red, or blue illumination. Total RNA was extracted from
100 mg of algal cultures with TRIzol reagent (Invitrogen) in
accordance with the manufacturer’s protocols. Nucleic acid
concentrations were measured spectrophotometrically at 260 nm.
The integrity and purity of the RNA were determined by 260/
280 nm ratios and separated by electrophoresis on a 1% agarose
formaldehyde gel. Reverse transcription (RT) was carried out using
a capacity RNA-to-cDNA kit (Applied Biosytems). Four photosyn-
thesis-related genes and one cell division-related gene in a fresh-
water algae C. vulgaris were selected as target genes. These select
genes are: (i) psaB (NCBI GeneID: 809130), which codes for the
photosystem I (PS I) reaction centre protein (ii) chlB (GeneID:
809139), which codes for light-independent protochlorophyllide
reductase subunit B (iii) rbcL (accession number: AF499684),
which codes for the large subunit of rubisco (iv) psbA, which codes
for the photosystem II (PS II) reaction protein D1 (v) minD, which
codes for cell division inhibitor protein. QRT-PCR analysis was per-
formed with SYBR-GreenÒ as the fluorescent. Gene specific primers
for psaB gene (psaB-F1 and psaB-R1), chlB gene (chlB-F1 and chlB-
R1), rbcL gene (rbcL-F1 and rbcL-R1), psbA gene (psbA-F1 and
psbA-R1), and minD (minD-F1 and minD-R1) were used for giving
rise to amplicons. The expression of each gene was normalized to
endogenous 18s rRNA gene expression with specific pair of primers
(18s-F1 and 18s-R1). The gene expression was calibrated using
2DDCt method. The range of expression was calibrated using
2DDCts–2DDCt+s, where s is the standard deviation of DCt value
(Ct = Threshold Cycle). All primers used for PCR are listed in
Table 1.
2.5. FAME analysis
After cultivation, algal biomass was harvested by centrifugation
(7000 rpm, 3 min) and then washed twice with DI water. The cell
pellet was freeze-dried at 40 °C for 3 days and used for lipid
extraction. Total lipids were extracted from 10 mg of lyophilized
biomass with a chloroform–methanol (2:1 v/v) solvent mixture
according to the modified Folch method (Folch et al., 1957). Ex-
tracted lipids were then converted into fatty acid methyl esters
(FAMEs) via the transesterification reaction (with methanol and
sulfuric acid as a catalyst) at 105 °C for 20 min. The FAMEs in the
organic phase were analyzed by gas chromatography (HP6890,
Agilent, USA), with a flame ionized detector (FID) and an INNO-
WAX capillary column (Agilent, USA, 30 m  0.32 mm  0.5 lm).
Table 1
All primers used in this study.
Target Primer name Nucleotide sequence (50 -30 )
chlB chlB-F1 50 -TGTTGCTGTTACACCGATGGG-30
chlB-R1 50 -GAGTGGCATCTCCAAAGACT-30
psbA psbA-F1 50 -GGTTATACAACGGTGGTCCT-30
psbA-R1 50 -CCTTGACCGATTGGGTAGAT-30
psaB psaB-F1 50 -ATTCCACGTTGCTTGGCAAGG-30
psaB-R1 50 -GCTGGTTGGAACGATGGTTGA-30
rbcL rbcL-F1 50 -GGACGACTGTATGGACTGATG-30
rbcL-R1 50 -CACGTATGCTGGTGGAATACG-30
minD minD-F1 50 -GGCTTGGTTATCGTGTTGCTC-30
minD-R1 50 -ACTGCCTCTTGTGCAGATGCA-30
18s RNA 18s-F1 50 -ACTTCACGAATCGCATGGCCT-30
18s-R1 50 -GACTTGCCCTCCAATTGATCC-30
The identification and quantification of fatty acids were deter-
mined by comparison with the retention times and peak areas of
standards.
2.6. ROS staining
In situ detection of the superoxide radical was performed by
algae cultures with nitroblue tetrazolium (NBT) (N6876, Sigma–
Aldrich) staining following the protocol described previously
(Rao and Davis, 1999). Three biological replications were per-
formed. Cultures grown in JM media for 3 days were centrifuged
and re-suspended in 0.2% NBT. After incubation for 12 h, cells were
re-centrifuged and re-suspended in 10 ml of 50% glacial acetic acid.
For quantification, sonicator was used to lyse the stained cells and
the extent of ROS was measured with the absorbance at 560 nm.
2.7. Statistical analysis
All experiments were performed with three replications. Data
obtained were analysis by one-way ANOVA following Duncan’s
multiple range test with the significance level (P < 0.05). The SPSS
version 17 (SPSS Inc., Chicago, IL, USA) program was used for all
statistical analyses.
3. Results and discussion
3.1. Effect of LED wavelength on algae growth
To test the effects of specific wavelength on the growth of mic-
roalgae, ubiquitous microalga C. vulgaris was chosen and its growth
characteristics cultivated under different three wavelengths of LED
were thoroughly examined. Microalgae have chlorophylls and
accessory pigments with maximum absorption peaks in the red
or blue part of light spectrum. For this reason, three different
wavelengths (450, 660 and 400–700 nm) representing blue
(450 nm), red (660 nm), and white (400–700 nm) as a control were
selected to determine the effects of major wavelengths necessary
for the photosynthesis on microalgal biology. Results showed that
specific wavelength could indeed affect microalgal growth
(Fig. 1a). Cell numbers were highly dependent on light wavelength,
since C. vulgaris produced the highest and the lowest cell number
under the red and blue light, respectively. The cell density of
C. vulgaris grown under red light was 1.5-fold higher than those
under blue light at the certain time point after the inoculation
(Fig. 1a). These results are consistent with other microalgal species
in that red wavelength turned out to be optimal for both Spirulina
platensis and Chlorella pyrenoidosa (Wang et al., 2007). Matthijs
et al. (1996) reported that red light led to enhanced chlorophyll
pigmentation, which could explain the results of a positive effect
of red light. Since red light had a similar influence on microalgal
growth in both C. pyenoidosa and S. platensis (Wang et al., 2007),
the responses upon light wavelength might be evolutionary well-
conserved between different microalgal species. On the other hand,
white light gave rise to middle extent of cell number, whereas blue
light consistently decrease the level of cell number. One possible
speculation is that blue light could suppress microalgal cell
division, thereby resulting in lower level of cell number. Further
study might be necessary to determine cellular mechanisms of
microalgae upon different wavelengths of light.
3.2. The effect of blue wavelength on the microalgal cell size
Interestingly, microalgal cell morphology, e.g. cell size, of
C. vulgaris was altered significantly in response to different wave-
length of light. Cell size of C. vulgaris cultivated under blue light
 D.G. Kim et al. / Bioresource Technology 159 (2014) 240–248
Fig. 1. Effects of three different LED wavelength including red (-s-), white (-d-) and blue (-.-) on C. vulgaris. (a) Cell density; (b) cell size; (c) correlation between cell
number and OD (optical density). Different letters over the bar represent significant difference (P < 0.05); (d) dry weight per cells cultured under different wavelength. Eight
blue (left) and five red wavelengths (right) were selected.
was consistently larger than those cultivated under red light. How-
ever, middle extent of cell size was observed under the white light
(Fig. 1b). Measurement of the cell size of C. vulgaris revealed that
microalgal cells grown under blue light have approximately a
60–70% increase in the diameter, compared to those under red
light (Fig. 1b).
To verify these observations further, two independent analyses
were performed. First method was based on the correlation
between optical density and cell number (Choi et al., 2011). The
correlation graphs between optical densities (OD) and cell number
were plotted to check any change in the correlation between blue
and red light. Consistent with the observation of increase in cell
sizes under blue light, the correlation curve was significantly
altered by the blue light (Fig. 1c). Higher OD values were consis-
tently obtained at certain cell number under the blue wavelength,
compared to those under red wavelength (Fig. 1c). The difference
in the correlations between blue and red light clearly reflects the
fact that there were significant changes in morphological feature
(differences in cell size) by changing light wavelength.
Additionally, the relative dry weight (g/cell) by taking cell
numbers into consideration was also calculated. To thoroughly
compare the effect of either blue or red light, eight (430, 435,
440, 445, 450, 455, 460, and 465 nm) and five (645, 650, 655,
660, and 665 nm) different LED wavelengths were selected from
blue and red light, respectively. Expectedly, dry weight per cells
could be fit into two categories: either blue or red light. Again, it
243
is evident that the dry weight per cell of C. vulgaris was altered
by the illumination of different wavelength of light (Fig. 1d).
Approximately, 30% increase in the dry weight per cell of C. vulgaris
was observed by changing the wavelength from red to blue
(Fig. 1d). Previous reports suggested similar effect of wavelength
on microalgal cells, though the evidences were fragmental without
providing a conclusive result. Lee and Palsson (1994) demon-
strated that red light led to smaller cells than those under the
full-spectrum white light due to the early autospore release upon
red light. Chlorella cells cultivated under blue light produced larger
and fewer autospores (Kowallik, 1963; Pirson and Kowallik, 1964),
whereas blue light delayed the Chlamydomonas reinhardtii cell divi-
sion (Münzner and Voigt, 1992; Oldenhof et al., 2004b). Interest-
ingly, Oldenhof et al. (2004a, 2006) recently demonstrated that
C. reinhardtii cells could be altered in cell cycle by shifting light
wavelength to either blue or red (Oldenhof et al., 2004a, 2006).
Furthermore, red or blue light could affect the division of chloro-
plast nucleoids and replication of chloroplast DNA in Dunaliella
salina (Zachleder et al., 1989). Even though all of these previous
studies were not intended to perform systematic comparison be-
tween blue and red light, they partially provided the evidences that
cell division could be accelerated or delayed upon red and blue
light. It is likely that differential rate of cell divisions or cell cycle
progression under either blue or red light might be underlying
mechanism for the observation of different cell size upon red or
blue light. Consistent with these results, the data in this study
244 D.G. Kim et al. / Bioresource Technology 159 (2014) 240–248
clearly indicate that C. vulgaris morphology of cell size was signif-
icantly transformed into bigger cells by the exposure of blue light,
compared to those under red light.
3.3. Investigation of possible molecular mechanisms of different C.
vulgaris responses under red, blue and white light
In order to determine molecular mechanisms of different C. vul-
garis responses under red, blue and white light, we next examined
the expression of genes possibly responsible for increased cell size
upon blue light. Based on altered cell morphology and possible
influence over the photosynthesis under the different wavelengths,
genes belonging to functional categories involved in either photo-
synthesis or cell division were selected. qRT-PCR was used to
investigate the relative expression of these genes under the condi-
tions between white, red, and blue illumination. Of select genes,
we could successfully identify the genes, which displayed differen-
tial expressions under white, red, and blue illumination. Identified
genes were psaB encoding for the photosystem I (PS I) reaction
center protein, chlB for light-independent protochlorophyllide
reductase subunit B, rbcL for the large subunit of rubisco, psbA
for the photosystem II (PS II) reaction protein D1, and minD for cell
division inhibitor protein.
Except for minD and psaB, all of genes displayed similar patterns
of expression in that the abundances of gene transcripts were grad-
ually decreased with the change of light wavelength from blue,
white and red (Fig. 2). Compared with those under red or white,
mRNA expressions of chlB, psbA, and rbcL were significantly higher
(P < 0.05) under the blue light. chlB is involved in light-indepen-
dent reaction for chlorophyll biosynthesis (Suzuki and Bauer,
1992) and rbcL encodes the large subunit of rubisco, which is the
key enzyme of the calvin cycle (Qian et al., 2009). psbA is required
for photosystem II encoding protein D1 (Suzuki and Bauer, 1992).
Since mRNA expressions of chlB, psbA, and rbcL were increased
upon the exposure to blue light in C. vulgaris, blue light might stim-
ulate the activation of enzyme(s) linked with photosynthesis.
Without doubt, blue light is more energetic radiation than red light
due to its shorter wavelength causing more stress to C. vulgaris
cells (Jeong et al., 2013; Menon et al., 2013). Due to this reason,
it is likely that C. vulgaris cells must adapt stressful radiation of
blue light, which might require high level of expression of photo-
synthesis-related genes such as chlB, psbA, and rbcL. In accordance
with this notion, it has been well accepted that photosynthesis,
particularly PS II, play an important role in regulating or adjusting
the light harvesting capacity (Briantais et al., 1979; Chow et al.,
1990; Sukenik et al., 1987). For this reason, it might be possible
to detect the increase of gene expressions involved in photosynthe-
sis, particularly PS II, under the blue illumination, as observed in
this study (Fig. 2).
On the other hand, psaB encodes a protein of the PS I reaction
center involved in binding of electron transfer components
(Murray et al., 2006). The results showed that gene (psaB) encoding
PS I protein transcribed differently from those linked with PS II.
Since both blue and red light could led to increased transcript for
psaB, compared to those under full-spectrum white light, it is likely
that psaB expression could be increased upon exposure of either of
two preferential wavelength of photosynthesis; red and blue light.
In addition, minD encodes the cell division inhibitor (Wakasugi
et al., 1997). Transcription of minD was significantly suppressed
under the red light, compared to those under both blue and white.
Consistent with the patterns of minD gene expression, increased
and decreased cell division was detected under the red and blue
light, respectively, thereby rendering differential C. vulgaris cell
sizes upon exposure of red and blue light. Therefore, since minD
expression and C. vulgaris cell sizes were well-correlated each
other under the red and blue light, these data provides molecular
evidence that minD serves as a negative regulator of cell sizes upon
different wavelength of light in C. vulgaris.
3.4. Determination of ROS stress level upon different wavelength
Because, theoretically, blue light has a feature of high energetic
wavelength, it is reasonable to hypothesize that C. vulgaris cells
could accumulate higher extent of stress under the blue light. To
further understanding of responsive stress mechanisms in microal-
gal cells upon specific wavelength of light, particularly blue light,
we examined the extent of cellular stress via the measurement of
reactive oxygen species (ROS). Generally, ROS are the typical
molecular mediators of various stresses in the cell of a wide variety
of living species (Apel and Hirt, 2004; Hamid Badawi et al., 2004).
To detect and quantify ROS generation in C. vulgaris cells upon dif-
ferent light wavelength, the standard protocol of nitroblue tetrazo-
lium (NBT) staining was utilized. The reactive oxygen species (ROS)
activity of C. vulgaris cultivated under three different wavelength of
light (red, blue and white) were thoroughly compared with three
biological replications (Fig. 3). Quantification demonstrated that
cultivation under blue light cells displayed significantly (P < 0.05)
higher extent of ROS than those under white or red light (Fig. 3).
The analyses on the level of ROS demonstrated that cells grown un-
der blue light had around a 40% increase of ROS, whereas cells
grown under red light showed more than 30% decrease of ROS level
(Fig. 3). These data clearly indicated that blue wavelength led to in-
crease the level of cellular ROS, whereas red light decreased the
ROS level in C. vulgaris.
Previously, it has been reported that shorter wavelengths such
as blue and green light deliver more energy for photosynthesis,
thereby causing photo-inhibition (Das et al., 2011; Jeong et al.,
2013). On the other hand, red light has an opposite feature of long-
er wavelength, possibly alleviating the effect of photo-inhibition
(Matthijs et al., 1996). However, there has been no direct or indi-
rect evidence demonstrating the involvement of ROS for these mic-
roalgal phenotypes under different wavelengths of light. In this
study, we first demonstrated that stress mechanism(s) associated
with ROS could help explain the photo-inhibition of microalgae
upon blue light. Moreover, ROS level was well-correlated with dif-
ferential C. vulgaris cell sizes upon exposure of red and blue light
(Figs. 1d and 3). Consequently, it is conceivable that increased
ROS generation via blue light illumination may be responsible for
increased cell size morphology in C. vulgaris. Similarly, microalga
Haematococcus pluvialis has a distinctive lifecycle, because it exhib-
its small-sized green cells under favorable environment and big-
ger-sized resting cells under unfavorable condition (Choi et al.,
2011). Though C. vulgaris has not much variation in cell size like
H. pluvialis, the results indicated that blue light induces oxidative
stress in C. vulgaris cells thereby causing the cellular transforma-
tion into bigger cells to cope with the unfavorable condition of high
energy irradiance from blue light.
3.5. Innovative wavelength shift approach to enhance productivities of
C. vulgaris culture
From the above results, we obtained novel results regarding to
phenotypic characteristics upon different light wavelengths and
discovered the possible mechanisms behind these phenotypic
characteristics by confirming differential gene expression as well
as proving altered ROS levels. Notably, cell morphology of sizes
was significantly changed depending on the wavelength of light.
Red light enhanced cell division giving rise to small cells, whereas
blue light led to increased cell size.
Since specific wavelength of light stimulates particular charac-
teristics of microalgal physiology of differential cell sizes, we
then next attempted to utilize these cellular features upon LED
 D.G. Kim et al. / Bioresource Technology 159 (2014) 240–248 245

3.0 1.6
(A) (chlB) (B) (psbA)
1.4
2.5

1.2
2.0
1.0

1.5 0.8

0.6
1.0

0.4
0.5
0.2

0.0 0.0
5 2.5
(C) (psaB) (D) (rbcL)
Relative transcriptional abundance

4 2.0

3 1.5

2 1.0

1 0.5

0 0.0
1.6 White Red Blue
(E) (minD)
1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0
White Red Blue

Fig. 2. Expression of ChlB (A), psbA (B), psaB (C), rbcL (D) and mind (E) from C. vulgaris cultivated under either red, blue, or white light.

wavelength into the biotechnological applications. We hypothe-
sized that different cell sizes possibly reflect different cellular
potentials containing different extent of nutrients or proteins for
following cell divisions. Based on that, we can speculate that gen-
eration of microalgal cells with bigger size during the beginning
stage of cultivation might help promote subsequent cell division
in the later growth stage, thereby leading to enhanced productivi-
ties for biomass as well as total FAME. This hypothesis prompted
us to propose that if we could manipulate microalgal cell sizes
properly via the appropriate illumination of LED wavelength, we
might be able to further enhance microalgal productivities.
In order to test the hypothesis, we adopted adequate manipula-
tion of LED wavelength illumination as the way to generate differ-
ent size of microalgal cells at different growth time points. The
experiments were performed via the shift of light wavelength at
different time points. The set of experiments was consisted of five
different light illuminations: continuous blue or red light; blue
light first and then shifted into red light or vice versa. Light wave-
length was shifted either blue or red after 24, 48, 72, and 96 h of
cultivation. The experimental results obtained from wavelength
shifts were designated as the form of R1B4 (1 day Red light and
then 4 days Blue light illumination) or B1R4 (1 day Blue light and
then 4 days Red illumination) and so on.
3.5.1. Change of chlorophyll contents under the wavelength shift
Chlorophyll content as an indicator of cell physiology was first
examined. The change of chlorophyll contents of C. vulgaris cells
upon different wavelength of red or blue light or under the
wavelength shifts were shown in Fig. 4. Notably, the content of
C. vulgaris chlorophyll was highly dependent on light wavelength.
246 D.G. Kim et al. / Bioresource Technology 159 (2014) 240–248
Fig. 3. ROS activity of C. vulgaris cultivated under either red, blue, or white light.
Different letters over the bar represent significant difference (P < 0.05).
For examples, blue and red lights resulted in high and low content
of chlorophyll, respectively. These data were consistent with our
previous observation on different cell sizes upon red or blue light,
since blue light gave rise to bigger cell size, possibly containing
higher cellular components including chlorophyll. Interestingly,
microalgal cultivations transferred from blue to red light resulted
in immediate decrease in chlorophyll contents (Fig. 4). After the
transfer to red light, the higher chlorophyll content under blue
light became almost same level with that under red light (Fig. 4).
The decreases of chlorophyll were happened within 24 h after
the transfer from blue to red light. These data again suggested that
microalgal cells with unique physiology could be produced by the
manipulation of different light wavelength.
3.5.2. Enhanced biomass productivity with the wavelength shift
Next, biomass productivities between continuous blue or red
light and wavelength shifts were thoroughly compared. Under
the continuous red light, C. vulgaris productivity was similar to
those under the white light, whereas blue light resulted in de-
creased production (Fig. 5). The results indicated that simple blue
light illumination was not supportive for C. vulgaris growth.
However, as expected, appropriate switch of wavelength could
lead to enhanced biomass production, compared to those under
3.2
3.0
2.8
Chlorophyll content(mg/L)
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1 2 3 4 5 6
Culture days
Fig. 4. Changes of chlorophyll content upon different light conditions. Chlorophyll
content under the continuous red (-d-) or blue light (-j-) in comparison with those
of wavelength shift (from blue to red) at different time points (2 days (-h-) or
3 days after inoculation (-s-)).
the continuous mono-color red or blue light (Fig. 5). Particularly,
blue light first and then switched into red light (B2R3 and B3R2)
displayed the best performance, compared to the others. Approxi-
mately 18–20% increase in both B2R3 and B3R2 were observed.
On the contrary, the deployment of red first and then shifted to
blue light (R1B4, R2B3, R3B2, R4B1) showed the decreased micro-
algal growth. A possible explanation for these observations is that
C. vulgaris cells favor red light for cell division generating small-
sized cells. These small-sized cells must have less ability or
potential for increasing cell volume, even under the following blue
illumination. In contrast, initial illumination of blue light induces
bigger sized cells with high potentials for subsequent division un-
der red light, thereby improving the overall production. Alterna-
tively, light wavelength of blue or red exerts an influence on
C. vulgaris cell cycle and thus leads to differential rate of progression
of cell cycle. For example, in C. reinhardtii, cells cultivated under blue
light have larger cell size due to the influential effect of blue light to
cell cycle, thereby having high potentials for subsequent cell division
(Oldenhof et al., 2006). In this regard, by manipulating light wave-
length appropriately from blue to red, it was possible to change
the progression of C. vulgaris cell cycle, which in turn resulted in
increased biomass productivity. Taken together, illustration of a
possible explanation for our results was provided in Fig. 6.
The only exception for this notion is found in B1R4 and B4R1.
We speculated that there must be the optimum time to switch
the wavelength (from blue to red light), since it was not effective
if applied at either too early or too late time points. With the aid
of adequate blue light illumination, microalgal cells become larger
and ready for following explosion of cell divisions. On the other
hand, red light must accelerate cell division as an environmental
cue. Therefore, red light was well-matched with late exponential
phase. Multiple lines of studies suggested that 0–48 h and
48–120 h after inoculation could be broadly categorized as lag
and exponential phase, respectively (Xu et al., 2013; Zwietering
et al., 1990). Consequently, the underlying cause of our observation
about the existence of optimum time (B2R3 and B3R2) to switch
the wavelength might be explained by the period for lag or expo-
nential phase. Additionally, it has been known that blue light
was involved in enzyme activation, thus regulating gene transcrip-
tion, energy-derivation and ultimately affecting cell cycle (Oldenhof
et al., 2006; Ruyters, 1984). Particularly, cyclin-dependent kinases
(CDKs) in C. vulgaris might mediate the key biological process of cell
cycle upon blue or red light, since CDKs play pivotal roles in regulat-
ing the cell cycle in all known eukaryotes by influencing a plethora of
Fig. 5. Biomass productivities obtained from either continuous mono-color illumi-
nation or wavelength shift. The experimental results obtained from wavelength
shifts were denoted by the form of R1B4 (1 day Red light and then 4 days Blue light
illumination) or B1R4 (1 day Blue light and then 4 days Red illumination) and so on.
Different letters over the bar represent significant difference (P < 0.05).
 D.G. Kim et al. / Bioresource Technology 159 (2014) 240–248 247

Fig. 6. Putative model explaining different morphological progressions of C. vulgaris upon different wavelength of light. Blue light can increase cell sizes, whereas red light
enhanced cell divisions. Consequently, we proposed a novel strategy for microalgal cultivation based on light wavelength change (from blue to red). By doing so, microalgal
cells with bigger size can be generated during the beginning stage of cultivation. Bigger microalgal cells must have a huge potential for cell division. Thus following red light
illumination then help promote subsequent cell division in the later growth stage, thereby leading to enhanced productivities for biomass as well as lipid production. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2
gene transcriptions. In this context, Oldenhof et al. (2004a, 2006) re- Fatty acids profiles (% of total lipids) of the C. vulgaris under either continuous mono-
ported that, in microalga C. reinhardtii, there is corresponding in- color illumination or wavelength shifts.
crease in CDK activity under blue light, which in turn affect
Fatty acid Light condition
multiple downstream genes for cell division or cell cycle progression.
Because of the importance of appropriate timing of CDK activation to Red R1B4* R2B3* R3B2* R4B1* White

ensure the commitment to cell cycle, it is not surprising to notice that C10:0 1.51 0.77 0.73 0.83 0.78 0.51
there is the optimal range of period to illuminate blue or red light for C14:0 1.11 1.23 1.32 1.42 1.10 1.63
C16:0 16.53 14.60 15.01 14.20 15.26 14.38
proper CDK function to improve biomass productivity. Further study
C17:1 11.23 12.88 12.62 13.10 12.27 12.28
might be necessary to pinpoint the involvement of CDKs in alteration C18:0 7.13 5.98 6.09 3.70 7.21 2.94
of microalgal biology upon different wavelength of light as observed C18:2 10.33 7.83 8.76 7.76 8.51 9.40
in this study. C18:3 29.70 33.29 32.51 33.47 33.41 30.30
To prove further, statistical analysis was also performed and TFAME/DW (%) 10.65 11.44 12.07 9.80 11.33 9.61

further suggested that B2R3 and B3R2 have the highest productiv- Blue* B1R4* B2R3* B3R2* B4R1*
ities, compared to the others (P < 0.05). Based on that, we con- C10:0 1.15 1.31 1.05 1.70 1.10
cluded that the novel strategy for microalgal cultivation via the C14:0 1.36 0.77 1.00 0.68 1.06
manipulation of light wavelength could be successfully demon- C16:0 14.49 16.26 15.78 16.54 15.28
C17:1 13.35 11.45 11.34 11.36 12.64
strated in C. vulgaris. Since the method based on the appropriate C18:0 3.55 10.86 6.46 8.99 4.19
manipulation of light wavelength could be easily expanded to C18:2 8.19 10.09 9.32 9.79 10.41
the other species of microalgae, we believe that this approach will C18:3 32.57 30.15 31.05 30.01 29.99
open the way to achieve further enhancement for microalgal TFAME/DW (%) 11.07 14.11 13.09 13.40 12.24
biotechnology. *
The experimental results obtained from wavelength shifts were designated as the
form of R1B4 (1 day Red light and then 4 days Blue light illumination) or B1R4
(1 day Blue light and then 4 days Red illumination) and so on.
3.5.3. Enhancement of biodiesel production with the wavelength shift
We further tested whether wavelength shift could increase fatty
continuous light illuminations due to the increased biomass pro-
acid production for biodiesel. Table 2 summarizes the fatty acids
ductivities (Table 2). Taken together, appropriate manipulation of
profiles and total FAME (TFAME) obtained from different light illu-
LED wavelength in consideration of microalgal biology could im-
minations: continuous blue or red light; R1B4–R4B1; B1R4–B4R1.
prove both microalgal growth as well as biodiesel production.
Fatty acid profiles were not altered significantly with the change
We believe that a novel discovery revealed in this study will help
of the wavelength, which is consistent with the previous report
push the limit for microalgal biotechnology leveraging its future
(Tang et al., 2011). However, TFAME was highly dependent upon
application.
light illumination. It should be noteworthy that overall FAME
yield(s) could be significantly increased via the switch of wave-
length, possibly owing to the efficient growth with the help of 4. Conclusion
sophisticated manipulation of light wavelength. Under the contin-
uous illumination of either white, red, or blue light, approximate In this study, the effect of specific light wavelength on C. vulga-
similar extent of TFAME were obtained (9.61%, 10.65% and ris biology was thoroughly investigated, particularly with red or
11.07%, respectively). However, TFAME obtained by appropriate blue light. Based on the discovery of the effect of light wavelengths
wavelength shift (from blue to red) was higher than those of on microalgal biology, the appropriate wavelength at different
248 D.G. Kim et al. / Bioresource Technology 159 (2014) 240–248
growth stages was conceived as a novel strategy for C. vulgaris cul-
tivation. With our innovative biotechnology process of wavelength
shift, we could achieve further increased productions in both bio-
mass and FAME. Consequently, these results will have significant
impact on future development of microalgal biotechnology.
Acknowledgements
This research was financially supported by the Ministry of
Trade, Industry & Energy (MOTIE), Korea Institute for Advancement
of Technology (KIAT) and Honam Institute for Regional Program
Evaluation through the Leading Industry Development for Eco-
nomic Region. This work was also supported by the Advanced Bio-
mass R&D Center (ABC) of Korea Grant funded by the Ministry of
Education, Science and Technology (ABC-2012-055032). In addi-
tion, this work was also supported by the Industrial Technology
Research Infrastructure Program (N0000004) funded by the Minis-
try of Trade, Industry and Energy (MTE, Korea).
References
Apel, K., Hirt, H., 2004. Reactive oxygen species: metabolism, oxidative stress, and
signal transduction. Annu. Rev. Plant Biol. 55, 373–399.
Baba, M., Kikuta, F., Suzuki, I., Watanabe, M.M., Shiraiwa, Y., 2012. Wavelength
specificity of growth, photosynthesis, and hydrocarbon production in the oil-
producing green alga Botryococcus braunii. Bioresour. Technol. 109, 266–270.
Briantais, J.-M., Vernotte, C., Picaud, M., Krause, G., 1979. A quantitative study of the
slow decline of chlorophyll a fluorescence in isolated chloroplasts. Biochim.
Biophys. Acta 548, 128–138.
Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306.
Choi, Y.-E., Yun, Y.-S., Park, J.M., Yang, J.-W., 2011. Multistage operation of airlift
photobioreactor for increased production of astaxanthin from Haematococcus
pluvialis. J. Microbiol. Biotechnol. 21, 1081–1087.
Chow, W.S., Melis, A., Anderson, J.M., 1990. Adjustments of photosystem
stoichiometry in chloroplasts improve the quantum efficiency of
photosynthesis. Proc. Natl. Acad. Sci. 87, 7502–7506.
Das, P., Lei, W., Aziz, S.S., Obbard, J.P., 2011. Enhanced algae growth in both
phototrophic and mixotrophic culture under blue light. Bioresour. Technol. 102,
3883–3887.
Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and
purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509.
Hamid Badawi, G., Yamauchi, Y., Shimada, E., Sasaki, R., Kawano, N., Tanaka, K.,
Tanaka, K., 2004. Enhanced tolerance to salt stress and water deficit by
overexpressing superoxide dismutase in tobacco (Nicotiana tabacum)
chloroplasts. Plant Sci. 166, 919–928.
Hill, J., Nelson, E., Tilman, D., Polasky, S., Tiffany, D., 2006. Environmental, economic,
and energetic costs and benefits of biodiesel and ethanol biofuels. Proc. Natl.
Acad. Sci. 103, 11206–11210.
Jeong, H., Lee, J., Cha, M., 2013. Energy efficient growth control of microalgae using
photobiological methods. Renew. Energ. 54, 161–165.
Kowallik, W., 1963. Die Zellteilung vonChlorella im Verlaufe einer Farblichtkultur.
Planta 60, 100–108.
Lee, C.G., Palsson, B.Ø., 1994. High-density algal photobioreactors using light-
emitting diodes. Biotechnol. Bioeng. 44, 1161–1167.
Matthijs, H.C., Balke, H., Van Hes, U.M., Kroon, B., Mur, L.R., Binot, R.A., 1996.
Application of light-emitting diodes in bioreactors: flashing light effects and
energy economy in algal culture (Chlorella pyrenoidosa). Biotechnol. Bioeng. 50,
98–107.
Menon, K.R., Balan, R., Suraishkumar, G., 2013. Stress induced lipid production in
Chlorella vulgaris: relationship with specific intracellular reactive species levels.
Biotechnol. Bioeng. 110, 1627–1636.
Münzner, P., Voigt, J., 1992. Blue light regulation of cell division in Chlamydomonas
reinhardtii. Plant physiol. 99, 1370–1375.
Murray, J.W., Duncan, J., Barber, J., 2006. CP43-like chlorophyll binding proteins:
structural and evolutionary implications. Trends Plant Sci. 11, 152–158.
Oldenhof, H., Bišová, K., van den Ende, H., Zachleder, V., 2004a. Effect of red and blue
light on the timing of cyclin-dependent kinase activity and the timing of cell
division in Chlamydomonas reinhardtii. Plant Physiol. Biochem. 42, 341–348.
Oldenhof, H., Zachleder, V., van den Ende, H., 2004b. Blue light delays commitment
to cell division in Chlamydomonas reinhardtii. Plant Biol. 6, 689–695.
Oldenhof, H., Zachleder, V., van den Ende, H., 2006. Blue- and red-light regulation of
the cell cycle in Chlamydomonas reinhardtii (Chlorophyta). Eur. J. Phycol. 41,
313–320.
Pirson, A., Kowallik, W., 1964. Spectral responses to light by unicellular plants.
Photochem. photobiol. 3, 489–497.
Qian, H., Chen, W., Li, J., Wang, J., Zhou, Z., Liu, W., Fu, Z., 2009. The effect of
exogenous nitric oxide on alleviating herbicide damage in Chlorella vulgaris.
Aquat. Toxicol. 92, 250–257.
Rao, M.V., Davis, K.R., 1999. Ozone-induced cell death occurs via two distinct
mechanisms in Arabidopsis: the role of salicylic acid. Plant J. 17, 603–614.
Ruyters, G., 1984. Effects of blue light on enzymes. In: Senger, H. (Ed.), Blue light
effects in Biological Systems. Springer Verlag, Berlin, pp. 283–301.
Sukenik, A., Bennett, J., Falkowski, P., 1987. Light-saturated photosynthesis-
limitation by electron transport or carbon fixation. Biochim. Biophys. Acta
891, 205–215.
Suzuki, J.Y., Bauer, C.E., 1992. Light-independent chlorophyll biosynthesis:
involvement of the chloroplast gene chlL (frxC). Plant Cell 4, 929–940.
Tang, H., Chen, M., Garcia, M., Abunasser, N., Ng, K., Salley, S.O., 2011. Culture of
microalgae Chlorella minutissima for biodiesel feedstock production. Biotechnol.
Bioeng. 108, 2280–2287.
Thompson, A.S., Rhodes, J.C., Pettman, I., 1988. Natural environmental research
council culture collection of algae and protozoa: catalogue of strains. Freshw.
Biol. Ambleside, 164.
Uduman, N., Qi, Y., Danquah, M.K., Forde, G.M., Hoadley, A., 2010. Dewatering of
microalgal cultures: a major bottleneck to algae-based fuels. J. Renew. Sust.
Energ. 2, 012701.
Wakasugi, T., Nagai, T., Kapoor, M., Sugita, M., Ito, M., Ito, S., Tsudzuki, J., Nakashima,
K., Tsudzuki, T., Suzuki, Y., 1997. Complete nucleotide sequence of the
chloroplast genome from the green alga Chlorella vulgaris: the existence of
genes possibly involved in chloroplast division. Proc. Natl. Acad. Sci. 94, 5967–
5972.
Wang, C.-Y., Fu, C.-C., Liu, Y.-C., 2007. Effects of using light-emitting diodes on the
cultivation of Spirulina platensis. Biochem. Eng. J. 37, 21–25.
Xu, B., Cheng, P., Yan, C., Pei, H., Hu, W., 2013. The effect of varying LED light sources
and influent carbon/nitrogen ratios on treatment of synthetic sanitary sewage
using Chlorella vulgaris. World J. Microbiol. Biotechnol., 1–12.
Zachleder, V., Kuptsova, E.S., Los, D.A., Cepák, V., Kubín, Š., Shapiguzov, J.M.,
Semenenko, V.E., 1989. Division of chloroplast nucleoids and replication of
chloroplast DNA during the cell cycle of Dunaliella salina grown under blue and
red light. Protoplasma 150, 160–167.
Zhao, Y.J., Hui, Z., Chao, X., Nie, E., Li, H.J., He, J., Zheng, Z., 2011. Efficiency of two-
stage combinations of subsurface vertical down-flow and up-flow constructed
wetland systems for treating variation in influent C/N ratios of domestic
wastewater. Ecol. Eng. 37, 1546–1554.
Zwietering, M., Jongenburger, I., Rombouts, F., Van’t Riet, K., 1990. Modeling of the
bacterial growth curve. Appl. Environ. Microbiol. 56, 1875–1881.