See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/283208610

The Effect of Light Stress and Other Culture Conditions on Photoinhibition

and Growth of Dunaliella tertiolecta

Article  in  Applied biochemistry and biotechnology · October 2015

DOI: 10.1007/s12010-015-1882-x

CITATIONS READS

8 315

7 authors, including:

Prayad Pokethitiyook Metha Meetam

Mahidol University Mahidol University
109 PUBLICATIONS   4,469 CITATIONS    24 PUBLICATIONS   490 CITATIONS   

SEE PROFILE SEE PROFILE

Kittisak Yokthongwattana Wanvisa Pugkaew

Mahidol University Mahidol University
34 PUBLICATIONS   377 CITATIONS    7 PUBLICATIONS   69 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Microalgae for biofuel, wastewater treatment and value added products View project

Microalgae View project

All content following this page was uploaded by Prayad Pokethitiyook on 30 October 2015.

The user has requested enhancement of the downloaded file.

Appl Biochem Biotechnol
DOI 10.1007/s12010-015-1882-x

The Effect of Light Stress and Other Culture Conditions

on Photoinhibition and Growth of Dunaliella tertiolecta

Jitpisut Seepratoomrosh 1,3,4 & Prayad Pokethitiyook 1,3 &

Metha Meetam 1,3 & Kittisak Yokthongwattana 2 &
Wenqiao Yuan 4 & Wanvisa Pugkaew 1,3,4 &
Kunn Kangvansaichol 5

Received: 22 July 2015 / Accepted: 1 October 2015

# Springer Science+Business Media New York 2015

Abstract This work aimed to examine the effects of high light stress as well as other
culture conditions including HCO3− concentration, temperature, salinity, and pre-acclimation
on photoinhibition and growth of halotolerant alga Dunaliella tertiolecta. Significant
photoinhibition of D. tertiolecta was observed during a short period of exposure
(6 hours) to high intensity of lights (1000, 1500, and 2000 μmol photons m−2 s−1);
however, after 2 days of continuous light exposure, the alga adapted to high light stress
and reached similar growth rates as low light exposure. The increase in HCO3− concen-
tration in the culture medium did not reduce photoinhibition, but the growth rate and
chlorophyll contents increased with increasing HCO3− concentrations. Temperature had
significant effects on photoinhibition. Combined high temperature and high light intensity
led to more serious photoinhibition and reduced cell growth rates, so did combined low
salinity and high light intensity. Pre-acclimation by 50, 200, or 500 μmol photons m−2 s−1
each for 1, 3, or 6 hours (a total of nine treatments) did not significantly influence
photoinhibition or cell growth of D. tertiolecta, probably because the acclimation periods
were not long enough.

* Prayad Pokethitiyook
prayad.pok@mahidol.ac.th

1
Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
2
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
3
Center for Environmental Health, Toxicology and Management of Chemicals, CHE: 3328 Si
Ayutthaya Road, Bangkok 10400, Thailand
4
Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC
27695, USA
5
PTT Research and Technology Institute, Phra Nakhon Si Ayutthaya 13170, Thailand
 Appl Biochem Biotechnol

Keywords Light stress . Photoinhibition . Pre-acclimation . Dunaliella tertiolecta . Biofuel .

Microalgae

Introduction

Microalgae have been widely recognized as a promising source of high value products
and biofuel [1, 2] such as β-carotene [3], omega-3 [4], EPA [5], DHA [6], bioethanol [7],
biogas [8], and biodiesel [9]. Use of microalgae as a potential biofuel source is favorable
because of their high oil contents, fast biomass accumulation, and no requirement for
large arable lands [10, 11]. Most microalgae are photoautotrophic and they are often
cultured under light exposure with the supply of CO2 or air [12, 13]. Light is an
important factor for photoautotrophic microorganisms. Light provides energy for photo-
synthesis process; however, too much light during noon time in Thailand can cause
damage to algal cells by degrading the proteins in light-harvesting complex. Excessive
light energy that is absorbed by the light-harvesting complexes may not be used for
photochemical reactions or be released as a heat, causing photoinhibition [14]. There are
wide variations in the nature of algal strains and their dependence on light intensity for
growth [15]. Khoeyi et al. [16] found that under low light intensity, Chlorella vulgaris
exhibited a slow growth, which increased when light intensity shifted from 37.5 to
62.5 μmol photons m −2 s −1, but with a further increase in light intensity to
100 μmol photons m−2 s−1, a decrease in biomass accumulation was observed. Pulz
[17] reported that the photosynthesis of most microalgae is saturated at about 30 % of the
total terrestrial solar radiation, i.e., 1700–2000 μmol photons m−2 s−1. Besides light, other
culture conditions were also found to influence algal growth rates. Kitaya et al. [18]
showed that the highest multiplication rate of Euglena gracilis occurred at temperatures of
27–31 °C, CO2 concentration of 4 %, and photo flux of about
100 μmol photons m−2 s−1, while the maximum specific growth rate of Amphidinium
sp. was obtained at a temperature of 26 °C, CO2 concentration of 0.05 %, and photo flux
of about 35 μmol photons m−2 s−1. It is apparent that optimum culture conditions of
different algal species vary widely.
Some common algal strains for biodiesel production are Chlorella sp., Dunaliella sp., and
Nannochloropsis sp. They are species with high tolerances for salt, temperature, and light.
They are also relatively easy to culture and have high cell growth and oil production rates [19,
20]. This study, however, focused on Dunaliella tertiolecta. It is a green alga (phylum
Chlorophyta, order Dunaliellales) [21] that possess ovoid, spherical, pyriform, fusiform, or
ellipsoid with size varying from 5 to 25 μm in length and from 3 to 13 μm in width [22, 23].
D. tertiolecta has been previously studied for various purposes including biodiesel production
[24], β-carotene accumulation [25], and also photosynthesis [26]. The advantage of this strain
is that it easily grows in a variety of environmental conditions, for example, a wide range of
salinity (from 0.5–5 M NaCl) [27] and temperature (from below 0 to around 45 °C) [23]. It was
also found to tolerate high light stress, although the growth rate and photosynthesis efficiency
of the alga were adversely affected by excessive light [28, 29].
In order to maximize biomass production of D. tertiolecta, the objectives of this work were
therefore to (1) examine the photoinhibition effects of high light intensity on D. tertiolecta and
(2) understand the effects of culture conditions, including HCO3− concentration, temperature,
salinity, and pre-acclimation on growth of D. tertiolecta under high light stress.
Appl Biochem Biotechnol

Materials and Methods

Microorganism and Culture Medium

D. tertiolecta (UTEX 999) was obtained from UTEX Culture Collection of Algae (the
University of Texas at Austin, TX, USA). D. tertiolecta was grown in artificial hypersaline
medium with the following recipe: 1.5 M NaCl, 5 mM KNO3, 40 mM Tris-HCl, pH 7.5, 5 mM
MgSO4, 0.3 mM CaCl2, 0.1 mM KH2PO4, and 2 μM FeCl3. The medium was also supple-
mented with a mixture of micronutrients (6 μM EDTA, 185 μM H3BO4, 7 μM MnC12,
0.8 μM ZnC12, 0.02 μM CoCl2, and 0.2 nm CuCl2) [30, 31]. The stock culture was kept at
25 °C under continuous illumination of 50 μmol photons m−2 s−1 provided by cool white
fluorescence lamps. Sterile air at the flow rate of 1 vv−1 m−1 (volume air per volume medium
per minute) was supplied to the bottom of the stock culture bottles.

Experimental Design

Experimental conditions of this work are summarized in Table 1. The following parameters
were studied. Light intensity 1000, 1500, and 2000 μmol photons m−2 s−1; initial concentration
of NaHCO3 50 and 100 mM. Temperature 30, 35, and 40 °C; NaCl concentration 0.5, 1, and
2 M. To study the effect of pre-acclimation, D. tertiolecta was pre-acclimated to three different
light intensities (50, 200, or 500 μmol photons m−2 s−1) each with three acclimation durations
(1, 3, or 6 h, a total of nine independent treatments) before being exposed to high light intensity
(2000 μmol photons m −2 s −1 ). The control condition used in this study was
50 μmol photons m−2 s−1 of light intensity, 25 mM HCO3− concentration, temperature at
25 °C, and 1.5 M NaCl salinity without pre-acclimation. All culture treatments were exposed
to low (50 μmol photons m−2 s−1) and high (2000 μmol photons m−2 s−1) light intensities
except for in the light intensity experiments where all four light intensities (50, 1000, 1500, and
2000 μmol photons m−2 s−1) were studied. In all experiments, D. tertiolecta was cultured
in 250-ml square laboratory glass bottles containing 200-ml hypersaline medium with
1 vv−1 m−1 of aeration rate with two replications. The initial optical density (OD at
680 nm) of all cultures was adjusted to 0.05 and a 1500-W halogen light was used to
provide illumination to the cultures.

Analytical Methods

Cultures were collected to measure Fv/Fm, chlorophyll contents, and specific growth rate at the
end of the 2-day culture period. Fv/Fm was also measured hourly in the first 6 h of cultivation,
using a fluorometer (Z985 Cuvette AquaPen, Qubit systems, Ontario, Canada). Fv/Fm is a

Table 1 The experimental conditions

Light intensity HCO3− Temperature Salinity Photoacclimation

(μmol photons m−2 s−1) concentration (°C) (M)
(mM)

Control 50 25 25 1.5 None

Test 1000, 1500, 2000 50, 100 30, 35, 40 0.5, 1, 2 50, 200, and 500 μmol photons
m−2 s−1 for 1, 3, and 6 h
 Appl Biochem Biotechnol

fluorescence parameter that provides an estimate of the maximum quantum efficiency of PS II

photochemistry. Normally, Fm (maximal fluorescence level) is generated during a short pulse
at high photosynthesis photon flux that drives most PS II reaction centers into Bclosed^ state.
F0 (minimal fluorescence level) is determined at very low photosynthesis photo flux density,
under which almost all PS II reaction center are in the Bopen^ state and ready for photochem-
istry. The difference between F0 and Fm is termed as Fv [32]. For chlorophyll (both a and b)
content measurement, D. tertiolecta was extracted with 80 % acetone (v/v) based on the
method introduced by Lichtenthaler and Buschmann [33]. Briefly, 1 ml of cells was collected
and centrifuged at 8000 rpm for 10 min, after that the supernatant was removed and the
concentrated cells were extracted with 80 % acetone (v/v) in the dark. Then the mixture was
centrifuged again, and the supernatant was measured for absorbance at the wavelengths of
646.8 and 663.2 nm using a spectrophotometer (Aquarius CE7200, Cecil instrument, Cam-
bridge, UK). Chlorophyll a (Ca) and chlorophyll b (Cb) contents were calculated using the
following equations:
Caðμg=mlÞ ¼ 12:25A663:2 2:79A646:8
Cbðμg=mlÞ ¼ 21:50A646:8 5:10A663:2

where A denotes the optical density and the subscripts represent the wavelength. Cell
density was measured using the same spectrophotometer at 680 nm and then calculated as
specific growth rate using the following equation:

1nðN 2 Þ−1nðN 1 Þ
μ¼
t2 −t1

where μ is the specific growth rate (day−1) and N1 and N2 are OD680 at time t1 (day) and t2
(day), respectively.

Statistical Analysis

The data were analyzed by two-way ANOVA in conjunction with Bonferroni’s test. Treat-
ments were considered significantly different at p < 0.05, represented by the different letters in
the figures. The uppercase letters were used for high light intensity while the lowercase letters
were used for low light intensity conditions.

Results and Discussion

The Effect of Light Stress

In healthy microalgal cultures, Fv/Fm ranges from 0.6 to 0.8 depending on the irradiance and
treatments [34]. Figure 1a, b shows Fv/Fm of D. tertiolecta after exposure to different light
stresses for 6 h and 2 days, respectively. Fv/Fm values were constant when exposed to low light
intensity (50 μmol photons m−2 s−1). However, when exposed to higher light intensities (1000,
1500, and 2000 μmol photons m−2 s−1), Fv/Fm decreased in the first 5 h of exposure and then
started to increase. After cultivation for 2 days, Fv/Fm of all light intensities was statistically
similar. Specific growth rates were also similar between all light intensities (Fig. 1c).
Simionato et al. [35] reported that during the exponential phase, Fv/Fm was stable and the
Appl Biochem Biotechnol

(A) 50 1000 1500 2000

(B)
1.0 1.0

0.8 0.8

0.6 0.6

Fv/Fm
Fv/Fm

0.4 0.4

0.2 0.2

0.0 0.0
0 1 2 3 4 5 6 50 1000 1500 2000
Exposure durations (hrs) Exposure light intensities ( mol m -2 s -1)
(C) (D)
2.0 20
Specific growth rate (day-1)

Chlorophyll (a+b) ( g/ml)

1.5 15 a

1.0 10

b
0.5 5 b b

0.0 0
50 1000 1500 2000 50 1000 1500 2000
Exposure light intensities ( mol m -2 s -1) Exposure light intensities ( mol m -2 s -1)

Fig. 1 (A) Fv/Fm in 6 h of exposure, (B) Fv/Fm, (C) specific growth rate, and (D) chlorophyll (a + b) content of
D. tertiolecta at the end of 2-day exposure to varying high light intensities. Different letters indicate significant
differences (p < 0.05)

specific growth rate of Nannochloropsis gaditana has very similar value for all light intensi-
ties. According to Sukenik et al. [26], photosynthesis rate when normalized to cell number
were virtually identical between low (70 μmol photons m − 2 s − 1 ) and high
(700 μmol photons m−2 s−1) light as a result of cell adaptation. Cell adaption in this study
can be seen from Fig. 1d, which shows significant decreases in chlorophyll contents under the
three high light intensities (1000, 1500, and 2000 μmol photons m−2 s−1) when compared with
the control (50 μmol photons m−2 s−1). However, there were no significant differences in
chlorophyll contents among the three high light intensities. A previous study also suggested
that cells growing in low light accumulated more chlorophylls than cell adapted in high light
intensities [35]. These results suggested that during the exponential growth phase D. tertiolecta
was able to adapt to light stress without affecting cell growth. The OD680 at the end of the 2-
day period was 0.783, 0.872, 0.785, and 0.805 for 50, 1000, 1500, and
2000 μmol photons m−2 s−1, respectively. A correlation of DW = 0.3077 * OD680,
R2 = 0.9815 was established. The corresponding cell dry weight concentrations were 0.241,
0.268, 0.242, and 0.248 g/l, which were relatively low compared to the literature because the
initial cell density was low to avoid light shading and the culture period was short.

The Effect of HCO3− Concentration

Light and carbon (mostly in the form of CO2) are major factors influencing the photosynthesis
process of algae [9]. In addition to CO2, D. tertiolecta can also uptake HCO3− as the dominant
inorganic carbon source [36]. In this experiment, it was hypothesized that the increase of
 Appl Biochem Biotechnol

HCO3− concentration in the culture medium from 25 to 100 mM may help alleviate the
photoinhibition and increase growth of D. tertiolecta under high light intensity. Figure 2a, b
shows that rising HCO3− concentration in the culture medium did not improve light response
(in term of Fv/Fm values) of D. tertiolecta. The specific growth rate (Fig. 2c) increased with
increasing HCO 3 − concentration under both low and high light intensities
(2000 μmol photons m−2 s−1). The chlorophyll contents (Fig. 2d) seemed to increase under
higher HCO3− concentration but statistically they were not significantly different, whereas the
results found by White et al. [37] showed that chlorophyll a increased when bicarbonate
increased. The total chlorophyll contents also reduced under high light intensity compared to
low light intensity. Chlorophylls are biochemical compounds that are easily degraded under
high light stress due to the generation of reactive oxygen species [38]. The highest OD680 of
HCO3− treatments was 1.080, corresponding to dry weight concentration of 0.332 g/l when
HCO3− concentration was 100 mM and exposed to high light intensity.

The Effect of Temperature

The optimal temperature for Dunaliella was found to be between 25 and 35 °C [39]. In this
study, Fv/Fm of cultures that were exposed to low light was similar under all temperatures, but
for cultures under high light intensity (2000 μmol photons m−2 s−1), Fv/Fm under high
temperature (35 and 40 °C) were significantly lower than under low temperature (25 and
30 °C) (Fig. 3a, b). Under high temperatures, photosynthesis can be inhibited by impaired

(A) (B)
1.0 25mM LL 50mM LL 100mM LL
25mM HL 50mM HL 100mM HL 1.0
0.8 Low light High light
0.8
0.6
Fv/Fm

0.6
Fv/Fm

0.4 0.4

0.2 0.2

0.0 0.0
0 1 2 3 4 5 6 25 50 100
Exposure durations (hrs) HCO3- concentrations (mM)

(C) (D)
20
2.0 Low light High light
Low light High light
Chlorophyll (a+b) ( g/ml)
Specific growth rate (day-1)

AB B
A 15
1.5

1.0 10

0.5 5

0.0 0
25 50 100 25 50 100
HCO3- concentrations (mM) HCO3- concentrations (mM)

Fig. 2 (A) Fv/Fm in 6 h of exposure, (B) Fv/Fm, (C) specific growth rate, and (D) chlorophyll (a + b) contents of
D. tertiolecta when cultured under varying HCO3− concentrations in 2-day exposure to low (LL,
50 μmol photons m−2 s−1) and high (HL, 2000 μmol photons m−2 s−1) light intensities. Different letters indicate
significant differences (p < 0.05)
Appl Biochem Biotechnol

electron transport that reduces photochemical efficiency of PS II and reduced Rubisco activity
due to generation of reactive oxygen species [40, 41]. Specific growth rates reached the highest
at 30 °C in both low and high light intensities. At 35 and 40 °C, specific growth rates were
significantly different between low light and high light intensities (Fig. 3c), which suggested
that the combined high temperature and high light intensity had adverse effects on cell growth.
The chlorophyll contents were lower when cells were exposed to high light or high temper-
atures (Fig. 3d). It probably explained the lower specific growth rates under those conditions.
The highest OD680 of temperature treatments was 0.861, corresponding to dry weight con-
centration of 0.265 g/l when temperature was 30 °C exposed to low light.

The Effect of Salinity

D. tertiolecta is a marine microalga that can thrive in a wide range of salinity, from brackish
water to sea water, or even in higher level of salinity in the hypersaline culture medium [23].
Figure 4a shows similar results as in other experiments that Fv/Fm decreased in the first 6 h of
exposure to high light. After 2 days of cultivation, Fv/Fm values were not significantly
different among cultures in different salinities when exposed to low light; however, salinity
did cause slightly different Fv/Fm when exposed to high light, e.g., culture of lower than 1.5 M
NaCl had smaller Fv/Fm (Fig. 4b). The increase in salinity to 2 M in the culture medium
reduced specific growth rate in both low light and high light intensities (Fig. 4c). Takagi et al.
[42] found similar results that cell concentration of D. tertiolecta decreased when NaCl

(A) (B)
25 C LL 30 C LL 35 C LL 40 C LL 1.0
1.0
25 C HL 30 C HL 35 C HL 40 C HL Low light High light
0.8
0.8

0.6 AB A
0.6
Fv/Fm
Fv/Fm

0.4 B
0.4

0.2 0.2
C
0.0 0.0
0 1 2 3 4 5 6 25 30 35 40
Exposure durations (hrs) Temperature ( C)

(C) Low light High light (D)

3 10
b
Specific growth rate (day-1)

A b
Chlorophyll (a+b) ( g/ml)

A Low light High light

2 a 8
B a b
1 6
b

0 4
25 30 35 40 A A c
-1 2 a
B
C
-2 C
0
25 30 35 40
Temperature ( C) Temperature ( C)

Fig. 3 (A) Fv/Fm in 6 h of exposure, (B) Fv/Fm, (C) specific growth rate, and (D) chlorophyll (a + b) content of
D. tertiolecta when cultured under varying temperatures in 2-day exposure to low (LL, 50 μmol photons m−2 s−1)
and high (HL, 2000 μmol photons m−2 s−1) light intensities. Different letters indicate significant differences
(p < 0.05)
 Appl Biochem Biotechnol

concentration increased from 1 to 2 M. High salinity also significantly decreased chlorophyll

content when exposed to low light (Fig. 4d). Salinity had no significant effects on chlorophylls
under high light intensity probably because light stress dominated chlorophyll contents
change. This study confirms that D. tertiolecta can grow under a wide range of NaCl
concentrations, from 0.5 to 2 M. However, the combination of high salinity and high light
intensity slightly elevated photoinhibition in D. tertiolecta than a sole stress from high light
intensity or high salinity. The highest OD680 of salinity treatments was 0.838, corresponding to
dry weight concentration of 0.258 g/l when salinity was 1 M at high light intensity.

The Effect of Pre-acclimation

Photoacclimation has been used as the adaptation mechanism allowing algae to maintain
optimum growth under a wide range of light intensities [43]. The study of green alga Chlorella
fusca found that photoacclimation was dependent on both intensity and duration of the light
supply [44]. In this experiment, D. tertiolecta cells were first grown under different light
intensities, e.g., 50, 200, or 500 μmol photons m−2 s−1 each for 1, 3, or 6 h (a total of nine
treatments), and then they were transferred to high light intensity at
2000 μmol photons m−2 s−1. Figure 5 shows that light response, growth rate, and chlorophyll
contents of D. tertiolecta grown under all conditions were not significantly different. These
results were different from published studies of genus Dunaliella and other algal strains, in
which the amount of thylakoids and total pigment were found to change due to
photoacclimation [45–47]. It might be that the alga in this experiment did not fully adapt to

(A) (B)
1.0 1.0
0.5M LL 1M LL 1.5M LL 2M LL Low light High light
0.8 0.5M HL 1M HL 1.5M HL 2M HL 0.8
AB B B
A
0.6 0.6
Fv/Fm
Fv/Fm

0.4 0.4

0.2 0.2

0.0 0.0
0 1 2 3 4 5 6 0.5 1 1.5 2
Exposure durations (hrs) Salinity (M)
(C) Low light High light (D)
2.0 20
Low light High light
Chlorophyll (a+b) ( g/ml)
Specific growth rate (day-1)

A A
a AB 15
1.5 a a b
B ab
b a

1.0 10 c

0.5 5

0.0 0
0.5 1 1.5 2 0.5 1 1.5 2
Salinity (M) Salinity (M)

Fig. 4 (A) Fv/Fm in 6 h of exposure, (B) Fv/Fm, (C) specific growth rate, and (D) chlorophyll (a + b) content of
D. tertiolecta when cultured under varying salinities in 2-day exposure to low (LL, 50 μmol photons m−2 s−1) and
high (HL, 2000 μmol photons m−2 s−1) light intensities. Different letters indicate significant differences (p < 0.05)
Appl Biochem Biotechnol

the tested light acclimation intensities within the exposure duration from 1 to 6 h. Longer
acclimation period might be needed, for example, Folkowski and Raven [48] stated that
photoacclimation of algae could be in a range of 26 to 100 h. The OD680 of pre-acclimation
treatments ranged from 0.654 to 0.782, corresponding to dry weight concentration of 0.201 to
0.241 g/l.
It has been a common belief that pre-acclimation can alleviate photoinhibition and may
offer a way to improve algal tolerance to light stress. For example, it might be possible that a
partial shade is applied in the morning for 1 or 3 h to allow the algal cells to acclimatize before
removing the shade and exposing them to full sunlight around noon. The results in this study
showed that doing so was not sufficient to alleviate the stress. Although longer pre-acclimation
may be able to help, it would not be practical in the real large-scale culture. The negative
results from this work still provide useful information about pre-acclimation effects on
photoinhibition of D. tertiolecta.
In all experiments, Fv/Fm was monitored during the first 6 h in order to simulate what
happens in an outdoor culture in which the algal cells may be exposed to full sunlight
(∼2000 μmol photons m−2 s−1) from 10 a.m. to 4 p.m. Although photoinhibition is likely to
occur only when the culture density is low (during the initial culture period), it is a situation
that should be avoided since overall algal productivity would be severely reduced or the
culture might crash out completely. As the culture grows denser, the cells tend to shade each
other, reducing light penetration and the possibility of photoinhibition; therefore,
photoinhibition was monitored only in the first 6 h of culture. However, some algal species

(A) (B)
1.0 control 1.0
50 E-1 h 50 E-3 h 50 E-6 h
1hr 3hrs 6hrs
0.8 200 E-1 h 200 E-3 h 200 E-6 h 0.8
500 E-1 h 500 E-3 h 500 E-6 h
0.6 0.6
Fv/Fm
Fv/Fm

0.4 0.4

0.2 0.2

0.0 0.0
0 1 2 3 4 5 6 control 50 200 500
Exposure durations (hrs) Acclimation light intensities ( E)

(C) (D) 5
2.0 1hr 3hrs 6hrs
Chlorophyll (a+b) ( g ml-1)

1hr 3hrs 6hrs

Specific growth rate (day-1)

4
1.5
3
1.0
2

0.5 1

0.0 0
control 50 200 500 control 50 200 500
Acclimation light intensities ( E) Acclimation light intensities ( E)

Fig. 5 a Fv/Fm in 6 h of exposure, b Fv/Fm, c specific growth rate, and d chlorophyll (a + b) contents of
D. tertiolecta when cultured under varying photoacclimation conditions in 2-day exposure to high light intensity
(2000 μmol photons m−2 s−1). Control in this experiment was the culture that was exposed to high light intensity
(2000 μmol photons m−2 s−1) and had no prior photoacclimation
 Appl Biochem Biotechnol

may be able to adapt to the high light stress, e.g., by antenna size reduction and non-
photochemical quenching. Thus, the 2-day assessment of light stress effect was performed
to see whether the cultures could recover (if they recover, it would suggest that the stress may
be temporary, e.g., only on the first day). In fact, in case of high temperature (40 °C), the
culture could not be recover after exposure to high light, suggesting that this condition must be
avoided at all costs. Growth after 2 days was not monitored because algae usually either fully
recover or completely die out after this point.

Conclusion

The Fv/Fm ratio of D. tertiolecta in short term exposure (within 6 h) greatly decreased with
increasing light intensity, indicating that D. tertiolecta encountered photoinhibition under high
light intensities. However, after 2 days of exposure, there were no significant differences in Fv/
Fm ratios or specific growth rates between low and high lights, indicating that D. tertiolecta
could adapt to a wide range of light intensity, from 50 up to 2000 μmol photons m−2 s−1. The
increase in HCO3− concentration did not alleviate photoinhibition of D. tertiolecta; however,
growth and chlorophyll contents increased with increasing HCO3− concentrations. Tempera-
ture had significant effects on photoinhibition. Combined high temperature and high light
intensity led to photoinhibition and reduced cell growth rates. Similarly, combined low salinity
and high light intensity caused photoinhibition and slower cell growth. Pre-acclimation of
algae to 50, 200, or 500 μmol photons m−2 s−1 each for 1, 3, or 6 h did not significantly lessen
photoinhibition or increase cell growth of D. tertiolecta, probably because longer acclimation
time was required.

Acknowledgments This research was supported by PTT Research and Technology Institute; the Royal Golden
Jubilee Ph.D. Program of Thailand (Thailand Research Fund); the Center for Environmental Health, Toxicology,
and Management of Chemicals under the Science & Technology Postgraduate Education and Research Devel-
opment Office (PERDO) of the Ministry of Education, Thailand; the US National Science Foundation (award #
CMMI-1239078); and the startup fund of North Carolina State University.

References

1. Markou, G., Vandamme, D., & Muylaert, K. (2014). Microalgal and cyanobacterial cultivation: the supply of
nutrients. Water Research, 65, 186–202.
2. Neto, A. M. P., et al. (2013). Improvement in microalgae lipid extraction using a sonication-assisted method.
Renewable Energy, 55, 525–531.
3. Leema, J. M., et al. (2010). High value pigment production from Arthrospira (Spirulina) platensis cultured in
seawater. Bioresource Technology, 101(23), 9221–9227.
4. Harun, R., et al. (2010). Bioprocess engineering of microalgae to produce a variety of consumer products.
Renewable and Sustainable Energy Reviews, 14(3), 1037–1047.
5. Cheng-Wu, Z., et al. (2001). An industrial-size flat plate glass reactor for mass production of
Nannochloropsis sp.(Eustigmatophyceae). Aquaculture, 195(1), 35–49.
6. Patil, V., et al. (2007). Fatty acid composition of 12 microalgae for possible use in aquaculture feed.
Aquaculture International, 15(1), 1–9.
7. Harun, R., Danquah, M. K., & Forde, G. M. (2010). Microalgal biomass as a fermentation feedstock for
bioethanol production. Journal of Chemical Technology and Biotechnology, 85(2), 199–203.
8. Vergara-Fernández, A., et al (2008). Evaluation of marine algae as a source of biogas in a two-stage
anaerobic reactor system. Biomass and Bioenergy, 32(4), 338–344.
Appl Biochem Biotechnol

9. Chisti, Y. (2007). Biodiesel from microalgae. Biotechnology Advances, 25(3), 294–306.

10. Terigar, B. G., & Theegala, C. S. (2014). Investigating the interdependence between cell density, biomass
productivity, and lipid productivity to maximize biofuel feedstock production from outdoor microalgal
cultures. Renewable Energy, 64, 238–243.
11. Teo, C. L., et al. (2014). Biodiesel production via lipase catalysed transesterification of microalgae lipids
from Tetraselmis sp. Renewable Energy, 68, 1–5.
12. Cheirsilp, B., & Torpee, S. (2012). Enhanced growth and lipid production of microalgae under mixotrophic
culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresource
Technology, 110, 510–516.
13. Ren, H.-Y., et al. (2014). Energy conversion analysis of microalgal lipid production under different culture
modes. Bioresource Technology, 166, 625–629.
14. Serôdio, J., Vieira, S., & Cruz, S. (2008). Photosynthetic activity, photoprotection and photoinhibition in
intertidal microphytobenthos as studied in situ using variable chlorophyll fluorescence. Continental Shelf
Research, 28(10), 1363–1375.
15. Tang, H., et al. (2011). Potential of microalgae oil from Dunaliella tertiolecta as a feedstock for biodiesel.
Applied Energy, 88(10), 3324–3330.
16. Khoeyi, Z. A., Seyfabadi, J., & Ramezanpour, Z. (2012). Effect of light intensity and photoperiod on
biomass and fatty acid composition of the microalgae, Chlorella vulgaris. Aquaculture International, 20(1),
41–49.
17. Pulz, O. (2001). Photobioreactors: production systems for phototrophic microorganisms. Applied
Microbiology and Biotechnology, 57(3), 287–293.
18. Kitaya, Y., et al. 2009. Effects of temperature, photosynthetic photon flux density, photoperiod and O2 and
CO2 concentrations on growth rates of the symbiotic dinoflagellate, Amphidinium sp. in Nineteenth
International Seaweed Symposium. . Springer.
19. Taher, H., et al. 2014. Growth of microalgae using CO2 enriched air for biodiesel production in supercritical
CO2. Renewable Energy
20. Ra, C. H., et al. (2015). Cultivation of four microalgae for biomass and oil production using a two-stage
culture strategy with salt stress. Renewable Energy, 80, 117–122.
21. Borowitzka, M. A., & Siva, C. J. (2007). The taxonomy of the genus Dunaliella (Chlorophyta, Dunaliellales)
with emphasis on the marine and halophilic species. Journal of Applied Phycology, 19(5), 567–590.
22. Hoshaw, R. W., & Maluf, L. Y. (1981). Ultrastructure of the green flagellate Dunaliella tertiolecta
(Chlorophyceae, Volvocales) with comparative notes on three other species. Phycologia, 20(2), 199–206.
23. Hosseini Tafreshi, A., & Shariati, M. (2009). Dunaliella biotechnology: methods and applications. Journal of
Applied Microbiology, 107(1), 14–35.
24. El Arroussi, H., et al. (2015). Improvement of the potential of Dunaliella tertiolecta as a source of biodiesel
by auxin treatment coupled to salt stress. Renewable Energy, 77, 15–19.
25. Fazeli, M., et al. (2006). Effects of salinity on β-carotene production by Dunaliella tertiolecta DCCBC26
isolated from the Urmia Salt Lake, north of Iran. Bioresource Technology, 97(18), 2453–2456.
26. Sukenik, A., et al. (1990). Adaptation of the photosynthetic apparatus to irradiance in Dunaliella tertiolecta.
A kinetic study. Plant Physiology, 92(4), 891–898.
27. Mishra, A., & Jha, B. (2011). Antioxidant response of the microalga Dunaliella salina under salt stress.
Botanica Marina, 54(2), 195–199.
28. Alizadeh, G., et al. 2011 The productivity and stability of plant cell for biotechnology purposes.
29. EonSeon, J. (2013). Comparison of the responses of two Dunaliella strains, Dunaliella salina CCAP 19/18
and Dunaliella bardawil to light intensity with special emphasis on carotenogenesis. Algae, 28(2), 203–211.
30. Pick, U., Karni, L., & Avron, M. (1986). Determination of ion content and ion fluxes in the halotolerant alga
Dunaliella salina. Plant Physiology, 81(1), 92–96.
31. Kim, J. H., Nemson, J. A., & Melis, A. (1993). Photosystem II reaction center damage and repair in
Dunaliella salina (green alga) (analysis under physiological and irradiance-stress conditions). Plant
Physiology, 103(1), 181–189.
32. Baker, N.R. and K. Oxborough. 2004. Chlorophyll fluorescence as a probe of photosynthetic productivity, in
chlorophyll a fluorescence. Springer. p. 65–82.
33. Lichtenthaler, H.K. and C. Buschmann, 2001. Chlorophylls and carotenoids: measurement and character-
ization by UV-VIS spectroscopy, in Current protocols in food analytical chemistry, M.M. Giusti and R.E.
Wrolstad, Editors.
34. Masojıdek, J., G. Torzillo, and M. Koblızek, 2013. Photosynthesis in microalgae, in Handbook of
microalgal culture: applied phycology and biotechnology, A. Richmond and Q. Hu, Editors. John
Wiley & Sons.
35. Simionato, D., et al. (2011). Acclimation of Nannochloropsis gaditana to different illumination regimes:
effects on lipids accumulation. Bioresource Technology, 102(10), 6026–6032.
 Appl Biochem Biotechnol

36. Amoroso, G., et al. (1998). Uptake of HCO3− and CO2 in cells and chloroplasts from the microalgae
Chlamydomonas reinhardtii and Dunaliella tertiolecta. Plant Physiology, 116(1), 193–201.
37. White, D., et al. (2013). The effect of sodium bicarbonate supplementation on growth and biochemical
composition of marine microalgae cultures. Journal of Applied Phycology, 25(1), 153–165.
38. Nixon, P. J., et al. (2005). FtsH-mediated repair of the photosystem II complex in response to light stress.
Journal of Experimental Botany, 56(411), 357–363.
39. Ben-Amotz, A. (1995). New mode of Dunaliella biotechnology: two-phase growth for β-carotene produc-
tion. Journal of Applied Phycology, 7(1), 65–68.
40. Silva, E. N., et al. (2010). Photosynthetic changes and protective mechanisms against oxidative damage
subjected to isolated and combined drought and heat stresses in Jatropha curcas plants. Journal of Plant
Physiology, 167(14), 1157–1164.
41. Liu, F., & Pang, S. J. (2010). Performances of growth, photochemical efficiency, and stress tolerance of
young sporophytes from seven populations of Saccharina japonica (Phaeophyta) under short-term heat stress.
Journal of Applied Phycology, 22(2), 221–229.
42. Takagi, M., & Yoshida, T. (2006). Effect of salt concentration on intracellular accumulation of lipids and
triacylglyceride in marine microalgae Dunaliella cells. Journal of Bioscience and Bioengineering, 101(3),
223–226.
43. Bracher, A. 1999. Photoacclimation of phytoplankton in different biogeochemical provinces of the Southern
Ocean and its significance for estimating primary production. Berichte zur Polarforschung (Reports on Polar
Research), 341.
44. Garcia-Mendoza, E., et al. (2002). Non-photochemical quenching of chlorophyll fluorescence in Chlorella
fusca acclimated to constant and dynamic light conditions. Photosynthesis Research, 74(3), 303–315.
45. Ritz, M., et al. (2000). Kinetics of photoacclimation in response to a shift to high light of the red alga
Rhodella violacea adapted to low irradiance. Plant Physiology, 123(4), 1415–1426.
46. Papadakis, I. A., Kotzabasis, K., & Lika, K. (2005). A cell-based model for the photoacclimation and CO2-
acclimation of the photosynthetic apparatus. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1708(2),
250–261.
47. Zou, N., & Richmond, A. (2000). Light-path length and population density in photoacclimation of
Nannochloropsis sp. (Eustigmatophyceae). Journal of Applied Phycology, 12(3–5), 349–354.
48. Falkowski, P.G. and J.A. Raven, 2013. Aquatic photosynthesis. Princeton University Press.

View publication stats