Algal Research 17 (2016) 61–66

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Algal Research

journal homepage: www.elsevier.com/locate/algal

Use of phenol-induced oxidative stress acclimation to stimulate cell

growth and biodiesel production by the oceanic microalga
Dunaliella salina
Kichul Cho a,c,1, Chi-Heon Lee b,c,1, Kyungjun Ko b,c,1, Yeon-Ji Lee b,c, Kil-Nam Kim c, Mi-Kyung Kim d,
Young-Ho Chung e, Daekyung Kim a,c,⁎, In-Kyu Yeo b,⁎⁎, Tatsuya Oda f
a
Korea University of Science & Technology, Daejeon 305-350, Republic of Korea
b
Department of Marine life Science, Jeju National University, Jeju 690-756, Republic of Korea
c
Jeju Center, Korea Basic Science Institute (KBSI), Jeju 690-756, Republic of Korea
d
Marine Science Research Center, Yeungnam University, Gyongsan 712-749, Republic of Korea
e
Drug & Disease Target Research Team, Korea Basic Science Institute, Daejeon 350-333, Republic of Korea
f
Graduate School of Fisheries Science & Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan

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

Article history: There is steadily increasing interest in the use of microalgae as a source of biomass for biodiesel conversion and
Received 22 December 2015 hydrocarbon based products. At the same time, microalgae have shown promise for the bioremediation of pollut-
Received in revised form 15 March 2016 ed water. This study evaluates the effect of prior phenol exposure on microalgal biomass production and quality
Accepted 23 April 2016
of biodiesel yielded by the oceanic microalga Dunaliella salina. Phenol had an EC50 of 155.03 mg L−1 on algal
Available online 6 May 2016
growth and caused lowered chlorophyll a/b ratios and biomass yields. Phenol also increased malondialdehyde
Keywords:
content and elevated superoxide dismutase enzyme activities indicative of phenol-induced oxidative stress.
Microalgae After prior exposure to phenol at 150 mg L−1 with subsequent transfer to culture without phenol, D. salina
Dunaliella cells increased 41% faster and had 26% higher lipid content than were obtained from the control group that
Phenol had no prior phenol exposure. Prior exposure to phenol altered fatty acid methyl ester compositions, increased
Oxidative stress levels of methyl linolenate (C18:3(n-3)) and γ-linolenic acid methyl ester (C18:3(n-6)), decreased levels of
Biodiesel cis-10-heptadecanoic acid methyl ester and methyl stearate (C18:0), and decreased cetane number, all in a
concentration-dependent manner. The results suggested that the use of prior acclimation by toxic chemicals
could potentially support efficient microalgal biomass production.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction photo bioreactor design, and management of environmental impacts

Microalgae are unicellular photosynthetic organisms responsible for
primary production and essential for trophic balance in aquatic ecosys-
tems [1]. Microalgae grow more rapidly and fix more carbon than ter-
restrial plants per unit area of incident solar radiation and produce a
variety of useful products such as biofuels, nutraceuticals, pharmaceuti-
cals, and are a viable hydrocarbon alternative for a number of others.
Thus, there is an increasing interest in developing efficient methods of
growing and harvesting microalgae for biomass conversion [2]. Efforts
in improve growth, yield and cost effectiveness of biomass production
and conversion have included use of wastewater, improvements in
⁎ Correspondence to: D. Kim, Jeju Center, Korea Basic Science Institute (KBSI), Jeju 690-
756, Republic of Korea.
⁎⁎ Corresponding author.
E-mail addresses: dkim@kbsi.re.kr (D. Kim), ikyeo99@jejunu.ac.kr (I.-K. Yeo).
1
These authors contributed equally to this work.
[3].
In this study, we focused on development of new strategies for bio-
mass production by investigating the effects of prior acclimation of
microalgae to toxic chemical stress on biomass conversion. Phenol is a
strong oxidant that is a waste product from varied industrial processes
[4]. For instance, whereas natural stream water contains phenol at
about 0.01 to 2.0 μg L−1, wastewater from petrol-processing plants con-
tains upwards of 40 mg L−1 of phenolic compounds [5,6]. Phenol is toxic
and can cause not only skin rashes, but also human cancers [7]. Phenol
has high solubility and chemical stability in water and is considered
one of the more important organic pollutants in the aquatic environ-
ment [8]. Accordingly, phenol is listed as a priority pollutant by the US
Environmental Protection Agency [9]. Phenols readily form phenoxy-
radicals which are strong oxidants and examples of what are more gen-
erally called reactive oxygen species (ROS) [10–12], which trigger oxi-
dative damage to aquatic organisms such as of peroxidation of lipids
in cell membranes [9,13]. Aerobic organisms have evolved antioxidant
enzymes such as superoxide dismutase (SOD), ascorbate peroxidase,

http://dx.doi.org/10.1016/j.algal.2016.04.023
2211-9264/© 2016 Elsevier B.V. All rights reserved.
62 K. Cho et al. / Algal Research 17 (2016) 61–66
catalase, and glutathione-S-transferase to deal with oxidative stress
[14].
Previous studies have shown that several algal species, including
Chlamydomonas debaryana, Chlorella luteoviridis, Desmodesmus
intermedius, Hindakia tetrachotoma, and Parachlorella kessleri, can
become acclimated to wastewater containing oxidants and show en-
hanced biomass production and increased antioxidant enzyme activi-
ties [15].
From these results, it is has been hypothesized that microalgae may
respond to dissolved chemical-exposures through enhanced antioxi-
dant defenses and that these responses may persist beyond the period
of chemical exposure.
Dunaliella sp. is a commercially cultured oceanic microalga used to
produce β-carotene, and because of its high tolerance for high salinity
and light, easy cultivation, and high growth rate, it is considered a prom-
ising source of microalgae for biodiesel production [16,17].
This study investigated the effect of phenol exposure on the growth
and oxidative stress responses of the oceanic microalga, Dunaliella sali-
na. The effects of prior phenol acclimation on biodiesel production prop-
erties were also evaluated.
2. Materials and methods
2.1. Algal cultivation
Dunaliella salina CCAP 19/20 was purchased from Yeungnam Univer-
sity. Each strain was inoculated in Na2SiO3·9H2O free sterilized F/2 (F2-
Si) medium [18]. Algal cultures were maintained in a plant growth
chamber (KG-8407, Vision, Korea) with a 12:12 h photoperiod at
26 °C and 105 μmol m− 2 s−1 of cool-white fluorescence light under
batch culture conditions. Cell populations were maintained in 50 mL
cultures in 100 mL Erlenmeyer flasks. Growth was monitored by absor-
bance at OD600, calibrated using a hemocytometer. All experiments used
cultures in the exponential growth phase.
2.2. EC50 toxicity test
EC50 toxicity tests were performed following OECD guidelines and
the method proposed by Moreno-Garrido [19,20]. Phenol was dissolved
in sterilized F/2 medium and mixed with D. salina cultures at 0, 100, 200,
and 300 μg mL−1 phenol in 6-well plates. Experimental cultures were
initiated at 2 × 104 cells mL−1 in 8 mL volumes per well. Cell concentra-
tions were measured using a Muse® cell analyzer (EMD Millipore Cor-
poration, USA), and EC50 values were calculated using following
equations using Excel 2007 (Microsoft, USA):
μ ¼ ð lnC e – lnC i Þ=ðt e –t i Þ
% I ¼ ½ðμ c –μ T Þ=μ c   100
where μ was the average specific growth rate (SGR) from day 0 day to
day 3, ti was the initial time, te was the ending time, and Ce and Ci
were cell concentrations at the initial and end times, respectively. The
value of %I was the percent inhibition of the SGR, μc and μT were the
SGR of the control and phenol-exposed groups, respectively.
2.3. Phenol acclimation and daily growth determination
To perform phenol-acclimation experiments, algal cells were seeded
at 2 × 104 cells mL− 1 in 50 mL F/2-Si medium with 0, 50, 100, and
150 mg L−1 phenol (Sigma Aldrich, USA) in 100 mL Erlenmeyer flasks.
The concentrations used were below the EC50 established from the ex-
periment in 2.2.
2.4. Analytical methods
2.4.1. Malondialdehyde (MDA) content determination
Following 10 days exposure to phenol, the degree of lipid peroxida-
tion was estimated by the production of malondialdehyde (MDA), mea-
sured by the thiobarbituric acid (TBA) assay [21]. Briefly, 1 mL algal
culture was removed and centrifuged at 13,000 ×g for 5 min. Following
removal of the supernatant, cells were resuspended in 300 μL of 50 mM
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer
(pH 7.4) with 3 μL of 10 mM butylated hydroxytoluene (BHT, added
as an antioxidant). After 10 min of incubation on ice, 600 μL of 0.5%
thiobarbituric acid (TBA) (w/v, in concentrated acetic acid) was added
to 200 μL of lysed cells and incubated for 60 min at 95 °C in a water
bath. After cooling to room temperature, MDA was measured
fluorometrically (532 excitation, 553 nm emission) in a Synergy micro-
plate reader (BioTek, USA) calibrated against an MDA concentration
curve.
2.4.2. Photosynthetic pigment determination
To determine chlorophyll a and b, and total carotenoids, we used
a method modified from Wellburn [22]. In brief, after 1 mL of algal
culture was centrifuged at 12,000 × g for 5 min. The supernatants
were removed and 1 mL of methanol was added to the pellets and in-
cubated at 60 °C for 50 min in a water bath. The lysates were clarified
by centrifugation at 10,000 × g for 3 min, and absorbance was mea-
sured at 470, 653, and 666 nm. To calculate chlorophyll a (Ca), chlo-
rophyll b (Cb) and total carotenoid (TCC), the following equations
were used [22].
C a ¼ 15:65 A666 –7:34A653
C b ¼ 27:05 A653 –11:21A666
TCC ¼ ð1000A470 –2:86C a –129:2C b Þ=221
where A666 , A653, and A470 are optical concentration at 666 nm,
653 nm, and 470 nm, respectively.
2.4.3. Antioxidant enzyme activities
Superoxide dismutase (SOD) activity of phenol-treated D. salina cul-
ture was measured using the SOD assay kit (Sigma Aldrich, USA). After
10 days of phenol exposure, a 3 mL aliquot of algal culture was centri-
fuged at 13,000 ×g for 5 min. The pellet was washed twice and 300 μL
of radio immunoprecipitation assay (RIPA) buffer (pH 8.0) was added
to the pellet for extraction. SOD activity was determined using supplied
WST-1 reagent following the manufacturer's protocol. Catalase (CAT)
activity was determined by CAT assay kit (Sigma Aldrich, USA) follow-
ing the kit protocol.
2.4.4. Cultivation under normal conditions and biomass determination
The 40 mL of D. salina cells previously grown at various concen-
trations of phenol were harvested by centrifugation at 3000 × g for
10 min and washed 3-times with distilled water. After resuspension
with F/2-Si medium, 10 mL of each phenol-exposed D. salina culture
was inoculated into 1.2 L of sterilized F/2-Si medium in 2 L Erlenmey-
er flasks to an initial cell concentration of 2.24 × 104 cells mL − 1 .
Flasks were aerated with 1.2 L min− 1 of filtered air. To determine
algal biomass, 10 mL of algal culture were centrifuged at 4000 × g
for 5 min and pellets were freeze-dried using a Coolsafe 110–4 Vac-
uum Concentrator (Labogene, Denmark). Lyophilized cells were
weighed using an XP-205 electronic balance (Mettler Toledo,
Switzerland).
2.4.5. Analysis of total lipid and fatty acid methyl ester (FAME)
To determine total lipid content, algal biomass from 10 day cultures
was harvested by centrifugation at 8000 ×g for 10 min. The pellets were
 K. Cho et al. / Algal Research 17 (2016) 61–66 63

washed three times with deionized water, and then freeze dried for 12 h
as described above. Total lipid was extracted using a method modified
from Bligh and Dyer and Chiu et al. [23,24]. Briefly, 80 mg of dried bio-
mass was precisely weighed and suspended in 20 mL of methanol/chlo-
roform (v/v, 2:1) and sonicated for 10 min in a test tube. The chloroform
phase was collected and dried under nitrogen gas. The remaining total
lipid residues were precisely weighed using an XP-205 electronic
balance.
FAMEs were determined by transmethylation of extracted total
lipids using the method of Cai et al [25]. Briefly, 3 mL of 0.5 N NaOH in
methanol was added to the extracted total lipid and boiled at 100 °C
in a JSWB-06 T Bath & Circulator (JSR, KOR) for 5 min and 1 mL of 95%
hexane (Sigma Aldrich, USA) was added to the mixture and mixed.
After centrifugation at 1000 ×g for 3 min, the upper hexane layer was
diluted three-fold for gas chromatographic analysis. FAME's were ana-
lyzed using a GC-2010 plus gas chromatograph fitted with a flame ion-
ization detector (FID) (Shimadzu, Japan) and a SP™-2560 Fused silica Fig. 1. Toxicity of phenol on the oceanic microalga Dunaliella salina. Algal cultures were
grown with varying concentrations of phenol for 3 d with a 12/12 h photoperiod at
capillary column (100 m × 0.25 mm × 0.2 μm film thickness). The
26 °C and 105 μmol m−2 s−1 of cool-white fluorescence light intensity. The initial cell
helium gas was used as a carrier at a flow rate of 1.03 mL min−1. The concentration was 2 × 104 cells mL−1. The EC50 is the concentration of phenol that
temperature of both injector and detector were 260 °C, and injection caused a 50% reduction in algal growth after 3 days.
volume was 1.0 μL with 100:1 of split ratio. FAME's were eluted using
an oven temperature program of 5 min for 100 °C, then a gradient
from 100 to 240 °C at 4 °C min−1 and then 240 °C for 20 min. FAME's Pseudokirchneriella subcapitata was 197 mg L− 1 [30,31]. There are
were identified by comparison of retention times to known standards many factors that may affect the uptake and toxicity of phenol by
(Sigma Aldrich, USA). microalgae, such as presence or absence of cell walls, variations in
surface to volume ratio, and varying adaptations to oxidative stress
2.4.6. Biodiesel properties determination [32,33]. D. salina lack a cell wall, although it is uncertain how this affects
Biodiesel properties were measured, including saponification value the toxicity from phenol.
(SV), iodine value (IV), cetane number (CN), and degree of unsaturation
(DU). Percentages of monounsaturated fatty acid (MUFA) and polyun-
saturated fatty acid (PUFA) were estimated from the FAME analysis.
The following calculations were used to estimate each parameter [15,
26,27]:

DU ¼ ðMUFA; wt%Þ þ ð2  PUFA; wt%Þ

IV ¼ Σð254  F  DÞ=M w

SV ¼ Σð560  F Þ=M w

CN ¼ ð46:3 þ 5458=SV Þ–ð0:225  IV Þ

where F is the percentage of each FAME and Mw is its molecular weight.

D is the number of double bonds in each FAME structure.

2.5. Statistical analysis

All the experiments were performed in triplicate and statistical anal-

ysis was performed by one-way ANOVA and subsequent t-tests using
Excel 2007. Experimental means were considered significantly different
from the controls at P b 0.05.

3. Results and discussion

3.1. EC50 toxicity tests under different phenol concentrations

The effective concentration test (EC50) is defined as the concentra-

tion of a toxic agent that causes a 50% response to the target organisms.
In this case, we measured the amount of phenol required to cause a 50%
inhibition of growth of D. salina following OECD Guidelines, using phe-
nol concentrations ranging from 0 to 300 mg L−1. Phenol significantly
affected the growth rate of D. salina and the EC50 value was estimated
to be 155 mg L−1 (Fig. 1.). Toxic effects of phenol on aquatic organisms
have been documented by many studies, especially in fish, shrimp, and
abalone [13,28,29]. In studies of microalgae, the phenol EC50 of
Entomoneis cf punctulata was found to be 74 mg L−1, and that from
Fig. 2. (A) Growth of Dunaliella salina treated with varying concentrations of phenol. Algal
cultures were grown with varying concentrations of phenol for 10 days with a 12/12 h
photoperiod at 26 °C and 105 μmol m−2 s−1 of cool-white fluorescence light.
(B) Cumulative biomass (mg L−1) of cells grown with varying concentrations of phenol
after 10 days of cultivation. Error bars represent average mean ± standard deviation
(SD) from triplicate experiments. Different letters represent significant differences
(P b 0.05).
64 K. Cho et al. / Algal Research 17 (2016) 61–66
3.2. Effect of different phenol concentrations on the growth of D. salina
We tested growth of D. salina at phenol concentrations below the
EC50 (0–150 mg L−1). As shown in Fig. 2A, algal growth was inhibited
in a concentration-dependent manner. The inhibition of algal growth
was observed as early as day 2 (early log-phase) and was observable
for the full 10 day period monitored. The biomass obtained after
10 days was affected by all phenol concentrations tested, with the
greatest level of inhibition observed at the highest concentration of phe-
nol tested (150 mg L− 1) (Fig. 2B). According to a previous study,
phenol-exposed microalga Chlorella pyrenoidosa showed morphologi-
cally visible membrane wrinkling due to an inhibition of the interaction
between the acyl chains of membrane phospholipids [34]. Furthermore,
the most likely explanation for phenol toxicity was phenoxy-radical re-
lated damage, for example, as reported for human epidermal
keratinocytes [35]. Therefore, the inhibitory effect of phenol was consid-
ered to be the result of phenol-derived radicals and we thus assessed
the relationship of phenol concentration to oxidative stress.
3.3. Effect of dissolved phenol on the photosynthetic pigment accumulation
To understand the effect of phenol toxicity on chloroplasts, photo-
synthetic pigments were determined after 10 days of cultivation.
While chlorophyll a, b, and total carotenoid content increased at 50
and 100 mg L−1 of phenol, the ratio of chlorophyll a/b decreased as
the phenol concentration increased (Table 1). The chlorophyll a/b
ratio is closely related to the light harvesting chlorophyll-protein com-
plex (LHC). Previous studies have shown that decreased chlorophyll a/
b was observed under copper-induced oxidative stress in green
microalgae Scenedesmus vacuolatus and Chlorella kessleri and is consid-
ered an adaptive response to radical [32]. These results suggest that
phenol can significantly inhibit the yield of photosynthesis and the
light harvesting ability of D. salina by inhibiting the amount of LHC in
chloroplasts. It is likely that phenol-induced phenoxy-radicals may
damage the chloroplast membrane. Therefore, dissolved phenol consid-
ered closely related with photosynthetic activity.
The total carotenoid content (TCC) was slightly elevated in algal cells
treated with phenol compared to the no-phenol control group. Previous
reports have shown that TCC plays an important role as an accessory
pigment and can defend against photochemical induction of ROS dam-
age [36]. Therefore, induction of algal carotenoid pigments may be a re-
sponse to phenol-induced phenoxy radicals [10,11,12].
3.4. Effect of phenol on the lipid peroxidation and antioxidant enzyme
activities
To evaluate degree of lipid peroxidation in algal cells, we measured
malondialdehyde (MDA) content as a proxy for the extent of lipid per-
oxidation in the cell membrane [37]. The degree of lipid peroxidation
of D. salina was determined after 10 days of phenol exposure. As
shown Table 2, malondialdehyde (MDA) content significantly increased
with rising phenol concentration. This result was most likely from acti-
vation of phenoxy radicals as previously described [10–12]. Previous
studies have shown that increased ROS can directly attack all types of
biomolecules such as nucleic acids, proteins, and polar lipids [38].
Therefore, the increase in MDA content indicated that phenol-derived
radicals may interfere with oxidative homeostasis. It has been suggested
that D. salina cells may permit penetration of phenoxy radicals by
regulation of permeability, which has been previously observed in
other organisms [39]. Phenol induced phenoxy radicals may damage
membrane-bound cellular organelles and affect growth and photosyn-
thetic pigment compositions in D. salina.
Previous studies have shown that phenol can denature proteins and
DNA by changing pH and macromolecular structure [40,41]. It has also
been shown that oxidative stress can significantly damage proteins
and DNA [40,42]. To prevent damage from oxidative stress, living
Table 1
Effects of different phenol concentrations on photosynthetic pigment accumulation after
10 days of cultivation at 26 °C, 12/12 h photoperiod, and 105 μmol m−2 s−1 of cool-white
fluorescent light.
Photosynthetic Phenol concentration (mg L−1)
pigments 0 50 100 150
Chlorophyll a 14.28 ± 0.61 16.76 ± 1.57 16.01 ± 1.05 14.28 ± 2.19
Chlorophyll b 5.38 ± 0.38 7.57 ± 0.37 8.58 ± 1.09 7.84 ± 1.26
Total carotenoids 4.83 ± 0.20 6.47 ± 0.48 6.24 ± 0.51 5.70 ± 0.63
Chlorophyll a/b 2.66 2.21 1.87 1.82
Values expressed as average mean ± standard deviation (SD) from triplicate experiments.
organisms have developed protective mechanisms such as antioxidant
enzymes. It has been shown that ROS can induce antioxidant enzymes
in a number of organisms [12,38]. To determine if antioxidant enzymes
are induced by phenol exposure, SOD and catalase activities were deter-
mined after 10 days of exposure to phenol in order to evaluate the re-
sponse of D. salina to oxidative stress. SOD is part of a group of
metallo-isoenzymes that scavenge superoxide radicals, converting
them to O2 and H2O2 [43]. Catalase also scavenges ROS [44]. In this reac-
tion, catalase reacts with hydrogen peroxide creating an oxygen-
abundant iron peroxide; the produced iron peroxide then reacts with
H2O2 forming non-toxic H2O and O2 as products [45]. Therefore, if
SOD activity is increased, hydrogen peroxide is produced, which then
increases catalase activity. As shown Table 2, SOD activity increased sig-
nificantly with increased phenol concentration. Catalase activity was
slightly increased but the increase was not significantly correlated
with phenol concentration. Similar results were reported for another
phenolic compound, pyrogallic acid, in the cyanobacterial species
Cylindrospermopsis raciborskii [46]. In that report, pyrogallic acid signif-
icantly inhibited algal growth and increased MDA content along with
SOD and catalase activities. Other reports on the oceanic microalga
Lingulodinium polyedrum have shown that phenol exposure increased
SOD and CAT activities and decreased ascorbate peroxidase and gluta-
thione S-transferase activity [47]. It has additionally been reported
that phenolic compounds inhibited peroxidase and polyphenol oxidase
in the fungus Rhizopus oryzae [48]. Therefore, it is likely that phenol-
derived radical damage affects cell enzymatic metabolism, and catalase
activity may remove hydrogen peroxide in D. salina cells.
3.5. Effect of prior phenol exposure on growth and lipid accumulation.
D. salina was cultivated in the presence of various concentrations of
phenol under standard culture conditions as described above. Following
phenol exposure (acclimation), cells were grown in the absence of phe-
nol for 10 days. As shown in Fig. 3A, the biomass of D. salina obtained
during the 10 days following the acclimation period was dependent
on the concentration of phenol during the acclimation period, with
higher phenol pre-exposure concentration resulting in higher biomass.
The total lipid accumulation (wt% per unit biomass) was unaffected but
the overall yield of lipid increased approximately 26% (Fig. 3B). The
Table 2
Changes in malondialdehyde content (A), superoxide dismutase (SOD) activity (B), and
catalase (CAT) activity of Dunaliella salina grown under varying concentrations of phenol
for 10 d at 26 °C, 12/12 h photoperiod, and 105 μmol m−2 s−1 of cool-white fluorescent
light.
Phenol concentration (mg L−1)
Parameters
0 50 100 150
MDA (nmol mg ) −1
0.81 ± 0.08 1.03 ± 0.02 1.16 ± 0.02 1.38 ± 0.09c
a b c
SOD (U mg−1) 1.77 ± 0.05a 2.52 ± 0.17b 2.73 ± 0.25c 3.63 ± 0.52c
CAT (μmol min−1 mg−1) 0.97 ± 0.13a 1.07 ± 0.07a 1.09 ± 0.03a 1.15 ± 0.26a
Values expressed as average mean ± standard deviation (SD) from triplicate experiments
and different letters denote significant differences (P b 0.05).
 K. Cho et al. / Algal Research 17 (2016) 61–66 65

Table 3
Effects of different phenol concentrations on fatty acid methyl ester (FAME) compositions
(wt%) of Dunaliella salina grown for 10 days at 26 °C, 12/12 h photoperiod, and 105 μmol
m−2 s−1 of cool-white fluorescent light.

Phenol concentration (mg L−1)

FAMEs
0 50 100 150

C14:0 0.23 ± 0.03 0.24 ± 0.03 0.22 ± 0.02 0.11 ± 0.03

C16:0 19.63 ± 1.38 19.38 ± 0.78 19.48 ± 0.99 11.00 ± 1.31
C16:1 0.28 ± 0.04 0.27 ± 0.03 0.32 ± 0.06 0.22 ± 0.03
C17:1 0.12 ± 0.04 0.09 ± 0.04 0.09 ± 0.03 0.03 ± 0.01
C18:0 1.83 ± 0.12 1.49 ± 0.36 0.61 ± 0.11 0.02 ± 0.01
C18:1(n-9),trans 0.25 ± 0.06 0.48 ± 0.11 0.49 ± 0.19 0.23 ± 0.05
C18:1(n-9),cis 1.60 ± 0.52 1.67 ± 0.29 1.87 ± 0.71 1.34 ± 0.05
C18:2(n-6),trans 20.91 ± 0.93 21.30 ± 0.87 19.96 ± 2.30 23.38 ± 3.68
C18:2(n-6),cis 4.56 ± 0.56 3.89 ± 0.39 4.88 ± 0.76 5.28 ± 0.73
C18:3(n-6) 4.45 ± 0.45 4.46 ± 0.18 5.15 ± 0.70 5.65 ± 0.97
C18:3(n-3) 45.58 ± 2.08 46.09 ± 5.81 46.38 ± 3.31 52.22 ± 6.85
C20:2 0.56 ± 0.17 0.64 ± 0.15 0.55 ± 0.01 0.52 ± 0.09

Values expressed as average mean ± standard deviation (SD) from triplicate experiments.

heptadecanoic acid methyl ester (C17:1) and methyl stearate (C18:0).

Fig. 3. (A) The biomass (mg mL−1); (B) accumulated total lipid content (% per biomass,
bars), and production (mg L−1, line) of 0–150 mg L−1 phenol-exposed Dunaliella salina
after 10 days of cultivation time under normal condition (sterilized F/2 medium). The
cultivation performed with a 12/12 h photoperiod at 26 °C and 105 μmol m−2 s−1 of
cool-white fluorescence light. Error bars represent average mean ± standard deviation
(SD) from triplicate experiments and different letters exhibit significant differences
(P b 0.05).
previous study reported that the acclimation of microalgae to the
wastewater can significantly increase algal growth along with enhanced
ascorbate peroxidase activity and carotenoid accumulation [15]. There-
fore, it was considered likely that the stimulation of growth by phenol-
exposure was derived from the induction of the antioxidant defense
system of D. salina against oxidative stress. The phenol degradation
pathway was considered as one possible mechanism for the increase
in growth. According to previous reports, phenol hydroxylase efficiently
converts absorbed phenol to catechol in the microalga Chlorella
pyrenoidosa [34]. Although more studies are required, these results sug-
gest that D. salina may absorb dissolved phenol and a related pathway
may convert the phenol to growth-related metabolites, and thus
phenol-acclimated D. salina exhibited concentration-dependent
growth.
3.6. Effect of phenol-exposure on the FAMEs and biodiesel properties of
D. salina
To evaluate biodiesel properties of phenol-acclimated D. salina
biomass, we harvested D. salina cells (acclimated with varying concen-
trations of phenol) after 10 days of cultivation in the absence of phenol
and determined FAME compositions. As shown Table 3, as phenol
concentration increased, higher levels of FAMEs including gamma-
methyl linolenate (C18:3(n-3)) and gamma-linolenic acid methyl
ester (C18:3(n-6)), were observed along with decreased cis-10-
The increase in unsaturated fatty acids, especially C18:3, under oxida-
tive stress was previously reported at a lower growth temperature,
higher light intensity, and conditions of nitrate depletion [49,50]. This
study found that phenol-induced stress can increase the ratio of unsat-
urated fatty acids and that the increased levels can be maintained under
normal culture conditions. That report and our results suggest that in-
creased unsaturated fatty acid levels are an important factor for stress
acclimation and adaptation of microalgae. Shekh et al. [50] hypothe-
sized that the fatty acid desaturase expression system protects algae
from oxidative stress by increasing the degree of disruption of the
lipid membrane. Therefore, phenol-induced stress may similarly affect
fatty acid desaturase expression.
The biodiesel property parameters were determined from the FAME
composition. Cetane number (CN) is an important biodiesel parameter
because it is related to nitrous oxide emissions, engine performance,
and combustion efficiency of diesel fuel [51]. Therefore, the biodiesel
property standard, ASTMD6751 recommends a CN of at least 47 for die-
sel used as an engine fuel [26]. The IV value is related to the degree of
unsaturation of the fatty acids and the cold flow properties of diesel.
The European standard, EN14214, recommends a maximum value of
120 [52]. From our results, the CNs and IVs of D. salina were little
changed by prior phenol exposure (Table 4), although there was a slight
trend towards lower CN and higher IV with increasing phenol concen-
tration during the acclimation period.
4. Conclusion
In this study, we evaluated the effect of phenol on growth and fuel
quality of biomass derived from the oceanic microalga D. salina. Our re-
sults suggested that, although phenol-induced oxidative stress occurred
along with apparent induction of antioxidant enzyme activities upon
Table 4
Effect of phenol concentration on biodiesel properties. (DU), degree of unsaturation; (SV),
saponification value; (IV), iodine value; (CN), cetane number. Dunaliella salina cells were
grown in media containing varying concentrations of phenol and harvested for biomass
after 10 days of cultivation at 26 °C, 12/12 h photoperiod, and 105 μmol m−2 s−1 of
cool-white fluorescent light.
Phenol concentration (mg L−1)
Parameters
0 50 100 150
DU 154.37 155.27 156.61 175.92
SV 194.11 194.06 194.13 192.74
IV 177.10 178.32 180.35 202.59
CN 34.57 34.30 33.84 29.04
66 K. Cho et al. / Algal Research 17 (2016) 61–66

exposure to phenol, algal biomass and lipid production were enhanced [24] S.Y. Chiu, C.Y. Kao, M.T. Tsai, S.C. Ong, C.H. Chen, C.S. Lin, Lipid accumulation and CO2
utilization of Nannochloropsis oculata in response to CO2 aeration, Bioresour.
when cells were grown under normal conditions following acclimation Technol. 100 (2009) 833–838.
to phenol. Increasing phenol in the media led to slight decreases in CN [25] T. Cai, X. Ge, S.Y. Park, Y. Li, Comparison of Synechocystis sp. PCC6803 and
and increases in IV. From these results, prior acclimation to phenol Nannochloropsis salina for lipid production using artificial seawater and nutrients
from anaerobic digestion effluent, Bioresour. Technol. 144 (2013) 255–260.
may be easily applicable for the production of high biomass and biodie- [26] E.C. Francisco, D.B. Neves, E. Jacob-Lopes, T.T. Franco, Microalgae as feedstock for
sel from microalgae. However, more studies into the improvement of biodiesel production: carbon dioxide sequestration, lipid production and biofuel
biodiesel quality in combination with other factors such as temperature, quality, J. Chem. Technol. Biotechnol. 85 (2010) 395–403.
[27] S.K. Mandotra, P. Kumar, M.R. Suseela, P.W. Ramteke, Fresh water green microalga
salinity and nitrogen concentrations are required for the effective use of Scenedesmus abundans: a potential feedstock for high quality biodiesel production,
phenol acclimation in industrial applications. Bioresour. Technol. 156 (2014) 42–47.
[28] J.S. Kim, P. Chin, Acute and chronic toxicity of phenol to mysid, Archaeomysis
kokuboi, Korean J. Fish. Aquat. Sci. 28 (1995) 87–97.
Acknowledgement
[29] L.B. Martello, C.S. Friedman, R.S. Tjeerdema, Combined effects of pentachlorophenol
and salinity stress on phagocytic and chemotactic function in two species of abalo-
This study was supported by a project fund (C34290) to D. Kim from ne, Aquat. Toxicol. 49 (2000) 213–225.
the Korea Basic Science Institute (KBSI). [30] M.S. Adams, J.L. Stauber, Development of a whole-sediment toxicity test using a
benthic marine microalga, Environ. Toxicol. Chem. 23 (2004) 1957–1968.
[31] V. Aruoja, M. Sihtmäe, H.C. Dubourguier, A. Kahru, Toxicity of 58 substituted anilines
Appendix A. Supplementary data and phenols to algae Pseudokirchneriella subcapitata and bacteria Vibrio fischeri:
comparison with published data and QSARs, Chemosphere 84 (2011) 1310–1320.
[32] S.E. Sabatini, Á.B. Juárez, M.R. Eppis, L. Bianchi, C.M. Luquet, M.D.C.R. de Molina, Ox-
Supplementary data to this article can be found online at http://dx. idative stress and antioxidant defenses in two green microalgae exposed to copper,
doi.org/10.1016/j.algal.2016.04.023. Ecotoxicol. Environ. Saf 72 (2009) 1200–1206.
[33] J.L. Levy, J.L. Stauber, D.F. Jolley, Sensitivity of marine microalgae to copper: the effect
of biotic factors on copper adsorption and toxicity, Sci. Total Environ. 387 (2007)
References 141–154.
[34] B. Das, T.K. Mandal, S. Patra, A comprehensive study on Chlorella pyrenoidosa for
[1] J.M. Ferris, R. Christian, Aquatic primary production in relation to microalgal re-
phenol degradation and its potential applicability as biodiesel feedstock and animal
sponses to changing light: a review, Aquat. Sci. 53 (1991) 187–217.
feed, Appl. Biochem. Biotechnol. 176 (2015) 1382–1401.
[2] S.S. Mostafa, E.A. Shalaby, G.I. Mahmoud, Cultivating microalgae in domestic waste-
[35] A.A. Shvedova, C. Kommineni, B.A. Jeffries, V. Castranova, Y.Y. Tyurina, V.A. Tyurin,
water for biodiesel production, Not. Sci. Biol. 4 (2012) 56–65.
E.A. Serbinova, J.P. Fabisiak, V.E. Kagan, Redox cycling of phenol induces oxidative
[3] Y. Chisti, Biodiesel from microalgae, Biotechnol. Adv. 25 (2007) 294–306.
stress in human epidermal keratinocytes, J. Invest. Dermatol. 114 (2000) 354–364.
[4] A.S. Ucisik, S. Trapp, Uptake, removal, accumulation, and phytotoxicity of phenol in
[36] A.A. Woodall, G. Britton, M.J. Jackson, Carotenoids and protection of phospholipids
willow trees (Salix viminalis), Environ. Toxicol. Chem. 25 (2006) 2455–2460.
in solution or in liposomes against oxidation by peroxyl radicals: relationship be-
[5] S. Das, S. Majumder, D. Mukherjee, Effect of phenol on ovarian secretion of 17β-
tween carotenoid structure and protective ability, Biochim. Biophys. Acta Gen.
estradiol in common carp Cyprinus carpio, Arch. Environ. Contam. Toxicol. 65
Subj. 1336 (1997) 575–586.
(2013) 132–141.
[37] G. Li, S. Wan, J. Zhou, Z. Yang, P. Qin, Leaf chlorophyll fluorescence, hyperspectral re-
[6] R.M. Bruce, J. Santodonato, M.W. Neal, Summary review of the health effects associ-
flectance, pigments content, malondialdehyde and proline accumulation responses
ated with phenol, Toxicol. Ind. Health 3 (1987) 535–568.
of castor bean (Ricinus communis L.) seedlings to salt stress levels, Ind. Crop. Prod.
[7] M. Bracher, C. Faller, W. Grötsch, R. Marshall, J. Spengler, Studies on the potential
31 (2010) 13–19.
mutagenicity of p-phenylenediamine in oxidative hair dye mixtures, Mutat. Res.
[38] C.M. Luna, C.A. González, V.S. Trippi, Oxidative damage caused by an excess of cop-
Genet. Toxicol. 241 (1990) 313–323.
per in oat leaves, Plant Cell Physiol. 35 (1994) 11–15.
[8] J.T. Barber, H.A. Sharma, H.E. Ensley, M.A. Polito, D.A. Thomas, Detoxification of phe-
[39] V.I. Lushchak, Adaptive response to oxidative stress: bacteria, fungi, plants and ani-
nol by the aquatic angiosperm, Lemma gibba, Chemosphere 31 (1995) 3567–3574.
mals, Comp. Biochem. Physiol. C 153 (2011) 175–190.
[9] J. Michałowicz, W. Duda, Phenols—sources and toxicity, Pol. J. Environ. Stud. 16
[40] R.J. Aitken, C. Krausz, Oxidative stress, DNA damage and the Y chromosome, Repro-
(2007) 347–362.
duction 122 (2001) 497–506.
[10] A. Bogadi-Šare, V. Brumen, R. Turk, V. Karačić, M. Zavalić, Genotoxic effects in
[41] A.E. Mirsky, L. Pauling, On the structure of native, denatured and coagulated pro-
workers exposed to benzene: with special reference to exposure biomarkers and
teins, Proc. Natl. Acad. Sci. U. S. A. 22 (1936) 439–447.
confounding factors, Ind. Health 35 (1997) 367–373.
[42] E. Cabiscol, J. Tamarit, J. Ros, Oxidative stress in bacteria and protein damage by re-
[11] J. Tuo, S.P. Wolff, S. Loft, H.E. Poulsen, Formation of nitrated and hydroxylated aro-
active oxygen species, Int. Microbiol. 3 (2010) 3–8.
matic compounds from benzene and peroxynitrite, a possible mechanism of ben-
[43] N. Mallick, F.H. Mohn, Reactive oxygen species: response of algal cells, J. Plant Phys-
zene genotoxicity, Free Radic. Res. 28 (1998) 369–375.
iol. 157 (2000) 183–193.
[12] A.R. Murray, E. Kisin, V. Castranova, C. Kommineni, M.R. Gunther, A.A. Shvedova,
[44] A.L. Dounce, A proposed mechanism for the catalatic ation of catalase, J. Theor. Biol.
Phenol-induced in vivo oxidative stress in skin: evidence for enhanced free radical
105 (1983) 553–567.
generation, thiol oxidation, and antioxidant depletion, Chem. Res. Toxicol. 20
[45] C. Michiels, M. Raes, O. Toussaint, J. Remacle, Importance of Se-glutathione peroxi-
(2007) 1769–1777.
dase, catalase, and Cu/Zn-SOD for cell survival against oxidative stress, Free Radic.
[13] N.C. Saha, F. Bhunia, A. Kaviraj, Toxicity of phenol to fish and aquatic ecosystems, B.
Biol. Med. 17 (1994) 235–248.
Environ. Contam. Toxicol. 63 (1999) 195–202.
[46] Z. Wu, J. Shi, S. Yang, The effect of pyrogallic acid on growth, oxidative stress, and
[14] R. Mittler, Oxidative stress, antioxidants and stress tolerance, Trends Plant Sci. 7
gene expression in Cylindrospermopsis raciborskii (cyanobacteria), Ecotoxicology
(2002) 405–410.
22 (2013) 271–278.
[15] O. Osundeko, A.P. Dean, H. Davies, J.K. Pittman, Acclimation of microalgae to waste-
[47] P.L.G. Martins, L.G. Marques, P. Colepicolo, Antioxidant enzymes are induced by phe-
water environments involves increased oxidative stress tolerance activity, Plant Cell
nol in the marine microalga Lingulodinium polyedrum, Ecotoxicol. Environ. Saf. 116
Physiol. 55 (2014) 1848–1857.
(2015) 84–89.
[16] E. Fakhry, D. Maghraby, Fatty acid composition and biodiesel characterization of
[48] C.G. Schmidt, L.M. Gonçalves, L. Prietto, H.S. Hackbart, E.B. Furlong, Antioxidant ac-
Dunaliella salina, J. Water Resour. Prot. 5 (2013) 894–899.
tivity and enzyme inhibition of phenolic acids from fermented rice bran with fungus
[17] H. Tang, N. Abunasser, M.E.D. Garcia, M. Chen, K.S. Ng, S.O. Salley, Potential of
Rizhopus oryzae, Food Chem. 146 (2014) 371–377.
microalgae oil from Dunaliella tertiolecta as a feedstock for biodiesel, Appl. Energy
[49] A.Y. Shekh, P. Shrivastava, K. Krishnamurthi, S.N. Mudliar, S.S. Devi, G.S. Kanade, S.K.
88 (2011) 3324–3330.
Lokhande, T. Chakrabarti, Stress-induced lipids are unsuitable as a direct biodiesel
[18] R.L.L. Guillard, Culture of Phytoplankton for Feeding Marine Invertebrates, in: W.L.
feedstock: a case study with Chlorella pyrenoidosa, Bioresour. Technol. 138 (2013)
Smith, M.H. Chanley (Eds.), Culture of Marine Invertebrates Animals, Plenum
382–386.
Press, New York 1975, pp. 29–60.
[50] A.Y. Shekh, P. Shrivastava, K. Krishnamurthi, S.N. Mudliar, S.S. Devi, G.S. Kanade, T.
[19] I. Moreno-Garrido, L.M. Lubián, A.M.V.M. Soares, Influence of cellular concentration
Chakrabarti, Stress enhances poly-unsaturation rich lipid accumulation in Chlorella
on determination of EC50 in microalgal growth inhibition tests, Ecotoxicol. Environ.
sp. and Chlamydomonas sp, Biomass. Bioenerg. 84 (2016) 59–66.
Saf. 47 (2000) 112–116.
[51] M.T. Arias-Penarands, E. Cristiani-Urbina, C.M. Montes-Horcasitas, F. Esparza-Garc´
[20] OECD (Organization for the Economic Cooperation and Development), OECD Guide-
ia, G. Torzillo, R.O. Canizares-Villanueva, Scenedesmus incrassatulus CLHE-Si01: a po-
line for Testing of Chemicals: alga, growth inhibition testAdopted June 7 1984 (201).
tential source of renewable lipid for high quality biodiesel production, Bioresour.
[21] R.L. Heath, L. Packer, Photoperoxidation in isolated chloroplasts: I. Kinetics and stoi-
Technol. 140 (2013) 158–164.
chiometry of fatty acid peroxidation, Arch. Biochem. Biophys. 125 (1968) 189–198.
[52] E.G. Giakoumis, A statistical investigation of biodiesel physical and chemical proper-
[22] A.R. Wellburn, The spectral determination of chlorophylls a and b, as well as total ca-
ties, and their correlation with the degree of unsaturation, Renew. Energy 50 (2013)
rotenoids, using various solvents with spectrophotometers of different resolution, J.
858–878.
Plant Physiol. 144 (1994) 307–313.
[23] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction and purification, Can. J.
Biochem. Physiol. 37 (1959) 911–917.