Appl Biochem Biotechnol (2012) 166:18431855

DOI 10.1007/s12010-012-9602-2

A Potential Commercial Source of Fucoxanthin Extracted

from the Microalga Phaeodactylum tricornutum

Sang Min Kim & Yu-Jin Jung & Oh-Nam Kwon &
Kwang Hyun Cha & Byung-Hun Um &
Donghwa Chung & Cheol-Ho Pan

Received: 7 April 2011 / Accepted: 5 September 2011 /

Published online: 28 February 2012
# Springer Science+Business Media, LLC 2012

Abstract Fucoxanthin, one of the main marine carotenoids, is abundant in macro- and
microalgae. Here, fucoxanthin was isolated and structurally identified as the major caroten-
oid in the diatom Phaeodactylum tricornutum through chromatographic and spectroscopic
methods, such as liquid chromatographypositive-ion atmospheric pressure chemical ioni-
zation/mass spectroscopy and nuclear magnetic resonance. This pigment was quantified by
reverse-phase high-performance liquid chromatography, and a number of extraction proce-
dures were assessed to investigate the effect of solvent type, extraction time, temperature,
and extraction method (maceration, ultrasound-assisted extraction, Soxhlet extraction, and
pressurized liquid extraction). Among the investigated solvents, ethanol provided the best
fucoxanthin extraction yield (15.71 mg/g freeze-dried sample weight). Fucoxanthin content
in the extracts produced by the different methods was quite constant (15.4216.51 mg/g
freeze-dried sample weight) but increased steeply based on the percentage of ethanol in
water, emphasizing the importance of ethanol in the extraction. The results indicate that P.
tricornutum is a rich source of fucoxanthin (at least ten times more abundant than that in
macroalgae) that is easily extracted with ethanol, suggesting potential applications in human
and animal food, health, and cosmetics.

Keywords Microalgae . Phaeodactylum tricornutum . Carotenoid . Fucoxanthin .

Extraction . Diatom

S. M. Kim : Y.-J. Jung : K. H. Cha : B.-H. Um : C.-H. Pan (*)

Functional Food Center, Korea Institute of Science and Technology (KIST), 679 Saimdang-ro,
Gangneung 210-340, Republic of Korea
e-mail: cheolpan@gmail.com

O.-N. Kwon
Research and Education Center for Marine Biology, Gangneung-Wonju National University, Gangneung
210-702, Republic of Korea

D. Chung
Department of Marine Food Science and Technology, Medical and Bio-Material Research Center,
Gangneung-Wonju National University, Gangneung 210-702, Republic of Korea
1844 Appl Biochem Biotechnol (2012) 166:18431855

Introduction

Marine microalgae constitute the largest group of living organisms in the ocean, with an
estimated range of 2105 to several million species [1]. Microalgae are important primary
producers in marine environments and play a critical role in supporting aquatic animals [2].
Various chemicals, including fatty acids, sterols, phenolic compounds, terpenes, enzymes,
polysaccharides, alkaloids, toxins, and pigments, have been isolated and structurally deter-
mined from an extensive investigation of marine microalgae culture [3]. Moreover, the
microalgal biotechnology industry has grown and diversified significantly since the 1970s.
Chlorella and Arthrospira species are two major microalgae produced in commercial large-
scale culture as nutrition sources for humans and animals. Dunaliella salina and Haemato-
coccus pluvialis are also produced on a commercial scale as sources of -carotene and
astaxanthin, respectively [4, 5].
Phaeodactylum tricornutum is a diatom that contains 36.4% crude protein, 26.1%
available carbohydrate, 18.0% lipid, 15.9% ash, and 0.25% neutral detergent fiber, on a
dry weight (dw) basis [6]. It is the only species in the genus Phaeodactylum that can exist in
different morphotypes (fusiform, triradiate, and oval) and change its cell shape based on
environmental stimuli [7]. In addition, P. tricornutum is one of two diatoms with its genome
sequenced (the other is Thalassiosira pseudonana) and serves as a model system for diatom
investigations. Efforts have been made in recent years to develop molecular techniques
based on nuclear transformation, including gene silencing and whole genome microarrays
[8]. During commercial use of this species, lipids, particularly eicosapentaenoic acid (EPA,
2.5 g in 100 g dw cells), have been the focus as a main beneficial nutrient, and a number of
studies have sought to increase the production yield of EPA and biomass [9, 10].
Fucoxanthin, a major marine carotenoid, occurs abundantly in some macro- and micro-
algae and contributes more than 10% of the estimated total production of carotenoids in
nature [11]. This pigment is bound to several proteins and, together with chlorophyll (Chl) a,
forms fucoxanthin-Chl a-protein complexes in the thylakoids, where it acts as a primary
carotenoid to harvest light and transfer energy [12]. This protein has been extensively
investigated in microalgae, including P. tricornutum, for its role in photosynthesis [13].
However, it is not known whether this pigment functions as a secondary carotenoid relating
to photo-oxidative protection. The fucoxanthin has been found to have a number of
therapeutic activities, including anticancer, antihypertensive, anti-inflammatory, and anti-
obesity effects [14, 15]. However, most of these studies were conducted with fucoxanthin
isolated from macroalgae, such as Laminaria japonica, Eisenia bicyclis, and Undaria
pinnatifida. Additionally, industrial production of this pigment has focused on macroalgae,
particularly the wasted parts of the brown alga L. japonica [16]. No studies have reported on
the commercial production of fucoxanthin derived from microalgae. Fortunately, while
searching for natural fucoxanthin sources in microalgae, P. tricornutum was found to contain
fucoxanthin as a main carotenoid.
Therefore, in the present study, we investigated extraction of fucoxanthin under various
conditions and suggest the potential use of P. tricornutum as a source of fucoxanthin. Briefly,
the effects of solvent, temperature, time, and extraction method on fucoxanthin extracted
from freeze-dried P. tricornutum were assessed, and fucoxanthin content was quantified by
reverse-phase high-performance liquid chromatography (HPLC). Furthermore, we compared
the fucoxanthin content in microalgae with that in macroalgae, and the possible combina-
tions of EPA and fucoxanthin production from pilot plant-scale outdoor culture are discussed
to suggest potential application of P. tricornutum as a natural source of highly valuable
fucoxanthin.
Appl Biochem Biotechnol (2012) 166:18431855 1845

Materials and Methods

Algal Materials, Culture Conditions, and Chemicals The marine microalga P. tricomutum
was kindly provided by the Korea Marine Microalgae Culture Center (B-007). The
algae were cultivated in 30-L plastic cylinders at 20 C, and air was continuously
supplied at 5 L/min by air-lift. Light was provided by 60-W fluorescent lamps at an
intensity of 2,500 lx. Algae were cultured in Conway medium [17] prepared from filter-
sterilized seawater, and the culture was continuously active during the 5 days after
onset. The cells were flocculated with 200 ppm Al2(SO4)3 (v/v) and then recovered
with centrifugation at 2,000 rpm using a basket centrifuge (Hanseong Co., Ansan,
Korea). The harvested biomass was frozen at 70 C and freeze dried for 2 days.
The macroalga E. bicyclis (Kijillman) Setchell (Laminariaceae) was harvested from the
east coast of South Korea, washed with fresh water for 12 h, and stored at 20 C until
use. All extraction solvents were of analytical grade and purchased from Daejung
(Gyonggi, Korea). Methanol and water for HPLC and liquid chromatographymass
spectrometry (LC-MS) were HPLC-grade solvents purchased from Fisher Scientific
(Pittsburgh, PA, USA). Methanol-d4 was purchased from the Cambridge Isotope Lab-
oratory (Andover, MA, USA).

LC-MS and HPLC Analysis Ethanol extracts were analyzed with a Varian HPLC-
hyphenated MS system (Palo Alto, CA, USA), consisting of a ProStar 410 autosampler,
two ProStar 210 pumps, and an 1,200 L triple quadrupole mass spectrometer. A YMC
carotenoid column (2504.6 mm i.d. with a 3-m particle size; Waters, Milford, MA, USA)
was used for the separation. The mobile phase consisted of methanol and water with a flow
rate of 1 mL/min at 40 C. In a gradient condition, methanol/water ratio was increased from
90:10 to 100:0 over 30 min, and then, 100% methanol was held for next 20 min. The
chromatogram was recorded at 210 and 445 nm. The MS conditions were as follows: the
positive-ion atmospheric pressure chemical ionization (APCI) mass spectra were acquired
from m/z 2001,000. The APCI interface section was set at 12 psi of drying gas at 150 C,
60 psi of nebulizing gas (N2), 17 psi of auxiliary gas, a corona current of 5 A, and a
housing temperature of 50 C. Varian MS workstation software (version 6.3) was used for
data acquisition and processing. For general quantification of fucoxanthin from the extract,
an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a
G1312A binary pump, a G1367B auto sampler, a G1315D PDA detector, a G1316A column
oven, and Chemstation software was used with the same solvent and gradient conditions as
for the LC-MS analysis. A diode array detector with a range of 200800 nm was used, and
the chromatogram was recorded at 445 nm. The fucoxanthin calibration curve, with a
concentration range of 10250 g/mL, was constructed by HPLC using fucoxanthin purified
by silica gel chromatography.

Fucoxanthin Isolation by Silica Gel Chromatography Freeze-dried powder of P. tricornu-

tum (20 g) was extracted with 1 L of 100% ethanol at room temperature for 3 h. The extract
solution was filtered with 3 M filter paper and concentrated with a rotary evaporator under
vacuum conditions to yield 3 g of extract. This extract was subjected to silica-gel adsorption
chromatography. Silica gel (50 g, 63200 mesh, Merck, Palo Alto, CA, USA) was slurry-
packed into a glass column (230 cm) in n-hexane:acetone (7:3, v/v). The ethanol extract
was adsorbed with the same weight of silica gel, placed on the top of the silica gel in the
column, and eluted with the same solvent in the dark. The red-colored fractions were
collected in test tubes, and the fractions containing fucoxanthin were combined together
1846 Appl Biochem Biotechnol (2012) 166:18431855

after HPLC analysis. The fucoxanthin was structurally confirmed by a variety of nuclear
magnetic resonance (NMR) analyses.

NMR Purified fucoxanthin (10 mg) from silica-gel chromatography was dissolved in 1 mL
of CD3OD and used for NMR spectroscopy. The 1H, 13C, and two-dimensional (2D) NMR
experiments were performed on a Varian 500 MHz NMR system with a carbon enhanced
cold probe (1H with 500 MHz, 13C with 125 MHz). Chemical shifts are expressed in (ppm)
referring to the solvent peaks H 3.31 and C 49.2 for CD3OD, and the coupling constant, J,
is in Hertz. 1H-1H correlation spectroscopy (COSY), 1H-13 C heteronuclear single quantum
coherence (HSQC), and heteronuclear multiple bond coherence (HMBC) were obtained by
employing conventional pulse sequences. Data were processed using the MestReNova
program (Mestrelab Research, Santiago de Compostela, Spain) and compared with data in
the literature.

Optimization of Fucoxanthin Extraction Extractions were performed under several different

conditions to determine the optimal conditions for fucoxanthin extraction. In all experi-
ments, 0.5 g dried powder of P. tricornutum was extracted with 25 mL of each tested-solvent
in a 50-mL flask, and the extract solution was filtered through a 0.42-m filter prior to
injection into the HPLC system. All extractions were performed independently in triplicate.
A series of conventional extraction solvents (acetone, ethanol, water, n-hexane, and ethyl
acetate) were used to screen the best solvent for fucoxanthin extraction. Normal maceration
extraction (MAC) was performed for 30 min at room temperature with each solvent. Under
the same conditions, the P. tricornutum dried powder was extracted with a series of ethanol
solutions (0100%) in water to assess the effect of ethanol content on extraction efficiency.
Additionally, the time-dependent fucoxanthin yield in 100% ethanol was assessed by
extracting for 260 min under MAC conditions. The effect of extraction temperature was
also assessed at 30, 50, and 70 C in two different aqueous ethanol solutions (50% and
100%) for 30 min.
Different physical processes [Soxhlet extraction (SOX), pressurized liquid extraction
(PLE), and ultrasound-assisted extraction (UAE)] were applied to fucoxanthin extraction
with ethanol as the extraction solvent and compared with MAC. For the SOX process, the
heating mantle temperature was set at 80 C, and when the Soxhlet process was
equilibrated with ethanol solvent in a 50-mL round flask, the sample was added to
the flask and maintained for 30 min. The flask was then immediately removed from the
mantle, and a portion of the extracted solvent was filtered for HPLC analysis. PLE was
performed with a fully automated ASE 200 system (Dionex, Sunnyvale, CA, USA) at
100 C, and a stainless steel cell (11 mL) was used with sea sand (particle size, 3050
mesh; Fisher Scientific) below the sample to avoid any void spaces. Static time was
30 min at 100 C and 1,500 psi. For the UAE experiment, a Flexonic-500 model
(Mirae Ultrasonic, Kyunggi, Korea) was used at a 70 KHz frequency. The extraction
was performed at room temperature for 30 min. Each extracted solution was quantified
by analytical HPLC.
To investigate the proportion of fucoxanthin in the biphasic system consisting of n-
hexane/ethanol/water020:60:40 (v/v/v), 10 mg of fucoxanthin was dissolved in 3 mL of
biphasic solution and mixed vigorously for 1 min. After 10 min, each phase was filtrated and
subjected to analytical HPLC.
To compare macroalgal fucoxanthin content, fresh E. bicyclis (0.5 g) was extracted with
100% ethanol (25 mL) for 30 min at room temperature, and the extracted solution was used
for quantification.
Appl Biochem Biotechnol (2012) 166:18431855 1847

Results and Discussion

Identification of the Major Carotenoid in P. tricornutum The P. tricornutum extract was

analyzed by HPLC-APCI-MS at 445 nm to detect carotenoids, and the UV and MS
chromatograms are presented in Fig. 1. The molecular mass of peak 1 was proposed as
fucoxanthin based on the fragment pattern at m/z 641, 659, and 581 corresponding to [M+H-
H2O]+, [M + H]+, and [M + H-H2O-AcOH]+, respectively (Fig. 1c). The UV-visible
spectrum of this peak showed a max at 448 nm, which supported this suggestion. Silica-
gel chromatography was conducted with the P. tricornutum ethanol extract in the dark to
identify the structural conformation of the predominant carotenoid in this species. A red
color band was clearly separated in the chromatography with n-hexane/acetone (7:3 solu-
tion; v/v). All fractions were analyzed by HPLC, and 33 mg of pure fucoxanthin could be
collected. The pure compound from the collected fractions was subjected to 1D and 2D
NMR spectroscopy. Complete assignments of the 1H and the 13C NMR spectra, including
stereochemistry, were possible by applying 2-D 1H-1H COSY, HMBC, and 1H-13 C HSQC
spectroscopy (Table 1). The 1H and 13C NMR spectra of the main carotenoid revealed
signals assignable to polyene containing acetyl, conjugated ketone, two quaternary germinal
dimethyls, two quaternary germinal oxygen methyls, four olefinic methyls, and allene

Fig. 1 Ultraviolet (UV) (a) and mass spectroscopy (MS) (b) chromatograms of the ethanol extract from P.
tricornutum using high-performance liquid chromatographypositive-ion atmospheric pressure chemical
ionizationMS analysis. The UV chromatogram was detected at 445 nm for carotenoid compounds, and the
MS chromatogram was scanned from m/z 1001,000 in the positive mode. The mass fragments of the main
peak at 19.7 min in the chromatograms were identical to those of fucoxanthin (c), and the chemical structure
of all-trans-fucoxanthin is presented with a numbering system (d)
1848 Appl Biochem Biotechnol (2012) 166:18431855

Table 1 Nuclear magnetic resonance spectroscopic data for fucoxanthin in CD3OD

13 1 13 1
Position C H (multiplicity, Position C H (multiplicity,
(125 MHz) J 0 Hz, 500 MHz) (125 MHz) J 0 Hz, 500 MHz)

1 34.95 1 35.31
2 47.27 1.50 (1H, d, m) 2 45.26 1.99 (1H, m)
47.27 1.28 (1H, m) 45.26 1.41 (1H, m)
3 63.00 3.70 (1H, d, m) 3 67.83 5.40 (1H, dd, J09.52, 13.70)
4 41.12 1.72 (1H, dd, J010.72, 13.71) 4 45.14 1.51 (1H, m)
41.12 2.28 (1H, d, t07.44, 7.44) 45.14 2.23 (1H, m)
5 66.43 5 71.43
6 67.45 6 116.73
7 40.29 2.63 (1H, d, J018.60) 7 202.23
40.29 3.85 (1H, d, J018.62)
8 199.65 8 102.82 6.11 (1H, s)
9 133.73 9 132.19
10 139.88 7.37 (1H, d, J011.01) 10 128.34 6.17 (1H, d, J011.38)
11 123.23 6.71 (1H, d, J010.76) 11 125.43 6.69 (1H, m)
12 145.38 6.86 (1H, d, J014.74) 12 137.10 6.41 (1H, d, J014.95)
13 135.35 13 137.73
14 136.69 6.53 (1H, d, J011.58) 14 132.10 6.33 (1H, d, J011.58)
15 129.19 6.73 (1H, m) 15 132.39 6.85 (1H, t, J012.75, 12.75)
16 23.47 1.05 (3H, s) 16 31.22 1.10 (3H, s)
17 27.18 0.96 (3H, s) 17 29.39 1.31 (3H, s)
18 19.80 1.22 (3H, s) 18 28.01 1.40 (3H, s)
19 10.34 1.95 (3H, s) 19 12.86 1.86 (3H, s)
20 11.29 2.03 (3H, s) 20 11.46 2.02 (3H, s)
21 170.90
22 19.80 2.05 (3H, s)

functionalities. These data were in agreement with those of fucoxanthin in the published
literature [11, 18]. Furthermore, a detailed comparison of these data, particularly focusing on
the olefinic protons in the 1H NMR spectra, revealed that the predominant fucoxanthin in the
P. tricornutum extract was all-trans fucoxanthin, among the naturally occurring geometrical
isomers of fucoxanthin (Fig. 1d) [19]. Therefore, all-trans fucoxanthin is the major carot-
enoid in P. tricornutum. However, two minor peaks (peaks 2 and 3) were also found in the
same extract with the same MS profiles. From the above results and the literature, these
peaks were believed to be naturally occurring fucoxanthin isomers [11].
A number of studies have analyzed pigments in this microalga. Chlorophylls, as well as
carotenoids, have been quantified from biomass cultured in external tubular photobioreac-
tors under various conditions [6], and an indirect method to rapidly estimate carotenoid
contents was developed [19]. Recently, circadian variations in the pigment content of P.
tricornutum were analyzed under different light regimens and suggested that diadinoxanthin
and -carotene are associated with photoprotection [20]. Additionally, pigment contents are
affected differently by the Fe(III) concentration and diadinoxanthin content, which increase
at low Fe(III) concentrations; the reverse occurs for the -carotene content [21]. Although
fucoxanthin is the predominant carotenoid in P. tricornutum, past studies focused more on
Appl Biochem Biotechnol (2012) 166:18431855 1849

diadinoxanthin and -carotene from a physiological aspect in response to internal or external

environmental stimuli, and no studies have reported fucoxanthin extraction from this species
as a valuable natural source for pharmaceutical application. Furthermore, fucoxanthin
content in most studies was quantified differently, i.e., femtogram/cell or microgram/liter.
Therefore, we focused on characterizing the ability to extract fucoxanthin from P. tricornu-
tum under various conditions for future commercial application, and the quantities are
expressed as milligrams per gram of freeze-dried sample in all experiments described below.

Effect of Solvent and Temperature on Fucoxanthin Extraction Selecting the proper solvent
to optimize extraction is very important, as it determines the similarities in the chemical
composition of the substances to be extracted [22]. Five solvents generally used to extract
plant, and marine materials were tested to investigate the best solvent for extracting
fucoxanthin from P. tricornutum (Fig. 2). Methanol was the first solvent generally used to
extract pigments, but because of its toxicity, it has been replaced by other solvents and was
omitted from this study. Extractions were conducted at room temperature for 30 min with. P.
tricornutum freeze-dried powder. The results showed that fucoxanthin yield was highly
dependent on the extraction solvent. Ethanol was the best solvent to maximize fucoxanthin
extraction (15.71 mg/g dw) among the five solvents tested, whereas water and n-hexane
were ineffective for extracting fucoxanthin. The result that n-hexane did not effectively
extract fucoxanthin was quite surprising, as n-hexane is the most commonly used solvent for
lutein extraction from microalgae during the commercial production of lutein [23]. Acetone,
the most widely used solvent for marine pigment extraction, yielded approximately one third
of the fucoxanthin extracted with ethanol (4.60 mg/g dw). In the case of ethyl acetate,
2.26 mg/g dw of fucoxanthin was extracted under the same conditions. Therefore, it could be
easily estimated that the fucoxanthin content in the acetone extract was lower than that of the
ethanol extract. In a previous study by the Rebolloso-Fuentes research group [6], total
carotenoid contents in the acetone extract were estimated to be 3.03 mg/g dw of P.
tricornutum, and 60% of the carotenoids were fucoxanthin, which means fucoxanthin was
present at 1.81 mg/g dw concentration in the acetone extract. A solvent effect in the
fucoxanthin extraction process has been discovered in different microalgae. The Porphyri-
dium purpureum extraction process was performed only with water, while other microalgae
were extracted with ethanol and methylene chloride together with water in an experiment in
which the active component was revealed as fucoxanthin. P. purpureum showed relatively
low antiproliferative activity against bronchopulmonary and epithelial cell lines, which
might have been caused by the use of an inadequate extraction solvent [24].

Fig. 2 The effect of solvents on

fucoxanthin extraction efficiency
from freeze-dried Phaeodactylum
tricornutum. The extraction was
conducted for 30 min at room
temperature, and the extracted
solutions were analyzed by high-
performance liquid chromatogra-
phy for quantification. Fucoxan-
thin content is expressed as mean
values with error bars from three
independent experiments
1850 Appl Biochem Biotechnol (2012) 166:18431855

Serial dilutions of ethanol in water were investigated for fucoxanthin extraction to reveal
the effect of ethanol (Fig. 3). With a 30-min extraction time at room temperature, fucoxan-
thin began to be extracted with a 50% aqueous ethanol solution (4.16 mg/g dw) and was
almost saturated in the 80% ethanol solution (approximately 15.01 mg/g dw), indicating that
the ethanol concentration in the extract solution was proportional to fucoxanthin yield.
Furthermore, 9.34 mg/g dw of fucoxanthin, which is approximately 57% of the fucoxanthin
yield extracted in 1 h, was obtained at room temperature within 2 min using 100% ethanol
(Fig. 4). Saturation was achieved at approximately 30 min. It was concluded that ethanol was
the optimal solvent to extract fucoxanthin from P. tricornutum, even though this solvent has
been used less commonly than acetone for microalgae pigment extraction. Generally, the
potential for a solvent to extract compounds from algae depends on the hydration and
permeability of the microalgal cell wall and the solubility of the target compound. However,
it is unclear how ethanol affects cell hydration, permeability, and integrity.
Fucoxanthin was extracted with 50% and 100% ethanol solutions at different temper-
atures (30, 50, and 70 C) to assess the effect of temperature on extraction yield (Fig. 5). As a
result, fucoxanthin concentrations increased with temperature in the 50% ethanol solution
extracted for 30 min, whereas the concentration remained relatively constant with an
increase in temperature for the 100% ethanol solution. However, the amount of extracted
fucoxanthin in the high temperature (70 C) and 50% ethanol condition was only 8.40 mg/
g dw, which was approximately 50% of the maximum extractable fucoxanthin using 100%
ethanol. Thus, it appeared that the extractability of fucoxanthin increased with increasing
temperature, but the temperature effect was overwhelmed by the high (100%) ethanol
concentration. Because fucoxanthin is bound to several proteins and chlorophyll a to form
fucoxanthinChl aprotein complexes, the increased temperature may help to detach these
bonds and increase extraction yield. This phenomenon occurred in the macroalgae L.
japonica during commercial-scale preparation of fucoxanthin [16]. However, ethanol con-
centration was more critical than extraction temperature to obtain a high yield in a short
period during P. tricornutum fucoxanthin extraction. This characteristic is beneficial in that
the extraction process requires less energy to increase temperature, and ethanol would be
relatively inexpensive when applying this microalga for commercial-scale fucoxanthin
preparation. An important consideration during the extraction process is fucoxanthin degra-
dation. Fucoxanthin is a quite unstable molecule and is sensitive to light, oxygen, and
temperature. Therefore, fucoxanthin can be degraded at high temperatures. The slight
decrease in fucoxanthin yield (3% compared to that at 30 C) at 70 C in 100% ethanol
solution might have occurred by degradation of fucoxanthin at high temperature.

Fig. 3 The effect of ethanol con-

centration in water on fucoxanthin
extraction efficiency from freeze-
dried Phaeodactylum tricornu-
tum. The extraction was con-
ducted for 30 min at room
temperature, and the extracted
solutions were analyzed by high-
performance liquid chromatogra-
phy for quantification. Fucoxan-
thin content is expressed as mean
values with error bars from three
independent experiments
Appl Biochem Biotechnol (2012) 166:18431855 1851

Fig. 4 Time-scale extraction of

fucoxanthin from freeze-dried
Phaeodactylum tricornutum. The
extraction was conducted using
100% ethanol at room tempera-
ture, and the extracted solutions
were analyzed by high-
performance liquid chromatogra-
phy for quantification. Fucoxan-
thin content is expressed as mean
values with error bars from three
independent experiments

Effect of Physical Processes During Fucoxanthin Extraction Another important aspect to

consider when extracting pigments from microalgae is disruption of cell integrity by
physical processes. The classical organic solvent extraction techniques use MAC, counter-
current extraction, PLE, SOX, ULE, and supercritical fluid extraction. Among them, the
SOX, PLE, and UAE methods were compared with MAC to extract fucoxanthin (Fig. 6).
Fucoxanthin content extracted by the SOX method with 100% ethanol at 80 C was
approximately 15.42 mg/g dw, which was similar to that obtained using the MAC method
(15.71 mg/g dw). Although the SOX method has several advantages compared to the
conventional MAC method, such as continuous contact between the sample and use of fresh
solvent during the entire procedure [25], this method did not produce any favorable effects
during fucoxanthin extraction.
PLE was applied at 100 C. A static time of 30 min was used during this extraction at
high pressure (1,500 psi). However, the results (16.51 mg/g dw) were similar to those of the
MAC method, indicating that a variety of advantages of the PLE method, including less
solvent use, shorter experiment time, oxygen- and light-free environment, and the high
pressure condition [26], did not affect the fucoxanthin extraction yield.
UAE decreases the extraction time significantly and increases extraction yields in many
marine materials [27]. Nevertheless, the fucoxanthin yield from UAE was also similar
(15.96 mg/g dw) to yields by other methods, indicating no beneficial effect of UAE.
Thus, the MAC method was sufficient to produce a maximum yield of fucoxanthin from
P. tricornutum. However, a number of studies have reported positive effects of PLE, SOX,

Fig. 5 The effect of temperature

on fucoxanthin extraction from
freeze-dried Phaeodactylum tri-
cornutum. Two different ethanol
solutions (50% and 100%) were
used for the 30-min extraction.
All extract solutions were ana-
lyzed by high-performance liquid
chromatography for quantifica-
tion. Fucoxanthin content is
expressed as mean values with
error bars from three independent
experiments
1852 Appl Biochem Biotechnol (2012) 166:18431855

Fig. 6 The effect of physical pro-

cesses on fucoxanthin extraction
efficiency from freeze-dried
Phaeodactylum tricornutum.
Soxhlet extraction (SOX), pres-
surized liquid extraction (PLE),
and ultrasound-assisted extraction
(UAE) were applied to extract
fucoxanthin

and UAE on pigment extraction from other microalgae. For example, PLE resulted in higher
extraction efficiency than MAC, SOX, and UAE when carotenoids and chlorophylls were
extracted from Chlorella vulgaris [28]. We conducted a UAE experiment in our laboratory to
mathematically optimize fucoxanthin extraction from P. tricornutum using response surface
methodology with ethanol concentration in water, temperature, and extraction time as
independent variables. Only the ethanol percentage was a significant factor at the 95%
significance level for extracting fucoxanthin (data not shown). This result agrees with the
extraction results described above. Therefore, the extraction efficiency of a biofunctional
material from microalgae is highly dependent on the species, extraction method, solvent, and
target compound. The diatom P. tricornutum has these characteristics of easy fucoxanthin
extractability, which can be an advantage in the use of this microalga as a natural source of
biofunctional fucoxanthin.

Quantification of Fucoxanthin in Macroalgae and Microalgae Although many studies have

isolated and quantified fucoxanthin from macroalgae, such as the brown algae E. bicyclis
and L. japonica [16, 2932], only a few studies have investigated fucoxanthin content in
microalgae (Table 2). In the present study, the maximum fucoxanthin yield was 16.33 mg/g
dw, approximately 1.63% of dried P. tricornutum, which was obtained by maceration for 1 h
with 100% ethanol at room temperature. In the marine diatom, Cylindrotheca closterium,
5.23 mg/g dw of fucoxanthin, which is about one third of that of P. tricornutum, was
produced using microwave-assisted extraction [3]. Fresh E. bicyclis was extracted under
the same conditions as a comparison, and the amount of fucoxanthin was 0.26 mg/g fresh
weight of sample. Because fresh macroalgae contain 80% water, the fucoxanthin content
was approximately 1.3 mg/g dw sample, which was quite low compared to that of P.
tricornutum. Fucoxanthin content in this seaweed was 0.08 mg/g fresh weight in another
study [16]. Fucoxanthin contents from a variety of macroalgae in the literature range from
0.02 (Hizikia fusiformis) to 0.58 mg/g fresh weight sample (Petalonia binghamiae) (Table 2),
suggesting that microalgae possess higher amounts of fucoxanthin than those in macroalgae.
As mentioned above, fucoxanthin fulfills a number of biofunctions and is a valuable natural
biomaterial; however, most sources of this pigment for commercial use have been macro-
algae, such as L. japonicus, U. pinnatifida, and E. bicyclis [16]. Therefore, if microalgae are
to be used as a commercial-scale source of fucoxanthin, continuous production will be
Appl Biochem Biotechnol (2012) 166:18431855 1853

Table 2 Fucoxanthin contents in microalgae and macroalgae

Species Fucoxanthin content Sample References

(mg/g sample) condition

Microalgae Phaeodactylum tricornutum 15.33a Dried In this study

Phaeodactylum tricornutum 1.81b Dried [6]
Cylindrotheca closterium 5.23b Dried [3]
Macroalgae Eisenia bicyclis 0.26 Fresh In this study
Eisenia bicyclis 0.08 Fresh [16]
Laminaria saccharina 0.24 Fresh [32]
Sargassum muricum 0.29 Fresh [32]
Fucus distichus 0.16 Fresh [32]
Petalonia binghamiae 0.430.58 Fresh [29]
Laminaria religiosa 0.24 Fresh [29]
Scytosiphon lomentaria 0.240.56 Fresh [29]
Undaria pinnatifida 0.32 Fresh [29]
Undaria pinnatifida 0.87 Dried [30]
Undaria pinnatifida 0.11 Fresh [16]
Laminaria japonica 0.12 Fresh [31]
Laminaria japonica 0.19 Fresh [16]
Sargassum fulvellum 0.06 Fresh [16]
Hizikia fusiformis 0.02 Fresh [16]
a
Extraction solvent was ethanol
b
Extraction solvent was acetone

necessary through cultivation. At present, lutein, zeaxanthin, and astaxanthin are three
xanthophylls produced from commercial-scale cultivation of microalgae. The chlorophycean
microalgae Muriellopsis sp. has a high lutein content (up to 35 mg/L culture) as well as a
high growth rate and standing cell density. Zeaxanthin was produced at levels up to 6 mg/
g dw from D. salina, and H. pluvialis accumulates astaxanthin at up to 4% of its dry mass
[33]. Therefore, a fucoxanthin yield of 16.33 mg/g dw from P. tricornutum is a suggested
amount for commercial-scale production if the growth rate and cell density are sufficient for
continuous biomass harvest. Regarding the P. tricornutum biomass, a maximum biomass of
25.4 g dry sample per liter was obtained from a split-cylinder photobioreactor [34]. In this
system, fucoxanthin can theoretically be produced at up to 414 mg/L according to our data.
Furthermore, a number of attempts to culture P. tricornutum focused on the pigment content
influenced by environmental conditions, such as light intensity and temperature. Addition-
ally, transformation and gene silencing techniques have been developed from this species.
Thus, the optimum fucoxanthin production conditions for a fucoxanthin-overproducing
mutant strain, such as zea1 generated from D. salina for zeaxanthin overproduction, are
expected to be developed in the near future. An important factor determining the market for
microalgal product development is the value of the target compound from the organism of
choice. As mentioned above, the main target compound for the commercial use of P.
tricornutum has been fatty acids, particularly EPA. A recent study reported that lipids were
successfully extracted from this microalga with ethanol and a partition process using a
biphasic system [35]. In that report, a water content of 40% (v/v) in the hydroalcoholic phase
gave the highest lipid recovery and n-hexane was added to hydroalcoholic phase to extract
1854 Appl Biochem Biotechnol (2012) 166:18431855

lipids from hydroalcoholic phase resulting in biphasic system of n-hexane/hydroalcoholic

phase (0.2, v/v). Most lipids could be recovered in n-hexane phase by several consecutive
extractions. Fortunately, the optimum solvent for lipid extraction was the same as that used
for fucoxanthin extraction, and a biphasic system can be applied for purifying fucoxanthin.
In order to investigate the possibility of the combined production of lipid and fucoxanthin,
fucoxanthin was dissolved in the same biphasic system, and then, the percentages of
fucoxanthin in each phase were analyzed by HPLC. In result, most fucoxanthin (over
99%) was present in the hydroalcoholic phase, implying lipid and fucoxanthin can be
separated by this biphasic system after same extraction process with 100% ethanol solvent.
Another point for consideration in the combined production of lipids and fucoxanthin is the
different extraction time between lipids and fucoxanthin. Longer time (generally over 10 h)
is required for lipid extraction, while fucoxanthin can be extracted within 1 h. However, in
many researches, fucoxanthin has been extracted for over 10 h from macroalgae, implying
that fucoxanthin is quite stable and has no problem in a long extraction time [30]. In these
respects, P. tricornutum can be used for combined production of fatty acids and fucoxanthin,
making it a beneficial organism for obtaining high-value natural compounds.

Acknowledgments This research was financially supported by the Ministry of Education, Science and
Technology (MEST), Gangwon Province, Gangneung City, Gangneung Science Industry Foundation (GSIF)
as the R&D Project for Gangneung science park promoting program.

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