b i o m a s s a n d b i o e n e r g y 5 9 ( 2 0 1 3 ) 2 7 9 e2 8 4

Available online at www.sciencedirect.com

http://www.elsevier.com/locate/biombioe

New mixture composition of organic solvents for

efficient extraction of lipids from Chlorella vulgaris

Jong-min Jeon a, Hee-Won Choi b, Guen-Chan Yoo b, Yong-Kuen Choi a,

Kwon-Young Choi c,d, Hyung-Yeon Park e, Sung-Hee Park c,d,
Yun-Gon Kim f, Hyung Joo Kim a, Sang Hyun Lee a, Yoo Kyung Lee g,
Yung-Hun Yang a,h,*
a
Department of Microbial Engineering, College of Engineering, Konkuk University, 1 Hwayang-dong, Gwangjin-gu,
Seoul 143-701, Republic of Korea
b
Paekahm High School, Shinjung-ro, Yangchun-gu, Seoul, Republic of Korea
c
School of Chemical and Biological Engineering, Seoul National University, Gwanak-gu, Seoul 151-742,
Republic of Korea
d
Institute of Molecular Biology and Genetics, Seoul National University, Gwanak-gu, Seoul 151-742,
Republic of Korea
e
Bio-MAX Institute, Seoul National University, Gwanak-gu, Seoul 151-742, Republic of Korea
f
Chemical Engineering, Soongsil University, 511 Sangdo-dong, Seoul 156-743, Republic of Korea
g
Division of Life Sciences, Korea Polar Research Institute, 12 Gaetbeol-ro, Yeonsu-gu, Incheon 406-840, Korea
h
Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul 143-701,
South Korea

article info abstract

Article history: To identify more effective but less toxic compositions of organic solvent mixtures for bio-
Received 18 February 2013 diesel production from Chlorella vulgaris, 15 different organic solvents were examined and
Accepted 16 September 2013 compared for their power of lipid extraction from algal biomass. When solvents were
Available online 14 October 2013 individually examined, methanol showed the best efficiency for the extraction of fatty acids,
followed by dichloromethane. Although chloroform resulted in yields which were equal to
Keywords: or greater than those of other solvents, it showed a very low fatty acid content and a high
Lipid extraction level of unknown impurities when analyzed by gas-chromatography (GC). Furthermore,
Mixture design solvent mixtures were applied in order to determine the optimal composition for a high lipid
Organic solvent extraction efficiency, using chloroform, methanol and dichloromethane. Through compar-
Algal biomass ison of compositions using the Bligh and Dyers method, the best composition was to be
Chlorella vulgaris methanol:dichloromethane (1:1). Our findings resulted in a 25% increase of lipid extraction
yield, together with C16, C16:2, C18:2 as major components from C. vulgaris.
ª 2013 Elsevier Ltd. All rights reserved.

1. Introduction such as plants, microalgae, animal fat and so on [1e3].

Although algae has been studied for a long time in the food
Algal biomass is one among the renewable resources, and is industry, the rapidly growing demand to replace petroleum
an alternative fuel source among many different biomasses based energy and the need for alternative energy sources, has

* Corresponding author. Department of Microbial Engineering, College of Engineering, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul
143-701, Republic of Korea. Tel.: þ82 2 450 3936; fax: þ82 2 3437 8360.
E-mail address: seokor@konkuk.ac.kr (Y.-H. Yang).
0961-9534/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biombioe.2013.09.009
280 b i o m a s s a n d b i o e n e r g y 5 9 ( 2 0 1 3 ) 2 7 9 e2 8 4

microwave assisted extraction methods have also been

Table 1 e Compositions of lipids extracted from C.
vulgaris using by three organic solvents. D.W.: dried developed [13,14].
weight. ND: not detected. (*): Fatty acid composition Although the health, security, and regulatory problems
(wt.%). Contents of fatty acid from D.W.: amount of associated with the use of organic solvents should be
sample measured and calculated by GC/amount of considered, in order to realize a commercialized process, the
sample measured from dry weight. economy of the cost of production and recovery should be
Fatty acids Extracted amounts of fatty acids (mg/g D.W.) considered with priority. As a result, to date, the recovery of
lipids from biomass has been largely dependent on extraction
Chloroform Methanol Dichloromethane
using halogenated hydrocarbons like chloroform with meth-
C13 0.64 (1.2%) 0.98 (0.8%) 0.91 (1.3%) anol [12,15,16]. However, there have not been many studies
C14 1.06 (2.0%) 1.52 (1.2%) 1.29 (1.8%)
which specifically and systemically investigate the effects of
C16 9.58 (18.5%) 22.99 (18.8%) 13.10 (18.1%)
chloroform and methanol composition, or the replacement of
C16:2 10.80 (20.9%) 29.41 (24.0%) 16.95 (23.4%)
C17 0.23 (0.4%) ND ND these solvents with others of a reduced toxicity [17e19]. Here,
C18 3.90 (7.5%) 0.75 (0.6%) 3.07 (4.2%) we report the screening of new solvents and mixture analysis
C18:1 ND 0.99 (0.8%) ND of chloroform based extraction methods to decrease the
C18:2 24.78 (47.9%) 62.04 (50.6%) 35.08 (48.4%) amount of chloroform, considering safety, cost-effectiveness,
C18:3 1.38 (2.7%) 3.96 (3.2%) 2.18 (3.0%) environmental implications of the entire operation, the
Total 51.78 (100%) 122.64 (100%) 72.42 (100%)
quantity of waste products, and the overall associated toxic-
Contents 13.2% 40.2% 15.9%
ities. As a result, we suggest possible compositions based on
of fatty
acids the result of mixture analysis, and propose a new alternative
from D.W. composition for the initial extraction in the production of algal
biofuels from Chlorella vulgaris.

encouraged scientists to reevaluate algal biomass as an

alternative source of fuel which can be sustainably produced
the future [2,4,5]. The production of algae is highly efficient
and avoids any collision with the growth of crops on land [6,7].
In addition, algal biomass is easy to process because of its
weaker cell wall compared to other cellulosic materials [8].
Typically, algae produce lipids between C14 and C20 in
length. The most common sources of biodiesel consist of
unsaturated C16 and C18 fatty acid methyl esters, suggesting
that algae biomass could be an excellent alternative source for
biodiesel production. Thus, various attempts to increase the
lipid content through changing culture methods or omics
approaches in algae have been reported [9e11].
In order to extract lipids from algal biomass, Soxhlet
extraction using hexane as the solvent, and Bligh and Dyer’s
method using a mixture of chloroform and methanol have
conventionally been used [12]. In addition, ionic liquid based
extraction of lipids, supercritical fluids like CO2 and
2. Materials and methods
2.1. Materials
Chloroform, sulfuric acid, methanol, and dichloromethane
were purchased from Biosesang (Seoul, Korea), other all
organic solvents were purchased from SigmaeAldrich (St.
Louis, USA). Commercial C. vulgaris was obtained from Dae-
sang Wellife (Korea). All chemicals used in this work were of
analytical grade, and were used without any further
purification.
2.2. Lipid extraction using organic solvent candidates
To 50 mg of C. vulgaris powder which was used as a biomass,
0.9 mL of each organic solvent was used for lipid extraction.

Table 2 e Mixture design and extracted lipid composition. C: ratio of chloroform, M: ratio of methanol and D: ratio of
dichloromethane.
Std C M D Extracted fatty acids (mg/g D.W.)
order
C16 C16:2 C18:2 Total lipid

1 1.00 0.00 0.00 5.6 (0.4) 3.3 (1.0) 8.2 (2.0) 18.7 (3.8)
2 0.67 0.33 0.00 13.8 (0.8) 15.0 (1.3) 31.0 (3.3) 66.3 (5.2)
3 0.67 0.00 0.33 8.3 (3.0) 5.5 (3.1) 13.3 (7.2) 31.3 (15.7)
4 0.33 0.67 0.00 12.2 (0.9) 13.0 (1.9) 26.8 (3.4) 56.7 (6.2)
5 0.33 0.33 0.33 15.1 (1.5) 17.6 (2.8) 36.1 (4.7) 76.7 (8.9)
6 0.33 0.00 0.67 9.8 (0.0) 8.4 (0.0) 19.8 (0.0) 43.3 (0.0)
7 0.00 1.00 0.00 8.3 (0.8) 7.0 (1.0) 14.3 (1.4) 33.0 (2.1)
8 0.00 0.67 0.33 13.6 (0.4) 14.9 (0.6) 30.6 (0.9) 65.1 (1.3)
9 0.00 0.33 0.67 15.4 (0.5) 18.4 (1.5) 37.6 (2.5) 79.3 (5.0)
10 0.00 0.00 1.00 7.2 (0.0) 4.6 (0.0) 11.3 (0.0) 26.8 (0.0)
11 0.67 0.17 0.17 13.6 (1.1) 15.8 (1.6) 33.0 (4.1) 70.1 (8.2)
12 0.17 0.67 0.17 8.7 (7.5) 15.5 (3.2) 32.1 (6.7) 62.5 (18.7)
13 0.17 0.17 0.67 13.2 (0.9) 13.9 (2.9) 28.2 (5.3 61.2 (11.0)
 b i o m a s s a n d b i o e n e r g y 5 9 ( 2 0 1 3 ) 2 7 9 e2 8 4
Fig. 1 e Comparison of extraction efficiencies for C. vulgaris
with different organic solvents. Lipid extraction
efficiencies of diverse organic solvents.
Initially the organic solvent added to the biomass were mixed
by vortexing and activated at 37  C for 1 h. The mixture was then
centrifuged at 1500 rpm for 5 min, and 0.45 mL of the liquid
phase was transferred to glass vials and the sample was evap-
orated and re-treated with 1 mL of chloroform for esterification.
2.3. Analytical techniques
The quantity and composition of fatty acids were determined
by gas chromatography using a slight modification of a pre-
viously described method [20,21]. Through this method, 1 mL
CHCl3 and 1 mL CH3OH/H2SO4 (85:15, v/v) were added to the
samples, and they were then incubated at 100  C for 2 h,
cooled to room temperature, and placed on ice for 5 min. After
adding 1 mL of cold distilled water, the samples were vortexed
thoroughly for 1 min, and were then centrifuged at 2000 rpm.
The organic phase was then extracted by pipette, and moved
to clean borosilicate glass tubes containing 1 mg of Na2SO4.
These samples were then analyzed by GC (Agilent, CA, USA)
equipped with a fused silica capillary column (Agilent HP-
FFAP, 30 m  0.32 mm, i.d. 0.25 mm film) containing a linear
temperature gradient (for fatty acid: at 140  C, 1 min, 5  C/min
to 235  C, hold for 5 min). The injector port temperature was
210  C. Mass spectra were obtained by electron impact ioni-
zation at 70 eV, and scan spectra were obtained within the
range of 100e600 m/z. Selected ion mode (SIM) was used for
the detection and fragmentation analysis of major products.
2.4. Design of experiments and mixture analysis
To develop a strategy for optimizing the organic solvent
composition and ratio for lipid extraction from C. vulgaris
powder, a mixture analysis model of three organic solvents in
the diverse ratio was developed and populated using a
281
Fig. 2 e Regression curve using linear solvation energy
relationship (LSER) of solvents with 5 parameters of
dipolarity/polarizability (p*), acidity (a), basicity (b), molar
volume (Vm/100), and Hildebrand solubility parameter (d2/
1000).
standard mixture analysis methodology [22], and the Minitab
V14 program (http://www.minitab.com). For the design of
mixture analysis experiments to populate the model, we used a
simplex lattice method augmented by the design of axial points
(0.17:0.17:0.67, 0.17:0.67:0.17, and 0.67:0.17:0.17 for chlor-
oform:methanol:dichloromethane). The degree of lattice for
this mixture analysis is three, and therefore, the experimental
design contained a set of all 13 combinations, where each value
of organic solvents was 0.00, 0.33, 0.67 and 1.00 respectively
(Table 2). The experimental data for response trace plots are
also shown in Table 2. To plot mixture contours, a mixture
regression using the model fitting method was applied with full
quadratic component terms initially included. In the data
analysis, the coefficients with p-value below 0.1 were used as
parameters (Table S3 in Supplementary data). Contour plots
were then generated as each of the chloroform, methanol and
dichloromethane components increased along the line from
the centroid (1.00:1.00:1.00) toward its pure component vertex
(1.00:0.00:0.00), while the ratio of the other components was
held constant.
3. Results and discussion
3.1. Preliminary screening of organic solvent for lipid
extraction
To screen new candidates for organic solvent extraction, 15
different organic solvents were selected, including chloroform
282 b i o m a s s a n d b i o e n e r g y 5 9 ( 2 0 1 3 ) 2 7 9 e2 8 4

Fig. 3 e Contour plots of three solvent mixtures for the amount of extracted fatty acid for C16 (A), C16:2 (B), C18 (C) and total
fatty acids (D). Mixture ratio follows: total volume of solvent mixtures is 0.9 mL.

and methanol (Table S1). Following the comparison on the

efficiency of extraction from algal biomass, methanol showed
the best efficiency for lipid extraction from C. vulgaris, fol-
lowed by dichloromethane and DMSO (Fig. 1). The comparison
between the measured weight after evaporation and calcu-
lated weights from GC analysis showed that the fraction of
lipids extracted by chloroform (13.2%) contained more impu-
rities (expected to be various pigments from C. vulgaris) than
methanol (40.2%) or dichloromethane (15.9%), resulting in a
lower recovery of lipids. When we profiled the extracted lipids
using each selected organic solvent, C16:0, C16:2 and C18:2
were commonly dominant in all examined solvents (Table 1).
As it is very difficult to directly describe the cumulative
lipid extraction efficiency by any simple parameter, we
determined the linear solvation energy relationship (LSER) by
solvatochromic parameters, (including dipolarity/polariz-
ability (p*), acidity (a) and basicity (b)) and molar volume (Vm/
100), and Hildebrand solubility parameter ðdH2 =1000Þ to un-
derstand the effect of a given solvent on extraction efficiency.
There have been many reports of the relationships between
solubilities, partition coefficients, toxicities and catalytic re- Fig. 4 e Comparison of lipid extraction efficiencies with
actions, and such determinations have been successfully selected mixtures of organic solvents. C: chloroform, M:
applied to many equilibrium and kinetic phenomena to methanol and D: dichloromethane.
 b i o m a s s a n d b i o e n e r g y 5 9 ( 2 0 1 3 ) 2 7 9 e2 8 4
describe their relationship [23,24]. By applying LSER to our
extraction systems, we found out the correlation between
extraction content and solvent property as shown below
(Fig. 2 and Table S2 in Supplementary data). Among these
parameters, molar volume (Vm/100) showed the highest cor-
relation to lipid content, and was determined to be the factor
which had the greatest effect on extraction efficiency.
logðlipid contentÞ ¼ 1:75  0:18p  0:52a  0:89b
 1:31ðVm =100Þ þ 1:53ðdH2 =1000Þ
n ¼ 12; R ¼ 0:88; SD ¼ 0:29
3.2. Mixture analysis of MeOH, CHCl3, and CH2Cl2
Mixture analysis was initiated because mixture of solvents
showed better extraction efficiencies than single solvent
systems (data not shown). To design mixture analysis,
methanol, dichloromethane and chloroform, which showed
good efficiency of lipid extraction in previous results, were
selected. Although DMSO was initially considered as a
mixture candidate, it was excluded because it has too high of
a boiling point, which could cause problems during subse-
quent evaporation and solvent recovery steps. To determine
the optimal organic solvent mixture ratio and contour plot
for lipid extraction from algal biomass, a mixture design
method was applied, as explained in Materials and methods
(Table 2). Through this method of multiple experimentation,
mixture analysis provides for a correlation between the
composition of three solvents and the amount of extracted
lipids. Other lipid correlations are detailed in Table S3 in
Supplementary data.
Yðmg=g D:W:Þ ¼ 17:5C þ 30:2M þ 29:2D þ 178:6CM þ 72:2CD
þ 193:1MD þ 137:6CMðC  MÞ
Y: extracted amount when 900 mL of total solvent mixture was
used.
C: ratio of chloroform, M: ratio of methanol, D: ratio of
dichloromethane, C þ M þ D ¼ 1.
Although there are some differences among C16, C16:2 and
C18:2 extractions, all three showed quite similar patterns in
terms of correlation of the amount of total lipids to lipid
extraction (Fig. 3). The contour plots showed the 1:1 ratio of
methanol and dichloromethane as being one of the optimal
ratios for the extraction of lipids from C. vulgaris. As initially
suggested, mixtures of solvents resulted in higher efficiencies
of lipid extraction than single solvent systems (Fig. 3).
3.3. Extraction of lipid with new organic solvent
composition
To confirm the optimized mixture ratio, seven points, such
as methanol, chloroform, dichloromethane individually,
and two each of methanolechloroform mixtures (1:1 and
1:3), and methanol dichloromethane mixtures (1:1 and 2:1),
were examined based on the prediction of mixture analysis
model. As we expected from the mixture analysis model,
which is expected to recover more lipid than
283
chloroform:methanol 1:1, showed an approximate 25%
higher efficiency (84.37 mg/g D.W.) than the method of Bligh
and Dyer (chloroform:methanol 1:1, 67.23 mg/g D.W.) for C.
vulgaris (Table 2 and Fig. 4). The overall patterns of lipid
recovery were quite similar, however, the amount of
recovered lipids were different for each solvent composition
(Table S4 in Supplementary data).
4. Conclusion
In this experiment, we applied systematic mixture analysis
and set up three solvent mixture models for lipid extraction
with methanol, dichloromethane and chloroform. As a result,
we determined a new optimum organic solvent mixture
composition of dichloromethane:methanol (1:1, v/v) for lipid
extraction from C. vulgaris. We applied new mixture compo-
sition for the extraction of lipid for C. vulgaris increasing the
lipid extraction yield by 25% from Bligh and Dyer’s method
from C. vulgaris.
Acknowledgment
The work was partially supported by a National Research
Foundation of Korea grant (NRF-2011-619-E0002), Basic Sci-
ence Research Program through the National Research Foun-
dation of Korea (NRF) funded by the Ministry of Education
(NRF-2013R1A1A2A10004690), Polar Academic Program (PAP,
PD13010) from KOPRI. This research was also partially sup-
ported by the Korea Ministry of Environment as a “Converging
Technology Project (201-101-007)” and as an “Eco-Innovation
Project (405-112-0382)”.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.biombioe.2013.09.009.
references
[1] Andersen O, Weinbach J-E. Residual animal fat and fish for
biodiesel production. Potentials in Norway. Biomass
Bioenergy 2010;34:1183e8.
[2] Ruan C-J, Xing W-H, Silva JATd. Potential of five plants
growing on unproductive agricultural lands as biodiesel
resources. Renew Energy 2011;41:191e9.
[3] Ahmad AL, Yasin NHM, Derek CJC, Lim JK. Microalgae as a
sustainable energy source for biodiesel production: a review.
Renew Sustain Energy Rev 2011;15:584e93.
[4] Lam MK, Lee KT. Microalgae biofuels: a critical review of
issues, problems and the way forward. Biotechnol Adv
2012;30:673e90.
[5] Chen CY, Yeh KL, Aisyah R, Lee DJ, Chang JS. Cultivation,
photobioreactor design and harvesting of microalgae for
biodiesel production: a critical review. Bioresour Technol
2011;102:71e81.
[6] Wahlen BD, Willis RM, Seefeldt LC. Biodiesel production by
simultaneous extraction and conversion of total lipids from
284 b i o m a s s a n d b i o e n e r g y 5 9 ( 2 0 1 3 ) 2 7 9 e2 8 4
microalgae, cyanobacteria, and wild mixed-cultures.
Bioresour Technol 2011;102:2724e30.
[7] Gong Y, Jiang M. Biodiesel production with microalgae as
feedstock: from strains to biodiesel. Biotechnol Lett
2011;33:1269e84.
[8] Eshaq FS, Ali MN, Mohd MK. Spirogyra biomass a renewable
source for biofuel (bioethanol) production. Int J Eng Sci
Technol 2011;2:7045e54.
[9] Beer LL, Boyd ES, Peters JW, Posewitz MC. Engineering algae
for biohydrogen and biofuel production. Curr Opin
Biotechnol 2009;20:264e71.
[10] Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M,
Seibert M, et al. Microalgal triacylglycerols as feedstocks for
biofuel production: perspectives and advances. Plant J
2008;54:621e39.
[11] Guarnieri MT, Nag A, Smolinski SL, Darzins A, Seibert M,
Pienkos PT. Examination of triacylglycerol biosynthetic
pathways via de novo transcriptomic and proteomic
analyses in an unsequenced microalga. PLoS One
2011;6:e25851.
[12] Bligh EG, Dyer WJ. A rapid method of total lipid extraction
and purification. Can J Biochem Physiol 1959;37:911e7.
[13] Demirbas A, Demirbas MF. Importance of algae oil as a
source of biodiesel. Energy Convers Manag 2011;52:163e70.
[14] Yang YH, Song E, Willemse J, Park SH, Kim WS, Kim EJ, et al.
A novel function of Streptomyces integration host factor
(sIHF) in the control of antibiotic production and sporulation
in Streptomyces coelicolor. Antonie Van Leeuwenhoek
2012;101:479e92.
[15] Ferraz TP, Fiuza MC, Dos Santos ML, Pontes De
Carvalho L, Soares NM. Comparison of six methods for
the extraction of lipids from serum in terms of
effectiveness and protein preservation. J Biochem Biophys
Methods 2004;58:187e93.
[16] Iverson SJ, Lang SL, Cooper MH. Comparison of the Bligh and
Dyer and Folch methods for total lipid determination in a
broad range of marine tissue. Lipids 2001;36:1283e7.
[17] Pruvost J, Van Vooren G, Le Gouic B, Couzinet-Mossion A,
Legrand J. Systematic investigation of biomass and lipid
productivity by microalgae in photobioreactors for biodiesel
application. Bioresour Technol 2011;102:150e8.
[18] Lee CM, Trevino B, Chaiyawat M. A simple and rapid solvent
extraction method for determining total lipids in fish tissue. J
AOAC Int 1996;79:487e92.
[19] Halim R, Gladman B, Danquah MK, Webley PA. Oil extraction
from microalgae for biodiesel production. Bioresour Technol
2011;102:178e85.
[20] Braunegg G, Sonnleitner B, Lafferty RM. A rapid gas
chromatographic method for the determination of poly-b-
hydroxybutyric acid in microbial biomass. Biotechnol Lett
1978;6:29e37.
[21] Indarti E, Majid MIA, Hashim R, Chong A. Direct FAME
synthesis for rapid total lipid analysis from fish oil and cod
liver oil. J Food Compost Anal 2003;18:161e70.
[22] Nowruzi K, Elkamel A, Scharer JM, Cossar D, Moo-Young M.
Development of a minimal defined medium for recombinant
human interleukin-3 production by Streptomyces lividans 66.
Biotechnol Bioeng 2008;99:214e22.
[23] Kamlet MJ, Doherty RM, Abboud JL, Abraham MH, Taft RW.
Linear solvation energy relationships: 36. Molecular
properties governing solubilities of organic nonelectrolytes
in water. J Pharm Sci 1986;75:338e49.
[24] Kamlet MJ, Doherty RM, Carr PW, Mackay D, Abraham MH,
Taft RW. Linear solvation energy relationships. 44. Parameter
estimation rules that allow accurate prediction of octanol/
water partition coefficients and other solubility and toxicity
properties of polychlorinated biphenyls and polycyclic
aromatic hydrocarbons. Environ Sci Technol 1988;22:503e9.