J Am Oil Chem Soc (2012) 89:189198

DOI 10.1007/s11746-011-1903-z

ORIGINAL PAPER

Optimization of an Analytical Procedure for Extraction

of Lipids from Microalgae
Eline Ryckebosch Koenraad Muylaert
Imogen Foubert

Received: 25 October 2010 / Revised: 17 June 2011 / Accepted: 1 July 2011 / Published online: 24 July 2011
 AOCS 2011

Abstract An optimized procedure for extraction of total

and non-polar lipids from microalgae is proposed. The
effects of solvent, pretreatment (lyophilization, inactivation
of lipases, and addition of antioxidants) and cell-disruption
(liquid nitrogen, sonication, and bead beating) on total lipid
content, lipid class, and fatty acid composition were
examined. Chloroformmethanol 1:1 was shown to be the
best solvent mixture for extraction of total lipids from
microalgae. When performing this extraction, lyophilized
algae can be used, no pretreatment with isopropanol to
inactivate the lipases is needed and addition of antioxidants
is not necessary. Furthermore, cell-disruption is not
essential, although in that case two extractions must be
performed in series to ensure that, irrespective of the
microalgal species, all lipids are extracted. Determination
of non-polar lipid content should be performed by separation of the total lipid extract on an SPE column.
Extraction using petroleum ether is only appropriate when
a bead beater is used for pretreatment.
Keywords Algae
Lipid extraction
Solvent mixture

Pretreatment
Cell-disruption
Fat content

E. Ryckebosch (&)
I. Foubert

K. U. Leuven Kulak, Research Unit Food and Lipids,
Department of Molecular and Microbial Systems,
Leuven Food and Nutrition Research Centre (LFoRCe),
Etienne Sabbelaan 53, 8500 Kortrijk, Belgium
e-mail: Eline.Ryckebosch@kuleuven-kortrijk.be
K. Muylaert
K. U. Leuven Kulak, Lab Aquatic Biology, Biology Department,
Etienne Sabbelaan 53, 8500 Kortrijk, Belgium

Introduction
Microalgae are sunlight-driven cell factories that convert
carbon dioxide into potential biofuels, foods, feed, and
high-value bioactives. Compared with traditional crops,
they have a high areal productivity, a relatively high oil
and protein content, and do not depend on arable land
and freshwater. Today, there is a strong interest in lipid
production using microalgae. Microalgae are theoretically
capable of producing much more lipids than any conventional crop and are, therefore, attractive as a potential
source of biodiesel. Because lipids from algae are often
rich in the long-chain omega-3 fatty acids EPA and
DHA, algae may also be a more sustainable source of
these fatty acids for use in food or feed compared with
fish oil.
Lipids have been recovered from microalgae via a
multitude of extraction methods described in the literature.
Because of the nature of microalgae, regular extraction
methods (used for example for food) may not be applicable. First of all, microalgae are single cells, each surrounded by an individual cell wall. Furthermore, they often
contain unusual lipid classes and fatty acids differing
from those in higher animal and plant organisms [1]. For
these reasons, it is necessary to have a profound look at
methods for extraction of lipids from microalgae.
The method of Folch et al. [2], which is mostly used for
the extraction of total lipids from microalgae, was originally optimized for isolation and purification of total lipids
from animal tissues. It uses chloroformmethanol 2:1 for
extraction of the lipids and water to remove non-lipid
substances from the extract. The method of Bligh and Dyer
[3], also often used for the extraction of total lipids from
microalgae, was originally optimized for extraction of
phospholipids from fish muscle. It uses chloroform

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methanol 1:2 followed by an extraction with chloroform.

Iverson et al. [4] compared the Bligh and Dyer and Folch
methods for determination of total lipids in marine tissue,
although marine algae were not investigated. Nevertheless,
their figures showed that the Bligh and Dyer method can
give rise to major underestimation for samples containing
more than 210% lipids. The unmodified Bligh and Dyer
extraction is thus not suitable for microalgae, because their
lipid content often exceeds 10%. Unfortunately, the
method is still used by several authors. Guckert et al. [5]
compared three solvent systems for extraction of lipids
from the green alga Chlorella. Soxhlet extraction (dichloromethanemethanol, 3 h reflux) recovered more non-polar
lipids than the other methods, but was equivalent to the
room temperature modified Bligh and Dyer extraction in
polar lipid recovery. The Soxhlet method, however, led to
significantly lower recovery of many polyunsaturated fatty
acids. The hexaneisopropanol method gave poor recovery
of all lipid classes. Lee et al. [6] also tested different solvent systems (chloroformmethanol 2:1, hexaneisopropanol 3:2, dichloroethaneethanol 1:1, and acetone
dichloromethane 1:1) for extraction of lipids from the
green alga Botryococcus braunii. The highest lipid content
was obtained with chloroformmethanol 2:1. Grima et al.
[7] compared the fatty acid profile of the biomass of
Isochrysis galbana, obtained by direct saponification, with
that of the lipids extracted by use of different solvent
systems followed by KOH-catalyzed saponification. The
solvent mixtures compared were chloroformmethanol
water 1:2:0.8, hexaneethanol 1:2.5, hexaneethanol 1:0.9,
butanol, ethanol, ethanolwater 1:1, and hexaneisopropanol 1:1.5. The highest yield was obtained with chloroformmethanolwater 1:2:0.8, by use of which 93.8% of
the fatty acids were extracted. Matyash et al. [8] proposed
the use of methyl-tert-butyl ether (MTBE) as an alternative
to the carcinogenic chloroform. Because of the low density
of MTBE, it forms the upper phase in the two-phase partitioning system, which makes it easier to collect. For
almost all major lipid classes this method results in
recoveries similar to or better than those of the golden
standards (Folch or Bligh and Dyer recipes). They tested
their method on E. coli, mouse brain, Caenorhabditis
elegans, and human blood plasma. The non-polar lipid
class was not considered.
For lipid extraction from microalgae, different pretreatment methods have also been used, the most important ones being lyophilization, inactivation of lipases, and
addition of antioxidants. Lyophilization increases the
surface area of the sample, leading to a better lipid
extraction [9]. Furthermore, lyophilized preparations do
not require special storage conditions and are easily
rehydrated [10]. Finally, lyophilized microalgae are easily
weighed and handled. Lipases potentially present in

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J Am Oil Chem Soc (2012) 89:189198

samples of plant or animal origin, which could cause

lipolysis, can be denatured by plunging the sample into
boiling water, isopropanol, or dilute acetic acid for short
periods [11]. Heat treatment can also deactivate these
enzymes [12]. When the microalgae are lyophilized,
removal of almost all water may, however, enable omission of this step, because lipases require a minimum
amount of water for full expression of their enzymatic
activity [13]. Precaution should also be taken to eliminate
oxidation of lipids during extraction, especially when the
sample contains polyunsaturated fatty acids. Use of an
antioxidant or antioxidant system may therefore prove
beneficial [12]. Natural tissue antioxidants, for example
tocopherols, afford some protection to lipid extracts, but it
is usually advisable to add further synthetic antioxidants,
for example butylhydroxytoluene (BHT), to storage solvents [11]. Algae are however, known to contain a large
amount of natural antioxidants [14], leading to a large
antioxidant capacity of microalgal extracts (our own
preliminary results). This may enable omission of addition of a synthetic antioxidant. Because microalgae consist of individual cells surrounded by thick cell walls,
appropriate cell-disruption before extraction may be necessary. Until now, few investigations have been performed on the effect of cell-disruption on total lipid
extraction. The lipid content of Botryococcus braunii was
determined by use of the solvent system chloroform
methanol 2:1 and six different cell-disruption methods
(none, sonication, homogenization, French press, beadbeating, lyophilization) [6]. The highest lipid content was
obtained by use of bead beating. Comparable research
was performed on different microalgae using the solvent
system chloroformmethanol 1:1 [15]. Large differences
in extraction efficiency were obtained, depending on
species (Botryococcus sp., Chlorella vulgaris, and
Scenedesmus sp.) and cell-disruption method (none,
autoclaving, bead-beating, microwaves, sonication, osmotic
shock).
The objective of this research was to optimize an analytical procedure for total lipid extraction from microalgae.
The focus was on solvent mixture, pretreatment, and celldisruption method. Not only the amount of extracted lipids
was considered, but also the potential effect of the
extraction procedure on degradation of the lipids, the lipid
class, and fatty acid composition. Because the extraction
procedure should be valid for all microalgae, the effect of
cell-disruption was studied using different microalgae
belonging to different taxonomic classes and with a different cell wall structure.
Because non-polar lipids are the most important in, e.g.,
the biodiesel industry, the possibility of using a direct
extraction technique to determine non-polar lipid content
was also investigated.

J Am Oil Chem Soc (2012) 89:189198

Materials
HPLC-grade chloroform, methanol, dichloromethane, and
acetone were purchased from Labscan (Boom, Meppel, The
Netherlands). GC-grade hexane, petroleum ether p.a. (bp
3050 C), diethyl ether p.a., acetic acid p.a., ethyl acetate
p.a., isopropanol p.a., ethanol p.a., sulfuric acid p.a., TBHQ,
anhydrous sodium sulfate, potassium chloride, rhodamine 6G,
sodium chloride, diethylamine, N,N-diisopropylethylamine,
ethanol p.a., and methyl-tert-butyl ether p.a. were purchased
from SigmaAldrich (Bornem, Belgium). Arachidic acid
(C20:0) was purchased from Nu-check Prep, (Minnesota,
USA). Bis(2-methoxyethyl)aminosulfur trifluoride was purchased from TCI Europe (Zwijndrecht, Belgium).

Experimental Procedures
Microalgae Cultivation and Harvest
Phaeodactylum tricornutum Pt1 8.6 was obtained from
CCMP2561 in the ProvasoliGuillard National Center for
Culture of Marine Phytoplankton. The cells were grown in
batch culture in 30-L Plexiglas columns, containing 25 L
WC medium [16] to which 30 g/L artificial sea salt
(Homarsel, Zoutman Industries, Roeselare, Belgium) was
added. The culture was continuously illuminated with two
fluorescent lamps (Sylvania Gro-Lux, 36 W) and aerated
with 0.3 lm-filtered air. Nannochloropsis salina SAG
40.85, Chlorella vulgaris SAG 211-11b, and Arthrospira
platensis SAG 85.79 were obtained from Sammlung von
Algenkulturen Goettingen (SAG), Germany. Scenedesmus
obliquus CCAP 276/3A was obtained from Culture Collection of Algae and Protozoa, UK. N. salina and C. vulgaris were grown in batch culture in 2-L glass bottles at
21.9 0.5 C in WC medium [16]. For N. salina 30 g/L
artificial sea salt was added. A. platensis was grown in 2 L
batch cultures at 21.9 0.5 C in Spirulina medium
(SAG, Germany). The cultures were illuminated 12 h/day
with fluorescent lamps (Philips, Master TL5 HE, 35 W/
840) and aerated continuously. Growth was monitored by
measuring the optical density at 550 nm. The microalgae
were harvested by centrifugation in the early stationary
phase (P. tricornutumday 14, N. salina, S. obliquusday
15, C. vulgarisday 9, A. platensisday 13). The pellet
obtained was either used directly (fresh algae) or lyophilized for further analysis.
Experimental Setup: Effect of Solvent Mixture
and Ratio

191

Therefore, the following solvents and mixtures, often used

in literature, were tested on C. vulgaris:

Total lipid content extracted with the different solvent

systems was compared.
Experimental Setup: Effect of Pretreatments
First, it was investigated whether lyophilization causes any
changes to the lipid composition of P. tricornutum. Therefore, the total lipid content, the lipid class composition, and
the total fatty acid composition of fresh and lyophilized algae
were compared. Lyophilization was performed on the microalgae pellet, stored at -80 C. Second, we tested whether
endogenic lipases cause lipolysis and their deactivation is
thus necessary before extraction. The effect of addition of
isopropanol (to deactivate the lipases [11]) to C. vulgaris on
the total lipid content and the lipid class composition was
investigated. Therefore, 1 mL isopropanol was added to
80100 mg microalgae (dry weight, DW) before the total
lipid extraction was performed, and the result was compared
with that from a sample with no isopropanol added. The
effect of addition of isopropanol was also determined for
P. tricornutum. Therefore, fresh and lyophilized biomass
were stored at 4 C with or without addition of isopropanol.
The free fatty acid (FFA) content of the microalgal biomass
was determined on days 0, 7, and 17. Finally, to examine
whether significant oxidation occurred during extraction, we
examined the effect of addition of the synthetic antioxidant
TBHQ on the total fatty acid composition of the alga
C. vulgaris to which eicosapentaenoic acid (EPA, C20:5n-3)
had been added. TBHQ was added at a concentration of
500 ppm to the methanol used during extraction.
Experimental Setup: Cell-Disruption Methods
We investigated whether algal cells need to be disrupted to
achieve a complete extraction and which method is best.
Therefore, three methods, often found in literature, were
tested on P. tricornutum:

We investigated which solvent mixture and which ratio

was best for extraction of lipids from microalgae.

Chloroformmethanol 1:1
Chloroformmethanol 2:1
Dichloromethaneethanol 1:1
Hexaneisopropanol 3:2
Acetone
Diethyl ether
Methyl-tert-butyl ethermethanol 10:3

Lyophilized microalgae were frozen in liquid nitrogen

and then thawed, three times.
Lyophilized microalgae were suspended in 0.5 mL
methanol and bead beaten twice (Qiagen Tissuelyser,
Venlo, The Netherlands) for 30 s at 60 Hz.

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J Am Oil Chem Soc (2012) 89:189198

Lyophilized microalgae were sonicated (sonication

bath EIA USCP 100SB) for 15 min after addition of
4 mL methanol, 2 mL chloroform, and 0.4 mL water.

Because an optimum procedure should be valid for all

microalgae, the effect of cell-disruption was studied using
different microalgae. The following microalgae were used,
because they belong to different taxonomic classes and
have a cell wall structure different from that of P. tricornutum (taxonomic class: Bacillariophyceaecell wall
structure: organic cell wall with up to 10 silica bands
embedded in its surface in the girdle region [17]):
N. salina (taxonomic class: Eustigmatophyceaecell
wall structure: algaenans containing trilaminar outer
cell wall (TLS) [18]);
S. obliquus (taxonomic class: Chlorophyceaecell
wall structure: glucose (major), galactose, mannose
rigid cell wall, glucose wall matrix [19]);
C. vulgaris (taxonomic class: Trebouxiophyceaecell
wall structure: no TLS [20], glucosamine rigid cell
wall, no fucose matrix [21]); and

A. platensis (taxonomic class: Cyanophyceaecell

wall structure: four-layered cell wall with a fibrillar
b-1,2-glucan inner layer, a murein one, another fibrillar
layer and a structured outer layer [22, 23]).
For time-saving reasons and because the different celldisruption methods resulted in no significant differences for
the lipid content of P. tricornutum (Section Effect of CellDistruption Method on Total Lipid Extraction) for the
remaining algae all cell-disruption methods were performed in series and compared with lyophilized algae
without further cell-disruption.
Analysis of Total Lipid ContentTesting Solvents
and Mixtures
The different solvents and mixtures described above were
tested. Briefly, the tested solvent or mixture (6 mL) was
added to 100 mg lyophilized microalgae and the tube was
vortex mixed for 30 s. The solvent or mixture (2 mL) and
water (2 mL) were then added and the tube was vortex
mixed again and subsequently centrifuged at 2,000 rpm
for 10 min. The aqueous layer was removed and the
solvent layer was transferred into a clear tube. The
remaining solids were re-extracted with 4 mL solvent or
mixture. The combined solvent layers were passed
through a layer of anhydrous sodium sulfate using
Whatman No. 1 filter paper in a funnel. The solvent
was removed by rotary evaporation at 40 C after which
the lipid content was determined gravimetrically. The
extraction was performed in quadruplicate. The resulting
percentage of extracted lipids is the sum of three
extractions performed in series.

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Analysis of Total Lipid ContentTesting

Pretreatment and Cell-Disruption
The different pretreatments and cell-disruption methods
were incorporated into the general chloroformmethanol
1:1 extraction method, which was selected as optimum
(section Effect of Solvent). Briefly, 400 mg fresh microalgae paste or 100 mg lyophilized microalgae was mixed
with 4 mL methanol. Chloroform (2 mL) and water
(0.4 mL) were added and the mixture was vortex mixed for
30 s. Chloroform (2 mL) and water (2 mL) were added and
the mixture was vortex mixed again. The tubes were centrifuged at 2,000 rpm for 10 min. The upper layer was
removed and the lower layer was transferred into a clear
tube. The remaining solids were re-extracted with 4 mL
chloroformmethanol 1:1. The combined solvent layers
were passed through a layer of anhydrous sodium sulfate
using Whatman No. 1 filter paper in a funnel. The solvent
was removed by rotary evaporation at 40 C after which
the lipid content was determined gravimetrically. The
extraction was performed in quadruplicate. Taking the high
lipid content of algae into account, the extraction was
repeated four times in series on the same algae, so possible
saturation of the solvent mixture with lipids could be
detected. Lipid content was determined separately for each
extraction.
Analysis of Non-polar Lipid Content
Non-polar lipids were extracted with petroleum ether [11,
12]. The extraction method was similar to the total lipid
extraction method, with replacement of the methanol and
chloroform with petroleum ether. No water was added,
unless otherwise stated. Considering the high non-polar
lipid content of algae, the extraction was repeated five
times in series on the same algae, so possible saturation of
the solvent with lipids could be detected. Lipid content was
determined separately for each extraction. The extractions
were performed in quadruplicate.
Analysis of Lipid Class Composition
To determine the effects of the pretreatments on lipid class
composition, thin-layer chromatography (TLC) as described by Christie [11] was performed. Briefly, lipids were
separated by spotting the total extract on a silica gel 60 F254
plate (Merck, Darmstadt, Germany). Non-polar lipids were
separated by use of hexanediethyl etheracetic acid
80:20:2 as mobile phase and polar lipids were separated by
use of ethyl acetateisopropanolchloroformmethanol
0.25% KCl in water 25:25:25:10:9 as mobile phase. Spots
were visualized by use of rhodamine 6G solution in ethanol
(0.5 g/L). Tentative identification of the lipid classes was

J Am Oil Chem Soc (2012) 89:189198

performed using standards that were spotted next to the

samples.
To determine the effects of cell-disruption methods on
lipid class composition, silica solid phase extraction (SPE)
(Refs. [11, 24], with slight adjustments) was performed.
Briefly, the SPE column was conditioned with 10 mL
chloroform. Then, approximately 10 mg lipids in 100 lL
chloroform was applied to the column. Elution with 10 mL
chloroform yielded the non-polar lipids (NL), 10 mL acetone gave the glycolipid fraction (GL), and 10 mL methanol yielded the phospholipids (PL). Each class was
determined gravimetrically. Quantitative determination of
lipid class composition was performed in triplicate.
Analysis of Fatty Acid Composition
To determine the effects of the pretreatments and the celldisruption methods on fatty acid composition, methylation
was performed according to Ref. [11], with slight adjustments. Briefly, 5 mg lipid sample was dissolved in 1 mL
toluene and 2 mL 1% sulfuric acid in methanol was added.
The mixture was left overnight in a stoppered tube at
50 C. Aqueous sodium chloride solution (5%, 5 mL) was
then added and the required methyl esters were extracted
with 3 mL hexane. Necessary dilutions were made before
injection for GC analysis.
The fatty acid methyl esters (FAMEs) obtained were
separated by gas chromatography with cold on-column
injection and flame ionization detection (FID) (Carlo Erba
Instruments, Interscience, Louvain-la-Neuve, Belgium;
GC8000 series instrument). An EC Wax column of length
30 m, ID 0.32 mm, film 0.25 lm (Grace) was used with
the following timetemperature program: 100180 C at
10 /min, 180215 C at 2 /min, 215 C (44 min). Peak
areas were quantified with Chromcard for Windows software (Interscience, Louvain-la-Neuve, Belgium). Standards (Nu-check, Elysian, USA) containing a total of 35
different FAMEs were analyzed for provisional peak
identification. Peak identification was confirmed by use of
GCMS (Trace GC, Thermo Scientific) using an Rxi-5 Sil
MS column of length 30 m, ID 0.25 mm, film 0.25 lm
(Restek, Belgium).
Analysis of FFA Content
The FFA content was determined by selective formation
of diethyl amide derivatives according to Kangani et al.
[25]. To do this, 0.45 mg arachidic acid (C20:0) in
chloroform (150 lL) was added as internal standard
before extraction. The extracted lipids were then dissolved
in 750 lL dichloromethane and transferred into a screwcapped tube. After addition of 10 lL diisopropylethylamine and 30 lL diethylamine, the solution was cooled to

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0 C. Bis(2-methoxyethyl)aminosulfur trifluoride (10 lL)

was added dropwise and the solution was vortex mixed
for 5 s. The solution was kept at 0 C for 5 min, subsequently warmed to room temperature, and kept there for
15 min. Water (2 mL) and hexane (4 mL) were added and
the tubes were vortex mixed for 1 min. After centrifugation for 10 min at 2,000 rpm, the organic layer was collected and transferred into a vial for GC analysis. A blank
analysis was performed by use of the same method, but
without addition of bis(2-methoxyethyl)aminosulfur
trifluoride.
The diethyl amide derivatives were analyzed by gas
chromatography with cold on-column injection and FID
(GC8000; Carlo Erba Instruments). An EC Wax column of
length 30 m, ID 0.32 mm, film 0.25 lm (Grace) was used
with the following temperature program: 100160 C at
10 /min, 160240 C at 2 /min, 240 C (7 min). Peak
areas were quantified with Chromcard for Windows software. The areas of the peaks that were not present in the
blank were summed and compared with the area of the
internal standard (C20:0).
Statistics
Results were statistically evaluated by use of one-way
analysis of variance (ANOVA) and a post-hoc Tukeys test
with a = 0.05 (Sigmaplot 11, Systat Software).

Results and Discussion

Effect of Solvent
The extracted total lipid percentage was highly dependent
on the solvent or mixture used (Fig. 1). Solvent mixtures
containing a polar and a non-polar solvent extracted a
greater amount of lipids. In these cases, the polar solvent
releases the lipids from their proteinlipid complexes, and
the lipids subsequently dissolve in the non-polar solvent
[26, 27]. Depending on the extent of release by the polar
solvent and the nature of the non-polar solvent, different
extracted percentages are obtained [28]. Chloroform
methanol 1:1 gave the highest lipid yield. Chloroform
methanol 2:1 extracted only 76.5% of the lipids extracted
with the 1:1 mixture. This is in contrast with the results of
Lee et al. [29], who stated that solvent ratio has no effect
on lipid recovery from fish when a high solvent-to-sample
ratio is used. Only the non-polar lipids are dissolved in the
relative non-polar diethyl ether, explaining the low percentage extracted when using this solvent [28]. Given its
highest lipid yield, chloroformmethanol 1:1 was thus used
to test the effects of pretreatments and cell-disruption
method.

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Table 2 Fatty acid composition as a percentage of total fatty acids
extracted (mean SD; n = 2) from lyophilized C. vulgaris to which
EPA was added, with (With TBHQ) or without (Without TBHQ)
addition of TBHQ

C16:0

Fig. 1 Effect of solvent mixture and/or ratio on the total amount of

lipids extracted (mean SD; n = 3; total of three extractions) from
C. vulgaris. Solvent systems: 1 chloroformmethanol 1:1, 2 dichloromethaneethanol 1:1, 3 hexaneisopropanol 3:2, 4 chloroform
methanol 2:1, 5 acetone, 6 diethyl ether, 7 methyl-tert-butyl ether
methanol 10:3. a, b show significance of difference

Table 1 Fatty acid composition as a percentage of total fatty acids

extracted (mean SD; n = 2) from fresh (No Lyo) and lyophilized
(Lyo) P. tricornutum
No Lyo

Lyo

C14:0

7.3 0.4

7.7 0.2

C16:0

26.9 0.1

27.9 0.4

C16:1
C16:3

48.2 0.3
3.2 0.1

48.7 0.1
3.0 0.1

C18:1

3.4 0.1

3.4 0.1

C20:5

11.0 0.2

9.3 0.5

Effect of Pretreatment
First, the effect of lyophilization was tested. Total lipids
extracted from fresh (24 2% of DW) and lyophilized
algae (26 1% of DW) were not significantly different.
Thus, in contrast with what Nielsen et al. [9] stated,
lyophilization does not lead to better lipid extraction. The
same lipid classes were detected with similar intensities by
use of TLC. The fatty acid composition, also, was not
significantly different (Table 1). This indicates that
lyophilization is a pretreatment that can be used without
altering the lipid composition. This is convenient, because
it makes the microalgae easier to handle.
Next, we tested whether endogenic lipases present cause
lipolysis and whether their deactivation is therefore necessary before extraction to eliminate any effect on lipid
content and composition. To do this, the effect of addition
of isopropanol (known to deactivate the lipases) on
the total lipid content and lipid class composition of

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Without TBHQ

With TBHQ

17.7 0.1

17.7 0.4

C16:1

9.8 0.5

9.4 0.3

C16:2

2.5 0.1

2.6 0.02

C16:3

13.5 0.8

13.2 0.3

C18:1

11.5 1.1

11.6 0.4

C18:2

5.6 0.2

5.4 0.2

C18:3n-3

30.2 1.5

28.9 0.8

C20:5n-3

9.2 1.9

11.2 1.8

C. vulgaris was investigated. Total extracted lipids of

lyophilized algae (28 1% of DW) and lyophilized algae
treated with isopropanol (29.6 0.9% of DW) were not
significantly different. The same lipid classes were detected
with similar intensities by use of TLC. No spot corresponding to the free fatty acid standard was detected.
Testing of P. tricornutum showed that lipases were active
in the fresh microalgal biomass that was not treated with
isopropanol: in this case the amount of FFA increased over
time. Lyophilization or treatment with isopropanol led to a
much smaller FFA content, suggesting deactivation of the
lipases by both techniques. The tests on both microalgal
species thus indicated that addition of isopropanol to
deactivate endogenic lipases is not necessary when using
lyophilized biomass. Most probably, the lipases have
already (partly) been deactivated by elimination of the
water during lyophilization, because lipases require a
minimum amount of water for full expression of their
enzymatic activity [13]. Furthermore, the microalgae were
extracted rapidly after harvest and lyophilization, leaving
the lipases limited time to work.
To examine whether significant oxidation occurs during
extraction, the effect of addition of the synthetic antioxidant TBHQ on the total fatty acid composition of the alga
C. vulgaris to which eicosapentaenoic acid (EPA, C20:
5n-3) was added, was examined. Although oxidation
of exogenous EPA may not be the same as oxidation of
endogenous complex lipids, more rapid oxidation of
exogenous added fatty acids is expected. Performing the
extraction procedure with or without addition of TBHQ
yielded no difference in the fatty acid profile of the alga nor
in the amount of EPA (Table 2). Therefore, because no
oxidation of exogenous EPA was observed, it is supposed
that, when using the same procedure, no oxidation of
endogenous fatty acids will take place either. Addition of
an antioxidant during lipid analysis is thus not necessary.
This can be explained by the large antioxidant capacity of

J Am Oil Chem Soc (2012) 89:189198

Fig. 2 Effect of cell-disruption method on total lipids extracted from,

and separate extractions of, P. tricornutum (mean SD; n = 4).
Cell-disruption methods: 1 none (fresh algae), 2 lyophilization,
3 lyophilization and sonication, 4 lyophilization and liquid nitrogen,
5 lyophilization and bead beating. a, b show significant differences
between amounts of total lipids extracted. x, y show significant
differences between amounts of lipids extracted during first extraction

microalgal extracts (our own preliminary results), confirming that the large amount of natural antioxidants, for
example tocopherols, carotenoids, polyphenols, , known
to be present in microalgae [14], protect the lipid extracts.
However, these results must be regarded with caution,
because for this test the algae were extracted rapidly
(\1 week) after harvest and lyophilization. When storage
time is significant, other results can be expected.
Effect of Cell-Disruption Method on Total Lipid
Extraction
The amount of total lipids extracted from P. tricornutum
(Fig. 2) was used as an indication of the efficiency of the
cell-disruption method used. No significant differences
could be detected between the different cell-disruption
methods. This indicates that the cell wall is penetrated or
dissolved by the solvents used, so it does not need to be
destroyed for optimum extraction. However, when looking
at the first extraction only, bead beating led to more efficient extraction of lipid, although the difference was rather
small (\7.5%). This result is consistent with the result of
Lee et al. [6], who extracted once only. During the second
extraction, a smaller amount of lipids is extracted when
bead beating is used as a cell-disruption method. It is most
likely that the same result would have been obtained using
microwaves or autoclaving, when a second extraction
would have been performed by Lee et al. [15]. Irrespective
of the cell-disruption method, more than 92% of the lipids
is extracted during the first extraction. Two extractions in
series extract more than 96% of the total lipids and is thus

195

Fig. 3 Lipid class composition of the first and second total lipid
extract from P. tricurnutum (mean SD; n = 4). a, b show significant differences. NL non-polar lipids, GL glycolipids, PL
phospholipids

sufficient, because more extractions make the procedure

too time-consuming. Ideally, one extraction only is sufficient to determine the complete lipid profile of the microalgae. This is, however, only possible if there is no
selective extraction of specific lipid classes or fatty acids
during the first or second extraction. To check this,
the lipid class composition of the first and second extracts
was determined (Fig. 3). No significant difference was
observed, meaning that no lipid class was favoured during
extraction with chloroformmethanol 1:1. Subsequently,
the fatty acid composition of the lipid classes obtained was
determined (Table 3). The fatty acid composition of the
non-polar lipids obtained during the first and second
extraction was not significantly different. The glycolipids
and phospholipids extracted during the first and second
extractions did not have the same fatty acid composition,
however. Some fatty acids were not present in the same
amounts in both extracts. Apparently, there is some
selectivity toward fatty acids during total lipid extraction.
However, there seems to be no trend toward (un)saturation
or chain length of the fatty acids extracted first. The differences are thus probably because of selectivity in lipid
class or molecular species extracted. Possibly these differences are not visible from Fig. 3, because of the high
variability or because the differences are at the molecular
species level (e.g., type of phospholipid or glycolipid);
these were not identified by the technique used. Taking
into account the biological variability and the time effort
necessary for a second extraction, the differences are
probably too small to conclude that a second extraction
must be performed when analyzing the fatty acid
composition.
To test whether this procedure is applicable to different
microalgae from different taxonomic classes and with
different cell wall structure and composition, the total lipid

123

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J Am Oil Chem Soc (2012) 89:189198

Table 3 Fatty acid composition as a percentage of total fatty acids (mean SD; n = 2) for the lipid classes from the first (Ex1) and second
(Ex2) total lipid extractions of P. tricornutum
Non-polar lipids
Ex1

Glycolipids
Ex2

Ex1

Phospholipids
Ex2

Ex1

Ex2

C14:0

5.3 0.4

5.3 0.00

1.2 0.2

1.2 1.2

14.4 3.6

9.6 1.1

C15:0

tr

tr

2.1 2.1

1.4 0.7

C16:0
C16:1b

29.9 1.2
46.5 0.3

30.7 0.1
45.9 0.2

9.0 1.7
23.3 3.4

18.1 2.1a
16.1 2.5

25.2 5.3
23.3 2.1

20.4 0.4
25.2 6.4

C16:2b

tr

tr

3.4 0.8

3.3 0.3

C16:3

tr

tr

22.5 2.8

19.0 2.1

tr

2.2 1.8

C16:4b

1.7 2.0

2.2 0.1

C18:0

tr

tr

1.6 1.1

6.5 0.4a

tr

1.8 0.9

C18:1b

7.6 0.4

7.8 0.1

1.6 0.2

3.6 0.2a

10.3 0.3

8.1 0.00a

C18:2

tr

tr

2.5 0.5

1.8 0.3

C18:3n-6

tr

tr

tr

tr

C18:3n-3

tr

tr

1.3 0.1

1.3 0.1

tr

tr

C20:4n-6

tr

tr

tr

tr

tr

tr

C20:3n-3

tr

tr

C20:5n-3

6.1 0.9

6.1 0.1

34.3 0.5

28.4 1.1a

12.3 7.8

18.0 6.9

C22:2b

tr

tr

C24:0

4.9 0.1

6.5 0.6

C22:6n-3

tr

tr

1.8 0.3

3.2 2.0

Indicates significantly different data

Indicates that the position of the double bond(s) could not be determined or a mixture of different isomers was detected

tr indicates that the component is \1%. indicates that the component was not detected

content of different microalgae was determined with or

without use of cell-disruption. The results are summarized
in Table 4. No significant difference was observed between
total lipid content with or without cell-disruption from
N. salina, C. vulgaris, and A. platensis when two extractions
in series were taken into account. As for P. tricornutum,
this indicates that the cell wall is penetrated or dissolved by
the solvents used. For N. salina, the first extraction is
slightly more efficient after extra cell-disruption whereas
for C. vulgaris cell-disruption results in a much greater
amount of lipids extracted during the first extraction. This
indicates that a different cell wall structure gives rise to
different infiltration by the solvents. C. vulgaris also forms
many extracellular polysaccharides [30], which possibly
have a large effect on infiltration of the solvents. For
A. platensis, the first extraction was slightly less efficient
when cell-disruption was performed. This can be explained
by the formation of a thick, sticky algaechloroform layer
after cell-disruption. Even after addition of extra chloroform, it was not possible to completely separate the chloroformlipid layer from the algae, leading to incomplete
recovery of the lipids. During the second extraction, this
phenomenon was not observed, leading to complete
recovery of the lipids. Thus, the same total (after two

123

extractions) amount of lipids was extracted as when no

cell-disruption was performed. For S. obliquus a small
difference was observed after two total lipid extractions. In
contrast with what might be expected, use of cell-disruption yielded a lower total lipid content. Most probably,
losses occurred during multiple manipulation of the algae
during the cell-disruption steps. The first extraction was
slightly more efficient after performing cell-disruption.
In conclusion it can be said that for the different microalgae, the first extraction yields a different percentage of
total lipids. Especially for C. vulgaris a second extraction
step is essential for accurate determination of lipid content
and comparison with other microalgae. To be able to
expand the method to microalgae in general, two extraction
steps must therefore be incorporated in the final procedure.
Effect of Cell-Disruption Method on Non-polar Lipid
Extraction
The amount of non-polar lipids extracted from P. tricornutum was used as an indication of the efficiency of the celldisruption method used during non-polar lipid extraction
(Fig. 4). Very large differences were detected between the
different cell-disruption methods. Bead beating was most

J Am Oil Chem Soc (2012) 89:189198

197

Table 4 Lipids (as a percentage of dry microalgal weight) extracted during the first (Ex1) and second (Ex2) total lipid extraction (mean SD;
n = 3)
Ex1
Lyo

Ex2
Lyo ? CD

Lyo

Total lipids
Lyo ? CD

Lyo

% 1st Ex
Lyo ? CD

Lyo

Lyo ? CD

N. salina

29.3 0.6

32.2 1.0 *

5.1 0.1

2.9 0.3 *

34.4 0.6

35.1 0.8

85.2 0.2

91.8 1.0 *

S. obliquus

23.6 0.4

26.0 0.4 *

6.1 0.4

2.7 0.01 *

29.7 0.1

28.7 0.4 *

79.6 1.4

90.5 0,2 *

C. vulgaris
A. platensis

10.2 0.4
11.3 1.1

18.1 1.7 *
8.9 0.6 *

9.6 2.2
1.6 0.7

3.6 0.4 *
2.7 0.8

19.9 1.6
13.2 2.1

21.7 2.0
11.6 0.8

51.8 7.1
87.8 3.2

83.3 0,5 *
76.6 5.9

Total lipids are the sum of Ex1 and Ex2. % 1st Ex is the percentage of the total lipids extracted during the first extraction. Lyo shows the results
for the lyophilized algae, Lyo ? CD shows the results for the lyophilized algae treated with liquid nitrogen, bead beater, and sonication
*Indicates significantly different result

Fig. 4 Effect of cell-disruption method on non-polar lipids extracted

from, and the separate extractions of, P. tricornutum (mean SD;
n = 4). Cell-disruption methods: 1 water (fresh algae), 2 none (fresh
algae), 3 lyophilization, 4 lyophilization and sonication, 5 lyophilization and liquid nitrogen, 6 lyophilization and bead beating.
a, b show significant differences between the total amounts of
extracted non-polar lipids. v, w show significant differences between
amounts of non-polar lipids obtained with one extraction

Conclusion

efficient. This indicates that petroleum ether cannot sufficiently penetrate the cell wall or dissolve components in the
cell wall, making it impossible to extract all the non-polar
lipids. Bead beating is the only technique that damages the
cell wall sufficiently to recover all the non-polar lipids.
When using bead beating, the first extraction released 92%
of the non-polar lipid content. The lipid class composition
of the total non-polar lipids extracted from lyophilized
bead-beaten algae was determined by use of SPE (Fig. 5). It
was observed that co-extraction of polar lipids occurs: 8.2%
of the extracted lipids were not non-polar lipids. Because
non-polar lipid extraction only can be performed by use of a
bead beater, it is very time consuming. Furthermore, it does
not extract the non-polar lipids only. Therefore, it is easier,
less time consuming, and more accurate to determine nonpolar lipids by separation of the total extract into lipid
classes by use of an SPE column.

An optimized analytical procedure was developed for

total lipid extraction from microalgae. The amount of
lipids extracted from microalgal biomass is highly
dependent on the solvent or mixture used. Chloroform
methanol 1:1 gave the highest lipid content and is thus
the preferred solvent mixture for determination of total
lipids. When performing this analysis, lyophilized algae
can be used, which makes weighing easier. There is no
need for pretreatment with isopropanol to inactivate the
lipases or for addition of antioxidants. No cell-disruption method is necessary. In general, two extractions
must be performed in series. For some microalgae
however, one extraction may be sufficient. Non-polar
lipids should, preferably, be determined by separation of
the total lipid extract on an SPE column. If extracting
with petroleum ether, bead beating is necessary as a
cell-disruption step.

Fig. 5 Lipid class composition of the lipids extracted with petroleum

ether from lyophilized bead-beaten P. tricornutum (mean SD;
n = 4)

123

198
Acknowledgments The research presented in this paper was
financially supported by the Institute for the Promotion of Innovation
by Science and TechnologyStrategic Basic Research (IWT-SBO)
project Sunlight and K.U.Leuven Kulak. We acknowledge IS-X
(Interscience, Louvain-la-Neuve, Belgium) for the use of the GCMS
at their demolab.

J Am Oil Chem Soc (2012) 89:189198

15.

16.
17.

References
1. Guschina IA, Harwood JL (2006) Lipids and lipid metabolism in
eukaryotic algae. Prog Lipid Res 45:160186
2. Folch J, Lees M, Stanley GHS (1957) A simple method for the
isolation and purification of total lipides from animal tissues.
J Biol Chem 226:497509
3. Bligh EG, Dyer WJ (1959) A rapid method of total lipid
extraction and purification. Can J Biochem Physiol 37:911917
4. Iverson SJ, Lang SLC, Cooper MH (2001) Comparison of the
Bligh and Dyer and Folch methods for total lipid determination in
a broad range of marine tissue. Lipids 36:12831287
5. Guckert JB, Cooksey KE, Jackson LL (1988) Lipid solvent systems are not equivalent for analysis of lipid classes in the micro
eukaryotic green alga, Chlorella. J Microbiol Methods 8:139149
6. Lee SJ, Yoon B-D, Oh H-M (1998) Rapid method for the
determination of lipid from the green alga Botryococcus braunii.
Biotechnol Tech 12:553556
7. Grima EM, Medina AR, Gimenez AG, Perez JAS, Camacho FG,
Sanchez JLG (1994) Comparison between extraction of lipids and
fatty acids from microalgal biomass. JAOCS 71:955959
8. Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A,
Schwudke D (2008) Lipid extraction by methyl-tert-butyl ether
for high-throughput lipidomics. J Lipid Res 49:11371146
9. Nielsen SS (2003) Food Analysis, 3rd edn. Kluwer Academic/
Plenum, New York
10. Ausborn M, Nuhn P, Schreier H (1992) Stabilization of liposomes by freezethawand lyophilization techniques: problems
and opportunities. Eur J Pharm Biopharm 38:133139
11. Christie WW (2003) Lipid Analysis: isolation, separation, identification and structural analysis of lipids, 3rd edn. The Oily
Press, Bridgewater
12. Wrolstad RE, Acree TE, Decker EA, Penner MH, Reid DS,
Schwartz SJ, Shoemaker CF, Smith DM, Sporns P (2005)
Handbook of food analytical chemistry. Wiley, Hoboken
13. Han D, Walde P, Luisi PL (1990) Dependence of lipase activity
on water content and enzyme concentration in reverse micelles.
Biocat Biotrans 4:153161
14. Li H-B, Cheng K-W, Wong C-C, Fan K-W, Chen F, Jiang Y
(2007) Evaluation of antioxidant capacity and total phenolic

123

18.

19.
20.

21.
22.

23.

24.

25.

26.

27.
28.

29.

30.

content of different fractions of selected microalgae. Food Chem

102:771776
Lee J-Y, Yoo C, Jun S-Y, Ahn C-Y, Oh H-M (2010) Comparison
of several methods for effective lipid extraction from microalgae.
Bioresour Technol 101:S75S77
Guillard RRL, Lorenzen CJ (1972) Yellow-green algae with
chlorophyllide c. J Phycol 8:1014
Borowitzka MA, Volcani BE (1978) The polymorphic diatom
Phaeodactylum tricornutum: ultrastructure of its morphotypes.
J Phycol 14:1021
Grossi V, Blokker P, Damste JSS (2001) Anaerobic biodegradation of lipids of the marine microalga Nannochloropsis salina.
Org Geochem 32:795808
Takeda H (1995) Cell wall sugars of some Scenedesmus species.
Phytochem 42:673675
Corre G, Templier J, Largeau C, Rousseau B, Berkaloff C (1996)
Influence of cell wall composition on the resistance of two
Chlorella species (Chlorophyta) to detergents. J Phycol
32:584590
Takeda H (1991) Sugar composition of the cell wall and the
taxonomy of Chlorella (Chlorophyceae). J Pycol 27:224232
van Eykelenburg C (1977) On the morphology and ultrastructure
of the cell wall of Spirulina platensis. Antonie van Leeuwenhoek
43:8999
van Eykelenburg C (1978) A glucan from the cell wall of the
cyanobacterium Spirulina platensis. Antonie van Leeuwenhoek
44:321327
Chen G-Q, Jiang Y, Chen F (2007) Fatty acid and lipid class
composition of the eicosapentaenoic acid-producing microalga
Nitzschia laevis. Food Chem 104:15801585
Kangani CO, Kelley DE, DeLany JP (2008) New method for GC/
FID and GCC-IRMS analysis of plasma free fatty acid concentration and isotopic enrichment. J Chrom B 873:95101
Spanner S (1973) Separation and analysis of phospholipids. In:
Ansell GB, Hawthorne JN, Dawson RMC (eds) Form and function of phospholipids. Elsevier, Amsterdam, pp 4365
Hanahan DJ (1960) Lipide Chemistry. Wiley, New York
Hamilton S, Hamilton RJ, Sewell PA (1992) Extraction of lipids
and derivative formation. In: Hamilton RJ, Hamilton S (eds)
Lipid analysis, a practical approach. Oxford University Press,
New York, pp 1364
Lee CM, Trevino B, Chaiyawat M (1996) A simple and rapid
solvent extraction method for determining total lipids in fish
tissue. J AOAC Int 79:487492
Henderson RK, Baker A, Parsons SA, Jefferson B (2008) Characterisation of algogenic organic matter extracted from cyanobacteria, green algae and diatoms. Wat Res 42:34353445