Eur. J. Lipid Sci. Technol. 107 (2005) 381–386
Gianpaolo Andricha
Ugo Nestib
Francesca Venturia
Angela Zinnaia
Roberto Fiorentinia
a
Dipartimento di Chimica e
Biotecnologie Agrarie,
Università di Pisa, Pisa, Italy
b
ICRAM, Istituto Centrale
per la Ricerca Scientifica e
Tecnologica Applicata al Mare,
Roma, Italy
DOI 10.1002/ejlt.200501130
381
Supercritical fluid extraction of bioactive lipids from
the microalga Nannochloropsis sp.
Marine microalgae are recognised as an important renewable source of bioactive lipids
with a high proportion of polyunsaturated fatty acids (PUFA), which have been shown
to be effective in preventing or treating several diseases. For the extraction of oil from
microalgae, supercritical CO2 (ScCO2) is regarded with interest, being safer than hexane and offering a negligible environmental impact, a short extraction time and a highquality final product. Whilst some experimental papers are available on the supercritical fluid extraction (SFE) of oil from microalgae, only limited information exists on
the kinetics of the process. In such a contest, a mathematical model able to describe
the kinetics of the SFE was applied to the recovery with ScCO2 of lipids from Nannochloropsis sp., a marine microalga commonly used in aquaculture and characterised
by a lipid fraction with a high PUFA content. The aim of this paper was to examine the
effect of operating conditions on the kinetics of the SFE, on process yields and on the
fatty acid composition of lipid extracts.
Keywords: Microalgae, Nannochloropsis, bioactive lipids, supercritical fluids, extraction kinetics.
Polyunsaturated fatty acids (PUFA), especially n-3 PUFA
such as a-linolenic acid (ALA, C18:3n-3), eicosapentaenoic acid (EPA, C20:5n-3), docosapentaenoic acid (DPA,
C22:5n-3), and docosahexaenoic acid (DHA, C22:6n-3),
have been shown to be effective in preventing or treating
several diseases including cardiovascular disorders,
cancer, type 2 diabetes, inflammatory bowel disorders,
asthma, arthritis, kidney and skin disorders, depression
and schizophrenia [1–5]. Moreover, many epidemiological
studies evidence that the current diet of the most economically advanced countries does not adequately cover
the daily requirement for such bioactive fatty acids.
Marine fish lipids are the main conventional source of n-3
PUFA used in functional food, nutraceuticals and pharmaceuticals, but microalgae are recognised as an additional important source [5]. When compared to fishery
and related food industries, microalgal cultivation presents the advantage to use an indefinitely renewable
resource having a negligible environmental impact.
Moreover, the growth of microalgae in batch or in photo-
Correspondence: Roberto Fiorentini, Dipartimento di Chimica e
Biotecnologie Agrarie, Università di Pisa, Via del Borghetto 80,
I-56124 Pisa, Italy.
E-mail: robfior@agr.unipi.it
Fax: 139-050-598614
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
bioreactors is carried out under highly controlled conditions, allowing the production of uncontaminated strains
having a standardised composition, with particular reference to their n-3 PUFA content [6]. On the other hand,
cultivation of microalgae is technically demanding and
costly.
Although the oil extraction from lipid-bearing biomasses
is normally carried out by using organic solvents, such as
hexane, chloroform, acetone and methanol, supercritical
CO2 (ScCO2) is regarded with interest as an industrial
process, being safer, offering mild operating conditions,
no environmental impact, shorter extraction time and a
high-quality final product without any trace of toxic solvent [7]. Some experimental papers on the supercritical
fluid extraction (SFE) of lipids from microalgal strains,
including Spirulina (Arthrospira) maxima, Spirulina
(Arthrospira) platensis, Botryococcus braunii, Chlorella
vulgaris, Ochronomas danica, Skeletonema costatum and
Isochrysis galbana, are available, but only limited information exists on the kinetics of the process and on the
influence of operating conditions on the fatty acid composition of lipid extracts [8–13]. In such a contest, a
mathematical model able to describe the kinetics of an
SFE process was applied to the extraction with ScCO2 of
lipids from Nannochloropsis sp., a marine microalga
commonly used in aquaculture either as individual diet or
as component of a mixed diet. It is characterised by a
particularly high lipid content and it is considered one of
the most promising EPA producers [14–19].
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Research Paper
1 Introduction
382
G. Andrich et al.
The aim of this paper was to examine the effect of operating conditions on the kinetics of the SFE, on process
yields and on the fatty acid composition of lipid extracts.
2 Materials and methods
2.1 Materials
Fresh biomass of the unicellular microalga Nannocloropsis sp., phototrophically grown and withdrawn during
the stationary growth phase, was supplied by Reed Mariculture Inc. (San Jose, CA, USA). Wet cells (82.8% of
moisture) were lyophilised, ground and sieved using a
standard sieve of 42 mesh of the Tyler series. Classified
material (particle size 0.37 mm) was stored under nitrogen at 220 7C until use.
2.2 Oil extraction
ScCO2 extractions of lipids were performed with commercial-grade CO2 using a pilot plant apparatus (Sitec,
Maur, Switzerland) described in a previous paper [20]. An
amount of 180 g microalga sample, mixed with 100 g of
glass microspheres (size about 3 mm), was utilised per
each run, with a working pressure (P) of 40, 55, or 70 MPa
and a temperature (T) of 40 or 55 7C. The flow rate of
ScCO2 (F) was maintained constant and equal to
10 kg h21 (Micro Motion Coriolis flow-meter; accuracy
60.2%; repeatability 60.05%), and the extraction time
was fixed to 6 h. Lipid fractions were collected after a
predetermined amount of co2, ranging from 0.8 to 60 kg,
had passed through the bed of microalgae. The extraction yields were determined gravimetrically. Extraction by
percolation with n-hexane for 6 h was also performed,
using a Soxhlet apparatus.
2.3 Fatty acid analysis
Lipid extracts were transmethylated with methanol-acetyl
chloride, as described by Cohen et al. [21], in the presence of an appropriate amount of heptadecanoic acid as
an internal standard. GLC analysis was performed using a
fused-silica gel capillary column, 25 m 6 0.32 mm, Carbowax 0.10–0.15 mm, heated at 195 7C. The temperature
of injector and flame-ionisation detector was fixed at
230 7C, and H2 was used as carrier gas (3 ml min21). Fatty
acid methyl esters were identified by co-chromatography
with authentic standards of Sigma-Aldrich S.r.l. (Milan,
Italy) and by calculation of the equivalent chain length
[22]. The data reported represent mean values with a
range of less than 5% (major peaks) or 10% (minor peaks)
of two independent samples, each analysed in duplicate.
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Lipid Sci. Technol. 107 (2005) 381–386
3 Results and discussion
3.1 Extraction kinetics
To optimise the extraction parameters, a kinetic approach
developed during a previous research activity was adopted [23–25]. This approach, based on the diffusion Fick’s
law, uses the following exponential equation to describe
the evolution of extracted oil over time (t):
Oe = H* [Os] (12e2kt)
(1)
where Oe is the amount (g) of oil extracted at a random
time, t, per gram of microalgal biomass submitted to SFE
(adimensional); H* is the adimensional constant, ranging
from 0 to 1, related to the equilibrium constant H, i.e.
H* = H/(H 1 1); [Os] is the amount (g) of oil present in 1 g
of starting material (adimensioal); and k is the kinetic
constant (s21).
The extraction rate (R) calculated as first derivative of the
exponential equation (1):
R = dOe/dt = H* [Os] k e2kt
(2)
reaches its maximum value (Rmax) at the beginning of the
extraction, when t is close to 0:
Rmax = k H* [Os]
(3)
According to Yu et al. [26], the value of Rmax (s21) was
assumed as an index to evaluate the efficiency of the SFE
system versus the oil fraction of microalgae. In particular,
while the constant k gives information on the kinetics of
the SFE, the product H* 6 [Os], which represents the
asymptotic value assumed by the extraction curve when
t ? ?, measures the maximal amount of oil theoretically
extractable under the working conditions adopted. In the
presence of a highly efficient SFE process, H* tends to 1,
and therefore the maximum amount of oil extractable per
gram of biomass is equal to the concentration of oil in the
starting material. The identification of the best fitting
values to be assigned to the equation parameters k and
H* 6 [Os] was carried out by a commercially available
statistical program (BURENL) described in a previous
paper [27].
Whilst Fig. 1 reports the experimental points and the theoretical evolution of the lipids extracted from Nannochloropsis sp. as a function of the run time and working
conditions (T and P) adopted, in Tab. 1 are reported the
values assumed by Rmax and by the functional parameters
H* 6 [Os] and k.
The equation parameter H* 6 [Os] assumes for all SFE
very similar values, which are also close to that obtained
when percolation with n-hexane is adopted. This means
that on the basis of the amount of oil extractable at
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Eur. J. Lipid Sci. Technol. 107 (2005) 381–386
Supercritical fluid extraction of lipids from Nannochloropsis
383
Fig. 1. SFE of lipids from Nannochloropsis sp.
Lipids extracted (mg g21 of lyophilised sample)
as a function of the run time and working conditions (pressure and temperature) adopted.
Experimental points [(d) 70 MPa; (n) 55 MPa;
(r) 40 MPa)] and theoretical evolution (–––).
Curve (.....) and experimental points (m) related
to the extraction run carried out by percolation
with n-hexane (52 7C and room pressure) are
also reported in the bottom part of the figure.
Tab. 1. SFE of lipids from Nannochloropsis sp. Maximum extraction rate (Rmax) and values of the equation parameters k
and H* 6 [Os] as a function of the temperature (T) and the pressure (P) adopted. Data in the last row refer to the traditional
extraction carried out by percolation with n-hexane. c.i. = confidence interval (p = 0.05); r = correlation coefficient.
T [ 7C]
P [MPa]
0
40
40
55
40
70
55
40
55
55
55
70
Soxhlet extraction
(Rmax 6 c.i.) 6 106 [s21]
(k 6 c.i.) 6 104 [s21]
(H* 6 [Os] 6 c.i.) 6 103
(adimensional)
r2
39.38 6 0.65
74.98 6 0.99
107.76 6 1.37
49.58 6 0.67
86.94 6 1.03
118.04 6 1.39
17.44 6 0.47
1.57 6 0.02
2.97 6 0.03
4.20 6 0.04
1.99 6 0.02
3.45 6 0.03
4.58 6 0.04
0.70 6 0.01
250.84 6 0.99
252.45 6 0.81
256.58 6 0.85
249.13 6 0.77
252.00 6 0.80
257.75 6 0.80
249.13 6 2.49
0.99
0.99
0.98
0.99
0.99
0.98
0.99
equilibrium (t ? ?), all processes (ScCO2 extractions
and percolation with n-hexane) are substantially equivalent. This result disagrees with those reported by Perretti
et al. [13] and Mendes et al. [12], related to the SFE of oil
from Isochrysis galbana and Spirulina (Arthrospira) maxima, respectively. These authors report in fact a higher
extraction yield when working with n-hexane instead of
ScCO2, albeit SFE was carried out under different conditions.
The values assumed by the kinetic constant k when
working at the same T reveal that P highly affects the
kinetics of SFE. In particular, for both T adopted, the value
of k increases markedly when P passes from 40 to
55 MPa, while the increases are lower passing from 55 to
70 MPa. Also the increase of T affects the kinetics of SFE
(see values of k when working at the same P), but this
influence appears relatively low. It is, however, the binomial P-T that affects, more than P and Talone, the kinetics
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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384
G. Andrich et al.
Eur. J. Lipid Sci. Technol. 107 (2005) 381–386
Tab. 2. Values of parameters involved in the Chrastil equation adopted to relate the maximum extraction rate (R*max) to the
density of ScCO2 (r). T = temperature (K); c.i. = confidence interval (p = 0.05); r = correlation coefficient.
Chrastil equation
a 6 c.i.
-(b 6 c.i.)
-(c 6 c.i.)
r2
R*max = ra e(b/T 1 c)
10.92 6 2.57
3506.57 6 1225.65
62.66 6 16.18
0.97
of the SFE process. In fact, the value of k increases three
times, passing from the mildest (40 7C and 40 MPa) to the
hardest (55 7C and 70 MPa) conditions.
Under all conditions adopted, the extraction with ScCO2
was faster than that carried out by percolation with nhexane, as testified by the values assumed by k and/or
Rmax. For example, when working under the hardest SFE
conditions (55 7C and 70 MPa), the rate at the beginning
of the extraction (Rmax) is about seven times that measured when performing the extraction with n-hexane.
To relate Rmax to the density (r) of ScCO2, the value of
which is influenced by both Tand P adopted, the equation
introduced by Chrastil in 1982 was adopted [28]. The
equation is reported in Tab. 2, together with the values of
the equation parameters a, b and c, calculated by applying the statistical program BURENL introduced above
and using the values of Rmax reported in Tab. 1. Each
value was previously expressed in g l21 by using the following mathematical relation:
R*max (g l21) = Rmax (s21) m r F21
(4)
where R*max is Rmax expressed in grams of extracted lipids
per litre of ScCO2 flowed through the bed of microalgae;
m is the amount of lyophilised microalgae submitted to
each SFE run (g); r is the density of ScCO2 at T and P
adopted (g l21); and F is the flow rate of ScCO2 (g s21).
Fig. 2. SFE of lipids from Nannochloropsis sp. Evolution
of R*max (g l21) versus the reduced density [rr =
r rc21 = real density/density at critical point (468 g l21)] of
ScCO2 as a function of three different temperatures (40,
50, 60 7C).
3.2 Fatty acid composition
In Fig. 2, the theoretical evolution of R*max is plotted
against the reduced density of ScCO2 as a function of
three different temperatures (40, 50, 60 7C).
Data reported in Tabs. 1 and 2 are of great practical interest, allowing the calculation of the extraction yield for any
value of P, T and t adopted. All data are in agreement with
those previously determined for the SFE of oil from some
oilseeds (sunflower, soybean, rapeseed) and obtained
under relatively close experimental conditions [23–25].
This consideration, together with the high values
assumed by the square of the correlation coefficient, testifies the suitability of the hypotheses introduced and
gives a measure of the validity of the kinetic model proposed.
Tab. 3 reports the fatty acid composition (wt-% of total
fatty acids) of Nannochloropsis lipids as a function of the
extraction conditions. Data tabled confirm that EPA is the
major fatty acid. Depending on the extraction conditions
adopted, its share of total fatty acids ranges from 29.4%
to 33.0%. Such values are lower than that reported by
Red Mariculture for the commercial product (37%). This
could be partially due to the fact that by using ScCO2, we
could have some losses in the most polar lipid fractions
(e.g. glycolipids) where EPA seems primarily located [29];
in addition, cultures of Nannochloropsis sp. withdrawn
during the stationary phase, like that used in this study,
are reported to have a lower EPA content than those
otherwise collected [17]. Other major fatty acids are, in
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Eur. J. Lipid Sci. Technol. 107 (2005) 381–386
Supercritical fluid extraction of lipids from Nannochloropsis
385
Tab. 3. Fatty acid composition of Nannochloropsis lipids as a function of extraction conditions (data expressed as wt-% of
total fatty acids).
ScCO2
40 7C
40 MPa
ScCO2
40 7C
55 MPa
ScCO2
40 7C
70 MPa
ScCO2
55 7C
40 MPa
ScCO2
55 7C
55 MPa
ScCO2
55 7C
70 MPa
Saturated
C14:0
C16:0
C18:0
Subtotal
3.5
19.8
2.2
25.5
5.1
19.1
2.0
26.2
5.0
18.9
2.0
25.9
4.6
19.0
2.1
25.7
5.7
17.8
1.8
25.3
6.5
17.8
1.8
26.1
4.3
19.2
2.1
25.6
Monoenoic
C14:1
C16:1n-7
C18:1n-9
Subtotal
6.0
14.2
3.8
24.0
5.9
12.9
3.0
21.8
6.8
12.7
3.2
22.7
6.8
13.0
3.4
23.2
6.1
11.4
2.6
20.1
6.9
11.0
2.9
20.8
5.5
12.9
3.5
21.9
Polyunsaturated
C18:2n-6
C18:3n-3 (ALA)
C20:4n-6 (ARA)
C20:5n-3 (EPA)
C22:5n-3 (DPA)
C22:6n-3 (DHA)
Subtotal
5.5
8.4
5.7
29.4
1.0
0.5
50.5
4.6
8.3
6.4
31.1
1.2
0.4
52.0
5.0
7.2
6.0
31.5
1.3
0.4
51.4
5.4
8.1
5.7
30.2
1.3
0.4
51.1
5.2
9.2
5.0
33.0
1.6
0.6
54.6
5.1
8.5
5.3
32.1
1.6
0.5
53.1
4.6
9.8
5.3
31.1
1.1
0.6
52.2
Total
Total n-3 PUFA
100.0
39.3
100.0
41.0
100.0
40.4
100.0
40.0
100.0
44.0
100.0
42.7
100.0
42.6
Fatty acids
decreasing order, C16:0, C16:1n-7 and C18:3n-3 (ALA),
while the long-chain n-3 PUFA content is always
approaching 40% of total fatty acids.
Data in Tab. 3 are in agreement with those reported by
Seto et al. [14] for Chlorella minutissima (later identified as
Nannochloropsis oculata), but disagree with those presented in other analytical works reporting for the same
microalga a substantially different fatty acid profile, with a
particularly low EPA level [16, 17]. On the other hand, the
fatty acid composition of microalgae is reported to be
highly changeable as a function of the culture conditions
adopted [30].
No particular differences were found in the fatty acid profiles of extracts when different SFE were performed, also
in comparison with the traditional extraction with n-hexane. Moreover, a slight increase in EPA and DPA, with a
corresponding decrease in C16:0, C16:1, C18:0 and
C18:1, seems perceivable while passing from the mildest
to the hardest SFE conditions. Such a trend needs, however, further investigations.
 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
n-hexane
(Soxhlet)
4 Conclusions
This research is part of a wider experimental project involving the SFE of bioactive lipids from microalgae and
having two main objectives: (a) to test the validity of the
mathematical model proposed to describe the kinetics of
extraction, the knowledge of which is required for process
optimisation and scaling-up; (b) to evaluate the economical feasibility of such a process. On the basis of the results
obtained, the kinetic model seems suitable, even if for its
generalisation more information is needed. While ScCO2
and n-hexane have been shown to be comparable on the
basis of the theoretical process yield (t ? ?) and the fatty
acid composition of the extracts, SFE proved to be much
faster. Although both solvents gave process yields lower
than expected, probably as a result of some losses in the
most polar lipid fractions, Nannochloropis sp. is confirmed
as a good source of bioactive fatty acids.
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[Received: January 4, 2005; accepted: April 19, 2005]
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