aagr.57.2009.2.17

Acta Agronomica Hungarica, 57(2), pp. 249–253 (2009)
DOI: 10.1556/AAgr.57.2009.2.17
Short communication
FAST AND UNAMBIGUOUS DETERMINATION OF EPA AND
DHA CONTENT IN OIL OF SELECTED STRAINS OF ALGAE
AND CYANOBACTERIA
B. CHRISTIAN1, B. LICHTI1, O. PULZ2, C. GREWE3 and B. LUCKAS1
1
INSTITUTE OF NUTRITION, FRIEDRICH-SCHILLER-UNIVERSITY OF JENA, JENA, GERMANY;
2
IGV FOR CEREAL PROCESSING LTD., NUTHETAL, GERMANY;
3
SALATA LTD, RITSCHENHAUSEN, GERMANY
Received: 7 January, 2009; accepted: 20 April, 2009
Microalgae may contain large quantities of high-quality EPA and DHA. Therefore,
they are considered as a potential source of these important fatty acids. Microalgae can be
grown autotrophically on cheap substrates with light. This type of cultivation can be used
to maximize the EPA and DHA content in microalgae, making the production of EPA and
DHA possible on a large scale. In the present study ten different microalgae were screened
for EPA and DHA contents as possible candidates for cultivation in bioreactors.
Key words: microalgae, fatty acids, GC, GC-MS
Introduction
Human physiology depends in various ways on polyunsaturated fatty
acids (PUFAs), either as components of membrane phospholipids in specific
tissues or as precursors of hormone-like compounds known as eicosanoids (Patil
and Gislerød, 2006; Jump, 2002), which have a number of nutraceutical and
pharmaceutical applications (Shahidi and Wanasundara, 1998; Horrocks and
Yeo, 1999). Eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid
(DHA, C22:6n-3) are important n-3 PUFAs, while arachidonic acid (AA,
C20:4n-6) is a vitally important n-6 PUFA. EPA and DHA show beneficial
effects in the courses of diseases like arthrosclerosis, cancer, rheumatic arthritis,
psoriasis and diseases of old age such as Alzheimer’s and age-related macular
degeneration (Devron et al., 1993; Simopoulos et al., 1999).
Fish oils are the major source of PUFAs, and considerable evidence has
indicated that the n-3 PUFAs in fish oils are actually derived via the marine food
chain from zooplankton that consume n-3 PUFA-synthesizing microalgae
(Yongmanitchai and Ward, 1989). Linoleic acid (LA, C18:2n-6) and α-linolenic
acid (ALA, C18:3n-3) are predominant in green vegetables and some plant oils.
0238–0161/$ 20.00©2009 Akadémiai Kiadó, Budapest
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B. CHRISTIAN et al.
Although some research (Carnielli et al., 1996; Salem et al., 1996) has
determined qualitatively that humans can convert the parent ALA to EPA and
then to DHA, the most recent consensus is the degree of conversion is
‘unreliable and restricted’ (Gerster, 1998).
As fish oil fails to meet the increasing demand for purified n-3 fatty acids,
the demand for alternative sources is increasing. Microalgae may contain large
quantities of high-quality EPA and they are considered a potential source of this
important fatty acid. Microalgae grow autotrophically on cheap substrates with
light. This mode of cultivation can be well controlled and provides a possibility
to maximize EPA production on a large scale. Numerous strategies have been
investigated for the commercial production of EPA by microalgae. These
include screening for high EPA-yielding microalgal strains, strain improvement
by genetic manipulation, optimization of culture conditions and development of
efficient cultivation systems (Wen and Chen, 2003). In the present study ten
different microalgae were screened for their EPA and DHA content. Problems
arising during the analysis of the fatty acid profile are also discussed.
Materials and methods
The microalgal strains were cultivated in 2 L glass bubble columns 8 cm in diameter, at a
temperature of 25°C, an aeration rate of 2 vvm (mixture of air and 2% carbon dioxide v/v) and a
light intensity of 70 µE m–2 s–1. BG-11 medium was used for the cyanobacterium (Allen, 1959),
and diatom f/2 medium for the diatoms, according to Guillard and Ryther (1962). Porphyridium
was cultivated in the medium reported by Sommerfield and Nichols (1970). The AF6 medium was
used for Chlorella sorokiniana, Scenedesmus pectinatus, Monodus subterraneus and Neochloris
oleoabundans according to Kato (1982), while Chlorella minutissima and Nannochloropsis sp.
were cultivated using an enriched natural seawater medium (Provasoli, 1968).
The procedure for sample preparation consisted of lipid extraction at room temperature with
chloroform, methanol and water according to the method of Bligh and Dyer (1959). Ten different
strains of microalgae were lyophilized and 40 mg of the freeze-dried powders were used for lipid
extraction with a mixture of 7.6 mL MeOH/CHCl3/H2O (2:1:0.8 v:v:v). After final removal of the
solvents in a gentle stream of nitrogen, a green, crude extract was obtained. The derivatization was
done by the introduction of 150 µL toluene and 100 µL trimethyl-sulphonium hydroxide (TMSH)
reagent. The vial was gently hand-shaken until a homogeneous solution was obtained.
The analysis of fatty acids with gas chromatography (GC) was carried out using a HewlettPackard HP 5890 Series II gas chromatograph (Agilent Technologies, Waldbronn, Germany)
equipped with a split/splitless injector and an Agilent 7673 series auto-sampler. Detection involved
a combination of flame ionization detection (FID) and mass spectrometry (MS) (MS Engine HP5989B, Agilent Technologies, Waldbronn, Germany). The separation of fatty acid methyl esters
was achieved on a SP2380 fused silica column (Supelco, Bellefonte, PA, USA) 60 m × 0.32 mm
i.d., 0.2 µm film thickness. The injection volume was 2 µL at a split ratio of 1:30.
Results
As a consequence of overloading the capillary column, the retention times
of the respective signals for EPA in the sample chromatograms (GC-FID) shifted
compared to the retention times of a standard solution containing several fatty
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EPA AND DHACONTENTS OF ALGAE AND CYANOBACTERIA
251
acids. Therefore, the algorithm for automatic integration included in the software
package was no longer able to denote these high area peaks as EPA. Thus, the
chromatograms of Phaeodactylum tricornutum, Nannochloropsis sp. and
Chlorella minutissima suggested the absence of EPA and the presence of C24:0
instead (Table 1). In order to remedy these deficiencies, all samples were
reanalyzed by GC-MS in scan mode (Fig. 1), when the presence or absence of
each fatty acid could be unambiguously determined. The results obtained with
GC-MS were used to modify and refine the algorithm for the automatic
integration of GC-FID. The exact quantitation of EPA and DHA was then
possible from the signals obtained with GC-FID (Table 2).
Table 1
Percentage of EPA in selected microalgae before and after consideration of GC-MS data
Algae
Thalassiosira punctigera
Neochloris oleoabundans
Phaeodactylum tricornutum
Nannochloropsis sp.
Nostoc sp.
Chlorella minutissima
Chlorella sorokiniana
Monodus subterraneus
Scenedesmus pectinatus
Porphyridium cruentum
Results (%)a
EPAb
EPAc
18.919
0.824
not detected
not detected
not detected
not detected
0.125
31.567
0.962
16.188
18.948
0.521
19.818
21.096
not detected
24.910
0.171
20.169
0.411
15.883
a
: percentage of total fatty acids; b: results obtained from GC-FID without consideration of GC-MS
data; c: results obtained from GC-FID with consideration of GC-MS data
Table 2
EPA and DHA content in selected microalgae µg/g dry weight
Algae
Thalassiosira punctigera
Neochloris oleoabundans
Phaeodactylum tricornutum
Nannochloropsis sp.
Nostoc sp.
Chlorella minutissima
Chlorella sorokiniana
Monodus subterraneus
Scenedesmus pectinatus
Porphyridium cruentum
Content (µg/g)
EPA
DHA
3441.8
189.5
12173.6
18170.4
not detected
30088.5
30.6
3170.4
159.2
2930.6
675.2
169.8
1121.0
118.1
114.2
111.7
87.1
99.1
115.8
91.8
Acta Agronomica Hungarica, 57, 2009
252
B. CHRISTIAN et al.
Fig. 1 (A) GC-MS of Chlorella minutissima – TIC; (B) mass spectrum of peaks at a retention time
of 33.3 min in chromatogram A; (C) GC-MS of an EPA standard solution (1766 µmol/L in
toluene) – TIC; (D) mass spectrum of peaks at a retention time of 33.5 min in chromatogram C
Discussion
It was shown that the retention times in fatty acid profiles may shift
depending on the amounts injected. Especially when large amounts are injected,
an overload may result, with higher retention times of the peaks. The algorithms
for automatic integration implemented in the software packages are then no
longer capable of denoting the corresponding signals correctly. In such cases, the
presence of each fatty acid should be confirmed by mass spectrometry and the
calibration files containing the retention times of each signal should then be
modified.
The screening of ten different microalgae for EPA and DHA contents
revealed three candidates with high amounts of these poly-unsaturated fatty
acids (PUFAs): Phaeodactylum tricornutum, Nannochloropsis sp. and Chlorella
minutissima. These three species of microalgae had contents between 12 and
30 mg/g for EPA and 112 to 1121 µg/g for DHA (Table 2). The EPA contents
obtained for the other seven species investigated were substantially lower (< 3.5
mg/g dry weight), while the DHA content in the other species ranged from
87 µg/g (Chlorella sorokiniana) to 675 µg/g (Thalassiosira punctigera).
Therefore, Phaeodactylum tricornutum, Nannochloropsis sp. and Chlorella
minutissima are promising candidates for the exploitation of “PUFA-rich” oils
through the cultivation of microalgae in bioreactors. Thalassiosira punctigera
would be another option if oils with a high percentage of DHA were favoured.
Acta Agronomica Hungarica, 57, 2009
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253
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Corresponding author: B. Christian
Phone: +49 (0) 36 41 – 94 96 54
Fax: +49 (0) 36 41 – 94 96 52
E-mail: b1chbe@uni-jena.de
Acta Agronomica Hungarica, 57, 2009