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 250 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 Acta Agronomica Hungarica, 57, 2009 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 EPA AND DHACONTENTS OF ALGAE AND CYANOBACTERIA 253 References Allen, M. B. (1959): Studies with Cyanidium caldarium, anomalously pigmented Chlorophyte. Arch. Microbiol., 32, 270–277. Bligh, E. G., Dyer, W. J. (1959): A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911–917. Carnielli, V. P., Wattimena, D. J. L., Luijendijk, I. H. T., Boerlage, A., Degenhart, H. J., Sauer, P. J. J. (1996): The very low birth weight premature infant is capable of synthesizing arachidonic and docosahexaenoic acids from linoleic and linolenic acids. Pediatr. Res., 40, 169–174. Devron, C. A., Baksaas, I., Krokan, H. E. (1993): Omega-3 Fatty Acids: Methods and Biological Effects. Birkhauser Verlag, Basel, 389 p. Gerster, H. (1998): Can adults adequately convert alpha-linolenic acid to eicosapentaenoic acid and docosahexaenoic acid. Int. J. Vitam. Nutr. Res., 68, 159–173. Guillard, R. R. L., Ryther, J. H. 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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
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